Regulation Of Autophagy By Heat Shock Protein 27 To Protect Human Coronary Artery Endothelial Cells From Oxidative Stress Injury

BackgroundCoronary atherosclerosis is an essential pathological foundation of coronary atherosclerotic heart disease,and endothelial dysfunction is an early step in the procession of coronary atherosclerosis.Among them,oxidative stress injury in the vascular endothelium is a significant mechanism leading to endothelial dysfunction:on the one hand,oxidative stress induces vascular inflammation and injury by activating transcription factors,upregulating adhesion molecules,stimulating chemokines,and recruiting inflammatory cells;on the other hand,the increase in intracellular reactive oxygen species（ROS）levels during oxidative stress can damage mitochondria,which subsequently release their contents,causing cellular injury by activating the Caspase cascade to trigger apoptosis.Therefore,suppressing oxidative stress-induced endothelial cell injury has important clinical implications for protecting endothelial function and slowing the progression of coronary atherosclerosis.Heat shock protein 27（HSP27）is a multifunctional protein that is induced and expressed under various stress conditions and can protect cells from stress injury.In recent years,studies have reported that HSP27 not only regulates cellular apoptosis,but also affects apoptosis in hepatoma cells or colon cancer cells under stress conditions by regulating autophagy,which is in favor of cell survival.Different extent of autophagy has varying effects on apoptosis under oxidative stress.On the one hand,moderate autophagy protects cells from oxidative stress injury by inhibiting apoptosis,pyroptosis and inflammatory response;on the other hand,excessive autophagy promotes apoptosis and aggravates oxidative stress injury.Therefore,it is important to explore the effects of autophagy on apoptosis induced by oxidative stress and the role of HSP27 in autophagy and apoptosis in order to elucidate the mechanisms of HSP27against oxidative stress injury in vascular endothelial cells.In the course of autophagy,as member of the ATG family,ATG7 encodes a unique ubiquitin-activating E1-like enzyme in autophagy system that contributes toward the activation of multiple autophagy-related proteins in two ubiquitin-like conjugation systems and plays essential roles in phagophore elongation and autophagosome formation.Moreover,studies also confirm that downregulating ATG7expression promotes apoptosis in acinar cells.It follows that ATG7 is able not only to regulate autophagy,but also affect apoptosis,which influences cell survival eventually.Therefore,intervention in ATG7-mediated autophagy may contribute to alleviate apoptosis induced by stress,which has a positive role in cell survival under oxidative stress conditions.And one previous study investigating the regulation of Drosophila eye morphology suggested a regulatory relationship between HSP27 and ATG7.Therefore,we speculate that HSP27 may inhibit apoptosis to alleviate cell injury by regulating ATG7 to promote autophagy,thereby exerting an anti-oxidative stress effect.ObjectivesIn the present study,HCAECs were selected for subjects in the study,and H₂O₂was used to construct a model of oxidative stress injury.Using this model,we explored injury mechanisms of oxidative stress on HCAECs and observe changes in HSP27 expression.We also investigated whether mechanisms of anti-oxidative stress effects of HSP27 are associated with autophagy or not,and further explored its molecular mechanisms in regulating autophagy.Methods1.HCAECs were treated with H₂O₂ to establish a model of oxidative stress injury.CCK-8 was employed to detect cell viability and LDH activity was measured using an LDH cytotoxicity assay kit.Intracellular ROS levels were examined using the DCFH-DA probe.Western blotting was performed to detect the expression of apoptosis-related proteins（Cleaved Caspase-3,Caspase-3,Bax,Bak,and Bcl-2）.Using these methods were performed to explore the effects of oxidative stress on HCAECs.2.Western blotting was used to measure HSP27 expression in HCAECs treated with H₂O₂.High and low HSP27 expression and NC models were constructed using lentiviral transfection technology and detected using western blotting and quantitative real-time PCR.Cell viability and LDH activity were detected using CCK-8 and LDH cytotoxicity assay kits respectively,in cells with high/low HSP27 expression.The expression of apoptosis-related proteins（Cleaved Caspase-3,Caspase-3,Bax,Bak,and Bcl-2）was detected using western blotting and TUNEL staining was used to detect apoptotic cells and assess the rate of apoptosis.These methods were used to explore the effects of HSP27 on oxidative stress-induced apoptosis in HCAECs.3.Western blotting was used to measure the expression of autophagy-related proteins（Beclin-1,LC3,and P62）in HCAECs treated with H₂O₂.Autophagosome morphology was observed microscopically using TEM.Immunofluorescence staining of LC3 were observed by the confocal laser microscopy to assess autophagy levels.Western blotting was used to analyze the expression of autophagy-related proteins（Beclin-1,LC3,and P62）in HCAECs pretreated with the autophagy inhibitor 3-MA,which were also subjected to immunofluorescence staining for LC3 to assess autophagy levels.After 3-MA pretreatment in HCAECs,cell viability,LDH activity,the relative expression of apoptosis-related proteins（Cleaved Caspase-3,Caspase-3,Bax,Bak,and Bcl-2）,and rates of cell apoptosis were measured to assess the effects of autophagy inhibition on apoptosis.The expression of autophagy-related proteins（LC3 and P62）was also examined after transfection with Pre HSP27/si HSP27.And immunofluorescence staining was used to observe LC3 expression in transfected cells.These methods were used to explore the effects of HSP27 on autophagy induced by oxidative stress in HCAECs.4.Western blotting was used to detect ATG7 expression in HCAECs under H₂O₂and in Pre HSP27-and si HSP27-transfected cells.A low ATG7 expression model was constructed using si RNA technology and ATG7 expression was detected using real-time PCR.Under H₂O₂ stimulation,detect cell viability,LDH activity,and the expression of autophagy-related proteins（LC3 and P62）and apoptosis-related proteins（Cleaved Caspase-3,Caspase-3,Bax,Bak,and Bcl-2）after downregulating ATG7 expression.At the same time,after transfecting si ATG7 plasmid in cells with high HSP27 expression,detect again the above indicators to explore whether the anti-oxidative stress effects of HSP27 were achieved by inhibiting apoptosis through regulating ATG7 to promote autophagy.Results1.Construction of oxidative stress injury model and effects of H₂O₂ on HCAECs:H₂O₂ decreased cell viability and increased LDH activity in a time-dependent manner,and caused intracellular ROS levels to be elevated obviously.Meanwhile,the Cleaved Caspase-3/Caspase-3 ratio,the levels of Bax and Bak were constantly elevated while the levels of Bcl-2 were constantly decreased with the prolongation of H₂O₂ treatment,indicating elevated levels of apoptosis.2.Effects of HSP27 on H₂O₂-induced apoptosis in HCAECs:HSP27 protein levels increased in a time-dependent manner in HCAECs stimulated with H₂O₂.Upon H₂O₂ stimulation,high expression of HSP27 increased cell viability,reduced LDH activity,the Cleaved Caspase-3/Caspase-3 ratio,and Bax and Bak expression,and increased Bcl-2 expression,which inhibited apoptosis.Conversely,upon H₂O₂stimulation,low expression of HSP27 further decreased cell viability,increased LDH activity,the Cleaved Caspase-3/Caspase-3 ratio,and Bax and Bak expression,and decreased Bcl-2 expression,which promoted apoptosis.3.Effects of HSP27 on H₂O₂-induced autophagy in HCAECs:H₂O₂ treatment increased Beclin-1 expression and the LC3-II/LC3-I ratio,and decreased P62expression.In H₂O₂-treated HCAECs,punctate accumulation of LC3 was observed by immunofluorescence staining and double-membrane structures called autophagosome were observed by TEM,suggested an increase in autophagy.Pretreating HCAECs with the autophagy inhibitor 3-MA decreased Beclin-1 expression and the LC3-II/LC3-I ratio while increasing P62 expression,reducing punctate accumulation of LC3 remarkably,indicating that autophagy caused by H₂O₂ was clearly inhibited.In addition,3-MA pretreatment under H₂O₂ stimulation further decreased cell viability,increased LDH activity,the Cleaved Caspase-3/Caspase-3 ratio,and Bax and Bak expression,and decreased Bcl-2 expression,which aggravated cell injury.High HSP27 expression further increased the LC3-II/L3C-I ratio and decreased P62expression under H₂O₂ stimulation,and increased punctate accumulation of LC3,which promoted autophagy.Conversely,low HSP27 expression decreased the LC3-II/L3C-I ratio and increased P62 expression under H₂O₂ stimulation,and led to a decrease in punctate accumulation of LC3,which inhibited autophagy.4.Effects of HSP27 on H₂O₂-induced apoptosis in HCAECs via the regulation of ATG7-mediated autophagy:During H₂O₂-induced HCAEC injury,ATG7 protein levels were markedly increased and high HSP27 expression further promoted ATG7 expression.Under H₂O₂ stimulation,downregulating ATG7 led to additional decrease in cell viability and further increase in LDH activity,and the LC3-II/LC3-I ratio was decreased while P62 levels were increased,and the Cleaved Caspase-3/Caspase-3 ratio,Bax and Bak levels were further increased while Bcl-2levels were further decreased;in addition,inhibiting ATG7 expression in HSP27high-expressing cells partially eliminated the increase in LC3-II/LC3-I ratio,the decrease in the P62 level,and the decrease in Cleaved Caspase-3/Caspase-3 ratio and levels of Bax and Bak,and the increase in the Bcl-2 level.ConclusionsH₂O₂ induced apoptosis,autophagy and HSP27 protein expression in HCAECs.Inhibiting autophagy further increased apoptosis and aggravated oxidative stress-induced HCAEC injury,whereas HSP27 inhibited apoptosis by regulating ATG7 to promote autophagy and thereby alleviate HCAEC injury caused by oxidative stress.Innovation and significanceSince coronary artery endothelial cell injury is the initial step in coronary atherosclerosis,so the ability of endothelial cells to combat oxidative stress is the key to attenuate endothelial cell injury,delaying or even preventing coronary atherosclerosis.This study first clarified the effects of autophagy on protective roles toward cells in oxidative stress response,and then clarified that HSP27 affected apoptosis to counteract oxidative stress injury of endothelial cells via promoting autophagy,and further revealed that this mechanism of oxidative stress injury of HSP27 was achieved by regulating ATG7.Together,the findings of this study provide a new scientific basis for the effects of HSP27 against oxidative stress injury and a new rational for prevention and treatment in the early stage of coronary atherosclerosis.