miR-92b-5p通过靶向HMGB1减轻糖尿病肾病大鼠肾损伤和炎症反应  

miR-92b-5p attenuates renal injury and inflammatory response in dia⁃betic nephropathy rats by targeting HMGB1

作　　者：庞欣[1] 王晓蒙 庞欣欣 张建伟[1] 王学敏[1] PANG Xin;WANG Xiao-meng;PANG Xin-xin;ZHANG Jian-wei;WANG Xue-min(Department of Nephrology,The Second Affiliated Hospital,Henan University of Traditional Chinese Medicine,Zhengzhou 450002,China)

机构地区：[1]河南中医药大学第二附属医院肾病科,河南郑州450002

出　　处：《中国病理生理杂志》2020年第9期1639-1646,共8页Chinese Journal of Pathophysiology

基　　金：河南省高等学校重点科研项目计划(No.19A360009);河南省中医院院级课题(No.2018YJKT01)。

摘　　要：目的:探讨微小RNA-92b-5p(miR-92b-5p)对糖尿病肾病(DN)大鼠肾损伤和炎症反应的作用及机制。方法:将大鼠分为对照组、DN组、慢病毒阴性对照(LV-NC)组、LV-miR-92b组、LV-高迁移率族蛋白B1(HMGB1)组和miR-92b+HMGB1组,每组15只。禁食12 h后,模型大鼠腹膜内注射60 mg/kg链脲佐菌素,对照大鼠腹膜内注射等体积柠檬酸盐缓冲液。3 d后,各处理组大鼠分别或联合静脉注射100μL LV-NC、LV-miR-92b和LVHMGB1(1×10^11 U/L),每周2次,连续8周。全自动生化分析仪检测尿蛋白、血糖、血尿素氮和血清肌酐;RT-qPCR检测miR-92b-5p和HMGB1 mRNA的表达;双萤光素酶报告基因实验验证miR-92b-5p和HMGB1的靶向关系;West⁃ern blot法检测肾组织中HMGB1蛋白表达;HE染色观察肾损伤;流式细胞术检测肾细胞凋亡;ELISA检测肾组织中白细胞介素6(IL-6)、IL-1β和肿瘤坏死因子α(TNF-α)的水平。结果:DN模型大鼠中miR-92b-5p低表达,HMGB1高表达;miR-92b-5p与HMGB1的3'端非翻译区存在结合位点,miR-92b-5p高表达显著抑制了含有野生型HMGB1质粒的萤光素酶活性(P<0.01),但对突变型HMGB1质粒的萤光素酶活性无影响。与DN组相比,LV-miR-92b组大鼠24 h尿蛋白、血糖、血尿素氮和血清肌酐水平均显著降低(P<0.01),肾小球系膜和基底膜小管增生、肿胀及炎性细胞浸润程度显著减轻,肾组织细胞凋亡率显著降低(P<0.01),肾组织中IL-6、IL-1β和TNF-α含量均显著减少(P<0.01)。共转染LV-HMGB1可逆转miR-92b-5p对DN大鼠的上述作用。结论:miR-92b-5p通过靶向下调HMGB1来减轻DN大鼠肾损伤和炎症反应。AIM:To investigate the effect of microRNA-92b-5p(miR-92b-5p)on renal injury and inflammatory response in diabetic nephropathy(DN)rats and its mechanism.METHODS:The rats were divided into control group,DN group,lentiviral negative control(LV-NC)group,LV-miR-92b group,LV-high mobility group protein B1(LV-HMGB1)group and miR-92b+HMGB1 group,with 15 rats in each group.After fasting for 12 h,the model rats were intraperitoneally injected with streptozotocin at dose of 60 mg/kg,and the control rats were intraperitoneally injected with an equal volume of citrate buffer.Three days later,the rats in each treatment group were intravenously injected with 100μL LV-NC,LV-miR-92b and LV-HMGB1(1×10^11 U/L)twice a week for 8 consecutive weeks.Urinary protein,blood glucose,blood urea nitrogen and serum creatinine were detected by an automatic biochemical analyzer.The expression of miR-92b-5p and HMGB1 mRNA was detected by RT-qPCR.The targeting relationship between miR-92b-5p and HMGB1 was verified by dual-luciferase reporter assay.HMGB1 expression in kidney tissue was detected by Western blot.The kidney damage was observed by HE staining.The apoptosis was detected by flow cytometry.The levels of interleukin-6(IL-6),IL-1β and tumor necrosis factor-α(TNF-α)in renal tissues were detected by ELISA.RESULTS:In DN model rats,miR-92b-5p was down-regulated,while HMGB1 was highly expressed.There was a binding site between miR-92b-5p and HMGB13'-untranslated region.High expression of miR-92b-5p inhibited the luciferase activity of the wild-type HMGB1 plasmid(P<0.01),but had no effect on the luciferase activity of the mutant HMGB1 plasmid.Compared with DN group,urinary protein,blood glucose,serum creatinine and blood urea nitrogen in LV-miR-92b group were significantly reduced(P<0.01).The degree of hyperplasia,swelling and inflammatory cell infiltration of glomerular mesangium and basement membrane tubules,the apoptosis rate of renal tissues,and the content of IL-6,IL-1β and TNF-α in renal tissues were significantly decreased(P<0.01).

关 键 词：糖尿病肾病 微小RNA-92b-5p 炎症 高迁移率族蛋白B1 

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