活性维生素D对高糖诱导的足细胞自噬相关蛋白Beclin-1和空泡分选蛋白34的调控作用研究

Regulatory effect of 1α,25（OH）2D3 on high glucose-induced podocyte autophagy associated proteins Beclin-1 and Vps34

目的探讨活性维生素D(VitD)对高糖诱导的足细胞自噬相关蛋白Beclin⁃1和空泡分选蛋白34(Vps34)的调控作用。方法(1)体外培养永生化小鼠足细胞,用高糖30 mmol/L培养0、3、6、12、24、36、48 h后,采用Western blot检测微管相关蛋白1轻链3Ⅱ(LC3⁃Ⅱ)、Beclin⁃1和Vps34的蛋白表达,激光共聚焦观察足细胞骨架蛋白Synatopodin、微管相关蛋白1轻链3(LC3)的表达变化;(2)1α,25二羟维生素D3[1α,25(OH)2D3](1、10、100 nmol/L)和高糖(HG,30 mmol/L)培养足细胞48 h,激光共聚焦观察Synatopodin、LC3⁃Ⅱ的表达变化,透射电镜下观察足细胞内自噬体的形成,Western blot及RT⁃qPCR半定量检测LC3⁃Ⅱ、Beclin⁃1和Vps34的蛋白和mRNA表达;(3)加入自噬抑制剂3⁃甲基腺嘌呤(3⁃MA,10 mmol/L)作用48 h后,Western blot检测LC3⁃Ⅱ、Beclin⁃1和Vps34的蛋白表达。结果足细胞经高糖处理后LC3⁃Ⅱ表达增强,Beclin⁃1和Vps34的蛋白和mRNA水平也逐渐升高,24 h达高峰,随后表达逐渐降低(P<0.05)。与高糖组相比,高糖+1α,25(OH)2D3(HG+VitD)组Beclin⁃1、Vps34蛋白和mRNA的表达呈浓度依赖性上调,抑制高糖长时刺激导致的自噬活性下降(P<0.05);与HG+VitD组相比,HG+VitD+3⁃MA组足细胞Beclin⁃1、Vps34蛋白和mRNA的表达明显降低(P<0.05)。结论足细胞在高糖刺激下早期自噬活性增强,长期刺激自噬抑制;1α,25(OH)2D3可能是一种自噬活化剂,通过调控自噬相关蛋白Beclin⁃1、Vps34水平,促进自噬溶酶体形成,改善高糖诱导的足细胞损伤。

Objective To explore the role of 1α,25(OH)2D3 on Beclin⁃1 and Vps34 of podocyte au⁃tophagy induced by high glucose.Methods(1)The immortalized mouse podocytes were cultured in vi⁃tro,and the expressions of LC3⁃II,Beclin⁃1 and Vps34 were detected by Western blot after culture for 0,3,6,12,24,36 and 48 h with high glucose 30 mmol/L.Laser confocal microscopy was used to observe the expres⁃sions of synatopodin and LC3⁃Ⅱ;(2)1α,25(OH)2D3(1 nmol/L,10 nmol/L,100 nmol/L)and high glu⁃cose(30 mmol/L)co⁃cultured podocytes for 48 h and observed the expressions of synatopodin and LC3.Au⁃tophagosomes were identified by transmission electron microscopy.The protein and mRNA expressions of LC3⁃II,Beclin⁃1 and Vps34 were detected by Western blot and RT⁃qPCR;(3)The protein expressions of LC3⁃II,Beclin⁃1 and Vps34 were detected by Western blot after adding autophagy inhibitor 3⁃MA(10 mmol/L).Results The expression of LC3⁃Ⅱ,Beclin⁃1 and Vps34 peaked at 24 hours after high glucose treatment and then decreased gradually(P<0.05).The levels of Beclin⁃1,Vps34 protein significantly increased in a dose⁃dependent manner in podocytes after treatment with 1α,25(OH)2D3 when compared with the high glucose group,which inhibited the decrease of autophagic activity induced by the stimulation of high glucose for a long time(P<0.05).In comparison with HG+VitD group,the cells exposed to HG+VitD+3⁃MA group showed down⁃regulated Beclin⁃1 and Vps34 expression(P<0.05).Conclusion The podocytes show ear⁃ly increased autophagy activity and long⁃term inhibition under high glucose stimulation.1α,25(OH)2D3 may be a kind of autophagic activators,which can promote autophagic lysosome formation and improve high glu⁃cose⁃induced autophagy inhibition by regulating the levels of autophagic associated protein Beclin⁃1 and Vps34.