Based On The Combination Of Components And Pharmacodynamics, The Tonifying Lung And Kidney Component Prescriptions Interfere With The Mechanism Of COPD Inflammation

Objective1.Efficacy evaluation of ECC-BYF group allocation:To evaluate the therapeutic effect of ECC-BYF and its group allocation on COPD rats,and initially reveal the compatibility laws using COPD rats.2.Discussion the mechanism of ECC-BYF in suppressing inflammatory response:To explore the possible mechanism of anti-inflammatory group allocation of ECC-BYF in suppressing inflammatory response to treat COPD based on transcriptomics,using LPS-induced macrophages inflammation model.MethodsExperiment 1228 SD rats were randomly divided into Normal group,Model group,ECC-BYF group,Buqi group(BQ),Bushen(BS),Huatan group(HT),Huoxue group(HX),Fuzheng Group(FZ),Quxie Group(QX),Buqi Huatan Group(BQHT),Buqi Huoxue Group(BQHX),Bushen Huatan Group(BSHT),Bushen Huoxue Group(BSHX),Fuzheng Huatan Group(FZHT),Fuzheng Huoxue Group(FZHX),Buqi Quxie Group(BQQX),Bushen Quxie Group(BSQX),Aminophylline Group(APL),N-Acetylcysteine Group(NAC),a total of 19 groups,12 rats in each group.COPD rat model of stable period was reproduced using cigarette smoke exposure combined with repeated bacterial infections from week 1 to week 8.The rats were oral gavaged with corresponding drug from week 9 to week 16.At last,the rats were sacrificed and samples were obtained at the end of week 16.Lung function was measured with WBP whole body plethysmography system and PFT measurement system.Pathological techniques were used to observe the lung tissue pathology and ultrastructure of the lung tissue,and to classify cells in bronchoalveolar lavage fluid(BALF).ELISA was used to detect the levels of Interleukin(IL)-1β,IL-6,Tumor necrosis factor(TNF)-α,etc.in serum and BALF,Collagen(COL)-1,COL-3,Matrix metalloprotein(MMP)-9,etc.in in serum and BALF,Mucin 5AC(MUC5AC),Aquaporins 5(AQP5)in BALF and lung tissue,vascular endothelial growth factor(VEGF),Tissue factor(TF)and Thrombomodulin(TM)in serum.Colorimetric method was used to detect the levels of oxidative stress products,such as malondialdehyde(MDA)and Lipid peroxide(LPO)in serum and lung tissue.Regional(R)value comprehensive evaluation method was used to comprehensively evaluate the therapeutic effect of ECC-BYF and its group allocation on COPD rats,and preliminary revealed the laws of ECC-BYF group allocation.Experiment 2Macrophages were divided into control group,LPS model group and ECC-BYF with different concentration groups.The effect of different concentrations of ECC-BYF on cell viability was detected by MTT method.The effect of different concentrations of ECC-BYF on the mRNA expression of IL-1β,IL-6,TNF-α and PTGS2 was detected by qPCR method,and then the appropriate concentration of ECC-BYF was selected.Macrophages were divided into control,model,and different compatibility groups(the same as experiment 1).The qPCR method was used to detect the effects of different groups on the mRNA expression of IL-1β,IL-6,TNF-αand PTGS2.The best compatibility group in suppressing inflammation response was assigned for the next experiment.According to above results,the macrophages were divided into control group,model group,ECC-BYF group,and BSHT group.Total RNA was extracted from the cells after 24 hours.The transcriptome of each group of cells was sequenced using the Illumina gene sequencing platform.Differentially expressed genes of cells in different groups were analyzed and screened.And then differentially expressed genes were annotated by GO and KEGG function analysis,enriched genes and related pathways were obtained subsequently.Furthermore,possible mechanisms of ECC-BYF and BSHT in suppressing inflammatory response were analyzed.ResultsExperiment 11 Effects on lung functionCompared vwith Normal group,Tidal Volume(VT),Peak Expiratory Flow(PEF),Minute Volume(MV),Forced Vital Capacity(FVC),Forced expiratory volume at 0.1s(FEV0.1)and FEV0.3 in the Model group significantly decreased(P<0.01).Lung function of rats in the ECC-BYF group and APL group was significantly improved when compared with Model group(P<0.01).2 Effects on histology of lung tissueCompared with Normal group,the lung tissue histology of Model group showed that basement membrane of bronchiolar thicken,alveolar wall rupture fusion,alveolar cavity and mean linear intercept(MLI)significantly increased(P<0.01),mean alveolar number(MAN)wassignificantly reduced(P<0.01).There were large number of inflammatory cells infiltration,showing emphysema signs.Compared with the model,lung tissue pathological signs of the ECC-BYF group and the APL group were significantly reduced(P<0.01).In terms of ultrastructure of lung tissue,respiratory membranes in Model group significantly thickened(P<0.01),the number of lamellar body and mitochondria decreased,ridges of mitochondrial were not so clear.ECC-BYF and the group except BS alleviated the respiratory membrane thickening(P<0.05,P<0.01),and the number of lamellar body and mitochondria increased in these groups.3 Effects on inflammatory responseCompared with Model group,the total number of cells,the percentage of neutrophils and lymphocytes in ECC-BYF,BSHT,and BQQX groups were significantly reduced(P<0.05,P<0.01);the total number of cells in FZ,QX,and FZHT groups,the percentage of neutrophils in BQ,BSHX,and FZHX,the percentage of lymphocytes in the BQ,FZHT,and BSQX groups significantly decreased(P<0.05,P<0.01).The levels of TNF-α and IL-6 in BALF of Model group were significantly increased(P<0.01),The distribution of ECC-BYF group had different effects on various inflammatory factors.The results of comprehensive evaluation of R values showed that the intensity of distribution of different groups to suppress inflammatory factors was APL,ECC-BYF,FZHX,NAC,BQHX,BQQX,BSHT,BSHX,BSQX(P<0.05,P<0.01).4 Effects on protease/anti-protease imbalanceCompared with Normal group,the levels of MMP-9 and MMP-12 in serum and BALF,the levels of COL-1,COL-3 in BALF of Model group were significantly increased,and TIMP-1 level in BALF were significantly decreased(P<0.05,P<0.01).The comprehensive evaluation results of the R values of various indicators showed that ECC-BYF,HX,FZ,FZHT,BQQX,BSQX regulated the protease/anti-protease imbalance significantly(P<0.05,P<0.01).5 Effects on oxidative stressCompared with Normal group,the levels of MDA and LPO in serum and lung tissue of Model group increased significantly(P<0.01).The comprehensive evaluation results of R values of various indicators showed that ECC-BYF,HT,FZ,BQHT significantly regulated oxidative/antioxidant imbalance in serum and lung tissue(P<0.05,P<0.01).6 Effects on mucus secretionCompared with Normal group,the levels of MUC5AC,AQP5 in BALF and MUC5AC in lung tissue of Model group increased significantly(P<0.01).The comprehensive evaluation results of the R values of various indicators showed that ECC-BYF,BSHT,BQHX,APL,and NAC significantly inhibited mucus hypersecretion(P<0.05,P<0.01).7 Effects on endothelial injury and repairCompared with Normal group,the levels of TF and VEGF in serum of Model group were significantly increased,and TM level was significantly decreased(P<0.01).The comprehensive evaluation results of the R values of various indicators showed that compatibility contained Paeonol,such as ECC-BYF,HX,FZ,BSHX,BQQX,had significant effects on regulating endothelial injury and repair(P<0.05,P<0.01).8 Effects on phenotype of T cellsCompared with Normal group,the number of CD4+T cells,CD8+T cells and the ratio of CD4+/CD8+ in blood of Model groups significantly decreased(P<0.01).Compared with Model group,the number of CD4+T cells,CD8+T cells in ECC-BYF,FZ,BS,BSHT,and BSHX groups significantly increased(P<0.05,P<0.01);the number of CD4+cells in BQHT,BQHX,FZHT,and APL groups,the number of CD8+cells in HT group,and the ratio of CD4+/CD8+in ECC-BYF,BQ,BQHT,BQHX,FZHT,BQQX,and APL groups significantly reduced(P<0.05,P<0.01).Experiment 21 Effects of ECC-BYF and its components on LPS-induced inflammatory response in macrophagesECC-BYF significantly reduced the mRNA expression of IL-1β,IL-6,TNF-α,and PTGS2 in LPS-induced macrophages,and the concentration of 160 μg/mL was much better(P<0.01);the mRNA expression IL-1β、PTGS2 in BQHT group,IL-1β、TNF-α、PTGS2 in BSHT and BSHX groups,IL-1β、IL-6、PTGS2 in BQQX and BSQX groups significantly decreased(P<0.05,P<0.01).2 Effects of ECC-BYF and BSHT on gene expression of LPS-induced macrophagesThe results of differentially expressed gene showed that there were 780 up-regulated differentially expressed genes and 677 down-regulated differentially expressed genes between Control group and Model group.There were 235 up-regulated differentially expressed genes and 276 down-regulated differentially expressed genes between ECC-BYF group and Model group.There were 99 differentially expressed genes were up-regulated and 144 differentially expressed genes were down-regulated between BSHT group and Model group.When compared all differentially expressed genes in all groups,we found that ECC-BYF regulated 186 genes of macrophages induced by LPS,and BSHT group regulated 110 genes.The GO enrichment analysis showed that the differentially expressed genes between Control group and Model group were enriched in 575 GO terms,including inflammatory response,innate immune response,intracellular signal transduction,and cell components.the differentially expressed genes between ECC-BYF group and Model group were enriched in 279 GO terms,mainly including inflammatory responses,cytokine-mediated signaling pathways,immune responses,and cellular components.Morever,differentially expressed genes between BSHT group and Model group were enriched in 111 GO terms,including inflammatory response,immune system processes,negative regulation of apoptotic processes,and cell components.KEGG pathway enrichment analysis showed that the differentially expressed genes between Control group and Model group were enriched in 63 pathways,such as cytokine-cytokine receptor interaction pathway,TNF signaling pathway,and NF-κB pathway.Differentially expressed genes between ECC-BYF group and Model group were enriched in 24 pathways,including Toll-like receptors,cytokine-cytokine receptor interaction pathway,TNF signaling pathway.Besides,differentially expressed genes between BSHT group and Model group were enriched in 14 pathways,like Toll-like receptor,MAPK pathway,cytokine-cytokine receptor interaction pathway.Conclusion1 ECC-BYF and its group distribution had different curative effects on different pharmacodynamic indexes on COPD rats treatment.The groups contained Icariin suppressed inflammatory response better.The groups contained Ginsenoside Rhl and Astragaloside showed benefit effects on regulating oxidation/antioxidation imbalance.The groups contained Paeonol had significant effects on regulating vascular injury and repair.The groups of ECC-BYF,BSHT,and BQHX had better effects on inhibiting mucus hypersecretion.FZHT,HX,FZ,BQQX,and BSQX groups regulated the protease/anti-protease imbalance and collagen deposition significantly.FZHT,ECC-BYF,and BQHT groups were able to regulate T cells phenotypes.2 ECC-BYF,BSQX,BQQX,and BSHT significantly inhibited the inflammatory response of macrophages induced by LPS.The ECC-BYF and BSHT were capable to down-regulate the genes related to inflammatory response in LPS-induced macrophages,which may via regulating the TNF mediated signaling pathway,NF-κB,and cytokine-cytokine receptor interaction pathway.