Kang, Sukyung
(Department of Food Science and Biotechnology of Animal Resources, Konkuk University)
,
Lee, Jae Sung
(Department of Animal Science and Technology, Konkuk University)
,
Lee, Hai Chon
(Wide River Institute of Immunology, Seoul National University)
,
Petriello, Michael C.
(Toxicology and Cancer Biology, College of Medicine, University of Kentucky)
,
Kim, Bae Yong
(Phylus Corporation)
,
Do, Jeong Tae
(Department of Animal Biotechnology, Konkuk University)
,
Lim, Dae-Seog
(Department of Biotechnology, CHA University)
,
Lee, Hong Gu
(Department of Animal Science and Technology, Konkuk University)
,
Han, Sung Gu
(Department of Food Science and Biotechnology of Animal Resources, Konkuk University)
Mastitis is a prevalent inflammatory disease that remains one of the main causes of poor quality of milk. Phytoncides are naturally occurring anti-inflammatory compounds derived from plants and trees. To determine if treatment with phytoncide could decrease the severity of lipopolysaccharide (LPS)-i...
Mastitis is a prevalent inflammatory disease that remains one of the main causes of poor quality of milk. Phytoncides are naturally occurring anti-inflammatory compounds derived from plants and trees. To determine if treatment with phytoncide could decrease the severity of lipopolysaccharide (LPS)-induced inflammatory responses, mammary alveolar epithelial cells (MAC-T) were pretreated with phytoncide (0.02% and 0.04% (v/v)) followed by LPS treatment (1 and 25 μg/ml). The results demonstrated that phytoncide downregulated LPS-induced pro-inflammatory cyclooxygenase-2 (COX-2) expression. Additionally, LPS-induced activation of ERK1/2, p38, and Akt was attenuated by phytoncide. Treatment of cells with known pharmacological inhibitors of ERK1/2 (PD98059), p38 (SB203580), and Akt (LY294002) confirmed the association of these signaling pathways with the observed alterations in COX-2 expression. Moreover, phytoncide attenuated LPS-induced NF-κB activation and superoxide production, and, finally, treatment with phytoncide increased Nrf2 activation. Results suggest that phytoncide can decrease LPS-induced inflammation in MAC-T cells.
Mastitis is a prevalent inflammatory disease that remains one of the main causes of poor quality of milk. Phytoncides are naturally occurring anti-inflammatory compounds derived from plants and trees. To determine if treatment with phytoncide could decrease the severity of lipopolysaccharide (LPS)-induced inflammatory responses, mammary alveolar epithelial cells (MAC-T) were pretreated with phytoncide (0.02% and 0.04% (v/v)) followed by LPS treatment (1 and 25 μg/ml). The results demonstrated that phytoncide downregulated LPS-induced pro-inflammatory cyclooxygenase-2 (COX-2) expression. Additionally, LPS-induced activation of ERK1/2, p38, and Akt was attenuated by phytoncide. Treatment of cells with known pharmacological inhibitors of ERK1/2 (PD98059), p38 (SB203580), and Akt (LY294002) confirmed the association of these signaling pathways with the observed alterations in COX-2 expression. Moreover, phytoncide attenuated LPS-induced NF-κB activation and superoxide production, and, finally, treatment with phytoncide increased Nrf2 activation. Results suggest that phytoncide can decrease LPS-induced inflammation in MAC-T cells.
Cell viability was determined using the MTT assay. Cells seeded in a 96-well plate were treated with phytoncide (0%, 0.01%, 0.02%, 0.04%, 0.08%, and 0.16% (v/v)) for 24 h or LPS (0, 1, and 25 μg/ml) for 12 h. Next, 10 μl of MTT solution (5 mg/ml in PBS) was added to each well and then incubated for 4 h.
Concentrations of phytoncide were chosen based on the cell viability data showing cell protection without cell death. Cells were treated with LPS at concentrations of 0, 1, and 25 μg/ml for various time points depending on each experimental settings. LPS concentrations were chosen based on previous studies by others using mammary epithelial cells [18,20].
MAC-T cells were treated with phytoncide (0.02% and 0.04% (v/v)) for 6 h followed by LPS treatment (1 μg/ml) for 12 h or 1 h for NF-κB and Nrf2, respectively. For nuclear translocation assay, cells were lysed in hypotonic buffer solution (20 mM Tris (pH 7.
Cells were grown to about 90% confluency and synchronized overnight in medium containing 1% FBS before initiation of cell treatments. The cells were treated with phytoncide at concentrations of 0%, 0.02%, and 0.04% (v/v) for 6 h followed by LPS treatment. Concentrations of phytoncide were chosen based on the cell viability data showing cell protection without cell death.
대상 데이터
Dihydroethidium (DHE) was purchased from Invitrogen (Carlsbad, CA, USA). Inhibitors such as PD98059, LY294002, and SB203580 were purchased from Santa Cruz Biotechnology.
이론/모형
Cell viability was determined using the MTT assay. Cells seeded in a 96-well plate were treated with phytoncide (0%, 0.
Aliquots of nuclear and cytosolic fractions were stored at −80℃ until use. The nuclear fraction was analyzed by SDS-PAGE and western blot assay for the NF-κB p65 and Nrf2 nuclear translocation. Lamin B, a nuclear fraction-specific housekeeping protein, was measured as a loading control.
성능/효과
5-1 h. However, pretreatment with phytoncide (0.02% and 0.04% (v/v)) significantly reduced the LPS-induced phosphorylation of ERK1/2, p38, and Akt (Fig. 2B), suggesting an anti-inflammatory effect of phytoncide.
Subsequently, mechanisms and cellular signaling pathways associated with protective effects of phytoncide were studied. Our data demonstrated that LPS increased production of reactive oxygen species (e.g., superoxide anion) and activated cellular signaling molecules (e.g., ERK1/2, p38 and Akt), whereas phytoncide blocked these cellular events. In fact, oxidative stress and activation of such signaling molecules are linked to NF-κB activation [34].
COX-2 expression was measured with cell lysates using SDS-PAGE and western blot analysis. Result showed that pretreatment of cells with inhibitors such as PD98059 (an ERK1/2 inhibitor) and LY294002 (an Akt inhibitor) markedly decreased the LPS-induced COX-2 expression (Figs. 3A and 3B).
Results from MTT assay showed that cell viability was not significantly perturbed at concentrations up to 0.04% phytoncide, compared with the control (Fig. 1A).
However, once stimulated, the p65 subunit is released from the heterodimer and translocates to the nucleus in order to bind to a specific region of the DNA [22]. Results from the nuclear translocation assay showed that LPS could increase the nuclear translocation of NF-κB p65 and this cellular event was attenuated by phytoncide in MAC-T cells (Fig. 5).
To further substantiate the signaling pathways associated with LPS-induced inflammatory responses, the expression of COX-2 in cells treated with pharmacological inhibitors for ERK1/2 (PD98059), p38 (SB203680), and Akt (LY294002) was determined. Cells were pretreated with the inhibitors at 20 and 40 μM for 1 h, followed by stimulation with LPS at 1 and 25 μg/ml for 12 h.
후속연구
Taken together, our data suggest that superoxide production, ERK1/2, p38, Akt, and NF-κB are mediators of LPS-induced COX-2 expression in MAC-T cells and phytoncide can attenuate the activation of these signaling molecules. This work highlights the protective effects of the pinecone-derived phytoncide extract against LPS-induced inflammatory responses, but more work needs to be completed to determine if the addition of this mixture in bovine feed or intra-mammary administration may protect the mammary gland against inflammation.
De UK, Mukherjee R. 2014. Activity of cyclooxygenase-2 and nitric oxide in milk leucocytes following intramammary inoculation of a bio-response modifier during bovine Staphylococcus aureus subclinical mastitis. Vet. Res. Commun. 38: 201-207.
Fujimori H, Hisama M, Shibayama H, Iwaki M. 2009. Protecting effect of phytoncide solution on normal human dermal fibroblasts against reactive oxygen species. J. Oleo Sci. 58: 429-436.
Giudice A, Arra C, Turco MC. 2010. Review of molecular mechanisms involved in the activation of the Nrf2-ARE signaling pathway by chemopreventive agents. Methods Mol. Biol. 647: 37-74.
Ha T, Liu L, Kelley J, Kao R, Williams D, Li C. 2011. Toll-like receptors: new players in myocardial ischemia/reperfusion injury. Antioxid. Redox Signal. 15: 1875-1893.
Han SG, Han SS, Toborek M, Hennig B. 2012. EGCG protects endothelial cells against PCB 126-induced inflammation through inhibition of AhR and induction of Nrf2-regulated genes. Toxicol. Appl. Pharmacol. 261: 181-188.
Hisama M, Matsuda S, Tanaka T, Shibayama H, Nomura M, Iwaki M. 2008. Suppression of mutagens-induced SOS response by phytoncide solution using Salmonella typhimurium TA1535/pSK1002 umu test. J. Oleo Sci. 57: 381-390.
Hu P, Huang P, Chen MW. 2013. Curcumin attenuates cyclooxygenase-2 expression via inhibition of the NF-kappaB pathway in lipopolysaccharide-stimulated human gingival fibroblasts. Cell Biol. Int. 37: 443-448.
Kang SN, Kim SE, Choi J, Park K, Goo JH, Sim DS, et al. 2015. Comparison of phytoncide with sirolimus as a novel drug candidate for drug-eluting stent. Biomaterials 44: 1-10.
Kang SR, Han DY, Park KI , Park HS, Cho YB, Lee HJ, et al. 2011. Suppressive effect on lipopolysaccharide-induced proinflammatory mediators by Citrus aurantium L. in macrophage RAW 264.7 cells via NF-kappaB signal pathway. Evid. Based Complement. Alternat. Med. 2011: 248592.
Kim HN, Kim DH, Kim EH, Lee MH, Kundu JK, Na HK, et al. 2014. Sulforaphane inhibits phorbol ester-stimulated IKK-NF-kappaB signaling and COX-2 expression in human mammary epithelial cells by targeting NF-kappaB activating kinase and ERK. Cancer Lett. 351: 41-49.
Lahouassa H, Moussay E, Rainard P, Riollet C. 2007. Differential cytokine and chemokine responses of bovine mammary epithelial cells to Staphylococcus aureus and Escherichia coli. Cytokine 38: 12-21.
Lee YK, Lee WS, Hwang JT, Kwon DY, Surh YJ, Park OJ. 2009. Curcumin exerts antidifferentiation effect through AMPKalpha-PPAR-gamma in 3T3-L1 adipocytes and antiproliferatory effect through AMPKalpha-COX-2 in cancer cells. J. Agric. Food Chem. 57: 305-310.
Li D, Zhang N, Cao Y, Zhang W, Su G, Sun Y, et al. 2013. Emodin ameliorates lipopolysaccharide-induced mastitis in mice by inhibiting activation of NF-kappaB and MAPKs signal pathways. Eur. J. Pharmacol. 705: 79-85.
Li F, Wang W, Cao Y, Liang D, Zhang W, Zhang Z, et al. 2014. Inhibitory effects of astragalin on lipopolysaccharide-induced inflammatory response in mouse mammary epithelial cells. J. Surg. Res. 192: 573-581.
Li Q, Nakadai A, Matsushima H, Miyazaki Y, Krensky AM, Kawada T, Morimoto K. 2006. Phytoncides (wood essential oils) induce human natural killer cell activity. Immunopharmacol. Immunotoxicol. 28: 319-333.
Liu M, Song S, Li H, Jiang X, Yin P, Wan C, et al. 2014. The protective effect of caffeic acid against inflammation injury of primary bovine mammary epithelial cells induced by lipopolysaccharide. J. Dairy Sci. 97: 2856-2865.
Niu M, Shen Y, Xu X, Yao Y, Fu C, Yan Z, et al. 2015. Piperlongumine selectively suppresses ABC-DLBCL through inhibition of NF-kappaB p65 subunit nuclear import. Biochem. Biophys. Res. Commun. 462: 326-331.
Parhiz H, Roohbakhsh A, Soltani F, Rezaee R, Iranshahi M. 2015. Antioxidant and anti-inflammatory properties of the citrus flavonoids hesperidin and hesperetin: an updated review of their molecular mechanisms and experimental models. Phytother. Res. 29: 323-331.
Reuven EM, Fink A, Shai Y. 2014. Regulation of innate immune responses by transmembrane interactions: lessons from the TLR family. Biochim. Biophys. Acta 1838: 1586-1593.
Ru K, Su F, Zheng Y, Zhang Y, Luo Y, Guo Z, et al. 2015. Inducible expression of enhanced green fluorescent protein by interleukin-1alpha, interleukin-1beta and Toll-like receptor 2 promoters in goat mammary epithelial cells in response to bacterial challenges. Vet. J. 203: 85-91.
Schukken Y, Chuff M, Moroni P, Gurjar A, Santisteban C, Welcome F, Zadoks R. 2012. The “other” gram-negative bacteria in mastitis: Klebsiella, Serratia, and more. Vet. Clin. North Am. Food Anim. Pract. 28: 239-256.
Seo KH, Park MJ, Ra JE, Han SI, Nam MH, Kim JH, et al. 2014. Saponarin from barley sprouts inhibits NF-kappaB and MAPK on LPS-induced RAW 264.7 cells. Food Funct. 5: 3005-3013.
Song X, Wang T, Zhang Z, Jiang H, Wang W, Cao Y, Zhang N. 2015. Leonurine exerts anti-inflammatory effect by regulating inflammatory signaling pathways and cytokines in LPS-induced mouse mastitis. Inflammation 38: 79-88.
Song X, Zhang W, Wang T, Jiang H, Zhang Z, Fu Y, et al. 2014. Geniposide plays an anti-inflammatory role via regulating TLR4 and downstream signaling pathways in lipopolysaccharide-induced mastitis in mice. Inflammation 37: 1588-1598.
Surh YJ, Chun KS, Cha HH, Han SS, Keum YS, Park KK, Lee SS. 2001. Molecular mechanisms underlying chemopreventive activities of anti-inflammatory phytochemicals: down-regulation of COX-2 and iNOS through suppression of NF-kappa B activation. Mutat. Res. 480-481: 243-268.
Tan KP, Kosuge K, Yang M, Ito S. 2008. NRF2 as a determinant of cellular resistance in retinoic acid cytotoxicity. Free Radic. Biol. Med. 45: 1663-1673.
Valko M, Rhodes CJ, Moncol J, Izakovic M, Mazur M. 2006. Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem. Biol. Interact. 160: 1-40.
Xie S, Liu B, Fu S, Wang W, Yin Y, Li N, et al. 2014. GLP-2 suppresses LPS-induced inflammation in macrophages by inhibiting ERK phosphorylation and NF-kappaB activation. Cell. Physiol. Biochem. 34: 590-602.
Zhao K, Song X, Huang Y, Yao J, Zhou M, Li Z, et al. 2014. Wogonin inhibits LPS-induced tumor angiogenesis via suppressing PI3K/Akt/NF-kappaB signaling. Eur. J. Pharmacol. 737: 57-69.
※ AI-Helper는 부적절한 답변을 할 수 있습니다.