目的 在高糖(high glucose，HG)环境下，探讨罗汉果甜苷Ⅴ(Mogroside Ⅴ，MV)对人肾小球系膜细胞(human glomerular mesangial cells，HRMC)焦亡的影响，通过硫氧还蛋白相互作用蛋白(thioredoxin-interacting protein，TXNIP)/NOD 样受体热蛋白结构域相关蛋白 3(NOD-like receptor protein 3，NLRP3)信号通路探索其肾脏保护机制。方法 用HG诱导HRMC细胞构建焦亡模型，盐酸二甲双胍(Metformin，Met)作为阳性对照。采用MTT法评估HG对HRMC细胞活力的影响;流式细胞术和超氧化物阴离子荧光探针法检测活性氧(reactive oxygen species，ROS)水平变化;透射电镜观察细胞结构变化;免疫荧光法和免疫印迹法(Western blot，WB)检测TXNIP、NLRP3蛋白表达水平;WB和实时荧光定量PCR(quantitative reverse transcription PCR，RT-qPCR)法检测焦亡相关因子GasderminD(GSDMD)、半胱氨酸蛋白酶-1(cysteinyl aspartate specific proteinase-1，Caspase-1)、凋亡相关斑点样蛋白(apoptosis-associated speck-like protein containing a CARD，ASC)及其下游炎症因子白细胞介素(interleukin，，IL)-18、IL-1β 的表达。沉默 TXNIP 基因后，WB 和 RT-qPCR 法检测 TXNIP/NLRP3信号通路及其下游细胞焦亡与炎症相关因子蛋白和mRNA的表达水平。结果 30 mmol/L的HG诱导48小时，HRMC细胞异常增殖(P＜0。05)，焦亡相关蛋白表达均不同程度增加，差异具有统计学意义(P＜0。05)。HG环境下HRMC细胞内ROS水平增高，MV治疗后能够降低胞内ROS水平;MV或Met干预能够逆转HG引起的TXNIP、NLRP3蛋白的高表达;HG组细胞中GSDMD、Caspase-1、ASC、IL-18、IL-1β蛋白和mRNA表达显著升高，差异有统计学意义(P＜0。05)，MV干预后逆转了 HG刺激后HRMC细胞焦亡相关蛋白和mRNA的高表达;HG处理HRMC导致细胞膜损伤，MV干预后能够抑制这种细胞膜损伤。MV和siRNA-TXNIP均能显著抑制HG引起的HRMC中TXNIP/NLRP3信号通路及细胞焦亡相关蛋白或mRNA的表达(P＜0。05)。结论 MV具有抑制细胞焦亡与炎症反应、降低ROS水平的作用，其抗焦亡和抗炎作用可能是通过TXNIP/NLRP3信号通路进行调控的。
The mechanism of Mogroside Ⅴ on pyroptosis of HRMC based on TXNIP/NLRP3 pathway
Objective To investigate the effect of Mogroside Ⅴ(MV)on pyroptosis in human glo-merular mesangial cells(HRMC)in high glucose(HG),and to explore its renal protective mechanism through thioredoxin-interacting protein(TXNIP)/NOD-like receptor protein 3(NLRP3)signaling pathway.Methods Pyroptosis model was constructed with HG-induced HRMC,and Metformin(Met)was used as a positive control.MTT method was measured to assess the effect of HG on HRMC cell viability.Flow cytometry and Dihydroethidium staining were used to detect reactive oxygen species levels.Transmission electron microscopy was used to observe cell structure.Immunofluorescence and Western blot(WB)were used to determine the expression levels of the TXNIP,NLRP3,and the pyroptosis related factors were detected by WB and RT-qPCR methods.After transfection TXNIP gene,WB and RT-qPCR methods were measured the expression levels of TXNIP,NLRP3 and pyroptosis and inflammation factors protein and mRNA.Results 30 mmol/L HG induced 48 hours,HRMC cells proliferated abnormally(P＜0.05),and the expression of pyroptosis proteins were increased to different degrees,with statistical significance(P＜0.05).The level of intracellular ROS was increased in HRMC cells by HG,and the treatment of MV was able to reduce the intracellular level of ROS,the intervention of MV or Met was able to reverse HG-induced high expression of TXNIP and NLRP3 proteins.GSDMD,Caspase-1,ASC,IL-18,IL-1 β protein and mRNA expression was significantly higher in the cells of HG group,the difference was statistically significant(P＜0.05),and MV intervention reversed the high expression of proteins and mRNAs related to pyroptosis in HRMC cells after HG stimulation;HG treatment of HRMC led to cell membrane damage,which could be inhibited by MV intervention.MV and siRNA-TXNIP significantly inhibited the expression of TXNIP/NLRP3 signaling pathway and cellular pyroptosis-related proteins or mRNAs in HG-induced HRMC(P＜0.05).Conclusion MV can inhibit pyroptosis,inflammatory and reduce reactive oxygen species levels,and its anti-pyroptosis and anti-inflammatory effects may be regulated through the TXNIP/NLRP3 signaling pathway.
diabetic nephropathyMogroside ⅤTXNIP/NLRP3 signal pathwaypyroptosis
万方数据
维普
