[보고서]태아기형의 예방 및 치료를 위한 천연물질 검색 및 작용기전 규명

남상윤

보고서 정보

주관연구기관

충북대학교
Chungbuk National University

연구책임자

남상윤

보고서유형

최종보고서

발행국가

대한민국

언어

한국어

발행년월

2022-06

과제시작연도

2022

주관부처

교육부
Ministry of Education

등록번호

TRKO202300007153

과제고유번호

1345352808

사업명

개인기초연구(교육부)

DB 구축일자

2023-09-19

키워드

니코틴.전배아배양계.태아 기형발생.천연예방물질.태반.nicotine.whole embryo culture system.fetal teratogenesis.natural preventive materials.placenta.

표 Whole embryo culture system
표 Representative teratogenic images in the mouse embryos exposed to various doses of nicotine. A) normal control. B) 0.06 mM nicotine. C) 0.15 mM nicotine. D) 0.6 mM nicotine. E) 0.8 mM nicotine. F) 0.9 mM nicotine. G) 1.0 mM nicotine. H) 1.2 mM nicotine
표 Changes of total morphological scores in nicotine-treated mouse embryos. Each value represents the mean ± SEM. *; versus the normal control group (p < 0.05)
표 Summary of morphological changes in cultured mouse embryos exposed to nicotines
표 Dose determination of methylcaconition (MC) (A) and mecamylamine (MA) (B) under 1 mM nicotine (N) treatment. Results are expressed as the mean ± SEM. Significantly different from control group at P<0.05 (*)
표 Expression levels of nicotinic acetylcholine receptor (nAchR) subtype mRNAs in the mouse embryos exposed to nicotine (0.6, 1.0, 1.2 mM) at embryonic day 8.5 for 2 days in vitro. The mRNA levels of nAchR subtype are significantly decreased by nicotine treatmens dose-depentdently embryos. Results are mean ± SEM (n=8). Significant differences between nicotine groups and control (Con; *) group were examined at p < 0.05. GAPDH was used as an internal standard to normalize target transcript expression
표 Nicotinic acetylcholine receptor (nAchR) α3 (A) and α4 mRNA expression levels in mouse embryos exposed to nicotine (1mM)and/or2.5 μM methylcaconitine (MC) or 25 μM mecamylamine (MA) μM at embryonic day 8.5 for 2 days in vitro. Results are mean ± SEM (n=8). Significant differences between comnited groups and control (Con; *) or nicotine alone (N; #)group were examined at p < 0.05. GAPDH was used as an internal standard to normalize target transcript expression
표 nicotinic acetylcholine receptor (nAchR) α5 (A) and α7 (B) mRNA expression levels in mouse embryos exposed to nicotine (1mM)and/orMC 2.5 μM methylcaconitine (ME) or 25 μM ME mecamylamine at embryonic day 8.5 for 2 days in vitro. Results are mean ± SEM (n=8). Significant differences between comnited groups and control (Con; *) or nicotine alone (N; #)group were examined at p < 0.05. GAPDH was used as an internal standard to normalize target transcript expression
표 nicotinic acetylcholine receptor (nAchR) β2 (A) and β4 (B) mRNA expression levels in mouse embryos exposed to nicotine (1mM)and/or2.5 μM methylcaconitine (MC) or 25 μM mecamylamine (MA) at embryonic day 8.5 for 2 days in vitro. Results are mean ± SEM (n=8). Significant differences between combined groups and control (Con; *) or nicotine alone (N; #)group were examined at p < 0.05. GAPDH was used as an internal standard to normalize target transcript expression
표 Gene expression levels of Bcl-xL(A) and caspase3 (B) in mouse embryos exposed to 0.6, 1, and 1.2 mM nicotine of at embryonic day 8.5 for 2 days in vitro. Results are mean ± SEM (n=8). Significant differences among each were examined at p < 0.05 (*) GAPDH was used as an internal standard to normalize target transcript expression
표 Gene expression levels of Bcl-xL(A)and caspase3 (B) in mouse embryos exposed to 1mM nicotine and/or 2.5 μM methylcaconitine (MC) or 25 μM mecamylamine (MA) at embryonic day 8.5 for 2 days in vitro. Results are mean ± SEM (n=8). Significant differences between combined groups and control (Con; *) or nicotine alone (N; #)group were examined at p<0.05. GAPDH was used as an internal standard to normalize target transcript expression
표 The reduction of chorioallantoic circulation induced by nicotine treatment. A: Maternal and fetal border in placenta from the impaired chorioallantoic attachment. A-a; Normal control group. Formation of the labyrinth in the placentas. A-b; Nicotine (1mM) group. Failure of chorioallantoic attachment. B: The enlarged structures of the boxed area in A. B-a; Normal control group. Maternal sinuses (yellow arrowheads) and embryonic nucleated blood vessels (white arrows) formed from the allantoic blood vessels. B-b; Nicotine (1mM)group with no detectable definitive labyrinth. gc, giant cells; sp, spongiotrophoblasts; la, labyrinth; ch, chorion; al, allantois. H&E. bar: 50μm
표 Nicotine-induced defective yolk sac vascularization in mouse embryos. A: Nicotine-induced yolk sac defects showing smaller size, pale color, rudimentary vasculature, lack of vitelline vessels (black arrows), and a ring of blood islands (asterisk). A-a; Normal control group. A-b; Nicotine (1mM) group. B: Histological examination of yolk sac wall stained with H Normal control group. B-b; Nicotine (1mM) group. EC; endodermal cells. MC; mesodermal cells
표 Nicotine induced teratogenesis via nAChR-apoptosis pathway and abnormal yolk sac and placental vasculogenesis
표 Growth changes in the cultured mouse embryos
표 Developmental changes in the cultured mouse embryos
표 Representative images of mouse embryos exposed to nicotine with/without lycopene. Normal control group (A). Embryos treated with 1 mM nicotine show typical anomalies such as open brain, reduced forebrain, deformed posterior trunk, and regressed forelimbs (B-D). Nicotine plus lycopene [1 × 10-6 μM (E,F) and 1 × 10-5 μM (G,H)] groups appear morphologically similar to the control group. Bar : 100 μm
표 Lycopene attenuates nicotine-induced defects of yolk sac vascularization in mouse embryos. [Ⅰ]: Nicotine-induced yolk sac defects showing smaller size, pale color, rudimentary vasculature, and a blood island at E10.5. [Ⅱ]: Histological examination of yolk sac wall stained with H control group. B; nicotine (1 mM) group. C,D; nicotine plus lycopene (1×10-6or1×10-5μM) group. sp; spongiotrophoblast, la; labyrinth, cp; chorionic plate, bv; blood vessel, EC; endothelial cell, MC; mesothelial cell, arrows; blood island, arrowheads; intracellular vacuole. Bar : 100 μm
표 Lycopene protects yolk sac placentas through anti-proinflammatory and anti-apoptotic effects. [Ⅰ]: TUNEL-positive cells observed in the nicotine-treated placentas. The enlarged structures of the boxed area. A,E; control group. B,F; nicotine (1 mM) group. C,G or D,H; nicotine plus lycopene (1×10-6or1×10-5μM) groups. [Ⅱ]A: Bcl-xL mRNA expression level. B: TNF-α mRNA expression level. The results are the means ± SEM. Significant differneces between lycopene(Ly) groups and the control (Con; a) or nicotine alone (N; b) group were examined at P< 0.05. Arrowheads; red blood cells, arrow; giant cells. Bar: 100 μm
표 Lycopene (Ly) decreases oxidative stress and improves antioxidant enzyme status in mouse embryos exposed to nicotine. A: Lipid peroxidation was evaluated by malondialdehyde (MDA) concentration. B: SOD activity levels. C: SOD1 mRNA expression levels. D: GPx1 mRNA expression levels. Results are means ± SEM. a; vs. control (P < 0.05). b; vs. nicotine (P < 0.05). GAPDH was used as an internal standard to normalize target transcript expression
표 Quantitative real-time PCR analysis of apoptosis-related genes (Bax, caspase3 and Bcl-xL) levels in mouse embryos exposed to nicotine (N;1mM) in the presence or absence of lycopene (Ly;1×10-6 and 1 × 10-5 μM) at E8.5 for two days in vitro. Results are means ± SEM. a; vs. control (Con; P < 0.05). b; vs. nicotine (P < 0.05). GAPDH was used as an internal standard to normalize target transcript expression
표 Quantitative real-time PCR analysis of NF-kB and TNF-α mRNA levels in the mouse embryos exposed to nicotine (N; 1 mM) and/or lycopene (Ly; 1 × 10-6 and 1 × 10-5 μM) at E8.5 for two days in vitro. Results are means ± SEM. a; vs. control (Con; P < 0.05). b; vs. nicotine (P < 0.05). GAPDH was used as an internal standard to normalize target transcript expression
표 Lycopene regulates expressions of growth factors in yolk sac placentas exposed to nicotine. A: Hif-1α mRNA expression levels. B: IGF-1 mRNA expression levels. C: α-SMA mRNA expression levels. D: VEGF-α mRNA expression levels. E: TGF-β1 mRNA expression levels. F and G: Western blot analyses of VEGF-α and TGF-β1, respectively. Results are means ± SEM. a; vs. control group (P < 0.05). b; vs. nicotine group (P < 0.05). GAPDH was used as an internal standard to normalize target gene or protein expression
표 Effects of nicotine and/or resveratrol on mouse organogenesis. Embryos treated with 1 mM nicotine show typical anomalies such as open brain, reduced forebrain, deformed posterior trunk, and regressed forelimbs (B1-B3). However, nicotine plus resveratrol groups appear morphologically similar to the control group. (A) Control. (B1-B3) 1 mM nicotine. (C1- C2) 1 mM nicotine plus resveratrol 1×10-8 μM. (D1-D2) 1 mM nicotine plus resveratrol 1×10-7 μ M
표 Morphological changes in the cultured mouse embryos exposed to nicotine and/or resveratrol
표 Protective effects of resveratrol against defects of yolk sac and placentas in mouse embryos treated with nicotine. (A trophoblast giant cell. sp; spongiotrophoblast. la; labyrinth. arrow; blood vessels
표 Resveratrol (1×10−8 and 1×10−7μM) attenuates nicotine-induced vascular defects in placentas via improvement of related gene expressions. (A) HIF-1α mRNA levels. (B) VEGF-α mRNA levels. (C) IGF-1 mRNA levels
표 Immunodetection of VEGF-α. (A) Control. (B) 1 mM nicotine. (C) 1 mM nicotine plus resveratrol 1×10-8 μM. (D) 1 mM nicotine plus resveratrol 1×10-7 μM. Bar: 100 uM
표 Immunodetection of IGF. (A) Control. (B) 1 mM nicotine. (C) 1 mM nicotine plus resveratrol 1×10-8 μM. (D) 1 mM nicotine plus resveratrol 1×10-7 μM. Bar: 100 uM
표 Resveratrol (1×10−8 and 1×10−7μM) decreases oxidative stress induced by nicotine and improves antioxidant enzyme status in mouse embryos. (A) Lipid peroxidation expressed as the malondialdehyde (MDA) level. (B) SOD1 mRNA levels. (C) GPx1 mRNA levels. (D) GPx4 mRNA levels
표 Expression levels of apoptotic genes Bax (A) and Bcl-xL (B) mRNAs in placentas exposed to nicotine (1 mM) and/or resveratrol (1×10−8 and 1×10−7μM)
표 Expression levels of TNFα (A) and SIRT 1 (B) mRNAs in placentas exposed to nicotine (1 mM) and/or resveratrol (1×10−8 and 1×10−7μM). Resveratrol enhances SIRT1-dependent cellular processes
표 Resveratrol protects yolk sac placentas through its anti-apoptotic effects. TUNEL-positive cells observed in the nicotine-treated placentas. The below enlarged structures are above boxed area (E-H). (A&E) Control. (B&F) 1 mM nicotine. (C&)G 1 mM nicotine plus resveratrol 1×10-8 μM. (D&H) 1 mM nicotine plus resveratrol 1×10-7 μM. Bar: 100 uM
표 Immunodetection of SIRT1. (A) Control. (B) 1 mM nicotine. (C) 1 mM nicotine plus resveratrol 1×10-8 μM. (D) 1 mM nicotine plus resveratrol 1×10-7 μM. Bar: 100 uM
표 Effects of nicotine and/or 4-o-methylhonokiol on mouse organogenesis. (A) control, (B1-B3) 1mM nicotine, (C1, C2) 1mM nicotine+4-o-methylhonokiol 1×10-4 μM, (D1, D2) 1mM nicotine+4-o-methylhonokiol 1×10-3 μM
표 Growth changes in the cultured mouse embryos exposed to 1mM nicotine (N) with or without 1×10 −4 or 1×10−3 μM of 4-o-methylhonokiol (MH)
표 Differentiation changes in the cultured mouse embryos exposed to 1mM nicotine (N) with or without 1×10−4 or 1×10−3 μM of 4-o-methylhonokiol (MH)
표 Protective effects of 4-o-methylhonokiol against defects yolk sac and placentas in mouse embryos treated with nicotine. (A) Control, (B) 1mM nicotine, (C) 1mM nicotine+ 4-o-methylhonokiol 1×10-4 μM, (D) 1mM nicotine+4-o-methylhonokiol 1×10-3 μM
표 4-o-methylhonokiol attenuates nicotine-induced vascular defects in placentas via improvement of related gene expressions. (A) HIF-1α mRNA expression levels in the placenta. (B) VEGF-α mRNA expression levels in the placenta.(C) IGF-1 mRNA expression levels in the placenta. . (D) TGF-β1 mRNA expression levels in the placenta.The results are the means ± SEM. Significant differences between MH-treated groups (1 mM N + MH 1×10−4 μM) and (1 mM N + MH 1×10−3 μM ) and normal control (Con; *) or nicotine alone (N; #) group were examined at p < 0.05. GAPDH was used as an internal standard to normalize the target transcript expression
표 Expression levels of IGF-1 and VEGF-α proteins in placentas and yolk sac exposed to nicotine (1 mM) and/or 4-o-methylhonokiol (MH, 1 × 10−4 or 1 × 10−3 μM) at embryonic day 8.5 for 2 days in vitro. The results are the means ± SEM. Significant differences between MH groups and the control (Con; *) or nicotine alone (N; #) group were examined at p < 0.05. GAPDH was used as an internal standard to normalize target transcript expression
표 Expression levels of apoptotic genes in placentas exposed to nicotine (1 mM) and/or 4-o-methylhonokiol (MH, 1 × 10−4 or 1 × 10−3 μM) at embryonic day 8.5 for 2 days in vitro. (A) Caspase 3 mRNA expression levels in the placenta. (B) Bax mRNA expression levels in the placenta. (C) Bcl-xL mRNA expression levels in the placenta. The results are the means ± SEM. Significant differences between MH-treated groups (1 mM N + MH10−4 μM) and (1 mM N + MH10−3 μM ) and normal control (Con; *) or nicotine alone (N; #) group were examined at p < 0.05. GAPDH was used as an internal standard to normalize the target transcript expression
표 Representative Images of mouse embryos exposed to nicotine and EGCG. Normal control embryo (A), embryo treated with 1 mM nicotine alone (B), embryo co-treated with 1 mM nicotine and 1 μM EGCG (C), embryo co-treated with 1 mM nicotine and 10 μM EGCG (D), embryo co-treated with 1 mM nicotine and 100 μM EGCG (E), and embryo co-treated with 1 mM nicotine and 1000 μM EGCG (F). Scale bar: 1 mm
표 Morphological evaluation according to items of mouse embryos exposed to nicotine and EGCG
표 Superoxide dismutase (SOD) activity levels (A) and lipid peroxidation measurements (B) in E8.5 mouse embryos exposed to nicotine and EGCG for 2 days in vitro . The results are presented the mean ± SEM (n=6). Statistically significant differences from control group (a) and nicotine group (b) at P value ＜0.05
표 Relative mRNA expressions of SOD1 (A), SOD2 (B), GPx1 (C) and GPx4 (D) in E8.5 mouse embryos exposed to nicotine and EGCG for 2 days in vitro. The results are presented the mean ± SEM (n=8). β-actin was used as an internal standard to normalize target transcript expression. Statistically significant differences from control group (a) and nicotine group (b) at P value ＜0.05
표 Relative mRNA expressions of HIF-1α in E8.5 mouse embryos exposed to nicotine and EGCG for 2 days in vitro . The results are presented the mean ± SEM (n=8). β-actin was used as an internal standard to normalize target transcript expression. Statistically significant differences from control group (a) and nicotine group (b) at P value ＜0.05
표 Relative mRNA expressions of TNF-1α (tumor necrosis factor-1α; A) and IL-1β (interleukin-1β; B) in E8.5 mouse embryos exposed to nicotine and EGCG for 2 days in vitro. The results are presented the mean ± SEM (n=8). β-actin was used as an internal standard to normalize target transcript expression. Statistically significant differences from control group (a) and nicotine group (b) at P value ＜0.05
표 Relative mRNA expressions of Bcl-χL(B-cell lymphoma-extra large) (A) and caspase 3 (B) in E8.5 mouse embryos exposed to nicotine and EGCG for 2 days in vitro . The results are presented the mean ± SEM (n=8). β-actin was used as an internal standard to normalize target transcript expression. Statistically significant differences from control group (a) and nicotine group (b) at P value ＜0.05
표 EGCG can prevent teratogenic effects induced by nicotine
표 Effects of nicotine and/or genistein on mouse organogenesis. (A) control, (B) 1mM nicotine, (C) 1mM nicotine+0.1μM genistein, (D) 1mM nicotine+1μM genistein, (E) 1mM nicotine+10μM genistein
표 Growth changes in the cultured mouse embryos exposed to 1mM nicotine with or without genistein
표 Differentiation changes in the cultured mouse embryos exposed to 1mM nicotine with or without genistein
표 Protective effects of genistein against defects yolk sac and placentas in mouse embryos treated with nicotine. (A) Control, (B) 1mM nicotine, (C) 1mM nicotine+0.1μM genistein, (D) 1mM nicotine+1μM genistein, (E) 1mM nicotine+10μM genistein

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태아기형의 예방 및 치료를 위한 천연물질 검색 및 작용기전 규명
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태아기형의 예방 및 치료를 위한 천연물질 검색 및 작용기전 규명 Examinations of preventive and therapeutic natural materials against fetal anomalies and their action mechanisms 원문보기

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