Molecular Mechanism Of NEXN Gene In Atherosclerosis By Regulation Pyrotosis

Objective:Coronary heart disease is a serious threat to human health.Atherosclerosis(atherosclerosis,AS)is the main pathological mechanism.A large number of studies have shown that AS is a series of chronic inflammatory reactions that occur after the injury of the inner wall of the blood vessels.Like other inflammatory diseases,the inflammatory reaction plays an important role in the process of AS development and development,which runs through the various pathological processes of AS.The inflammatory cysteine aspartate proteinase(Caspase)plays an important role in AS inflammation.Cysteine aspartic proteinase-1 is a key molecule in pyrotosis(a newly discovered programmed mode of death).We speculate that the process of AS disease is associated with cell death.It is found that a major inducement of oxidized low density lipoprotein(OX-LDL),the important inducement of AS,can cause the microfilament breakage,disorder and absence of the cytoskeleton F-actin.The binding protein NEXN of F-actin is increased in AS plaques,but the mechanism is unknown.The purpose of this study is to explore the role and mechanism of NEXN in the pathological process of AS from the angle of pyrotosis and the cell model established by OX-LDL stimulating cells to provide an experimental basis for elucidating the pathological mechanism of AS.Methods:In the process of atherosclerosis,the cells that are deposited in atherosclerotic plaques are mainly endothelial cells,macrophages and smooth muscle cells.Therefore,human acute monocytic leukemia cells(THP-1)were studied.1.OX-LDL induced THP-1 cells pyrotosis1.1 Detection of OX-LDL induced THP-1 pyrotosisAfter THP-1 cells were induced by lipopolysaccharide(LPS)and oxidized low density lipoprotein(OX-LDL),the incidence of cell pyroptosis was detected by EB and EthD-2 staining,the cytotoxicity of lactate dehydrogenase(LDH)release test was detected,and polymerase chain reaction(PCR),protein immunoblotting(Western blot,WB)Detection of focal death related molecules:Caspase-1 and IL-1 beta expression.1.2 The pyrotosis inhibition testCytotoxicity was detected by lactate dehydrogenase(LDH)release assay after treatment of THP-1 cells with Caspase-1 inhibitor Z-YVAD-FMK.2.Effect of NEXN on cell death in THP-1 cells induced by OX-LDL2.1 The expression of NEXN in OX-LDL induced THP-1 cells.After THP-1 were treated with lipopolysaccharide(LPS)and oxidized low density lipoprotein(OX-LDL),the rate of pyroptosis,the release of lactate dehydrogenase(LDH),NEXN,and the expression of Caspase-1 and IL-1 beta were detected.2.2 In OX-LDL induced THP-1 cells,siRNA was interfered with NEXN and pyrotosis was detected.In order to further explore the role of NEXN in the OX-LDL induced the pyrotosis of THP-1,we interfere with NEXN in the OX-LDL induced THP-1 cells established by THP-1,and detect the incidence of pyroptosis,the release of lactate dehydrogenase(LDH),NEXN,and the related components of pyrotosis:Caspase-1,IL-1 beta expression.2.3 Detection of cell death after overexpression of NEXN in OX-LDL induced THP-1 cells.In order to further clarify the role of NEXN in AS,we overexpress NEXN in the THP-1 cells induced by OX-LDL to detect the incidence of pyroptosis,the release of lactate dehydrogenase(LDH),NEXN,and the expression of Caspase-1 and IL-1 beta.2.4 After overexpression of NEXN in OX-LDL induced THP-1 cells,Caspase-1was inhibited at the same time,and the pyrotosis was detected.Results:1.After induction of THP-1 cells with 40mg/ml concentration of OX-LDL,the rate of pyrotosis of OX-LDL cells was 15% higher than that of control group.2.OX-LDL induced the Caspase-1 mRNA transcriptional level was 2.71±0.32 times as high as that in control groupin THP-1 cell cells,and the level of IL-1 beta mRNA was 2.71±0.46 times as high as that in control group,and the protein expression level also increased in varying degrees.3.OX-LDL induced pyrotosis in THP-1 cells inhibited by Caspase-1inhibitor,and the incidence of pyrotosis was reduced by 22%.4.in OX-LDL induced THP-1 cells,the transcription level of NEXN mRNA was 8.31 times higher than that of control group,and the level of protein expression was also increased.5.After siRNA interference induced by OX-LDL in THP-1 cells,the incidence ofpyrotosis was 35% higher than that in siNC group,LDH was 12.6% higher than that in siNC group,IL-1 beta was higher than that of siNC group,and cleaved caspase-1 protein expression was also increased.6.After the overexpression of NEXN in THP-1 cells induced by OX-LDL,the rate of pyrotosis was 14% lower than that of the vehicle group,and the LDH was 29.8% lower than that in the vehicle group.The IL-1 beta was reduced by20.2ng/ml in the vehicle group and the expression of cleaved caspase-1 protein was also reduced.Conclusion:1.In OX-LDL induced THP-1 cells,cell death occurs and plays an important role.2.In OX-LDL induced THP-1 cells,we overexpress NEXN,what result in the decrease of pyrotosis;we interfere the transcription of NEXN,and that result in the increase of pyrotosis;indicating that NEXN may participate in the process of pyrotosis through Caspase-1,and NEXN may inhibit the pyrotosis.