Experimental Study On The Effect Of Berberine Oncoronary Endothelial Cells In Kawasaki Disease

Objective: The present study was designed to investigate whether the injury of human coronary artery endothelial cells(HCAECs)can be induced by serum of patients with KD,the underlying mechanism on berberine(BBR)can protect human coronary artery endothelial cells(HCAECs)from injury induced by serum of patients with KD.Methods:1.Incubated Human coronary artery endothelial cells with healthy children and KD patient serum samples and grouped.Added berberine(BBR)extract to the two groups of cell samples for culture and grouping.2.Analysed the cell cycle and apoptosis by flow cytometry.3.Analysed markers of endothelial cell injury by western blotting.4.Detected differentially expressed proteins in each group with Quantitative proteomic analysis.5.Performed bioinformatics with the Cytoscape plug-in ClueGO and ReactomeFIViz software.6.Validated the differentially expressed proteins in cells with healthy children and KD patient serum samples with or without BBR treatment by western blotting.Results:1.Examined apoptosis ratio with flow cytometry.Before BBR treament,the HCAECs under KD condition(67.6%)had a higher apoptosis ratio compared to those exposed to serum of normal children(52.8%).Following BBR treament,the KD-induced apoptosis was notable suppressed.2.Examined cell cycle progression with flow cytometry.Before BBR treament,the cell cycle progression in HCAECs showed cells exposed to KD serum led to down-regulation of G0/G1 phase cells and up-regulation of S phase cells,suggesting KD can promote the progression of cell cycle in HACECs.Following BBR treatment,the level of G0/G1 phase cells induced by KD was significantly increased(from 67.5% to 80.2%)while the percentage of G2/M phase and S phase(15.8% to 7.0%)cells were decreased.3.Examined the levels of injury markers,THBD,VWF,and EDN1 in vascular endothelial cells to explore whether BBR could repair the damage of coronary artery in KD patients.Expressions of all the markers were up-regualted in cells exposed to KD serum(P ? 0.01).Following BBR treatment,the levels of THBD,VEF,and EDN1 were significantly decreased.4.Investigated the mechanisms by which BBR exerts protective effects in HCAECs against KD-induced damage by iTRAQ and MS/MS analysis with HCAECs of control and KD groups with or without BBR treatment.A threshold change of 1.5-fold was used as the stardard to identify the differentially expressed proteins.The proteins of KD groups with or without BBR treatment were differentially expressed?5.Built an interaction network with a Cytoscape software to clarify the relationship among the differentially expressed proteins.The differentially expressed proteins from the KD group with or without BBR treatment were divided into six different clusters labelled with different colours according to their functions.Without BBR treatment,ER related proteins,such as HSP90B1,CALR,P4 HB,HSPA5,VCP,and vascular endothelial cell injury related proteins,such as VWF and FGG,were composed in the network hub.Following BBR treatment,VCP,CALR,VWF,and FGG were exluded in the hub.6.Categorised the identified differentially expressed proteins according to their biological functions.Five biological processes including blood coagulation,proteinactivation cascade,response to ER stress,tricarboxylic acid metabolic process,and extracellular matrix organization were enriched in the KD group.However,following BBR treatment,only blood coagulation,single organism cell adhesion,and hydrogen peroxide catabolic processes were enriched.Furthermore,the main differentially expressed proteins were involved in the response to ER stress,ERAD pathway,protein exit from ER processes,regulation of blood vessel size and negative regulation of blood coagulation.7.Examined the levels of the ER stress associated proteins to confirm the regulative effect of BBR on KD-induced ER stress.KD induced the expressions of the ER stress associated proteins in HCAECs and BBR could reverse the KD-induced increased expressions.Conclusion: Serum in KD can promote apoptosis in HCAECs and elicite their injury,while these processes can be reversed by treatment with BBR.These results suggest that ER stress may be involved in the pathogenesis of KD and BBR may be a promising therapeutic method for patients with KD.