结合网络药理学分析p53信号通路在麦门冬汤改善肺纤维化小鼠模型中的作用

Integration of Network Pharmacology Analysis Investigating the Functional Role of the p53 Signaling Pathway in the Improvement of Pulmonary Fibrosis in A Mouse Model by Maimendong Decoction

目的探讨麦门冬汤对肺纤维化小鼠模型的干预作用及其药理机制。方法将C57BL/6小鼠随机分成对照组、模型组、干预组,每组6只,模型组小鼠经气管滴注博来霉素建立肺纤维化小鼠模型,干预组造模24 h后麦门冬汤灌胃给药20 d,观察小鼠一般情况,HE染色观察肺组织的病理变化,免疫组化和Western blot分析α-平滑肌肌动蛋白(alpha smooth muscle actin,α-SMA)和钙黏蛋白(epithelial cadherin,E-cadherin)的表达情况。网络药理分析麦门冬汤干预肺纤维化的潜在机制。其后通过转化生长因子(transforming growth factor,TGF)-β1诱导小鼠胚胎成纤维细胞NIH3T3构建肺纤维化细胞模型,麦门冬汤含药血清和(或)p53抑制剂(pifithrin-α,PFT-α)干预后,荧光定量PCR和Western blot检测p53、α-SMA、E-cadherin和I型胶原蛋白(collagen type I,Collagen I)的表达情况。结果与对照组比较,肺纤维化小鼠模型肺组织均出现大面积实变区和大量炎性细胞浸润,α-SMA表达增加,E-cadherin降低(P<0.01),麦门冬汤干预后上述症状得到显著改善。网络药理学分析发现麦门冬汤的差异药靶基因主要富集在p53信号通路。细胞实验则显示麦门冬汤组含药血清干预可使肺纤维化细胞模型中α-SMA下调,E-cadherin和p53表达上调(P<0.01)。PFT-α干预实验中,PFT-α干预后对照组和麦门冬汤组中p53和E-cadherin表达降低,α-SMA和Collagen I表达升高(P<0.01)。结论麦门冬汤可通过调节p53信号通路改善肺纤维化进展。

Objective To investigate the interventional effects of Maimendong decoction on a mouse model of pulmonary fibrosis and the underlying pharmacological mechanisms.Methods C57BL/6 mice were randomly allocated into three groups:control group,model group,and treatment group,with 6 mice in each group.A pulmonary fibrosis mouse model was established by tracheal instillation of bleomycin in the model group,and 24 hours after the model was established,the intervention group was given Maimendong decoction by gavage for 20 days.The pulmonary fibrosis mouse model was established by tracheal instillation of bleomycin in the model group.Twenty-four hours after the model was established,mice in the treatment group were given Maimendong decoction by gavage for 20 days.The general condition of the mice was observed,and the pathological alterations in lung tissue were examined by HE staining.The expression of alpha smooth muscle actin(α-SMA)and epithelial cadherin(E-cadherin)was analyzed by immunohistochemistry staining and Immunoblotting.Network pharmacology analysis was performed to explore the potential mechanism of Maimendong decoction in improving pulmonary fibrosis.Furthermore,a pulmonary fibroblast model was established by inducing mice embryonic fibroblast NIH3T3 with transforming growth factor-β1(TGF-β1).After treatment with Maimendong Decoction-containing serum and/or p53 inhibitor(Pifithrin-α,PFT-α),the expression of p53,α-SMA,E-cadherin,and collagen type I(Collagen I)was detected by real-time PCR and Immunoblotting.Results Compared with the control group,the lung tissues of mice in the pulmonary fibrosis model group showed extensive consolidation areas,abundant infiltration of inflammatory cells,increased expression ofα-SMA,and decreased expression of E-cadherin(P<0.01).These features were significantly improved after Maimendong decoction treatment.Network pharmacological analysis revealed that the differentially targeted genes of Maimendong decoction were mainly enriched in the p53 signaling pathway.Cell experiments showed that the treatment with Maimendong decoction-containing serum led to the downregulation ofα-SMA and upregulation of E-cadherin and p53 in the pulmonary fibroblast model(P<0.01).In the PFT-αtreatment experiment,the expression of p53 and E-cadherin was reduced in both the control and Maimendong decoction groups,while the expression ofα-SMA and Collagen I was increased(P<0.01).Conclusion Maimendong decoction can improve the progression of pulmonary fibrosis by regulating the p53 signaling pathway.