TRPM7参与诱发心肌纤维化作用及药物干预

Effects of TRPM7 on Cardiac Fibrosis and Drug Intervention

目的:研究脂多糖(LPS)引起的成纤维细胞上TRPM7通道的上调和细胞增殖间的关系,并利用黄芪甲苷进行干预。方法:分离大鼠乳鼠心脏成纤维细胞,分成7组,正常组、模型组(LPS 0.1μg·mL^(-1)、1μg·ml^(-1))、TRPM7通道阻断剂组(carvacrol 500μmoL·L^(-1)、2-APB 100μmoL·L^(-1))、黄芪甲苷(ASGⅣ)组(1μmoL·L^(-1)、10μmoL·L^(-1)),后4组在加入1μg·mL^(-1)LPS的同时,分别给予500μmoL·L^(-1)carvacrol、100μmoL·L^(-1)2-APB、1μmoL·L^(-1)ASGⅣ、10μmoL·L^(-1)ASGⅣ,孵育72h,通过全细胞膜片钳技术检测TRPM7电流的变化,利用RT-PCR和Western Blot方法分别测定TRPM7通道的基因和蛋白的表达,利用MTT方法测定成纤维细胞的增殖能力。结果:与正常组相比,LPS两个组的TRPM7电流大小及基因、蛋白表达明显升高(P<0.01);与模型组相比,carvacrol、2-APB通道阻断剂组的电流大小、基因和蛋白表达显著降低(P<0.01);此外,ASGⅣ对电流、基因和蛋白表达也有不同程度的抑制作用(P<0.05)。模型组的成纤维细胞增殖能力明显强于其他组(P<0.01),通道阻断剂组及黄芪甲苷组均能不同程度抑制细胞的异常增殖(P<0.01)。结论:脂多糖(LPS)能够使成纤维细胞上TRPM7电流大小及通道表达上调,且该上调能增强细胞增殖能力,而ASGⅣ能通过下调TRPM7表达抑制成纤维细胞的增殖。

Objective：To investigate the effects of Lipopolysaccharide（LPS） and the inhibitory effects of astragaloside Ⅳ（ASG Ⅳ）on the proliferation of cardiac fibroblasts through TRPM7 channel.Methods：Cardiac fibroblasts obtained from neonatal SD rats were cultured in vitro divided into 7 groups,control group,model groups（LPS 0.1μg ·mL-（-1）,1μg· mL-（-1））,TRPM7 channel blocker groups（carvacrol 500μmoL·L-（-1）,2-APB 100μmoL·L-（-1））,ASG Ⅳ treatment groups（1μmoL·L-（-1）,10μmoL·L-（-1））.The treatment groups were administered with 1 μg·mL-1 LPS and treated respectively with 500μmoL·L-（-1） carvacrol,100μmoL·L-（-1）2-APB,1μmoL·L-（-1） ASG Ⅳ,10μmoL·L-（-1） ASG Ⅳ.Each group was incubated for 72 hours.The changes of gene and protein expression,channel current,cell proliferation before and after administration were measured respectively by RT-PCR,western blot,whole cell patch clamp and MTT.Results：RT-PCR and western blot results showed that LPS had significantly upregulated the gene and protein expression of TRPM7（P0.01） and carvacrol,2-APB,ASG Ⅳ inhibited the increase on different levels.Patch clamp data revealed that there was a decrease of TRPM7 current in LPS ＋carvacrol group,LPS＋2-APB group and LPS＋ASG Ⅳ group compared with model group.Furthermore,ASG Ⅳ markly prevented the proliferation of cardiac fibroblasts induced by LPS（P0.01）.Conclusion：We demonstrate that TRPM7 channels contributed to LPS induced cardiac fibroblasts proliferation and suggest that this contribution may be suppressed by ASG Ⅳ through inbibition of TRPM7 channel.