| Objective: Kawasaki Disease(KD)is a common systemic vasculitis syndrome in children under 5 years old,of which the main complication is coronary artery disease.The pathogenesis of KD is still not completely clear.In recent years,a large number of clinical and basic studies have shown that miRNAs are involved in the pathophysiological process of a variety of diseases.Studies on KD related miRNAs are still rare,and their specific mechanism of action is still in the preliminary exploration stage.This study is based on the network pharmacology to investigate the role and mechanism of Berberine(BBR)in alleviating the inflammation of Human coronary endothelial cells(HCAECs)by constructing in vivo animal models and in vitro cell models,and to investigate the role of target genes in endothelial injury of KD coronary artery.Methods: 1.The network pharmacology study of BBR in Coptidis Rhizoma(CR)for the treatment of KD: Through literature mining and retrieval of multiple databases,the compounds,drug targets and genes targeting KD of CR were collected.STRING was used to construct the target protein interaction network between CR and KD,and DAVID online tool was used to perform GO and KEGG pathway enrichment analysis for key targets.The core target proteins were verified by WB.2.Study on the effect and mechanism of BBR on KD coronary arteritis mice: In this part,C57 wild-type mice were extracted by single intritoneal injection of Lactobacillus cascasi cell wall to construct KD coronary arteritis mice model,and blank control group,KD model group,infliximab treatment group and BBR treatment group were set up respectively.The expression levels of Smad3 and miR-18a-5p in mice heart tissue were detected by QPCR and WB.3.Study on the mechanism of BBR in alleviating the inflammation of KD coronary endothelial cell: The expression of Smad3,miR-18a-5p and other related targets were detected by QPCR,WB and other methods.Dual luciferase assay was used to verify the relationship between miR-18a-5p and Smad3.The methods of CCK8 and Transwell were used to study the effect of BBR on the function of KD coronary artery endothelial cells.Results: 1.The network pharmacology study of BBR in the treatment of KD by CR: A total of 9 compounds,369 relative drug targets and 624 KD target genes were collected in this database.Network analysis showed that 41 targets might be the therapeutic targets of CR against KD.GO and KEGG enrichment analysis showed that the biological processes were most significant in response to hormones,response to inorganic substances,enzyme linked receptor protein signaling,response to cancer signaling,toll-like receptor signaling,and pancreatic cancer signaling.The results of WB indicated that the expressions of CASP3,PTGS2 and SRC proteins were up-regulated,while the expressions of AKT1 and ERK proteins were down-regulated after BBR treatment.2.The effect and mechanism of BBR on KD coronary arteritis mice: Compared with blank control group,mice in KD model group had more infiltration of inflammatory cells around the coronary arteries,less smooth coronary wall and more obvious damage of endothelial cells.Compared with KD model group,inflammatory cell infiltration around the coronary artery was reduced in BBR group and Infliximab group.Compared with KD model group,the scores of coronary arteritis,aortitis and myocarditis were all lower in BBR group.Compared with blank control group,the contents of IL-1β and TNF-α in KD model group were significantly increased,while the contents of IL-1β and TNF-α in Infliximab group and BBR group were significantly decreased.The results of QPCR showed that compared with KD model group,the expression of miR-18a-5p in BBR group showed a downward trend,the expression of Smad3 in BBR group was significantly increased.The results of WB showed that the expression of Smad3 protein was increased in KD model group,and significantly decreased in BBR group,with statistically significant difference.3.KD serum induces HCAECs to construct in vitro cell model to explore the molecular mechanism of BBR therapy for KD: This part of the study included 20 children with KD: 14 males(70%)and 6 females(30%),aged(31.20±16.99)months.20 cases in normal control group: 11 males(55%)and 9 females(45%),aged(39.25±13.76)months,and the differences were not statistically significant.The results of QPCR indicated that,compared with healthy children,the serum of KD children treated with HCAECs 48 h,the expressions of miR-18a-5p,miR-18b-5p,miR-4735-3p,miR-129-1-3,miR-129-2-3p,miR-16-5p and miR-195-5p were significantly decreased.The expression levels of Smad1,Smad2,Smad3,Smad4,Smad5 and Smad6 were significantly increased,and the differences were statistically significant.The results of dual luciferase assay showed that miR-18a-5p regulation of Smad3 might occur in the nucleus of HCAECs,and the expression of Smad3 in the nucleus of HCAECs was significantly increased compared with that in the cytoplasm of HCAECs,the expression of miR-18a-5p was significantly decreased.There were many mutation sites between them,and all three mutated simultaneously.Hsa-miR-18a-5p regulates the expression of luciferase with Smad3 3’UTR,down 32.89%.After the mutation of binding site,the regulatory relationship disappeared.On the base of KD serum treatment,the expression of miR-18a-5p was significantly increased after the addition of miR-18a-5p mimics.The results of CCK8 showed that the proliferation of HCAECs after KD serum treatment was significantly decreased compared with the blank control.Compared with BBR treatment alone,the addition of KD serum and miR-18a-5p mimics at the same time significantly reduced cell proliferation.The results of QPCR showed that the expression of miR-18a-5p was significantly increased after BBR and miR-18a-5p mimics were added simultaneously on the base of KD serum treatment.The results of WB showed that compared with blank control,after KD serum treatment,the protein expression of Smad3 and Bax in HCAECs increased,while the protein expression of Bcl-2 decreased.On the base of KD serum treatment,adding BBR,the protein expression of Smad3 decreased,while the protein expression of Bax and Bcl-2 increased.On the base of KD serum addition,compared with miR-18a-5p mimics alone and BBR at the same time,the protein expression of Smad3 and Bax decreased,while the protein expression of Bcl-2 increased.The results of Transwell showed that compared with blank control,cell migration was significantly decreased after KD serum treatment.On the base of KD serum treatment,the addition of BBR and miR-18a-5p mimics at the same time significantly reduced cell migration.In addition to KD serum,compared with BBR alone,miR-18a-5p mimics treatment at the same time significantly reduced cell migration.Conclusions: 1.Through the verification of "drug-compound-target-pathway" and related target cells,it was found that the mechanism of BBR in CR treating KD may be related to the expression of CASP3,PTGS2,SRC,AKT1 and ERK.2.BBR may alleviate the inflammatory response of coronary artery endothelial cells in KD mice through miR-18a-5p/Smad3 axis to play its drug effect.3.Mi R-18a-5p may be a potential biomarker for KD diagnosis.Mi R-18a-5p/Smad3 signaling pathway is involved in the process of KD vascular endothelial injury,and BBR may reduce endothelial inflammatory response through this pathway. |
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