基于自噬途径探讨黄芪甲苷抑制糖尿病肾病系膜细胞NLRP3炎症小体活化通路及机制

Inhibitory Effect and Mechanism of Astragaloside IV on NLRP3Inflammasome Activation Pathway in Mesangial Cells of Diabetic Nephropathy Based on the Autophagy Pathway

目的:观察黄芪甲苷对高糖刺激的小鼠肾小球系膜细胞(SV40)核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎症小体活化通路的抑制作用,并基于线粒体自噬途径探究其作用机制。方法:小鼠SV40细胞分别在含有0~80μmol/L黄芪甲苷的DMEM培养基中体外培养48 h,通过细胞活力测试筛选出适宜的黄芪甲苷浓度进行后续实验。实验分为正常对照组(葡萄糖5.6 mmol/L)、高糖组(葡萄糖30 mmol/L)、黄芪甲苷低剂量组(葡萄糖30 mmol/L+黄芪甲苷10μmol/L)、黄芪甲苷中剂量组(葡萄糖30 mmol/L+黄芪甲苷20μmol/L)、黄芪甲苷高剂量组(葡萄糖30 mmol/L+黄芪甲苷40μmol/L),观察黄芪甲苷对小鼠SV40细胞的影响。结果:CCK-8检测结果提示黄芪甲苷浓度达到80μmol/L时小鼠SV40细胞活力明显下降,故本次研究分别选用10、20、40μmol/L作为低、中、高剂量黄芪甲苷进行后续实验。与正常对照组比较,高糖会降低小鼠SV40细胞活力并促进细胞凋亡,并可增加活性氧(ROS)与炎症因子水平,扩大炎症与氧化应激反应,LC3、Beclin1表达水平明显下调,自噬受到抑制。与高糖组比较,黄芪甲苷中、高剂量组细胞凋亡、炎症因子、ROS水平和NLRP3、Caspase-1表达水平均降低(P<0.05或P<0.01),细胞活力、LC3、Beclin-1表达水平均升高(P<0.05或P<0.01)。透射电镜观察发现高糖会减少自噬体产生,黄芪甲苷组自噬体的数目明显多于高糖组。结论:高糖可导致小鼠SV40细胞处于微炎症状态,扩大氧化应激反应并激活NLRP3炎症小体,黄芪甲苷可以抑制NLRP3表达,并提高线粒体自噬,起到减缓DN疾病进展的作用。

Objective:To observe the inhibitory effect of astragalosideⅣon the inflammasome activation pathway of nucleotide binding oligomerization domain like receptor protein 3(NLRP3)in mouse glomerular mesangial cells(SV40)stimulated by high glucose,and to explore its mechanism based on mitochondrial autophagy pathway.Methods:Mouse SV40 cells were cultured in DMEM medium containing 0-80μmol/L astragalosideⅣfor 48 h in vitro,and the appropriate concentration of astragalosideⅣwas selected by cell viability test for subsequent experiments.The experiment was divided into normal control group,high glucose group,astragalosideⅣlow-dose group,astragalosideⅣmedium-dose group,and astragalosideⅣhigh-dose group.The effects of astragalosideⅣon mouse SV40 cells were observed.Results:The results of CCK-8 indicated that the activity of SV40 cells in mice decreased significantly when the concentration of astragalosideⅣreached 80μmol/L.Therefore,this study selected 10,20,40μmol/L as low,medium and high-doses of astragalosideⅣfor follow-up experiments.Compared with the normal control group,high glucose could reduced the vitality of mouse SV40 cells and promoted apoptosis,increased the levels of reactive oxygen species(ROS)and inflammatory factors,enlarged the response of inflammation and oxidative stress,significantly downregulated the expression levels of LC3 and Beclin1,and inhibited autophagy.Compared with the high glucose group,the levels of apoptosis,inflammatory factors and ROS,NLRP3 and caspase-1 decreased in astragalosideⅣmedium and high-dose group(P<0.05 or P<0.01),and the cell viability,LC3 and Beclin1 expression increased(P<0.05 or P<0.01).Transmission electron microscopy showed that high glucose would reduce the production of autophagosomes,and the number of autophagosomes in the astragalosideⅣmedium and high-dose group was significantly higher than that in the high glucose group.Conclusion:High glucose can cause mouse SV40 cells to be in a micro-inflammatory state,expand the oxidative stress response and activate the NLRP3 inflammasome.AstragalosideⅣcan inhibit the expression of NLRP3,increase mitochondrial autophagy and slow down the progression of DN disease.