通过SRC3敲低改善糖尿病肾病肾功能紊乱的实验研究  

Experimental Study on Improvement of Renal Dysfunction in Diabetic Kidney Disease by SRC3 Knockdown

作　　者：周鑫帝 甘淳 陈婉冰 李秋[1] ZHOU Xin-di;GAN Chun;CHEN Wan-bing;LI Qiu(Children’s Hospital of Chongqing Medical University,National Clinical Research Center for Child Health and Disease,Key Laboratory of Child Development and Disorders(Ministry of Education),Chongqing Key Laboratory of Pediatrics,Chongqing 400014,China)

机构地区：[1]重庆医科大学附属儿童医院、国家儿童健康与疾病临床医学研究中心、儿童发育疾病研究教育部重点实验室儿科学重庆市重点实验室,重庆400014

出　　处：《中国生物工程杂志》2023年第7期23-35,共13页China Biotechnology

基　　金：国家自然科学基金(82170720);重庆市自然科学基金(博士后基金)(cstc2021jcyj-bshX0109)资助项目。

摘　　要：目的:研究类固醇受体共激活因子3(steroid receptor coactivator 3,SRC3)参与糖尿病肾病(diabetic kidney disease, DKD)肾功能紊乱及足细胞损伤的分子机制。方法:分析临床DKD肾活检样本SRC3的表达与足细胞损伤的相关性,并通过对C57BL/6及SRC3-/-小鼠注射链脲霉素(streptozotocin, STZ)诱导DKD疾病模型,检测空腹血糖、血清肌酐、血清尿素氮、24 h尿量、尿白蛋白肌酐比以评估肾功能,苏木精-伊红染色法、糖原染色法观察肾小球病理改变,透射电镜观察肾小球超微结构变化;免疫荧光染色及Western blot分析SRC3、足细胞标志物WT1、Podocin、Nephrin、凋亡相关蛋白Caspase3在DKD肾脏中的表达;酶联免疫吸附测定检测足细胞和系膜细胞上清液炎症因子TNF-α、IL-1β的水平。结果:STZ成功诱导了DKD小鼠模型,可以模拟DKD患者肾小球损伤;SRC3在DKD患者及小鼠肾脏中高表达,伴随足细胞标志物表达降低,提示SRC3蛋白表达水平与肾小球损伤程度呈正相关;小鼠敲除SRC3基因能够改善STZ诱导的小鼠肾功能紊乱,缓解足细胞损伤;体外实验也证实了沉默SRC3可缓解高糖环境刺激下足细胞的损伤;同时,敲降SRC3能够减少高糖环境下足细胞与系膜细胞炎症因子的产生。结论:SRC3蛋白在DKD患者、小鼠肾脏以及高糖环境的足细胞中高表达,证实了SRC3与DKD肾小球损伤呈正相关,进一步研究证实,在高糖刺激下SRC3蛋白高表达可促进足细胞与系膜细胞炎症因子产生。Objective:To investigate the mechanism of steroid receptor coactivator 3(SRC3)on renal dysfunction and podocyte injury in the diabetic kidney disease(DKD).Methods:Streptozotocin(STZ)was injected into C57BL/6 mice to induce DKD disease model.Fasting blood glucose(FBG),serum creatinine(Scr),blood urea nitrogen(BUN),24-hour urine and urine albumin creatinine ratio(UACR)were detected to assess renal function.The glomerular pathology change and glomerular ultrastructure were observed by HE,PAS staining and transmission electron microscopy(TEM).Immunofluorescence was used to observe the relationship between SRC3 and glomerular injury in DKD patients and mice,and Western blot was used to detect the expression levels of SRC3,Podocin and WT1 in DKD glomeruli to verify the relationship between SRC3 and DKD mouse.SRC3-/-DKD mouse model was induced by STZ injection,and the effect of SRC3 on kidney injury was investigated by renal function,pathological structure,and ultrastructural changes compared with WT DKD mice.Western blot and immunofluorescence were used to detect the levels of SRC3 and podocyte markers WT1,Podocin and Nephrin in SRC3-/-DKD and WT DKD mice.Next,SRC3-silenced RNA and SRC3-overexpressed podocytes were constructed,and the negative control(NC),high glucose group(HG),high glucose+SRC3 silenced group(HG+si-SRC3),and overexpression of SRC3 group(SRC3OE)were set.Western blot was used to detect the levels of SRC3,WT1,Podocin and apoptosis-related protein Caspase3,and immunofluorescence was used to detect the levels of SRC3 in podocytes under HG environment.To verify the effect of SRC3 on DKD glomerular injury under high glucose environment,all the groups including Normal,HG,HG+si-SRC3 and SRC3OE were set to determine the relationship between SRC3 and the expression of inflammatory cytokines by detecting the levels of TNF-αand IL-1βof supernatant in both podocyte and mesangial cells.Results:Compared with WT group,FBG,BUN and Scr in WT+STZ group were significantly increased(P<0.001,P<0.001,P<0.001).The 24 h urin

关 键 词：类固醇受体共激活因子3 糖尿病肾病 肾功能紊乱 炎症 足细胞损伤 

分 类 号：Q819[生物学—生物工程]