Paeoniflorin Exerts Neuroprotective Effects And Mechanism Study Of Vascular Dementia Rats Via Activating Cannabinoid Receptor Type 2

Objective:Vascular dementia?VD?is a type of cognitive dysfunction syndrome caused by cerebrovascular disease.After Alzheimer's disease,the incidence of the disease is about 20%of all dementia types.Microglia cells?MG?are important immune cells in the central nervous system?CNS?and are the main effector cells in the neuroinflammatory response.After stress or injury,the M1 or M2 phenotype and its polarization transition of MG play a key role in relieving the inflammatory response of CNS disease and promoting the repair of nerve cells.We have studied more than 100ingredients and extracts used in the traditional Chinese medicine and found that paeoniflorin?PF?was a potent activator of Cannabinoid receptor type 2?CB₂R?,which protected hippocampal neurons of cerebral ischemia and reperfusion in rats by activiating CB₂R.By simulating pathological physiology characteristic of VD,in view of biological characteristics of M1or M2 phenotypes,and taking CB₂R as the target of intervention,the study aimed at exploring the mechanism of PF activation of CB₂R to regulate polarization transition of MG phenotypes and its neuroprotective mechanism.This study will provide new targets and clinical improvement for VD secondary neuroinflammation,in addition to new ideas and drug treatment for clinical prevention or treatment of VD.Method:1.Four-vessel occlusion?4-VO?was carried out in qualified rats according to the method described by Pulsinelli et al.One week after surgery,90 rats of dementia were selected,based on the results from the Morris water maze with the success criterion.In addition to the sham-operated group?n=16?,successful model rats were randomly assigned into six groups:model group?n=16?,paeoniflorin high dose group?40 mg/kg,n=16?,paeoniflorin medium dose group?20 mg/kg,n=16?,paeoniflorin low dose group?10 mg/kg,n=16?,Nimodipine group?20mg/kg,n=10?,HU308 group?CB₂R selective agonist,3 mg/kg,n=16?.With the exception of nimodipine,all drugs were dissolved in Tween 80and dimethyl sulfoxide?DMSO?,and then was diluted by saline?Tween 80:DMSO:saline=1:1:18?,all dose groups of rats were intraperitoneally administered at a volume of 5 ml/kg at a time of 15 min after permanent four-vessel occlusion and then with the same dose once a day for 28 days.The sham-operated group and the model group of rats were administered intraperitoneally with an equal volume of mixed solution?Tween 80:DMSO:saline=1:1:18?.The Morris water maze was used to test the spatial learning and memory ability of the animals during the last 5 days?24–28 days?of administration,and HE staining was used to observe the changes in the pathological damage of the hippocampal CA1 region,and the concentration of IL-1??IL-6 and TNF-?in the hippocampus of rats was detected by enzyme-linked immunosorbent assay?ELISA?,and the protein expression of CB₂R was detected by Western blotting.2.The modeling method was the same as above except that in the sham-operated group?n=24?,120 rats of dementia were randomly divided into 5 groups?n=24/group?:model group,PF group?PF 40 mg/kg?,PF+AM630 group?PF 40 mg/kg combined CB₂R selective antagonist AM630 3 mg/kg?,AM630 group?AM630 3 mg/kg?,HU308 3mg/kg group?HU308 3 mg/kg?.AM630 was administered intraperitoneally 15 min prior to the PF administration each time,and the rest was the same as above.The Morris water maze was used to test the spatial learning and memory ability of the animals during the last 5 days?24–28 days?of administration,and light microscope and electron microscope was used to evaluate the effects on the pathological damage of the hippocampal CA1 region and the entorhinal cortex.The expression rule of CB₂R and key molecules related to the M1/M2 polarization of microglia/macrophages were assessed by RT-qPCR,ELISA and Western blotting,respectively.Immunofluorescence double labeling was performed with M1-type marker CD68 and M2-type marker CD206 to evaluatethe phenotypic polarization of microglia/macrophages.The content of nitrite in the hippocampus tissues of rats were determined by the Griess reaction.In addition,Western blotting was used to detect the rule of protein expression of the mTOR/NF-?B and PI3K/Akt signaling pathway.3.The BV2 cells were cultured in vitro with CoCl₂?a hypoxia-inducing agent?to establish an oxygen glucose deprivation?OGD?model that mimics in vivo hypoxic/ischemic conditions.According to different drug treatments,cells were divided into 6 groups:vehicle control group,OGD group?CoCl₂ 160?M?,PF group?CoCl₂ 160?M+PF 50?M?,PF+AM630 group?Co Cl₂ 160?M+PF 50?M+AM630 2?M?,AM630 group?CoCl₂ 160?M+AM630 2?M?and HU308 group?CoCl₂ 160?M+HU308 10?M?.Immunofluorescence staining was used to observe the co-expression of Iba1 and CB₂R,CD68 and CD206;ELISA was used to detect the phenotypes of M1 marker and M2 marker and NO metabolites was detected by Griess method;Furthermore,Western blotting was used to assesse the protein expression rule of the mTOR/NF-?B and PI3K/Akt signaling pathway.Result:1.Administration of PF could enhance the learning and memory ability of VD rats induced by 4-VO,inhibit the release of proinflammatory cytokines IL-1?,TNF-?and IL-6 in hippocampus,improve the neuronal pathological changes in hippocampal CA1 region,and then protect the nervous system.2.PF could reduce the histopathological damage in the hippocampal CA1 region and entorhinal cortex of VD rats and improve the ultrastructural changes in the hippocampal CA1 region by up-regulating CB2R mRNA and protein expression;Moreover,PF could induce M1-type of microglia/macrophages to M2-type in the hippocampus of rats.In addition to its inhibitory property against proinflammatory M1-phenotype mediator expression,such as CD68,IL-1?,TNF-?,IL-6,iNOS and NO,PF dramatically up-regulated expression of anti-inflammatory cytokines M2-phenotype CD206,IL-10,arginase-1,TGF-?1 and Ym1.Importantly,AM630,CB₂R selective antagonist,abolished these beneficial effects produced by PF on learning memory and hippocampus structure in rats,as well as the polarization of microglia/macrophages to the M2 phenotype.Additionally,Administration of PF significantly inhibited cerebral hypoperfusion-induced mTOR/NF-?B proinflammatory pathway and enhanced PI3K/Akt anti-inflammatory pathway.Effects of PF on these signaling pathways were effectively attenuated when rats were co-treated with PF and AM630,but HU308 had similar neuroprotective effects,indicating that the mTOR/NF-?B and PI3K/Akt signaling pathways were involved in the PF effects through CB₂R activation.3.The study also revealed in the OGD model that PF could significantly increase the protein expression of CB₂R,inhibit the activation of BV2 cells,and also induce the conversion of M1 to M2 phenotype of MG,characterized by the inhibitory expression of M1 phenotype markers CD68,IL-1?,TNF-?,IL-6,iNOS,and NO,while M2 phenotype markers CD206,IL-10 and TGF-?1 expression were further increased;PF significantly inhibited mTOR/NF-?B and PI3K/Akt proinflammatory pathway.Effects of PF on these signaling pathways were effectively attenuated when BV2 cells were co-treated with PF and AM630,and the effect of CB₂R selective agonist HU308 on BV2 cells was similar to that of PF.Conclusion:PF induces the conversion of microglia M1 to M2 phenotype of vascular dementia rats and plays a neuroprotective role by activating CB₂R,and the mechanism may be related to the regulation of m TOR/NF-?B and PI3K/Akt signaling pathways.