miR-21通过下调PPAR-α参与脂质代谢紊乱并促进糖尿病大鼠肾组织及肾小管上皮细胞纤维化病变  

Pathogenic role of microRNA-21 in lipid metabolism disorder to promote fibrotic lesions in renal tissues and tubular epithelial cells of diabetic rats through downregulation of PPAR-α

作　　者：向珈谊 张会芳 梁露群 周星丞 王丹 毛彦稳 张小欢 王圆圆 郭兵 XIANG Jia-yi;ZHANG Hui-fang;LIANG Lu-qun;ZHOU Xing-cheng;WANG Dan;MAO Yan-wen;ZHANG Xiao-huan;WANG Yuan-yuan;GUO Bing(Department of Pathophysiology,Guizhou Medical University,Guizhou Provincial Key Laboratory of Pathogenesis and Drug Research on Common Chronic Diseases,Guiyang 550025,China)

机构地区：[1]贵州医科大学基础医学院病理生理教研室,贵州省常见慢性疾病发病机制及药物研究重点实验室,贵州贵阳550025

出　　处：《中国病理生理杂志》2021年第10期1858-1867,共10页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金项目(No.81760131,No.81860135);贵州省科技创新平台计划(黔科中引地[2019]4008号)。

摘　　要：目的:探讨微小RNA-21(microRNA-21,miR-21)下调过氧化物酶体增殖物激活受体α(peroxisome proliferators-activated receptor-α,PPAR-α)参与脂质代谢紊乱从而促进糖尿病(diabetes mellitus,DM)大鼠肾组织及大鼠肾小管上皮细胞纤维化病变的机制。方法:12只SD大鼠随机均分为正常对照(normal control,NC)组和DM组。DM组大鼠腹腔注射链脲佐菌素复制1型糖尿病模型。于模型复制成功后第10周处死大鼠,收集各组大鼠血清和处死前24 h的尿液检测生化指标,HE及天狼星红染色观察肾脏组织病理改变和纤维化病变;real-time PCR检测miR-21及PPAR-αmRNA在肾组织中的表达;ELISA检测白细胞介素18(interleukin-18,IL-18)的表达水平。以高糖培养的肾小管上皮细胞(NRK52E)为研究对象,敲减miR-21的表达或给予PPAR-α特异性激动剂非诺贝特干预,采用流式细胞术检测非诺贝特干预后细胞凋亡情况。Western blot检测各组肾脏组织及NRK52E细胞中PPAR-α、IL-6、Bax、纤连蛋白(fibronectin,FN)、转化生长因子β1(transforming growth factor-β1,TGF-β1)和α-平滑肌肌动(α-smooth muscle motility,α-SMA)蛋白的表达。双萤光素酶报告基因实验验证miR-21对PPAR-α的转录调控作用。结果:与NC组比较,DM组大鼠血糖、甘油三酯、总胆固醇和24 h尿蛋白均升高(P<0.05);HE和天狼星红染色结果显示DM组肾小管间质纤维化增加,胶原阳性染色增多,胶原容积分数(collagen volume fraction,CVF)升高(P<0.05);DM组肾组织miR-21表达显著升高,PPAR-αmRNA和蛋白的表达显著减少(P<0.05),IL-18水平及Bax、IL-6、TGF-β1、α-SMA和FN蛋白的表达增多(P<0.05)。双萤光素酶报告基因实验表明,miR-21模拟物可以显著抑制PPAR-α的转录活性,同时下调其蛋白表达水平(P<0.05);miR-21抑制物可逆转高糖对PPAR-α蛋白的抑制作用,并降低FN、IL-6和Bax蛋白的表达(P<0.05);与溶剂对照组相比,非诺贝特可降低高糖诱导的细胞凋亡(P<0.05)。结论:�AIM:To explore the mechanism by which microRNA-21(miR-21)down-regulates peroxisome proliferators-activated receptor-α(PPAR-α)is involved in lipid metabolism disorders to promote fibrotic lesions in diabetic rat kidney tissue and rat renal tubular epithelial cells.METHODS:SD rats were randomly divided into normal control(NC)group and diabetes meuitus(DM)group,with 6 rats in each group. After fasting for 12 h,the type one diabetes mellitus(T1 DM)model of rat was established by intraperitoneal injection of streptozocin(STZ). The rats were sacrificed at the 10 th week after the successful replication of the model. Serum and urine samples(24 h)were collected to detect biochemical indexes. The pathological changes and fibrosis of renal tissues were observed by HE and Sirius red staining. The expression of miR-21 and PPAR-α mRNA in renal tissues was detected by real-time PCR. The protein level of interleukin-18(IL-18)was measured by ELISA. High glucose cultured renal tubular epithelial cells(NRK52 E)were used as the study subjects,and the expression of miR-21 was knocked down or fenofibrate,a PPAR-α specific agonist,was administered. Apoptosis was analyzed by flow cytometry after fenofibrate intervention. Western blot was performed to detect the protein expression of PPAR-α,IL-6,Bax,fibronectin(FN),transforming growth factor-β1(TGF-β1),and α-smooth muscle actin(α-SMA)in the renal tissues and NRK52 E cells of different groups. Dual luciferase reporter assay was used to verify the role of miR-21 on PPAR-α transcriptional regulation.RESULTS:Compared with NC group,blood glucose,triglyceride,total cholesterol and 24 h urinary protein were all increased in DM group(P<0. 05). HE and Sirius red staining showed increased tubulointerstitial fibrosis and increased collagen positive staining,with increased collagen volume fraction(CVF)in DM group(P<0. 05). miR-21 expression in DM group was significantly increased(P<0. 05). The mRNA and protein expression of PPAR-α were significantly decreased(P<0. 05). The protein expres

关 键 词：糖尿病肾病 微小RNA-21 过氧化物酶体增殖物激活受体Α 白细胞介素6 细胞凋亡 

分 类 号：R587.2[医药卫生—内分泌] R363.2[医药卫生—内科学]