敲低高迁移率族蛋白B1(HMGB1)抑制高糖诱导的大鼠肾小球系膜细胞增殖并促进其凋亡

Knockdown of HMGB1 inhibits proliferation and promotes apoptosis of rat mesangial cells induced by high glucose

目的探讨敲低高迁移率族蛋白B1(HMGB1)水平对高糖培养大鼠肾小球系膜细胞(GMC)增殖的影响及机制。方法培养大鼠GMC,分为正常组、高糖处理组、阴性对照小干扰RNA联合高糖处理组(siRNA-NC-高糖组)、HMGB1小干涉RNA联合高糖处理组(siRNA-HMGB1-高糖组)。正常组GMC用正常DMEM培养基培养;高糖处理组GMC换用高糖DMEM培养基培养;siRNA-HMGB1-高糖组GMC转染siRNA-HMGB1序列6 h后,用高糖培养液培养24 h;siRNA-NC-高糖组GMC转染siRNA-NC序列6 h后,用高糖培养液培养24 h。实时荧光定量PCR检测GMC中HMGB1 mRNA水平,MTT法检测GMC增殖情况,流式细胞术检测GMC凋亡情况,Western blot法检测GMC中HMGB1、核因子κBp65(NF-κBp65)和核因子κB抑制物α(IκBα)蛋白水平,ELISA检测细胞上清液中白细胞介素1β(IL-1β)、IL-6和肿瘤坏死因子α(TNF-α)水平。结果与siRNA-NC-高糖组或单纯高糖组相比,siRNA-HMGB1-高糖组GMC中HMGB1 mRNA水平降低,在处理24、48、72、96 h,GMC增殖活性和细胞凋亡率降低;敲低GMC的HMGB1水平后,细胞NF-κBp65蛋白水平降低、IκBα蛋白水平增加,且上清液中IL-1β、IL-6和TNF-α水平降低。结论敲低GMC的HMGB1水平抑制高糖诱导的GMC增殖,并促进其凋亡,可能与抑制NF-κB/IκBα通路有关。

Objective To investigate the effect of knockdown of high mobility group protein B1(HMGB1)on the proliferation of rat mesangial cells(GMCs)cultured in high glucose(HG)and its mechanism.Methods Rat GMCs was cultured and divided into normal group,high glucose treatment group,negative control small interfering RNA combined with high glucose treatment group(siRNA-NC-HG group)and HMGB1 small interference RNA combined with high glucose treatment group(siRNA-HMGB1-HG group).GMCs in the normal group were cultured in normal DMEM medium.GMCs in the HG treatment group were cultured with HG-DMEM medium.The GMCs in the siRNA-HMGB1-HG group,after transfected with siRNA-HMGB1 sequence for 6 hours,were cultured with high glucose medium for 24 hours.GMCs in the siRNA-NC-HG group,after transfected with siRNA-NC sequence for 6 hours,were cultured in HG medium for 24 hours.HMGB1 mRNA expression levels of GMCs were detected by real-time quantitative PCR.MTT assay was used to detect the proliferation of GMCs.Flow cytometry was performed to assess the apoptosis of GMCs.Western blot analysis was used to detect the protein levels of HMGB1,NF-κBp65 and nuclear factor kappa B inhibitor alpha(IκBα).ELISA was used to detect the levels of interleukin-1β(IL-1β),IL-6 and tumor necrosis factorα(TNF-α)in the cell supernatants.Results Compared with the siRNA-NC-HG group or HG treatment group,HMGB1 mRNA level decreased in GMCs in the siRNA-HMGB1-HG group,and after 24-,48-,72-and 96-hour treatment,the proliferation activity and apoptosis rate of GMCs decreased.After knock-down of HMGB1 level of GMCs,the level of NF-κBp65 protein decreased,the level of IκBαprotein increased,and the levels of IL-1β,IL-6 and TNF-αin the supernatant decreased.Conclusion Knockdown of HMGB1 inhibits proliferation and promotes apoptosis of GMCs induced by HG,which may be related to the inhibition of NF-κB/IκB-αpathway.