白藜芦醇改善高糖引起肾小球系膜细胞损伤的作用研究

Effect of resveratrol on glomerular mesangial cell injury induced by high glucose

目的:探讨白藜芦醇(RSV)对体外高糖刺激引起大鼠肾小球系膜细胞(RGMCs)损伤的影响及其潜在分子保护机制。方法:取处于对数生长期的RGMCs,分为甘露醇高渗透压对照组(OC)、正常对照组(NG)、高糖组(HG)以及高糖RSV处理组,分别处理24、48 h。试剂盒检测不同时间各组细胞上清液中LDH的水平;Western印迹法检测不同时间各组凋亡相关B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)、半胱氨酸天冬氨酸蛋白酶(Caspase)-1、Caspase-1活性片段(p20)、Gasdermin E(GSDME)、GSDME活性片段(GSDME-N)、Caspase-3、Caspase-3活性片段(p17)、炎性相关细胞因子白细胞介素-1β(IL-1β)及其活化形式(mIL-1β)的表达;ELISA检测不同时间各组细胞培养上清IL-1β的水平。结果:LDH结果显示,RSV可降低高糖刺激48 h后细胞的死亡率(F=31.48,P<0.05)。Western印迹法结果显示,仅在RSV处理24 h后,Bax表达较HG组显著降低,同时伴随Bcl-2表达升高(F=143.1,P<0.05);RSV处理24 h后HG组p20、mIL-1β表达降低(F=26.74、25.76,均P<0.05);此外,RSV处理48 h后p17、GSDME-N的表达较HG组明显下降(F=25.84、18.49,均P<0.05)。ELISA结果显示,与HG组相比,RSV处理48 h后上清中IL-1β的水平显著降低(F=80.22,P<0.01)。上述指标在OC组及NG组间的变化均无统计学意义(均P>0.05)。结论:RSV可通过减少炎症因子的产生及抑制细胞早期凋亡、晚期焦亡过程来改善高糖引起的RGMCs损伤。

Objective:To investigate the effect of resveratrol(RSV)on the injury of rat glomerular mesangial cells(RGMCs)induced by high glucose in vitro and its potential molecular protection mechanism.Methods:RGMCs in logarithmic growth phase were divided into mannitol hypertonic control group(OC),normal control group(NG),high glucose group(HG)and high glucose with RSV treatment group,and RGMCs were cultured for 24 h and 48 h respectively.The levels of LDH in the supernatant of each group were detected by the LDH release assay kit.The protein levels of apoptosis related B-cell lymphoma-2 gene(Bcl-2),Bcl-2 associated X protein(Bax),cysteine-containing aspartate specific protease 1(Caspase-1),Caspase-1 activated fragment(p20),Gasdermin E(GSDME),Gasdermin E activated fragment(GSDME-N),Caspase-3,Caspase-3 activated fragment(p17),inflammatory related cytokines interleukin-1β(IL-1β)and mature form of IL-1β(mIL-1β)were detected by Western blotting analysis.The levels of IL-1βin the supernatant were detected by ELISA analysis.Results:LDH assay showed that RSV reduced the cell death rate after 48 h of high glucose stimulation(F=31.48,P<0.05).Western blotting analysis showed that the expression of Bax in RSV treatment group was significantly lower than that in HG group only at 24 h and the expression of Bcl-2 was increased(F=143.1,P<0.05).RSV also decreased the expression of p20 and mIL-1βin HG group at 24 h(F=26.74,25.76,all P<0.05).Moreover,RSV significantly decreased the expression of p17 and GSDME-N in HG group at 48 h(F=25.84,18.49,all P<0.05).ELISA analysis showed that IL-1βlevel in the supernatant of RGMCs with RSV treatment for 48 h was significantly reduced than that in HG group(F=80.22,P<0.01).There were no significant difference in above related indexes between OC group and NG group(all P>0.05).Conclusion:RSV can improve the damage in RGMCs induced by high glucose via reducing the production of inflammatory factors,inhibiting the early apoptotic and late pyroptosis.