Maiwei Yangfei Decoction Mitigate Pulmonary Fibrosis Via Regulating P53 To Inhibit Fibroblast Senescence

Objective:Pulmonary fibrosis is a respiratory disease which is progressive and fatal.Apart from lung transplantation,there is still a lack of effective methods.Pirfenidone and nintedanib can only delay the decline rate of forced vital capacity and reduce the fatality rate to a certain extent,but it comes with many disadvantages such as the excessive side effects and high price.Clinical observation shows that Maiwei Yangfei decoction(MWYF),which has a good safety,can improve the clinical symptoms and quality of life of patients with pulmonary fibrosis.In previous research,MWYF has an anti-pulmonary fibrosis effect,but its molecular mechanism is still unclear.This study intends to analyze MWYF components,to clarify its therapeutic effect on mice with pulmonary fibrosis,and to further explore whether MWYF inhibits the aging of pulmonary fibroblasts through p53 acting as an anti-pulmonary fibrosis mechanism.Methods:1.Component analysis of MWYF:The pieces of MWYF are boiled with pure water for three times,then filtered and concentrated to obtain a certain concentration of water decoction;samples and standards were prepared and detected by UPLC/LTQ-Orbitrap-MS,then the total ion current diagram(positive and negative ions),retention time,relative molecular mass,peak intensity and other information were obtained.The qualitative analysis of MWYF was conducted by comparing with standard products.2.Ameliorative effect of MWYF on pulmonary fibrosis in mice:72 mice were equally divided into blank group,model group,low-dose MWYF group(20g/kg/d),medium-dose MWYF group(40 g/kg/day),high-dose MWYF group(60g/kg/day)and pirfenidone group(0.3g/kg/day).The mouse model of pulmonary fibrosis was induced by instillation of bleomycin using an aerosol intratracheal dosing device,and the mice were given pirfenidone and different concentrations of MWYF by gavage for 21 days.The degree of improvement of pulmonary fibrosis in mice was evaluated by histopathological staining,pulmonary function,chest Micro CT testing,immunohistochemistry and ELISA.3.Network pharmacology research of MWYF:The TCMSP,SwissTargetPrediction,TCMID,and SEA databases were used to find all the active ingredients and targets of MWYF,and the targets obtained from all the ingredients were the potential therapeutic targets of MWYF after combination and deduplication.Through the OMIM,GeneCards Suite,CTD and TTD databases,the targets related to pulmonary fibrosis diseases were searched,and the results of the four databases were combined to eliminate duplicates then used as potential targets for pulmonary fibrosis.The common targets of pulmonary fibrosis and MWYF were used to make PPI maps through the STRING platform,and topological analysis was performed to select the core targets.GO and KEGG enrichment analysis was conducted by the DAVID bioinformatics database.4.Transcriptomics study of MWYF:Fresh lung tissues from three mice respectively from blank group,model group and the MWYF high-dose group were taken for transcriptomics sequencing.After obtaining high-quality sequencing data,Lianchuan Biological Cloud Platform was used to perform sample correlation analysis,differential gene expression analysis and signal pathway enrichment analysis to quickly identify candidate genes and discover potential mechanisms of action.At last,some genes in the results of differential gene cluster heat map were verified by RTPCR.5.MWYF inhibits the aging of lung fibroblast in vitro and in vivo:①In vivo:β-galactosidase staining was used to observe the aging of lung tissue in mice,and Western Blot was used to detect the expression of the p53,p21 and p16 which are senescence-related proteins in lung tissue.The co-staining of fibroblast marker α-SMA and senescence-related proteins p53,p21 and p16 in lung tissue was observed by immunofluorescence.②In vitro:The primary lung fibroblasts of mice were extracted and identified by immunohistochemistry,then the dose of MWYF freeze-dried powder was determined by CCK-8.Primary lung fibroblasts were divided into blank group,model group,MWYF low-dose group(400 μg/mL),MWYF medium-dose group(600 μg/mL)and MWYF high-dose group(800 μg/mL).The cell senescence model was established by the intervention of doxorubicin for 48 hours,and the medium containing different concentrations of MWYF freeze-dried powder was intervened for 24 hours,then the senescence of lung fibroblasts was observed by β-galactosidase staining.Western Blot was conducted to detect the expression of senescence-related proteins p53,p21,p16,and SASP in lung fibroblasts.6.MWYF inhibited the senescence of lung fibroblast by p53:The overexpressed and silent p53 plasmids were constructed to over-express and silence p53 genes of lung fibroblasts.After determining the success of plasmid construction and excluding the effect of plasmids on cells,the aging of lung fibroblasts was observed by β-galactosidase staining,and the expression of agingrelated proteins p53,p21 and p16 in lung fibroblasts was detected by Western Blot.Results:1.Component analysis of MWYF:After the qualitative analysis of MWYF was conducted by UPLC/LTQ-Orbitrap-MS,9 substances were identified.Ephedrine hydrochloride,liquiritin,and schisandrin were identified in the positive ion mode;ferulic acid,aurantiamarin,lobetyolin,rosmarinic acid,baicalin,and glycyrrhizic acid were identified in the negative ion mode.2.Ameliorative effect of WMYF on pulmonary fibrosis in mice:MWYF can increase the weight of mice,reduce respiratory resistance,and enhance respiratory compliance.It can also reduce the extent of inflammation and fibrosis and decrease the expression of Collagen Ⅰ,Collagen Ⅲ,P YD,and HYP in lung tissue.Among them,the MWYF high-dose group and the pirfenidone group had the most obvious effects.3.Network pharmacology research of MWYF:260 potential targets,and 1657 pulmonary fibrosisrelated targets in MWYF were obtained through database analysis.124 common targets were obtained after merging and deduplication.19 core targets were finally screened by using the STRING platform.Though KEGG enrichment analysis,it was found that the p53 signaling pathway is quite different,which may be one of the potential mechanisms.4.Transcriptomic research of MWYF:The results of Pearson correlation coefficient and PCA principal component analysis showed that the differences within the three groups were small and the distribution was relatively concentrated,indicating that the differentially expressed genes were reliable.The analysis of differential genes showed that compared with the control group,957 genes were up-regulated and 876 genes were down-regulated in the model group.690 genes were upregulated and 1043 genes were down-regulated in the MWYF high-dose group when compared with the model group.The result of KEGG enrichment analysis indicated that the p53 signaling pathway had high significance.Through hierarchical clustering analysis of genes which expressed differentially in p53 signaling pathway,it was found that MWYF can regulate the expression levels of Trp53(p53),Cdk1,Cdkn1a(p21),Cdkn2a(p16),Ccnb1,Ccnd2 and other cell cycle genes.Finally,we selected the p53,p21,Cdkl and Ccnd2 genes with obvious differential expression in the clustering results,and verified their expression at the gene level by RT-PCR.Then we found that they were consistent with the transcriptomic sequencing results.5.MWYF inhibits the senescence of lung fibroblast in vivo and in vitro:In vivo,it was found that MWYF can reduce β-galactosidase activity in lung tissue and decrease the expression of p16,p21 and p53.We also found that MWYF could inhibit the senescence of lung fibroblast in lung tissue by double fluorescent labeling.In vitro,MWYF can reduce β-galactosidase activity in lung fibroblasts,decrease the expression of p16,p21 and p53,and inhibit the expression of SASP.The results both in vivo and vitro showed that the lyophilized powder in the high-dose MWYF group had the most obvious effect.6.MWYF inhibits the senescence of lung fibroblast through p53:After silencing p53 gene in lung fibroblasts by plasmid vector,it was found that the effect of MWYF on reducing senescence of lung fibroblasts was inhibited,and the ability to decrease the expression of p21 and p16 was inhibited.After the p53 gene was overexpressed in lung fibroblasts by plasmid vector,it was found that the effect of MWYF on suppressing the senescence of lung fibroblasts was not inhibited,and the ability to decrease the expression of p21 and p16 proteins was also not inhibited.Conclusions:1.MWYF can play an anti-pulmonary fibrosis role by improving the lung function of mice,alleviating lung inflammation and reducing collagen deposition in lung tissues.2.MWYF can inhibit the senescence of lung fibroblasts to play an anti-pulmonary fibrosis role both in vivo and in vitro.3.MWYF can inhibit the senescence of lung fibroblasts to play an anti-pulmonary fibrosis role through the p53 signaling pathway.