青藤碱阻断miR-21/ADAMTS-1信号通路改善博莱霉素A5诱导的大鼠肺纤维化  

Sinomenine ameliorates bleomycin A5-induced pulmonary fibrosis by blocking the miR-21/ADAMTS-1 signaling pathway in rats

作　　者：刘理静 钱红 孟庆欣 张翔 魏英民 贺兼斌 LIU Ljing;QIAN Hong;MENG Qingxin;ZHANG Xiang;WEI Yingmin;HE Jianbin(Department of Clinical Medicine,School of Medicine,Changsha Social Work College,Changsha 410004;Department of Respiratory and Critical Care Medicine,First People's Hospital of Huaihua,The Fourth Affiliated Hospital of Jishou University,Huaihua 418000,China)

机构地区：[1]长沙民政职业技术学院医学院临床医学系,湖南长沙410004 [2]吉首大学附属第四医院,怀化市第一人民医院呼吸与危重症医学科,湖南怀化418000

出　　处：《细胞与分子免疫学杂志》2023年第8期721-728,共8页Chinese Journal of Cellular and Molecular Immunology

基　　金：湖南省教育厅资助科研项目(21C1593);长沙民政职业技术学院教授、博士科研项目(2020JB11)。

摘　　要：目的 探讨青藤碱对博莱霉素A5诱导的大鼠肺纤维化(PF)的影响及分子机制。方法 培养MRC-5细胞,采用噻唑蓝(MTT)法检测细胞增殖活性,以确定青藤碱最佳处理浓度与时间,选取80μmol/L青藤碱处理MRC-5细胞48 h,或者miR-21模拟物(miR-21 mimic)或1型血小板应答蛋白基序的解整联蛋白样金属蛋白酶1(ADAMTS-1)小干扰RNA转染MRC-5细胞后,再用青藤碱处理,采用实时荧光定量PCR, Western blot法分别检测miR-21、 ADAMTS-1、 1型胶原蛋白(Col1)和3型胶原蛋白(Col3)表达。将SD大鼠随机分为对照组、青藤碱组和青藤碱联合miR-21激动剂(miR-21 agomir)组,每组10只。通过博莱霉素A5一次性注入大鼠气管内构建PF模型,分别给予9 g/L氯化钠溶液、青藤碱、青藤碱联合miR-21 agomir处理,28 d处死大鼠,分离肺组织行HE和Masson染色,实时荧光定量PCR、 Western blot法检测ADAMTS-1、 Col1和Col3表达,ELISA测定血清1型前胶原羧基端前肽(P1CP)和3型前胶原氨基端前肽(P3NP)水平。结果 青藤碱下调MRC-5细胞miR-21表达,升高ADAMTS-1水平,促进Col1和Col3降解,干扰miR-21/ADAMTS-1信号通路部分逆转青藤碱对Col1、 Col3降解的促进作用。青藤碱减少SD大鼠肺泡炎与PF评分,降低血清P1CP、 P3NP水平,上调肺组织ADAMTS-1表达,下调Col1、 Col3表达,这些作用被miR-21 agomir逆转。结论 青藤碱阻断miR-21/ADAMTS-1途径促进Col1和Col3降解,抑制大鼠PF。Objective To explore the impact of sinomenine on bleomycin A5-induced pulmonary fibrosis(PF)in rats and the underlying mechanism.Methods MRC-5 cells were cultured and treated with sinomenine to determine its optimal concentration and time through the MTT assay.Subsequently,MRC-5 cells were incubated with 80μmol/L sinomenine for 48 hours or transfected with miR-21 mimic/a disintegrin-like and metalloproteinase with thrombospondin type 1 motif(ADAMTS-1)siRNA prior to sinomenine treatment.The expression of miR-21,ADAMTS-1,collagen type 1(Coll)and collagen type 3(Col3)was detected by quantitative real-time PCR(qRT-PCR)and/or Western blot analysis.Thirty SD rats were randomly divided into control group,sinomenine group and sinomenine combined with miR-21 agomir group,with 10 animals in each group.Bleomycin A5 were intratracheally administered to establish the PF model.Then,rats in control group,sinomenine group and sinomenine+miR-21 agomir group were treated with 9 g/L sodium chloride solution,sinomenine and sinomenine+miR-21 agomir,respectively.On day 28,all rats were sacrificed.HE and Masson staining was performed in pulmonary tissue.The expression of ADAMTS-1,Coll and Col3 in pulmonary tissue were detected by qRT-PCR and/or Western blot analysis.ELISA was used to measure serum procollagen type 1 carboxyterminal propeptide(P1CP)and procollagen type 3 aminoterminal propeptide(P3NP)levels.Results Administration of sinomenine decreased miR-21 levels,up-regulated ADAMTS-1 expression,and promoted Coll and Col3 degradation in MRC-5 cells.Importantly,interfering with the miR-21/ADAMTS-1 signaling pathway partially reversed the promotive effect of sinomenine on Coll and Col3 degradation.Treatment of SD rats with sinomenine reduced alveoltis and PF scores,decreased serum PICP and P3NP levels,up-regulated pulmonary ADAMTS-1 expression,and down-regulated Coll and Col3 expression.However,these effects were reversed by miR-21 agomir.Conclusion Sinomenine promotes Coll and Col3 degradation and inhibits PF in rats by miR-21/ADAMT

关 键 词：青藤碱 肺纤维化 微小RNA21(miR-21) 1型血小板应答蛋白基序的解整联蛋白样金属蛋白酶1(ADAMTS-1) 1型胶原蛋白(Col1) 3型胶原蛋白(Col3) 

分 类 号：R285[医药卫生—中药学] R392.5[医药卫生—中医学] R563R441.8