芪地糖肾方抑制IRE1α/XBP1s改善高糖诱导足细胞内质网应激的研究

Mechanism of Qiditangshen Formula for improving high glucose-induced endoplasmic reticulum stress in MPC5 cell by inhibiting IRE1α/XBP1s

目的观察芪地糖肾方通过肌醇依赖酶1α(IRE1α)/剪接型x-框结合蛋白1(XBP1s)抑制高糖诱导的条件永生化小鼠足细胞(MPC5)内质网应激(ERS)的作用。方法以MPC5细胞为研究对象,采用CCK-8法检测细胞活力筛选高糖造模浓度和时间以及中药干预浓度。将MPC5细胞分为正常组、模型组、芪地糖肾方组,分别予完全培养基、35 mmol/L高糖培养基、35 mmol/L高糖培养基+芪地糖肾方冻干粉溶液培养,采用Western blot法检测足细胞中硫氧还蛋白相互作用蛋白(TXNIP)、NOD样受体热蛋白结构域相关蛋白3(NLRP3)及ERS相关通路IRE1α、p-IRE1α、XBP1s、蛋白激酶RNA样ER激酶(PERK)、p-PERK、激活转录因子6(ATF6)蛋白表达变化。结果与正常组比较,模型组MPC5细胞p-IRE1α/IRE1α、p-PERK/PERK、XBP1s、TXNIP、NLRP3蛋白表达水平升高(P<0.01,P<0.05),ATF6表达升高,但差异无统计学意义(P>0.05);与模型组相比,芪地糖肾方组能够显著下调高糖诱导的MPC5细胞p-IRE1α/IRE1α、XBP1s、TXNIP、NLRP3蛋白表达水平(P<0.01,P<0.05),但p-PERK/PERK、ATF6蛋白表达差异无统计学意义(P>0.05)。结论芪地糖肾方可以通过抑制IRE1α/XBP1s途径过度活化,减少下游TXNIP和NLRP3过度表达,从而改善高糖诱导的足细胞内质网应激。

Objective To explore whether Qiditangshen Formula can inhibit MPC5 endoplasmic reticulum stress(ERS)induced by high glucose through IRE1α/XBP1s pathway.Methods MPC5 cells were studied and CCK-8 method was used to screen the concentration and time of high glucose model and the best concentration of Qiditangshen Formula intervention.MPC5 cells were divided into control group,model group and Qiditangshen group.They were cultured in complete medium,35 mmol/L high sugar medium,35 mmol/L high sugar medium+Qiditangshen,respectively.Western blot was used to detect the protein expression changes of TXNIP,NLRP3 and endoplasmic reticulum stress related pathway,such as p-IRE1α,IRE1α,XBP1s,p-PERK,PERK,ATF6 protein in podocytes.Results Compared with the control group,the expression levels of p-IRE1α/IRE1α,p-PERK/PERK,XBP1s,TXNIP,NLRP3 proteins were significantly higher in the model group of MPC5 cells(P<0.01,P<0.05),the expression of ATF6 had an upward trend but the difference had no statistical significance(P>0.05).Compared with the model group,Qiditangshen Formula group could significantly reduce p-IRE1α/IRE1α,XBP1s,TXNIP,NLRP3 proteins expression level of MPC5 cells induced by high glucose(P<0.01,P<0.05),but the differences in expression of p-PERK/PERK and ATF6protein had no statistical significancece(P>0.05).Conclusion Qiditangshen Formula can improve the ERS of MPC5 cells induced by high glucose,and the mechanism is closely related to the inhibition of the overexpression of IRE1α/XBP1s pathway and TXNIP,NLRP3 proteins downstream.