The Study Of The Role Of Melatonin In Palmitic Acid-induced Pyroptosis In Vascular Endothelial Cells

Objective: Coronary artery disease-related cardiovascular disease caused by atherosclerosis(AS)has become one of the main causes of death in developed and developing countries in the 21 st century.In view that the high incidence and death of AS has brought about the economic and living burden to the entire society,it is urgent to find effective measures on the prevention and treatment of AS.Inflammation is closely related to the occurrence and development of AS,and endothelial injury is the initial factor.In recent years,more and more studies have shown that immune response is closely related to AS.Pyroptosis,a form of programmed cell death different from apoptosis,is an important part of innate immunity,which induces the occurrence of AS by causing inflammation and AS-related cell pyroptosis.The importance of vascular endothelial cells to vascular homeostasis is self-evident.Therefore,actively seeking new mechanisms or drug targets to reduce or inhibit endothelial cell pyroptosis and inflammation is the focus of prevention and treatment of AS.As a sensor of innate immunity,the inflammasome NLRP3 is involved in the occurrence of cardiovascular disease.Under the stimulation of pathogenic factors,its activation can cause the classical pathway caspase-1-mediated cell pyroptosis,thereby starting the occurrence of AS.Therefore,inhibiting the activation of NLRP3 has become an important target for inhibiting endothelial cell pyroptosis and the development direction of drug inhibitors in recent years.Melatonin is an indoleamine hormone secreted mainly by the human pineal gland with the function of regulating the circadian rhythm of the human body.Its chemical name is N-acetyl-5 methoxytryptamine.A large number of studies have shown that melatonin has powerful effects in scavenging free radicals,anti-aging,inhibiting tumor growth,anti-oxidative stress,and anti-mitochondrial apoptosis.Studies in our laboratory have confirmed that melatonin can significantly improve endothelial injury induced by low shear stress.Further exploration of its effect on pyroptosis in vascular endothelial cells and related molecular mechanisms is of great value for the prevention and treatment of AS,and provide a new basis for clinical drug development for AS.Therefore,this experiment mainly used palmitic acid to induce endothelial cell injury,and used melatonin to intervene to observe its effect on cell viability and changes in the expression levels of pyroptosis-related molecules.Methods: The inverted phase contrast microscope was used to observe the morphological changes of vascular endothelial cells induced by different concentrations of palmitic acid and the intervention of melatonin.The morphology of vascular endothelial cells was observed by scanning electron microscope.The CCK8 method was used to detect the effects of different concentrations of palmitic acid on the viability of EA.hy926 for 24 hours and the effect of melatonin intervention on the viability of EA.hy926 cells.Western Blotting method was used to detect the relative protein expression levels of NLRP3,pro-caspase-1,caspase-1p20,GSDMD-N in vascular endothelial cells induced by palmitic acid at different concentrations for 24 hours.Western Blotting method was used to detect the relative protein expression levels of NLRP3,pro-caspase-1,caspase-1 and GSDMD-N in vascular endothelial cells of the blank control group,palmitic acid treatment group,and melatonin intervention group.Results: Observed under different fields of inverted microscope: compared with the control group,palmitic acid of different concentrations can induce endothelial cell death,reduce cell density,and cause changes in endothelial cell morphology,especially when the PA concentration is higher than 400μM to 500μM,the number of cell death increases significantly and the cell morphology changed significantly.The results of scanning electron microscopy showed that compared with the control group,palmitic acid can induce the typical pyroptosis of endothelial cells(Pyroptotic body).CCK8 method detection found that PA can significantly reduce the cells viability of EA.hy926,and as the concentration of PA increases,the cell viability decreases more significantly.After melatonin intervention,it can improve the viability of endothelial cells.Western Blotting found that PA can significantly increase the relative protein expression levels of NLRP3,pro-caspase-1,GSDMD-N and caspase-1p20 in vascular endothelial cells,and the protein expression level increases with increasing concentration.The relative protein expression levels of NLRP3,pro-caspase-1,GSDMD-N,caspase-1 in PA-induced vascular endothelial cells have no obvious change,compared with the control group and while there is a significant difference between the PA group and the melatonin group.Conclusion: Palmitic acid has a significant inhibitory effect on the viability of vascular endothelial cells.In addition,palmitic acid can increase the expression levels of pyroptosis-related proteins such as NLRP3,caspase-1 and GSDMD-N in vascular endothelial cells,which is concentration-dependent and melatonin can sharply inhibit palmitic acid-induced pyroptosis in vascular endothelial cells,thereby reducing endothelial cell damage.