Protective Mechanism Of Berberine On Human Coronary Endothelial Cell Injury Induced By Intermittent High Glucose

Objective:In this study,a model was established for the damage of human coronary endothelial cells(HCAECs)by high glucose fluctuation,to investigate the protective effect of Berberine on human coronary endothelial cells injury induced by intermittent high glucose and explore the possible molecular mechanism.Methods:HCAECs were cultured and divided into 5 groups:Group A was the normal blood glucose group(5.5mmol/L glucose,NG);Group B was the persistent hyperglycemia group(25mmol/L glucose,PHG);Group C was the intermittent high glucose group(5.5mmol/L and 25mmol/L glucose fluctuated every 24 hours,IHG);Group D was the fluctuation of hyperglycemia+ Berberine intervention group(5.5mmol/L and 25mmol/L glucose +50?mol/L Berberine fluctuated every 24 hours,IHG+BBR);Group E was a fluctuating hypertonic environment(without glucose,mannitol with the same concentration as IHG was added to maintain the same fluctuating hypertonic environment as IHG,OC).The cells of each group were changed into maintenance solution containing 2%serum concentration once every 24 hours,and were co-cultured for 7 days for later experiments.The cell viability of HCAECs was determined by MTT assay;TUNEL staining and flow cytometry were used to detect apoptosis;The levels of oxidative stress markers such as reactive oxygen species(ROS),malonic dialdehyde(MDA)and superoxide dismutase(SOD)were measured with corresponding detection kits;The levels of interleukin-10(IL-10),interleukin-1?(IL-1?),tumor necrosis factor-?(TNF-?)and human monocyte chemotactic protein-1(MCP-1)were measured by ELISA;Western blotting was used to detect the expressions of Caspase-3,NADPH oxidase 4(Nox4),nuclear factor kappa-B p65(NF-?B p65),advanced glycation end products(AGEs)and the receptor of advanced glycation end products(RAGE);mRNA expression levels of IL-10,IL-1?,TNF-?,MCP-1,Caspase-3,Nox4,NF-?B p65,AGEs and RAGE were detected by qRT-PCR.Results:1.Cell viability:Compared with the NG group,the PHG group decreased cell viability(P<0.05).Compared with the PHG group,the IHG group showed a more significant decrease in cell viability(P<0.05).After intervention with Berberine,the cell viability of IHG+BBR group was significantly enhanced compared with that of IHG group(P<0.05).2.Apoptosis:Apoptosis was detected by TUNEL assay and flow cytometry at the cell level,and transcription and protein expression levels of Caspase-3,the apoptotic effector molecule,were detected by qRT-PCR and Western blotting at the molecular level.Compared with the NG group,apoptosis was significantly increased in the PHG group(P<0.05),and apoptosis was more significant in the IHG group(P<0.05).Berberine was added into the intermittent high glucose,and the apoptosis of cells in the IHG+BBR group was significantly reduced compared with that in the IHG group(P<0.05).However,no signi:ficant apoptosis was observed in OC group(P>0.05).3.Inflammatory response:Compared with the NG group and the OC group,the levels of IL-1?,TNF-? and MCP-1 in the PHG and the IHG groups were significantly increased,while the levels of IL-10 expression were significantly decreased(P<0.05).Compared with the PHG group,the IHG group showed a larger variation(P<0.05).However,after Berberine was added into the IHG group,the expression levels of IL-10,TNF-a and MCP-1 in the IHG+BBR group were significantly down-regulated and the expression levels of IL-10 were increased compared with the IHG group(P<0.05),indicating that the inflammatory reaction was significantly inhibited.4.Oxidative stress:Compared with the NG group and the OC group,ROS,Nox4 and MDA contents increased in the PHG group and the IHG group,while SOD activity decreased(P<0.05).However,comparing the PHG group with the IHG group,ROS,Nox4 and MDA levels in the IHG group were significantly higher than those in the PHG group,and SOD activity was significantly decreased(P<0.05).Compared with the IHG group,the production of ROS,Nox4 and MDA in the IHG+BBR group decreased,and SOD activity showed a significant increase trend(P<0.05).5.Expression of NF-?B p65:The protein and mRNA levels of NF-?B p65 were significantly higher in the IHG group than in the PHG group(P<0.05).However,the levels of NF-?B p65 protein and mRNA in the IHG+BBR group were significantly lower than those in the IHG group(P<0.05).6.AGEs and RAGE generation:Compared with the PHG group,the protein and mRNA expression levels of AGEs and RAGE in the IHG group were increased(P<0.05).Berberine intervention was added into the IHG group,and the protein and mRNA expression levels of AGEs and RAGE in the IHG+BBR group were significantly lower than those in the IHG group(P<0.05).Conclusions:1.Compared with persistent hyperglycemia,fluctuating hyperglycemia is more likely to cause inflammatory reactions and oxidative stress in human coronary endothelial cells,reduce cell viability,promote the generation of advanced glycation end products and the receptor of advanced glycation end products,activate the NF-?B signaling pathway,and promote apoptosis.The effect of fluctuating high glucose on human coronary endothelial cells was more obvious,and the damage was independent of osmotic pressure.2.Berberine has a significant protective effect on human coronary endothelial cells induced by fluctuating hyperglycemia.This mechanism may be related to the fact that Berberine inhibits the generation of advanced glycation end products and the receptor of advanced glycation end products,reduces the expression of NF-?B p65,and alleviates the inflammatory response and oxidative stress of human coronary endothelial cells.