| Chronic inflammation is one of the main pathological changes in AD.Recent studies have shown that the main cause of chronic inflammation is inflammation resolution disorder,which is mediated by specialized pro-resolving lipid mediators(SPMs).The results of previous studies have confirmed that Ma R1 which is one of the SPMs can improve the survival of neurons and enhance the phagocytosis of Aβ42 by microglia,but Ma R1 is significantly reduced in the brain and entorhinal cortex(ENT)of AD patients.The decrease of Ma R1 may cause Aβ clearance disorder,excessive deposition and then aggravate the inflammatory response and lead to chronic inflammation.Therefore,stimulating resolution of inflammation by Ma R1 may be a new therapy for AD.Aim: We want to investigate the effects of Ma R1 on the chemotaxis,activation,phenotype of microglia and the effects of Ma R1 on learning and memory behavior and brain pathology in AD mice.Ultimately we want to elucidate the protective mechanisms of Ma R1.Methods:PartⅠIn vitro experiments:1.Study on the chemotactic effect of Ma R1 on microglia: primary neurons were planted at the bottom of the six multiwell plates and the transwell cell culture inserts were placed in the six multiwell plates.The microglia labelled with green fluorescent marker CFSE were seeded in the transwell cell culture inserts.Aβ42 and/or different concentrations of Ma R1 were used to treat the transwell co-cultured cells.After 6,16,24 hours,the chemotactic CFSE positive microglia including the cells in the culture medium and loading on the plate bottoms were collected and analysed by flow cytometry.2.The effect of Ma R1 on the phenotype of microglia and the activation of "on/off" signal: microglia and neurons were co-cultured for 24 hours and then treated with Aβ42and/or Ma R1.Six hours later,flow cytometry was used to analyse the phenotypic marker CD40,CD183 and the regulated marker of microglia activation CD200,CD200 R.The changes of inflammatory cytokines and chemokines in the supernatant of the co-culture medium were tested by cytometric beads array(CBA).The altered protein pathways were analysed by proteomics and the outcomes were verified by western blot.Part Ⅱ In vivo experiment:Forty male C57BL6/J mice aged three to four months were randomly divided into Vehicle group,Aβ42 group,Ma R1 group and Aβ42+Ma R1 group.Seven days after the models were established,the behavioral changes of the mice were evaluated by Morris water maze and the expression of CD11 b,GFAP and Fluoro-Jade B in the hippocampus and cortex were observed.The inflammatory cytokines and chemokine were detected by CBA and ELISA.The protein pathways detected by proteomics in vitro experiment were examined by western blot.Results:PartⅠIn vitro experiment:1.Compared with the Vehicle group,Aβ42 increased the chemotaxis of microglia.Ma R1 decreased the chemotaxis of microglia with or without Aβ42 stimulation(P<0.05).2.In the co-cultured cell models,the expression of CD200 R in Aβ42 group was lower than that in Vehicle group and Ma R1 could increase the expression of CD200 R reduced by Aβ42(P<0.05).However,there was no significant difference in CD200,CD40,CD183 expression among the four groups.3.In the co-cultured cell models,Aβ42 stimulation increased the secretion of pro-inflammatory cytokines TNF-ɑ,IL-6,chemokine MCP-1 and decreased the secretion of anti-inflammatory cytokine IL-10(P<0.05).Ma R1 decreased the secretion of TNF-ɑ,IL-6induced by Aβ42(P<0.05).Compared with the Aβ42 group,the secretion of chemokine MCP-1 decreased and the secretion of anti-inflammatory cytokine IL-10 increased in Aβ42+Ma R1 group,but it was not statistically significant.Ma R1 decreased IL-6 even without Aβ42 stimulation(P< 0.01).4.Quantitative proteomics revealed the molecular changes in the four differentstimulation groups.Compared with the Vehicle group,p-PI3K/total-PI3 K,p-AKT/total-AKT decreased and p-p38/total-p38,p-ERK/total-ERK,p-m TOR/total-m TOR,caspase3,p75 NTR,Cdc42 increased in the Aβ42 group(P<0.05).Compared with the Aβ42 group,p-PI3K/ total-PI3 K,p-AKT /total-AKT,p-ERK/total-ERK increased and p-p38/total-p38,p-m TOR/total-m TOR,caspase3,p75 NTR,Cdc42 decreased in the Aβ42+Ma R1 group(P<0.05).The results of western blot validation were consistent with the results of proteomics.Part Ⅱ In vivo experiment:1.Praxiology: the escape latency of all the groups of mice gradually shortened through five consecutive training days.Differences gradually became larger in the mean latency between the four groups of mice.The average escape latency of Aβ42 group was longer than that of Vehicle group on the fifth day(P<0.05).The average escape latency of Aβ42+Ma R1 group obviously decreased compared with that of Aβ42 group on the fifth day(P<0.05).The average escape latency of mice treated with Ma R1 alone had no significant difference with that of Vehicle group.In the probe trial test the Aβ42-injected mice showed significant memory deficits with decreased numbers of platform crossing times and shorter time staying in the target quadrant compared with the Vehicle group and Ma R1 group(P<0.001).Ma R1 treatment improved the cognition decline induced by Aβ42 injection and it was manifested by significant increase in platform crossing times and longer time staying in the target quadrant(P<0.05).2.Pathology:(1)The neurodegenerative neurons were revealed by FJB staining.The numbers of FJB-positive neuronal cells were significantly increased in the hippocampus and cortex of Aβ42-injected mice with or without Ma R1 treatment compared with the Vehicle and Ma R1 group(P<0.001).Ma R1 significantly reduced the numbers of FJB-positive neurons induced by Aβ42 in the hippocampus and cortex(P<0.001).(2)The feature of neuroinflammation is microglia and astrocyte activation.CD11 b and GFAP positive staining cells stand for active microglia and astrocytes respectively.The injection of Aβ42 protein increased the numbers of CD11 b and GFAP positive cells in the Aβ42 and Aβ42+Ma R1 group compared with the Vehicle and Ma R1 group(P<0.01).Treatment of Ma R1 reduced the numbers of CD11 b and GFAP positive cells induced by Aβ42(P<0.01).The numbers ofCD11 b and GFAP positive cells didn′t show any difference between the Vehicle and Ma R1 group.3.Secretion of inflammatory cytokines and chemokines: the pro-inflammatory cytokines TNF-ɑ and IL-6 increased in the brain of AD mice and decreased after Ma R1treatment(P<0.05).MCP-1 also increased in the brain of AD mice and it decreased in the presence of Ma R1(P<0.05).TNF-ɑ,IL-6 and MCP-1 didn′t show any difference between the Vehicle and Ma R1 group.IL-2 and IL-10 increased in the brain of AD mice(P<0.05).In the hippocampus,Ma R1 increased IL-2 and IL-10 without Aβ42 stimulation(P<0.05).In the cortex,the concentrations of IL-2 and IL-10 in Aβ42+Ma R1 group were higher than those of Vehicle group(P<0.05).IL-4,interferon(IFN)-γ and IL-17 A didn′t show any difference among the four groups.4.Changes of protein pathways: p-PI3K/total-PI3 K and p-AKT/total-AKT significantly decreased(P<0.001)and p-p38/total-p38,p-m TOR/total-m TOR,caspase3 increased in the presence of Aβ42(P<0.01).Ma R1 could reverse these changes induced by Aβ42(P<0.05).The p-ERK/total-ERK was increased in the brain of AD mice and increased more obviously after Ma R1 treatment(P<0.05).Ma R1 could increase p-ERK/total-ERK expression even without Aβ42 stimulation(P<0.01).Conclusion:1.Ma R1 inhibited the chemotaxis and activation of microglia.2.Ma R1 decreased the secretion of pro-inflammatory cytokines and chemokine and increased the secretion of anti-inflammatory cytokines.3.Ma R1 played a neuroprotective role by affecting cell survival,autophagy,axon formation,apoptosis inhibiting proteins and inflammation related proteins.4.Ma R1 attenuated glia activation and neuronal degeneration and improved cognitive decline in AD mouse model. |
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