| Purpose:Pulmonary fibrosis is an adverse consequence of many interstitial lung diseases.At present,there are limited clinical treatment methods.This study discusses the intervention effect of MBHD on the angiogenesis phenotype of pulmonary fibrosis,demonstrates its gain effect through clinical trials,and excavates its specific mechanism and new therapeutic targets through basic experiments,providing new research directions and ideas for the future development of pulmonary fibrosis treatment.Method: Part 1: Research on biological information analysisDEGs was obtained by performing differential expression analysis on two datasets of patients with pulmonary fibrosis,GSE 53845 and GSE 110147,and enrichment analysis was performed on DO,KEGG,GO,GSEA and GSVA to explore their gene function,pathway and disease enrichment.The risk model of IPF was built by LASSO regression model,and the disease characteristic genes were screened,and the model was tested by ROC curve.A molecular regulatory network was constructed for visual analysis,and finally the screened key gene targets were verified by RT-q PCR.Part 2 Experimental study:Experiment 1: To investigate the effects of MBHD combined with NINTEDANIB on angiogenesis-related factors in rats with pulmonary fibrosis and pathological observation and analysis.Forty healthy male Wistar rats(200 20 g)were randomly divided into five groups: the blank group,the model group,the NINTEDANIB group,the MBHD group,and the NINTEDANIB+MBHD group.The rats model was established by tracheal intubation and bleomycin infusion.Three days later,rats were given corresponding drugs by gavage,and they were killed after 28 days of anesthesia.He,Masson and immunohistochemistry were used to analyze the lung tissue sections.The related indexes of fibronectin,collagen type I,α-SMA,Notch 1,DLL 4,FGF and PDGF were detected by Western Blotting and q PCR.To investigate the effect on angiogenesis-related factors and pathological observation in rats with pulmonary fibrosis.Experiment 2: To investigate the intervention effect of MBHD-containing serum combined with NINTEDANIB on pulmonary microvascular endothelial cells.The pulmonary microvascular endothelial cells were randomly divided into five groups: a blank group,a TGF-β model group,NINTEDANIB group,MBHD group and NINTEDANIB+MBHD group.The drug-containing serum was used for intervention culture,and the sprouting,proliferation,migration and tube forming processes of the pulmonary microvascular endothelial cells in the angiogenesis process were detected and analyzed through Edu,scratch,Transwell and tube forming experiments.Western Blotting and q PCR were used to detect fibronectin,type I collagen,α-SMA,Notch 1,DLL 4,FGF and PDGF.To explore the intervention mechanism of the combination of MBHD and NINTEDANIB on pulmonary fibrosis.Experiment 3: To explore the mechanism of Notch pathway in the regulation of angiogenesis of pulmonary microvascular endothelial cells by MBHD.The pulmonary microvascular endothelial cells were randomly divided into seven groups: the blank group,TGFβ model group,NINTEDANIB group,MBHD group,NINTEDANIB + MBHD group,NINTEDANIB + MBHD group +pc DNA 3.1-NC group,NINTEDANIB + MBHD group +pc DNA 3.1-Notch 1 group.Notch 1 was overexpressed by transfection,and a Rescue test was designed.The Edu,scratch,Transwell,tube-forming experiment and Western Blotting tests further verified the regulatory effects and mechanisms of Notch pathway in the angiogenesis of pulmonary microvascular endothelial cells.Part 3 Clinical Studies:A clinical controlled trial was designed and conducted in a randomized,parallel-controlled,and open-label manner.The rats were randomly divided into a clinical group and a control group according to the ratio of 1:1 using random number table,for clinical treatment and observation.The control group was treated with NINTEDANIB according to the guidelines,and the experimental group was treated with MBHD based on the treatment of the control group.After one month,the follow-up data were entered,and the data sets of the whole analysis population and the population in line with the protocol were established.The general data of the patients,the efficacy comparison and the safety comparison were analyzed and evaluated.Result:Part 1: Research on biological information analysisThrough the analysis of letters,it was found that pulmonary fibrosis was closely related to the biological processes and phenotypes such as inflammatory reaction,extracellular matrix deposition,and angiogenesis.The gene targets related to angiogenesis were screened,and 39 downregulated genes and 78 up-regulated genes related to angiogenesis were found in the pulmonary fibrosis group.Pulmonary fibrosis was associated with angiogenesis and allograft rejection processes as determined by GSVA analysis.The AUC values of the lung fibrosis risk model established by the Lasso regression model were 1 and 0.9380.A PPI network between the characteristic genes of pulmonary fibrosis was established,and the TF gene network and mi RNA gene network related to CCR7,CD24,MDK,CXCL12,IL1R2,CXCL14,CCL19,and IL13RA2 were screened out from the top 8 genes.The analysis of the correlation between immune function and immune cell infiltration showed that there were significant differences in the degree of immune cell infiltration such as B cell,plasma cell and T cell CD8.Finally,through PCR verification,the key genes of CCR 7,CD 24,MDK,CXCL 14 and CCL 19 were finally selected,laying a foundation for disease diagnosis,condition evaluation and selection of new drug treatment targets.Part 2: In vivo and in vitro experimental studyExperiment 1: HE,Masson and immunohistochemistry analyses of pathological pictures in each group showed that the combination of MBHD and NINTEDANIB could effectively reduce the number of inflammatory cells,collagen fiber content and fibronectin area in the tissues of pulmonary fibrosis.The results of Western Blotting and q PCR showed that the MBHD combined with NINTEDANIB could effectively reduce the expression levels of relevant indicators of pulmonary fibrosis such as Fibronectin,Collagen I and α-SMA,inhibit the expression of angiogenesis-related regulatory factors PDGF and FGF,and reduce the expression of Notch1 receptor and DLL4 ligand in Notch pathway.The progression of pulmonary fibrosis was slowed by inhibition of angiogenesis.Experiment 2: The combination of MBHD and NINTEDANIB could inhibit angiogenesis by reducing the processes of budding,proliferation,migration and tube formation of pulmonary microvascular endothelial cells,and reduce the expression of fibronectin,Collagen I,and α-SMA collagen related to pulmonary fibrosis,and effectively inhibit the production of fibrin.Experiment 3: The specific role of Notch pathway in the angiogenesis was further verified through the rescue test,and it was found that Notch pathway could regulate angiogenesis by inhibiting the migration and budding of pulmonary microvascular endothelial cells and shortening the tubeforming process.Part 3: Clinical studiesIn clinical trials,the combination of NINTEDANIB and MBHD can effectively improve the symptoms of patients,reduce the scores of single symptoms(cough and shortness of breath)in traditional Chinese medicine,reduce the level of symptom score,activity score and total score in the St.George’s questionnaire,and improve body resistance.There was no statistical difference in adverse reaction events compared with the control group.Conclusion:The combination of MBHD with NINTEDANIB had a certain gain,and achieved the inhibitory effect on pulmonary fibrosis by regulating the angiogenesis in the process of pulmonary fibrosis.MBHD could interfere with Notch pathway and inhibit the sprouting and migration links in the process of angiogenesis,further clarifying its mechanism of action. |
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