雷帕霉素对高糖诱导的肾系膜细胞自噬抑制、氧化损伤和衰老的影响

目的探讨雷帕霉素及3-甲基腺嘌呤(3-MA)对高糖诱导的原代大鼠系膜细胞自噬、氧化损伤及衰老的影响。方法从大鼠肾分离培养原代肾小球系膜细胞(GMCs),分为正常对照组、高糖组、高糖+雷帕霉素(自噬增强剂)及高糖+3-甲基腺嘌呤(自噬抑制剂)干预组。在细胞培养24 h、72 h及10 d时,Western blotting观测自噬标志物-自噬相关基因LC3及泛素结合蛋白p62/SQSTMI的表达变化;硫代巴比妥酸法检测细胞脂质损伤产物-丙二醛(MDA)水平,2,4-二硝基苯肼(DNPH)比色法测定细胞蛋白质损伤产物-羰基含量,通过进行衰老相关β-半乳糖苷酶活性(SA-β-gal)染色及衰老相关异染色质斑块(SAHF)分析检测细胞的衰老程度。结果与对照组相比,细胞经高糖处理72 h和10 d后,自噬标志物LC3表达降低,p62/SQSTMI升高,MDA及羰基含量上升(均P<0.05);处理72 h细胞SA-β-gal染色阳性率增加(P<0.05),细胞核异染色质斑块形成增加。高糖培养的细胞用雷帕霉素干预72 h和10 d后,LC3表达升高,p62/SQSTMI表达下降,MDA及蛋白质羰基含量均下降(均P<0.05),72 h后细胞SA-β-gal染色阳性率下降(P<0.05),细胞核异染色质斑块形成减少;高糖培养的细胞用3-MA干预72 h和10 d后,LC3表达下降,p62/SQSTMI表达升高,MDA及蛋白质羰基含量升高(均P<0.05),72 h细胞SA-β-gal染色阳性率上升(P<0.05),细胞核斑块形成增多。结论高糖可抑制系膜细胞自噬功能,促进系膜细胞氧化损伤,加速细胞衰老;雷帕霉素干预可减轻高糖对系膜细胞自噬功能抑制,减少氧化损伤,延缓衰老;3-MA加重高糖对系膜细胞自噬功能的抑制,进一步加重细胞氧化损伤,加速衰老。

Effect of rapamycin on high glucose-induced autophagy impairment, oxidative stress and premature senescence in rat mesangial cells in vitro

Objective To investigate the effects of rapamycin and 3-methyladenine on autophagy impairment,oxidative stress and premature senescence induced by high-glucose in primarily cultured rat mesangial cells.Methods Rat glomerular mesangial cells(GMCs) were isolated and cultured in normal glucose,high glucose,high glucose with 3-methyladenine(3-MA),or high glucose with rapamycin.At 24 h,72 h and 10 days of culture,the cells were examined for expression levels of autophagy markers LC3 and p62/SQSTM1,malondialdehyde(MDA) and protein carbonyl,β-galactosidase(SA-β-gal) activity and heterochromatin foci(SAHF).Results Compared with those of normal cell culture,the cells exposed to high glucose for 72 h and 10 days showed down-regulated LC3 expression,up-regulated p62/SQSTM1 expression,elevated MDA and protein carbonyl levels,and increased SAHF formation and percentage of SA-β-gal-positive cells.These changes were reversed in GMCs exposed to high glucose and rapamycin for 72 h and 10 days,but exacerbated in cells incubated with 3-MA.Conclusion High glucose can suppress autophagic function of rat GMCs to result in oxidative damage and cell senescence.Rapamycin can attenuate autophagy impairment,oxidative damage and senescence induced by high glucose,whereas 3-MA can further aggravate high glucose-induced cell injuries in rat GMCs.