Study On The Mechanism Of Qingluoyin Regulating TGF-β₁/Smads Pathway To Intervene In Apoptosis In Acute Exacerbation Of Idiopathic Pulmonary Fibrosi

Purpose:This study aims to explore the research hotspots of idiopathic pulmonary fibrosis and its acute exacerbation through literature research,as well as the mechanism of action of traditional Chinese medicine compound Qingluo Yin in treating acute exacerbation of idiopathic pulmonary fibrosis;Innovate the modeling method for animal models of acute exacerbation of idiopathic pulmonary fibrosis through experimental research,explore the regulation of TGF-β₁/Samds signaling pathway by Qingluo Yin,exert its anti apoptotic effect,and further improve the efficacy evaluation and mechanism research of Qingluo Yin;Propose an innovative theory of"etiology,pathogenesis,syndrome differentiation,and treatment"in traditional Chinese medicine for acute exacerbation of idiopathic pulmonary fibrosis through literature research.Method:1.Based on bibliometrics,evidence-based medicine and network pharmacology,explore idiopathic pulmonary fibrosis and traditional Chinese medicine Qingluo Decoction:bibliometrics,evidence-based medicine adopts CNKI,Wanfang data knowledge service platform,Weibo,China Biomedical Literature Service System（Sinomed）,Baidu Academic,Google Scholar data sources,According to the research purposes of bibliometrics and evidence-based medicine,the retrieval and inclusion criteria were set,and the data were analyzed by using Citespace software,Rev Man 5.3 software,stata15.1 software,and ADDIS1.16.6 software,respectively,to obtain the literature visualization results and mesh meta-analysis results.Network pharmacology uses TCMSP database and BATMAN-TCM database to obtain effective drug targets,Dis Ge NET database and Gene Cards database to obtain disease target genes,Venny2.1 tool to obtain intersection targets,Cytascape 3.7.1software to build"effective drug components-intersection genes"action network,and STRING database to build protein-protein interaction network of targets,The DAVID Bioinformatics Resources database was used to enrich the GO function and KEGG pathway of key target genes.2.Establishment of an animal model of acute exacerbation of idiopathic pulmonary fibrosis induced by bleomycin and lipopolysaccharide in rats:150 healthy male Wister rats were randomly divided into negative control group（Control group）,remission phase model group（IPF group）,acute exacerbation phase model group（BLM group）,low dose lipopolysaccharide group（LPS-low group）,medium dose lipopolysaccharide group（LPS-mid group）,and high dose lipopolysaccharide group（LPS-high group）,The acute exacerbation model was induced by the first intratracheal injection of bleomycin（BLM）and the second intraperitoneal injection of lipopolysaccharide（LPS）（0 d,BLM,5 mg/m L;28 d,LPS,2.5mg/m L,5 mg/m L,5 mg/m L）in the BLM group,except that the BLM group was induced by two consecutive intratracheal injections of bleomycin（BLM）（0 d,5 mg/m L;28 d,7mg/m L）;The samples were taken at 31d,35d and 42d after the establishment of the model to observe the survival of the rats,the ratio of wet to dry weight（W/D）of lung tissue,lung coefficient,HE staining,Masson staining,lung CT,lung function,arterial blood gas analysis,bronchoalveolar lavage fluid（BALF）component analysis（total cell count,neutrophil count,TNF-αMass concentration）,serum IL-6 mass concentration,hydroxyproline（HYP）content in lung tissue,lung tissueαSMA immunohistochemical analysis index.3.Qingluo Yin regulates TGF-β₁/Smads signal transduction pathway and the mechanism of intervention in AE-IPF rat apoptosis:315 male Wistar rats were injected with bleomycin through the air tube for the first time and lipopolysaccharide for the second time to establish AE-IPF model.The rats were divided into negative control group（Control group）,remission phase model group（IPF group）,acute exacerbation phase model group（AE-IPF group）,low dose group of traditional Chinese medicine（QLY-low group）,medium dose group of traditional Chinese medicine（QLY-mid group）The high-dose group（QLY-high group）and hormone group（PNS group）of traditional Chinese medicine were given prednisone acetate by gavage in the hormone group,and the middle,high and low doses of traditional Chinese medicine compound Qingluo Decoction were given in the traditional Chinese medicine group.Lung tissue biopsy,HE staining,Masson staining,lung function detection,arterial blood gas analysis,hydroxyproline determination,bronchoalveolar lavage and other methods were used to evaluate the lung function and degree of pulmonary fibrosis in rats,and immunohistochemistry,Western-Blot,quantitative RT-PCR and other methods were used to detect the lung tissue in ratsα-SMA、TGF-β₁、TGFβRⅠ、TGFβRⅡ,Smad2,Smad4,Smad7,Bax,Bcl-2,Fas,Fas L,Cas Pase-3 for detection and analysis of TGF-β₁/Smads signal transduction pathway and apoptosis-related protein intervene in the process of acute exacerbation of pulmonary fibrosis in rats.4.Qingluo Yin regulates TGF-β₁/Study on the mechanism of Smads signal transduction pathway interfering with the apoptosis of alveolar epithelial cells:50 Wistar rats,20 of which were used for the preparation of drug-containing serum and 30 for the primary extraction of cells.Prepare Qingluoyin medicated serum.The drug efficacy evaluation cells were divided into control group,model group and QLY group,and the pathway mechanism validation cells were divided into model group,agonist group and QLY-Agonist group.The primary cells were identified and evaluated by immunofluorescence method,the law of cell apoptosis was analyzed by ELISA,and the cells were detected by Western-Blot,Quantitative RT-PCR and other methodsα-SMA and SP-C were tested to evaluate the effectiveness of the drug,and TGF-β₁、TGFβRⅠ、TGFβRⅡ,Smad2,Smad4,Smad7,Bax,Bcl-2,Fas,Fas L,Cas Pase3cells were tested to verify that Qingluo Yin can regulate TGF-β₁/Smads signal transduction pathway interferes with apoptosis of alveolar epithelial cells.5.Based on the theory of collateral disease and the existing research results of the research group,the author uses the theoretical analysis methods with dialectical thinking,such as induction and deduction,analysis and synthesis,abstraction and concrete,to explore and innovate the cause,mechanism,syndrome and treatment of AE-IPF,and provide a theoretical basis for the experimental research and clinical practice of AE-IPF.Results:1.Based on bibliometrics,evidence-based medicine and network pharmacology to explore idiopathic pulmonary fibrosis and traditional Chinese medicine compound Qingluo Decoction:（1）The bibliometrics research results show that the overall number of IPF research in the field of traditional Chinese medicine is on the rise,and researchers from different research institutions cooperate less,and there are certain academic barriers,but the research topics are diverse,involving treatment research,literature research,clinical research,and animal experimental research,Among them,the prevention and treatment of IPF based on the theory of collateral diseases in traditional Chinese medicine is a research hotspot.（2）Evidence-based medicine research results show that the traditional Chinese medicine compound prescription based on TCM collateral disease theory is superior to western medicine in the treatment of IPF,with a high total effective rate.TCM collateral disease theory can be used as the guiding ideology for the treatment of IPF;Intervention measures The traditional Chinese medicine compound is mainly composed of pungent medicine,insect medicine,rattan medicine,and tonic medicine,which embodies the general method of treating collateral diseases by means of dredging;There is some bias in the study,which needs further verification.（3）The results of network pharmacology research show that the active ingredients in Qingluo Decoction,a traditional Chinese medicine compound,are diverse and have broad intervention targets for AE-IPF.It can participate in a variety of biological processes and intervene in AE-IPF process through a variety of ways.For example,it represents the active ingredients:baicalin,quercetin,robin,gene target:SRC,STAT3,EGCR,biological process:drug reaction,protein phosphorylation,apoptosis process,and pathway:PI3K-Akt signal pathway,apoptosis,Th17cell differentiation.2.The establishment of an animal model of acute exacerbation of idiopathic pulmonary fibrosis induced by bleomycin and lipopolysaccharide in rats:（1）The rats in the model group showed obvious shortness of breath,dyspnea,cyanosis of claw lips,decreased body mass,and increased mortality,among which the LPS-mid group and BLM group showed more consistent performance（P>0.05）;（2）The W/D value and lung coefficient of rats in the model group were significantly increased,and acute pulmonary edema appeared,among which the LPS-mid group and BLM group were more consistent（P>0.05）;（3）The pathological changes in the model group were consistent with the classic pathological manifestations of the disease,and the LPS-mid group was more consistent with the BLM group（P>0.05）;（4）In the model group,the lung CT showed patchy cotton-like shadow and irregular density increase shadow,especially in LPS-high group;（5）The lung function and blood gas analysis structure of the model group showed a downward trend,and the performance of LPS-mid group was more consistent with that of BLM group（P>0.05）;（6）The total number of cells and neutrophils in rat alveolar lavage fluid increased,and IL-6 and TNF increased-αThe mass concentration increased,and the performance of LPS-mid group was more consistent with that of BLM group（P>0.05）;（7）The content of HYP in the lung tissue of rats in the model group,α-The expression of SMA was increased,and the expression of LPS-mid group was more consistent with that of BLM group（P>0.05）.3.Qingluo Yin regulates TGF-β₁/Smads signal transduction pathway and the mechanism of intervention in AE-IPF rat apoptosis:（1）Compared with rats in AE-IPF group,rats in QLY-mid group had obvious remission of disease symptoms and low mortality,which was not significantly different from those in PNS group;（2）The results of lung tissue observation,lung wet-dry weight ratio,lung coefficient and pulmonary fibrosis score（Masson staining）showed that compared with AE-IPF group,the lung tissue texture of QLY-mid group was softer,there were less ecchymosis and ecchymosis,and the degree of pulmonary edema and pulmonary fibrosis was lighter（P<0.05）,but it was similar to PNS group in lung tissue appearance,pulmonary edema,alveolitis and pulmonary fibrosis（P>0.05）;（3）The results of alveolitis score（HE staining）and bronchoalveolar lavage fluid composition analysis showed that the inflammation in QLY-mid group was lighter than that in AE-IPF group,with significant difference（P<0.05）,but there was no significant difference between QLY-mid group and PNS group（P>0.05）;（4）The analysis of pulmonary function and arterial blood gas showed that Pa O₂ in QLY-mid group and PNS group was similar to that in Control group（P>0.05）,and drug treatment was effective in improving the hypoxia state of rats;（5）The content of HYP and the expression ofα-SMA in lung tissue are important indicators reflecting the severity of pulmonary fibrosis in this study.The results showed that there was no difference between QLY-M group and PNS group（P>0.05）,but there was significant difference between the two groups and AE-IPF group（P<0.05）;（6）Through the localization,quantitative analysis and m RNA expression quantitative analysis of key cytokine proteins in TGF-β₁/Smads signal transduction pathway and cell apoptosis by immunohistochemistry,Western-Blot method and q PCR method,it can be seen that the expression of TGF-β₁、TGFβRⅠ、TGFβRⅡ、Smad2、Smad4、Smad7、Bax、Bcl-2、Fas、Fas L、Cas Pase-3 protein and m RNA in QLY-mid group and PNS group is significantly different from that in AE-IPF group（P<0.05）,and the analysis results between QLY-mid group and PNS group are consistent（P>0.05）.4.Qingluo Yin regulates TGF-β₁ Study on the mechanism of signal transduction pathway interfering with apoptosis of alveolar epithelial cells:（1）Most of alveolar epithelial cells were cubic or round,and a few were flat or wide.Immunofluorescence results showed that SP-C expression was positive,and Vimentin was positive compared with normal cells;（2）The expression ofα-SMA、SP-C protein and m RNA in QLY group was significantly different from that in Control group and Model group（P<0.05）;（3）There were significant differences in the expression of TGF-β₁、TGFβRⅠ、TGFβRⅡ、Smad2、Smad4、Smad7、Bax、Bcl-2、Fas、Fas L、Cas Pase-3 protein and m RNA between QLY-Agonist group and Model group and Agonist group（P<0.05）;（4）There were significant differences in cell apoptosis between QLY group and Control group and Model group（P<0.05）;There was significant difference in apoptosis between QLY-Agonist group and Model group and Agonist group（P<0.05）.5.The"pathogenesis syndrome and treatment"of AE-IPF described in this study is based on the collateral disease theory.Based on the existing research,the innovative discussion of its theory is carried out.Combined with the theories of"three-dimensional network","control and leveling"and"collateral for use"of collateral disease,it is expounded that the pathogenesis of AE-IPF’s collateral disease is lung and collateral disorders due to abnormal natural environment,psychosocial stress,abnormal living and living conditions,accumulation of metabolic products and animal trauma of golden blade insects,The syndrome types of collateral diseases are lung collateral injury syndrome,lung collateral pathogenic stagnation syndrome,and lung collateral deficiency syndrome.In the treatment,the collaterals should be unblocked to remove pathogenic factors,and the collaterals should be replenished to help control,and the collaterals should be unblocked to remove pathogenic factors,while repairing the lung collateral function and mobilizing its self-stabilizing function.Conclusion:1.Research on bibliometrics,evidence-based medicine and network pharmacology shows that IPF is a research hotspot in the field of traditional Chinese medicine,and traditional Chinese medicine compound has good efficacy in the treatment of IPF,TGF-β₁/Smads signal transduction pathway and cell apoptosis can be used as the research target for the intervention of traditional Chinese medicine Qingluoyin on AE-IPF.2.The AE-IPF disease model of rats can be made by successively injecting bleomycin through the air tube and intraperitoneal injection of lipopolysaccharide.The specific dosage is:bleomycin concentration:5mg/m L,dose:0.1m L/100g;LPS concentration:5mg/m L,dose:0.5m L/100g;AE-IPF was the most significant at 31 days.Compared with twice injection of bleomycin through gas tube,this method is simpler.3.Traditional Chinese medicine compound Qingluo Yin interferes with AE-IPF by TGF-β₁/The regulation of Smads signal transduction pathway is the center,and the result of multi-target interaction with anti-apoptosis as the synergy.The medium dose has the best effect,which can reduce the death of AE-IPF,improve clinical symptoms,improve lung function and blood oxygen,alleviate acute inflammation in the lung,repair damaged tissue,and inhibit the process of EMT and ECM.4.The traditional Chinese medicine Qingluo Yin can regulate TGF-β₁/Smads signal transduction pathway interferes with apoptosis of alveolar epithelial cells in AE-IPF rats,namely TGF-β₁/Smads signal transduction pathway can inhibit the expression of proapoptotic factors Fas,Fas L,Bax,Cas Pase3,and promote the expression of anti-apoptotic factor Bcl-2.5.The collateral disease of AE-IPF is caused by abnormal natural environment,psychosocial stress,abnormal living conditions,accumulation of metabolic products and trauma of golden blade insects.The pathogenesis of collateral disease is lung collateral disorder,and the syndrome types of collateral disease are lung collateral injury syndrome,lung collateral pathogen stagnation syndrome,and lung collateral deficiency syndrome.In the treatment,the collateral should be unblocked to remove the pathogenic factors,and the collateral should be replenished to support the control.The collateral dredging method should be used to remove the pathogenic factors,while repairing the lung collateral function and mobilizing its self-stabilizing function.