Study On The Mechanism Of Dahuang(?)chongwan In Treating Pulmonary Fibrosis Based On Rho/ROCK Signaling Pathwa

Research purposesBased on the theory of "pulmonary collateral disease",the tutor Professor Zhang Lishan proposed the hypothesis that "dry blood exists in the lung collaterals of patients with IPF ",and believes that "dry blood in the lung collaterals is the cause of pulmonary fibrosis,and extracellular matrix deposition is the essence of dried blood".This study firstly explored the efficacy and safety of traditional Chinese medicine based on the "activating blood and dredging collaterals" method in the treatment of IPF;Secondly,the antifibrotic effect and mechanism of Dahuang Zhechong Pill,was investigated on transforming growth factor-β1(TGF-β1)-induced human alveolar epithelial cell line(A549)and on BLM-induced pulmonary fibrotic rats,in order to enrich the academic connotation of IPF prevention and treatment by removing the lung and collaterals.Research method1.In terms of systematic review,by searching China National Academic Literature Database(CNKI),Wanfang Digital Journal Database,China Biomedical Literature Database(CBM),VIP Chinese Science and Technology Periodical Database(VIP),PubMed,The Cochrane Library and Embase,etc.The search period was from the establishment of the database to February 15,2022.Randomized controlled trials(RCTs)comparing traditional Chinese medicine with conventional western medicine in the treatment of idiopathic pulmonary fibrosis were collected.The Cochrane risk bias assessment tool was used to evaluate the quality of the included studies.Meta-analysis was performed with Review Manager 5.4 software,and publication bias was tested using an inverted funnel plot.2.In terms of animal experiments,it was divided into blank control group(Control,n=10),model group(Model,n=14),pirfenidone group(PFD,n=14),and Dahuang Zhechong Pill highdose group(DHZC-H,n=14),middle-dose Dahuang Zhechong Pill group(DHZC-M,n=14),and Dahuang Zhechong Pill low-dose group(DHZC-L,n=14).Analysis of pathological changes in lung tissue by HE and Masson’s trichrome staining;Immunofluorescence detection to analyze the secretion of α-SMA in lung tissue;Elisa to detect serum TGF-β1,TNF-α,IL-6 levels;RT-PCR to detect TGF-β1,α-SMA and ROCK1 mRNA expressions;Western The expression of TGF-β1,p-RhoA,ROCK1,p-MLC,α-SMA,Collagen Ⅰ/Ⅲ,CTGF protein in lung tissue was detected by blot.3.In terms of cell experiments,it was divided into blank control group(DMS-C),model group(DMS-MOD),western medicine group(DMS-PFD),model+Dahuang Zhechong Pill containing high-dose serum group(DMS-H),model+Dahuang Zhechong Pills drug serum medium dose group(DMS-M),model+Dahuang Zhechong Pills drug serum low dose group(DMS-L).A549 cells in logarithmic growth phase were cultured normally,and the morphological changes of A549 cells were observed under electron microscope after TGF-β1(5ng/ml)induction for 48 hours;and the effect of drug serum containing Dahuang Zhechong Pill on the proliferation of A549 cells after TGF-β1 intervention was observed by cck-8 method;E-cadherin,Fibronectin,α-SMA and ROCK1 mRNA were detected by RT-PCR and CollagenⅠ/Ⅲ mRNA expression;Western blot detection of Smad2,p-Smad2,p-RhoA,ROCK1,p-MLC and α-SMA protein expression.Research result1.Systematic review of "Huoxue Tongluo" traditional Chinese medicine in the treatment of IPFA total of 21 RCTs were included in this study,with a total of 1550 patients,795 in the experimental group and 755 in the control group.The sample size ranged from 37 to 140,with an average sample size of 74.According to different treatment methods,it was divided into three subgroups for analysis:traditional Chinese medicine+hormones,traditional Chinese medicine+N-acetylcysteine,and traditional Chinese medicine+other.The main outcome indicators were total effective rate,FEV1%,DLCO%,PaO2,PaCO2,6-MWT,HRCT score,TGF-β1,TNF-α,SGRQ score,TCM symptom score and adverse reactions.Overall response rate:Overall response rate was reported in 17 studies with a total of 1254 patients.Among them,the clinical efficacy of traditional Chinese medicine+hormone therapy was better(RR=1.28,95%CI[1.15,1.41],P<0.000 01);the clinical efficacy of traditional Chinese medicine+N-acetylcysteine was better(RR=1.40,95%CI[1.04,1.89],P=0.03);Chinese medicine+other treatments had better clinical efficacy(RR=1.45,95%CI[1.15,1.83],P=0.002).DLCO%:DLCO%was reported in 13 studies with a total of 950 patients.Among them,the clinical effect of traditional Chinese medicine+hormone therapy was better(MD=2.89,95%CI[0.52,5.27],P=0.02);the clinical effect of traditional Chinese medicine+N-acetylcysteine was better(MD=4.98,95%CI[1.49,8.47],P=0.005);Chinese medicine+other treatments had better clinical efficacy(MD=4.95,95%CI[1.68,8.22],P=0.003).6-MWT:Seven studies reported 6-MWT with a total of 434 patients.Among them,the clinical effect of traditional Chinese medicine+hormone therapy was better(MD=22.77,95%CI[12.84,32.70],P<0.000 01);the clinical effect of traditional Chinese medicine+Nacetylcysteine was better(MD=45.83,95%CI[14.73,76.94],P=0.004);Chinese medicine+other treatments had better clinical efficacy(MD=45.18,95%CI[19.97,70.39],P=0.0004).Adverse events:Adverse events were reported in 8 studies with a total of 531 patients.Among them,the clinical effect of traditional Chinese medicine+hormone therapy was better(MD=0.16,95%CI[0.05,0.50],P=0.002);the clinical effect of traditional Chinese medicine+N-acetylcysteine was better(MD=0.36,95%CI[0.15,0.85],P=0.02);Chinese medicine+other treatments had better clinical efficacy(MD=17.00,95%CI[1.03,281.50],P=0.05).2.Results of animal experimentsHE and Masson staining results:The lung tissue structure of Control rats was relatively ideal,the alveolar septum was thin,and there was no inflammatory reaction;the lung tissue of Model rats was severely inflamed,the alveolar septum was significantly thickened,and inflammatory cells were infiltrated.Compared with Model,in DHZC-H,DHZC-M,DHZC-L,less inflammatory cells,collagen fiber deposition and better alveolar structure were observed in rat lung tissue.Immunofluorescence staining results:Compared with the Model,the lung tissue of the Control rat was normal,showing a very small amount of green staining,while in DHZC-H,DHZC-M,and DHZC-L,less α-SMA secretion and less α-SMA were observed in the rat lung tissue.Alveolar structure was better(P<0.05 or P<0.01).ELISA results,compared with Control,the serum expression of TGF-β1,TNF-α,IL-6 in Model was significantly(P<0.05 or P<0.01);compared with Model,in DHZC-H,DHZC-M,DHZCL,the above expression of inflammatory factors in rat serum decreased to different degrees(P<0.05 or P<0.01).RT-PCR results:Compared with Control,the expression of TGF-β1,α-SMA and ROCK1 mRNA in Model lung tissue was significantly(P<0.05 or P<0.01);compared with Model,DHZC-H,DHZC-M,DHZC-L,the expression of the above genes decreased in different degrees in lung tissue(P<0.05 or P<0.01).Western blot results:Compared with Control,the expression of TGF-β1,p-RhoA,ROCK1,pMLC,a-SMA,Collagen Ⅰ/Ⅲ,CTGF protein in the lung tissue of Model increased(P<0.05 or P<0.01);In comparison,among DHZC-H,DHZC-M,and DHZC-L,the expression of the above proteins in lung tissue decreased to varying degrees(P<0.05 or P<0.01).And when the dose is 0.45g/kg/d,the therapeutic effect is better.3.Cell Experiment ResultsInverted microscope:After being induced by TGF-β1 for 48 h,the morphology of A549 cells in DMS-MOD gradually changed from cobblestone-like in close contact to elongated spindle-like or spindle-like shape.Similar in shape.cck-8 results:At 24,48,and 72 h,compared with DMS-C,the proliferation rate of A549 cells in DMS-MOD was inhibited to varying degrees(P<0.05 or P<0.01);compared with DMSMOD,DHZC-H,DHZC-M,DHZC-L in A549 cells OD value decreased in different degrees(P<0.05 or P<0.01).And when the dose was 1.80g/kg/d,the inhibitory effect was the best.RT-PCR results:Compared with DMS-C.the expression of E-cadherin mRNA in A549 cells in DMS-MOD decreased significantly(P<0.01),while the expression of Fibronectin,αSMA,ROCK1,and Collagen Ⅰ/Ⅲ mRNA increased to varying degrees(P<0.01).P<0.05 or P<0.01);compared with Model,in DHZC-H,DHZC-M,DHZC-L,except for E-cadherin,the expression of other genes in A549 cells decreased to varying degrees(P<0.05 or P<0.01).Western blot results:Compared with DMS-C,the expression of Smad2,p-Smad2,p-RhoA,ROCK1,p-MLC and α-SMA proteins in A549 cells in DMS-MOD increased to varying degrees(P<0.05 or P<0.01);Compared with Model,in DHZC-H,DHZC-M,DHZC-L,the expression of the above proteins in A549 cells decreased to varying degrees(P<0.05 or P<0.01).Analysis conclusion1.The traditional Chinese medicine compound prescription based on the method of"activating blood and dredging collaterals" has good curative effect and good safety in the treatment of idiopathic pulmonary fibrosis.Clinical evidence-based basis.2.Dahuang Zhechong Pill can inhibit the excessive activation of Rho/ROCK signaling pathway,reduce the expression of inflammatory factors and extracellular matrix components,and delay the progression of bleomycin-induced pulmonary fibrosis in rats.3.The medicated serum of Dahuang Zhechong Pill can inhibit the proliferation rate of A549 cells induced by TGF-β1,and by inhibiting the excessive activation of Rho/ROCK signaling pathway,it can reduce the epithelial-mesenchymal transition,the expression of extracellular matrix components,and delay TGF-β1-induced fibrosis progression in A549 cells.