| Neuropathic pain,a type of chronic pain,develops because of peripheral nerve injury and various kinds of diseases,such as infection,diabetes and cancer.During pathological pain,the activation of the endocannabinoid system leads to effective anti-allodynia and modifies a variety of spinal microglial responses.ATP and its receptor play an important role in the transmission and modulation of nociceptive information.P2Y122 and P2Y13receptor expressed in dorsal horn microglia and these two receptors are involved in the pathogenesis of neuropathic pain following peripheral nerve injury.The previous research provided that intrathecal administration CB2 receptor agonist AM1241 can suppress the increased expression of P2Y122 and P2Y13 receptor in chronic constriction injury?CCI?rats.However,it is not known whether CB2 receptor activation can influence ADP?S-evoked P2Y12 and P2Y13 receptors expression and the release of inflammatory cytokines from cultured dorsal spinal cord microglia cells.For this reason,we examined the effects of AM1241 on the expression of P2Y12/P2Y13 receptors and the release of inflammatory cytokines from ADP?S-activated dorsal spinal cord microglia cells.Objective:To explore whether AM1241 can influence ADP?S-evoked P2Y12 and P2Y13 receptors mRNA expression and the release of inflammatory cytokines from cultured dorsal spinal cord microglia cells.Methods:1.Primary cultures of microglia were prepared from dorsal spinal cord of new born SD rats?<3d?.The cells were seeded on 12-well plates at a cell density of 5×105/well for 48h.These cells were randomized into four groups?n=4?:normal group?High glucose medium?,ADP?S?10-55 mol/L,3h?group,AM1241(10-7,10-6,10-55 mol/L,1h)+ADP?S?10-55 mol/L,3h?group,AM630(10-7,10-6,10-55 mol/L,1h)+AM1241?10-55 mol/L,1h?+ADP?S(10-5mol/L,3h)group.The expression of Iba-1,P2Y12/P2Y13 receptor mRNA from microglia cells by using RTFQ-PCR.2.Primary cultures of microglia were prepared from dorsal spinal cord of new born SD rats?<3d?.The cells were seeded on 12-well plates at a cell density of 5×105/well for 48h.These cells were randomized into four groups?n=4?:normal group?High glucose medium?,ADP?S?10-55 mol/L,3h?group,AM1241?10-55 mol/L,1h?+ADP?S?10-55 mol/L,3h?group,AM630?10-55 mol/L,1h?+AM1241?10-55 mol/L,1h?+ADP?S?10-55 mol/L,3h?group.The expression of IL-1?,IL-6 and TNF-?in microglia cells were evaluated by using RTFQ-PCR.The release of IL-1?,IL-6 and TNF-?from microglia cells were evaluated by using ELISA.3.Primary cultures of microglia were prepared from dorsal spinal cord of new born SD rats?<3d?.These cells were randomized into three groups?n=4?:normal group,ADP?S?10-55 mol/L?group,MRS2211?10-55 mol/L,20min?+ADP?S?10-55 mol/L?group,AM1241?10-55 mol/L,10min?+ADP?S?10-55 mol/L?group,The changes of[Ca2+]i in microglia cells were measured by laser scanning confocal microscopy using fluo-4 AM as a calcium fluorescent indicator.Results:1.In cultured spinal dorsal horn microglia cells,compared with the control,stimulation of microglia cells with ADP?S?10-55 mol/L,3h?led to a robust increase of Iba-1 mRNA?P<0.05?.ADP?S-induced the increased expression of Iba-1 mRNA was not influenced after pretreatment with AM1241(10-7,10-6,10-55 mol/L)for 1h.2.In cultured spinal dorsal horn microglia cells,compared with the control,stimulation of microglia cells with ADP?S(10-5 mol/L,3h)led to a robust increase expression of P2Y12 and P2Y13 receptors mRNA?P<0.05?.ADP?S-induced the increased expression of P2Y12 and P2Y13 receptors mRNA was significantly inhibited after pretreatment withAM1241 at 10-55 mol/L for 1h?P<0.05?.Furthermore,the inhibitory effects of AM1241on ADP?S-evoked P2Y12 and P2Y13 receptors mRNA expression were significantlyreversed after administration of CB2 receptor antagonist AM630 at 10-55 mol/L for 1h?P<0.05?.3.In cultured spinal dorsal horn microglia cells,compared with the control,stimulation ofmicroglia cells with ADP?S(10-5 mol/L,3h)induced the obvious increased expressionof IL-1?,IL-6 and TNF-??P<0.05?.ADP?S-induced the increased expression of IL-1?,IL-6 and TNF-?were obvious inhibited after pretreatment with AM1241 at 10-55 mol/Lfor 1 h?P<0.05?.Furthermore,the inhibitory effects of AM1241 on ADP?S-evokedIL-1?,IL-6 and TNF-?expression were significantly reversed after administration ofCB2 receptor antagonist AM630 at 10-55 mol/L for 1 h?P<0.05?.4.In cultured spinal dorsal horn microglia cells,compared with the control,stimulation ofmicroglia cells with ADP?S(10-5 mol/L,3h)evoked the release of IL-1?,IL-6 andTNF-?release?P<0.05?.ADP?S-evoked IL-1?,IL-6 and TNF-?release were obviousinhibited after pretreatment with AM1241 at 10-55 mol/L for 1h?P<0.05?.Furthermore,the inhibitory effects of AM1241 on ADP?S-evoked IL-1?,IL-6 and TNF-?releasewere significantly reversed after administration of AM630 at 10-55 mol/L for 1h?P<0.05?.5.ADP?S(10-5 mol/L)causes[Ca2+]i increase in cultured dorsal spinal cord microglia cells.The rapid elevation of[Ca2+]i in microglia by ADP?S was blocked by P2Y13receptor antagonist MRS2211(10-5 mol/L,20min).However,ADP?S(10-5mol/L)-induced Ca2+mobilization was not impaired after AM1241?10-55 mol/L,10min?pretreatment.Conclusion:1.AM1241 significantly suppressed ADP?S-evoked the increased expression of P2Y12 and P2Y13 receptors mRNA in cultured rat dorsal spinal cord microglia cells.2.AM1241 significantly suppressed ADP?S-evoked the increased expression and release of IL-1?,IL-6 and TNF-?from cultured rat dorsal spinal cord microglia cells. |
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