Study Of Estrogen Against Atherosclerosis By Enhancing Autophagy

Objective:To investigate the effect of estrogen on macrophages in the inflammatory state through autophagy.Methods:1.1 Inflammation model preparation:The logarithmic phase of RAW 264.7 cells cultured in vitro.RAW 264.7 cells were divided into control group and different concentrations of LPS group（10 ng/ml,100 ng/ml,1000 ng/ml,10000 ng/ml）.The supernatants of RAW 264.7 cells were collected after reatmented 24 h or 48 h.The content of IL-1βin the cell supernatant was measured by ELISA kit.1.2 Determine the optimal concentration of estradiol:RAW 264.7 cells were cultured in vitro,and pretreated with different concentrations of E2(10^-9m M,10^-8m M,10^-7m M,10^-6m M)for 1 h.After incubation with LPS（1000 ng/ml）for 24 h,the IL-1βin the cell supernatant was measured by ELISA kit.1.3 Estradiol may play an anti-inflammatory role by mediating autophagy:RAW264.7 cells were cultured in vitro and divided into control group,LPS group,LPS+E2 group,LPS+E2+3-MA group.After 24 h of treatment,the levels of IL-1βand IL-18 in the supernatant of RAW 264.7 cells were measured by ELISA kit.1.4 Estradiol can improve the survival of macrophages by mediating autophagy:RAW 264.7 cells were cultured in vitro and divided into control group,LPS group,E2 group,LPS+E2 group and LPS+E2+3-MA group.After 24 h,the survival of the macrophages was determined by CCK-8.1.5 E2 can exert anti-inflammatory effects and improve macrophage survival by enhancing autophagy:RAW 264.7 cells were cultured in vitro,and divided into control group,LPS group,E2 group,LPS+E2 group.After 24 h,the expression levels of autophagy-related proteins SQSTM1/P62 was measured by Western blot.Results:1.LPS can stimulate RAW 264.7 cells to secrete inflammatory mediators（IL-1β）in a concentration-dependent manner.Elisa results showed that the content of IL-1βincreased with the increase of LPS concentration within 24 h.However,the content of IL-1βdecreased with the extension of time.2.The content of IL-1βin RAW 264.7 cells treated with LPS and E2decreased significantly（p<0.05）.The optimal concentration of E2 was 10^-8m M.3.The anti-inflammatory effect of E2 was decreased after the addition of3-methyladenine（3-MA）（p<0.05）,which suggested that the anti-inflammatory effect of estrogen was related to autophagy.4.E2 improved the survival of RAW 264.7 cells in the inflammatory state,and the ability of E2 to increase the survival of RAW 264.7 cells was reduced after the addition of 3-MA（p<0.05）,which suggested that the effect of estrogen to increase the survival of RAW 264.7 cells was related to autophagy.5.In LPS group,P62 was up-regulated,while in LPS+E2 group,P62 was down-regulated.indicating that the autophagy is enhanced.Furthermore,it is confirmed that E2 could play an anti-inflammatory role and improve the survival rate of macrophages by enhancing autophagy.Conclusion:1.A certain concentration of LPS can induce RAW 264.7 cells to construct an inflammation model.2.E2 may play an anti-inflammatory role by enhancing autophagy.3.E2 can improve the survival rate of macrophages in inflammatory state by enhancing autophagy.