The Neuroprotective Role And Mechanism Of Microglia Cannabinoid Receptor 2 In Postoperative Cognitive Dysfunction

Background: Postoperative cognitive dysfunction(POCD)is a severe postoperative complication characterized by deterioration of memory and execution ability.It can persist for several months,which seriously affects the quality of life of patients and places a heavy burden on the family and society.Therefore,it is urgent to identify effective interventions to reduce POCD,which requires a better understanding of the mechanism.Several pathophysiological mechanisms are considered to be involved in the pathogenesis of POCD,among which the hippocampal neuroinflammation has been shown to be a critical mechanism in POCD.Postoperative cognitive decline can be improved by anti-inflammatory treatment.Microglia,as a key participant in the inflammatory response,once activated can develop classically activated M1 phenotype and alternative activated M2 phenotype resulting in distinct effects.M1-type cells play an important role in inflammation by secreting pro-inflammatory cytokines and mediators,and aggravate neurological deficits.M2-type cells suppress inflammation by secreting mainly anti-inflammatory cytokines and neurotrophic factors,and promote tissue recovery.M1/M2 type microglia collectively maintain central nerve system(CNS)inflammatory homeostasis.The inhibition of M1 microglia polarization along with promoting M2 microglia polarization may be a viable treatment strategy for the treatment of inflammation-related diseases.The cannabinoid receptor 2(CB2R)of the endocannabinoid(e CB)system was initially thought to be the "peripheral cannabinoid receptor".However,recent studies have demonstrated the presence and the central effects of CB2 R in the brain.Accumulating evidence suggests that the up-regulation of microglia CB2 R expression in CNS diseases closely associates with their activation.The activation of CB2 R mediates central anti-inflammatory and immunomodulatory effects through modulation of microglia function.It remains unclear about the effects of microglia CB2 R on postoperative microglia polarization and cognitive function.Extracellular signal-regulated kinase1/2(ERK1/2)and nuclear factor-κB(NF-κB)are key signaling pathways in the inflammatory response and regulated by the CB2 R.However,the role of ERK1/2/NF-κB signaling pathway in the regulation of inflammation and microglia polarization by microglia CB2 R has not been reported.In this study,we firstly investigated the effects of microglia CB2 R activation on microglia phenotype and inflammatory response after LPS stimulation.Second,we utilized a CB2 R agonist encapsulated-poly(lactic-co-glycolic acid)drug delivery system with the aim of preferential activation of microglia CB2 R,and then evaluated its effects on microglia polarization,inflammation and postoperative cognitive function in vitro and in vivo.Finally,the role of ERK1/2/NF-κB signaling in microglial CB2R-mediated regulation of microglial function and inflammation was investigated.Methods: 1)In vitro inflammation model was established by LPS.After pharmacological agitation of CB2 R,the expression of CB2 R was detected by western blot,the expressions of M1-type microglia markers(CD68,i NOS)and M2-type microglia markers(CD206,Arg-1)were detected by RT-PCR and immunofluorescence staining,and the levels of IL-1β and TNF-α in culture medium were detected by ELISA;2)Inflammatory responses in vitro were induced using LPS after overexpression of CB2 R by transfection with p EGFP-C1-Cnr2 plasmid.The expressions of M1-type and M2-type microglia markers were detected by RT-PCR and immunofluorescence staining,and the IL-1β and TNF-α levels in culture medium were detected by ELISA;3)The JWH133 encapsulated-PLGA nanoparticles(JWHNPs)were synthesized by nanoprecipitation and characterized by DLS.The cytotoxicity and uptake levels of JWHNPs were assessed by CCK-8 and fluorescence staining.The inflammatory responses were induced by LPS stimulation in vitro.The expressions of M1-type and M2-type microglia markers were detected by RT-PCR and immunofluorescence staining,and the IL-1β and TNF-α levels in the culture medium after JWHNPs treatment were assessed by ELISA;4)The POCD model was established in 8–10-week-old C57BL/6mice with left-sided open tibial intramedullary fixation surgery under 3 % sevoflurane anesthesia.JWHNPs were stereotactically injected in the hippocampal CA1 region,and hippocampal tissues were obtained 3 days after surgery.The expressions of M1 and M2 microglia markers were assessed by RT-PCR and immunofluorescence staining,and the levels of IL-1β,TNF-α,and IL-6 in the hippocampus were measured by ELSIA.The motor ability,spatial memory,contextual,and tone-related fear memory of mice were assessed by open field test,Y-maze,and fear-conditioning test on postoperative day 3;5)The expressions of ERK1/2,p-ERK1/2,p-65 and p-p65 in the ERK1/2/NF-κB signaling pathway after LPS stimulation were detected by western blot.After pharmacological inhibition of ERK1/2 and NF-κB pathways,respectively,followed by LPS stimulation,the expressions of M1-type and M2-type microglia markers were assessed by RT-PCR and immunofluorescence staining,and the levels of pro-inflammatory cytokines after LPS treatment were assessed by ELISA.In addition,the effects of the inhibition of ERK1/2 signaling pathway on the expressions of p-65 and p-p65 were assessed by western blot.Results: LPS stimulation in vitro induced a down-regulation of CB2 R expression in BV2 cells,a significant increase in the expressions of M1-type microglia markers i NOS and CD68,and secretion of IL-1β and TNF-α.Pharmacological activation of CB2 R decreased the expressions of M1-type microglia markers after inflammatory stimulation,as well as promoting the expressions of M2-type microglia markers,and reducing the secretion of IL-1β and TNF-α.Overexpression of CB2 R by transfection with p EGFP-C1-Cnr2 plasmid significantly reduced LPS stimulation-induced expressions of M1-type microglia markers,increased the levels of M2-type microglia markers,and reduced the levels of inflammation.CB2 R agonist loaded-PLGA nanoparticles(JWHNPs)were synthesized by nanoprecipitation method.DLS result showed that JWHNPs were spherical particles with a diameter of 261.5 ± 16.02 nm and a zeta potential of-24.20 ± 2.20 m V.JWHNPs at concentrations of 0-50 μg/ml showed favorable biosafety and phagocytosis level in BV2 cells.JWHNPs showed a similar decrease in LPS-induced levels of M1-type microglia markers and inflammation,as well as increased the expressions of M2-type microglia markers.Stereotactic injection of JWHNPs in the hippocampal CA1 region to activate microglia CB2 R in the model of open tibial intramedullary fixation significantly inhibited M1-type microglia polarization induced by surgical trauma and promoted M2-type polarization,thereby improving neuroinflammation.In addition,the hippocampal-dependent spatial memory and contextual related-fear memory of mice were significantly improved by JWHNPs after surgery,however there was no effect on postoperative motor ability and non-hippocampal-dependent tone-related fear memory.Further exploration of the mechanism revealed that microglial CB2 R activation inhibited LPS-induced the up-regulation of expressions of ERK1/2,p-ERK1/2,p-65 and p-p65.Inhibition of ERK1/2 and NF-κB signaling by PD98059 and Bay11-7085,respectively,inhibited microglia M1-type microglia polarization induced by LPS stimulation,promoted polarization to M2-type,and reduced pro-inflammatory cytokine secretion.In addition,inhibition of ERK1/2 suppressed LPS-induced activation of NF-κB signaling pathway.Conclusion: These results suggest that activation of microglia CB2 R modulates microglia polarization shift through ERK1/2/NF-κB signaling pathway to ameliorate in vitro LPS and surgery-induced inflammation and thus provides cognitive protection.Collectively,our findings reveal an important role of microglia in the central pathogenesis of POCD and suggest that microglia CB2 R may be a potential therapeutic target for POCD.