[보고서]바이오활성가스를 이용한 염증성 대사질환 제어 원천기술 개발

정헌택

[국가R&D연구보고서] 바이오활성가스를 이용한 염증성 대사질환 제어 원천기술 개발
Development of core techniques for inflammatory metabolic disease using Bio-Active Gas 원문보기

보고서 정보
주관연구기관	울산대학교 University of Ulsan
연구책임자	정헌택
보고서유형	최종보고서
발행국가	대한민국
언어	한국어
발행년월	2018-01
과제시작연도	2016
주관부처	과학기술정보통신부 Ministry of Science and ICT
연구관리전문기관	한국연구재단 National Research Foundation of Korea
등록번호	TRKO201900027284
과제고유번호	1711042254
사업명	바이오.의료기술개발
DB 구축일자	2020-09-19
키워드	바이오활성가스.대사증후군.인플라마좀.소포체스트레스.당뇨병.Bio-active gas (BAG).metabolic syndrome.inflammasome.endoplasmic reticulum stress.Type 2 disbetes.

초록 ▼

- 동물 모델에서 CO가 GSK3 활성을 억제함으로써 ER stress에 의한 염증을 억제함을 확인
- CO가 항염증 기능이 있는 TTP 발현을 유도하며, 발현된 TTP가 염증성 사이토카인들의 mRNA 분해를 촉진하여 염증을 억제함을 확인함
- CO가 고지방 식이에 의하여 유도된 leptin 저항성을 완화시킴을 확인
- 고지방 식이와 streptozotocin에 의하여 당뇨병이 유도된 생쥐에 CO 가스를 투입시 사이토카인 생산 및 염증이 억제됨을 확인
- 고지방식이로 유도된 비만쥐 모델에서 quercetin이 지방간 염증을 억제함을 확인
- Cilostazol과 probucol이 지방간질환을 개선함을 확인
- CO가 FGF21 발현 유도를 통하여 고지방식으로 유도된 간질환을 개선함을 확인
- 테로스틸벤 4’-베타-글루코시드(Pterostilbene 4’-b-Glucoside, 4-PG)가 염증성장질환을 개선시킴을 확인
- 케르세틴이 HO-1유도 작용을 통하여 비만성 시상하부 염증 개선 효과 확인
- CO가 고지방식이로 증가된 간의 지방을 분해하는 효과 in vivo확인

(출처 : 보고서 요약서 3p)

Abstract ▼

Purpose & Contents
Purpose
‣ This study is designed to develop a preventive and/or curing technique for metabolic diseases.
‣ The pathway of pathophysiology of metabolic diseases starts with high fat diet results in metabolic diseases.
‣ During the course of the way, there are endoplasmic reticulum stress due to high calory in various tissues including adipose tissue and metabolic inflammation due to ER Stress.
‣ We will try to block the pathophysiologic pathway or treat the metabolic diseases by using Bio-Active Gas (BAG)
Contents
‣ Blocking inflammasome assembly and formation of inflammatory mediators
- Inhibition of inflammasome priming and activation with BAG
- Attenuation of pro-inflammatory cyto-chemokines with BAG
- Potentiation of anti-inflammatory cytokines with BAG
‣ Inhibition of type 2 diabetes induced by inflammatory cytokines
- inhibition of insulin resistance caused by inflammatory mediators
- Cytoprotection of pancreatic β-cells
- Technique for the delivery of BAG to animals
‣ Suppressing inflammation and type 2 diabetes in high fat diet (HFD) feeding animals with BAG
- Inhibition of inflammation in adipose tissues caused by HFD
- Inhibition of obesity caused by HFD
- Inhibition of insulin resistance and type 2 diabetes caused by HFD
‣ In vitro validation analysis for inhibitory effects of BAG-producing compounds on inflammation and insulin resistance
- Validation analysis for inhibition of inflammasome priming
- Validation analysis for inhibition of insulin resistance
- Validation analysis for cytoprotection of pancreatic β-cells
‣ In vivo validation analysis for inhibitory effects of BAG-producing compounds on type 2 diabetes
- Technique for the delivery of BAG-producing compound to animals
- Validation analysis for inhibition of inflammation caused by HFD
- Validation analysis for inhibition of obesity and type 2 diabetes caused by HFD

Results
- CO suppressed the ER-stress-induced inflammation through inhibition of GSK3 activity in animal model.
- CO induced the expression of TTP and, in turn, the TTP suppressed inflammation through enhancing the decay of inflammatory cytokine mRNAs.
- CO attenuated the leptin-resistance induced by high fat diet.
- CO suppressed the production of cytokines and the inflammation in diabetic mouse induced by high fat diet and streptozotocin.
- Quercetin inhibited fatty liver inflammation in high fat diet-fed obese mouse model.
- Cilostazol and probucol attenuated the fatty liver disease in animal model
- CO induced FGF21 expression and attenuated fatty liver disease induced by high fat diet.
- Pterostilbene 4’-b-Glucoside (4-PG) attenuated the DSS-induced colitis in animal model.
- Quercetin induced HO-1 expression and attenuated the inflammatory response of lipid-laden microglia.
- CO enhanced the lipolysis in high fat diet-fed fatty liver animal model.

Expected Contribution
‣ Establishment of blocking technique of inflammation in HFD fed animal based on comprehensive knowledge of the pathophysiological pathway of metabolic syndrome with BAG
‣ Establishment of preserving pancreatic β-cells from ER Stress and meta-inflammation with BAG
‣ Establishment of background technique for the amelioration of m-inflammation and treatment of metabolic syndrome with minimal side effects by BAG

(출처 : SUMMARY 6p)

목차 Contents

표지 ... 1
제 출 문 ... 2
보고서 요약서 ... 3
국문 요약문 ... 4
SUMMARY ... 6
Contents ... 8
목차 ... 8
제1장. 연구개발과제의 개요 ... 9
제2장. 국내외 기술 개발 현황 ... 15
제3장. 연구 수행 내용 및 성과 ... 24
제4장. 연구개발성과의 활용계획 ... 134
1절 연구개발결과의 활용방안 ... 134
2절 기대성과 ... 134
제5장. 연구 과정에서 수집한 해외 과학기술 정보 ... 136
제6장. 연구개발성과의 보안등급 ... 136
제7장. 국가과학기술종합정보시스템에 등록한 연구시설·장비 현황 ... 136
제8장. 연구개발과제 수행에 따른 연구실 등의 안전 조치 이행 실적 ... 137
제9장. 연구개발과제의 대표적 연구 실적 ... 138
제10장. 기타 사항 ... 139
제11장. 참고 문헌 ... 139
끝페이지 ... 140

표/그림 (132)

표 비만 및 대사질환의 관계
표 대표적 대사질환 치료제의 특징 및 부작용
표 대사성 만성질환과 관련된 대사질환
표 인플라마솜의 형성과 활성화 기전
표 인플라마솜의 활성화와 관련된 대사질환
표 식이유도 인슐린 저항성에 있어서 JNK1의 작용 기전
표 CO reverses endoplasmic reticulum (ER) stress-induced leptin resistance in SK-N-AS-Ob-Rb cells. A: SK-N-AS-Ob-Rb cells were preincubated with CO-releasing molecule (CORM)-2 at indicated concentrations (0–20 μM) for 2 h and then treated with tunicamycin (TM; 2 μg/ml) or vehicle for 4 h and then stimulated with leptin (10 μg/ml) for 15 min. B: Chinese hamster ovary (CHO)-K1 cells were treated with IL-6 (25 ng/ml) and leptin (10 μg/ml); CHO-K1-Ob-Rb cells were incubated with leptin at indicated concentrations (1–10 μg/ml) for 15 min. C: CHO-K1-Ob-Rb cells were treated with thapsigargin (TG; 1 μM), TM (2 μg/ml), CORM-2 (20 μM), TG (1 μM) + CORM-2 (20 μM), or TM (2 μg/ml) + CORM-2 (20 μM) for 4 h, and expression levels of Ob-R were analyzed by Western blotting; β-actin served as the standard. D: SK-N-AS-Ob-Rb cells were preincubated with 4-phenylbutyric acid (4-PBA; 5 mM) or CORM-2 (20 μM) for 2 h and then treated with TM (2 μg/ml) or vehicle for 4 h and subsequently stimulated with leptin (10 μg/ml) for 15 min. A, B, and D: phospho-(p-)STAT3 and STAT3 levels were analyzed by Western blotting. Densitometric analysis of p-STAT3 relative to STAT3 expression is shown; n = 3. ***P < 0.001, **P <0.01
표 CO induces activation of the protein kinase R-like endoplasmic reticulum kinase (PERK) branch of the ER stress response in SK-N-AS-Ob-Rb cells. A and B: SK-N-AS-Ob-Rb cells were incubated with CORM-2 (20 μM) or TM (2 μg/ml) or TG (1 μM) for indicated periods of time, and expression levels of p-PERK, p-eIF2α (eukaryotic initiation factor 2α), and p-IRE1α (inositol-requiring transmembrane kinase/endonuclease-1α) were analyzed by Western blotting. The unphosphorylated forms of each protein served as the respective standard. C: SK-N-AS-Ob-Rb cells were incubated with CORM-2 (20 μM) or TG (1 μM) for 6 h, and expression levels of X-box binding protein-1 (Xbp-1) and p50 activating transcription factor 6 (ATF6) were analyzed by Western blotting. D: SK-N-AS-Ob-Rb cells were incubated with CORM-2 (20 μM), TG (1 μM), or TM (2 μg) for 6 h, and expression levels of glucose-regulated protein (GRP)78 and C/EBP homologous protein (CHOP) were analyzed by Western blotting; β-actin served as a standard
표 CO induces activation of PERK branch of ER stress in SK-N-AS-Ob-Rb cells. A and B: SK-N-AS-Ob-Rb cells were preincubated with CORM-2 (20 μM) for 2 h and then treated with TG (1 μM, 4h), and expression levels of p-PERK and p-IRE1α were analyzed by Western blotting. Dephospho forms served as the standard. C: SK-N-AS-Ob-Rb cells were preincubated with CORM-2 (20 μM) for 2 h and then treated with TG (1 μM, 4 h), and expression levels of Xbp-1 and p50 ATF6 were analyzed by Western blotting; β-actin served as the standard (D and E). SK-N-AS-Ob-Rb cells were transfected with control siRNA or PERK siRNA (50 nM). D: 36 h after transfection, Western blotting analysis was performed for PERK protein expression; β-actin served as the standard. E: 36 h after transfection, cells were preincubated with CORM-2 (20 μM) for 2 h and then treated with TG (1 μM, 4 h), and expression levels of p-IRE1α were analyzed by Western blotting and quantified by densitometry.; n = 3. P < 0.05
표 IRE1 is involved in ER stress-induced leptin resistance in SK-N-AS-Ob-Rb Cells. A and B: SK-N-AS-Ob-Rb cells were transfected with with control (si Con) or IRE1α siRNA (si IRE1) (50 nM). A: 36 h after transfection, Western blotting analysis was performed for IRE1α protein expression; β-actin served as the standard. B: 36 h after transfection, cells treated with TM (2 μg/ml, 4h) and stimulated with leptin (10 μg/ml) for 15 min had Western blotting analysis performed for p-STAT3 and STAT3 expression. C: IRE1+/+ and IRE1−/− murine embryo fibroblasts (MEF) Ob-Rb cells were preincubated with CORM-2 (20 μM) for 2 h and then treated with TM (2 μ g/ml, 4 h), and then expression levels of p-STAT3 were analyzed by Western blotting and quantified by densitometry; n = 3. *P < 0.05
표 CO reverses palmitate-induced leptin resistance in SK-N-AS-Ob-Rb cells. SK-N-AS-Ob-Rb cells were incubated with palmitate at indicated concentrations for 6 h (A), or preincubated with CORM-2 (20 μM) for 2 h (B), and then treated with palmitate (200 μM, 6 h), and expression levels of p-PERK, p-IRE1α, and CHOP were analyzed by Western blotting. C: SK-N-AS-Ob-Rb cells were preincubated with palmitate at the indicated concentrations for 6 h, and then treated with leptin (10 μg/ml) for 15 min. Cell lysates were subjected to Western blotting analysis for p-STAT3 and STAT3 expression. D: SK-N-AS-Ob-Rb cells were preincubated with CORM-2 (20 μM) for 2 h and then treated with palmitate (200 μM/ml, 6 h) and then stimulated with eptin (10 μ g/ml) for 15 min. p-STAT3 and STAT3 levels were analyzed by Western blotting
표 Inhaled CO reduces body weight, food intake, adipose pads, and serum leptin induced by high-fat diet (HFD). A: after feeding mice HFD or normal diet (ND; control) for 16 wk, mice inhaled CO (250 ppm) for 2 h each day for 10 wk. Inhalation of CO in diet-induced obese mice decreased body weight. In contrast to high-fat group, there was no effect of CO inhalation on the normal diet group. ***P < 0.001. B: food intake was decreased by CO inhalation in the high-fat group. *P < 0.05. C, D, and E: after feeding mice HFD or ND for 8 wk, mice inhaled CO (250 ppm) for 2 h each day for 4 wk. Inhalation of CO in diet-induced obese mice decreased epididymal and perirenal fat content and serum leptin levels. In contrast to HFD group, there was no effect of CO inhalation on ND group; n = 5. ***P < 0.001
표 CO reverses HFD-induced-leptin resistance by modulating the ER stress response in mice. Mice were fed HFD or ND for 16 wk. Beginning at the 6th week, mice inhaled CO (250 ppm) for 2 h each day for subsequent 10 wk. Western blot analyses were performed for p-PERK (A), and p-IRE1α (B). The corresponding dephospho forms served as standard. C: additionally, mice in each group were stimulated with leptin. p-STAT3 and STAT3 levels were analyzed by Western blotting. Relative protein expression was quantified by densitometry; n = 3. *P < 0.05, **P < 0.01
표 Hemin-induced BAG reduces transcript of inflammatory cytokines and the proteins release in cocultured adipocyte/macrophages. 3T3-L1 adipocytes were cocultured with Raw264.7 macrophages with/without pretreatment of hemin for 24h. ZnPP was used as HO-1 inhibitor. Levels of TNF-α, IL-6, and adiponectin mRNAs were measured by qRT-PCR (A). Total RNAs were isolated and analyzed the level of TNF-α, IL-6, and adiponectin mRNAs, 36B4 was used as control gene (B). The protein TNF-α, IL-6 levels were detected by ELISA (B). Experiment was set up in triplicate. * P<0.05,#P<0.005,##P<0.001(compared with control)
표 HO-1 and Nrf-2 expression in cocultured adipocytes/macrophages. Adipocytes/macrophages were cocultured (A) in the presence or absence of hemin (1 or 10 μM) (B) for 24h in serum-free media. Total RNAs were isolated and analyzed the level of HO-1, Nrf-2 by qRT-PCR, 36B4 was used as control gene. Experiment was set up in duplicate. * P < 0.05, ## P < 0.001 (compared with control)
표 HO-1 induction alters macrophage M1/M2 polarization in cocultured adipocytes/macrophages. 3T3-L1 adipocytes were cocultured with Raw264.7 macrophages with/without pretreated with hemin for 24h. ZnPP was used as HO-1 inhibitor. Total RNAs were isolated and analyzed the level of IL-4 (A), M1 marker (CD274) (B) and M2 markers (Mrc1, Clec10a), (C) by qRT-PCR, b-actin was used as control gene. Experiment was set up in duplicate. *P<0.05, **P<0.01, #P<0.005 (compared with control)
표 HO-1 induction inhibits NF-kB activation in adipocytes or macrophages treated with macrophage- or adipocyte-conditioned medium (CM). 3T3-L1 adipocytes or Raw264.7 macrophages were treated with Raw macrophage conditioned medium (Raw-CM) or adipocyte-conditioned medium (Adipo-CM) with/without hemin pretreatment for 1h. ZnPP was used as HO-1 inhibitor. The expression of IkBa was detected by Western blotting, b-actin was used as control
표 CO producer (CORM2) reduces transcript of inflammatory cytokines and enhances M-2 macrophages in cocultured adipocyte/macrophages. 3T3-L1 adipocytes were cocultured with Raw264.7 macrophages with/without pretreatment of CORM2 for 24h. RuCl3 was used as control. Levels of TNF-α, IL-6 mRNAs were measured by qRT-PCR or ELISA (A). M2-macropahge marker M1 (CD274) and M2 markers (Mrc1, Clec10a) were measured by qRT-PCR (B), b-actin was used as control gene. Experiment was set up in triplicate. * P<0.05,#P<0.005,##P<0.001 (compared with control)
표 Inhaled CO reduces expression of inflammatory cytokine mRNAs in obese adipose tissue. Mice were fed an HFD or a normal diet (ND; control) for 16w, and from the sixth week of the feeding, mice were inhaled CO (250ppm) for 2h each day for 10 wk. RT-PCR analyzed inflammatory cytokines (MCP-1, IL-6)
표 ER stress induces the production of IL-1b in vitro and in vivo.(A)Human leukemic monocyte cells (U937) were treated with the chemical ER-stress inducing compounds tunicamycin (TM, 10 mg/ml) for the indicated time intervals and the secretion of IL-1b was quantified by ELISA. Mouse primary peritoneal macrophages (B), primary bone marrow-derived macrophages (BMDM) (C) were treated with TM for 18 h at the indicated concentrations and the secretion of IL-1b was quantified by ELISA. (I) C57BL/6 mice (N=6) were injected with TM (3 mg/kg, i.p.). Mice were sacrificed, and then blood was collected. Serum IL-1b was quantified by ELISA. Liver tissue was collected for the analysis of CHOP mRNA by RT-PCR. 18s rRNA served as the standard (lowerpanel). U937 cells(E) or BMDM(F were treated with palmitate for 24 hours at the indicated concent rations, or with 200mM at the indicated times, and the secretion of IL-1b was quantified by ELISA. Data represent mean +/- S.D of three independent determinations. *P<0.05,**P<0.01,***P<0.001.Blots shown are representative of three independent experiments
표 ER stress induces the expression of pro-IL1b mRNA via the activation of NF-kB. primary peritoneal macrophages (A-B) and Raw264.7 cells (C) were treated with TM and then mRNA expression for pro-IL-1b, NLRP3, CHOP, and XBP-1(s) were determined by PCR. (D) Raw264.7 cells were treated with the saturated fatty acids such as palmitate (C16:0) or equivalent monounsaturated fatty acids (oleate, C18:1) for 6h at the indicated concentration and mRNA expression for pro-IL-1b was determined by RT-PCR. (E) U937 leukemia cells were treated with TM (10 mg/ml), and then the level of RelB/p65 and p50 in cytosolic(upperpanel) and nuclear(lowerpanel) extracts was determined by Western immunoblotanalysis .b-actin was served as the standard. Lamin A/C was served as the standard for nuclear extracts. (F) Peritoneal macrophages were treated with TM (10 mg/ml) for 6 hrs. The expression of RelB/p65 and p50 subunits of NF-kB was determined in cytosolic and nuclear extracts. b-actin was served as the standard. Lamin A/C was served as the standard for nuclear extracts. (G) U937 (leftpanel) and THP-1 leukemia cells(rightpanel) were treated with TM (10 mg/ml) in the absence or presence of the NF-kB translocation inhibitor SN50 (20 mM). The expression of pro-IL-1b mRNA was determined by RT-PCR. GAPDH or 18s rRNA was served as the standard. Blots shown are representative of three independent experiments
표 ER Stress stimulates pro-caspase-1 activation. U937 leukemia cells (A), differentiated adipocytes (B) and primary macrophages (C) were treated with TM (10 mg/ml) for the indicated times, or at the indicated doses for 18 h. The expression of pro-caspase-1 and the formation of cleaved caspase-1 were determined by Western Immunoblot analysis. b-actin (A) or a-tubulin (B-C) served as the standard. Additionally, IL-1b secretion was determined in the supernatant of U937 cells. Non-specific immunoreactivity was used as the standard for secreted protein (A). Blots shown are representative of three independent experiments
표 Chemical chaperone treatment attenuates ER stress-mediated IL-1b production. U937 cells (A-C) were treated with TM (10 mg/ml) for 18 hrs, in the absence or presence of 4-PBA (0.5 mM). (A) The level of pro-IL-1b and CHOP mRNA was analyzed by PCR. GAPDH served as the standard. (B) The nuclear translocation of the p50 subunit of NF-kB was determined by Western immunoblot analysis. b-actin was served as the standard. Lamin A/C was served as the standard for nuclear extracts. (C) IL-1b secretion in U937 supernatants was determined by ELISA. (D-E) U937 cells were treated with TM (10 mg/ml) for 18 hrs, in the absence or presence of TUDCA (0.1 mM) or NAC (25 mM). IL-1b secretion in U937 supernatants was determined by ELISA. (F) C57BL/6 mice were pre-injected with TUDCA (N=10, 500 mg/kg/day, i.p.), or pre-treated with 4-PBA (N=10,1gram/kg/dayoralgavage) for 20days. Attheend of the 20day pre-treatment, mice were injected 20days later with TM(3mg/kg,i.p.). Mice were sacrificed at 6h, post-injection and then blood was collected for the analysis of serum IL-1b (left and center panels). Liver tissue was collected for the analysis of pro-IL-1b, NLRP3, and CHOP mRNA expression levels in the liver by RT-PCR. 18s RNA was served as the standard (Figure 4F, rightpanel). U937 cells were pre-treated for 1 hr with (G) 4-PBA (1 mM), (H) TUDCA, or for 5 min with NAC (I) (at the indicated concentrations), and then treated with palmitate (200 mM) for 18 hrs. IL-1b secretion in U937 supernatants was determined by ELISA. Data represent mean +/- S.D of three independent determinations. *P<0.05,**P<0.01.Blots shown are representative of three independent experiments
표 ER stress-mediated ROS accumulation leads to the activation of NLRP3 inflammasome through TXNIP. (A-B) U937 cells were treated with TM (10 mg/ml) for the indicated times and then stained with DCF-DA. ROS production was evaluated using DCF-DA fluorescence measured by confocal microscopy (A), or flow cytometric assays (B). U937 cells were pre-treated with CORM-2 (40 mM) or NAC (5 mM) and then treated with TM (10 mg/ml) for the indicated times (C,leftpanel ) or for 9hours (C, rightpanel). (C-D) U937 cells were treated with TM for the indicated times (C), or for 9 hours (D) in the absence (C) or presence (D) of NAC (5 mM). Lysates were immunoprecipitated with anti-TXNIP and then immunoblotted with anti-NLRP3. IgG was served as the immunoprecipitation control. (E) U937 cells were transfected with specific siRNA against TXNIP or control siRNA (scramble), and the level of endogenous TXNIP was measured by Western blot analysis (upperpanel). Under these conditions, the amount of secreted IL-1b was measured from cultured media of U937 cells in the absence or presence of TM treatment (10 mg/ml) (lowerpanel).(F)Peritoneal macrophages isolated from TXNIP knockout mice (Txnip-/-)were subjected to TM treatment (10 mg/ml), and the production of IL-1b was measured by ELISA. (I) NLRP3-/-mice (N=6) were exposed to TM(3mg/kgi.p.). Serum was collected 6hrs post-injection and evaluated for the secretion of IL-1b by ELISA. Data represent mean +/- S.D of three independent determinations. *P<0.05,**P<0.01. Blots shown are representative of three independent experiments
표 The HO-1/CO system inhibits pro-IL-1b synthesis and IL-1b maturation. U937 cells (A-C) or peritoneal macrophages (G) were treated with TM (10 mg/ml for 18 hours) in the absence or presence of cobalt-protoporphyrin-IX (CoPP) (1-10 mM, as indicated). (A) The secretion of IL-1b was determined by ELISA; (B, G) pro-IL-1 b and NLRP3 mRNA expression was determined by RT-PCR, with GAPDH or 18s rRNA as the standard; (C) The activation (cleavage) of caspase-1, with b-actin as the standard; were determined by Western analysis; (D-F, H) were treated with TM in the absence or presence of CORM-2 (40 mM). (D) The secretion of IL-1 b was determined by ELISA; (E, H) pro-IL-1b and NLRP3 mRNA expression was determined by RT-PCR, with GAPDH or 18s rRNA as the standard; (F) The activation (cleavage) of caspase-1, with b-tubulin as the standard; were determined by Western analysis; (I) U937 cells were pre-treated with hemoglobin (Hb)(1 h) and then treated with were treated with TM in the absence or presence of CORM-2 (40 mM). Data represent mean +/- S.D of three independent determinations. *P<0.05,**P<0.01.Blots shown are representative of three independent experiments
표 CO inhalation inhibits inflammasome formation in High-fat diet mice and STZ-induced diabetic model
표 Nicotine induces the expression of both HO-1 and TTP in macrophages. (A-D)RAW264.7cellswerepretreatedwiththeindicatedconcentrationofnicotinefor20minandincubatedwith1 μg/mlLPSfor24h.(A)CellsupernatantswereanalyzedforTNF-αbyELISA.Values are mean ± SD (n = 3). ***p<0.001.(B)ThelevelsofTNF-α, TTP, and HO-1 were measured by semi-qRT-PCR and the levels of HO-1 were measured by Western blot assays. The representative band is shown (C). Scion Image software was used for densitometry analysis of the HO-1 protein band (D). The relative density of the sample lane was divided by the control lane (β-actin). Values are mean ± SEM (n = 3). ***p<0.001.(E)RAW264.7cellsweretreatedwith1mMnicotineforindicatedtime.ThelevelsofHO-1andTTPwerea nalyzedbysemi-RT-PCR
표 Inhibition of HO-1 activity attenuates the effect of nicotine on the TTP induction in macrophages. (A) RAW 264.7 cells were pretreated with the indicated concentration of ZnPP for 30 min and further incubated in the presence or absence of 1 mM nicotine for 24 h. The levels of TTP and HO-1 were analyzed by semi-qRT-PCR. (B) Peritoneal macrophages isolated from wild-type Balb/c mice were pretreated with ZnPP (20μM) or SnPP (50μM) for 30min and further incubated with 1 mM nicotine for 24h. The levels of TTP and HO-1 were analyzed by semi-qRT-PCR. (C) RAW 264.7 cells were pretreated with ZnPP (20μM) for 30min and further incubated with 1 mM nicotine and 1 μg/ml LPS for 24 h. The levels of TTP, HO-1, and TNF-a were analyzed by semi-qRT-PCR
표 HO-1 gene silencing blocks the effects of nicotine on TTP induction in macrophages. (A) RAW 264.7 cells were transfected with siRNA against HO-1 (HO-1-siRNA) or scrambled siRNA (scRNA). After treatment with 1 mM nicotine and 1 μg/ml LPS for 24 h, the levels of TTP, HO-1, and TNF-a were analyzed by semi-qRT-PCR. (B-D) Peritoneal macrophages were harvested from HO-1 KO and wild-type mice. (B & C) Cells were treated with 1 mM nicotine for 24 h. The level of TTP and HO-1 were determined by semi-qRT-PCR (B) and Western blot assays (C). (D) Cells were treated with 1 mM nicotine and 1 μg/ml LPS for 24 h and TTP levels were analyzed by semi-qRT-PCR
표 Induction of HO-1 increases TTP levels in macrophages. (A&B) RAW264.7cells were treated with the indicated concent ration of hemin for 18h. HO-1 levels were an analyzed by Western blot(A) and semi-qRT-PCR(B) and TTP levels were analyzed by semi-qRT-PCR (B). (C) RAW 264.7cells were pretreated with 20μM ZnPP for 30min and further incubated with 10μM hemin for 18h. The levels of TTP and HO-1 were analyzed by semi-qRT-PCR. (D&E) RAW 264.7cells were treated with theindicated concent ration of CoPP for 18h. HO-1 levels were analyzed by Westernblot (D) and semi-qRT-PCR (E) and TTP levels were analyzed by semi-qRT-PCR (E). (F) RAW 264.7cells were pretreated with 20μM ZnPP for 30min and further incubated with 10μM CoPP for 18h. The levels of TTP and HO-1 were analyzed by semi-qRT-PCR
표 STAT3 is required for HO-1-induced TTP expression in macrophages. (A-C) Nicotine-HO-1 enhances STAT3 phosphorylation. (A) RAW 264.7 cells were treated with 1 mM nicotine for the indicated times and the levels of total STAT3 and phospho-STAT3 were analyzed by Western blot assays. (B) RAW 264.7 cells were pretreated with 20 μM ZnPP for 30 min and further incubated with the indicated concentration of nicotine for 24 h. The levels of total STAT3 and phospho-STAT3 were analyzed by Western blot assays. (C) RAW 264.7 cells were transfected with HO-1-siRNA. After treatment with 1 mM nicotine for 24 h, the levels of STAT3, phospho-STAT3, and HO-1 were analyzed by Western blot assays. (D) Peritoneal macrophages were harvested from HO-1 KO and wild-type mice. Cells were treated with 1 mM nicotine for 24 h. The level of HO-1, STAT3, and phospho-STAT3 were determined by Western blot assays. (E-H) STAT3 is required for nicotine-induced TTP expression. RAW 264.7 cells were transfected with STAT3-siRNA. (E) The level of STAT3 was analyzed by Western blot assays. (F) Cells were incubated with 10μM CoPP for 18h and the levels of TTP, HO-1, and STAT3 were analyzed by semi-qRT-PCR. (G) After treatment with 1 mM nicotine for 24 h, the levels of TTP, STAT3, and HO-1 were analyzed by semi-qRT-PCR. (H) After treatment with 1 mM nicotine and 1 μg/ml LPS for 24 h, the levels of TTP, STAT3, HO-1, and TNF-α were analyzed by semi-qRT-PCR
표 TTP deficiency does not affect nicotine-induced HO-1 expression in the LPS-induced endotoxemia mouse model. (A-E) Wild type (WT) and TTP knockout (TTP KO) mice were pretreated with nicotine (400 μg/kg, i.p.) for 2h and administrated with LPS (12.5 mg/kg, i.p.) for 24h. (A-D) Liver tissues were analyzed for the levels of HO-1, STAT3, and TNF-α by semi-qRT-PCR. One representative band is shown (A **p<0.01.ns, not significant. (E) Blood serum was analyzed for the TNF-α levels by ELISA. Values are mean ± SEM (n = 3). **p<0.01 .(F-H) WT and TTP KO mice were treated with nicotine (400 μg/kg, i.p.) for 24 h. Liver tissues were analyzed for the levels of HO-1, STAT-3, and phosphor-STAT3 by Western blot assays. One representative band is shown (F). Scion Image software was used for densitometry analysis of pSTAT3 and HO-1 band was done by using Scion Image software (G & H). The relative density of the sample lanes was divided by STAT3 and actin, respectively. The expression levels obtained from mice without nicotine treatment were set to 1. Values are mean ± SEM (n = 3). **p<0.01
표 HO-1 deficiency blocks the effect of nicotine on TTP expression and TNF-a production in the LPS-induced endotoxemia mouse model. Wild type and HO-1 KO mice were pretreated with nicotine (400 μg/kg, i.p.) for 2h and administrated with LPS (12.5 mg/kg, i.p.) for 24h. (A-D) Liver tissues were analyzed for TTP and TNF-α mRNA levels by semi-qRT-PCR. One representative band is shown (A **p<0.01;***p<0.001.ns,notsignificant.(E) Blood serum was analyzed for the level of TNF-α by ELISA. Values are mean ± SEM (n = 3). ***p<0.001.ns, not significant
표 Time- and dose-dependent increases of mitochondrial proteins by quercetin. (A) HepG2 cells (4 × 105cells/well) were exposed for indicated times (0,0.5,1,3,5h) (B) to various concentrations (0,5,10,15,25μM) of quercetin. Expressions of COX-IV protein were determined by Western blotting. Results are expressed as mean ± SE of three independent experiments. p˂0.05 compared with β-actin
표 Time- and dose-dependent increases of mitochondrial DNA copy number by quercetin. (A) HepG2 cells (4 × 105cells/well) were exposed for indicated times (0,0.5,1,3,5h) (B) to various concentrations (0,5,10,15,25μM) of quercetin. Expression of mitochondrial DNA complex II(succinate-ubiquinoneoxidoreductase) was quantified by realtime PCR(n=3)
표 Expression levels of HO-1 expression by HepG2 cells exposed to quercetin. Expression levels of HO-1 mRNA and protein level were determined after HepG2 cells (4 × 105cells/well) were exposed for indicated times (0,0.5,1,3,5h) (B) to various concentrations (0,5,10,15,25μM) of quercetin. Expressions of HO-1 mRNA and protein were determined by reverse transcription PCR (A and C) and Western blotting (B and D) (n=3). Results are expressed as mean ± SE of three independent experiments. p˂0.05 compared with GAPDH and β-actin
표 Decreased expression of mitochondrial biogenesis activators due to inhibition of HO-1/CO, and restoration of cell survival by quercetin. HepG2 cells (4 × 105cells/well) were exposed to quercetin 15μM, quercetin and snpp(A), and Hb(B) for 3h as described in panels A and B. Expressions of markers of mitochondrial biogenesis (PGC-1, NRF-1 and TFAM) in mRNA were determined by reverse transcription PCR (n=3). Results are expressed as mean ± SE of three independent experiments. p˂0.05 compared with GAPDH
표 Decreased expression of mitochondrial protein due to inhibition of HO-1/CO, and restoration of cell survival by quercetin. HepG2 cells (4 × 105cells/well) were exposed to quercetin 15μM, quercetin and snpp 20μM (A), and Hb 20μg (B) for 3h as described in panels A and B. Expression of mitochondrial protein COX-IV was determined by Western blotting (n=3). Results are expressed as mean ± SE of three independent experiments. p˂0.05 compared with β-actin
표 Effects of inhibition of HO-1/CO system on quercetin-induced induction of mitochondrial DNA copy number. HepG2 cells (4 × 105cells/well) were exposed to quercetin 15μM, quercetin and snpp 20μM (A), and Hb 20μg (B) for 3h as described inpanels A and B. Expression of mitochondrial DNA complexII(succinate-ubiquinoneoxidoreductase) was quantified by real time PCR(n=3)
표 Time-dependent effect of high glucose (25mM) on COX-IV expression in HepG2 cells. HepG2 cells adapted to low glucose (5 mM) were treated with high glucose (25mM) for indicated times (0, 2, 3, 6, 12 h), and expression of mitochondrial protein COX-IV was determined by Western blotting (n=3). Results are expressed as mean ± SE of three independent experiments. p˂0.05 compared with β-actin
표 Effects of HO-1/CO on quercetin-induced rescue of mitochondrial integrity by high glucose. HepG2 cells adapted to low glucose (5 mM) were treated with high glucose (25mM), quercetin 15 μM, quercetin and snpp 20 μM (A), and Hb μg (B) for 3h as described in panels A and B. Expression of markers of mitochondrial biogenesis (PGC-1, NRF-1 and TFAM) in mRNA were determined by reverse transcription PCR (n=3). Results are expressed as mean ± SE of three independent experiments. p˂0.05 compared with GAPDH
표 NO induces mitochondrial biogenesis through the induction of HO-1. (A to E) HepG2 cells were pretreated in the absence or presence of SnPP (20 μM), a competitive inhibitor of HO activity, and then treated with 100 μM SNAP for 12 h. (FtoH) HepG2 cells were transfected with HO-1-specifics iRNA or controls iRNA(con), and then treated with SNAP(100μM) for 12 h. (A,F) The expression of PGC-1α, NRF-1, TFAM mRNA were measured by RT-PCR. GAPDH was served as the standard. (B,G)There lative mtDNA content was measured by realtime-PCR. mtDNA content was normalized to nuclearDNA (nDNA,β-actin gene) content. Control values were normalized to 1 arbitrary unit.(C) Mitochondrial mass was assessed by using MitoTracker® Red CMXRos staining (red). Nuclei were stained with Hoechst dye (blue). Images of fluorescence were analyzed by confocal microscopy. (D,H) The expression of CI(complexI,NDUFB8), CIII(complexIII,UQCRC1) and CIV(complexIV, COXIV) were analyzed by Western blotting. b-actin served as the standard. (E) ATP levels in cells treated with SNAP in the absence or presence of SnPP were measured. Expression in untreated cells was assigned the value of 1.GAPDH and β-actin were used as a loading control for RT-PCR or Western experiments, respectively. All experiments were performed in triplicate, and representative data are shown. Data are expressed as mean ± SEM. *P<0.05 compared with untreated control cells (oruntreated, control siRNA cells);†P<0.05 compared with cells treated with SNAP alone (or SNAP-treated, control siRNA cells)
표 NO induced mitochondrial biogenesis through HO-dependent mechanisms in vivo. (AtoD) C57BL/6 mice were pre-injected with the HO inhibitor, SnPP(50μmol/kg) or saline, for 6 h prior to injection of SNAP (3 mg/kg). After 24 hrs post-injection livers were excised and analyzed for mitochondrial biogenesis in mice. (A) Expression of TFAM mRNA was measured by RT-PCR, with 18s rRNA as the standard. (B) Total hepatic DNA was isolated and used to amplify along mtDNA fragment(8636bp). The mtDNA content was measured by Expand Long Template PCR. Relative amounts of mtDNA and nDNA(18s) contents were compared. (C) The expression level of CIV (complexIV, COXIV) was analyzed by Western blotting, with a-tubulin serving as the standard. (D)Mitochondrial mass was assessed by using MitoTracker® Red CMXRos staining (red) in liver sections. Fluorescent-stained were analyzed by confocal microscopy. All experiments were performed in triplicate (n=5/group), and representative data are shown. Quantitative data are expressed as mean ± SEM. *P<0.05 compared with the un-injected control group; †P<0.05 relative to mice injected only with SNAP
표 NO induces HO-1 expression through a cGMP-dependent pathway. (A to E) HepG2 cells were pretreated in the absence or presence of ODQ (1 μM), an inhibitor of sGC, and then treated with SNAP (100 μM) for 12 h. (F to H) HepG2 cells were treated with 10 μM of 8-Br-cGMP (cGMP) for 12 h after pretreatment in the absence or presence of 20 μM SnPP. (A, F) PGC-1α, NRF-1, TFAM mRNA levels. (B, G) mtDNA content. (C) Fluorescence intensity of MitoTracker Red (red) and Hoechst (blue). (D, H) CI (complex I), CIII (complex III) and CIV (complex IV) protein levels. (E) ATP levels. GAPDH and β-actin were used as a loading control for RT-PCR or Western experiments, respectively. All experiments were performed in triplicate, and representative data are shown. Data are expressed as mean ± SEM. *P<0.05 compared with untreated control cells; †P<0.05 compared with cells treated with SNAP or cGMP (8-Br-cGMP) alone
표 Resveratrol induces mitochondrial biogenesis through the sequential production of NO and CO. (A to E) HepG2 cells were treated with 1 μM resveratrol for 12 h after pretreatment in the absence or presence of 20 μM of SnPP. (F to I) C57BL/6 Mice, resveratrol (20 mg/kg/d) was given once daily for 7 days by i.p. injection, with or without SnPP pretreatment as indicated. Liver tissue were excised and analyzed for mitochondrial biogenesis in mice. (J to M) HepG2 cells were transfected with control siRNA (con) or HO-1 siRNA to knockdown HO-1 levels. Cells were treated with 1μM of resveratrol for 12h. siRNA were subjected to RT-PCR and western blot to confirm HO-1 siRNA efficiency. (A, F, J) Expression of PGC-1α, NRF-1 and/or TFAM was measured by RT-PCR. (B, L) The relative mtDNA content was measured by real-time PCR. (C, H, K) The expression level of CI (complex I), CIII (complex III) and CIV (complex IV) protein were analyzed by Western blotting. (D, I, M) Mitochondrial mass was assessed by using MitoTracker® Red CMXRos staining (red). Nuclei were stained with Hoechst dye (blue). Images of fluorescence were analyzed by confocal microscopy. (E) ATP levels in cells. (G) A long mtDNA fragment (8636 bp) was selected for amplification. The mtDNA content was measured by Expand Long Template PCR. All experiments were performed three times independently, and representative data are shown. Data are expressed as mean ± SEM. *P<0.05 compared to control group (or untreated, control siRNA cells); †P<0.05 compared to resveratrol group (or resveratrol -treated, control siRNA cells)
표 Resveratrol-induced mitochondrial biogenesis and HO-1 expression are mediated by an eNOS-cGMP-dependent pathway. (A to B) HepG2 cells were treated with 1 μM resveratrol for 12 h after pretreatment in the absence or presence of 1 mM L-NAME for 1 h. (C to F) WT and eNOS-/- mice, resveratrol (20 mg/kg/d) was given once daily for 7 days by i.p. injection. Liver tissue were excised and analyzed for mitochondrial biogenesis in mice. (G to H) HepG2 cells were treated with 1 μM resveratrol for 12 h after pretreatment in the absence or presence of 1 μM ODQ for 1 h. (A, C, G) Expression of PGC-1α, NRF-1, TFAM and/or HO-1 were measured by RT-PCR. (B, E, H) The expression levels of CI (complex I), CIII (complex III) and CIV (complex IV) protein were analyzed by Western blotting. (D) A long mtDNA fragment (8636 bp) was selected for amplification. The mtDNA content was measured by Expand Long Template PCR. (F) Mitochondrial mass was assessed by using MitoTracker® Red CMXRos staining (red). All experiments were performed three times independently, and representative data are shown. Data are expressed as mean ± SEM. *P<0.05 compared to control group (or un-injected WT mice); †P<0.05 compared to resveratrol group
표 CO directly activates mitochondrial biogenesis associated transcriptional coactivators and increases mitochondrial DNA and protein. (A to G) HepG2 cells were treated with 10 μM 8-Br-cGMP or 20 μM CoPP for 12 h after pretreatment with in the absence or presence of 20 μM Hb, a CO scavenger. (A, B) PGC-1α, NRF-1 and TFAM mRNA levels. (C, D) CI (complex I), CIII (complex III) and CIV (complex IV) protein levels, and (E, F) mtDNA content. (G) Fluorescence intensity of MitoTracker Red (red) and Hoechst (blue). All experiments were performed three times independently, and representative data are shown. GAPDH and β-actin were used as a loading control in each experiment. Data are expressed as mean ± SEM. *P<0.05 compared with untreated control cells; †P<0.05 compared with cells treated with 8-Br-cGMP or CoPP
표 Nrf2-Akt activation is involved in mitochondrial biogenesis induced by the HO-1/CO system. (A to D) HepG2 cells were transfected with control siRNA (con) or Nrf2 siRNA, and then treated with 20 μM CoPP or 20 μM CORM for 12 h. (E to G) HepG2 cells were treated with 20 μM CoPP or 20 μM CORM-3 for 12 h after pretreatment in the absence or presence of 25 μM LY (LY294002), a PI-3K/Akt inhibitor. (A) The effectiveness of Nrf2 knockdown was confirmed by Western blot. (B, E) Expression of PGC-1α, NRF-1 and/or TFAM mRNA. (C, G) mtDNA content. (D, F) CI (complex I), CIII (complex III) and CIV (complex IV) protein levels. Experiments were performed three times independently, and representative data are shown. GAPDH and β-actin were used as a loading control in each experiment. Data are expressed as mean ± SEM. *P<0.05 compared with untreated control cells; †P<0.05 compared with cells treated with CoPP or CORM-3 alone
표 Resveratrol induces mitochondrial biogenesis and anti-inflammatory response through a pathway involving HO-1/CO. (A to B) HepG2 cells were pretreated with 1 μM resveratrol for 6 h in the absence or presence of 20 μM of SnPP, and then stimulated for 18 h with LPS (100ng/ml). (A) Expression of PGC-1α, NRF-1, TFAM were measured by RT-PCR. (B) The expression levels of CI (complex I), CIII (complex III) and CIV (complex IV) protein were analyzed by Western blotting. (C to E) HepG2 cells were pretreated with 1 μM resveratrol for 1h in the absence or presence of 20 μM of SnPP, and then stimulated for 24h with LPS (10μM/ml). (C) ROS levels were assayed for carboxy-H2DCFDA by flow cytometry. Percentage of carboxy-H2DCFDA positive cells for indicated conditions. (D) TNFa, IL-1b and iNOS gene expression was determined by real-time RT-PCR. (E) NO production in the medium was observed by using Griess reagents. Results were divided by the corresponding control. Expression in untreated cells was assigned the value of 1. All experiments were performed three times independently, and representative data are shown. GAPDH and β-actin were used as a loading control in each experiment. Data are expressed as mean ± SEM. *P<0.05 compared with untreated control cells; †P<0.05 compared with cells treated with LPS alone. #P<0.05 compared with cells treated with LPS+resveratrol
표 Resveratrol protects mice against hepatic injury by stimulation of mitochondrial biogenesis. (A-I) C57BL/6 Mice were injected with resveratrol in the presence of LPS treatment, with or without SnPP pretreatment as indicated. Liver tissues were excised and analyzed for mitochondrial biogenesis in mice. (A) Expression of PGC-1α, NRF-1 and/or TFAM mRNA were measured by RT-PCR (B) A long mtDNA fragment (8636 bp), was selected for amplification. The mtDNA content was measured by Expand Long Template PCR. Relative amounts of mtDNA and nDNA (18s) contents were compared. (C) The expression level of complex I, complex II, complex IV was analyzed by Western blotting (D) Mitochondrial mass was assessed by using MitoTracker® Red CMXRos staining (red) in liver sections. (E) ROS levels were assayed for carboxy-H2DCFDA (green). Fluorescent-stained were analyzed by confocal microscopy. (F) Citrate synthase (CS) activity. (G) TNFa, IL-1b, IL-6, CCL5 and iNOS gene expression was determined by real-time RT-PCR. Hepatic injury was assessed by determining liver tissue myeloperoxidase (MPO) levels (H) and serum levels of alanine aminotransferase (ALT) (I). All experiments were performed in triplicate (n=5/group), and representative data are shown. Quantitative data are expressed as mean ± SEM. *P<0.05 compared with the un-injected control group; †P<0.05 relative to mice injected only with LPS; #P<0.05 relative to mice injected with LPS+resveratrol. (a: control , b: LPS, c: LPS+resveratrol, d: LPS+ resveratrol+SnPP)
표 CORM-2 induces TTP promoter activity and TTP mRNA in macrophages. RAW264.7cells were incubated with different concentration of CORM-2 for 4 h. A, TTP mRNA expression was determined by semi-quantitative RT-PCR. GAPDH was used as internal controls. B, RAW264.7 cells were transfected with pGL3/mTTPP-1309 containing the mouse TTP promoter. Aafter 24h, cells were treated with different concentration of CORM-2 for 4h and luciferase activity was determined. The levels of firefly luciferase activity were normalized to the Renilla luciferase activity. The relative luciferase activities are presented as a fold increase over no-treated cells. Each bar represents the mean± S.D. of three independent experiments (*, p<0.05;**,p<0.01)
표 TTP deficiency blocks anti-inflammatory function of CORM-2 in macrophages. Peritoneal macrophages were harvested from WT and TTP KO mice. Cells were treated with 1 mg/ml LPS in the presence or absence of 10 μM CORM-2. A, TNF-a in the supernatant of cells was determined by ELISA. Data shown were mean ± S.D. of three independent experiments (**, p<0.01).B,TheexpressionlevelsofTTPandTNF-a mRNA were determined by semi-quantitative RT-PCR. GAPDH was used as internal controls. C, Expression of TNF-_ mRNA in macrophages was determined by quantitative real-time PCR at indicated times after the addition of 5 μg/ml actinomycin D. Results shown on the graph represent mean ± S.D. of three independent experiments (***, p<0.001)
표 TTP deficiency blocks the anti-inflammatory function of CO in DSS-induced colitis. After the treatment of 2% (w/v) DSS for 7 days, WT and TTP KO mice were exposed to CO at a concentration of 250 ppm for 5 days and analyzed for colitis. A, Clinical scores were assessed as described in Materials and Methods. B and C, Colon length. B, Representative images of five conducted in each group. C, Data are mean ± S.D. for 5 mice (*, p<0.05; **, p<0.01). D, Representative H&E sections of each group from colons of WT and TTP KO mice. Scale bars: 100 μ m. E, Neutrophil infiltration into the colon, quantified by measuring MPO activity. F, Colonic cytokine/chemokine mRNA levels, analyzed by semi-quantitative RT-PCR. Data represents one experiment out of 5 independent experiments with similar results
표 Pretreatment of mice with CO gas inhalation ameliorates liver I/R injury via AKT-GSK3 β activation. Mice were subjected to 90 minutes liver warm ischemia, followed by 6 h reperfusion. (a) Hepatocellular function was evaluated by sALT (IU/L). (b) Representative liver histology of ischemic liver lobes. (c) Liver neutrophil accumulation, assessed by MPO activity. Data represent the mean ± standard deviation (SD) ( = 4–6 samples/group). ∗∗<0.01. (d) Hepatic HMGB1 expression in liver tissue was assessed by Western blot analysis at 1 h and 6 h of reperfusion. Total cell lysates were analyzed for HMGB1 and β-actin protein levels by Western blot analysis. (e) Western-blot analysis of phospho (p)-GSK3β (Ser 9), p-GS (Ser641), and p-Akt in HepG2 cells after treatment with CORM2 (50 μM) at the indicated times. (f) RAW264.7 cells were stimulated with 10 ng/mL of LPS for 30 minutes in the absence or presence of CORM2 and the ROS scavenger, N-acetyl-cysteine (NAC). Total cell lysates were analyzed for phosphorylated GS, GSK3β, and Akt as well as total GS, GSK3β, Akt, and β-actin protein levels by Western immunoblot analysis. (g) Mice were subjected to 90 minutes of liver warm ischemia, followed by 1 h or 6 h reperfusion. Liver tissue was analyzed by Western blotting of p-Akt and total Akt. β -Actin served as the standard
표 Inhibition of GSK3β by CO inhalation ameliorates liver I/R injury. Mice were sham-operated or subjected to 90 minutes hepatic warm ischemia followed by 1 hour reperfusion. Recipients were treated with air or CO gas (250 ppm) inhalation. (a) Liver samples, harvested 1 hour later, were subjected to Western blot analysis of phospho (p)-GS (GS 641), p-GSK3β (S9), p-JNK, and p-p38. β-Actin was used as an internal control. ∗<0.05, ∗∗∗<0.001. (b) Immunohistochemical staining of GSK3 inhibition. Mice were sacrificed, liver tissues were harvested, and the tissue slices were processed for formalin-fixed paraffin embedding. Hematoxylin counterstaining after 3-amino-9-ethylcarbazole-based immunohistochemical staining was used to detect GS 641 ((A)–(C), red/brown) and phosphorylation of GSK3β S9 ((D)–(F), red/brown). ((c) and (d)) Quantitative RT-PCR-assisted detection of TNF-α, IL-6, CXCL10, and IL-10 gene expression at 1 hour or 6 hours in liver tissue. Data were normalized to 18S gene expression. Data shown represent the mean ± S.D. (=4-5/group), ∗< 0.05
표 PI3 K blockade restores liver I/R injury in mice pretreated with CO inhalation. Mice were treated with CO gas (CO), LY294002 (LY), or both or vehicle at 30 minutes prior to the liver ischemia insult, as described in Materials and Methods. Liver samples were harvested at 6 hours after-reperfusion. (a) Proteins were analyzed by Western blotting with Abs against phosphorylated or total GSK3β and β-actin. Sixty minutes ischemia time was used to show the effect of PI3 K inhibition in liver I/R injury. ∗P<0.05 , ∗∗P<0.01 . (b) Average sALT levels in different experimental groups were measured. sALT levels were measured at 6 hours of reperfusion. (c) Representative liver histology (H&E staining) is shown. To establish the functional relationship between PI3 K and GSK3β, CO inhalation was administered 12 hr and LY 30 minutes prior to the ischemic insult and 6 hr after reperfusion. (d) Liver neutrophil accumulation, assessed by MPO activity. Data represent mean ± S.D. (N = 4–6 samples/group). ∗∗P< 0.01 , ∗∗∗P<0.001.
표 CO-induced PI3 K/Akt-dependent GSK3 inactivation regulates TLR4 responses and affects the ability of CREB and NF-κB-p65 to associate with CBP. RAW264.7 cells were stimulated with 100 ng/mL of LPS for 1 hour in the absence or presence of CORM2 and the PI3 K inhibitor (LY294002). (a) Total cell lysates were analyzed for phosphorylated GS, GSK3β, Akt, and CREB as well as total GS, GSK3β, Akt, CREB, and β-actin protein levels by Western immunoblot analysis. (b) The mRNA expression of TNF-α and IL-10 was determined by semiquantitative RT-PCR. GAPDH was used as an internal control. ((c) and (d)) RAW264.7 cells were stimulated with 0.1 μg/mL of LPS for 1 hour in the absence or presence of CORM2 (50 μM) and nuclear extracts were obtained. Interaction of CBP with NF-κB p65 and CREB was assessed by immunoprecipitation of CBP followed by immunoblotting for NF-κB or CREB. Total CBP served as the input standard
표 CO induces activation of the protein kinase R-like endoplasmic reticulum kinase (PERK) branch of the ER stress response in SK-N-AS-Ob-Rb cells. A and B: SK-N-AS-Ob-Rb cells were incubated with CORM-2 (20 μM) or TM (2 μg/ml) or TG (1 μM) for indicated periods of time, and expression levels of p-PERK, p-eIF2α (eukaryotic initiation factor 2α), and p-IRE1α (inositol-requiring transmembrane kinase/endonuclease-1α) were analyzed by Western blotting. The unphosphorylated forms of each protein served as the respective standard. C: SK-N-AS-Ob-Rb cells were incubated with CORM-2 (20 μM) or TG (1 μM) for 6 h, and expression levels of X-box binding protein-1 (Xbp-1) and p50 activating transcription factor 6 (ATF6) were analyzed by Western blotting. D: SK-N-AS-Ob-Rb cells were incubated with CORM-2 (20 μM), TG (1 μM), or TM (2 μg) for 6 h, and expression levels of glucose-regulated protein (GRP)78 and C/EBP homologous protein (CHOP) were analyzed by Western blotting; β-actin served as a standard
표 CO induces activation of PERK branch of ER stress in SK-N-AS-Ob-Rb cells. A and B: SK-N-AS-Ob-Rb cells were preincubated with CORM-2 (20 μM) for 2 h and then treated with TG (1 μM, 4h), and expression levels of p-PERK and p-IRE1α were analyzed by Western blotting. Dephospho forms served as the standard. C: SK-N-AS-Ob-Rb cells were preincubated with CORM-2 (20 μM) for 2 h and then treated with TG (1 μM, 4 h), and expression levels of Xbp-1 and p50 ATF6 were analyzed by Western blotting; β-actin served as the standard (D and E). SK-N-AS-Ob-Rb cells were transfected with control siRNA or PERK siRNA (50 nM). D: 36 h after transfection, Western blotting analysis was performed for PERK protein expression; β-actin served as the standard. E: 36 h after transfection, cells were preincubated with CORM-2 (20 μM) for 2 h and then treated with TG (1 μM, 4 h), and expression levels of p-IRE1α were analyzed by Western blotting and quantified by densitometry.; n = 3. P < 0.05
표 IRE1 is involved in ER stress-induced leptin resistance in SK-N-AS-Ob-Rb Cells. A and B: SK-N-AS-Ob-Rb cells were transfected with with control (si Con) or IRE1α siRNA (si IRE1) (50 nM). A: 36 h after transfection, Western blotting analysis was performed for IRE1α protein expression; β-actin served as the standard. B: 36 h after transfection, cells treated with TM (2 μg/ml, 4h) and stimulated with leptin (10 μg/ml) for 15 min had Western blotting analysis performed for p-STAT3 and STAT3 expression. C: IRE1+/+ and IRE1−/− murine embryo fibroblasts (MEF) Ob-Rb cells were preincubated with CORM-2 (20 μM) for 2 h and then treated with TM (2 μg/ml, 4 h), and then expression levels of p-STAT3 were analyzed by Western blotting and quantified by densitometry; n = 3. *P < 0.05
표 CO reverses palmitate-induced leptin resistance in SK-N-AS-Ob-Rb cells. SK-N-AS-Ob-Rb cells were incubated with palmitate at indicated concentrations for 6 h (A), or preincubated with CORM-2 (20 μM) for 2 h (B), and then treated with palmitate (200 μM, 6 h), and expression levels of p-PERK, p-IRE1α, and CHOP were analyzed by Western blotting. C: SK-N-AS-Ob-Rb cells were preincubated with palmitate at the indicated concentrations for 6 h, and then treated with leptin (10 μg/ml) for 15 min. Cell lysates were subjected to Western blotting analysis for p-STAT3 and STAT3 expression. D: SK-N-AS-Ob-Rb cells were preincubated with CORM-2 (20 μM) for 2 h and then treated with palmitate (200 μM/ml, 6 h) and then stimulated with leptin (10 μg/ml) for 15 min. p-STAT3 and STAT3 levels were analyzed by Western blotting
표 Inhaled CO reduces body weight, food intake, adipose pads, and serum leptin induced by high-fat diet (HFD). A: after feeding mice HFD or normal diet (ND; control) for 16 wk, mice inhaled CO (250 ppm) for 2 h each day for 10 wk. Inhalation of CO in diet-induced obese mice decreased body weight. In contrast to high-fat group, there was no effect of CO inhalation on the normal diet group. ***P < 0.001. B: food intake was decreased by CO inhalation in the high-fat group. *P < 0.05. C, D, and E: after feeding mice HFD or ND for 8 wk, mice inhaled CO (250 ppm) for 2 h each day for 4 wk. Inhalation of CO in diet-induced obese mice decreased epididymal and perirenal fat content and serum leptin levels. In contrast to HFD group, there was no effect of CO inhalation on ND group; n = 5. ***P < 0.001
표 CO reverses HFD-induced-leptin resistance by modulating the ER stress response in mice. Mice were fed HFD or ND for 16 wk. Beginning at the 6th week, mice inhaled CO (250 ppm) for 2 h each day for subsequent 10 wk. Western blot analyses were performed for p-PERK (A), and p-IRE1α (B). The corresponding dephospho forms served as standard. C: additionally, mice in each group were stimulated with leptin. p-STAT3 and STAT3 levels were analyzed by Western blotting. Relative protein expression was quantified by densitometry; n = 3. *P < 0.05, **P < 0.01
표 The HO-1/CO system inhibits pro-IL-1b synthesis and IL-1b maturation. U937 cells (A-C) or peritoneal macrophages (G) were treated with TM (10 mg/ml for 18 hours) in the absence or presence of cobalt-protoporphyrin-IX (CoPP) (1-10 mM, as indicated). (A) The secretion of IL-1b was determined by ELISA; (B, G) pro-IL-1 b and NLRP3 mRNA expression was determined by RT-PCR, with GAPDH or 18s rRNA as the standard; (C) The activation (cleavage) of caspase-1, with b-actin as the standard; were determined by Western analysis; (D-F, H) were treated with TM in the absence or presence of CORM-2 (40 mM). (D) The secretion of IL-1 b was determined by ELISA; (E, H) pro-IL-1b and NLRP3 mRNA expression was determined by RT-PCR, with GAPDH or 18s rRNA as the standard; (F) The activation (cleavage) of caspase-1, with b-tubulin as the standard; were determined by Western analysis; (I) U937 cells were pre-treated with hemoglobin (Hb)(1 h) and then treated with were treated with TM in the absence or presence of CORM-2 (40 mM). Data represent mean +/- S.D of three independent determinations. *P<0.05,**P<0.01.Blots shown are representative of three independent experiments
표 CO inhalation inhibits inflammasome formation in High-fat diet mice and STZ-induced diabetic model
표 CO attenuates DSS-induced experimental colitis as measured by survival, body weight, and colon length.Mice were administrated with 3% DSS with drinking water for 6 days and CO (250 ppm) inhalation for 4 h daily and injection of LiCl (200mg/kg, i.p) was performed daily for more 4 or 6 days. (a) Survival percent was measured at day 12. (b) Body weight was measured at day 10. (c) and (d) Colonlength.(c) Representative images of 3 tests conducted in each group. (d) Data are mean ± SD for 3 mice. Con: Control, DSS: dextran sufate sodium salt and Data represents mean ± SEM, ∗P < 0.05, ∗∗P < 0.01
표 COattenuates experimental colitis asmeasuredbyhistology incolonand inflammatory cytokines in colon.Mice were administrated with 3% DSS with drinking water for 6 days and CO (250 ppm) inhalation for 4 h daily and injection of LiCl (200mg/kg, i.p) was performed daily for more 4 days. (a) ColonsectionsweresubjectedtoH&Estaining.(b)TNF-α,iNOS,andIL-10mRNAlevelswere detected from colon tissue by RT-PCR. (c) iNOS and pGSK-3β protein levels were measured from colon tissue by western blotting
표 CO attenuates experimental colitis as measured by histology in colon and inflammatory cytokines in MLNs, and BMMs. To detect the levels of cytokines in MLNs and BMM cells, mice were treated with 3% DSS solution for 6 days and isolated MLN and BMM cells were treated with CORM2 (100 uM) or LiCl (20mM)for6h.ThelevelsofTNF-α,iNOS,andIL-10mRNAwereperformedbyusingRT-PCRin MLN cells (d) and BMMs (e) and the levels of pGSK-3β were detected with western blot in MLN (f) and BMM (g) cells. Also, mRNA levels of GATA-3 (h) and t-bet (i) were performed by using real-time RT-PCR in MLN cells. Data represents mean ± SEM, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001
표 CO attenuates expression of GSK-3β signaling in human macrophage cell lines and MLNs. (a) U937cells were incubated with CORM2 (100 uM) for various time periods (0, 10, 20, 30, and 60 min) (b) Cells were treated with CORM2inadosedependentmanner(0,10, 20, 30, 50, and 100uM) for 30 min. (c) U937cells were preincubated with CORM2 (100 uM) and LiCl (20mM)for30minandthenstimulated with LPS (1 ug/mL) for 30 min. (d) MLN cells were preincubated with CORM2 (100 uM) and LiCl (20mM)for30minandthenstimulated with LPS (1 ug/mL) for 30 min. Protein expressions of pGSK-3β and pIkB were detected by western blotting
표 CO downregulates TNF-α and iNOS expression via inhibition of GSK-3βsignaling. (a) to (c) U937 cells were pretreated with CORM2 (100 uM) and LiCl(20mM) for 30 min followed by stimulation with LPS(1ug/mL) for 24 h. (a) TNF-α and iNOS mRNA expression was measured by RT-PCR, (b) iNOS protein expression was measured by western blotting, and (c) supernatant was collected and TNF-α protein level was measured by ELISA. (d) to (e)MLN cells were pre-incubated with CORM2 (100 uM) and LiCl (20mM)for30minandthen stimulated with LPS (1 ug/mL) for 24 h. (d) iNOS and TNF-α mRNA expression was measured by RT-PCR, and (e) Supernatant was used to measure TNF-α protein level by ELISA. The representative band or blot is shown. Data represents mean ± SEM, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001
표 Cilostazol reduces I/R-induced hepatic injury. (A-E) Mice were subjected to 60 minutes liver warm ischemia, followed by 6 h reperfusion. Hepatic injury was assessed by determining serum levels of ALT (A), TNF-a (B) and liver tissue myeloperoxidase (MPO) levels (C). Representative liver histology (H&E staining) is shown (D). Liver tissue mtDNA damage was analyzed by long PCR of long mtDNA (8636-bp) fragment. Short (316-bp) fragment served as the standard (E). Citrate synthase activity (F) and ATP production (G) in liver tissue were measured. All experiments were performed in triplicate (n = 7/group), and representative data are shown. Quantitative data are expressed as the mean±SEM. P<0.05,**P<0.01,***P<0.001
표 Cilostazol induces mitochondrial biogenesis. (A-F) HepG2 cells were treated with cilostazol at the indicated dose and time. Expression levels of NRF-1, PGC-1a, and TFAM mRNA were measured by real-time RT-PCR (A, B). COX III, COX IV, PGC-1a and TFAM protein levels were measured by western blotting (C, D). The relative mtDNA content was measured by real-time PCR (E, F). Mitochondrial mass was assessed by MitoTracker Red CMXRos staining (red) (G). Nuclei were stained with Hoechst dye (blue). Images of fluorescence were analyzed by confocal microscopy. All experiments were performed in triplicate, and representative data are shown. Quantitative data are expressed as mean±SEM. **P<0.01,***P<0.001
표 Cilostazol induces mitochondrial biogenesis through the induction of HO-1 and reducing ROS production. (A-D) HepG2 cells were pretreated in the absence or presence of ZnPP (10mM), and then treated with 10mM cilostazol for 12 h. Expression levels of NRF-1, PGC-1a, and TFAM mRNA were measured by real-time RT-PCR (A). The relative mtDNA content was measured by real-time PCR. mtDNA content was normalized to nDNA (b-actin gene) content (B). Mitochondrial mass was assessed by using MitoTracker Red (red). Nuclei were stained with Hoechst dye (blue). Images of fluorescence were analyzed by confocal microscopy (C). HepG2 cells were pretreated in the absence or presence of ZnPP (10 mM), treated with 10 mM cilostazol for 6 h, and then treated with 100 mM H2O2 for 30 min. Intracellular ROS production was measured by flow cytometry using CM-H2DCFDA (D). (E, F) WT and HO-1 KO mice, cilostazol (10mg/kg/day) was given once daily for 3 days by i.p. injection. Liver tissues were excised and analyzed for mitochondrial biogenesis in mice. Expression of PGC-1a, NRF-1, TFAM mRNA (E) and mtDNA content (F) were measured by real-time RT-PCR. (G-I) HepG2 cells were transfected with control siRNA (con) or Nrf2 siRNA to knockdown Nrf2 levels. Cells were treated with 10mM cilostazol for 12 h. siRNA were subjected to western blot and RT-PCR to confirm Nrf2 siRNA efficiency (G). Expression of HO-1 (G), PGC-1a, NRF-1, TFAM mRNA (H) was measured by RT-PCR. mtDNA content were measured by real-time RT-PCR (I). All experiments were performed in triplicate (n = 7/group), and representative data are shown. Quantitative data are expressed as mean±SEM. ** P<0.01, *** P<0.001
표 Cilostazol reduces I/R-induced hepatic injury in Nrf2 WT and KO mice. (A-E) Wild type (WT) and Nrf2 KO mice, cilostazol (10 mg/kg/day) was given once daily for 3 days by i.p. injection. Mice were subjected to 60 minutes liver warm ischemia, followed by 6 h reperfusion. Liver tissues were excised and analyzed for mitochondrial biogenesis in mice. Expression of HO-1 protein was measured by Western blot analysis (A). Expression of NRF-1, PGC-1a, TFAM mRNA and mtDNA content were measured by real-time RT-PCR (B, C). Hepatocellular function was evaluated by serum levels of ALT (D). Representative liver histology (H&E staining) is shown (E). All experiments were performed in triplicate (n = 7/group), and representative data are shown. Quantitative data are expressed as mean±SEM. *P<0.05,**P<0.01,***P<0.001
표 Cilostazol increases HO-1 expression and Nrf2 activation in dose- and time dependent manner. HepG2 cells were treated with cilostazol at the indicated dose and time. Expression levels of HO-1 mRNA and protein were measured by RT-PCR and western blotting (A, B). Cytosol (CE) and Nuclear (NE) accumulation of Nrf-2 was confirmed by Western blotting. Lamin A/C and β -actin were used as a loading control (C)
표 Carbon monoxide protects liver ischemia/reperfusion injury in mice. Mice were sham-operated or subjected to 60 minutes liver warm ischemia followed by 6 hour reperfusion. Mice were treated with air or CO gas (250 ppm) inhalation. (A) sALT levels (U/L) were measured in blood samples retrieved after IRI. (B) Liver neutrophil accumulation, assessed by MPO level. (C) H&E staining of liver samples from air- and CO-inhaled mice after IR and Sham mice. (D) Quantitative RT-PCR-assisted detection of TNF-α, IL-6, iNOS, and IL-1β gene expression at 6 hours in liver tissue. Data were normalized to GAPDH gene expression. SEM. (N=4-6/group), **P<0.01,***P<0.001
표 Carbon monoxide induces SIRT1 expression after hepatic ischemia/reperfusion (I/R). Liver samples were harvested at 6 hours after liver IRI. (A) Western blot analysis of SIRT1, acetylated-p65, and p53. β-actin was used as an internal control. (B) SIRT1 mRNA expression, as detected by quantitative RT-PCR in liver tissue. Data were normalized to GAPDH mRNA levels. (C) qRT-PCR analysis of miR34a expression in liver tissue. Data were normalized to RNU6B. Data shown represent the mean ± SEM. (N=4-6/group), ***P<0.001,**P<0.01
표 P53 inhibitor, PFT administration ameliorates liver I/R injury in mice. Mice were subjected to 60 minutes liver warm ischemia, followed by 4 h reperfusion. Liver samples were harvested from unclamped mice (sham) or mice clamped and pretreated with DMSO (IR), Pifithrim-α (IR-PFT). (A) Hepatocellular function was evaluated by sALT (U/L). (B) MPO level, an index of neutrophil infiltration. (C) H&E staining of liver sections from each group. (D) Hepatic HMGB1 expression in liver tissue was assessed by western blot analysis. (E) Semi- and quantitative RT-PCR-assisted detection of TNF-α, IL-6, iNOS, CXCL10, and IL-1β gene expression in liver samples. Data were normalized to GAPDH gene expression. Data represent the mean ± SEM (N=4-6 samples/group). *P<0.05,**P<0.01
표 PFT induces SIRT1 expression after hepatic ischemia/reperfusion (I/R). Liver samples were harvested at 4 hours after liver IRI. (A) Hepatic expression of SIRT1 and Ac-p65 were evaluated in IR lobes from DMSO (IR) and PFT (PFT-IR) injected mice versus sham mice by immunoblotting. (B) SIRT1 mRNA expression, as detected by semi- and quantitative RT-PCR in liver tissue. Data were normalized to GAPDH mRNA levels. (C) Immunohistochemical staining of SIRT1. (D) Expression of miR34a was evaluated in liver tissue by quantitative RT-PCR. Data were normalized to RNU6B. Data shown represent the mean ± SEM. (N=4-6/group), *P<0.05
표 PFT increases SIRT1 induction, promotes anti-apoptotic functions. Hepatic expression of acetylated p53, cleaved caspase 3, Bcl2, Bcl-xL and actin were evaluated in IR lobes from DMSO (IR) and PFT (PFT-IR) injected mice versus sham mice by immunoblotting
표 The protective effect of p53 inhibition by PFT is dependent on the miR34a/SIRT1 pathway. RAW264.7 cells were stimulated or not with 1 μg/ml of LPS for 1 hour in the absence or presence of PFT. (A) Expression of miR34a was evaluated by quantitative RT-PCR. Data were normalized to RNU6B. (B) Cell lysates were analyzed by western blotting with Abs against SIRT1, acetylated-p65, or β-actin. SIRT1 mRNA expression was also evaluated by semi- and quantitative RT-PCR. (C-F) After silencing of SIRT1 with siRNA in RAW264.7 cells, mRNA expression of SIRT1(C and D), protein levels of ac-p65 (E), and inflammation (TNF-α, IL-6, iNOS, and IL-1β) (F) were evaluated. The mRNA levels were normalized to GAPDH mRNA levels. Data shown represent the mean ± SEM. (N=3), *P<0.05.**P<0.01,***P<0.001
표 PFT reduces inflammatory response in macrophages. RAW264.7 cells were stimulated or not with 1 μg/ml of LPS for 1 hour in the absence or presence of PFT. The gene expression of TNF-α, IL-6, iNOS, and IL-1β was evaluated by semi- and quantitative RT-PCR. Data were normalized to GAPDH gene expression. Data shown represent the mean ± SEM. (N=3), *P<0.05.**P<0.01,***P<0.001
표 Carbon monoxide reduces p53 level, inhibiting p53-dependent induction of the miR-34a/SIRT1/p53 pathway in hepatocytes. (A and B) AML12 hepatocytes were transfected with p53 responsible element reporter constructs (p53-luc) for 36 hours. Cells were pretreated with CORM in a dose dependent manner and stimulated with 1mM of H2O2 or 250uM of Cocl2. After 6h, luciferase activity was measured. (C) AML12 hepatocytes were pretreated with CORM in a dose dependent manner and stimulated with 250uM of Cocl2. Cell lysates were analyzed by western blotting with Abs against p53 and β-actin. (D-E) AML12 hepatocytes were transfected with p53 expressing plasmid for 36 hrs and treated with 30 μM with treated with CORM2. D, Expression of miR34a was evaluated by quantitative RT-PCR. E, SIRT1 mRNA expression was also evaluated by quantitative RT-PCR. (F) AML12 hepatocytes were co-transfected with p53 responsible element reporter constructs (p53-luc) and p53 expressing plasmids for 36 hours and then treated with CORM2. After 6h, luciferase activity was measured. (G and H) AML12 hepatocytes were preincubated with CORM2 for 1 hour and stimulated with 1mM of H2O2. G, Expression of miR34a was evaluated by quantitative RT-PCR. H, Western blotting of SIRT1, Ac-p53, or β-actin. I. EX527 or siSIRT1 was used as SIRT1 inhibition or SIRT1 knockdown. miR34a expression data were normalized to RNU6B and mRNA expression were normalized to GAPDH mRNA levels. Data shown represent the mean ± SEM. (N=3), *P<0.05.**P<0.01,***P<0.001
표 CO decreases miR34a expression and increases SIRT1 expression. (A and B) AML12 hepatocytes were transfected with miR34a mimic for 36 hours. Cells were pretreated with CORM2. After 6h, luciferase activity was measured. Expression of miR34a was evaluated by quantitative RT-PCR. Data were normalized to RNU6B. (C) Cell lysates were analyzed by western blotting with Abs against SIRT1 and β-actin
표 CoPP의 공급에 따른 렙틴 수용체 결핍(db/db) 쥐의 몸무게와 혈당량 변화와 CoPP 투여에 따른 HO-1의 발현 관찰. (A) CoPP(3mg/kg once a day total 6weeks)가 복강 내 투여된 각 그룹 쥐의 6주간의 몸무게 변화. (B) CoPP(3mg/kg once a day total 6weeks)가 복강 내 투여된 각 그룹 쥐의 6주간의 혈당량 변화. (C) CoPP(3mg/kg once a day total 6weeks)가 복강 내 투여된 각 그룹 쥐의 간조직을 채취, 단백질 시료로 만든 후, HO-1 단백질에 대한 발현 정도를 Western blot으로 확인함
표 소포체 스트레스로 유도된 GSK3β 활성화는 배양된 대식세포에서 IL-1β과 TNF-α의 메신저 RNA 발현을 조절
표 소포체 스트레스로 유도되는 IRE1α의 활성화는 두 개의 다른 downstream 분자인 GSK-3β와 XBP-1을 조절
표 GSK3β 활성화는 소포체 스트레스와 TLR4 신호에 의한 IL-1β와 TNF-α의 전사를 조절
표 GSK3β는 TNF-α의 유전자 발현을 매개하는 소포체 스트레스와 TLR4 신호에 의한 XBP-1 splicing을 억제
표 IRE1α-의존적인 XBP-1 splicing은 소포체 스트레스로 유도된 TNF-α 유전자 활성화와 연관
표 화학적 샤페론은 소포체 스트레스로 유도되는 IL-1β와 TNF-α 전사를 감소
표 소포체 스트레스 동안 염증성 사이토카인의 조절에서 GSK3β와 XBP-1의 연관된 역할
표 Effects of quercetin on hepatic inflammatory responses and macrophage phenotypes in HFD-fed obese mice. (A) Inflammatory cytokines (TNFa, IL-6, and MCP-1) and (B) an anti-inflammatory cytokine (IL-10) in liver were determined as described in Methods. Results are means ± SEM of six mice per group. *<0.05, **<0.01, ***<0.001 compared with obese mice fed an HFD. (C) F4/80 as a macrophage marker and (D-E) M1 and M2 macrophages mRNAs in livers were determined by real time-qPCR. The intensity of the bands was densitometrically measured and normalized to GAPDH transcripts. Results are means ± SEM of six mice per group. *<0.05, **<0.01 compared with obese mice fed an HFD
표 Effect of HO-1 inhibition on quercetin action in cocultures of lipid-laden hepatocytes with macrophages. Raw 264.7 macrophages were cocultured with the lipid-laden hepatocytes for 12h in the presence or absence of quercetin or Znpp. (A) HO-1 protein was measured by western blotting in macrophages. Results are means ± SEM of two experiments with duplicate determinations. *<0.05, ***<0.001 compared with no treatment. (B) MCP-1 production detected by ELISA in cocultured lipid-laden hepatocytes and macrophages. Results are means ± SEM of triplicate samples. *<0.05, ***<0.001 compared with quercetin treatment. (C) M1 macrophage mRNAs in cocultured lipid-laden hepatocytes and macrophages determined by real time-qPCR. Results are means ± SEM of triplicate samples. *<0.05, **<0.01, ***<0.001 compared with quercetin treatment
표 Effect of Nrf2 deficiency on quercetin action in LPS-stimulated macrophages (A) HO-1 protein was measured by western blotting in peritoneal macrophages of Nrf2 KO and WT mice. Results are means ± SEM of three independent experiments. **<0.01 compared with LPS treatment. (B) M1/M2 marker mRNAs were determined by real time-qPCR. The intensity of the bands was densitometrically measured and normalized to GAPDH transcripts. Results are means ± SEM of two experiments with duplicate determinations. *<0.05, ***<0.001 compared with LPS treatment
표 CO administration increases TTP level in lung tissue. (A) Basic experimental protocol used to test the effect of CO administration on TTP level. Wild type (TTP WT) and TTP−/− (TTP KO) mice were exposed to CO at a concentration of 250 ppm for 5 days and acute lung injury was induced by intranasal administration of LPS (2.5 mg/kg body weight). After treatment with LPS for 24 h, lung tissues were collected and TTP level was determined by (B) western blot and (C) quantitative real-time PCR. Values are mean ± SD (n = 5) from two separate experiments, with 5 mice per group per experiment. *, p<0.05
표 TTP deficiency abrogates the protective effects of CO against LPS-induced lung injury. Wild type (TTP WT) and TTP−/− (TTP KO) mice were treated as described in the legend of Figure 1. (A and B) After treatment with LPS for 24 h, BAL fluids were collected and analyzed for (A) total cells and (B) total protein. (C) Representative FACS plots and (D) the percentages of CD11b+Ly6G+neutrophilsinBALfluidsofWTandTTPKOmice.(EandF)Histologicalevaluationofthetherapeuticpo tentialofCOagainstLPS-inducedlunginjuryinWTandTTPKOmice.(E)Representativeimageswithmagnifiedinse tsofhematoxylinandeosin-stainedlungsectionsfromfourexperimentalgroups.(F)Histopathologicallungin juryscoreforfourexperimentalgroups.Thescorerepresentstheaverageoftwoindependentinvestigatorswhor eadeachH&E-stainedlungsection.Allvaluesaremean±SD from two separate experiments, with 5 mice per group per experiment.* **,p<0.001
표 TTP deficiency blocks the inhibitory effects of CO on LPS-induced lung inflammation. Wild type (TTP WT) and TTP−/− (TTP KO) mice were treated as described in the legend of Figure 1. (A-C) Levels of proinflammatory cytokines in BAL fluid. After treatment with LPS for 24 h, BAL fluids were collected and analyzed for levels of the proinflammatory cytokines, (A) TNF-α, (B) IL6, and (C) IL1β by ELISA. (D-I) Levels of proinflammatory cytokines in lung homogenates. After treatment with LPS for 24 h, lung homogenates were analyzed for levels of the proinflammatory cytokines, (D and G) TNF-α, (E and H) IL6, and (F and I) IL1β by (D-F) ELISA or (G-I) quantitative real-time PCR. All values are mean ± SD from two separate experiments, with 5 mice per group per experiment. ***, p<0.001
표 CO enhances degradation of inflammatory cytokine mRNAs in a TTP-dependent manner. Wild type (TTP WT) and TTP−/− (TTP KO) mice were treated as described in the legend of Figure 1. After treatment with LPS for 24 h, BAL fluids were collected, and the expression of (A) TNF-α and (B) IL1β mRNAs in BAL cells was determined by quantitative real-time PCR and mRNA half-life was calculated from the non-linear regression of the mRNA levels at the indicated times after the addition of 5 mg/ml of actinomycin D. All values are mean ± SD from two separate experiment, with 3 mice per group per experiment. *, p<0.05
표 Effect of combinatorial treatment of cilostazol (CZ) and probucol (PB) on HO-1 expression. HepG2 cells were treated with cilostazol at various concentrations (0, 0.1, 0.3, 1, and 3 μM) with or without probucol (0.1 μM) for 4 h and HO-1 mRNA levels were detected by RT-PCR (A) and real-time RT-PCR (B). Primary hepatocytes were treated with probucol (0.1 μM) and cilostazol (3 μM) individually, as well as with their combination for 4 h and HO-1 mRNA and protein levels were detected by RT-PCR (C) and Western blot analysis (D), respectively. Quantitative data are expressed as means ± SE, n=3. **: P<0.01, ***: P<0.001 and #: probucol (0.1 μM) and cilostazol (3 μM) have a synergistic effect on HO-1 expression
표 Effect of combinatorial treatment of cilostazol (CZ) and probucol (PB) on mitochondrial biogenesis related genes expression. HepG2 cells were treated with cilostazol at various concentrations (0, 0.1, 0.3, 1, and 3 μM) with or without probucol (0.1 μM) for 4 h and PGC-1 α, TFAM, NRF-1 mRNA expression were detected by RT-PCR (A) and real-time RT-PCR (B, C, and D). Primary hepatocytes isolated from C57BL/6 mice were treated with probucol (0.1 μM) and cilostazol (3 μM) individually, as well as with their combination for 4 h. Mitochondrial biogenesis-related genes PGC-1α, TFAM, and NRF-1 were measured by RT-PCR (E), and mitochondrial-related proteins PGC-1α, COX III, and COX IV were measured by Western bloting (F). Primary hepatocytes from HO-1 WT mice were pretreated in the absence or presence of ZnPP (10 μM) and then were treated with probucol (0.1 μM) and cilostazol (3μM) individually or in combination for 4 h and then the expression of mitochondrial biogenesis-related genes PGC-1α, TFAM, and NRF-1 was detected by RT-PCR (G). Furthermore, primary hepatocytes from HO-1 KO mice were also analyzed by RT-PCR to detect mitochondrial biogenesis-related genes expression (H). Bar graphs, right panels of (A), (E) and (F), as well as lower panels of (G) and (H) are summary data of normalized densitometric ratios. Quantitative data are expressed as means ± SE, n=3. *: P<0.05, **: P<0.01, ***: P<0.001 vs. cells without treatment; ### vs. cells treated with probucol; $$$ vs. cells treated with cilostazol
표 Combinatorial treatment of cilostazol (CZ) and probucol (PB) ameliorates tunicamycin (TM)-induced mitochondria dysfunction. Primary hepatocytes were pretreated with probucol (0.1 μM) and cilostazol (3 μM) individually, as well as with their combination for 30 min followed by stimulation of tunicamycin (10 μg/ml) for 18 h. Mitochondrial mass was assessed by using Mito Tracker Red (red). Nuclei were stained with Hoechst dye (blue). Images of fluorescence were analyzed by confocal microscopy (A). The relative mtDNA content was measured by real-time PCR. mtDNA content was normalized by nDNA content (B). ATP production was measured in primary hepatocytes isolated from HO-1 WT and KO mice (C). The expression of HO-1 and mitochondrial biogenesis-related genes PGC-1α, TFAM, and NRF-1, as well as related proteins PGC-1α, COX III, and COX IV were measured by RT-PCR (D) and Western blot analysis (E). RT-PCR was also performed in HO-1 KO mice to detect mitochondrial biogenesis-related genes PGC-1α, TFAM, and NRF-1 (F). HepG2 cells were transfected with scRNA and siHO-1 for 24h. Then cells were pretreated with probucol(0.1 μM) and cilostazol(3 μM) individually or combinatorially for 30 min followed by stimulation of tunicamycin (10 μg/ml) for another 18h. RT-PCR was performed to detect HO-1, PGC-1α, TFAM, and NRF-1 mRNA level (G). Bar graphs, right panels of (D), (E) and (F), are summary data of normalized densitometry ratios. Quantitative data are expressed as means ± SE, n=3. *: P<0.05, **: P<0.01, ***: P<0.001 vs. cells without treatment; #: P<0.05, ###: P<0.001 vs. cells treated with tunicamycin; $$$: P<0.001 vs. cells treated with probucol and tunicamycin; &: P<0.05, &&&: P<0.001 vs. cells treated with cilostazol and tunicamycin
$Metformin induces expression of TTP in both p53 wild-type and p53 mutant breast cancer cells. a, b Metformin increases TTP levels in p53 wild-type human breast cancer MCF7 cells. MCF7 cells were treated a with the indicated concentrations of metformin for 24 h or b with 6 mM metformin for the indicated length of time. The levels of TTP were measured by semi-qRT-PCR (a, b, top) and Western blotting (a, b, bottom). c Metformin induces TTP promoter activity in p53 wild-type MCF7 cells. MCF7 cells were transfected with pGL3/TTPp-1343 containing the TTP promoter. After treatment with 6 mM metformin for 24 h, luciferase activity was measured. The expression levels obtained from pGL3-transfected cells without metformin treatment were set to 1. Data are presented as the mean ± SD (n = 3). ***p\0.001. d Metformin induces p53 phosphorylation in p53 wild-type MCF7 cells but not p53 mutant MDA-MB-231 cells. MCF7 and MDA-MB-231 cells were treated with 6 mM metformin for the indicated length of time. The levels of p53 and phospho-p53 (p-p53) were measured by Western blotting. e, f Metformin increases TTP levels in p53 mutant human breast cancer MDA-MB-231 cells. MDA-MB-231 cells were treated e with the indicated concentrations of metformin for 24 h or f with 6 mM metformin for the indicated length of time. The levels of TTP were measured by semi-qRT-PCR (e, f top) and Western blotting (e, f bottom). g Metformin induces TTP promoter activity in MDA-MB- 231 cells. MDA-MB-231 cells were transfected with pGL3/TTPp- 1343 containing the TTP promoter. After treatment with 6 mM metformin for 24 h, luciferase activity was measured. The expression levels obtained from pGL3-transfected cells without metformin treatment were set to 1. Data are presented as the mean ± SD (n = 3). ***p\0.001$ 표 Metformin induces expression of TTP in both p53 wild-type and p53 mutant breast cancer cells. a, b Metformin increases TTP levels in p53 wild-type human breast cancer MCF7 cells. MCF7 cells were treated a with the indicated concentrations of metformin for 24 h or b with 6 mM metformin for the indicated length of time. The levels of TTP were measured by semi-qRT-PCR (a, b, top) and Western blotting (a, b, bottom). c Metformin induces TTP promoter activity in p53 wild-type MCF7 cells. MCF7 cells were transfected with pGL3/TTPp-1343 containing the TTP promoter. After treatment with 6 mM metformin for 24 h, luciferase activity was measured. The expression levels obtained from pGL3-transfected cells without metformin treatment were set to 1. Data are presented as the mean ± SD (n = 3). ***p\0.001. d Metformin induces p53 phosphorylation in p53 wild-type MCF7 cells but not p53 mutant MDA-MB-231 cells. MCF7 and MDA-MB-231 cells were treated with 6 mM metformin for the indicated length of time. The levels of p53 and phospho-p53 (p-p53) were measured by Western blotting. e, f Metformin increases TTP levels in p53 mutant human breast cancer MDA-MB-231 cells. MDA-MB-231 cells were treated e with the indicated concentrations of metformin for 24 h or f with 6 mM metformin for the indicated length of time. The levels of TTP were measured by semi-qRT-PCR (e, f top) and Western blotting (e, f bottom). g Metformin induces TTP promoter activity in MDA-MB- 231 cells. MDA-MB-231 cells were transfected with pGL3/TTPp- 1343 containing the TTP promoter. After treatment with 6 mM metformin for 24 h, luciferase activity was measured. The expression levels obtained from pGL3-transfected cells without metformin treatment were set to 1. Data are presented as the mean ± SD (n = 3). ***p\0.001
$TTP mediates the antiproliferative function of metformin in both p53 wildtype and p53 mutant breast cancer cells. a–-d Metformin inhibits proliferation of both p53 wild-type MCF7 cells and p53 mutant MDA-MB-231 cells. a, c MCF7 and b, d MDAMB- 231 cells were treated (a, b) with the indicated concentrations of metformin for 24 h and (c, d) with 6 mM metformin for 24 and 48 h. Cell viability was assessed by measuring absorbance at 490 nm using an MTS cell proliferation assay. The values obtained with mock-treated cells were set to 100. Values are the mean ± SD (n = 3). *p\0.05, **p\0.01, ***p\0.001. e–-h Inhibition of TTP attenuates the antiproliferative effects of metformin in both MCF7 and MDA-MB-231 cells. MCF7 and MDA-MB-231 cells were transfected with e, g, h pcDNA6/TTP or f, g, h TTPspecific siRNA (TTP-siRNA). scRNA and pcDNA6 were used as negative controls. After treatment with 6 mM metformin for 24 h, cell viability was assessed by measuring the absorbance at 490 nm using an MTS cell proliferation assay. The values obtained with mocktreated cells were set to 100. Values are the mean ± SD (n = 3). *p\0.05, **p\0.01, ***p\0.001. ns not significant$ 표 TTP mediates the antiproliferative function of metformin in both p53 wildtype and p53 mutant breast cancer cells. a–-d Metformin inhibits proliferation of both p53 wild-type MCF7 cells and p53 mutant MDA-MB-231 cells. a, c MCF7 and b, d MDAMB- 231 cells were treated (a, b) with the indicated concentrations of metformin for 24 h and (c, d) with 6 mM metformin for 24 and 48 h. Cell viability was assessed by measuring absorbance at 490 nm using an MTS cell proliferation assay. The values obtained with mock-treated cells were set to 100. Values are the mean ± SD (n = 3). *p\0.05, **p\0.01, ***p\0.001. e–-h Inhibition of TTP attenuates the antiproliferative effects of metformin in both MCF7 and MDA-MB-231 cells. MCF7 and MDA-MB-231 cells were transfected with e, g, h pcDNA6/TTP or f, g, h TTPspecific siRNA (TTP-siRNA). scRNA and pcDNA6 were used as negative controls. After treatment with 6 mM metformin for 24 h, cell viability was assessed by measuring the absorbance at 490 nm using an MTS cell proliferation assay. The values obtained with mocktreated cells were set to 100. Values are the mean ± SD (n = 3). *p\0.05, **p\0.01, ***p\0.001. ns not significant
$Metformin induces TTP expression through down-regulation of c-Myc. a Metformin treatment increases phospho-AMPK (pAMPK) but decreases c-Myc and phospho-STAT3 (pSTAT3) in MCF7 and MDA-MB-231 cells. MCF7 and MDA-MB-231 cells were treated with 6 mM metformin for the indicated length of time, and the levels of TTP, STAT3, pSTAT3, AMPK, pAMPK, and c-Myc were measured by Western blotting. b, c inhibition of c-Myc by siRNA enhances TTP promoter activity in MCF7 and MDA-MB-231 cells. b MCF7 and c MDA-MB-231 cells were transfected with pGL3/ TTPp-1343 containing the TTP promoter. After treatment with 6 mM metformin for 24 h, luciferase activity was measured. The expression levels obtained from pGL3-transfected cells without metformin treatment were set to 1. Data are presented as the mean ± SD (n = 3). *p\0.05, **p\0.01, ***p\0.001. d Metformin increases TTP expression in an AMPK-dependent manner. MCF7 and MDA-MB-231 cells were treated with 6 mM metformin with or without 20 lM Compound C for 24 h. The levels of TTP, AMPK, and pAMPK were measured by Western blotting. e, f Overexpression of c-Myc blocks the effect of metformin on TTP induction. MDA-MB- 231 cells were transfected with pcDNA3/c-Myc or control pcDNA3. Cells were treated with 6 mM metformin for 24 h. e c-Myc and TTP levels were measured by quantitative RT-PCR. The values obtained with pcDNA-transfected and mock-treated cells were set to 1. Data are presented as the mean ± SD (n = 3). ***p\0.001. ns not significant. g, h Overexpression of c-Myc attenuates the antiproliferative effect of metformin in MCF7 and MDA-MB-231 cells. g MCF7 and h MDA-MB-231 cells were transfected with pcDNA3/c- Myc or control pcDNA3. Cells were treated with 6 mM metformin for 24 h. Cell viability was assessed by measuring the absorbance at 490 nm using an MTS cell proliferation assay. The values obtained with mock-treated cells were set to 100. Data are presented as the mean ± SD (n = 3). **p<0.01, ***p<0.001. ns not significant$ 표 Metformin induces TTP expression through down-regulation of c-Myc. a Metformin treatment increases phospho-AMPK (pAMPK) but decreases c-Myc and phospho-STAT3 (pSTAT3) in MCF7 and MDA-MB-231 cells. MCF7 and MDA-MB-231 cells were treated with 6 mM metformin for the indicated length of time, and the levels of TTP, STAT3, pSTAT3, AMPK, pAMPK, and c-Myc were measured by Western blotting. b, c inhibition of c-Myc by siRNA enhances TTP promoter activity in MCF7 and MDA-MB-231 cells. b MCF7 and c MDA-MB-231 cells were transfected with pGL3/ TTPp-1343 containing the TTP promoter. After treatment with 6 mM metformin for 24 h, luciferase activity was measured. The expression levels obtained from pGL3-transfected cells without metformin treatment were set to 1. Data are presented as the mean ± SD (n = 3). *p\0.05, **p\0.01, ***p\0.001. d Metformin increases TTP expression in an AMPK-dependent manner. MCF7 and MDA-MB-231 cells were treated with 6 mM metformin with or without 20 lM Compound C for 24 h. The levels of TTP, AMPK, and pAMPK were measured by Western blotting. e, f Overexpression of c-Myc blocks the effect of metformin on TTP induction. MDA-MB- 231 cells were transfected with pcDNA3/c-Myc or control pcDNA3. Cells were treated with 6 mM metformin for 24 h. e c-Myc and TTP levels were measured by quantitative RT-PCR. The values obtained with pcDNA-transfected and mock-treated cells were set to 1. Data are presented as the mean ± SD (n = 3). ***p\0.001. ns not significant. g, h Overexpression of c-Myc attenuates the antiproliferative effect of metformin in MCF7 and MDA-MB-231 cells. g MCF7 and h MDA-MB-231 cells were transfected with pcDNA3/c- Myc or control pcDNA3. Cells were treated with 6 mM metformin for 24 h. Cell viability was assessed by measuring the absorbance at 490 nm using an MTS cell proliferation assay. The values obtained with mock-treated cells were set to 100. Data are presented as the mean ± SD (n = 3). **p<0.01, ***p<0.001. ns not significant
표 (a, b, e, f) THP-1 cells were treated with 20 μM CORM-2 for indicated times and various concentrations of CORM-2 or RuCl2 for 12 h. (c and g) THP-1 cells were exposed with CO gas (250 ppm) for indicated time points. (d and h) THP-1 cells were incubated with CORM-2 and/or the transcriptional inhibitor actinomycin D (Act D; 1 μg mL−1) and cycloheximide (CHX; 5 μg mL−1) for 12 h. (i) THP-1 cells were pretreated with CORM-2 at the indicated concentrations for 30 min, and then treated with LPS (1 μg mL−1) for 12 h. (j and k) THP-1 cells were incubated with CORM-2, LPS, or IL-10 at the indicated concentrations for 12 h.a, b, c, d, j: The levels of pyrin mRNA were analyzed by quantitative real-time PCR. GAPDH served as the standard.e, f, g, h, i, k: The expression of pyrin was determined by western blot analysis. β-actin served as the standard. The relative band density on western blots was normalized to the β-actin loading control and was quantitated. All experiments were performed in triplicate, and representative data are shown. Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.005
표 (a) THP-1 cells were stimulated with LPS (1 μg mL−1) in the absence or presence of CORM-2 (20 μM) at indicated time, and then ATP (5 mM) was added for the last 30 min
표 (a–d) THP-1 cells were transfected with pyrin siRNA or control siRNA (con), pre-treated with or without 20 μM CORM-2 for 30 min, and then treated with LPS (1 μg mL−1) for 12 h and ATP (5 mM) was added for the last 30 min. (a) The knockdown of pyrin by siRNA was confirmed by quantitative real-time PCR. (b) The pro-caspase-1, cleaved caspase-1 (p10), pro-IL-1β, and IL-1β p17 in the supernatants (Sup) and the cell lysates (Lys) were analyzed by western blot analysis. Secreted IL-1β (c) and TNF-α (d) in the supernatants were measured by ELISA. (e) THP-1 cells were exposed with LPS (1 μg mL−1) + ATP (5 mM) in the absence or presence of CORM-2 (20 μM). The interaction of pyrin and pro-caspase-1 was analyzed by immunoprecipitation (IP) of anti-Pyrin followed by western blot (WB) for anti-pro-caspase-1. To evaluate the binding ASC and NLRP3, cell lysates were performed IP with anti-ASC and then followed by WB for anti-NLRP3. Total cell lysates were used as input controls. (f and g) THP-1 cells were transfected with pyrin siRNA or control siRNA (con), cells were exposed with 20 μM of CORM-2 for 12 h. The levels of pyrin mRNA (f) and protein (g) were measured. GAPDH and β-actin were used as a loading control for quantitative real-time PCR or western blot analysis, respectively. The relative band density on western blots was normalized to the β-actin loading control and was quantitated. All experiments were performed in triplicate, and representative data are shown. Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.005
표 (a–e) C57BL/6 mice were exposed to air or CO gas (250 ppm) for 4 h each day for 5 days and then injected with LPS (10 mg kg−1, i.p.) for 24 h. The levels of pyrin and IL-10 were detected in the lung (a and c) and spleen (b) tissue. Protein expression was measured using western blot analysis. β-actin served as the standard. IL-10 (d) and IL-1β (e) in the lung, spleen tissue, and serum measured by ELISA. (f–j) C57BL/6 mice were exposed to air or CO gas (250 ppm) for 4 h each day for 5 days and then subjected to intranasal administration of LPS (2.5 mg kg−1). (f) The levels of pyrin and IL-10 in the lung after 24 h. (g and h). Lung sections were stained with H **P < 0.01; ***P < 0.005
표 Schema representing the downregulation of IL-1β production by CO via suppressing the inflammasome through induction of pyrin. CO increases pyrin expression in an IL-10-dependent manner. The increased pyrin downregulates IL-1β production via suppressing the NLRP3 inflammasome and associated caspase-1 cleavage
표 Effect of quercetin on the inflammatory response of lipid-laden microglia. Microglia were pretreated with quercetin (5, 10μM) for 2h, and then treated with palmitic acid (300 μM) for 48h. (A and B) The levels of MCP-1, IL-6 and IL-1b in the conditioned-medium were measured by ELISA. (C) The amounts of nitrite in the conditioned-medium were measured by the Griess method. (D) The transcription levels of CD11b were measured by RT-PCR analysis. Data are the means ± SEM of three independent experiments performed in triplicate. *P<0.05, **P<0.01, ***P<0.001 compared with control
표 Effect of quercetin on inflammatory response and HO-1 inhibition in lipid-laden microglia. Microglia were pretreated with quercetin (5, 10 μM) for 2h, then treated with palmitic acid (300μM). (A) The expression of HO-1 protein was analyzed by 11% SDS-PAGE and Western blotting with antibody against HO-1 protein. (B) The transcription levels of inflammatory cytokines (MCP-1, TNFa, IL-1b) were measured by RT-PCR analysis. (C) The expression of IkBa protein was analyzed by 11% SDS-PAGE and western blotting with antibody against IkBa protein. Data are the means ± SEM of three independent experiments performed in triplicate. *P<0.05, **P<0.01 compared with control
표 Effect of quercetin supplementation on obesity-induced hypothalamic inflammation in obese mice fed an HFD. B6/SJL mice were fed a low fat diet (LFD, 10% of calories from fat), high fat diet (HFD, 60% of calories from fat) or HFD+0.05% quercetin for 8 weeks. The hypothalamuses were then isolated from LFD (n=5), HFD (n=5) and quercetin fed HFD (n=5). (A) Expression of Plin-2, (B) inflammatory cytokine (TNFa, IL-1b, and MCP-1) genes, (C) markers of microglia activation (Iba-1 and CD11b), (D) expression of HO-1, (E) SOCS3, (F) HSP72 and HSP70 genes in hypothalamus from LFD (n=5), HFD (n=5), and HFD+0.05% quercetin (n=5) fed mice. Values are means ± SEM *P<0.05, **P<0.01, significantly different from LFD control (LFD vs. HFD), HFD control (HFD vs. HFD+0.05% quercetin)
표 FGF21 Wild type과 knockout 생쥐에 고지방식이와 CO을 먹인 후 간손상의 변화 관찰
표 FGF21 Wild type과 knockout 생쥐에 고지방식이와 CO을 먹인 후 몸무게 변화
표 FGF21 Wild type과 knockout 생쥐에 고지방식이와 CO을 먹인 후 ITT와 GTT 측정
표 간의 무게, Triglyceride양, serum ALT와 AST측정
표 간세포에서 CO에 의한 FGF21 발현 양 측정
표 간세포에서 CO에 의한 FGF21 발현 양 조절의 기전 관찰
표 FGF21은 CO/PERK/eIF2a/ATF4의 신호전달 경로를 통하여 증가
표 ER stress에 의한 간손상에서 FGF21의 역할
표 Tunicamycin에 의한 비알콜성간질환은 FGF21의 과발현으로 간손상회복됨 Tunicamycin (TM)
표 비알콜성 간질환에서 FGF21을 통한 염증성 싸이토카인 조절
표 CO에 의한 고지방식이에 따른 손상된 지방조직 회복
표 CO에 의한 고지방식이에 따른 각 지방조직의 무게 감소
표 CO에 의한 고지방식이에 따른 지방조직의 조절
표 CO에 의한 고지방식이에 따른 간 지방 분해에 관련된 유전자의 조절
표 CO에 의한 갈색지방화에 관여 하는 유전자 조절
표 CO에 의한 지방분해와 갈색지방화의 증가
표 4-PG induces TTP mRNA and protein in RAW264.7 cells. (a) Chemical structure of pterostilbene (PTER) and pterostilbene 4′-glucoside (4-PG). (b–d) RAW264.7 cells were treated at various concentrations of 4-PG (0, 1, 5, 10, and 20 M) and PTER(10 M) for 12 h. The mRNA and protein expression of TTP were determined by RT-PCR (b), real-time PCR (c) and Western blot (d), respectively. (e–g) RAW264.7 cells were treated with 4-PG (10 M) and PTER (10 M) in indicated time points (0, 2, 4, 8, and 12 h). The mRNA and protein expression of TTP were analyzed by RT-PCR (e), real-time PCR (f) as well as Western blot (g), respectively. (h) To compare the effect of 4-PG and PTER on TTP protein expression, RAW264.7 cells were treated with 4-PG (10 M) and PTER (10 M) for 12 h. The protein level of TTP was measured by Western blot assay. ImageJ software was used for densitometry analysis. GAPDH and -actin were used as internal controls. Data are expressed as means ±SE, =3. ∗: <0.05, ∗∗: <0.01, ∗∗∗: <0.001 versus cells without treatment
표 4-PG decreases LPS-induced proinflammatory cytokine expression via enhancement of TTP in RAW264.7 cells. RAW264.7 cells were pretreated with 4-PG (10 μM) for 6 h followed by the stimulation of LPS (100 ng/ml) for 4 h. (a) The mRNA expression of IL-6, TNF-α, and TTP was measured by RT-PCR. (b, c) The secreted protein level of IL-6 and TNF-α in the supernatant of cultured cells was determined by ELISA. (d) Effects of 4-PG on reactive oxygen species (ROS) production were detected in RAW 264.7 cells. RAW264.7 cells were preincubated with or without 4-PG (10 μM) for 6 h and N-acetyl-L-cysteine (NAC, 1 mM) for 30 min followed by the stimulation of LPS (100 ng/ml) for another 4 h. The intracellular level of ROS was stained by H2DCFDA, and the production of ROS was measured by flow cytometry. The fold change of fluorescence intensity is presented as means ± SE, n = 3. ∗∗∗P < 0 001. To confirm that the anti-inflammatory effect of 4-PG was mediated by TTP, RAW264.7 cells were transfected with scramble RNA (scRNA) and siRNA against TTP (siTTP) for 24 hours. Then, cells were pretreated with 4-PG (10 μM) for 6 h followed by the challenge of LPS (100 ng/ml) for 4 h. (e, f) The mRNA expression of TTP and TNF-α was analyzed by real-time PCR, respectively. GAPDH was used as internal control. (g, h) To determine the mRNA-decaying activity of TTP on inflammatory cytokines, bone marrow-derived macrophage (BMDM) extracted from Ttp+/+ and Ttp−/− mice was pretreated with 4-PG (10 μM) for 6 h followed by the stimulation of LPS (100 ng/ml) for 4 h. Then, the mRNA level of IL-6 (g) and TNF- α (h) was measured by real-time PCR at indicated times (0, 30, 60, and 120 min) after the addition of 5 μg/ml actinomycin D. Data are expressed as means ± SE, n = 3. ∗∗P < 0 01, ∗∗∗P < 0 001. ns: not significant
표 TTP deficiency abolished the anti-inflammatory effect of 4-PG in a DSS-induced colitis model. To construct an acute colitis animal model, 10 weeks Ttp+/+ and Ttp−/− mice were administrated with 3% DSS dissolved in drinking water for 7 days in the presence or absence of 4-PG (10 mg/kg, i.p. injection). Then, the mice from 4-PG and DSS+ 4-PG groups were still administrated with 4-PG (10 mg/kg, i.p. injection) for another 3 days. After 10 days’ treatment, the mice were sacrificed, and the entire colon was removed from the cecum to the anus. (a) Representative images of 6 tests conducted in each group. (b) Bar graph represents the colon length from each group. (c) Colon tissues were excised, and representative colon histology was shown by hematoxylin and eosin staining. (d) Neutrophil infiltration into the colon was measured by the expression of myeloperoxide (MPO) in colon tissues. The protein level of proinflammatory cytokines, IL-6 (e) and TNF-α (f), were detected by ELISA kit from serum. (g, h) The mRNA and protein levels of TTP in colon tissues from Ttp+/+ and Ttp−/− mice were detected by RT-PCR and Western blot. 18S RNA and β-actin were used as internal controls. Data are expressed as means ± SE, n = 6. ∗P < 0 05, ∗∗P < 0 01, ∗∗∗P < 0 001. ns: not significant

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