[1]张玉,周曦,易龙,等.二氢杨梅素通过TGF-β1/Smad信号通路抑制肝星状细胞活化的作用研究[J].第三军医大学学报,2018,40(04):282-298.
 ZHANG Yu,ZHOU Xi,YI Long,et al.Dihydromyricetin attenuates activation of hepatic stellate cells through TGF-β1/Smad signaling pathway[J].J Third Mil Med Univ,2018,40(04):282-298.
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二氢杨梅素通过TGF-β1/Smad信号通路抑制肝星状细胞活化的作用研究(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
40卷
期数:
2018年第04期
页码:
282-298
栏目:
基础医学
出版日期:
2018-02-28

文章信息/Info

Title:
Dihydromyricetin attenuates activation of hepatic stellate cells through TGF-β1/Smad signaling pathway
作者:
张玉周曦易龙彭山珊张乾勇糜漫天
陆军军医大学(第三军医大学)军事预防医学系营养与食品安全研究中心,重庆市医学营养研究中心,重庆市营养与食品安全重点实验室
Author(s):
ZHANG Yu ZHOU Xi YI Long PENG Shanshan ZHANG Qianyong MI Mantian

Department of Nutrition and Food Hygiene, Center of Natrition and Food Safety, Chongqing Center of Medical Nutrition, Chongqing Key Laboratory of Nutrition and Food Safety, Faculty of Military Preventive Medicine, Army Medical University (Third Military Medical University), Chongqing, 400038, China

关键词:
二氢杨梅素肝星状细胞TGF-&beta1 Smad蛋白
Keywords:
dihydromyricetin hepatic stellate cells transforming growth factor-&beta1 Smad protein
分类号:
R322.47;R914.4;R966
文献标志码:
A
摘要:

目的   观察二氢杨梅素(dihydromyricetin, DHM)对肝星状细胞(hepatic stellate cells,HSC)活化的影响,并探讨TGF-β1/Smad信号通路在其中的作用及机制。方法    以大鼠肝星状细胞株HSC-T6为研究对象,TGF-β1(5 ng/mL)处理2 h后,再以不同浓度的DHM处理 24 h。CCK-8法检测细胞活力,流式细胞仪检测细胞凋亡和细胞周期,ELISA法检测细胞分泌MMP-1、TIMP-1和COL-Ⅰ的水平,免疫荧光细胞化学法检测α平滑肌肌动蛋白(α-SMA)的表达,qRT-PCR法检测MMP-1、TIMP-1和COL-Ⅰ、COL-Ⅲ、Smad2、Smad3、Smad4、Smad7的mRNA表达,Western blot检测Smad2/3、p-Smad2/3、Smad7、AMPK、p-AMPK蛋白表达。结果      CCK-8法检测结果显示,40 μmol/L以上DHM单独处理能明显抑制HSC-T6细胞增殖活力,并能显著抑制活化的HSCT6细胞增殖活力的增加,流式细胞仪检测结果表明,DHM能够明显促进活化的HSC-T6细胞凋亡,TGF-β1能够促进HSC-T6细胞G0/G1期的比例降低、S期和G2/M期的比例增加(P<0.05),而DHM能够抑制TGF-β1对细胞周期的影响,ELISA检测结果表明,DHM能够显著抑制活化的HSC-T6细胞上清液中TIMP-1和COL-Ⅰ水平增高及MMP-1水平下降(P<0.05),免疫荧光细胞化学法检测显示,HSC-T6细胞表达HSC活化特定抗原α-SMA,TGF-β1处理后表达增加,而DHM能够抑制α-SMA表达,qRT-PCR结果显示,DHM能够显著影响活化的HSC-T6细胞的细胞外基质相关基因MMP-1、TIMP-1和COL-Ⅰ、COL-Ⅲ、Smad2、Smad3、Smad4、Smad7等的mRNA表达,Western blot检测结果提示,DHM抑制活化的HSCT6细胞AMPK磷酸化下降、Smad2/3的磷酸化水平升高及Smad7的蛋白表达下降,而AMPK抑制剂Compound C处理后,DHM的作用被显著抑制。结论     DHM能够显著抑制HSC-T6细胞的活化,该作用可能通过促进AMPK的磷酸化并抑制TGF-β1/Smad信号通路介导的细胞外基质产生而实现。

Abstract:

Objective     To investigate the effects of dihydromyricetin (DHM) on the activation of hepatic stellate cells (HSC) and explore the role of transforming growth factor-β1 (TGF-β1)/Smad signaling pathway in the regulation of HSC activation by DHM. Methods     Rat hepatic stellate cell line (HSC-T6) was treated with TGF-β1 (5 ng/mL) for 2 h and then with different concentrations of DHM for 24 h. CCK-8 essay was used to detect the cell viability, and the cell apoptosis and cell cycle were analyzed with flow cytometry. The levels of MMP-1, TIMP-1 and COL-Ⅰ in the supernatants were measured using enzyme-linked immunosorbent assay (ELISA). The expression of α-smooth muscle actin (α-SMA) in the cells was detected by immunofluorescence cytochemistry. The mRNA expression levels of MMP-1, TIMP-1, COL-Ⅰ, COL-Ⅲ, Smad2, Smad3, Smad4 and Smad7 were detected by qRT-PCR, and Western blotting was performed to detect the protein expression levels of Smad2/3, p-Smad2/3, Smad7, AMPK and p-AMPK. Results     CCK-8 assay showed that treatment with DHM alone at the concentration above 40 μmol/L significantly lowered the viability of HSC-T6 cells. DHM significantly inhibited the increased cell viability and promoted apoptosis of activated HSC-T6 cells. TGF-β1 treatment caused increase in the cell percentage of G0/G1 phase and decreases in the cell percentages of S and G2/M phases in HSCT6 cells, and this effect was obviously inhibited by DHM treatment. DHM also significantly inhibited the increase in TIMP-1 and COL-Ⅰ levels and the decrease in MMP-1 level in the supernatant of the activated HSC-T6 cells. Immunofluorescence cytochemistry assay showed that HSC-T6 cells expressed α-SMA, a specific antigen of activated HSCs; TGF-β1 treatment increased α-SMA expression in the cells, and this effect was suppressed by DHM treatment. DHM caused significant changes in the mRNA expressions of MMP-1, TIMP-1, COL-Ⅰ, COL-Ⅲ, Smad2, Smad3, Smad4 and Smad7 in activated HSC-T6 cells. TGFβ1 decreased the phosphorylation of AMPK and Smad7 and increased the phosphorylation of Smad2/3, and these effects were obviously suppressed by DHM treatment; the effect of DHM was significantly abolished by pretreatment of the AMPK inhibitor Compound C. Conclusion     The effect of DHM against hepatic fibrosis might be mediated by phosphorylation of AMPK and inhibition of extracellular matrix production through TGF-β1/Smad signaling pathway.

参考文献/References:

[1]ESLAMPARAST T, TANDON P, RAMAN M. Dietary composition independent of weight loss in the management of nonalcoholic fatty liver disease[J]. Nutrients, 2017, 9(8). DOI: 10.3390/nu9080800.
[2]DE OLIVEIRA D S B, RAMOS L F, Moraes K. Molecular interplays in hepatic stellate cells: apoptosis, senescence, and phenotype reversion as cellular connections that modulate liver fibrosis[J]. Cell Biol Int, 2017, 41(9): 946-959.DOI: 10.1002/cbin.10790.
[3]YOSHIDA K, MATSUZAKI K. Differential regulation of TGFbeta/Smad signaling in hepatic stellate cells between acute and chronic liver injuries[J]. Front Physiol, 2012, 3: 53. DOI: 10.3389/fphys.2012.00053.
[4]YAMANAKA Y, GINGERY A, OKI G, et al. Blocking fibrotic signaling in fibroblasts from patients with carpal tunnel syndrome[J]. J Cell Physiol, 2017. DOI: 10.1002/jcp.25901. [Epub ahead of print]
[5]艾志波, 张荣华, 闫国和, 等. 鳖甲煎改良方对大鼠肝纤维化的作用及其机制研究[J]. 第三军医大学学报, 2011, 33(3): 274-277.
AI Z B, ZHANG R H, YAN G H, et al. Effect of modified turtle shell decoction on hepatic fibrosis in rats and its mechanism[J]. J Third Mil Med Univ, 2011, 33(3): 274-277.
[6]HUANG Y, ZHAO J, JIAN W, et al. Effects of verapamil on the pharmacokinetics of dihydromyricetin in rats and its potential mechanism[J]. Xenobiotica, 2017: 1-6. DOI: 10.1080/00498254.2017.1366576.[Epub ahead of print]
[7]CHEN S, ZHAO X, WAN J, et al. Dihydromyricetin improves glucose and lipid metabolism and exerts antiinflammatory effects in nonalcoholic fatty liver disease: A randomized controlled trial[J]. Pharmacol Res, 2015, 99: 74-81. DOI: 10.1016/j.phrs.2015.05.009.
[8]SHI L, ZHANG T, ZHOU Y, et al. Dihydromyricetin improves skeletal muscle insulin sensitivity by inducing autophagy via the AMPKPGC1alphaSirt3 signaling pathway[J]. Endocrine, 2015, 50(2): 378-389. DOI: 10.1007/s1202001505995.
[9]ZHOU Q, CHEN K, LIU P, et al. Dihydromyricetin stimulates irisin secretion partially via the PGC1alpha pathway[J]. Mol Cell Endocrinol, 2015, 412: 349-357. DOI: 10.1016/j.mce.2015.05.036.
[10]CHEN S, ZHAO X, WAN J, et al. Dihydromyricetin improves glucose and lipid metabolism and exerts antiinflammatory effects in nonalcoholic fatty liver disease: A randomized controlled trial[J]. Pharmacol Res, 2015, 99: 74-81. DOI: 10.1016/j.phrs.2015.05.009.
[11]ZHOU B, ZHAO Y, WANG X, et al. Unravelling the inhibitory effect of dihydromyricetin on heterocyclic aromatic amines formation[J]. J Sci Food Agric, 2017. DOI: 10.1002/jsfa.8682. [Epub ahead of print]
[12]PERUMAL N, PERUMAL M, HALAGOWDER D, et al. Morin attenuates diethylnitrosamineinduced rat liver fibrosis and hepatic stellate cell activation by coordinated regulation of Hippo/Yap and TGFbeta1/Smad signaling[J]. Biochimie, 2017, 140: 10-19. DOI: 10.1016/j.biochi.2017.05.017
[13]唐静, 戴立里, 呙琳琳, 等. 肝纤维化中丹参素对TGFβ_1/Smads/ERK信号通路的影响及其相互关系[J]. 第三军医大学学报, 2011, 33(11): 1159-1164.
TANG J, DAI L L, GUO L L, et al. Tanshinol inhibits TGFβ1/Smads/ERK signaling pathways in rat hepatic stellate cells[J]. J Third Mil Med Univ, 2011, 33(11): 1159-1164.
[14]KOBAYASHI T, KIM H, LIU X, et al. Matrix metalloproteinase-9 activates TGFbeta and stimulates fibroblast contraction of collagen gels[J]. Am J Physiol Lung Cell Mol Physiol, 2014, 306(11): L1006-L1015.  DOI: 10.1152/ajplung.00015.2014.
[15]SHI L, ZHANG T, ZHOU Y, et al. Dihydromyricetin improves skeletal muscle insulin sensitivity by inducing autophagy via the AMPKPGC1alphaSirt3 signaling pathway[J]. Endocrine, 2015, 50(2): 378-389. DOI: 10.1007/s12020-015-0599-5. 

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更新日期/Last Update: 2018-02-28