摘要
目的探索内质网应激(endoplasmic reticulum stress,ERS)作用对肝星状细胞活化的影响及可能机制。方法利用衣霉素(tunicamycin,Tun)对正常培养的大鼠肝星状细胞HSC-T6进行处理,处理浓度分别为0(空白对照组),0.1,0.5,1.0,2.0,4.0μg/ml。处理24 h,收集细胞,提取RNA和蛋白质,分别利用real-time PCR和Western blot检测内质网应激相关基因分子伴侣葡萄糖调节蛋白78(glucose-regulated protein 78,GRP78)、CCAAT增强子结合同源蛋白(CCAAT/enhancer-binding homologous protein,CHOP),肝纤维化相关基因α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、组织金属蛋白酶抑制剂1(tissue inhibitor of metalloproteinases 1,TIMP1)、Ⅰ型胶原蛋白(collagen typeⅠalpha 1,Col1A1),及TGF-β/Smad通路相关基因TGF-β1、Smad4在RNA转录或蛋白表达水平的变化。结果real-time PCR检测结果表明,与空白对照组相比,Tun处理能够显著上调内质网应激相关基因Grp78、CHOP的转录水平(P<0.01);肝纤维化相关基因α-SMA、TIMP1的转录水平在Tun浓度为2.0μg/ml最高,但Col1A1的转录水平在Tun处理后有所下降(P<0.01或P<0.05);TGF-β/Smad通路相关基因TGF-β1在Tun浓度为2.0μg/ml和4.0μg/ml显著上升(P<0.01或P<0.05)。Western blot检测表明,Tun处理后,与空白对照相比内质网应激相关基因Grp78、CHOP的蛋白水平显著升高(P<0.01或P<0.05);α-SMA的蛋白水平在Tun浓度为1.0μg/ml和2.0μg/ml时明显上调(P<0.01或P<0.05);TGF-β1/Smad通路相关基因Smad4在Tun处理后亦明显上调(P<0.01或P<0.05)。结论内质网应激可促进肝星状细胞的活化,其机制可能与TGF-β1/Smad信号通路相关。
Objective To investigate the effect of endoplasmic reticulum stress(ERS)on the activation of rat hepatic stellate cell HSC-T6.Methods Rat hepatic stellate cell HSC-T6 cells were treated by different concentrations of Tunicamycin(Tun,0,0.1,0.5,1.0,2.0,4.0μg/ml),and 0μg/ml was chosen as blank control group.Cells were collected 24 h after treatment to extract RNA and protein,and the transcription and translation levels of related genes,ERS-related genes Grp78(glucose-regulated protein 78),CHOP(CCAAT/enhancer-binding homologous protein),liver fibrosis related genesα-SMA(α-smooth muscle actin),TIMP1(tissue inhibitor of metalloproteinases 1),Col1A1(collagen typeⅠalpha 1),and TGF-β/Smad pathway related genes TGF-β1,Smad4,were detected by real-time PCR and Western blot,respectively.Results Real-time PCR results indicated that,compared with blank control group,ERS-related genes Grp78 and CHOP were upregulated significantly in transcription level after Tun treatment(P<0.01),α-SMA and TIMP1 levels were the highest at the concentration of 2.0μg/ml Tun,the transcription level of Col1A1 was decreased(P<0.01 or P<0.05),and TGF-β/Smad related gene TGF-β1 was upregulated dramatically when the concentration of Tun were 2.0μg/ml and 4.0μg/ml(P<0.01 or P<0.05).Western blot results indicated that Tun treatment significantly increased the expression of ERS-related genes Grp78,CHOP,liver fibrosis geneα-SMA,and TGF-β1/Smad signal pathway related gene Smad4(P<0.01 or P<0.05).Conclusion Endoplasmic reticulum stress can promote the activation of hepatic stellate cells,and its mechanism may be related to TGF-β1/Smad signal pathway.
作者
史晓燕
范妤
段丽芳
闫曙光
李京涛
席奇
SHI Xiaoyan;FAN Yu;DUAN Lifang;YAN Shuguang;LI Jingtao;XI Qi(Department of Biochemistry,Basic Medical College of Shaanxi University of Chinese Medicine,Xianyang 712046,China;Department of Liver Disease,Affiliated Hospital of Shaanxi University of Chinese Medicine)
出处
《山西医科大学学报》
CAS
2019年第8期1054-1059,共6页
Journal of Shanxi Medical University
基金
国家自然科学基金资助项目(81704073)
国家自然科学基金资助项目(81703925)
陕西省中医药管理局基础面上项目(JCMS030)
陕西省自然科学基础研究计划青年项目(2017JQ8018)
陕西中医药大学胰、肝疾病的分子机制及中医药防治创新团队项目(2019-YL14)
关键词
内质网应激
肝星状细胞
HSC-T6
衣霉素
endoplasmic reticulum stress
hepatic stellate cell
HSC-T6
tunicamycin
