Effects Of Knocking Down Bmal1 Gene On Autophagy And Apoptosis Of Human Umbilical Vein Endothelial Cells

Background:Bioclock widely exists in living organisms and plays an important role in maintaining the normal physiological function of the body.In recent studies,the relationship between the circadian clock and the cardiovascular diseases has been widespread emphasized.The disorder of the biological clock will influence on physiological activities such as lipid metabolism and blood pressure,and cause vascular damage.However,there are few reports about the effect of the core clock gene,Bmal1,on vascular endothelial autophagy and apoptosis.Objective and significance:To investigate the role of core clock gene,Bmal1 in autophagy and apoptosis of human umbilical vein endothelial cell(HUVEC).It is expected to provide new ideas for the bridge between clock genes and vascular injury.Methods:In this study,HUVEC was synchronized by treatment with 1μM dexamethasone(DEX)for 1 h.Cellular mRNA was extracted every 4 h and lasted for 24 h.The circadian expressive profile of Bmal1 gene in HUVEC was determined by real-time quantitative PCR to obtain the peak time point of Bmall gene expression.SiRNA interference(liposome 2000 Transfection)was performed at different time points of Bmall gene expression and the peak time point was used to observe the knockdown efficiency.Keeping unchanged condition of the Bmall gene knockdown experiment,MTT was conducted to check up the effect of transfection for gene knockdown on endothelial cell activity.Monodansylcadaverine(MDC)staining and fluorescence microscopy were used to observe changes in cell autophagy.Real-time PCR was used to detect the expression of Beclin-1,P62,LC3 in endothelial cells after knocking down Bmall gene while Western blot was used to detect the relative expression of autophagy-related proteins p62 and LC3-Ⅱ/LC3-Ⅰ.The Hoechst staining was used to take pictures under fluorescence microscope to observe the changes of endothelial cell apoptosis after knocking down Bmall gene.Real-time PCR was also used to detect the expression of apoptosis-related genes such as Bax,Bcl-2,Caspase-3 changes after knocking down Bmall gene and Western blot was used to detect the relative expression levels of Bcl-2,Bax and Bcl-2/Bax.ROS(Reactive oxygen species,ROS)fluorescent probe was used to detect changes in reactive oxygen species levels in endothelial cells after knocking down Bmal1 gene.Results:The Bmall gene expression showed a circadian rhythm in HUVEC,which conformed to the cosine function curve,and the peak time appeared at time CT12.After transfecting cells with liposome 2000-siBmall,MTT assay displayed that the transfection process did not cause significant changes in endothelial cell activity.In views of autophagy,MDC staining revealed that compared with the negative control(NC),the autophagy level of endothelial cells increased after knocking down the Bmal1 gene,and the expression of autophagy-related genes Beclin-1 and LC3 was significantly increased by qPCR while the expression of p62 gene was decreased slightly.The protein test results showed that the expression of p62 was significantly reduced,and LC3-II/LC3-I was significantly increased.In terms of apoptosis,cells with Bmal1 knockdown were more likely stained by Hoechst test,showing densely stained nucleus.Gene expression tests showed the increased expression of apoptosis-related genes Bax,Caspase3 and Bcl-2.Cell protein detection found that the pro-apoptotic protein Bax did not change significantly,but the expression of the anti-apoptotic protein Bcl-2 was significantly reduced,and the Bcl-2/Bax ratio was significantly reduced.ROS staining revealed that Bmall gene knockdown promoted endothelial superoxide production in cells.Conclusion:Bmall gene possess circadian rhythmic expression in the vascular endothelial cell.Down-regulation of Bmall expression enchance autophagy and apoptosis levels of vascular endothelial cells,which may be related to the increase of cell superoxide.