Effect Of Ethyl Pyruvate On Autophagy And Apoptosis Of THP-1 Derived Foam Cells

Objective:As we all know, macrophages phagocytose oxidized low density lipoprotein (ox-LDL) and transform into foam cells. Foam cell death may lead to the formation of lipid core, plaque progression and plaque rupture. Cell death modes include:autophagic cell death, apoptosis and necrosis. Ethyl pyruvate (EP) is a derivative of pyruvate. Multiple studies have shown that the drug, by antagonizing high mobility protein (HMGB1), can inhibit cell autophagy and apoptosis therefore reduct cell death, and thus play a protective effect on organs. In this study, we observed the effect of EP on autophagy and apoptosis of foam cells and tried to explore the possible underlying molecular pathways..Methods:1. Construction of THP-1 derived foam cell model:THP-1 cells were treated with PMA for 24h and changed into macrophages. Then we incubated these THP-derived macrophages with different concentrations of ox-LDL (0mg/L～150mg/L) for 24 hours. After that, we detected lipids distribution in foam cells with oil red O staining and determined cell growth activity by MTT.2.We gave these THP-derived macrophages different concentrations of ox-LDL (0mg/L～150mg/L) for 24 hours and detected levels of autophagy and apoptosis of THP-derived foam cells by flow cytometry. We did all these to select the optimal model building condition.3. EP’s effects on autophagy and apoptosis of THP-derived foam cells:We used the optimum concentration of ox-LDL selected by former procedures to stimulate THP-1 cells to foam cells. Then we used different concentrations EP (5mM～20mM) for intervention to observe its effects on foam autophagy and apoptosis. We dividedThe THP-1 derived foam cells into five groups ①the control group:this is a group without any intervention factor; ②foam cells group:100mg/L of ox-LDL was added;③ low concentration EP group:5mM EP was added with 100mg/L ox-LDL;④ moderate concentration EP group:lOmM EP was added with 100mg/L ox-LDL;⑤ high concentration EP group:20mM EP was added with 100mg/L ox-LDL. We incubated both the cells and the reagents for 24 hours, then deteced the levels of autophagy and apoptosis of the cells with western blot, flow cytometry, immunefluorescence and real-time PCR technique. We did all these in order to evaluate effects of different concentrations of EP on THP-1 derived foam cells’ autophagy and apoptosis.4. We detected the possible molecular mechanism of EP’s effects on autophagy and apoptosis of THP-derived foam cells. We used immunofluorescence and real-time PCR to detect mRNA expression levels of HMGB1, Beclinl, RAGE and TNF-a in ox-LDL group and ox-LDL+20mM group. Through comparision of the two groups we explored the possible mechanism of EP’s effects on autophagy and apoptosis levels of foam cells.Results:1. When ox-LDL was 25mg/L, the survival rate of the THP-derived foam cells was down to 87.18% ± 5.34%(compared with control group P<0.05). With the ox-LDL concentration increased, cell viability decreased. Oil red O staining showed a large number of lipid droplets within the foam cells.2. Autophagy and apoptosis levels of THP-1 derived foam cell increased significantly consistent with ox-LDL concentrations. Autophagy rates of the Omg/L group,25mg/L group,50mg/L group, 100mg/L group,150mg/L group were respectively 0.05%± 0.01%,1.28%±0.32%,2.09%±0.17%,5.01%±0.33%,5.41%±0.27%; apoptosis rates were 10.01%±0.19%,20.36%±3.42%,29.83%±6.27%,49.29%±10.66%, 62.70%±1.95%. But between 100mg/L group and 150mg/L group, the difference was not statistically significant.3.Flow cytometry, real-time PCR, Western blot and other detection methods confirmed that with EP concentration increasing, levels of autophagy decreased(P <0.05). Flow cytometry showed that the apoptosis rate of foam cells was significantly increased compared to the control group (48.77%±7.22 VS 5.87±1.03) (P<0.05).The apoptosis rates of different concentrations of EP group(5mM,1 OmM,20mM) were lower than the foam cells significantly,26.12%±3.99%,12.92%±1.32% 9.46%±0.53%(P<0.05). With the EP concentration going higher, rate of apoptosis dropped.4. Immunofluorescence staining, real-time PCR showed that the HMGB1, Beclinl, RAGE and TNF-a expression level of foam cell group were all lower than the ox-LDL group.Conclusion:1. PMA can induce differentiation of THP-1 cells into adherent macrophages after incubating for 24h, then foam cell model can be successfully built after incubating with ox-LDL for 24h. This is a simple and reproducible method, which can establish a reliable experimental model for the study of arterial sclerosis in vitro;2. In the ox-LDL-induced THP-1 derived foam cells, autophagy and apoptosis levels were significantly higher than the control group;3. EP can inhibit foam cell autophagy and apoptosis in a dose-dependent way;4.EP may reduce foam cell autophagy through HMGB1-Beclinl intereaction and HMGB1-RAGE passway; EP may reduce foam cell apoptosis through inhibiting the HMGB1-TLR4-TNF-a passway.