Effect Of Bushenhuoxue Compound On IL-6 And STAT3 Protein In Rats With Chronic Heart Failur

Purpose:In order to explore the effects of Bushen Huoxue Compound on IL-6 and STAT3proteins in rats with chronic heart failure from histological and molecular levels,this studyadopted multiple methods--network pharmacology,filtering effective composition and key targets,and rats with chronic heart failure model establishment.Material and method:1 To explore the mechanism of Bushen Huoxue Compound in the treatment of chronic heart failure based on network pharmacology and molecular docking.The active ingredients and targets of Bushen Huoxue Compound were searched by TCMSP,and disease-related targets were collected through Gene Cards,OMIM,TTD,Pharm Gkb,and Drug Bank databases.Constructing a"compound-target"network and an intersecting target-protein interaction network by Cytoscape 3.6.1 software and the STRING platform respectively,Cytoscape performs topological analysis of the network to obtain core genes.After performing GO enrichment analysis and KEGG pathway through R language,the core genes were finally verified by molecular docking with the main active components.2 The effect of Bushen Huoxue Compound on IL-6 and STAT3 protein in chronic heart failure rats.80 SPF male SD rats were purchased,20 of which were randomly selected to be not ligated as the sham operation group and the remaining were ligated to the left anterior descending coronary artery（LAD）to cause myocardial infarction.After 4 weeks of exhaustive swimming and diet reduction to form the model of chronic heart failure,echocardiography was used to detect and calculate the ejection fraction（EF）.And the model is deemed to be completed once EF is not higher than 45%.Then rats were divided into three groups:model,kidney-invigorating and blood-activating,and captopril.According to the conversion of human and mouse measurements,the Bushen Huoxue compound was given by gavage at a dose of 9.2g·kg^-1·d^-1;the captopril group was given Captopril at a dose of12.5mg·kg^-1·d^-1;The sham operation group and the model group were given equal volume of distilled water by gavage,once a day,3 ml each time;the above four groups were all given medication for 4 weeks.After 4 weeks,electrocardiogram and echocardiography were used to detect and observe the changes of cardiac function again.After the above administration and ultrasonic detection,all the rats were put to death and the materials were taken immediately.Then enzyme-linked immunosorbent assay（ELISA）method was used to detect the content of N-Terminal Pro-Brain Natriuretic Peptide（NT-pro BNP）and IL-6 in rat serum,and RT-PCR（reverse transcription polymerase chain reaction）to detect the expression of STAT3m RNA in rat heart tissue was detected by reverse transcription-polymerase chain reaction（RT-PCR）method,and WB（Western blotting）to detect the content of STAT3 in rat heart tissue.Results:1 There were 109 effective constituents and 97 targets of Bushen Huoxue Compound in the treatment of CHF through Network pharmacology,and 17 core genes such as STAT3,EGF,TNF,VEGFA,MMP9,MAPK1 through topology analysis.GO enrichment analysis involved a total of 2298 biological processes（BP）,324 cellular components（CC）,and 145 molecular functions（MF）.KEGG pathway enrichment analysis screened 155 CHF-related pathways,mainly involving TNF,IL-7,HIF-1 and other signaling pathways.Molecular docking results showed that the main active compounds were able to bind to the core targets with good affinity.2 Ultrasound changes in rats after four weeks of medication:Compared with the model group,there were differences in LVDD,LVSD,EF,and FS in the sham operation group（P<0.05）.the LVDD and LVSD in the Bushen Huoxue group and the captopril group were improved after treatment,as well as the FS and EF values,and the difference was statistically significant（P<0.05）.However,in the comparison between the captopril and Bushen Huoxue group,the levels of LVDD,LVSD,EF and FS did not change much,indicating that two groups had similar effects on the cardiac function of CHF rats（P>0.05）.Also,based on the obtained data,CHF did not meet the standard of sham operation,although the cardiac function recovered significantly after medication.3 ELISA method to detect NT-pro BNP in rat serum:Compared with the sham operation group,the serum NT-pro BNP content of the model group increased（P<0.05）;The concentration of NT-pro BNP in the captopril group decreased（P<0.05）than the model group;there was no significant difference between the two drug groups（P>0.05）,suggesting that the heart failure in rats treated with drugs was significantly improved.4 Serum IL-6 concentration detected by ELISA:Compared with the sham operation group,the IL-6 content of the model group was significantly increased（P<0.05）.And the level of IL-6 was significantly down-regulated（P<0.05）than the model group.There was no significant difference in the level of IL-6 between the two drug groups（P>0.05）.5 RT-PCR method to detect the content of STAT3 m RNA in rat heart tissue:compared with the sham operation group,the expression level of STAT3 m RNA in the model group was significantly increased（P<0.05）;compared with the model group,the expression level of STAT3 m RNA in the Bushen Huoxue group and the captopril group was significantly decreased（P<0.05）;compared with the captopril group,there was no significant difference in the Bushen Huoxue group（P>0.05）.6 The expression of STAT3 protein in rat heart tissue was detected by WB method:compared with the sham operation group,the STAT3 protein content in the model group was significantly increased（P<0.05）;The protein content was significantly decreased（P<0.05）;compared with the captopril group,there was no significant difference in the kidney-tonifying and blood-activating group（P>0.05）.7 Observation of myocardial morphological changes in rats by HE staining:It was observed under HE staining that the myocardial cells in the sham-operated group were regularly arranged,the longitudinal section was elongated,the distance between the nuclei was even,and the network was seen as a whole.In the model group,the myocardial cells were arranged disorderly,the cells were hypertrophic,the distance between the nuclei was unequal,some nuclei appeared pyknosis,and the myofibrils were not obvious,indicating that the myocardium was seriously damaged.Compared with the model group,the kidney-tonifying and blood-activating group and captopril group had more regular arrangement of myocardial cells,less fibrous connective tissue,and less cell hypertrophy,indicating that the medication intervention had obvious effect.Conclusion:1 The Bushen Huoxue Compound may contain quercetin,luteolin,kaempferol,β-carotene and other components,participate in oxidative stress,lipopolysaccharide and other biological functions and effects,and regulate TNF,IL-17 and other pathways by acting on STAT3,EGF,TNF,VEGFA and other targets.2 Bushen Huoxue Compound can reduce myocardial fibrosis by down-regulating IL-6 and inhibit the expression of STAT3 protein to achieve the effect of treating chronic heart failure.3 This experiment proves that Bushen Huoxue Compound can improve the cardiac function of rats,reduce the concentration of NT-pro BNP in the serum of rats,and relieve the symptoms of chronic heart failure.