Effects Of Clozapine On Oxidative Stress Injury Of Human Umbilical Vein Endothelial Cell Induced By High Glucose

Background and PurposeClozapine is the primary treatment for patients with refractory schizophrenia.Studies have reported that clozapine use increases the risk of venous thromboembolism,but the specific biological mechanism remains unclear.Long-term use of clozapine will cause increased blood glucose,intracellular high glucose will change the polyol pathway,hexosylamine pathway,Protein kinase C（PKC）pathway and Advanced glycation end products（AGEs）accumulation,leading to the accumulation of reactive oxygen（ROS）free radicals,and induce oxidative stress injury and apoptosis of vascular endothelial cells.Endothelial cell injury is the initial factor of venous thromboembolism.In this study,A model of oxidative stress injury of human umbilical vein endothelial cell（HUVEC）induced by high glucose was established in vitro to analyze the effect of clozapine on HUVEC induced by high glucose,and to explore whether clozapine might induce oxidative stress injury of vascular endothelial cells,thus causing the occurrence and development of venous thromboembolism.Materials and MethodsFresh neonatal umbilical cords were obtained,and 0.1%Ⅰcollagenase was infused into umbilical vein for digestion,separation and culture of HUVEC.The morphology and growth status of HUVEC were observed by electron microscopy,the cells were identified by immunofluorescence method,and the cell purity was detected by flow cytometry.CCK-8 kit assay was used to determine the activity difference of DMEM/F12 with different concentrations(10、20、30、40、50、60mmol·L^-1)of glucose after co-incubation with HUVEC for 24,48 and 72 hours,respectively.According to the results,the optimal glucose concentration and incubation time were selected as the model building conditions.CCK-8 kit was used to detect the effects of clozapine of different concentrations(0,250,500,1000,1500,2000,3000ng·m L^-1)on HUVEC activity and the effects of clozapine of different concentrations on HUVEC activity under high glucose condition.The effects of clozapine at different concentrations on the levels of ROS,MDA,SOD and NO secreted by HUVEC under high glucose condition were detected by ELISA.Results1.The isolated cultured HUVEC grew adherently under an inverted microscope and were arranged like"pebbles".The expression of v WF was positive by immunofluorescence,and the purity of the cells was 97.7%by flow cytometry.2.CCK-8 assay showed that HUVEC activity（OD value）decreased gradually with the extension of treatment time,and the difference was statistically significant（P<0.001）.At the same time,compared with the control group,HUVEC activity increased at 10mmol·L^-1glucose（P>0.05）;After 24 hours of treatment,HUVEC activity decreased in a concentration-dependent manner with 20-60mmol·L^-1glucose（P<0.05）;After 48 hours of treatment,HUVEC activity decreased in a concentration-dependent manner with 40 to 60mmol·L^-1glucose（P<0.05）;After 72hours of treatment,HUVEC activity was not significant among all glucose concentrations（P>0.05）.The high glucose injury model of HUVEC in vitro was successfully established.Finally,30mmol·L^-1glucose was selected as the modeling condition for 24 hours for subsequent experiments.3.Compared with the control group,HUVEC activity was not significantly affected by clozapine incubation at different concentrations for 24 hours（P>0.05）;Compared with high glucose group,HUVEC activity was not significantly affected by clozapine co-incubated with high glucose model at different concentrations for 24 hours（P>0.05）.4.After HUVEC was treated with high glucose and clozapine for 24 hours,the ROS levels in the 500-3000ng·m L^-1clozapine group were significantly different from that of high glucose model group（P<0.05）;the MDA level in the 1000-2000ng·m L^-1clozapine group were significantly different from that of high glucose model group（P<0.05）;The changes of SOD and NO levels were not statistically significant compared with the high glucose model group（P>0.05）.Conclusion1.With the increase of glucose concentration and the extension of action time,HUVEC activity decreased gradually.The mechanism of HUVEC activity may be that the increase of intracellular glucose level leads to the increase of oxidative stress level,which then activates several metabolic pathways to promote apoptosis.2.In vitro,the addition of 250-3000 ng·m L-1 clozapine had no significant effect on HUVEC activity compared with the untreated group.Compared with the high glucose model group without clozapine,the addition of 250-3000 ng·m L-1clozapine had no significant effect on HUVEC activity.3.In vitro,clozapine can reduce the elevated ROS and MDA levels caused by high glucose,and play a certain protective role against oxidative stress injury of vascular endothelial cells.