| Background and purpose: Atherosclerosis is a chronic inflammatory response that increases the risk of cardiovascular disease,which is the leading cause of death worldwide.Its pathological characteristics are mainly endothelial dysfunction and gradual accumulation of lipids,necrotic cell debris and extracellular matrix proteins on the intima of blood vessels,which eventually lead to vascular occlusion or thrombotic complications.Many infiltrating immune cells have been found in atherosclerotic lesions,and these immune cells are activated during local inflammation or immune response to antigenic substances.And found in the local tissues and surrounding systems of atherosclerotic blood vessels including tumor necrosis factor(TNF-a),IL-1a,IL-1b,IL-4,interferon-c(IFN-c)and IL-18 And other inflammatory cytokines.Among them,the expression level of IL-18 is higher in atherosclerotic plaques.IL-18 acts by binding to specific receptors on target cell membranes to activate corresponding signaling pathways,and IL-18 binding protein(IL-18BP)It can reduce the risk of atherosclerotic plaque formation,suggesting that IL-18 may play an important role in the pathogenesis of atherosclerosis.Interleukin 18(IL-18)treated vascular smooth muscle cells(VSMC),explored the expression of TNF-?,IL-6,IL-8 in vascular smooth muscle cells by IL-18,and preliminary explored possible molecular mechanisms for arteries Research on the prevention and treatment of atherosclerosis provides new mechanisms.Methods:(1)Mouse vascular smooth muscle cells(VSMC),kindly donated by Chen Bing,a teacher of the Department of Biochemistry,were transfected with SV40 large T antigen and immortalized VSMC cell lines.Mouse vascular smooth muscle cells(VSMCs)were stored in the laboratory,and mouse VSMCs were resuscitated from liquid nitrogen and cultured and passaged;(2)Mouse IL-18(0,3,10,30,100 ng / ml)with varying concentration was treated with VSMC for 12 h,TRIZOl was added to lyse the cells and total RNA was extracted,and the target gene(TNF-?(IL-6,IL-6,IL-8)expression,and analyzed the correlation of the concentration of dosing treatment;adding IL-18(30ng / ml)at different time(0,2,6,12,18,24h)to treat VSMC,the same The method was used to amplify the expression of the target gene and analyze the time correlation of the drug treatment.(3)IL-18(30ng / ml)treatment VSMCs were cultured for 3 days,cell supernatants were collected,and the expression of target proteins(IL-6,IL-8,TNF-?)in the VSMC supernatants was analyzed by ELISA.(4)At different time(0,10,30,60,120m),IL-18(30ng / ml)was added to treat VSMCs,total cell proteins were extracted,and the expressions of pp38 and p AKT in p38 MAPK and PI3 K / AKT signaling pathways were analyzed by Western Blot method.Variety.(5)Specific inhibitors SB203580(10?M,p38 MAPK inhibitor)and LY294002(50?M,PI3 K / AKT inhibitor)were used to treat VSMC cells,and total cell proteins were extracted.Western Blot method was used to analyze the expression changes of pp38 and p AKT,respectively,while ELISA method The effect of VSMC supernatant on the expression of IL-18-induced cytokines(IL-6,IL-8,TNF-?)was analyzed.(6)VSMCs were treated with IL-18(0,3,10,30,100 ng /ml)for 72 h to determine the appropriate IL-18 concentration.(7)VSMC(0,1,2,3,4,5d)treated with IL-18(30 ng / ml),cell survival rate of each group was detected by MTS,and cell cycle and cells of each treatment group were detected by flow cytometry.Changes in apoptosis;total cell proteins were extracted,and changes in cell cycle and apoptosis-related proteins were obtained by Western blotting.Results:(1)Real-time PCR results showed that IL-18 could increase the expression of TNF-?,IL-6,and IL-8 m RNA levels in VSMCs,and was positively correlated with the concentration of IL-18(p <0.05);IL-18(30ng / ml)can increase the expression of TNF-?,IL-6,and IL-8 m RNA in VSMCs,and the expression is positively correlated with time within 24 hours(p <0.05).That is,the m RNA expression levels of TNF-?,IL-6,and IL-8 are dose-and time-dependent with IL-18.(2)Analysis of the results of ELISA method found that IL-18 can significantly increase the expression of TNF-?,IL-6,and IL-8 protein levels in VSMC supernatants(p <0.05).(3)Analysis of the results of Western blot showed that IL-18 can increase the expression levels of pp38 and p AKT in VSMC,and the peak values?were 60 min and 120 min,respectively.(4)SB203580 reduced the expression of pp38 in IL-18 treated cells,and LY294002 decreased the expression of p AKT in IL-18 treated cells.That is,SB203580 and LY294002 specifically blocked IL-18-activated p38 MAPK and PI3 K / AKT signaling pathways,respectively.SB203580(10 ? M,p38 MAPK inhibitor)partially blocks IL-18-induced expression of TNF-?,IL-6,and IL-8;LY294002(50 ?M,PI3 K /AKT inhibitor)partially blocks IL-18-induced TNF-?,IL-6,IL-8 expression(p<0.05).(5)The effect of IL-18 concentration on VSMC cell survival was not significant;(6)MTS test results showed that IL-18(30ng / ml)treated VSMC after1 d,2d,3d,4d,and 5d.The effect of cell survival rate was not significant.(7)The results of cell cycle analysis showed that there was no significant difference in the proportion of VSMC cells in G1,S,and G2 phases at five time points compared with the control group after IL-18 treatment of VSMCs for 1d,2d,3d,4d,and 5d..(8)Analysis of apoptosis induced by IL-18 in VSMC cells: There was no significant difference in the effects of apoptosis induced by IL-18(30ng / ml)on VSMCs in the experimental group(1d,2d,3d,4d,5d).(9)Western Blot results showed that Cyclin B1,Cyclin E,and p21 had no statistically significant changes after IL-18 treatment;IL-18 treatment partially down-regulated the apoptotic protein Bcl-2 and partially up-regulated the apoptotic protein Bax.Conclusion:IL-18 has no significant inhibitory effect on vascular smooth muscle cell proliferation,but IL-18 increases the expression of TNF-?,IL-6 and IL-8 in vascular smooth muscle cells in vitro,and its mechanism depends in part on the activation of p38 and PI3K/AKT signaling pathways.The results may help clarify some of the mechanisms of IL-18 in the occurrence and development of atherosclerosis,and lay the foundation for finding new prevention and treatment measures for atherosclerosis. |
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