探讨培土益肺颗粒抑制大鼠特发性肺纤维化(IPF)的机制,将50只无特异性病原体(SPF)级雄性Wistar大鼠随机分为空白组和模型组。模型组大鼠气管内输注5mg·kg~(-1)博莱霉素(BLM)诱导IPF,随机分为模型组,吡非尼酮组,和高剂量,中等剂量,低剂量组给予培土益肺颗粒治疗。建模24小时后,治疗组给予培土益肺颗粒或吡非尼酮灌胃作为阳性对照药物;同时,模型组给予等量生理盐水.治疗21天后,收集肺组织样本进行分析.采用苏木精-伊红(HE)染色和Masson三色染色评估肺组织的病理变化。蛋白激酶B(Akt)的表达水平,哺乳动物雷帕霉素靶蛋白(mTOR),它们的磷酸化形式,通过Westernblot(WB)测定了螯合体1(p62)。荧光定量实时聚合酶链反应(RT-qPCR)用于测量Beclin-1,微管相关蛋白1A/1B轻链3B(LC3B)的信使核糖核酸(mRNA)表达水平,p62进行免疫组织化学以评估肺组织样品中Beclin-1和LC3B的蛋白质表达水平。结果:空白组大鼠肺组织结构正常,无明显胶原沉积。相比之下,模型组大鼠肺泡间隔增厚,并有明显的炎症改变和胶原沉积。与模型组大鼠相比,培土益肺颗粒或吡非尼酮治疗的肺组织结构明显改善,组织学观察到炎症和胶原沉积减少。此外,与空白组相比,p62及其mRNA的表达,模型组肺组织中p-Akt和p-mTOR蛋白明显升高,而Beclin-1、LC3B及其mRNA水平显著降低。与模型组相比,p62及其mRNA的表达,吡非尼酮组、培土益肺颗粒高、中剂量组肺组织中p-Akt和p-mTOR均显著降低,而Beclin-1、LC3B及其mRNA表达显著增加。以上结果表明培土益肺颗粒可通过抑制磷酸肌醇3-激酶(PI3K)/Akt/mTOR信号通路改善肺组织自噬水平,延缓IPF疾病的发展。
To investigate the mechanism by which Peitu Yifei Granules inhibit idiopathic pulmonary fibrosis(IPF) in rats, fifty specific-pathogen-free(SPF) grade male Wistar rats were randomly divided into blank group and modeling group. IPF was induced in the modeling group rats by tracheal infusion of 5 mg·kg~(-1) bleomycin(BLM) and then randomly divided into model group, pirfenidone group, and high-dose, medium-dose, and low-dose groups treated with Peitu Yifei Granules. After 24 hours of modeling, the treatment groups received intragastric administration of either Peitu Yifei Granules or pirfenidone as a positive control drug; meanwhile, the model group received an equal volume of normal saline. After 21 days of treatment administration, lung tissue samples were collected for analysis. Pathological changes in lung tissues were assessed using hematoxylin-eosin(HE) staining and Masson\'s trichrome staining. The expression levels of protein kinase B(Akt), mammalian target of rapamycin(mTOR), their phosphorylated forms, and sequestosome 1(
p62) were determined through Western blot(WB). Fluorescent quantitative real-time polymerase chain reaction(RT-qPCR) was used to measure messenger ribonucleic acid(mRNA) expression levels of Beclin-1, microtubule-associated proteins 1A/1B light chain 3B(LC3B), and
p62. Immunohistochemistry was performed to assess protein expression levels of Beclin-1 and LC3B in lung tissue samples. RESULTS:: demonstrated that lung tissue structure appeared normal without significant collagen deposition in the blank group rats. In contrast, rats from the model group exhibited thickened alveolar septa along with evident inflammatory changes and collagen deposition. Compared to the model group rats, those treated with Peitu Yifei Granules or pirfenidone showed significantly improved lung tissue structure with reduced inflammation and collagen deposition observed histologically. Furthermore, compared with those of the blank group, the expressions of
p62 and its mRNA, p-Akt and p-mTOR protein in lung tissues of the model group were significantly increased, while Beclin-1, LC3B and their mRNA levels were significantly decreased. Compared with those of the model group, the expressions of
p62 and its mRNA, p-Akt and p-mTOR in lung tissues of the pirfenidone group and Peitu Yifei Granules high-dose and medium-dose groups were significantly decreased, while Beclin-1, LC3B and their mRNA expressions were significantly increased. The above results indicate that Peitu Yifei Granules can improve autophagy levels in lung tissues by inhibiting the phosphoinositide 3-kinase(PI3K)/Akt/mTOR signaling pathway and delay the development of IPF disease.