| Objective:The aim of this research is to investigate the clinical effectiveness and underlying mechanism of "Yiqi Qingxin" therapy for treating Viral Myocarditis(VMC)in children.The study hopes to provide a novel diagnostic approach and treatment plan for VMC in children using traditional Chinese medicine.Methods:1.Sixty children with viral myocarditis(VMC)were collected and randomly divided into a control group and an experimental group,with 30 cases in each group.The control group received routine basic treatment for 7 days(including Xinyanping injection and creatine phosphate injection),while the experimental group received Yiqi Qingxin decoction orally based on the control group’s treatment for 7 days.The clinical symptoms and changes in myocardial enzyme spectrum,adverse reactions,and comparison between the two groups were observed before and after treatment.2.Using network pharmacology method,the target and mechanism of the core drugs Bitter Sophora Root and Panax ginseng in Yiqi Qingxin decoction for treating VMC were analyzed.The active ingredients and targets of Bitter Sophora Root and Panax ginseng were screened from databases such as TCMSP.Relevant treatment targets for VMC were searched in databases such as OMIM and Genecards using "viral myocarditis" as a keyword.A Venn diagram was used to analyze the targets of Bitter Sophora Root and Panax ginseng and VMC,and a "Chinese medicine-component-target-disease" network model was constructed.STRING 11.0 database was used to construct a protein-protein interaction(PPI)network graph.Cytoscape 3.8.2 software and its Network Analyzer and cytoHubba functions were used for further analysis of the PPI network.Metascape platform was used for GO and KEGG enrichment analysis of intersecting targets,and R language was used to visualize the data.3.Forty male Balb/c mice were randomly divided into a control group,a VMC group,a high-dose group(275mg/kg)of Bitter Sophora Root total alkaloid and Panax ginseng total saponin combination(KX combination),and a low-dose group(138mg/kg)of KX combination,with 10 mice in each group.Except for the control group,all other groups were injected with 0.1ml of CVB3 virus liquid containing 100TCID50 via the abdominal cavity of each animal to establish the VMC model.Two hours after virus injection,high and low doses of KX combination were given by gavage once a day for 7 days,and pure water(10ml/kg)was given to the control group and the VMC group at the same time.Animal status and body weight were monitored daily during the experiment.After the last administration for 2 hours,the mice were anesthetized and processed.ELISA kits were used to detect CK-MB,LDH,cTn-I,AST,IL-6,IL-1β,TNF-α,hs-CRP activity or content in mouse serum of each group according to the instructions.HE staining was used to observe the morphological changes in myocardial tissue,and western blotting and qRT-PCR were used to detect changes in the NF-κB pathway in myocardial tissue.4.Forty-eight male Balb/c mice were randomly divided into a control group,a self-immune myocarditis model(EAM)group,a high-dose group(275mg/kg)of KX combination,and a low-dose group(138mg/kg)of KX combination,with 12 mice in each group.Except for the control group,all other groups were immunized with porcine myocardial myosin subcutaneously(back,bilateral inguinal region)on day 0 and day 7 to establish EAM.Starting from day 0 of immunization,KX combination at high and low doses were given orally once a day for 21 consecutive days for all treatment groups,while pure water(10ml/kg)was given to the control group and the EAM group at the same time every day.Body weight changes were monitored weekly,and after the last administration for 2 hours,the mice were anesthetized and processed.Splenic cell suspensions were prepared and used for flow cytometry to detect the proportion of TH17 and Treg cells.ELISA kits were used to measure CK-MB,LDH,cTn-I,AST,IL-6,IL-1β,TNF-α,hs-CRP,IL-17,IL-21,IL-2,and IL-10 activity or concentration in mouse serum according to the instructions.HE staining was used to observe the morphological changes in myocardial tissue,and western blotting was used to detect changes in the JAK2/STAT3 pathway in myocardial tissue.Results:1.A total of 60 pediatric cases were collected in the clinical study,with 30 cases in the control group and 30 cases in the experimental group.In the experimental group,there were 17 males and 13 females,while in the control group,there were 15 males and 15 females.All patients were between the ages of 1 and 14 years.After treatment,compared with before treatment,the symptom scores of both groups significantly decreased(P<0.05);after treatment,the scores for sighing,fatigue,and fever in the experimental group significantly decreased compared to the control group(P<0.05).After treatment in both groups,the levels of myocardial enzymes in the serum of the pediatric patients significantly decreased(P<0.05);after treatment,the levels of CK-MB,LDH,and HDBH in the serum of the pediatric patients in the experimental group significantly decreased compared to the control group(P<0.05).Among the pediatric patients in the experimental group,the total effective rate reached 93.33%;in the control group,the total effective rate was 86.66%.There was a significant difference in the total effective rate between the two groups(P<0.05),and no significant adverse reactions were observed.2.Network pharmacology analysis showed that a total of 37 chemical components were obtained from Sophora flavescens and Panax ginseng,and 151 common targets for the treatment of viral myocarditis(VMC)were identified.Both Sophora flavescens and Panax ginseng contain multiple components for the treatment of VMC,including MOL011398(ginsenoside Re),MOL011398(ginsenoside Rg1),MOL011398(ginsenoside Rbl),MOL006634(matrine),etc.,all of which have high Degree values,representing a high correlation with VMC.The targets with high Degree values included PTGS2,HSP90AA1,PIK3CG,NFKBIA,JAK2,STAT1,IL6,and TNF.GO and KEGG enrichment analysis showed that the intersecting targets were enriched in 1900 biological processes,mainly involving the response processes to lipopolysaccharides,bacterial molecules,cell movement and migration processes,and inflammatory response processes.The intersecting targets were enriched in 156 molecular functions,including cytokine-receptor binding,DNA transcription factor binding,kinase and phosphatase binding,etc.The intersecting targets were enriched in 52 cellular component expression processes,mainly involving membrane rafts,cytoplasmic-nuclear envelope regions,extracellular matrix regions.KEGG enrichment analysis revealed 302 signaling pathways,and the common targets were mainly enriched in the IL-17 signaling pathway,TNF signaling pathway,T cell receptor signaling pathway,TH17 cell differentiation signaling pathway,etc.3.The experimental results of the VMC model in mice showed that the mice exhibited obvious myocardial inflammation and damage,and the activity of myocardial enzymes and the content of inflammatory factors(TNF-α,IL-6,IL-1β,HS-CRP)in the serum were significantly increased,and the TAB 1/NF-κB pathway in the myocardium was activated.Treatment with the KX compound in VMC mice reduced the cardiac index of the mice and improved the levels of myocardial enzymes and inflammatory factors in a dose-dependent manner,reduced the expression level of TAB 1 and NF-κB pathway-related mRNA and protein expression levels.4.The experimental results of the EAM showed that there was still inflammation,infiltration of inflammatory cells,and significant myocardial cell injury in the EAM mice’s hearts.Additionally,the expression of inflammatory cytokines(IL-6,IL-1β,TNFa,HS-CRP)in the serum was significantly increased.Furthermore,the JAK2/STAT3 pathway in the myocardium was activated,the proportion of TH17 cells in the spleen and their related cytokines in the serum increased,the proportion of Treg cells and their related cytokines in the serum decreased.Treatment with the KX compound in EAM mice reduced the expression of inflammatory cytokines IL-6,IL-1β,TNF-α,HS-CRP,and cytokines IL-17 and IL-21 in a dosedependent manner,and reduced the proportion of TH17 cells in the spleen.At the same time,it also increased the expression of IL-10,IL-2,and the proportion of Treg cells,inhibited the expression of JAK2,p-STAT3,RORy protein,and promoted FOXP3 protein expression.Conclusion:Based on conventional treatment,Yiqi Qingxin Decoction can effectively alleviate clinical symptoms and reduce the severity of VMC in pediatric patients.It can also lower the concentration of myocardial enzymes in the serum,with no significant adverse reactions observed.This may be due to Yiqi Qingxin Decoction’s regulation of the TAB1/NFκB pathway and JAK2/STAT3 pathway,balancing TH17/Treg cell ratios and improving inflammation and damage in the body.This reflects Yiqi Qingxin Decoction’s multi-target and multi-pathway characteristics in treating VMC,providing a new differential diagnosis method and a safe and effective treatment plan for children with VMC using traditional Chinese medicine. |
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