基于CaSR表达及AKT磷酸化水平的花旗泽仁改善胰岛素抵抗作用机制

目的基于CaSR表达及AKT磷酸化水平考察花旗泽仁改善胰岛素抵抗的作用机制。方法采用高浓度胰岛素体外诱导培养法建立IR L02肝细胞模型,并通过葡萄糖消耗实验对细胞模型进行鉴定。采用免疫荧光法和qRT-PCR技术检测CaSR表达的细胞定位以及CaSR mRNA表达,同时采用Western blot技术检测CaSR蛋白、磷酸化AKT(Ser473和Thr308)蛋白表达。结果高浓度胰岛素诱导后的L02肝细胞模型葡萄糖消耗量和摄取率明显下降(P<0.01)。免疫荧光实验结果表明,模型对照组的CaSR分布较稀疏,相对应的,给予花旗泽仁后的CaSR分布较密集。通过qRT-PCR实验发现,模型对照组的CaSR mRNA表达显著下调(P<0.01);与模型对照组比较,花旗泽仁组、阳性对照组中CaSR mRNA表达明显上调(P<0.01)。同时Western blot检测结果表明,模型对照组的CaSR和磷酸化AKT(Thr308和Ser473)蛋白表达显著下降(P<0.01),与模型对照组比较,花旗泽仁组、阳性对照组中CaSR和磷酸化AKT(Thr308和Ser473)蛋白表达明显升高(P<0.01,P<0.05)。结论该实验证明了花旗泽仁通过影响PI3K通路改善胰岛素抵抗,可能与通过调节CaSR,激活PI3K/AKT信号通路AKT上的2个(Thr308,Ser473)磷酸化位点的蛋白表达有关。

Action mechanism of Huaqizeren in improving insulin resistance based on CaSR expression and AKT phosphorylated level

AIM To investigate the action mechanism of Huaqizeren in improving insulin resistance based on CaSR expression and AKT phosphorylated level.METHODS The insulin resistance cell models of L02 hepatocytes induced by high-concentration insulin in vitro culture was identified by a glucose consumption experiment.Immunofluorescence and qRT-PCR were used to detect the localization of CaSR expression and CaSR mRNA expression.Meanwhile,Western blot was used to detect CaSR protein and phosphorylated AKT(Ser473 and Thr308)protein expression levels.RESULTS The insulin resistance L02 hepatocyte models displayed significantly decreased glucose consumption and uptake rate(P<0.01).The results of immunofluorescence experiments showed relatively sparse CaSR distribution in the model control group,but more dense distribution in the group intervened with Huaqizeren.The qRT-PCR experiments revealed significantly decreased CaSR mRNA expression levels in the model control group(P<0.01);but significantly increased CaSR mRNA expression levels in both Huaqizeren group and the positive control group in contrast to the model control group(P<0.01).Simultaneously,Western blot results showed significantly reduced CaSR and phosphorylated AKT(Thr308 and Ser473)protein expressions in the model control group(P<0.01),but significantly increased such protein expressions in both Huaqizeren group and the positive group when in comparison with the model group(P<0.01,P<0.05).CONCLUSION Huaqizeren’s efficacy confirmed in the experiment in insulin resistance improvement through its influence on the PI3K pathway and CaSR activation,associates with the protein expression of two phosphorylation sites(Thr308,Ser473)on the AKT of the PI3K/AKT signaling pathway.