| Purpose : Observe the effectiveness and safety of Buyang Huanwu Decoction in the treatment of chronic obstructive pulmonary disease complicated with pulmonary hypertension(COPD-PH)through clinical research,used network pharmacology to explore its therapeutic mechanism and main pathways,and verify it through animal experiments.Material and method:1.Clinical study of Buyang Huanwu Decoction in the treatment of COPD-PHThis study used a prospective,randomized,double-blind,and controlled research method to observe patients with COPD-PH(Qi deficiency and blood stasis syndrome)who were admitted to the outpatient inpatient department of the Affiliated Hospital of Liaoning University of Traditional Chinese Medicine from June 2019 to August 2020.A total of 67 patients were enrolled and randomly divided into treatment group and control group.The treatment group was given Buyang Huanwu Decoction granules,and the control group was given Buyang Huanwu Decoction simulation granules with the same shape,smell and appearance as the treatment group.Two groups were treated with oxygen therapy,anti-infective therapy,diuretics,anticoagulation and other treatments based on the actual condition of the patients.The course of treatment was 4 weeks,and the 12 th week was followed up.Set pulmonary function and echocardiogram as screening indicators,pulmonary function,echocardiogram,TCM syndrome total score,individual symptom score,COPD assessment test(CAT score),6-minute walk test,Borg scale,BODE score,arterial blood gas(ABG),NT-pro BNP,D-dimer,and fibrinogen level were effective observation indicators.The effectiveness of Buyang Huanwu Decoction in the treatment of COPD-PH was evaluated,and adverse events occurred during the entire study were recorded.Observe the effectiveness of the drug Safety,to evaluate the compliance and safety of patients in this study.2.Based on network pharmacology to explore the mechanism of Buyang Huanwu Decoction in the treatment of COPD-PHObtain the active ingredients and targets of Buyang Huanwu Decoction through TCMSP or BATMAN-TCM database,and obtain disease targets of COPD-PH from the Gene Cards database,and extract cross-targets as disease targets of COPD-PH.Map drug targets and disease targets,construct protein interaction PPI network,perform gene GO function enrichment analysis and KEGG pathway enrichment analysis,and screen the main pathways.3.Based on PI3K/Akt signaling pathway to explore the effect of Buyang Huanwu Decoction on vascular remodeling in rats with COPD-PH3.1 Experiment 1 Study on the intervention effect of Buyang Huanwu Decoction on vascular remodeling in rats with COPD-PHThe rats were randomly divided into a blank group,a model group,chinese medicine group,western medicine group,and integrated Chinese and western medicine group.After one week of responsive feeding,the other four groups except the blank group were used lipopolysaccharide,smoking and hypoxia to create a rat model of COPD-PH,and the experiment was performed by the method of modeling and administration.Chinese medicine group was given 13.02g/(kg·d)Buyang Huanwu Decoction by gavage;western medicine group was given 12.6ug/(kg·d)beraprost sodium tablets by gavage;integrated Chinese and western medicine group was given 13.02g/(kg·d))Buyang Huanwu Decoction+12.6ug/(kg·d)beraprost sodium tablets by gavage;the three groups were configured into equal volumes of drug solutions according to the actual situation;the normal group and the model group were gavage with equal volumes of normal saline.After modeling and gavage for 4 weeks,the rats were subjected to mean pulmonary artery pressure(m PAP),right heart hypertrophy index(RV/LV+S),HE staining,and observation of pulmonary vessel wall thickness/vascular diameter percentage(WT%)and vessel wall area /Total vascular area percentage(WA%),to judge whether the model was successful.After preparing tissue specimens,apoptosis was detected by TUNEL method,and phosphorylation expression of key targets of PI3K/Akt pathway in pulmonary arterioles was observed by immunohistochemical method.3.2 Experiment 2 Based on PI3K/Akt signaling pathway to explore the regulation mechanism of Buyang Huanwu Decoction on PASMCs cell proliferation and apoptosisTake out the rat pulmonary artery,separate and culture PASMCs,identify PASMCs by?-SMA antibody immunofluorescence method,detect cell proliferation activity by CCK-8method,detect cell apoptosis rate by flow cytometry,and western-blot method detected the phosphorylation expression of key targets in the PI3K/Akt pathway,m TOR,p27kip1 and PCNA that affect cell proliferation,and Bcl-2,Bax,Cyt c,Caspase-3,and caspase-9 that affect cell apoptosis.Results:1.Clinical study of Buyang Huanwu Decoction in the treatment of COPD-PH1.1 Comparison of pulmonary function results before and after treatment in the two groups There was no significant difference in pulmonary function(Percentage of predicted FEV1 value,FEV1/FVC)between the two groups of patients before treatment(P>0.05),and they was comparable.After treatment,they were improved compared with before treatment,and the difference was statistically significant(P<0.05);and there was no significant difference between the treatment group and the control group(P > 0.05).1.2 Comparison of the results of echocardiography between the two groups of patients before and after treatmentThere was no significant difference in the results of the two groups before treatment(P>0.05),and they was comparable;the indicators of the treatment group after treatment are significantly better than those before treatment,the difference was statistically significant(P<0.05).After treatment,the indicators of the treatment group are better than those of the control group,and the difference was statistically significant(P<0.05),there was no significant difference between the control group S and RVFAC before treatment(P>0.05),The remaining indicators were significantly better than before treatment,and the difference was statistically significant(P<0.05).1.3 Comparison of the efficacy of TCM syndromes between the two groups of patients after treatmentAfter treatment,6 cases(20%)were clinically controlled in the treatment group,12cases(40%)were markedly effective,10 cases(33.33%)were effective,and 2 cases(6.67%)were ineffective.The control group was clinically controlled in 2 cases(6.67%),and 10cases(33.33%)were markedly effective,12 cases(40%)were effective,and 6 cases(20%)were ineffective.The therapeutic effect of TCM syndromes after treatment in the treatment group was better than that in the control group,and the difference was statistically significant (P<0.05).1.4 Comparison of TCM syndromes total scores between the two groups before and after treatmentBefore treatment,there was no significant difference in TCM syndrome scores between the two groups of patients(P>0.05),and they were comparable.After treatment,the two groups had improved compared with before treatment,and the treatment group was better than the control group during the same period,and the difference was statistically significant(P<0.05);there was no statistically significant difference between the 12 th week and the 4th week in the treatment group(P>0.05).1.5 Comparison of individual symptom scores before and after treatment in the two groupsBefore treatment,the two groups of patients was no significant difference in the symptoms of wheezing,shortness of breath,expectoration,fatigue,chest tightness or pain,fixed pain and dim complexion(P>0.05),and they were comparable.After treatment,the two groups of patients were comparable.The scores were significantly improved compared to before treatment,and the scores of the treatment group were better than those of the control group during the same period,the difference was statistically significant(P<0.05);there was no difference in the symptom scores between the two groups at the 12 th week and the 4th week statistical significance(P>0.05).1.6 Comparison of CAT scores between the two groups before and after treatmentThe difference in CAT scores between the two groups of patients before treatment was no statistically significant(P>0.05),and they were comparable.After treatment,the scores of the two groups were significantly improved compared with those before treatment,and the scores of the treatment group were better than those of the control group during the same period.The difference was statistically significant(P<0.05);there was no significant difference in the symptom scores between the two groups at the 12 th week and the 4th week(P>0.05).1.7 Comparison of 6-minute walking distance before and after treatment in the two groupsThere was no statistically significant difference in 6-minute walking distance between the two groups of patients before treatment(P>0.05),and they were comparable.After treatment,both groups improved compared with before treatment,and the treatment group was significantly better than the control group during the same period,and the difference was statistically significant(P<0.05);there was no significant difference between the 12 th week and the 4th week in the treatment group(P>0.05).1.8 Comparison of the Borg scale before and after treatment in the two groupsThere was no significant difference in the Borg scale between the two groups of patients before treatment(P>0.05),and they were comparable;after treatment,they were improved compared with before treatment,and the treatment group was significantly better than the control group during the same period,and the difference was statistically significant(P<0.05);There was no significant difference between the 12 th week and the 4th week in the treatment group(P>0.05).1.9 Comparison of BODE scores before and after treatment in the two groups.The difference in BODE scores between the two groups of patients before treatment was no statistically significant(P>0.05),and they were comparable.After treatment,they were improved compared with before treatment,and the treatment group was significantly better than the control group,the difference was statistically significant(P<0.05).1.10 Comparison of arterial blood gas before and after treatment in the two groupsThere was no significant difference in arterial blood gas(PaO2,PaCO2)between the two groups of patients before treatment(P>0.05),and they were comparable.After treatment,they were improved compared with before treatment,and the treatment group was significantly better than the control group,and the difference was statistically significant(P<0.05).1.11 Comparison of NT-pro BNP before and after treatment in the two groupsThere was no statistically significant difference in NT-pro BNP between the two groups of patients before treatment(P>0.05),and they were comparable.After treatment,they were improved compared to before treatment,and the treatment group was significantly better than the control group,and the difference was statistically significant(P< 0.05).1.12 Comparison of D-dimer before and after treatment in the two groupsThe difference in D-dimer between the two groups of patients before treatment was not statistically significant(P>0.05),and they were comparable;after treatment,they were improved compared to before treatment,and the treatment group was significantly better than the control group,and the difference was statistically significant(P<0.05).1.13 Comparison of FIB before and after treatment in the two groups.The difference in FIB between the two groups of patients before treatment was no statistically significant(P>0.05),and they were comparable.After treatment,they were improved compared with before treatment.The treatment group was significantly better than the control group,and the difference was statistically significant(P<0.05).1.14 Safety indicatorsRoutine blood test,Routine urinalysis,liver and kidney function,and electrocardiogram were monitored before and after treatment of the two groups of patients.Some patients showed large right ventricular manifestations on the electrocardiogram,and some indicators showed abnormalities without clinical significance,and no obvious abnormalities were found.2.Research on the mechanism of Buyang Huanwu Decoction in the treatment of COPD-PH based on network pharmacologyA total of 96 effective compounds were obtained by applying network pharmacology technology,a total of 1140 corresponding targets were obtained,and 203 disease targets were obtained according to preset conditions.GO enrichment analysis yielded BP1416,CC26 and MF68 with significant.KEGG pathway analysis revealed 104 significant pathways.Quercetin,luteolin,beta-carotene,kaempferol and baicalein are the main components,ALB,IL6,VEGFA,Akt1,IL1 B,etc.are the main targets,and AGE-RAGE,Relaxin,PI3K-Akt and others were the main pathways.After analysis,it was concluded that the PI3K-Akt pathway plays an important role in this disease.3.Based on PI3K/Akt signaling pathway to explore the effect of Buyang Huanwu Decoction on vascular remodeling in rats with COPD-PH3.1 Experiment 1 Study on the intervention effect of Buyang Huanwu Decoction on vascular remodeling in rats with COPD-PH3.1.1 After tracheal injection of LPS,smoking combined with hypoxia for 4 weeks,m PAP,right heart hypertrophy index,HE staining and pulmonary arteriole revascularization indexes WT% and WA% of the model group were significantly abnormal compared with the normal group,and the difference was statistically significant(P<0.05 or P<0.01),successfully created an animal model of COPD-PH.3.1.2 The general living conditions of COPD-PH rats in Chinese medicine group,western medicine group and integrative medicine group were significantly better than those of the control group,m PAP,right heart hypertrophy index,HE staining and pulmonary arteriole vascular remodeling index WT% and WA% levels Compared with the model group,the difference was statistically significant(P < 0.05 or P < 0.01).There was no significant difference between chinese medicine group and western medicine group(P > 0.05).The integrative medicine group was better than chinese medicine group and western medicine group.The difference was statistically significant(P<0.05 or P<0.01).3.1.3 TUNEL results of pulmonary arteriolesCompared with the blank control group,the number of apoptosis in the model group was significantly reduced,and the number of apoptosis in chinese medicine group,western medicine group and integrative medicine group was significantly increased,and the difference was statistically significant(P<0.01);compared with the model group,the number of apoptosis in chinese medicine group,western medicine group and the integrative medicine group increased,and the difference was statistically significant(P < 0.01);compared with chinese medicine group,western medicine group had no significant difference in the number of apoptosis,and the difference was no statistically significant(P>0.05)),the number of apoptosis in the integrative medicine group increased,and the difference was statistically significant(P<0.01);compared with western medicine group,the number of apoptosis in the integrative medicine group increased significantly,and the difference was statistically significant(P<0.01).3.1.4 Immunohistochemical method to detect PI3K/Akt pathway related indicators of pulmonary arteriolesCompared with the blank control group,the expression of p-PI3 K and p-Akt in the model group,chinese medicine group,western medicine group and integrative medicine group was up-regulated,and the difference was statistically significant(P<0.01);compared with the model group,the expression of p-PI3 K and p-Akt was down-regulated in chinese medicine group,western medicine group and integrative medicine group,and the difference was statistically significant(P<0.01);compared with chinese medicine group,the expression of p-PI3 K and p-Akt in the western medicine group had no significant difference(P>0.05),the expression of p-PI3 K and p-Akt in the integrative medicine group was down-regulated,and the difference was statistically significant(P<0.01).And compared with western medicine group,the expression of p-PI3 K and p-Akt in integrated traditional Chinese and Western medicine group was down regulated,and the difference was statistically significant(P <0.01).3.2 Experiment 2 Based on PI3K/Akt signaling pathway to explore the regulation mechanism of Buyang Huanwu Decoction on PASMCs cell proliferation and apoptosis3.2.1 Results of identification of PASMCs by immunofluorescenceThe cells all showed ?-SMA positive green fluorescence,and obvious myofilaments were seen,which were arranged in a filamentous spindle parallel to the cells.PASMCs were successfully extracted from the rat pulmonary artery.3.2.2 Western-blot method detects the protein expression results of p-PI3 K and p-Akt in each group of PASMCsCompared with the blank control group,the expression of p-PI3 K and p-Akt in the model group,chinese medicine group,western medicine group and integrative medicine group was up-regulated,and the difference was statistically significant(P<0.01);compared with the model group,the expression of p-PI3 K and p-Akt was down-regulated in chinese medicine group,western medicine group and integrative medicine group,and the difference was statistically significant(P<0.01);compared with chinese medicine group,the expression of p-PI3 K and p-Akt in western medicine group had no significant difference(P>0.05)),the expression of p-PI3 K and p-Akt in integrative medicine group was down-regulated,and the difference was statistically significant(P<0.01).And compared with western medicine group,the expression of p-PI3 K and p-Akt in integrated traditional Chinese and Western medicine group was down regulated,and the difference was statistically significant(P < 0.01).3.2.3 CCK-8 cell proliferation test resultsCompared with the blank control group,the cell proliferation activity of the model group,chinese medicine group,western medicine group and integrative medicine group was significantly enhanced,and the difference was statistically significant(P<0.01);compared with the model group,the cell proliferation activity of chinese medicine group,western medicine group and integrative medicine group was significantly reduced,and the difference was statistically significant(P<0.01);compared with chinese medicine group,there was no significant difference in the cell proliferation activity of western medicine group,while the cell proliferation activity of integrative medicine group was significantly reduced,and the difference was statistically significant(P<0.01);compared with western medicine group,the cell proliferation activity of the integrative medicine group was significantly reduced,and the difference was statistically significant(P<0.01).3.2.4 Western-blot method detects p-m TOR,PCNA,p27kip1 protein expression results of each group of PASMCsCompared with the blank control group,the expressions of p-m TOR and PCNA were significantly up-regulated in the model group,chinese medicine group,western medicine group,and integrative medicine group,and the expression of p27kip1 was significantly down-regulated,the difference was statistically significant(P<0.01);compared with the model group,The expression of p-m TOR and PCNA in chinese medicine group,western medicine group and integrative medicine group was significantly down-regulated,and the expression of p27kip1 was significantly up-regulated,the difference was statistically significant(P<0.01);compared with chinese medicine group,the expression of p-m TOR and PCNA in the integrative medicine group was significantly down-regulated,and the expression of p27kip1 was significantly up-regulated,and the difference was statistically significant(P<0.01).The expressions of p-m TOR,PCNA and p27kip1 in western medicine group were not significantly different from those in chinese medicine group(P>0.05);compared with western medicine group,the expression of p-m TOR and PCNA in the integrative medicine group was significantly down-regulated,and the expression of p27kip1 was significantly up-regulated,and the difference was statistically significant(P<0.01).3.2.5 Flow cytometry detection of apoptosis rate resultsCompared with the blank control group,the apoptosis rate of the model group decreased,and the difference was statistically significant(P < 0.05);the apoptosis rate of chinese medicine group,western medicine group and the integrative medicine group increased,and the difference was statistically significant(P <0.01);compared with the model group,the apoptosis rate of chinese medicine group,western medicine group and the integrative medicine group increased,and the difference was statistically significant(P<0.01).Compared with chinese medicine group,there was no significant change in the apoptosis rate in western medicine group,and the difference was not statistically significant(P>0.05).The apoptosis rate in the integrative medicine group increased,and the difference was statistically significant(P<0.01);compared with western medicine group,the apoptosis rate integrative medicine group increased,and the difference was statistically significant(P<0.01).3.2.6 Western-blot method to detect the Bcl-2,Bax,Cyt C,caspase-9 and caspase-3 protein expression results of each group of PASMCsCompared with the blank control group,the expression of Bcl-2 in the model group was significantly up-regulated,the expression of Bcl-2 in chinese medicine group,western medicine group and the integrative medicine group was significantly down-regulated,and the expression of Bax,Cyt C,caspase-9 and caspase-3 in the model group was significantly down-regulated.The expressions of Bax,Cyt C,caspase-9 and caspase-3 in chinese medicine group,western medicine group and integrative medicine group were significantly up-regulated,and the difference was statistically significant(P<0.01);compared with the model group,the expression of Bcl-2 in chinese medicine group,western medicine group and the integrative medicine group was significantly down-regulated;the expression of Bax,Cyt C,caspase-9 and caspase-3 was significantly up-regulated,and the difference was statistically significant(P<0.01);compared with the chinese medicine group,the expressions of Bcl-2,Bax,Cyt C,caspase-9 and caspase-3 in the western medicine group were not significantly different from those in the Chinese medicine group(P>0.05);Compared with the western medicine group,Bcl-2 was significantly down-regulated in the integrative medicine group,and Bax,Cyt C,caspase-9 and caspase-3 were significantly up-regulated,and the difference was statistically significant(P<0.01).Conclusion:1.Buyang Huanwu Decoction can improve the clinical symptoms such as shortness of breath and shortness of breath in COPD-PH patients with Qi deficiency and blood stasis,improve hypoxia,improve cardiopulmonary function,increase exercise endurance,and improve quality of life.The mechanism may be closely related to alleviating pulmonary vasoconstriction and spasm caused by hypoxia,hypercapnia and respiratory acidosis,reducing blood viscosity,preventing in situ thrombosis,etc.,and had high clinical safety.2.The effective ingredients such as quercetin,luteolin and kaempferol in Buyang Huanwu Decoction can act on ALB,IL6,VEGFA and other targets,and participate in cells by regulating signal pathways such as AGE-RAGE,PI3K-Akt,and TNF.A variety of biological processes such as apoptosis,cell proliferation,and inflammatory response achieve the effect of treating COPD-PH.3.Buyang Huanwu Decoction can regulate PI3K/Akt signaling pathway to inhibit PASMCs proliferation,promote PASMCs apoptosis,inhibit vascular remodeling,and reduce COPD-PH pulmonary artery pressure. |
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