Syndrome Distribution Of Patients With Pulmonary Fibrosis And Transcriptomic Study On The Intervention Of Dahuang(?)chongwan On Pulmonary Fibrosi

Objective:This study investigates the characteristics of pulmonary fibrosis and its associated factors,as well as the prevalence of blood stasis in patients with pulmonary fibrosis,based on the distribution of evidence patterns in patients with post-infection pulmonary fibrosis.In addition,this study also established a diagnostic model of blood stasis evidence in patients with pulmonary fibrosis.The aim is to characterize the distribution of evidence in patients with pulmonary fibrosis and to provide objective criteria for the identification of blood stasis evidence in pulmonary fibrosis.Meta-analysis provides an objective evaluation of the clinical efficacy of the combination of blood-promoting and blood stasis removing Chinese medicines and western medicines in the treatment of IPF,to provide an evidence-based basis for the use of blood-vitalizing and blood-stasis-removing methods in the clinical treatment of IPF.The experimental part was validated by transcriptome sequencing technology and experiments to further investigate the mechanism of action of Dahuang Zhechong pills in the treatment of pulmonary fibrosis.Methods:1 285 patients with pulmonary fibrosis who met the diagnostic criteria were included.The general medical history data of the patients were collected,while the four Chinese medicine consultations of the patients were collected by two experts of associate chief physicians or above to determine the traditional Chinese medicine syndrome,and the data were statistically analyzed using SPSS26.0 software to analyze the relevant factors affecting the distribution of the traditional Chinese medicine syndrome.And the Logistic regression analysis was used to establish the blood stasis evidence of pulmonary fibrosis.2.This study was conducted in Pubmed,Ovid Medline,Scopus,Embase,Web of Science,CochranceLibrary,Chinese Biomedical Database,Chinese National Knowledge Infrastructure,Chongqing VIP information,ClinicalTrials.gov,International ClinicalTrials Registry(ICTRP)and Chinese ClinicalTrials Registry(ChiCTR)were used for literature search.The search period is from the beginning of database construction to November 17,2022.In addition,this study also screened the included literature and references for related meta-analyses.Search is limited to English and Chinese.Randomized controlled trials(RCTS)on the treatment of IPF with Chinese patent drugs for promoting blood circulation and removing blood stasis were collected,and the literature needed for this study was screened out according to the inclusion criteria.We used the Cochrane collaboration’s tool for assessing the risk of bias in randomized trials to assess the risk of bias of included studies.Data was extracted from the studies included,and meta-analysis was performed on the extracted data using Revman5.4.1.Weighted mean difference(WMD)was used for continuous variables,and relative risk(RR)was used as the effect size for binary variables.According to the results of the I2 test,the fixed effects model or random effects model was selected for meta-analysis.P<0.05 was considered to be statistically significant.Subgroup analysis and meta-regression were used to analyze the source of heterogeneity.In addition,funnel plot egger’s and Begg’s test,and GRADE grading was used to detect publication bias and to evaluate the quality and sensitivity of the evidence.3 Intratracheal drip of bleomycin method to establish the animal model of pulmonary fibrosis in rats was set Control group,the BLM group,and Dahuang Zhechong pill(DHZCP)group.The survival and body weight of the rats were recorded.The lung function of the rats was detected 28 days after modeling,and then the rats were put to death.HE and Masson staining were performed on the fixed part of the lung tissue,and the inflammation and fibrosis of the lung tissue of rats were evaluated by the improved Ashcroft score and alveolar inflammation score.The transcriptional profiles of mRNAs from different groups of rat lung tissues were sequenced and analyzed by high-throughput transcriptome sequencing technology,and the differentially expressed genes were screened and GO,KEGG,and Hallmark enrichment analyses were performed to obtain the key pathways and targets of Dahuang Zhechong pill for lung fibrosis intervention and select some of these for experimental verification.The Westen blot assay detects the epithelial-mesenchymal transition(EMT)-associated proteins E-cadherin(E-cad),Ncadherin(N-cad),α-smooth muscle actin(α-SMA),fibronectin(Fn),the apoptosis-associated protein Cleaved-Caspase-3,transforming growth factor-beta(TGF-β)/Hedgehog signaling pathway-associated protein TGF-β1,glioma-associated oncogene(Glil),sonic hedgehog(Shh),smoothened receptor(Smo)and hypoxia-inducible factor(HIF-1)signaling pathway-associated protein HIF-1α,immunofluorescence detection of lung tissue α-SMA expression and surfactant protein C(SP-C)and B cell lymphoma-2 associated X protein(Bcl-2).A TGF-β1-induced EMT model of RLE-6TN cells was established and set Control group,TGF-β1 group,DHZCP group,Smo agonist SAG group,and DHZCP+SAG group for intervention.qRT PCR and Western blot were used to detect the expression of Gli1,Shh,Smo mRNA and protein,and Western blot was used to detect the expression of E-cad and N-cad in each group.A CoCl2-induced hypoxia model of RLE-6TN cells was established and divided into the Control group,TGF-β1 group,DHZCP group,HIF-1α agonist DMOG group,and DHZCP+DMOG group for each intervention.CCK-8 method was used to detect cell viability.TUNEL staining was used to detect apoptosis.Western blot was used to detect the expression of Bcl-2 and B lymphocytoma-2-associated X protein(Bax).Western blot,qRT PCR,as well as immunofluorescence assay,were used to detect the expression of HIF-1α in each group.Results:1 1 The results of this study show that the deficiency of qi in lung and blood stasis syndrome,deficiency of qi in the lung,phlegm-damp(turbid)and blood stasis syndrome,and deficiency of qi in lung syndrome were most common in pulmonary fibrosis.The main syndromes in pulmonary fibrosis were deficiency of qi in lung and deficiency of both qi and yin in lung,while the secondary syndromes were mainly blood stasis syndrome,and phlegm-damp(turbid)syndrome.The proportion of blood stasis syndrome was the highest,which shows the prevalence of blood stasis syndrome in the pulmonary fibrosis population.The distribution of syndrome was associated with gender,mMRC dyspnoea grade,FVC%,FEV1%,DLCO%,traction bronchiectasis,ILD-GAP grade,and the diagnosis of pulmonary fibrosis.Diagnostic models of blood stasis syndrome for pulmonary fibrosis and different diagnostic classifications of pulmonary fibrosis were established based on the Logistic regression analysis.After the goodness-of-fit test and ROC test,the model had good calibration ability and high predictive value.2 Overall,26 studies were enrolled in the review.2 studies evaluated the efficacy of CPM plus N-acetylcysteine(NAC)and NAC alone.Meta-analysis of partial arterial oxygen concentration(PaO2)(MD=14.21,95%CI[12.90,15.52],P<0.00001)was in favor of CPM plus NAC treatment.When comparing CPM plus conventional western medicine(CWM)with CWM,only 1 study was included,suggesting that CPM plus CWM had a superior effect in diffusing lung capacity of carbon monoxide(DLCO)(MD=1.54,95%CI[0.61,2.47],P=0.001).16 studies compared CPM plus glucocorticoids(GC)with GC,and the meta-analysis showed that forced vital capacity(FVC)(MD=0.45,95%CI[0.37,0.54],P<0.00001),total lung capacity(TLC)(MD=0.45,95%CI[0.29,0.61],P=0.00001),DLCO(MD=4.04,95%CI[3.49,4.59],P<0.00001),DLCO%(MD=4.21,95%CI[0.97,7.45],P=0.01),FEV1/FVC(MD=10.75,95%CI[5.31,16.19],P=0.0001),PaO2(MD=2.77,95%CI[10.97,14.56],P<0.00001),6MWT(MD=67.32,95%CI[42.67,91.97],P<0.00001),the incidence of reinfection(RR=0.56,95%CI[0.42,0.75],P<0.0001)improved more in the CPM plus GC group.No serious adverse events were observed in the intervention group.3 Experimental part 3.1 The survival rate of rats in the BLM group was reduced compared to Control,and the survival rate of rats in the DHZCP group was increased compared to the BLM group,but the difference was not statistically significant(P>0.05).Compared with the Control group,the BLM group had significantly lower body weight,lung function FVC,FEV0.4,and lung dynamic compliance(Cydn),and significantly higher inspiratory resistance(Ri),alveolitis score,and modified Ashcroft score;compared with the BLM group,the DHZCP group had significantly higher FVC,FEV0.4 and Cydn,and significantly lower Ri,alveolitis score and modified Ashcroft score.The differences were statistically significant(P<0.05),while there was no significant difference in expiratory resistance(Re)between the groups(P>0.05).3.2 The results of the enrichment analysis of 209 DEGs indicated that Dahuang Zhechong pill may play an anti-fibrotic role through the regulation of signaling pathways like TGF-β,Hedgehog,NF-κB,HIF-1 signaling pathways,affecting pathological aspects such as EMT,cellular senescence,apoptosis,platelet activation,myofibroblast activation,and hypoxia.3.3 In the lung tissue of rats,E-cad was decreased and N-cad,α-SMA,Fn,Cleaved-Caspase-3,Shh,Smo,Gli1,HIF-1α were increased in the BLM group compared to the Control group;while N-cad,α-SMA,Fn,Cleaved-Caspase-3,Shh,Smo,Glil,HIF-1α decreased and E-cad increased in the DHZCP group compared to the BLM group;the difference was statistically significant(P<0.05).IF staining of α-SMA showed that the expression of α-SMA was increased in the BLM group compared to the Control group and decreased in the DHZCP group compared to the BLM group.IF co-localization of SP-C and Bcl-2 showed that the expression of Bcl-2 protein in the SP-C positive region was decreased in the BLM group compared to the Control group;the expression of Bcl-2 in the SP-C positive region was increased in the DHZCP group compared to the BLM group.In the TGF-β1-induced EMT model of RLE-6TN cells,compared to the Control group,the mRNA and protein expression of Glil,Shh,and Smo were increased,the expression of E-cad protein was decreased,and the expression of N-cad protein was increased in the TGF-β1 group;whereas compared with the TGF-β1 group,the mRNA and protein expression of Gli1,Shh,and Smo decreased,the expression of E-cad protein increased and the expression of N-cad protein decreased in the DHZCP group;furthermore,compared with the DHZCP group,the expression of E-cad protein was increased and N-cad protein was decreased in the DHZCP+SAG group;the differences between the groups were statistically significant(P<0.05).In the CoCl2-induced hypoxia model of RLE-6TN cells,compared to the Control group,the CoCl2 group showed significantly lower cell viability,significantly increased mRNA and protein expression of HIF-1α,significantly increased expression of Bax protein,while significantly decreased expression of Bcl-2(P<0.05)and increased number of TUNEL-positive cells;whereas,compared with the CoCl2 group,the DHZCP group showed significantly higher cell viability,reduced mRNA and protein expression of HIF-1α,significantly decreased expression of Bax protein,significantly increased expression of Bcl-2(P<0.05),and decreased number of TUNEL-positive cells;compared with the DHZCP group,the expression of Bcl-2 was significantly lower in the DHZCP+DMOG group compared with the DHZCP group(P<0.05),and the number of TUNEL-positive cells was increased,but the difference of the expression of Bax compared with DHZCP group was not statistically significant(P>0.05).Conclusion:1 Blood stasis evidence is prevalent in the pulmonary fibrosis population,and the diagnostic model of pulmonary fibrosis in patients with pulmonary fibrosis and different diagnostic classifications of pulmonary fibrosis with blood stasis syndrome has certain diagnostic properties,laying the foundation for further research on the objectification of the diagnosis of blood stasis syndrome in patients with pulmonary fibrosis.2 The results of the Meta-analysis showed that blood promoting and blood stasis removing Chinese medicines improved lung function,6MWT,arterial blood gas analysis and the incidence of reinfection in IPF patients with good safety.3 Dahuang Zhechong pill can reduce the inflammation and pulmonary fibrosis caused by bleomycin and improve the lung function of rats.Dahuang Zhechong pill may play an anti-fibrotic role by modulating signaling pathways such as the TGF-β signaling pathway,Hedgehog signaling pathway,NF-κB signaling pathway,and HIF-1 signaling pathway,and affect pathological aspects such as EMT,cell senescence,platelet activation,myofibroblast activation,hypoxia,and apoptosis.Dahuang Zhechong pill can improve lung fibrosis by inhibiting the activation of the HIF-1 signaling pathway to reduce the apoptosis of type Ⅱ alveolar epithelial cells and simultaneously reducing the activation of the TGF-β1/Hedgehog signaling pathway to alleviate EMT.