冬虫夏草提取物对肺炎小鼠肺泡上皮细胞的自噬作用及机制

Effect and mechanism of Cordyceps sinensis extract on autophagy of alveolar epithelial cells in pneumonia mice

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.