氟非尼酮通过调控mTOR信号通路抑制百草枯中毒致肺纤维化的作用及机制研究  

Fluorofenidone attenuates paraquat-induced pulmonary fibrosis by inhibiting the mTOR pathway

作　　者：蒋菲娅 蒋宇 王彤彤 刘文[1] JIANG Feiya;JIANG Yu;WANG Tongtong;LIU Wen(Department of Parmacy,First Hospital Affiliated to Hunan Normal University,Changsha 410000,China;Emergency Medical Research Institute,First Hospital Affiliated to Hunan Normal University Changsha 410000,China)

机构地区：[1]湖南师范大学附属第一医院药学部,长沙410000 [2]湖南师范大学附属第一医院急救研究所,长沙410000

出　　处：《免疫学杂志》2021年第12期1013-1020,共8页Immunological Journal

基　　金：湖南省卫生和计划生育委员会科研项目(B2017076);长沙市科技厅项目(kq1901051);湖南省自然科学基金(2021JJ80028);湖南省中医药管理局重点课题(201922)。

摘　　要：目的研究氟非尼酮(AKF-PD)对百草枯(PQ)中毒致肺纤维化的影响及其mTOR信号通路的调控作用,探讨氟非尼酮对百草枯中毒致肺纤维化的作用及机制。方法将SD大鼠随机分为对照组、PQ组、PQ+AKF-PD组、AKF-PD组。通过苏木精-伊红(HE)染色和Masson染色观察各组肺组织形态变化,分光光度法检测SOD、MDA、HYP含量,蛋白质免疫印迹(Western blot)分析TGF-β1、E-Cadherin、α-SMA蛋白表达水平;将肺上皮细胞(HPAEpiC)分为对照组、PQ组、PQ+AKF-PD组、AKF-PD组,用百草枯诱导肺上皮细胞发生上皮间质转化,给予1 mmol/L氟非尼酮干预24 h后,通过CCK-8试剂盒检测细胞增殖活力,ELISA试剂盒检测炎症细胞因子IL-1β、IL-6、TNF-α的含量,Western blot检测肺纤维化程度相关蛋白TGF-β1、ECadherin、α-SMA的表达水平以及mTOR信号通路相关蛋白mTOR和S6K1的磷酸化水平。结果与对照组相比,PQ组大鼠肺组织炎症反应明显,MDA、HYP含量以及TGF-β1、α-SMA蛋白表达水平显著升高(P<0.01),而SOD含量以及E-Cadherin蛋白表达水平显著降低(P<0.01);与PQ组相比,PQ+AKF-PD组大鼠肺组织炎症反应明显减轻,MDA、HYP含量以及TGF-β1、α-SMA蛋白表达水平显著降低(P<0.01),而SOD含量以及E-Cadherin表达水平显著升高(P<0.01);与对照组相比,PQ组细胞增殖活力显著下降(P<0.05),炎症细胞因子IL-1β、IL-6、TNF-α的含量显著升高(P<0.01),TGF-β1、α-SMA蛋白表达水平以及mTOR和S6K1的磷酸化水平显著增加(P<0.01),E-Cadherin的蛋白表达水平显著降低(P<0.01);与PQ组相比,PQ+AKF-PD组细胞增殖活力显著升高(P<0.05),炎症细胞因子IL-1β、IL-6、TNF-α的含量显著降低(P<0.01),TGF-β1、α-SMA蛋白表达水平以及mTOR和S6K1的磷酸化水平显著降低(P<0.01),而E-Cadherin的蛋白表达水平显著增加(P<0.05)。结论AKF-PD可能通过抑制mTOR信号通路,减轻百草枯诱导的炎症反应和肺纤维化程度。The purpose of this study was to investigate the effect of fluorofenidone(AKF-PD)on pulmonary fibrosis caused by paraquat(PQ)poisoning and the underlying mechanism.SD rats were randomly divided into control group,PQ group,PQ+AKF-PD group,and AKF-PD group.The morphological changes of lung tissues in each group were observed by hematoxylin-eosin(HE)staining and Masson staining,SOD,MDA,and HYP content were detected by spectrophotometry,and the expression of TGF-β1,E-Cadherin,andα-SMA were analyzed by Western blotting.HPAEpiC cells were treated with AKFPD in the presence or absence of PQ,then the CCK-8 kit was employed to detect cell proliferation activity,the ELISA kits were used to detect the levels of IL-1β,IL-6,and TNF-α,while Western blot was used to detect the expression levels of pulmonary fibrosis related proteins TGF-β1,E-Cadherin,α-SMA,and the mTOR signaling pathway related proteins mTOR and S6K1.Data showed that compared with the control group,the lung tissue of the PQ group demonstrated obvious inflammatory responses,higher levels of of MDA,HYP,TGF-β1 andαSMA(P<0.01),while lower levels of SOD and E-Cadherin protein expression(P<0.01).Compared with the PQ group,the level of alveolitis and pulmonary fibrosis in the PQ+AKFPD group was significantly reduced,the MDA,HYP content,and the TGF-β1 andα-SMA protein levels were significantly reduced(P<0.01),while the SOD content and E-Cadherin expression level were significantly increased(P<0.01);compared with the control group,the cell viability of PQ group was significantly decreased(P<0.05),and the levels of inflammatory cytokines IL-1β,IL-6,and TNF-αwere significantly increased(P<0.01),the expression of TGF-β1,α-SMA,p-mTOR/mTOR and p-S6K1/S6K1 increased significantly(P<0.01),and the expression of ECadherin decreased significantly(P<0.01);compared with the PQ group,the cell viability of PQ+AKF-PD group was significantly increased(P<0.05),the content of inflammatory cytokines IL-1β,IL-6,and TNF-OL were significantly decreased(P<0.01),the expression o

关 键 词：氟非尼酮 百草枯 肺纤维化 MTOR信号通路 

分 类 号：R285.5[医药卫生—中药学]