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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 study the relationship between the up-regulation of TRPM7 channel on fibroblasts induced by lipopolysaccharide (LPS) and cell proliferation, and to use astragaloside for intervention. Methods: Rat cardiac fibroblasts were isolated and divided into 7 groups: normal group, model group (LPS 0.1μg · mL -1, 1μg · ml -1), TRPM7 channel blocker group carvacrol 500μmol·L -1, 2-APB 100μmol·L -1), ASGⅣ (1μmol·L -1, 10μmol·L -1) The rats in the last four groups were treated with 500 microg x L (-1) carvacrol, 100 microg L - (-1) 2-APB and 1 microg L (-1) ASG IV , 10μmoL · L -1 ASGⅣ for 72h. The changes of TRPM7 currents were detected by whole-cell patch clamp technique. The expression of TRPM7 channel genes and proteins were determined by RT-PCR and Western Blot. Fibroblast proliferation ability. Results: Compared with the normal group, the TRPM7 currents and the expression of genes and proteins were significantly increased in both LPS groups (P <0.01). Compared with the model group, the currents of the carvacrol and 2-APB channel blockers, (P <0.01). In addition, ASGⅣ also inhibited the current, gene and protein expression to a certain extent (P <0.05). The proliferation of fibroblasts in model group was significantly stronger than that in other groups (P <0.01). Both channel blocker and astragaloside group inhibited cell proliferation to some extent (P <0.01). CONCLUSION: Lipopolysaccharide (LPS) can upregulate TRPM7 currents and channels in fibroblasts, and this upregulation enhances cell proliferation. ASGIV can inhibit the proliferation of fibroblasts by down-regulating TRPM7 expression.