The Effect Of Gap Junction Of Atorvastatin In Against The Injury Of Endothelial Cells Induced By Oxidized Low Density Lipoprotein In Vitro.

ObjectiveTo investigate the effects of atorvastatin on injury of human umbilical veinendothelial cells (HUVECs) induced by oxidized low density lipoprotein(ox-LDL)which influences the expression of connexin43and connexin40.MethodsHUVECs line routine passage, the cultured endothelial cells were dividedinto four groups:①control group: give DMEM high glucose cell culture mediumcontaining10%fetal bovine serum and cultured for24hours;②ox-LDL in group:HUVECs were cultured30min in10%fetal bovine DMEM high glucose cellculture medium of serum, then the ox-LDL of50mg/L was added to cellculture medium of serum for24hours③blocker group:the heptanol of50umol/L was added to DMEM high glucose cell culture medium of10%fetal bovineserum for30min,then ox-LDL of50mg/L was added to cell culture medium ofserum for24hours;④atorvastatin group: first Atorvastatin0.01,0.1,1.0umol/L were added to10%FBS DMEM high glucose cell culture medium for30min,then added to50mg/L ox-LDL cultured for24h. HUVECs morphology observedunder an inverted microscope, then itratereductase determination NOcontent,immunocytochemical determination the connexin43and connexin40protein positioning expression, western blot detect connexin43and40protein semiquantitative expression.Results(1) HUVECs morphological observation under an inverted microscope:Normal control group HUVECs form of flat, polygonal, long or fusiform forms,the cell outline a clear, rich and clear cytoplasm, cell fusion into a single layeradherent growth, forming a typical "cobblestone"Structure; ox-LDL groupHUVECs cells were shrunken into a spherical cell gap increases, morevacuoles in the cytoplasm,"cobblestone" structure is completely destroyed;blocker group and normal control group cells morphologically similar, and cansee "cobblestone"structure, even visible shrinkage of spherical cells;atorvastatin groups visible cell shrinkage was the number of spherical graduallyreduced with the increase in cell concentration, cell gap was significantlyreduced compared with ox-LDL group.(2) The nitrate enzymatic determination of NO content:compared with thecontrol group, ox-LDL group, atorvastatin concentration group and the blockingagent group HUVECs supernatant NO content significantly reduce(P<0.01);compared with ox-LDL group: the NO content of blocker group wassignificantly higher (P<0.01); atorvastatin groups in a dose-dependent increasethe content of NO content (P<0.05, p <0.01).(3) Immunocytochemistry determination connexin43, connexin40proteinlocalization and expression of ox-LDL group cells: only weak staining in normalcontrol cells, membrane was stained mild yellow, connexin43, connexin40asmall amount of expression;ox-LDL cells appar shrinkage, and membrane wasstained brown, connexin43and connexin40plenty of expression; theconnexin43, connexin40expression of blocker group and atorvastatin groupswas somewhere in between, and atorvastatin each group in a dose-dependentdecrease the expression of connexin43, connexin40. (4) western blot detect connexin43,40the protein semiquantitativeexpression: compared with the control group: ox-LDL group,Blocker group,atorvastatin groups connexin43the40protein quantitative expression wassignificantly increased (P<0.01); compared with ox-LDL group:blocker group,atorvastatin groups connexin43,40protein expression was significantly lower(P<0.05), and atorvastatin each concentration group in a dose-dependentreduction in connexin43,40protein.Conclusion(1) Atorvastatin dose-dependent inhibition of ox-LDL-induced HUVECsconnexin43and40protein increase.(2) Atorvastatin protect endothelial gap junctions by means of reducing theconnexin43and40, which play a statin lipid anti-atherosclerosis role.