吡非尼酮抑制大鼠气管瘢痕狭窄和成纤维细胞功能的实验研究  

Experimental study of pirfenidone inhibiting tracheal scar stenosis and fibroblast function in rats

作　　者：汪玮 吴平[1] 李鑫 唐瑶云[1] 樊玉华 方兴[1] WANG Wei;WU Ping;LI Xin;TANG Yaoyun;FAN Yuhua;FANG Xing(Department of Otolaryngology Head and Neck Surgery,Hunan Province Key Laboratory of Otolaryngology Critical Diseases,Hunan Province Clinical Research Center of Laryngopharyngeal and Voice Diseases,Xiangya Hospital,Central South University,Changsha 410008,China)

机构地区：[1]中南大学湘雅医院耳鼻咽喉头颈外科耳鼻咽喉重大疾病研究湖南省重点实验室湖南省咽喉嗓音疾病临床研究中心,湖南长沙410008

出　　处：《中国耳鼻咽喉颅底外科杂志》2020年第3期272-277,共6页Chinese Journal of Otorhinolaryngology-skull Base Surgery

基　　金：国家自然科学青年基金(81302355)。

摘　　要：目的探讨吡非尼酮(PFD)对大鼠气管瘢痕狭窄及成纤维细胞功能的影响。方法将20只SD雄性大鼠通过气管切开和气管损伤手术构建气管狭窄模型,随机分成2组,实验组(10只)予以PFD胶囊粉末经口灌喂,50 mg/(只·d);对照组(10只)予以无菌水经口灌喂,5 mL/(只·d),两组连续灌喂10 d。手术14 d后HE染色检测气管瘢痕厚度,免疫组织化学检测TGFβ1、Collagen I和α-SMA的表达情况。使用不同浓度的PFD处理RFL-6细胞,CCK-8法检测细胞存活率,划痕和Transwell实验检测细胞侵袭转移能力,Western blot法检测蛋白表达水平。结果HE染色显示PFD实验组大鼠气管瘢痕厚度为(337.5±33.5)μm,明显低于对照组瘢痕厚度(537.0±38.8)μm(P<0.05);免疫组织化学结果显示TGFβ1、Collagen I和α-SMA在实验组的表达强度明显低于对照组,两组比较差异均具有统计学意义(P<0.05)。细胞实验结果显示PFD能抑制RFL-6细胞的生长,浓度为1.5mM时效果明显(P<0.05);PFD能减弱细胞的划痕愈合以及穿出Transwell小室的能力,与对照组比较差异均具有统计学意义(P<0.05)。Western blot检测显示PFD能明显下调RFL-6细胞中TGFβ1、Collagen I和α-SMA的表达(P<0.05)。结论PFD能显著拮抗大鼠气管瘢痕形成,并能抑制成纤维细胞的增殖和转化、迁移愈合能力以及细胞外基质的分泌能力。Objective To investigate the effect of pirfenidone(PFD)on tracheal scar stenosis and fibroblast function in rats.Methods 20 SD male rats were undergone tracheotomy and tracheal injury surgery to establish tracheal stenosis model,and then randomly divided into two groups.The rats of experimental group(n=10)were fed with PFD capsule powder by gavage at 50 mg/case/day,and those of control group were treated with sterile water at 5 ml/case/day.All rats in both groups were continuously administered for 10 days.Fourteen days after surgery,the thickness of tracheal scar was detected on HE staining,and the expressions of TGFβ1,Collagen I andα-SMA in scar tissue were detected by immunohistochemistry.Survival rates of RFL-6 cells treated with PFD of different concentrations were measured by CCK-8,cell invasion and metastasis ability was determined by scratch and transwell experiments,and protein expression level was detected by Western blot.Results HE staining showed that the thickness of tracheal scar of the experimental group was(337.5±33.5)μm,which was significantly lower than that of the control group[(537.0±38.8)μm](P<0.05).Immunohistochemical results showed that the expression intensities of TGFβ1,Collagen I andα-SMA in the experimental group were significantly weaker than those in the control group(all P<0.05).PFD inhibited the growth of RFL-6 cells with most obvious effect at the concentration of 1.5 mM(P<0.05).Moreover,PFD reduced the scratch healing of cells and the ability to penetrate the transwell cell(P<0.05).Western blot revealed significantly down-regulated expressions of TGFβ1,collagen I andα-SMA in RFL-6 cells by PFD(all P<0.05).Conclusion PFD can significantly attenuate tracheal scar formation in rats,and inhibit the function of proliferation,transformation,migration,healing and the secretion of extracellular matrix in fibroblasts.

关 键 词：喉气管狭窄 吡非尼酮 转化生长因子Β1 成纤维细胞 

分 类 号：R768.11[医药卫生—耳鼻咽喉科]