乌索酸增强自噬改善高糖诱导的足细胞损伤  

Attenuation of high glucose induced podocyte injury by ursolic acid up- regulating autophagy level

作　　者：徐莉[1] 范秋灵[1] 汪旭[1] 李琳[1] 卢新星[1] 岳媛[1] 曹旭[1] 刘佳[1] 赵雪[1] 王力宁[1] 

机构地区：[1]中国医科大学附属第一医院肾内科,沈阳110001

出　　处：《中华肾脏病杂志》2015年第11期820-827,共8页Chinese Journal of Nephrology

基　　金：基金项目：国家自然科学基金（81270808）;辽宁省科技厅社会发展攻关计划（2012225019）;辽宁省高等学校重大科技平台免疫皮肤病学重点实验室自主创新课题基金（201303）

摘　　要：目的探讨乌索酸是否通过改善高糖状态下的足细胞白噬抑制，发挥其肾脏保护作用。方法体外高糖培养小鼠条件永恒足细胞株，加入P13K抑制剂LY294002及乌索酸进行干预。RT．qPCR法检测细胞内微小RNA一21（miR．21）和磷酸酶基因（PTEN）的mRNA表达。Western印迹法检测磷脂酰肌醇-3激酶（P13K）／蛋白激酶B（Akt）／哺乳动物雷帕霉素靶蛋白（mTOR）信号通路相关蛋白，白噬相关蛋白及足细胞标志蛋白的表达变化。采用荧光显微镜观察足细胞标志蛋白、内源性微管相关蛋白LC3的表达变化。透射电镜下观察足细胞内白噬体的形成。结果与对照组相比，高糖组足细胞自噬相关蛋白LC3II、Beclinl的表达降低，泛素结合蛋白p62（p62／SQSTMl）表达增高；足细胞标志蛋白synaptopodin、podocin及nephrin表达降低；同时伴miR-21表达上调，PTEN的mRNA和蛋白表达均下调，p85-P13K、磷酸化（P）-Akt、p-mTOR表达增加（均P〈0．01）。加入LY294002及乌索酸干预后，p85-P13K、磷酸化（P）-Akt、p-mTOR表达减少；足细胞自噬相关蛋白LC3II、Beclin1的表达增加，p62／SQSTM1表达降低；足细胞标志蛋白synaptopodin、podocin及nephrin表达降低（均P〈0．01），但LY294002对足细胞内miR-21和PTEN的表达无影响。乌索酸可抑制细胞内miR-21的过表达，上调PTEN表达（均P〈0．05）。结论高糖可抑制足细胞自噬，促进足细胞损伤。乌索酸干预可减轻高糖刺激下的足细胞自噬抑制，缓解足细胞损伤，其保护机制可能是通过抑制足细胞内miR-21过表达，上调PTEN表达，抑制P13K／Akt—mTOR信号通路的异常活化实现的。Objective To investigate the effects of ursolic acid （UA） on autophagy and podocyte injury induced by high glucose. Methods Conditionally immortalized murine podocyte were cultured in high glucose, the effect of PI3K inhibitor LY294002 and ursolic acid treatment were observed. The miR- 21 expression was detected using RT- qPCR. The activation of PTEN-PI3K/Akt/ mTOR pathway, expression of autophagy-related protein and podocyte marker protein were determined by Western blot. Immunofluorescence staining showed the expression of podocyte marker protein and endogenous accumulation of LC3. Autophagosomes were observed using electron microscopy. Results Compared with normal control group, the cells exposed to high glucose condition showed down- regulated synaptopodin, podocin and nephrin expression （P 〈 0.01）, up-regulated miR-21 expression （P 〈 0.01）, down-regulated PTEN expression （P 〈 0.01）, up-regulated p85-P13K, phospho（p）-Akt, p-mTOR, p62/SQSTMI, expression and down-regulated LC3II and Beclinl expression （all P 〈 0.01）. Ursolic acid and LY294002 promoted synaptopodin, podocin and nephrin expression （all P 〈 0.01）, up- regulated LC3II, Beclinl expression and down-regulated p62/SQSTM1 expression （all P 〈 0.01）, down- regulated p85-PI3K, p-Akt, p-mTOR expression （all P 〈 0.01）. However, LY294002 did not affect the expression of miR- 21 and PTEN. Ursolic acid inhibited miR-21 expression and upregulated PTEN level. Conclusions The podocyte injury is associated with defective autophagy level under high glucose condition. Ursolic acid could reduce podocyte injury by increasing autophagy level via inhibition of miR-21 expression and PTEN/Akt/mTOR pathway.

关 键 词：高血糖症 足细胞 自噬 乌索酸 MIR-21 磷酸酶基因 

分 类 号：R692[医药卫生—泌尿科学]