华蟾素对转化生长因子-β1诱导肝星状细胞激活的作用及机制研究  

Effects and mechanism of cinobufotalin on transforming growth factor-β1 induced activation of hepatic stellate cells

作　　者：汪玉倩 徐可[2] 詹月萍 陈轶斐 殷佩浩[1,2] WANG Yu-qian;XU Ke;ZHAN Yue-ping;CHEN Yi-fei;YIN Pei-hao(Department of General Surgery,Puluo Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai 200062,China;Interventional Cancer Institute of Chinese Integrative Medicine,Puluo Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai 200062,China;College of Pharmacy,East China University of Science and Technology,Shanghai 200062,China)

机构地区：[1]上海中医药大学附属普陀医院普外科,上海200062 [2]上海中医药大学附属普陀医院中西医结合肿瘤介入研究所,上海200062 [3]华东理工大学药学院,上海200062

出　　处：《中国临床药理学杂志》2022年第13期1452-1456,共5页The Chinese Journal of Clinical Pharmacology

基　　金：国家自然科学基金资助项目(81873137);上海市普陀区中心医院院级基金资助项目(2020362A);上海市自然科学基金资助项目(20ZR1450500);上海中医药大学“研究生创新培养”专项科研基金资助项目(2020年-2022年)。

摘　　要：目的研究华蟾素对人肝星状细胞(HSCs)LX-2活化的影响及其可能的分子机制。方法将LX-2细胞分为空白组,对照组和华蟾素低、中、高实验组。空白组予以完全培养基进行培养,对照组予以含10 ng·mL^(-1)转化生长因子-β1(TGF-β1)的培养基处理,华蟾素低、中、高实验组在对照组的基础上分别予以0.5,1和2 mg·mL^(-1)华蟾素处理。以细胞计数试剂盒-8(CCK-8)实验检测华蟾素对人肝星状细胞系LX-2细胞增殖的影响;以蛋白质印迹(Western blot)法检测各组细胞中α-平滑肌动蛋白(α-SMA)和I型胶原(Collagen-Ⅰ)蛋白的表达;以实时荧光定量聚合酶链反应(RT-qPCR)检测α-SMA和Collagen-ⅠmRNA的表达情况;以免疫荧光法检测各组细胞α-SMA蛋白的表达。结果Western blot和RT-PCR结果显示随着TGF-β1浓度的递增,α-SMA、Collagen-Ⅰ在LX-2细胞中的表达增强,差异有统计学意义(P<0.01);表明TGF-β1可诱导LX-2细胞活化增殖。CCK-8实验测得华蟾素对于LX-2细胞的半数抑制浓度为2 mg·mL^(-1)。Western blot结果显示对照组,华蟾素低、中、高实验组α-SMA蛋白表达水平分别为1.00±0.04,0.62±0.03,0.48±0.01和0.36±0.01,Collagen-Ⅰ蛋白表达水平分别为1.00±0.04,0.52±0.03,0.18±0.01和0.06±0.01;免疫荧光结果显示空白组,对照组,华蟾素低、中、高实验组α-SMA表达水平分别为1.00±0.01,19.84±0.04,4.85±0.02,2.15±0.03,1.11±0.02;RT-PCR结果显示对照组和华蟾素低、中、高实验组α-SMA mRNA表达水平分别为1.00±0.04,0.68±0.01,0.34±0.02,0.25±0.03,Collagen-Ⅰ表达水平分别为1.00±0.04,0.69±0.01,0.21±0.03,0.11±0.02,以上结果华蟾素低、中、高实验组与对照组比较,差异均有统计学意义(均P<0.05)。结论华蟾素可抑制TGF-β1诱导的LX-2细胞的增殖活化,从而抑制肝纤维化。Objective To investigate the effect of cinobufotalin on the activation of LX-2 in human hepatic stellate cells(HSCs)and its possible molecular mechanism.Methods The human HSC line LX-2 were divided into blank group,control group,and cinobufotalin low,medium and high experimental groups.The blank group was cultured with complete medium,the control group was treated with medium containing 10 ng·mL^(-1) transforming growth factor-β1(TGF-β1),and the cinobufotalin low,medium and high experimental groups were treated with 0.5,1 and 2 mg·mL^(-1) cinobufotalin on the basis of the control group,respectively.Cell counting kit-8(CCK-8)experiment was used to detect the effect of different concentrations of cinobufotalin on the proliferation of human hepatic stellate cell line LX-2 cells;Western blot was used to detect the expression ofα-smooth muscle actin(α-SMA)and collagen typeⅠ(Collagen-Ⅰ)protein in each group of cells;real-time fluorescent quantitative polymerase chain reaction(RT-PCR)was used to detectα-SMA and Collagen-ⅠmR NA expression.Immunofluorescence method was used to detect the expression ofα-SMA protein in each group of cells.Results Western blot and RT-PCR results showed that with the increasing concentration of TGF-β1,the protein expressions ofα-SMA and Collagen-Ⅰin LX-2 cells were enhanced,and the difference was statistically significant(P<0.01);indicating that TGF-β1 could induce the activation and proliferation of LX-2 cells.The CCK-8 assay showed that the median inhibitory concentration of cinobufotalin on LX-2 cells was about 2 mg·mL^(-1).Western blot results showed that the expression levels ofα-SMA protein in the control group,the cinobufotalin low,medium and high experimental groups were 1.00±0.04,0.62±0.03,0.48±0.01 and 0.36±0.01,the levels of Collagen-Ⅰprotein were 1.00±0.04,0.52±0.03,0.18±0.01 and0.06±0.01;the results of immunofluorescence showed that the expression levels ofα-SMA in the blank group,control group,and cinobufotalin low,medium and high experimental gro

关 键 词：人肝星状细胞 华蟾素 转化生长因子-Β1 肝纤维化 细胞增殖 

分 类 号：R97[医药卫生—药品]