Immunological Regulation And Mechanism Study Of Cannabinoid Receptors On The Development Of Experimental Autoimmune Encephalomyelitis In Mice

Immunological regulation and mechanism study of cannabinoid receptor on the development of experimental autoimmune encephalomyelitis in micePart I The dynamic change of cannabinoid receptors in experimental autoimmune encephalomyelitisObjective:To observe the dynamic change of cannabinoid 1 receptor (CB1R) and cannabinoid 2 receptor (CB2R) in both central nervous system (CNS) and spleens during the course of experimental autoimmune encephalomyelitis (EAE). Methods: Mice were grouped randomly as normal group, complete Freunds adjuvant (CFA) group and EAE group. Clinical scores and body weight were recorded during the course of EAE. Brains, spinal cords and spleens in different groups were obtained. We detected the expression of CB1R/CB2R by SYBR Green-based real time RT-PCR and western-blot technique. Results:The expressioin of CB1R/CB2R was detectable in both CNS and periphery. CB1R decreased while CB2R increased significantly in CNS during the course of EAE; CB1R had negative correlation with clinical scores. Conclusion:CB1R could play a prominent role in neuroprotection, while CB2R could participate in the immune-regulation in the CNS.Part II The role of cannabinoid receptors on the development of EAEObjective:To observe the role and mechanism of cannabinoid receptors on the development of EAE by using the selective antagonists of CB1R or CB2R. Methods: EAE mice were grouped randomly as vehicle control group, SRI group and SR2 group. Clinical score and body weight were recorded during the course of EAE. Brains, spinal cords and spleens in different groups were obtained. The expression of CB1R/CB2R, the splenic mononuclear cell proliferation, the level of cytokines and chemokines were measured by SYBR Green-based real time RT-PCR, western-blot technique, MTT and ELISA, respectively. Results:SRI exhibited an increased clinical score from day 4 to day 12 p.i. and corresponded to a decrease in body weight from day 5 to day 28 p.i.. SR2 exhibited an increase of clinical score from day 17 to 28 p.i.. Both SRI and SR2 influenced the dynamic balance between CB1R and CB2R. In addition, SR1 could up-regulate the level of Th1/Th17 and inflammatory cytokines, such as IL-1?, IL-6, TNF-?, as well as chemokine MCP-1. SR2 could up-regulate the level of Th1/Th17 and down regulate the level of Th2 cytokine. Conclusion:SRI accelerates onset of disease while SR2 deteriorates EAE clinical severity after day 17 p.i.. Both SRI and SR2 participate in the immune regulation in the CNS and the periphery. SR2 induced antigen specific T cell proliferation during the course of EAE.Part III The dynamic change of cannabinoid receptors on neurons and glial cells in vitro and in vivo1. The expression of cannabinoid receptors on primary neurons and glial cells in vitroObjective:To observe the expression of cannabinoid receptors on primary cultured neurons, astrocytes and microglia. Methods:We first prepared hippocampal neurons from C57BL/6 (B6) mice embryos (E18) and glia cells from B6 mice neonatal cortex (postnatal day 1). The purity of neurons was>98%by using anti-NeuN antibodies, and the purity of astrocytes and microglia was>98%by using anti-GFAP antibodies or anti-CD 11b antibodies, respectively. The expression of CB1R and CB2R was measured with the SYBR Green based real time RT-PCR, western-blot and immunostaining. Results:CB1R expressed on both neurons and astrocytes, CB2R expressed on neurons, astrocytes and microglia. Conclusion:Different nervous cells express different CBR.2. The dynamic change of cannabinoid receptors on neurons and glial cells of EAE mice in vivoObjective:To observe the dynamic expression of cannabinoid receptors on neurons, astrocytes and microglia of EAE, to clarify the mechanism of CBR in the course of development of EAE. Methods:EAE mice were grouped randomly as vehicle control group, SRI group and SR2 group. The dynamic change of CB1R and CB2R in different nervous cells was measured by immunostaining. The expression of CB1R and CB2R were observed in cingulate gyrus for neurons, dentate gyrus for astrocytes and corpus fimbriatum for microglia. Results:CB1R was decreased in neurons, while increased in microglia during the course of EAE. CB2R was upregulated in neurons, astrocytes and microglia during the course of EAE. SRI could upregulate the expression of CB2R on microglial cells, SR2 could elevate the expression of CB1R on neurons. Conclusion:The different cellular location and expression of CB1R or CB2R demonstrated the different roles of CBR on the development of EAE.Part?The immunoregulation of cannabinoid receptors on activated BV2 microgliaObjective:To observe the immunoregulation of CBR on activated BV2 microglia. Methods:After activation of BV2 microglia with IFN-?, BV2 microglia were exposed to nonselective cannabinoid receptor agonist WIN55,212-2 for 1h, then incubated with SR1 or SR2, respectively. The expression of CB1R and CB2R, the levels of cytokines and chemokines, cell proliferation were measured using SYBR Green-based real time RT-PCR, western-blot, ELISA and MTT, respectively. Results: IFN-?activated BV2 microglia to express CB1R and CB2R. SRI promoted the production of IFN-?, IL-6, TNF-?, NO, inhibited the secretion of IL-17; SR2 stimulated the production of Th1/Th17 cytokines and inflammatory cytokines, such as: IL-1?, IL-6, TNF-a as well as NO, decreased the production of Th2 cytokines and chemokine CX3CL1. SR2 could promote cell proliferation. Conclusion:SR1 or SR2 stimulates Th1/Th17 and inflammatory cytokines, SR2 inhibits Th2 cytokines.Summary1?Neurons and microglia, but not astrocytes, express CB1R. Astrocytes, neurons and microglia express CB2R.2?The expression of CB1R in the CNS decreased during the EAE, while CB2R increased.3?Both SR1 and SR2 influence the balance of CB1R/CB2R expression.4?SR1 promoted the onset of EAE, accompanied by increasing of Th1 and inflammatory cytokines; SR2 deteriorated the EAE, accompanied by increasing of Th1/Th17 as well as inflammatory cytokines and decreasing of Th2 cytokines.5?SR1 and/or SR2 regulated the level of Th1/Th17/Th2, inflammatory cytokines and chemokines of BV2 microglia by cell proliferation and NO release.