CAJ | 학술논문

目的研究高糖引起足细胞自噬变化及其相关的信号机制.方法培养的足细胞被分为6组,正常浓度葡萄糖(NG)组、高浓度葡萄糖(HG)组、NG+雷帕霉素(Rap)组、HG+Rap组、NG+LY294002组和HG+LY294002组.观察自噬增强剂Rap和PI3K抑制剂LY294002对高糖条件下培养的足细胞自噬和凋亡的影响.电镜和吖啶橙染色观察细胞内自噬体的形成；Western印迹检测自噬标志蛋白微管相关蛋白1轻链3(LC3)和自噬血管基因Beclin-1的表达；通过阻断自噬的信号通路观察磷脂酰肌醇3激酶-蛋白激酶B-哺乳动物雷帕霉素靶蛋白(PI3K-AKT-mTOR)相关蛋白AKT和mTOR的磷酸化水平的改变.结果高糖可导致足细胞凋亡增加,促进足细胞内自噬体和自噬相关蛋白表达增加(均P＜ 0.05).与高糖组相比,HG+ Rap组LC3-Ⅱ和Beclin-1的表达增加(均P＜0.05);LY294002部分抑制高糖导致的LC3-Ⅱ和Beclin-1表达增加(均P＜0.05).与高糖组相比,HG+ LY294002组足细胞内AKT磷酸化的水平增加(P＜0.05),mTOR的磷酸化水平降低(P＜0.01);HG+ LY294002组足细胞的AKT和mTOR磷酸化水平较高糖组均降低(均P＜0.05).结论高糖可促进足细胞的自噬和凋亡,推测高糖诱导的足细胞自噬作用部分通过PI3K-AKT-mTOR信号通路调节实现的.
목적 연구고당인기족세포자서변화급기상관적신호궤제.방법 배양적족세포피분위6조,정상농도포도당(NG)조、고농도포도당(HG)조、NG+뢰파매소(Rap)조、HG+Rap조、NG+LY294002조화HG+LY294002조.관찰자서증강제Rap화PI3K억제제LY294002대고당조건하배양적족세포자서화조망적영향.전경화아정등염색관찰세포내자서체적형성；Western인적검측자서표지단백미관상관단백1경련3(LC3)화자서혈관기인Beclin-1적표체；통과조단자서적신호통로관찰린지선기순3격매-단백격매B-포유동물뢰파매소파단백(PI3K-AKT-mTOR)상관단백AKT화mTOR적린산화수평적개변.결과 고당가도치족세포조망증가,촉진족세포내자서체화자서상관단백표체증가(균P＜ 0.05).여고당조상비,HG+ Rap조LC3-Ⅱ화Beclin-1적표체증가(균P＜0.05);LY294002부분억제고당도치적LC3-Ⅱ화Beclin-1표체증가(균P＜0.05).여고당조상비,HG+ LY294002조족세포내AKT린산화적수평증가(P＜0.05),mTOR적린산화수평강저(P＜0.01);HG+ LY294002조족세포적AKT화mTOR린산화수평교고당조균강저(균P＜0.05).결론 고당가촉진족세포적자서화조망,추측고당유도적족세포자서작용부분통과PI3K-AKT-mTOR신호통로조절실현적.
Objective To evaluate the effects of high glucose on autophagy and apoptosis of podocyte and explore the signaling pathway in high glucose-induce podocyte autophagy.Methods Differentiated mouse podocytes were exposed to high glucose(30 mmol/L) or rapamycin (autophagy enhancer,1 μg/L) or LY294002 (a selective PI3K inhibitor,50 mmol/L) for 24 h.The formations of autophagy were observed by electron microscopy and acridine orange staining.Apoptosis was evaluated by flow cytometry.The expression of autophagy protein LC3-Ⅱ/Ⅰ and Beclin-1 as well as the phosphorylation of AKT and mTOR were examined by Western blotting analysis.Results High glucose induced podocytes apoptosis,increased autophagy and the expression of autophagy-associated proteins (all P ＜ 0.05).Rapamycin further increased the expression of LC3-Ⅱ and Beclin-1 protein (all P ＜ 0.05),but LY294002 inhibited partialiy the protein expression of LC3-Ⅱ and Beclin-1 induced by high glucose (both P ＜ 0.05).Treatment with rapamycin increased the phosphorylation of AKT,but reduced that of mTOR in podocytes.Moreover,LY294002 inhibited phosphorylation of both AKT and mTOR (both P ＜ 0.05).Conclusions High glucose promotes podocyte autophagy and apoptosis.High glucose-induced autophagy is mediated partly through PI3K-AKT-mTOR signaling pathway.