加味济生肾气汤调控HIF-1α/Notch通路改善缺氧抗肝硬化的机制研究  

Study on the mechanism of Jiawei Jisheng Shenqi Decoction regulating HIF-1α/Notch pathway to improve hypoxia and antagonize liver cirrhosis

作　　者：磨奕玲 周晓玲 刘琳 孙东琪 吴腾 罗艺 阮博文 王月明 贾瑶 MO Yiling;ZHOU Xiaoling;LIU Lin;SUN Dongqi;WU Teng;LUO Yi;RUAN Bowen;WANG Yueming;JIA Yao(Guangxi University of Chinese Medicine,Nanning 530001,China;the First Affiliated Hospital of Guangxi University of Chinese Medicine,Nanning 530001;Liuzhou Tratidonal Chinese Medical Hospital,Liuzhou 545000)

机构地区：[1]广西中医药大学,南宁530001 [2]广西中医药大学第一附属医院,南宁530001 [3]柳州市中医医院,广西柳州545000

出　　处：《中国比较医学杂志》2025年第2期1-12,共12页Chinese Journal of Comparative Medicine

基　　金：国家自然科学基金(81760855);广西自然科学基金(2025GXNSFAA069382);广西中医药大学研究生教育创新计划项目(YCSW2024404,YCBZ2024145);柳州市科技计划项目(2023YRZ0102)。

摘　　要：目的探究加味济生肾气汤改善缺氧微环境抗肝硬化的机制。方法体内实验用CCl4诱导制备大鼠肝硬化模型,设置正常组、模型组、秋水仙碱组和加味济生肾气汤低、中、高剂量(JWJSSQ low/medium/high-dose)组。HE染色和Masson染色观察各组大鼠肝组织病理改变;试剂盒检测各组大鼠血清肝功能变化;ELISA检测各组大鼠透明质酸(hyaluronic acid,HA)、层粘连蛋白(laminin,LN)、Ⅲ型前胶原(procollagenⅢ,PCⅢ)、Ⅳ型胶原(collagen typeⅣ,COL4)水平;Western blot检测大鼠缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)、Notch1、Jagged1、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)蛋白表达。体外实验以HSC-T6细胞为研究对象,CCK-8法筛选出CoCl2培养细胞最适宜(100μmol/L、200μmol/L、400μmol/L、600μmol/L和800μmol/L)浓度及含药血清(5%、10%、15%和20%)最佳干预浓度;划痕试验检测各组细胞迁移能力;流式细胞术检测各组细胞凋亡率变化;Western blot检测各组细胞内HIF-1α、Notch1、Jagged1、α-SMA、基质金属蛋白酶9(MMP9)、基质金属蛋白酶抑制剂-1(TIMP-1)蛋白表达情况。结果体内实验:与正常组比较,模型组大鼠肝肿胀、炎性细胞浸润和胶原沉积明显增多,假小叶出现,血清ALT、AST、HA、LN、PCⅢ、COL4水平明显增高,ALB显著降低,肝组织HIF-1α、Notch1、Jagged1、α-SMA蛋白表达明显升高(P<0.01),与模型组比较,各治疗组大鼠肝肿胀、炎性细胞浸润和胶原沉积明显减少,纤维化程度减轻,血清ALT、AST、HA、LN、PCIII、COL4水平明显降低,ALB显著增高,肝组织HIF-1α、Notch1、Jagged1、α-SMA蛋白表达均有不同程度降低(P<0.05);体外实验:缺氧能够促进HSC-T6迁移,减少HSC-T6凋亡,并提高HIF-1α、Notch1、Jagged1、α-SMA、TIMP-1蛋白表达,降低MMP9蛋白表达(P<0.01),加味济生肾气汤含药血清能够抑制HSC-T6迁移,促进HSC-T6凋亡,并降低HIF-1α、Notch1、Jagged1、α-SMA、TIMP-1蛋白表�Objective To explore the mechanism of Jiawei Jisheng Shenqi Decoction in improving the hypoxic microenvironment and antagonizing liver cirrhosis.MethodsIn vivo experiments were conducted using a rat model of carbon tetrachloride(CCL4)-induced liver cirrhosis.Rats were divided into normal,model,colchicine,JWJSSQ low-dose,JWJSSQ medium-dose,and JWJSSQ high-dose group.Pathological changes in liver tissues in each group were examined by hematoxylin and eosin(HE)and Masson staining,changes in serum liver function were detected using test kits,levels of hyaluronic acid(HA),laminin(LN),procollagenⅢ(PCⅢ),and collagen typeⅣ(COL4)were detected by enzyme-linked immunosorbent assay(ELISA),and protein expression levels of hypoxia-inducible factor-1α(HIF-1α),Notch1,Jagged1,andα-smooth muscle actin(α-SMA)were detected by Western blot.In vitro experiments were conducted in HSC-T6 cells,and the optimal concentration of CoCl2(100μmol/L,200μmol/L,400μmol/L,600μmol/L and 800μmol/L)in the cultured cells and the optimal concentration of drug-containing serum(5%,10%,15%,20%)were determined by Cell Counting Kit-8(CCK-8)assay.The migration ability of cells in each group was detected by scratch testing,and changes in the apoptosis rates were determined by flow cytometry.Protein expression levels of HIF-1α,Notch1,Jagged1,α-SMA,matrix metallopeptidase 9(MMP9),and tissue inhibitor of metalloproteinases 1(TIMP-1)were detected by Western blot.ResultsIn the in vivo experiments,liver swelling,inflammatory cell infiltration,collagen deposition,and the appearance of pseudolobules were significantly increased in the model group compared with those in the normal group.Serum levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),HA,LN,PCⅢ,and COL4 were significantly increased and albumin(ALB)was significantly decreased in the model group,while liver levels of HIF-1α,Notch1,Jagged1,andα-SMA proteins were significantly increased(P<0.01).Liver swelling,inflammatory cell infiltration,and collagen deposition were sign

关 键 词：肝硬化 肝星状细胞 加味济生肾气汤 缺氧微环境 缺氧诱导因子-1Α 

分 类 号：R-33[医药卫生]