糖尿病肾病通过转录激活因子3诱导小鼠足细胞凋亡机制

Activating transcription factor 3 downregulate YAP induced podocyte apoptosis

目的探究高糖刺激下转录激活因子3(ATF3)在足细胞中的凋亡效应及其可能的分子机制。方法通过免疫荧光染色及激光共聚焦观察糖尿病db/db小鼠模型肾组织足细胞中ATF3表达情况;体外培养足细胞分化成熟后,用高糖(HG)培养基(30 mmol/L)分别培养足细胞0、1、2、4、6 h,通过荧光定量PCR和Western blot检测足细胞中ATF3的表达;过表达足细胞ATF3后,通过荧光定量PCR及Westernblot检测过表达效率,通过Annexin V-APC/V450双染色检测细胞凋亡情况,荧光定量PCR及Western blot检测凋亡相关因子BAX、Bcl-2的变化;免疫荧光染色和激光共聚焦观察HG诱导足细胞核内ATF3表达,及通过Western blot检测核蛋白ATF3在HG刺激下表达变化;荧光定量PCR及Western blot检测YAP表达情况。结果糖尿病db/db小鼠肾组织足细胞中ATF3表达增加;HG干预下,足细胞ATF3表达逐渐升高,2 h增高最明显,随后下调,6 h基本恢复基线水平;过表达足细胞ATF3后,足细胞凋亡比率增加,凋亡相关因子BAX上调,Bcl-2下调;HG干预后诱导足细胞核内ATF3表达增多;过表达ATF3后,足细胞YAP表达下调。结论高糖诱导足细胞ATF3表达增多,增多的ATF3通过下调足细胞凋亡生理拮抗剂Yes相关蛋白(YAP)介导足细胞凋亡。

Objective To explore the role and mechanism of activating transcription factor 3(ATF3) in HG-treated podocyte apoptosis. Methods Laser confocal microscopy and Western blotting were used to observed the expression of ATF3 in the db/db mouse of diabetic kidney disease. The conditionally immortalized mouse podoyte cell line was cultured in vitro and exposed to HG(30 mmol/L) for different time points. Real-time quantity PCR and Western blotting were used to analyze the expression of ATF3; Cell apoptosis in ATF3 over-expression podocyte was observed by flow cytometry. Real-time quantity PCR and Western blotting were used to analyze the expression of BAX and Bcl-2. Immunofluorescent staining and Western blotting were used to evaluate the change of nuclear localization of ATF3. After podocyte overexpression ATF3, real-time quantity PCR and Western blotting were used to analyze the change of YAP. Results The expression of ATF3 was up-regulated in the podocyte of db/db mouse. ATF3 mRNA and protein increased in HG-treated pdocytes for 1,2,4 hour, and ATF3 activation peaked at 2 hours and diminished 6 hours. After overexpression of ATF3, the podocyte apoptosis ratio was up-regulated, BAX mRNA and protein were also up-regulated, Bcl-2 mRNA and protein were down-regulated. The nuclear localization of ATF3 increased in HG-treated podocyte; YAP mRNA and protein were down-regulated in overexpression ATF3 podocyte. Conclusion HG up-regulate ATF3 expression, and ATF3 could downregulate YAP to induce podocyte apoptosis.