Mechanism Of Nicotine On Autophagy In Vascular Endothelial Cells

Objective:Smoking as an independent risk factor for cardiovascular damage is a serious hazard to human health.This experiment simulates smoking on cardiovascular damage model in vivo through studying the effect of nicotine on vascular endothelial cells in vitro,and then discusses the mechanism of nicotine-induced vascular endothelial cell injury,by defining the influence of nicotine on autophagic flux and apoptosis to reveal the relationship between nicotine and the vascular endothelial cell dysfunction.Methods:The human umbilical vein endothelial cells cultured with nicotine were simulated the body smoking model.1.To detect the effect of nicotine on apoptosis and autophagy in HUVECs.HUVECs were randomly divided into several groups:the control group and nicotine(6×10^-6 M)/chloroquine group（CQ）,chloroquine and nicotine group（CQ+NIC）.The apoptosis protein expression level of cleaved caspase-9,cleaved caspase-8,cleaved caspase-3 and the autophagosome marker protein of LC3Ⅱand the autophagy-related protein expression level were detected by Western Blot;the cell apoptosis rate was detected by Flow Cytometry.2.To detect the signaling pathway of nicotine inhibits autophagy.HUVECs were randomly assigned:the control group,nicotine in different time（15 min,30 min,60 min）andα7nAChR silencing group（α7nAChR siRNA+NIC）.The activation of AMPKαand ERK1/2,JNK,p38 MAPK were detected by Western Blot;after using the AMPKαsiRNA and ERK1/2/p38 MAPK inhibitor.The protein expression level of SQSTM1/p62 and LAMP2 was detected by Western Blot.3.To detect whether ROS inhibits the autophagosome clearance of autophagic flux.HUVECs were randomly assigned:The control group,nicotine group（NIC）,ROS scavenger group（NAC+NIC）orα7n AChR silencing group（α7nAChR siRNA+NIC）.the intracellular production of ROS in HUVECs was detected by Flow Cytometry;the protein expression level of SQSTM1/p62 and LAMP2 were detected by Western Blot.Results:1.Nicotine promoted the apoptosis of HUVECs by increasing cleaved caspase-8（exogenous apoptosis pathway）and cleaved caspase-3 activation and cell apoptosis;nicotine decreased Beclin-1,LAMP2 and increased LC3Ⅱ,SQSTM1/p62 protein expression and then mainly inhibited the autophagosome clearance of autophagic flux and blocked autophagic flux.After nicotine treatment,the protein expression of cleaved caspase-9 had no significant difference（P>0.05）,cleaved caspase-8 and cleaved caspase-3 protein expression level were obviously increased（P<0.01）,as well the apoptosis rate of HUVECs significantly increased（P<0.01）.The expression of LC3Ⅱprotein level also significantly increased after treatment with nicotine（P<0.01）.Treatment with CQ resulted in a significant increase in LC3Ⅱlevel（P<0.01）,while nicotine induced comparable increased of LC3Ⅱin CQ-treated groups manifested no increase compared to CQ treated control.In nicotine group,the change of SQSTM1/p62 protein expression was similar to the change of LC3Ⅱ（P<0.05）.However,compared with the control group,the Beclin-1 and LAMP2 protein expression were decreased（P<0.05）.2.Nicotine activated the AMPKα,ERK1/2/p38 MAPK signaling pathway of HUVECs viaα7nAChR（the endothelial cell member recoptor of nicotine）,while the avtivation signal did not affect the inhibition of nicotine on autophagic flux.After nicotine treatment,AMPKαand ERK1/2/p38 MAPK were activated at different time point,among them,the phosphorylation of AMPKαhad the highest level at 30 min（P<0.01）,while the phosphorylation of with ERK1/2/p38 MAPK had the highest level at 15 min（P<0.01）.Compared with nicotine treated for 30 min of 15 min,the phosphorylation of with ERK1/2/p38 MAPK obviously decreased after pretreated withα7n AChR siRNA（P<0.01）.However,the phosphorylation of JNK was no significant difference between control group and nicotine group in 60 min（P>0.05）.Compared with nicotine group,AMPKαsilencing had no effct on the protein expression of SQSTM1/p62 and LAMP2（P>0.05）;morever,the protein expression of SQSTM1/p62 and LAMP2 had no significant difference after pre-treatment with SB203580 and PD98059,the inhibitor of p38 MAPK and ERK1/2（P>0.05）;after usingα7nAChR siRNA,the protein expression of SQSTM1/p62 and LAMP2 were contrary to nicotine group（P<0.05）.3.Nicotine inhibited the autophagosome clearance of autophagic flux by the overproduction of ROS in HUVECs.Nicotine induced the overproduction of ROS in HUVECs compared with normal control（P<0.01）.Compared with nicotine group,pre-treatment with the scavenger of ROS induced the significantly decreased of SQSTM1/p62 protein expression（P<0.01）,while NAC-induced the protein expression level of LAMP2 obviously increased（P<0.01）.Conclusion:1.Nicotine induces the apoptosis of HUVECs in the exogenous and non-endogenous mitochondrial apoptosis pathway.Nicotine has regulation effects on autophagy in HUVECs,the formation and degradation of autophagosome were all blocked,eapecially impaires the autophagosome clearance.2.Nicotine can increase the phosphorylation of AMPKαand ERK1/2/p38 MAPK by binding toα7nACh R.However,nicotine-induced the impaired autophagosome clearance was independent of the activation of AMPKαand ERK1/2/p38 MAPK.3.Nicotine inhibits the autophasosome clearance by the overproduction of ROS in HUVECs.