Evaluation Of Clinical Efficacy Of Lung-breeding Live Blood To Treat Pulmonary Fibrosis And Experimental Study Of Intervention Of Notch Pathway Affects Pulmonary Fibrosis Cell Proliferation

Pulmonary fibrosis(PF)is mainly characterized by excessive accumulation of extracellular matrix(ECM).The causes of pulmonary fibrosis are diverse and the prognosis is poor.The results of the study confirm that PF can be caused by a variety of causes,but the exact and detailed etiology has not been known so far.GEO is currently the most non-tumor database used by researchers,from which researchers can publicly obtain a large number of microarray gene expression data sets,and the need for people to integrate gene expression data sets to obtain more accurate results continues to grow.However,no previous studies have involved comprehensive bioinformatics analysis of PF.Only a few differentially expressed genes have been found in PF,and the functional annotation of DEGs and the interactions between proteins are still unclear.According to the theory of traditional Chinese medicine,PF is classified into the categories of "pulmonary paralysis and lung dysentery",and it is considered that "lung qi and yin deficiency,blood stasis and blocking of the lungs" are important pathogenesis of PF,and its core pathogenesis is "lung qi deficiency-Blood stasis-phlegm turbid condensation-phlegm bruise qi-deficiency and spleen and kidney." Yang fei Huo xue Decoction is a clinical prescription of Professor Wen Canhui,a psychiatrist.Modern pharmacological studies have confirmed that the above-mentioned drug ingredients can inhibit PF to varying degrees.Yang fei Huo xue Decoction has theoretical basis and clinical efficacy and clinical efficacy.Traditional Chinese medicine treatment relies on "differentiation based on syndrome differentiation" and focuses on the implementation of the law.The specific prescription medication is based on the experience of doctors and "differs from person to person." The clinical evidence for treating PF with"nourishing lungs and activating blood" is quite effective.In order to enrich,but the reported results are not consistent,this article collects clinical randomized controlled trials(RCTs)and uses evidence-based medicine as the guide.The aim is to explore the clinical application of"yang fei huo xue" and its specific effect on PF patients.Clinicians provide objective theoretical basis.The regulation of abnormal fibroblasts(FB)/myofibroblasts(MFB)is mainly in response to transforming growth factor-β1(TGF-β1).Studies have confirmed that TGF-β1 signaling is closely related to fibrotic diseases such as lung,liver and kidney.Therefore,TGF-β1 has become a popular star target for studying tissue fibrosis.The mechanism of "Lung-Living and Activating Blood" method for the effective prevention and treatment of PF may include inflammatory immune response,micro angiogenesis,apoptosis,and the effects of multiple signaling pathways.It is worth noting that the effects of the above mechanisms are mostly related to the Notch signaling pathway.In view of this,this research is based on the research of the National Natural Science Foundation of China and the Doctoral Program of the Ministry of Education.First,it conducts a Meta-analysis of the clinical effects of lung nourishing and blood circulation methods,and uses bioinformatics analysis methods to screen and distinguish PF-related genes.Based on the systematic analysis of the expressed genes,the effects of the Notch pathway on the Yang fei Huo xue formula-containing serum on the phenotype of lung fibroblasts induced by TGF-β were further explored.Method1.Meta analysis:retrieve the research progress of traditional Chinese medicine for treating PF,extract the synonymous expressions related to "nourishing lungs and promoting blood circulation" and oral type of decoction or proprietary Chinese medicine,and perform in-depth and comprehensive article retrieval based on the extracted keywords and search the database Including Chinese databases such as CNKI,WanFang,VIP,CBM,and English databases such as Pubmed,EMbase,Web of Science,Cochrane Library,etc.The"Data Extraction Table"was compiled with reference to the method recommended by the Cochrane Collaboration Network,and the data were extracted independently,and the bias risk assessment of the RCT was based on the Cochrane Collaboration Network.Cochrane collaboration network RevMan5.3 was used for Meta-analysis.The Egger and Begg methods were used to test for publication bias,respectively.Sensitivity analysis was performed by removing individual studies.The GRADE system was used to evaluate the results.2.Bioinformatics analysis:According to the consensus statement of the American Thoracic Society and the European Respiratory Society,all PF patients included in this study met the diagnostic criteria of PF.Lung tissue samples from the normal control group were taken from patients without PF,including lung cancer patients and lung transplant patients.GSE110147 microarray data was downloaded from the Gene Expression Omnibus database.The samples included 22 patients with PF,10 patients with nonspecific interstitial pneumonia,5 patients with mixed PF-NSIP,and 11 normal volunteers.In this study,only 22 cases of PF were selected as the PF group,and 11 normal volunteers were selected as the control group.GE02R tool was used to analyze the differential expression of GEO sample data.The differentially expressed genes were further analyzed by GO enrichment analysis and KEGG pathway analysis,and DAVID online tools were used to perform GO and KEGG pathway enrichment analysis to obtain rich biological processes and pathways(P<0.01 is the threshold).Finally,we systematically analyze the relationships between proteins by constructing a PPI network,use the STRING online tool to analyze DEG’s PPI network,and cluster by k-means.3.Cell function experiment:This study used the animal-containing serum of the rabbit that raised the lung living blood and the removed side,and carried out the experiment of the original lung fibroblast.Experiment one successfully cultured the primary pulmonary fibroblasts by cell walling method,screened the optimal concentration and time of TGF-beta 1 inducing pulmonary fibroblast proliferation,and established the proliferation model of pulmonary fibroblasts,and then screened the best way for the drug to inhibit the proliferation model of pulmonary fibroblasts for follow-up experiments.Experiment Ⅱ used Western blot to detect protein expression levels such as Notch pathway and its cross-talk pathway Wnt key proteins Wnt key proteinS Notch1,NICD1,Jagged1,Hes1,nuclei beta-cateninin,p-GSK-3 beta,etc.of each given group.Experiment THREE was conducted by streaming cytometry,Western blot and immunofluorescent method to carry out qualitative and quantitative testing of the drug’s cycle and apoptosis effect on the cycle and apoptosis of lung fibroblasts.Experiment Ⅳ is to further prove that the drug through the Notch pathway has played a inhibitory effect on proliferation,after the NID1 slow virus transfection,Western blot detection NICD1 protein expression,NICD1 slow virus transfection mold success,the drug acts on the transfection model,The qualitative or quantitative study of cell proliferation,cell cycle,apoptosis,and Wnt pathway-related proteins in the NICD1 slow viral transfection model was carried out by the corresponding experimental methods respectively.Result1.1046 related documents were detected initially,190 documents were deleted after rechecking,and 856 were left.After layer-by-layer screening exclusion,43 articles were finally included for Meta-analysis of relevant clinical studies.The heterogeneity test result was[P=0.53,I2=0%],suggesting that there were no heterogeneity among the 22 studies.Therefore,a fixed effects model(Fixed Effects,FE)was selected for statistical analysis,and the absolute variable was selected as the result variable index.The degree of risk difference was RD.The statistical results were[RD=0.20,95%CI(0.16,0.24),P<0.00001],suggesting that the application of the method of nourishing lungs and promoting blood circulation can effectively improve clinical effectiveness.Subgroup analysis showed that the lung function related indicators Dlco,TLC,VC,FEV1,FVC,FEVI/FVC,and blood gas analysis related indicators PaO2 and PaCO2 were significantly improved,and 6MWT,dyspnea score,TCM syndromes(symptoms))The improvement of points and other indicators also shows that the method of nourishing lungs and activating blood is better than the control group in statistical significance.At the same time,the method of nourishing lungs and promoting blood circulation has a good reversal effect on laboratory serum indicators such as TNF-α,IV-C,Ig,and CD.In addition,the use of this method has an effect on specific indicators related to SGRQ,respiratory symptoms,and activity.Limits,disease effects,and overall scores all significantly improved.2.Using the GE02R tool to analyze the PF and DEGS of the control group in the GES110147 expression profile data set,a total of 707 DEGS met the selection criteria,478 genes were up-regulated,and 229 genes were down-regulated;DEGS performed GO and After pathway analysis,up-regulated DEGS-enriched GO BP entries are mainly related to ciliated components,ciliary movement,DNA duplex expansion,extracellular matrix tissue,mRNA splicing via spliceosome,osteoblast differentiation,RNA secondary structure expansion,Microtubule-based movement,protein-targeted vacuoles,and collagen catabolism,while down-regulated DEGS-enriched GO BP entries are mainly related to positive regulation of gene expression,cell response to tumor necrosis factor,and cell response to cadmium ions,Vasoconstriction,cell response to zinc ions,negative growth regulation,neutrophil chemotaxis and mitochondrial electron transport,cytochrome c transmission to oxygen and angiogenesis are related;KEGG analysis shows that the pathway that upregulates DEGs enrichment is mainly related to RNA Transport,adhesion,ECM-receptor interaction,platelet activation and protein digestion and absorption are related.The pathway of down-regulating DEGS enrichment is mainly related to oxidative phosphorylation,myocardial contraction,and element absorption,and also includes chemokine signaling pathways,Parkinson’s disease,cell adhesion molecules,and non-alcoholic fatty liver disease;based on data from the STRING 11.0 database A PPI network was constructed,and five genes in down-regulated genes participated in chemokine signaling pathways(CCL21,CXCL10,GNG11,XCL1,ARRB1).3.The primary culture of third-generation cell immunohistomydization visible waveform protein expression positive,more than 90%of cells dyed brown,cell morphology is clear,most of the shuttle-shaped changes,indicating that purified to the third generation of primary cells are fibroblasts,can be used for follow-up experiments;And at different points in time to observe the proliferation of cells,the results are as follows:TGF-beta]in the various phases of the pulmonary fibroblast savecell proliferation,compared with the control group,there is a significant difference(P 0.01);And the most statistically significant at 12h.4.Compared with the blank control group,the model group Notch 1,NICD1,Jagged1,Hes],nucleothal beta-cateninin,p-GSK-3 beta expression were significantly increased;The setting of the disassembling group also had an effect on the inhibition effect of each protein,and the inhibition effect of each protein had corresponding advantage targets.4.The setting of the demolition group also has an effect on the inhibition effect of each protein,the inhibition effect of each protein has the corresponding advantage target,the whole party-10%group on Notch1,NICD1,Jagged1,Hes1,nucleus beta-catenin p-GSK-3 beta has a regulatory effect the effective targets of the lung-10%group are Notch1,NICD1,Jagged 1,Hesl,intranuclear beta-catenin,and live blood-10%The active regulatory proteins are Notch1,Jagged1,Hes1,intranuclear beta-catenin,p-GSK-3 beta.5.The effect of the drug on apoptosis and cycle found that the prosthesis group promoted the expression of Caspase3 to varying degrees and the apoptosis of cells to varying degrees.The effect of the drug on the cycle of pulmonary fibroblasts found that compared with the model group,the whole square-10%group and the pulmonary-10%group significantly increased the proportion of G1 cells,reduced the percentage of G2 and S cells,and inhibited cell division.In addition,the drug on the lung fibroblast cycle-related protein CyclinD1 has different degrees of inhibition,reflecting different degrees of cycle inhibition,the overall data analysis results show that the inhibition of pulmonary-10%group of pulmonary fibroblasts is mainly through cycle inhibition,and the inhibition of active blood group on pulmonary fibroblast proliferation is mainly by promoting cell apoptosis.6.Immunofluorescent detection showed that slow virus transfection efficiency is high,high-dose lung-raising blood-containing serum can significantly reduce cell proliferation after treatment of 24h,36h,48h.The drug effect is not time-sensitive,the inhibition effect of the action 36h on cell proliferation is most obvious,the various dosing groups promote the apoptosis of cells and the expression of Caspase3 to varying degrees,increase the percentage of G1 cells,inhibit the proliferation of cells.Conclusion1.Evidence-based medical evidence shows that lung nourishing and blood circulation can improve clinical symptoms and some objective indicators of PF patients;2.Bioinformatics combined with literature traceability analysis found that the gene-differentiated expression of pulmonary fibrosis participated in ECM formation,and the effect of lung-breeding blood method on ECM suggested the possible target of the corresponding group;3.Yangfei Huoxue Decoction-containing serum can inhibit the proliferation of lung fibroblasts induced by TGF-β1;4.The lung-raising living blood prescription and its antidote-containing serum can inhibit the activation of Notch and the ghetto signaling pathway Wnt,regulate cell cycle and promote apoptosis,and regulate the protophaficell proliferation phenotypes.Innovation1.Firstly,Meta provide evidence-based analysis of the clinical efficacy PF nourishing lung and activating blood circulation therapy,then,explore the genes and signaling pathways related to PF from bioinformatics mining data,and finally,complete the phenotypic function verification from cell experiments,and demonstrate the subject of this study from multidimensional aspects;2.Based on the analysis in the previous section,we explored the changes of Notch signaling pathway affecting the cell cycle,apoptosis and proliferation of lung fibroblasts through TGF-β1 induction of pulmonary fibrosis model in rat primary cells.