| Objective:To explore the mechanism of PEA by interfering with hippocampal NF-κB signaling pathway to improve the depression-like behavior of chronic unpredictable mild stress（CUMS）combined with lipopolysaccharide（LPS）model in rats.Methods:Forty healthy male Sprague-Dawley（SD）rats were fed adaptively for 7 days（to weigh up to 180-200g）,according to their weight,randomly divided into 4 groups（10rats/group）:normal control group（NC）,model control group（MC）,fluoxetine group（FLU）and PEA group.In NC group,5 animals/cage,other groups were housed in solitary cage.LPS was injected intraperitoneally on the 4th day of the experiment,FLU and PEA were administered by gavage on the 5th day（24 hours after LPS injection）.LPS was injected intraperitoneally every 3 days,and PEA and FLU were administered intragastrically once a day for 28 consecutive days.In the fifth week of the experiment,after 24 hours of lipopolysaccharide injection,the related behavioral and physiological indicators tests were performed,including body weight,open-field test（OFT）,sucrose preference test（SPT）,novelty suppressed feeding test（NSFT）and physical state score（PSS）.After the last behavioral test,tissue samples were prepared for subsequent experimental testing.Immunopositive expression of microglia marker ionized calcium binding adaptor molecule 1（Iba 1）and astrocyte marker glial fibrillary acidic protein（GFAP）in the rat hippocampal slices were observed by immunohistochemistry（IHC）.Western blot（WB）was used to detect the protein levels nuclear transcription factorκB p65（NF-κB p65）,the phosphorylated nuclear transcription factorκB p65（p-NF-κB p65）,phosphorylated nuclear factorκB inhibitorα（p-IκBα）,arginase-1（Arg-1）and chitinase 3like protein 3（Ym-1）in the hippocampus of rats.The enzyme linked immunosorbent assay（ELISA）was used to detect the contention of tumor necrosis factor-α（TNF-α）and interleukin-1β（IL-1β）,Interleukin-6（IL-6）,Interleukin-4（IL-4）,Interleukin-10（IL-10）,Prostaglandin E2（PGE2）and cyclooxygenase-2（COX-2）in the hippocampus of rats.Colorimetric method and nitrate reductase method were used to detect the contention of inducible nitric oxide synthase（i NOS）and nitric oxide（NO）in the hippocampus of rats.Results:1.The basic body weight and depression like behavioral indicators of the rats in each group were similar at the beginning of the experiment and there was no difference.In the fifth week of the experiment,compared with the NC group,the weight of rats in the MC group was decreased significantly,and standing times,locomotion time and distance were decreased significantly,immobility time is increased significantly in the OFT,the sucrose preference and physical state scores were decreased significantly,the latency of feeding was decreased significantly in the NSFT,and the rats had yellow and dull hair,unresponsive and slow movement;compared with the MC group,the weight,standing times,locomotion time and distance,sucrose preference rate and physical state scores of rats in the FLU group and the PEA group were increased significantly,the immobility time and latency of feeding were decreased significantly,and the rats had snow-white and shiny hair,quick reactions and actions.2.Immunohistochemical results showed that,after 5 weeks of experiment,the number of positive cells of the microglia marker Iba 1 in the hippocampus of the NC group was less,the cell body was small and irregularly round,the number of neurites was large,and the branches were long and thin;compared with the NC group,the number of Iba 1 positive cells of microglia in the MC group was increased significantly,the cell body was enlarged and round significantly,and the number of neurites was decreased significantly,and the branches were short and thick;compared with the MC group,the number of Iba 1 positive cells in the FLU group and the PEA group was decreased significantly,and the cell body was smaller round or oval shape,the number of neurites was increased significantly,and the branches were long and thin.Meanwhile,the number of positive cells for the astrocyte marker GFAP was more in the hippocampus of the NC group,the cell body was smaller and star-shaped,and the number of neurites was larger and slender;compared with the NC group,the number of GFAP positive cells in the MC group was decreased significantly,and the cell body was obvious.Compared with the MC group,the number of GFAP positive cells of the FLU group and the PEA group were increased significantly,the cell body decreased,the number of neurites increased,and the branches were longer.3.Western blot analysis showed that,after 5 weeks of experiment,compared with the NC group,the protein levels of NF-κB p65,p-NF-κB p65 and p-IκBαin the hippocampus of the MC group were increased significantly,the protein levels of Arg-1and Ym-1 were significantly reduced;compared with the MC group,the protein levels of NF-κB p65,p-NF-κB p65 and p-IκBαin the hippocampus in the FLU group and the PEA group were decreased significantly,the protein levels of Arg-1 and Ym-1 were increased significantly.4.Enzyme linked immunosorbent assay showed that,after 5 weeks of the experiment,compared with the NC group,the contents of TNF-α,IL-1β,IL-6,PGE2and COX-2 of the hippocampus in the MC group were all significantly increased,and the contents of IL-4 and IL-10 were decreased significantly;compared with the MC group,the contents of TNF-α,IL-1β,IL-6,PGE2and COX-2 of the hippocampus of rats in the FLU group and PEA group were decreased significantly,and the contents of IL-4 and IL-10 were increased significantly.5.Colorimetric method and nitrate reductase experiment results showed that,after5 weeks of experiment,compared with the NC group,the content of i NOS and NO of the hippocampus in the MC group were increased significantly;compared with the MC group,the contents of i NOS and NO of the hippocampus in the FLU group and the PEA group were decreased significantly.Conclusion:PEA can improve the depression-like behaviors of the rats induced by CUMS combined with LPS.The mechanism may be related to the inhibition of NF-κB pathway activation,the decreased overexpression of downstream inflammatory factors,and the inhibition of microglia over activation,and activation of astrocyte,as well as the restoration of cytokine balance,etc. |
