Effects Of Simvastatin On The Autophagy Of LPS-induced Human Neutrophils And Release Of Inflammatory Mediators

Part one: Effects of simvastatin on the autophagy of human neutrophils induced by LPSObjective To investigate the effects of simvastatin on autophagy of neutrophils activated by LPSMethods Peripheral blood neutrophils(n = 10)from healthy adults were divided into five groups:GroupⅠ(Control group),group Ⅱ(LPS group),group Ⅲ(LPS + simvastatin group),group Ⅳ(LPS + simvastatin + 3-MA group)and groupⅤ(LPS + 3-MA group).groupⅠwas blank control group,groupⅡ,Ⅲ,Ⅳ,Ⅴwere added lipopolysaccharide(final concentration of 1ug / ml),cultured 12 h,thencentrifugedandstorage each cell culture supernatant in a refrigerator at-80 ℃,Group II was given equal volume of PBS,groupⅢwas added simvastatin(final concentration of6uM),groupⅣ wasaddedsimvastatin(Final concentration of 6uM)and 3-MA(final concentration of 5m M),groupⅤwasadded3-MA(final concentration of 5uM)only,Cultured for 24 hours,then centrifugedandstorage each cell culture supernatant in a refrigerator at-80℃。the expression of beclin-1 mRNA was detected by RT-PCR,the expression of Beclin 1 and LC3-II protein was detected by Western Blot.Results Compared with group Ⅰ:the expression of Beclin 1 mRNA and protein、LC3-II protein expression in group Ⅱ、Ⅳ、Ⅴhas no obvious difference(P > 0.05),and the expression of Beclin 1 mRNA and protein、LC3-II protein were increasedingroupⅢ(P<0.05);Compared with group Ⅱ:the expression of Beclin 1 mRNA and protein、LC3-II protein expression in groupⅢincreased(P> 0.05),groupⅣandⅤhaveno significant differenceswithgroupⅡin the expression ofBeclin 1 mRNA and protein、LC3-II protein(P> 0.05).Compared with group Ⅲ,the expression of Beclin 1 mRNA and protein、LC3-II protein in groupⅣⅤweredecreased(P <0.05).Conclusion Simvastatin can enhance the autophagy activity of neutrophils induced by LPS.LPS does not significantly promote autophagy for neutrophils,and 3-MA can inhibit the autophagy effect of simvastatin.Part two: Effects of simvastatin on the inflammatory mediators released by LPS-induced human neutrophilsObjective To investigate the effects of simvastatin on inflammatory factors produced by LPS-activated neutrophils and to explore whether the anti-inflammatory effect of simvastatin is related to autophagy.Methods Take out the frozensupernatantfrom-80 ℃ refrigerator,room temperature melting.The concentrations of TNF-α and IL-6 in the supernatant were measured by ELISA.Results Compared with group Ⅰ,the levels of TNF-α and IL-6 in group Ⅱ,Ⅳ andⅤ were increased after treatment with LPS for 12 h(P <0.05),whilethe levels of TNF-αand IL-6 in groupⅢ hadno significantly difference(P> 0.05);Compared with group Ⅱ,the levels of TNF-α and IL-6 in group Ⅲweredecreased(P <0.05),the levels of TNF-α and IL-6 in group Ⅳ and Ⅴ were not significantly different(P> 0.05);Compared with groupⅢ,the levels of TNF-α and IL-6 in groupⅡ、Ⅳ、Ⅴwereincreased(P <0.05).Conclusion LPS can promote neutrophils release of inflammatory mediators,simvastatin makes LPS activated neutrophils,release IL-6 and TNF-α.When Autophagy inhibitor 3-MA inhibitestheautophagy of neutrophils,the level of of IL-6andTNFαingroupⅣ hadno differencewithgroup Ⅱ,and use 3-MA processing LPS activation of neutrophils,also can be observed in the IL-6,TNF alpha level no difference with the groupⅡ,suggest autophagy can affect the release of inflammatory mediators and theanti-inflammatory effect of simvastatin,may be related to its increase of autophagy.