艾拉莫德经Smad3/p300通路抑制转化生长因子β_(1)介导的人肺成纤维细胞活化及胶原分泌  

Iguratimod inhibits transforming growth factor-β_(1) induced human lung fibroblast activation and collagen secretion via the Smad3/p300 pathway

作　　者：朱富 冯媛[2] 林浩博[2] 张光峰[2] 张晓[2] Zhu Fu;Feng Yuan;Lin Haobo;Zhang Guangfeng;Zhang Xiao(School of Medicine,South China University of Technology,Guangzhou 510006,China;Department of Rheumatology,Guangdong Provincial People's Hospital,Guangzhou 510080,China)

机构地区：[1]华南理工大学医学院,广州510006 [2]广东省人民医院风湿免疫科,广州510080

出　　处：《中华风湿病学杂志》2021年第11期721-726,I0002,I0003,共8页Chinese Journal of Rheumatology

基　　金：国家自然科学基金(81771734);广东省医学科研基金(A2019139);广州市科技计划(201804010343)。

摘　　要：目的研究艾拉莫德是否能够抑制TGF-β_(1)诱导的人肺成纤维细胞(HLFs)活化及胶原分泌。方法在体内,建立小鼠肺纤维化模型,分为3组:正常对照组、博莱霉素组和博莱霉素+艾拉莫德组,苏木精-伊红(HE)染色观察肺形态,马松染色观察肺内胶原积累程度,组织免疫荧光检测肺内肌成纤维细胞标志蛋白纤维连接蛋白(fibronectin)和平滑肌22蛋白(SM22),氯胺-T法检测肺组织羟脯氨酸含量。在体外,用原代人肺成纤维细胞(pHLFs)进行细胞实验评估艾拉莫德对TGF-β_(1)刺激的影响,分为4组:正常对照组、艾拉莫德组、TGF-β_(1)组、TGF-β_(1)+艾拉莫德组,流式细胞术检测细胞的凋亡情况,实时荧光定量(qRT)-PCR检测细胞Ⅰ型胶原蛋白和Ⅲ型胶原蛋白的mRNA表达水平,蛋白质印迹法和细胞免疫荧光检测细胞α-平滑肌肌动蛋白(α-SMA)、fibronectin、p-Smad2和p-Smad3以及转录辅助激活因子p300的蛋白水平。数据均采用单因素方差分析,两两比较用LSD-t检验或Kruskal-Wallis检验。结果羟脯氨酸含量:正常对照组为(0.552±0.075)μg/mg,博莱霉素组为(1.293±0.081)μg/mg,博莱霉素+艾拉莫德组(0.833±0.053)μg/mg(F=169.672,P<0.01),艾拉莫德降低了小鼠肺组织羟脯氨酸含量,减少了肺内胶原积累,从而降低了博莱霉素诱导的肺纤维化程度,改善了小鼠肺部病理改变。在细胞实验中,艾拉莫德对细胞凋亡比例差异无统计学意义(F=0.83,P=0.54);Ⅰ型胶原蛋白mRNA相对表达量:正常对照组为(100.4±1.2),TGF-β_(1)组为(299.0±13.0),TGF-β_(1)+艾拉莫德组(202.5±7.0)(F=468.7,P<0.01);Ⅲ型胶原蛋白mRNA相对表达量:正常对照组为(99.8±1.9),TGF-β_(1)组为(350.6±8.0),TGF-β_(1)+艾拉莫德组(220.3±9.9)(F=468.7,P<0.01);α-SMA蛋白相对表达量:正常对照组为(0.193±0.038),TGF-β_(1)组为(0.530±0.061),TGF-β_(1)+艾拉莫德组为(0.410±0.065)(F=35.620,P<0.01);Fibronectin蛋白相对表达量:正常对照组为(0.200±0.020),TObjective To investigate the effect of iguratimod(IGU)on transforming growth factor-β_(1)(TGF-β_(1))-induced primary human lung fibroblasts(pHLFs)activation and collagen secretion.Methods Mice pulmonary fibrosis(PF)models were established in vivo and were divided into three groups:the control group(CTR group),the Bleomycin(BLM)group and the BLM+IGU group,hematoxylin-eosin(HE)staining was used to observe lung morphology,and Masson staining was used to observe the degree of collagen accumulation in lung.Fibronectin and smooth muscle 22(SM22)were detected by immunofluorescence,and the content of hydroxyproline in lung tissue was detected by chloramine-T method.In vitro,pHLFs were used to assess the effect of IGU on TGF-β_(1) stimulation in four groups:CTR group,IGU group,TGF-β_(1) group and TGF-β_(1)+IGU group,the apoptosis of cells was detected by flow cytometry,and the mRNA expression of collagen type Ⅰ(COL-Ⅰ)and collagen type Ⅲ(COL-Ⅲ)was detected by quantitative real-time polymerase chain reaction(qRT-PCR).The protein levels ofα-smooth muscle actin(α-SMA),fibronectin,p-Smad2,p-Smad3 and transcription coactivator p300 were detected by Western blot and immunofluorescence.One-way ANOVA was used for all data,and LSD-t test or Kruskal-Wallis test was used for pair comparison.Results The content of hydroxyproline in CTR group,the BLM group and the BLM+IGU group was(0.552±0.075)μg/mg,(1.293±0.081)μg/mg and(0.833±0.053)μg/mg(F=169.672,P<0.01)respectively.IGU reduced the content of hydroxyproline in the lung tissue of mice,reduced the accumulation of collagen in the lung,and thus reduced the degree of BLM-induced pulmonary fibrosis,and improved the pathological changes in the lung of mice.In cell experiments,IGU had no significant effect on apoptosis(F=0.83,P=0.54).The relative expression levels of COL-ⅠmRNA in the CTR group,TGF-β_(1) group and TGF-β_(1)+IGU group were(100.4±1.2),(299.0±13.0)and(202.5±7.0)respectively(F=468.7,P<0.01).The relative expression levels of COL-Ⅲ mRNA in the CTR gro

关 键 词：成纤维细胞 转化生长因子β_(1) 艾拉莫德 肺纤维化 

分 类 号：R965[医药卫生—药理学]