The Role And Relationship Of Autophagy And PARP-1 In VSMCs Under Oxidative Stress

Objective:Atherosclerosis is a kind of serious disease endangering human health.Oxidative stress has played an important role in development of AS,excessive ROS is an important promoting factor of atherosclerosis in the process of oxidative stress.Autophagy is a catabolic pathway for bulk destruction of long-lived proteins and organelles via lysosomes,which is involved in the maintenance of normal cellular homeostasis and prosurvival mechanism.Autophagy plays a crucial role in the pathogenesis of atherosclerosis,which involves in the occurrence and development of AS.At present,a growing body of evidence suggests ROS in AS is known as inducer of autophagy and play an important role in the formation of autophagy.Autophagy is an evolutionary conserved process involved in the degradation of long-lived proteins and dysfunctional organelles.Therefore,autophagy is usually thought of as a survival mechanism.While when survival mechanisms fail,death programs are activated in response to oxidative stress.In recent years,it has become accepted that autophagy,in addition to its role in cell survival,can also lead to cell death（referred to as type II cell death）.Therefore,autophagy has a dual role in determining the cell fate.At present,research about the role of autophagy in oxidative stress are different,and the specific mechanism remains unclear.It’s of important significance to further study on autophagy mechanism for prevention and treatment of AS。PARP-1 is a nuclear enzyme that widely presents in eukaryotic cells which can be activated by DNA damage induced by ROS or RNS.PARP-1 overactivation can modify target protein（histone,transcription factor and itself）by poly ADP-ribosylation using intracellular NAD+as a substrate to in order to exert different biological effects.Increasing evidence suggests PARP-1 activation participates in pathogenesis of atherosclerosis,heart failure and other cardiovascular diseases.The inhibition of PARP-1 activation can effectively delay the development of atherosclerosis and increase the stability of atherosclerotic plaque through several mechanisms.PARP-1 can regulate transcription factor of macrophages,endothelial cells and vascular smooth muscle cells to involve in AS.Recent studies have found that PARP-1 activation not only causes cell death,but also may involve in autophagy through the AMPK-mTOR signaling pathway.But the research about the relationship between PARP-1 and autophagy in cardiovascular field is rare.PARP-1 is known to be implicated in a number of physiological and pathological processes,leading to various biological consequences including cell death（either apoptotic or non-apoptotic/necrotic cell death）.Recent studies have found that PARP-1 may mediate a new non-caspase dependent cell death type.PARP-1 may involve in various signal pathways of cell death,but the specific mechanism remains unclear.VSMCs is the major components of the blood vessel wall and its proliferation and migration involve in in atherosclerosis and restenosis process.At present,the study on the regulation of PARP-1 to VSMCs autophagy under oxidative stress has not been reported.Therefore,we aim to explore the relationship of between PARP-1 and autophagy and potential mechanisms by H₂O₂-stimulated oxidative stress model.The role of PARP-1 is very complex,so we further explore the role of PARP-1 in VSMCs death under oxidative stress.The study on mechanism that PARP-1 and autophagy involve in AS has important significance for the prevention and treatment of diseases.Methods:Primary VSMCs isolated from WT mice was applied in our study.1.VSMCs was isolated using an explant technique from the thoracic aorta of mice.Immunofluorescence techniques was conducted to identify VSMCs.2、H₂O₂ was used to form oxidative stress model of VSMCs.3.LC3 and Beclin-1 were detected by western-blot to evaluate the autophagy level;PARP-1 and activated product PAR were detected to observe the activation of PARP-1;p-AMPK、p-p70S6K were detected to observe the pathway linking PARP-1 and autophagy.4.Cell viability was quantified by MTT method and AnnexinV/PI binding assay.5.PARP-1 small interfering RNA（siRNA）was used to knock down PARP-1.6.The fluorescent probe of CM-H2DCFDA coupled with flow cytometry were used to detect intracellular ROS generation.7.Caspase-3 Colorimetric Assay was used for caspase-3 activity.Results:1.The induction of autophagy and its role in VSMCs survival under oxidative stress.H₂O₂ could induce autophagy in a time dependent manner,and the autophagic marker protein LC3-II and beclin-1 expression increased.Lysosomal inhibitor-hydroxychloroquine sulfate（HCQ）further increased the expression of LC3-II,suggesting that H₂O₂ could induce autophagic flux.The autophagy inhibitor3-MA decreased the expression of LC3 and beclin1 protein,we further studied the effect of autophagy inhibition on H₂O₂-mediated cell death.The result showed autophagy inhibition by 3-MA significantly increased cell viability than H₂O₂ group that suggested the ability of VSMCs in autophagy inhibition group against oxidative stress increased.In addition,we found intracellular ROS production was significantly increased by stimulation H₂O₂,while the antioxidant N-acetylcysteine could weaken the up-regulation of ROS.Moreover,NAC could significantly suppress the autophagy protein expression induced by H₂O₂.This suggested ROS involved in the induction of autophagy.2.The activation of PARP-1 and its modulation of autophagy in VSMCs under oxidative stress.The expression of PAR polymer,a direct result of PARP-1activation,was increased after H₂O₂ exposure.The activation of PARP-1 was ROS dependent,because the PAR polymer expression was inhibited by NAD.Compared with the control siRNA group,the expression of LC3-II decreased in the PARP-1silencing VSMCs after H₂O₂ stimulation.Furthermore,the p-AMPK expression decreased and the p-p70S6K expression restored.3.The role of PARP-1 in VSMCs survival under oxidative stress.PARP-1 gene silencing of VSMCs protected cell against H₂O₂-mediated cell death and the apoptosis significantly reduced.PARP-1inhibition markedly reduced caspase-3 activation compared with control group.Conclusion:1.H₂O₂ could induce autophagy in VSMCs,and the inhibition of autophagy could protect VSMCs against oxidative stress-mediated cell death.2.H₂O₂promoted the generation of ROS that further participate in the induction of autophagy and activation of PARP-1 in VSMCs.3.PARP-1 may involve in H₂O₂-yinduced autophagy via AMPK-mTOR pathway,and PARP-1 inhibition protected against apoptosis in VSMCs in response to H₂O₂.