基于钙敏感受体探讨花旗泽仁改善胰岛素抵抗的作用机制

Discussion on the Mechanism of Action of HQZR in Improving Insulin Resistance Based on Calcium Sensitive Receptor

目的通过观察花旗泽仁对胰岛素抵抗(IR)细胞中钙敏感受体(CaSR)基因、蛋白表达及AKT活性的影响,探究花旗泽仁改善IR的分子机制。方法采用肿瘤坏死因子(TNF-α)体外诱导培养法建立L6肌细胞IR模型;采用免疫荧光技术检测CaSR在L6肌细胞中的分布;采用qRT-PCR检测L6肌细胞内CaSR基因表达水平;采用Western blot法检测L6肌细胞内CaSR蛋白、及磷酸化AKT(Ser473和Thr308)蛋白表达水平,并观察CaSR激动剂对L6肌细胞IR模型AKT活性的影响。结果与正常对照组比较,模型对照组细胞的葡萄糖消耗量明显降低(P<0.05)、CaSR在模型组中的分布较稀疏、mRNA表达水平显著降低(P=0.000)、蛋白表达量显著降低(P=0.000)、细胞中磷酸化AKT(Ser473和Thr308)蛋白表达量明显下降(P=0.000);与模型对照组比较,花旗泽仁组CaSR的分布较密集、mRNA表达水平明显升高(P<0.01)、蛋白表达量明显增加(P=0.000)、细胞中磷酸化AKT(Ser473和Thr308)表达量明显上升(P<0.01)。结论L6肌细胞发生IR时,CaSR mRNA及蛋白表达量降低,AKT活性被抑制,CaSR激动剂可增强AKT活性,提示CaSR可以调控AKT;花旗泽仁可能通过上调CaSR表达水平,激活与IR关系密切的PI 3K/AKT信号通路中的AKT活性,从而改善IR。

Objective To investigate the effect of HQZR on the CaSR gene,protein expression and AKT activity in insulin-resistant cells,and to explore the molecular mechanism of the improvement of IR by HQZR.Methods Using IR-induced factor necrosis factor(TNF-α)in vitro,we established an IR model of L6 myocytes.Using immunofluorescence,we detected the distribution of CaSR in L6 myocytes.Using qRT-PCR,we detected CaSR in L6 myocytes Gene expression level.Western blot was used to detect the expression of CaSR protein and phosphorylated AKT(Ser473 and Thr308)protein in L6 myocytes,and the effect of CaSR agonist on AKT activity of L6 myocardium IR model was observed.Results Compared with the blank control group,the glucose consumption of the model group was significantly lower(P<0.05).The distribution of CaSR in the model group was sparse.The mRNA expression level was significantly decreased(P=0.000),and the protein expression was significantly decreased(P=0.000).The expression of phosphorylated AKT(Ser473 and Thr308)protein in cells decreased significantly(P=0.000).Compared with the model group,the distribution of CaSR in the Citigroup was more dense,the mRNA expression level was significantly increased(P<0.01).The protein expression was significantly increased(P=0.000),and the phosphorylated AKT(Ser473 and Thr308)expression in the cells.The amount increased significantly(P<0.01).Conclusion When insulin resistance occurs in L6 muscle cells.CaSR mRNA and protein expression are decreased,AKT activity is inhibited.CaSR agonists can enhance AKT activity,suggesting that CaSR can regulate AKT.HQZR may up-regulate CaSR expression level,activate insulin it resists AKT activity in the closely related PI 3K/AKT signaling pathway,thereby improving insulin resistance.