六味地黄汤通过miR-210/HIF-1α信号通路对CoCl_(2)诱导的HK-2细胞上皮间质转化的影响和机制研究  

Effects and mechanism of Liuwei Dihuang Decoction on CoCl_(2)-induced epithelial-mesenchymal transition of HK-2 cells through the miR-210/HIF-1αsignaling pathway

作　　者：张海英 王晖 潘佳俊 高海波 赵欣然 刘春燕[1] 唐群[1] ZHANG Haiying;WANG Hui;PAN Jiajun;GAO Haibo;ZHAO Xinran;LIU Chunyan;TANG Qun(Medical College,Hunan University of Chinese Medicine,Changsha,Hunan 410208,China)

机构地区：[1]湖南中医药大学医学院,湖南长沙410208

出　　处：《湖南中医药大学学报》2023年第4期619-626,共8页Journal of Hunan University of Chinese Medicine

基　　金：湖南省自然科学基金项目(2021JJ30506);湖南省教育厅项目(21A0242,20C1398);长沙市自然科学基金项目(kq2014089);湖南中医药大学研究生科研创新项目(2021CX32)。

摘　　要：目的观察六味地黄汤(Liuwei Dihuang Decoction,LWDHD)调控miR-210/HIF-1α信号通路对CoCl_(2)诱导的HK-2细胞上皮间质转化(epithelial mesenchymal transdifferentiation,EMT)的作用和机制。方法体外培养HK-2细胞,分对照组(N组)、模型组(M组)、LWDHD血清组(LW组)、空白血清+miR-210过表达组(LV-miR-210组)、空白血清+mi R-210沉默组(LV-antimi R-210组)、空白血清+miR-210阴性对照组(LV-miR-210 NC组)、LWDHD血清+miR-210过表达组(LW+LV-miR-210组)、LWDHD血清+mi R-210沉默组(LW+LV-anti-miR-210组)、LWDHD血清+miR-210阴性对照组(LW+LV-miR-210 NC组),除N组外,其他各组加入CoCl_(2)处理。CCK-8法检测不同浓度LWDHD、CoCl_(2)在24 h后对HK-2细胞活性的影响并选择最佳干预浓度。细胞免疫荧光和Western blot法检测各组HK-2细胞中缺氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)、β-联蛋白(β-catenin)、波形蛋白(Vimentin)、上皮钙黏素(E-cadherin)蛋白表达情况;qPCR法检测miR-210、HIF-1α、β-catenin、Vimentin m RNA表达情况。通过慢病毒感染HK-2细胞,实现miR-210的过表达和抑制,并加入CoCl_(2),观察miR-210、HIF-1α、β-catenin、Vimentin蛋白和mRNA表达以及LWDHD的干预作用。结果CCK-8结果显示,LWDHD、CoCl_(2)最佳干预浓度分别为10%、200μmol/L。与N组相比,M组细胞形态由铺路石样向长梭形改变,HIF-1α、β-catenin、Vimentin蛋白和mRNA表达升高(P<0.05,P<0.01),mi R-210m RNA表达升高(P<0.01),E-cadherin蛋白表达降低(P<0.01);与M组相比,LW组HIF-1α、β-catenin、Vimentin蛋白和mRNA表达降低(P<0.05,P<0.01),miR-210 mRNA表达降低(P<0.05),E-cadherin蛋白表达升高(P<0.01)。与LV-miR-210 NC组相比,LVanti-miR-210组HIF-1α、β-catenin、Vimentin蛋白和mRNA表达降低(P<0.05,P<0.01);与LV-miR-210 NC组相比,LV-miR-210组HIF-1α、β-catenin、Vimentin蛋白和mRNA表达升高(P<0.05,P<0.01)。在慢病毒转染各组的基础上,加入LWDHD处理后,LWDHD可降低各组HIF-1α、β-catenin、VimentObjective To observe the effects and mechanism of Liuwei Dihuang Decoction(LWDHD)regulating epithelial mesenchymal transition(EMT)of HK-2 cells induced by CoCl_(2) through miR-210/HIF-1α signaling pathway.Methods HK-2 cells were cultured in vitro and divided into normal group(N group),model group(M group),LWDHD medicated serum group(LW group),blank serum+over-espressed miR-210 group(LV-miR-210 group),blank serum+silent miR-210 group(LV-anti-miR-210 group),blank serum+miR-210 negative group(LV-miR-210 NC group),LWDHD medicated serum+over-expressed miR-210 group(LW+LV-miR-210 group),LWDHD medicated serum+silent miR-210 group(LW+LV-anti-miR-210 group)and LWDHD medicated serum+miR-210 negative group(LW+LV-miR-210 NC group).Except for N group,the other groups were treated with CoCl_(2).CCK-8 was used to detect the effect of different concentrations of LWDHD and CoCl_(2) on the activity of HK-2 cells after 24 hours,and the best intervention concentration was selected.Cell immunofluorescence and Western blot were used to determine hypoxia-inducible factor-1α(HIF-1),β-catenin,Vimentin,and E-cadherin protein expression levels.miR-210,HIF-1α,β-catenin and Vimentin mRNA expression levels were measured by qPCR.By lentivirus transfection of HK-2 cells,miR-210 was overexpressed and inhibited.Then CoCl_(2) was added to the medium.The miR-210,HIF-1α,β-catenin,Vimentin protein and mRNA expression of the regulation effect of LWDHD were observed.Results The CCK-8 showed that the optimal intervention concentrations of LWDHD and CoCl_(2) were 10% and 200μmol/L,respectively.Compared with N group,the morphology of cells in group M changed from the shape of paving stone to the shape of long spindle.HIF-1α,β-catenin,Vimentin protein and mRNA expression increased(P<0.05,P<0.01)while miR-210 mRNA expression increased(P<0.01)and E-cadherin protein expression decreased(P<0.01).Compared with M group,LW group can significantly reduce the expression of HIF-1α,β-catenin,Vimentin protein and mRNA(P<0.05,P<0.01),down-regulate the expre

关 键 词：肾纤维化 六味地黄汤 上皮间质转化 MIR-210 缺氧诱导因子1Α HK-2细胞 

分 类 号：R259[医药卫生—中西医结合]