新肾病1号方调控miR-199b-5p抑制肾小管上皮间充质转化改善肾纤维化的作用研究  

Improved-Nephropathy Formula 1 inhibits epithelial mesenchymal transformation of renal tubelar and improves renal fibrosis by regulating miR199b-5p

作　　者：曾聪聪[1] 俞文秀 王成功 姜程曦 程锦国[1] ZENG Congcong;YU Wenxiu;WANG Chenggong;JIANG Chengxi;CHENG Jinguo(Department of Traditional Chinese Medicine,The First Affiliated Hospital of Wenzhou Medical University,Wenzhou 325000,China;Wenzhou Medical University,Wenzhou 325000,China;School of Pharmaceutical Sciences,Wenzhou Medical University,Wenzhou 325000,China)

机构地区：[1]温州医科大学附属第一医院中医科,浙江温州325000 [2]温州医科大学,浙江温州325000 [3]温州医科大学药学院,浙江温州325000

出　　处：《中草药》2024年第5期1600-1608,共9页Chinese Traditional and Herbal Drugs

基　　金：浙江省科学技术厅研发攻关计划立项项目(2022C03160);全国名老中医药专家学术传承工作室项目(国中医药办人教函[2021]270号)。

摘　　要：目的研究miR-199b-5p在新肾病1号方抗肾纤维化中的作用。方法36只大鼠随机分为假手术组、模型组及新肾病1号方低、中、高剂量(9.69、19.38、38.76 g/kg)组和氯沙坦(50 mg/kg)组,每组6只。除假手术组外,其余各组采用左侧单侧输尿管梗阻方法建立大鼠肾纤维化模型,给予药物干预14 d后,检测各组大鼠肾功能;苏木素-伊红(HE)和Masson染色观察肾脏组织病理变化;对假手术组、模型组和新肾病1号方高剂量组大鼠肾脏组织进行miRNA测序,筛选差异表达miRNAs,qRT-PCR验证,并进行靶基因的基因本体(gene ontology,GO)功能及京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)通路富集分析,生物信息学分析miRNAs与上皮间质转化(epithelial-mesenchymal transition,EMT)、肾纤维化的关联性。免疫荧光法和Western blotting检测肾组织EMT相关蛋白表达情况。结果与模型组比较,新肾病1号方组大鼠血清肌酐(serum creatinine,SCr)和血清尿素氮(blood urea nitrogen,BUN)水平均明显降低(P<0.05、0.01),肾组织病理变化有所改善。miRNA测序分析发现,miR-199b-5p在模型组表达上调,而在新肾病1号方高剂量组表达下调,qRT-PCR验证了其mRNA相对表达量与测序结果基本一致;miR-199b-5p的靶基因中与EMT、肾纤维化相关功能的90个。与模型组比较,新肾病1号方组肾组织中EMT相关蛋白α-平滑肌肌动蛋白(α-smooth muscle actin,αSMA)和波形蛋白(Vimentin)表达下调(P<0.05、0.001),E-钙黏蛋白(E-cadherin)和紧密连接蛋白-1(Zonula occludens1,ZO-1)表达上调(P<0.05)。结论新肾病1号方可能通过下调miR-199b-5p表达,抑制EMT,从而发挥抗肾纤维化的作用。Objective To investigate the role of miR-199b-5p in the treatment of anti-renal fibrosis of Improved-Nephropathy Formula 1(新肾病1号方,N1F).Methods A total of 36 rats were randomly divided into sham group,model group,N1F low-,medium-and high-dose(9.69,19.38,38.76 g/kg)groups and losartan(50 mg/kg)group,with six rats in each group.Except the sham group,renal fibrosis model was established by left unilateral ureteral obstruction method in the other groups.After 14 d of drug intervention,renal function was detected in each group of rats.The pathological changes of renal tissue were observed by hematoxylin-eosin(HE)and Masson staining.Renal tissues of rats in sham group,model group and N1F high-dose group were sequenced by miRNA,screened for differentially expressed miRNAs and verified by qRT-PCR,gene ontology(GO)function and Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analysis of target genes were performed.The correlation between miRNAs and epithelialmesenchymal transition(EMT)and renal fibrosis was analyzed by bioinformatics.The expressions of EMT-related proteins in renal tissue were detected by immunofluorescence and Western blotting.Results Compared with model group,serum creatinine(SCr)and blood urea nitrogen(BUN)levels were significantly decreased(P<0.05,0.01),the pathological changes of renal tissue were improved in N1F groups.miRNA sequencing analysis showed that the expression of miR-199b-5p was up-regulated in model group,but downregulated in N1F high-dose group.qRT-PCR verified that the relative mRNA expression level was basically consistent with the sequencing results.A total of 90 target genes in miR-199b-5p were related to EMT and renal fibrosis.Compared with model group,the expressions of EMT-related proteinsα-smooth muscle actin(α-SMA)and Vimentin in renal tissue were down-regulated(P<0.05,0.001),but the expressions of E-cadherin and Zonula occludens-1(ZO-1)were up-regulated in N1F groups(P<0.05).Conclusion N1F may inhibit EMT by down-regulating the expression of miR-199b-

关 键 词：新肾病1号方 上皮间充质转化 肾纤维化 RNA测序 miR-199b-5p 柴胡皂苷 黄芪甲苷 盐酸小檗碱 

分 类 号：R285.5[医药卫生—中药学]