基于Wnt/β-catenin信号通路研究扶肝化纤汤抑制HSC-T6活化的作用  

Mechanism of Fugan Huaqian Decoction inhibiting HSC-T6 activation via Wnt/β-catenin signaling pathway

作　　者：肖政华 石以石则 杨庆万 李婷婷 李珊珊 钟燕 XIAO Zhenghua;SHIYI Shize;YANG Qingwan;LI Tingting;LI Shanshan;ZHONG Yan(Department of Gastroenterology,The Second Affliated Hospital of Guizhou University of Traditional Chinese Medicine,Guiyang 550001,Guizhou,China;The Second Clinical Medical College of Guizhou University of Traditional Chinese Medicine,Guiyang 550001,Guizhou,China;Department of Gastroenterology,Meishan Hospital of Traditional Chinese Medicine,Meishan 620000,Sichuan,China;Department of Traditional Chinese Medicine,First People's Hospital of Lanzhou New Area,Lanzhou New Area 730300,Gansu,China)

机构地区：[1]贵州中医药大学第二附属医院消化内科,贵州贵阳550001 [2]贵州中医药大学第二临床医学院,贵州贵阳550001 [3]眉山市中医医院消化内科,四川眉山620000 [4]兰州新区第一人民医院中医科,甘肃兰州730300

出　　处：《山东大学学报（医学版）》2023年第6期41-46,共6页Journal of Shandong University:Health Sciences

基　　金：国家自然科学基金(81660790)。

摘　　要：目的探讨扶肝化纤汤抑制大鼠肝星状细胞系HSC-T6增殖的作用机制。方法健康清洁级雄性SD大鼠40只,予扶肝化纤汤等效量灌胃,制备含药血清及正常血清。CCK8法检测不同含药血清浓度(5%、8%、10%、15%、20%)对HSC-T6增殖率的影响,选择最佳作用浓度。将HSC-T6分为空白组(15%胎牛血清)、模型组[转化生长因子-β1(TGF-β1)+15%正常血清)]和治疗组(TGF-β1+15%含药血清),Western blotting、qt-PCR检测Wnt1、β-catenin、Cyclin D1蛋白和mRNA表达,细胞免疫荧光染色法检测α-SMA表达。结果镜下观察治疗组HSC-T6细胞数量减少,形态变圆,体积缩小,悬浮细胞增多。CCK8法检测结果显示,扶肝化纤汤含药血清抑制TGF-β1诱导活化的HSC-T6细胞增殖(P<0.01),其中15%、20%浓度效果最佳,但15%与20%两组间比较差异无统计学意义(P>0.05)。Western blotting、qt-PCR检测结果显示,与模型组比较,治疗组Wnt1、β-catenin、Cyclin D1的mRNA和蛋白表达下降(P<0.01)。细胞免疫荧光染色法结果显示,模型组胞质内α-SMA表达增加,治疗组与模型组比较胞质内α-SMA表达减少。结论扶肝化纤汤含药血清可抑制HSC-T6活化增殖,降低活化后HSC-T6中Wnt1、β-catenin、Cyclin D1的蛋白及mRNA表达;扶肝化纤汤可能通过抑制Wnt/β-catenin信号通路的激活从而防治HF。Objective To explore the inhibitory effect of Fugan Huaqian Decoction on hepatic stellate cell line HSC-T6.Methods A total of 40 healthy and clean male SD rats were given equivalent amount of Fugan Huaxian Decoction by gavage to prepare drug-containing serum and normal serum.The effects of different drug-containing serum concentrations(5%,8%,10%,15%,20%)on the proliferation rate of HSC-T6 cells were detected with CCK8 assay to determine the optimal acting concentration.HSC-T6 cells were divided into blank group(15%fetal bovine serum),model group[transforming growth factor-β1(TGF-β1)+15%normal serum]and treatment group(TGF-β1+15%medicated serum).Protein and mRNA expressions of Wnt1,β-catenin and Cyclin D1 were detected with Western blotting and q-PCR,respectively.Expression ofα-SMA was determined with immunofluorescence staining.Results In the treatment group,the number of cells decreased,the morphology turned round,the volume shrank,and the number of suspension cells increased.CCK8 assay showed that drug-containing serum inhibited TGF-β1-induced proliferation of HSC-T6 cells(P<0.01),and the best effect was observed at 15%and 20%concentration,but there was no significant difference between the two groups(P>0.05).Western blotting and q-PCR showed that compared with the model group,the treatment group had decreased mRNA and protein expressions of Wnt1,β-catenin and Cyclin D1(P<0.01).Immunofluorescence staining showed that the cytoplasmic expression ofα-SMA increased in the model group,but decreased in the treatment group.Conclusion Serum containing Fugan Huaqian Decoction can inhibit HSC-T6 activation and proliferation,and decrease the protein and mRNA expressions of Wnt1,β-catenin and Cyclin D1 in activated HSC-T6 cells.Fugan Huaqian Decoction may prevent and treat hepatic fibrosis by inhibiting the activation of Wnt/β-catenin signaling pathway.

关 键 词：扶肝化纤汤 肝纤维化 肝星状细胞 Wnt1蛋白 Β-CATENIN蛋白 

分 类 号：R575[医药卫生—消化系统]