Effects And Mechanisms Of Resolvin D1 On Autophagy In Microglia

Part ? Effects of resolvin D1 on autophagy in microglia Objective To study whether resolvin D1(RvD1)could induce autophagy in microglia and the effects of RvD1 on autophagy influx.Methods Mice microglial cell line BV-2 was taken as the research object.The experiments were divided into three parts: A,B and C.Part A was to select the optimal concentration of RvD1 in promoting microglia autophagy.The cells were stimulated with vehicle or RvD1(10nM,50 nM,100nM)for 2 hours.Whole cell lysates were harvested and detected by western blotting for LC3,a marker of autophagy.Part B was to study on the optimum time of RvD1 in promoting microglia autophagy.The cells were treated with vehicle or RvD1(50nM)for different time(15min,30 min,1h,2h).Total protein was extracted and measured by western blotting for ULK1,BECN1,P62 and LC3.Part C was to demonstrate if RvD1 induced a dynamic autophagic flux.We used LY294002(LY)to prevent the initial steps in the formation of autophagosomes,and chloroquine(CQ)to inhibite the later steps of autophagy.The cells were divided into four groups: control,RvD1(50nM),CQ(20?M)+RvD1 and LY(10?M)+ RvD1 group.CQ was added 22 hours before RvD1 and LY was 2 hours before RvD1.Whole cell lysates were harvested and detected by western blotting for LC3 and P62.Transmission electron microscopy analysis and immunofluorescence were performed to observe the formation of autophagosomes.Results In part A,compared with control group,RvD1 of 10,50 and 100 nM could increase the ratio of LC3-II/LC-I in microglia,especially at 50 nM.Part B showed that the expression of ULK1,BECN1 and P62 did not change significantly at the early stage of RvD1 stimulation(15 minutes,30 minutes),while the LC3-II/LC-I ratio and degradation of P62 increased significantly at the later stage of RvD1 stimulation(1 hour,2 hours),especially at 2 hours.Part C showed that the presence of LY failed to significantly change LC3 or P62 protein levels in cells treated with RvD1.While cotreatment with CQ increased LC3-II/LC3-I ratio significantly.Transmission electron microscopy analysis and immunofluorescence confirmed that RvD1 indeed triggered autophagosomes formation.Conclusion RvD1 could promote autophagy in microglia.The optimum concentration of RvD1 was 50 nM and the optimum culture time was 2 hours.RvD1 could promote autophagosomes formation and the fusion of autophagosomes and lysosomes.Part ? The relationship between autophagy in microglia by resolvin D1 and m TOR pathwayObjective To study whether RvD1 activated microglia autophagy through m TOR pathway.Methods Mice microglial cell line BV-2 was taken as the research object.The experiments were divided into three parts: A,B and C.Part A was to study the effect of RvD1 on mammalian target of rapamycin(m TOR)pathway.The cells were treated with vehicle or RvD1(50n M)for different time(15min,30 min,2h,4h,6h).Whole cell lysates were extracted and measured by western blotting for protein levels of m TOR,P-m TOR,ULK1,P-ULK1,4E-BP1,P-4E-BP1,S6 K and P-S6 K.Part B was to study whether the activation of microglia autophagy by RvD1 was related to m TOR pathway.Rapamycin(Rapa),an inhibitor of m TOR,and 3BDO,an agonist,were used in this part.The cells were divided into four groups: control,RvD1(50n M),Rapa(50n M)and 3BDO(50?M)group.6 hours later,whole cell lysates were harvested and detected by western blotting for the levels of P62,LC3,mTOR,P-mTOR,ULK1,P-ULK1,4E-BP1,P-4E-BP1,S6 K and P-S6 K.Part C was to investigate whether RvD1 regulated intracellular calcium level.The experiments were divided into four groups: control,arachidonic acid(AA,50?M),RvD1(50n M)and AA+RvD1 group.The cells were primed with AA or vehicle for 2 hours and cultured with RvD1 or vehicle for another 2 hours.The intracellular calcium level was detected by flow cytometry and the expression of P-CAMK2 and CAMK2 was detected by Western blotting.Results Part A showed that,compared with the control group,RvD1 had no significant effect on the protein levels of m TOR,P-m TOR,ULK1,P-ULK1,4E-BP1,P-4E-BP1,S6 K or P-S6 K at the early stage of 15 min or 30 min.But at the later stage of 2h,4h and 6h,the phosphorylated proteins of m TOR,ULK1,4E-BP1 and S6 K were elevated by RvD1,especially at 6h.Part B showed that,compared with the control group,the LC3-II/LC-I ratio and phosphorylation levels of ULK1,4E-BP1,S6 K of RvD1 group were significantly higher.In Rapa group,phosphorylation levels of ULK1,4E-BP1 and S6 K decreased,but the LC3-II/LC-I ratio and degradation of P62 increased significantly.The 3BDO group showed the opposite effects.Part C showed that RvD1 alone did not affect the intracellular calcium level and the expression of P-CAMK2 and CAMK2.AA could significantly increase intracellular calcium level and phosphorylation of CAMK2.Compared with AA group,the intracellular calcium level and phosphorylation of CAMK2 in AA+RvD1 group decreased significantly.Conclusion RvD1 could activate m TOR pathway at the later stage.The activation of microglia autophagy by RvD1 might be through m TOR-independent pathway.