转化生长因子-β1受体依赖的心肌纤维化伴随大电导钙激活钾通道的表达和功能下调  

Reduced Expression and Function of Big Conductance Ca^(2+)-activated K^(+) Channel in Transforming Growth Factor-β1 Induced Myocardial Fibrosis

作　　者：李磊 董亚辉 杨探 李秋芳 聂永梅 李光 于风旭 LI Lei;DONG Yahui;YANG Tan;LI Qiufang;NIE Yongmei;LI Guang;YU Fengxu(Department of Cardiovascular Surgery,The Affiliated Hospital of Southwest Medical University,Luzhou(646000),Sichuan,China)

机构地区：[1]西南医科大学附属医院心脏大血管外科,四川省泸州市646000 [2]西南医科大学心血管医学研究所医学电生理学教育部重点实验室医学电生理四川省重点实验室

出　　处：《中国循环杂志》2021年第6期612-618,共7页Chinese Circulation Journal

基　　金：四川省科技厅资助项目(2018JY0080);泸州市人民政府-西南医科大学科技战略合作项目(2018LZXNYD-ZK40);四川省心血管疾病防治协同创新中心项目(xtcx2019-4);泸州市人民政府-西南医科大学联合项目(2017-LH003)。

摘　　要：目的:探讨大电导钙激活钾通道(BKCa)是否参与转化生长因子-β1(TGF-β1)诱导的心肌纤维化过程,以及在这一过程中的作用机制。方法:分离培养乳大鼠心室成纤维细胞,并根据TGF-β1的不同浓度(5 ng/ml组、10 ng/ml组),以及在10 ng/ml TGF-β1基础上加入不同浓度TGF-β1受体特异阻断剂SB431542(0.1μmol/L组、1μmol/L组、10μmol/L组)进行分组,处理后提取蛋白及RNA,应用蛋白免疫印迹(Western blot)和实时荧光定量PCR(qRT-PCR)检测各组TGF-β1受体蛋白,α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原(CollagenⅠ)、Ⅲ型胶原(CollagenⅢ)、BKCa蛋白和信使RNA(mRNA)的表达情况。单通道及全细胞膜片钳技术记录TGF-β1作用24 h前后的BKCa电流特点及其变化情况。结果:TGF-β15 ng/ml组和10 ng/ml组均上调TGF-β1受体蛋白,α-SMA、CollagenⅠ、CollagenⅢ蛋白和mRNA的表达,10 ng/ml组TGF-β1下调BKCa蛋白和mRNA的表达。与TGF-β110 ng/ml组相比,在10 ng/ml TGF-β1基础上加入SB4315421μmol/L组、10μmol/L组下调TGF-β1受体蛋白,α-SMA、CollagenⅠ、CollagenⅢ蛋白和mRNA的表达,加入SB43154210μmol/L组上调BKCa蛋白和mRNA的表达,差异均具有统计学意义。此外,单通道及全细胞膜片钳技术记录结果表明TGF-β1预处理24 h后可明显抑制成纤维细胞上BKCa电流。结论:BKCa可能参与了TGF-β1诱导乳大鼠心室成纤维细胞纤维化通路,其机制可能是TGF-β1抑制了成纤维细胞上BKCa的电流。这提示BKCa可能是防治心肌纤维化的潜在靶点。Objectives:To investigate whether big conductance Ca^(2+)-activated K^(+)channel(BKCa)is involved in transforming growth factor-β1(TGF-β1)-induced myocardial fibrosis and the underlying mechanisms.Methods:New natal rat ventricular fibroblasts were isolated,cultured,and passed to the first generation.According to the different concentrations of TGF-β1(5 ng/ml,10 ng/ml)and the specific blocker of TGF-β1 receptor SB431542(0.1μmol/L,1μmol/L,10μmol/L),the ventricular fibroblasts of new natal rats were divided into different groups.Western blot and realtime reverse transcription-polymerase chain reaction were used to detect the protein and mRNA expressions of TGF-β1 receptor protein,α-SMA,collagenⅠ,collagenⅢ,BKCa.Single-channel and whole-cell patch-clamp techniques were used to evalute BKCa current changes before and after 24 hours TGF-β1 treatment.Results:TGF-β15 ng/ml group and 10 ng/ml group significantly up-regulated the expression of TGF-β1 receptor protein,the expression ofα-SMA,collagenⅠ,collagen Ⅲ at protein and mRNA levels.TGF-β110 ng/ml group substantially down-regulated the expression of BKCa at protein and mRNA levels.Additionally,SB4315421μmol/L group and 10μmol/L group significantly down-regulated the expression of TGF-β1 receptor protein,the expression ofα-SMA,collagenⅠ,collagen Ⅲ at protein and mRNA levels compared to TGF-β110 ng/ml group.Significantly up-regulated expression of BKCa at protein and mRNA levels was observed in 10μmol/L SB431542 group.Single-channel and whole-cell patch clamps recordings evidenced significant deactivation of BKCa channel currents in fibroblasts pretreated with TGF-β1 for 24 hours.Conclusions:This study indicates that BKCa channels of rat ventricular fibroblasts is involved in TGF-β1 induced fibrosis in that TGF-β1 could inhibit BKCa currents of the fibroblasts.Therefore,it is suggested that the BKCa channel could serve as a potential target for preventing and treating myocardial fibrosis.

关 键 词：心肌纤维化 转化生长因子Β1 转化生长因子β1受体阻断剂SB431542 大电导钙激活钾通道 乳大鼠心室成纤维细胞 

分 类 号：R541[医药卫生—心血管疾病]