摘要
目的探讨冬虫夏草提取物(CSE)介导哺乳动物雷帕霉素靶蛋白复合物1(mTORC1)/转录因子EB(TFEB)通路对间质性肺炎小鼠肺泡上皮细胞自噬的作用。方法75只C57BL/6雄性小鼠,随机选择15只作为正常组,另外60只用一次性鼻腔滴注博来霉素诱导间质性肺炎模型,后随机分为模型组、CSE低剂量组(20 mg·kg^(-1)·d^(-1))、CSE中剂量组(40 mg·kg^(-1)·d^(-1))和CSE高剂量组(80 mg·kg^(-1)·d^(-1)),每组各15只,正常组和模型组灌服等量生理盐水,连续28 d。检测肺组织湿干质量比(W/D);检测肺泡灌洗液中肿瘤坏死因子-α(TNF-α)、白介素6(IL-6)、白介素1β(IL-1β)水平;染色观察各组小鼠肺脏组织形态及纤维化情况;检测肺组织细胞凋亡水平;检测Ⅱ型肺泡上皮细胞中mTORC1、磷酸化哺乳动物雷帕霉素靶蛋白复合物1(p-mTORC1)、TFEB、p62、自噬微管相关蛋白轻链3Ⅱ(LC3Ⅱ)、自噬微管相关蛋白轻链3Ⅰ(LC3Ⅰ)和α-平滑肌肌动蛋白(α-SMA)蛋白表达水平。结果与正常组比较,模型组小鼠肺脏水肿,W/D值显著提高(P<0.05);肺组织结构不完整,炎性渗出较多、胶原纤维严重沉积并见有大量细胞凋亡,肺灌洗液中TNF-α、IL-6、IL-1β水平及Ⅱ型肺泡上皮细胞中p-mTORC1、p62、α-SMA蛋白表达水平显著升高,LC3Ⅱ/LC3Ⅰ及TFEB蛋白表达水平显著降低(P<0.05);与模型组比较,CSE低、中和高剂量组小鼠间质性肺炎病理症状得到显著改善,TNF-α、IL-6和IL-1β水平及Ⅱ型肺泡上皮细胞中p-mTORC1、p62和α-SMA显著降低,LC3Ⅱ/LC3Ⅰ及TFEB蛋白表达水平显著升高(P<0.05),且表现出剂量依赖性。结论CSE可减轻小鼠间质性肺炎的病理损伤,降低细胞凋亡及促炎因子的表达,改善肺脏组织纤维化,其机制可能与抑制mTORC1活性、促使TFEB参与肺泡上皮细胞自噬的发生有关。
Objective To investigate the effect of Cordyceps sinensis extract(CSE)mediating mammalian rapamycin target protein complex 1(mTORC1)/transcription factor EB(TFEB)pathway on autophagy of alveolar epithelial cells in mice with interstitial pneumonia,Methods Fifteen of 75 C57BL/6 male mice were randomly selected as the normal group,and another 60 mice were treated with a single nasal drip of bleomycin to induce interstitial pneumonia model,then they were randomly divided into model group,CSE low-dose group(20 mg·kg^(-1)·d^(-1)),CSE medium-dose group(40 mg·kg·d^(-1))and CSE high-dose group(80 mg·kg^(-1)·d^(-1)),with 15 mice in each group.The normal group and model group were given the same amount of normal saline by irrigation for consecutive 28 days.The wet-dry weight ratio of lung tissue(W/D)was detected.The expression of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and interleukin-1β(IL-1β)in alveolar lavage fluid was detected.The pulmonary morphology and fibrosis of each group was observed by staining.The level of apoptosis in lung tissue,the protein levels of mTORCl,phosphorylated mammalian target complex 1(p-MTORC1),TFEB,p62,autophagy microtubule-associated protein light chain 3Ⅱ(LC3Ⅱ),autophagy microtubule-associated protein light chain 3Ⅰ(LC3Ⅰ)andα-smooth muscle actin(α-SMA)in typeⅡalveolar epithelial cells were detected.Results Compared with the normal group,the lung edema and W/D value in model group were significantly increased(P<0.05).The lung tissue structure was incomplete,with more inflammatory exudation,more serious deposition of collagen fibers and much more cell apoptosis.The levels of TNF-α,IL-6 and IL-1βin lung lavage fluid and the protein expression levels of p-mTORCl,P62 andα-SMA in typeⅡalveolar epithelial cells were significantly increased.The protein levels of LC3Ⅱ/LC3Ⅰand TFEB were significantly decreased(P<0.05).Compared with the model group,the pathological symptoms of interstitial pneumonia in CSE low,medium and high dose groups were significantly improved,the TNF-α,IL-6,IL-1αand p-mTORC1,p62 and a-SMA levels decreased significantly,and the expression levels of LC3Ⅱ/LC3Ⅰand TFEB protein increased significantly(P<0.05).The effect showed a dose dependence.Conclusion CSE can reduce the pathological damage of interstitial pneumonia in mice,reduce the expression of apoptosis and pro-inflammatory factors,and improve pulmonary fibrosis.The mechanism may be related to the inhibition of mTORC1 activity and the participation of TFEB in the occurrence of alveolar epithelial cell autophagy.
作者
邢双丽
赵明娟
金荣
XING Shuangli;ZHAO Mingjuan;JIN Rong(The Second District,Department of Respiratory and Critical Care Medicine,Central Hospital of Jiaozuo Coal(Group)Lmited Liability Company,Jiaozuo 454100,China)
出处
《西北药学杂志》
CAS
2023年第1期72-77,共6页
Northwest Pharmaceutical Journal
基金
河南省医学科技攻关计划联合项目(编号:LHGJ20190341)。
