摘要
目的:基于mTORC2-AKT-IRF4信号探讨保金尘肺方抑制巨噬细胞M2极化改善二氧化硅(SiO2)诱导矽肺作用机制.方法:32只SPF级雄性SD大鼠随机分为正常组、模型组、汉防己甲素组(27 mg·kg-1·d-1)、保金尘肺方组(9.72 g·kg-1·d-1),每组8只.采用一次性非暴露式气管滴注SiO2混悬液(50 mg/mL)制备矽肺大鼠,于造模第7周予对应药物灌胃治疗,共2周.检测大鼠肺功能;HE染色和Masson染色观察肺组织病理改变和胶原沉积;免疫组化法检测Ⅰ型胶原蛋白(COL-Ⅰ)、Ⅲ型胶原蛋白(COL-Ⅲ)、CD68、CD206和转化生长因子-β1(TGF-β1)的表达;免疫印迹法检测CD206、精氨酸酶(ARG)-1、哺乳动物雷帕霉素靶蛋白(mTOR)、AKT、p-mTORser2481、p-AKTser473和干扰素调节因子4(IRF4)蛋白表达;Real-time PCR检测CD206和ARG-1 mRNA表达.结果:与正常组比较,模型组大鼠肺功能VC、TV、Cdyn和Cchord均显著下降(P＜0.01);大鼠肺泡结构破坏,大量炎性细胞浸润;肺组织COL-Ⅰ、COL-Ⅲ以及M2巨噬细胞标志物CD68、CD206和促纤维化因子TGF-β 1的表达显著升高(P＜0.01).与模型组比较,保金尘肺方组大鼠肺功能VC、TV、Cdyn和Cchord均显著升高(P＜0.05,P＜0.01),汉防己甲素组VC、TV、Cdyn均显著升高(P＜0.01).保金尘肺方组和汉防己甲素组肺组织病理明显改善,COL-Ⅰ、COL-Ⅲ及M2巨噬细胞标志物CD68、CD206和促纤维化因子TGF-β 1表达显著降低(P＜0.01).此外,IL-4诱导肺泡巨噬细胞M2极化标志物CD206、ARG-1 mRNA和蛋白,以及mTORC2信号蛋白p-mTORser2481、p-AKTser473、IRF4表达显著升高(P＜0.05,P＜0.01);而保金尘肺方高浓度可以显著抑制IL-4诱导肺泡巨噬细胞的M2标志物的mRNA和蛋白及mTORC2信号磷酸化蛋白表达(P＜0.05,P＜0.01).结论:保金尘肺方可以改善SiO2诱导的矽肺大鼠肺纤维化,其机制可能与抑制mTOR/AKT通路,阻抑巨噬细胞M2极化有关.
Abstract
Objective:To explore the effect and mechanisms of Baojin Chenfei Formula(BCF)on SiO2-induced silicosis by inhibiting M2 polarization of macrophages via regulating mTORC2-AKT-IRF4 signal.Methods:Thirty-two SPF male SD rats were randomly divided into control group,model group,tetrandrine group(27 mg·kg-1·d-1),and BCF group(9.72 g·kg-1·d-1),8 in each group.The experimental silicosis model was established by intratracheal injection with SiO2 suspension(50 mg/mL).From the week 7,silicosis rats were treated with tetrandrine or BCF for 2 weeks.The changes of pulmonary function were detected.The pathological changes and collagen deposition of lung tissue were analyzed by HE and Masson staining.The protein levels of COL-Ⅰ,COL-Ⅲ,CD68,CD206 and TGF-β1 expressions were detected by immunohistochemistry.The protein levels of CD206,ARG-1,mTOR,AKT,p-mTORser2481,p-AKTser473 and IRF4 were detected by Western blot.The mRNA levels of CD206 and ARG-1 expressions were detected by Real time PCR.Results:Compared with the normal group,the pulmonary function including VC,TV,Cdyn and Cchord were significantly decreased in the model group(P＜0.01);the alveolar structure of model rats was damaged and a large number of inflammatory cells were infiltrated;the protein levels of COL-Ⅰ,COL-Ⅲ,CD68,CD206 and TGF-β 1 were significantly increased in the model group(P＜0.01).Compared with the model group,the pulmonary function TV,VC,Cchord and Cydn were significantly increased(P＜0.05,P＜0.01);the pathological changes of the lung tissue were improved;the expressions of COL-Ⅰ,COL-Ⅲ,macrophage markers and fibrosis promoting factor was significantly reduced in BCF-treated rats(P＜0.01).In addition,the mRNA and protein of CD206,ARG-1,as well as the protein levels of p-mTORser2481,p-AKTser473,and IRF4,the M2 polarization markers significantly increased in macrophages induced by IL-4(P＜0.05,P＜0.01);these changes were significantly inhibited by BCF treatment of high concentration(P＜0.05,P＜0.01).Conclusion:BCF could improve pulmonary fibrosis in silicosis rat induced by SiO2,and its mechanism may be related to the inhibition of M2 polarization of macrophages via suppressing mTOR signal.
基金项目
河南省重点研发与推广专项(科技攻关)(212102310356)
国家自然科学基金(81904170)
国家自然科学基金(82105048)
河南省中医药科学研究专项(20-21ZYZD01)
国家科技期刊平台
NSTL
万方数据