摘要
目的探讨miR-34a介导Notch信号通路对糖尿病肾病(DN)足细胞损伤及凋亡的影响。方法体外实验:通过RT-PCR法检测高糖(30 mmol/L)环境下足细胞中miR-34a表达水平,构建miR-34a过表达足细胞系(miR-34a组)及其阴性对照(miR-NC组),使用荧光素酶实验验证miR-34a与Notch 1的靶向关系,构建Notch 1过表达足细胞系(Notch 1组)和miR-34a、Notch 1均表达升高细胞系(miR-34+Notch 1组);采用CCK-8法检测足细胞存活情况,流式细胞法检测细胞凋亡情况,Western blot法检测细胞凋亡关蛋白水平。体内实验:通过高脂饮食和链脲佐菌素建立DN小鼠模型并分为模型组、miR-34a组(n=15/组),另选15只不做干预小鼠为对照组,miR-34a组和模型组小鼠分别尾静脉注射agomir-34a[80 mg/(kg·d)]、agomir-NC[80 mg/(kg·d)],连续注射3 d,4周后,HE染色、TUNEL法分别观察肾组织病理、凋亡情况,Western blot检测肾组织凋亡相关蛋白和Notch 1蛋白表达水平。结果高糖环境下足细胞中miR-34a表达水平低于正常糖诱导下足细胞和高渗透压下作用下的足细胞(P<0.05),miR-34a组足细胞中Notch 1表达水平低于miR-NC组(P<0.05);Notch 1 wt/miR-34a组荧光素酶活力值低于Notch 1 wt组(P<0.05),而Notch 1 mut/miR-34a组和Notch 1 mut/miR-NC组荧光素酶活力值差异无统计学意义(P>0.05);miR-34a组足细胞的A值高于miR-NC组(P<0.05),而细胞凋亡率和caspase-3、caspase-9、Bax/Bcl-2蛋白水平均低于miR-NC组(P<0.05);Notch 1组足细胞的A值低于miR-NC组(P<0.05),而细胞凋亡率和caspase-3、caspase-9、Bax/Bcl-2蛋白水平均高于miR-NC组(P<0.05);miR-34a+Notch 1组足细胞的A值低于miR-34a组(P<0.05),而细胞凋亡率和caspase-3、caspase-9、Bax/Bcl-2蛋白水平均高于miR-34a组(P<0.05)。对照组小鼠肾组织结构清晰完整,模型组小鼠肾小球肿胀、体积增大,miR-34a组小鼠肾组织病变程度得到改善;模型组小鼠肾组织miR-34a表达水平低于对照组(P<0.05),miR-34a组小鼠的肾组织miR-34a表达水平高于对照组(P<0.05);模型组小鼠肾组织凋亡指数和caspase-3、caspase-9、Notch 1蛋白水平高于对照组(P<0.05),miR-34a组小鼠肾组织凋亡指数和caspase-3、caspase-9、Notch 1蛋白水平低于模型组(P<0.05)。结论miR-34a可通过靶向作用Notch 1改善DN足细胞损伤和凋亡。
Objective To explore the effects of miR-34a on injury and apoptosis of podocytes in diabetic nephropathy(DN)and the role of Notch signaling pathway in mediating its effects.Methods The expression of miR-34a in podocytes exposed to high glucose(30 mmol/L)was detected using RT-PCR.A podocyte line with miR-34a overexpression was constructed,and the miRNA-target relationship between miR-34a and Notch 1 was verified with luciferase assay.The effects of overexpression of Notch 1 and both miR-34a and Notch 1 on podocyte survival and apoptosis were evaluated using CCK-8 and flow cytometry and by detecting apoptosis-related proteins using Western blotting.In a DN mouse model established by high-fat diet and streptozotocin,the effect of tail vein injection of agomir-34a and agomir-NC on pathology and apoptosis in the renal tissues were observed with HE staining and TUNEL staining,and the renal expressions of apoptosis-related proteins and Notch 1protein were detected with Western blotting.Results High glucose exposure significantly lowered miR-34a expression in cultured human podocytes(P<0.05).The expression of Notch 1 was significantly lowered in miR-34a-overexpressing podocytes as compared with the cells with miR-NC transfection(P<0.05).Luciferase assay confirmed the mRNA-target relationship between miR-34a and Notch 1(P<0.05).MiR-34a overexpression obviously promoted podocyte survival(P<0.05),reduced Notch 1 expression,and lowered apoptosis rate and the protein expressions of caspase-3,caspase-9 and Bax/Bcl-2levels in the cells(P<0.05),while the reverse changes were observed in Notch 1-overexpressing podocytes(P<0.05).In DN mouse models,treatment with miR-34a obviously alleviated renal pathologies.Compared with that in the control group,the expression level of miR-34a in the renal tissues was significantly lowered in DN model group(P<0.05)and increased in miR-34a group(P<0.05).The mice in the model group showed significantly higher apoptosis index of the renal tissues with increased expressions of caspase-3,caspase-9 and Notch 1(P<0.05),which were lowered by treatment with miR-34a(P<0.05).Conclusion MiR-34a is capable of improving podocyte injury and apoptosis in DN mice by targeted downregulation of Notch 1.
作者
王欢岚
刘红
张燕敏
陈伟栋
WANG Huanlan;LIU Hong;ZHANG Yanmin;CHEN Weidong(Department of Nephrology,Wuhan First Hospital(Wuhan Hospital of Integrated Traditional Chinese and Western Medicine),Wuhan 430022,China)
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2022年第12期1839-1845,共7页
Journal of Southern Medical University
基金
湖北省卫生健康委员会中医药科研项目(ZY2019Q024)。
