抑制TGF-β_(1)/Smads通路中Smad7泛素化降解改善小鼠肺纤维化的作用机制  

Mechanism of inhibiting Smad7 ubiquitination and degradation in TGF-β_(1)/Smads pathway to improve pulmonary fibrosis in mice

作　　者：杨珣 邵松军[1,3] 崔妙雅 李霜 张湘燕 饶珊珊[1,3] YANG Xun;SHAO Songjun;CUI Miaoya;LI Shuang;ZHANG Xiangyan;RAO Shanshan(Department of Respiratory and Critical Medicine,Guizhou Provincial People’s Hospital,Guiyang 550002,China;不详)

机构地区：[1]贵州省人民医院呼吸与危重症医学科,贵州贵阳550002 [2]贵州航天医院呼吸与危重症医学科 [3]国家卫生健康委员会肺脏免疫性疾病诊治重点实验室 [4]贵黔国际总医院呼吸与危重症医学科

出　　处：《山东医药》2022年第17期47-51,共5页Shandong Medical Journal

基　　金：中国医学科学院中央级公益性科研院所基本科研业务费专项资金资助项目(2019PT320003);贵州省科技计划项目(黔科合基础2018-1098);贵州省人民医院博士基金项目(GZSYBS-2018-01);贵州省科技计划项目(黔科合支撑2021-一般054);贵州省科技计划项目(黔科合基础-ZK-2021-一般346)。

摘　　要：目的探讨抑制TGF-β_(1)/Smads通路中Smad7泛素化降解改善小鼠肺纤维化的作用机制。方法将30只雄性C57小鼠按随机数表法分为对照组、模型组及MG132干预组,每组10只。模型组及MG132干预组采用一次性气管滴入博来霉素构建肺纤维化模型,空白组小鼠不做处理。MG132干预组于造模第2天起给予Smad7泛素蛋白酶体抑制剂MG132腹腔注射,对照组及模型组给予等量生理盐水腹腔注射。28 d后观察各组小鼠肺纤维化程度(HE染色观察肺组织形态学、Masson染色观察肺组织胶原含量、ELISA检测肺组织羟脯氨酸含量);Western blotting法检测小鼠肺组织α-SMA、Smad7、CollagenⅠ蛋白表达;qRT-PCR法检测小鼠肺组织α-SMA、Smad7、CollagenⅠ、TGF-β_(1) mRNA表达;免疫沉淀Smad7蛋白,免疫印迹检测各组小鼠肺组织中Smad7泛素化水平。结果HE染色结果显示,对照组小鼠肺组织结构完整,模型组小鼠肺组织正常结构遭到破坏,MG132干预组小鼠肺组织结构破坏程度减轻;Masson染色结果显示,对照组镜下蓝紫色面积(胶原纤维沉积)最小,模型组镜下见大片蓝紫色视野,MG132干预组蓝紫色视野面积较模型组缩小;羟脯氨酸含量检测结果显示,小鼠肺组织羟脯氨酸含量模型组>MG132干预组>对照组(P均<0.05)。各组小鼠肺组织α-SMA、CollagenⅠ蛋白表达比较,模型组>MG132干预组>对照组;Smad7蛋白表达比较,对照组>MG132干预组>模型组(P均<0.05)。各组小鼠肺组织α-SMA、Smad7、CollagenⅠ、TGF-β_(1) mRNA表达比较,模型组>MG132干预组>对照组(P均<0.05)。各组小鼠肺组织Smad7泛素化水平比较,模型组>MG132干预组>对照组。结论抑制Smad7泛素化降解可通过上调Smad7蛋白水平增强Smad7对TGF-β1/Smads信号通路的负向调控作用,减轻肺纤维化病变。Objective To investigate the mechanism of inhibiting Smad7 ubiquitination and degradation in TGF-β_(1)/Smads pathway to improve pulmonary fibrosis in mice.Methods Thirty male C57 mice were randomly divided into the control group,model group(BLM group)and intervention group(BLM+MG132 group),with 10 mice in each group.The pulmonary fibrosis model of BLM group and BLM+MG132 group was constructed by dropping BLM(5 mg/kg)into trachea at once.The mice in BLM+MG132 group were intraperitoneally injected with MG132(0.1 mg/kg)once a day from the second day after modeling until the 28th day.After 28 days,mice in each group were killed under anesthesia,and the lungs of mice were taken for further detection.HE staining was used to observe the morphological changes of the lung tissues under light microscope.Masson staining was used to observe the collagen content.The content of hydroxyproline in the lung tissues of mice was detected by ELISA to evaluate the degree of pulmonary fibrosis.Western blotting was used to detect the protein expression levels of α-SMA,Smad7 and Collagen Ⅰ in the lung tissues of mice.The mRNA expression levels of α-SMA,Smad7,Collagen Ⅰ and TGF-β1 in the lung tissues were detected by qRT-PCR.Immunoprecipitation(IP)of Smad7 protein and immunoblotting(IB)were used to detect the ubiquitination level of Smad7 in lung tissues of mice in each group.Results HE staining results showed that the lung tissue of mice in the control group was intact,but the lung tissue of mice in the BLM group was damaged,and the damage degree of lung tissue of mice in the BLM+MG132 group was reduced.Masson staining results showed that the blue-purple area(collagen fiber deposition)under microscope was the smallest in the control group,and large blue-purple field was observed in the BLM group,while the blue-purple field area in the BLM+MG132 group was smaller than that in the model group.The results of hydroxyproline content detection showed that the hydroxyproline content in lung tissues of mice in the BLM group was higher t

关 键 词：肺纤维化 MG132 泛素化 SMAD7 TGF-β_(1)/Smads信号通路 

分 类 号：R563.19[医药卫生—呼吸系统] R363.2[医药卫生—内科学]