The Role Of Cannabinoid Receptors In Regulating The Function Of P2X_2,3 Receptors In Dorsal Root Ganglion Neurons In Neuropathic Pain

Neuropathic pain is a kind of chronic pain that is complicate and difficult to cure.As a neurotransmitter,cannabinoid is involved in a variety of physiological and pathological processes.There are two subtypes of cannabinoid receptors,cannabinoid receptor 1（CB1R）and cannabioid receptor 2（CB2R）.Cannabinoid receptors are widely expressed in the central and peripheral nervous system related to nociceptive information transmission.Previous studies have shown that cannabinoids have significant analgesic effects on neuropathic pain.Increased excitability of dorsal root ganglion（DRG）neurons is the key to the development of neuropathic pain.DRG neurons,the primary sensory neurons,receive sensory information and transfer it to the spinal cord.Previous research showed that 50% DRG neurons express CB1 R,and CB2 R is slightly expressed in DRG neurons and satellite cells.In addition,activation of purinergic P2 X receptors in the peripheral nervous system contributes to the development of neuropathic pain.DRG neurons express multiple P2 X receptors,including P2X1–6.Among them,P2X3 receptors were present predominantly in a subpopulation of small-diameter sensory neurons related with nociceptive reception.Previous studies have shown that the function of P2 X receptor is regulated by protein kinase A.On the other hand,activation of cannabinoid receptors can regulate the activity of cAMP-PKA signal.Whether cannabinoid receptors activation modulates the function of P2 X receptors in DRG neurons remains unclear.The present study is aimed to explore: 1)The role of cannabinoid receptors in the generation and development of neuropathic pain,and in the expression of P2 X receptor in DRG of CCI rat.2)Whether cannabinoid receptors activation modulates the function of P2 X receptors in cultured DRG neurons.Objective:1.To investigate the role of cannabinoid receptors in the generation and development of neuropathic pain and its effect on the expression of P2 X receptors.2.To observe the effects of cannabinoids on [Ca²⁺]i induced by P2 X receptors activation in cultured DRG neurons.Methods:1.In vivo:Fifty healthy male SD rats（7⁸ weeks）were divided into 5 groups:（1）sham group（n=10）;（2）CCI group（intrathecal injection of DMSO saline,n=10）;（3）CP55940 + CCI group（intrathecal injection of 0.05 mg/kg CP55940,n=10）;（4）AM251 + CP55940 + CCI group（intrathecal injection of 0.05 mg/kg AM251,followed by 0.05 mg/kg CP55940 in 15 min later,n=10）;（5）AM630 + CP55940 + CCI group（intrathecal injection of 0.05 mg/kg AM630,followed by 0.05mg/kg CP55940 in 15 min later,n=10）.Paw withdrawal thermal latency（PWTL）and paw withdrawal mechanical threshold（PWMT）were measured.The expression of CB1 R,CB2R,P2X2 R,P2X3R was detected by western blot.2.In vitro: Dorsal root ganglion neurons of 2³w SD rats were cultured and divided into 7 groups:（1）ATP group（100 μmol/L）;（2）EGTA（pre-incubate 20 min）+ ATP（100 μmol/L）group;（3）TNP-ATP（10 μmol/L,pre-incubate 10 min）+ ATP（100 μmol/L）group;（4）CP55940（0.001,0.01,0.1,1 μmol/L pre-incubate 10 min）+ ATP（100 μmol/L）group;（5）AM251（10 μmol/L,pre-incubate 10 min）+ CP55940（1 μmol/L,pre-incubate 10 min）+ ATP（100 μmol/L）group;（6）AM630（10 μmol/L,pre-incubate 10 min）+ CP55940（1 μmol/L,pre-incubate 10 min）+ ATP（100 μmol/L）group;（7）Fsk（10 μmol/L,pre-incubate 10 min）+ CP55940（1 μmol/L,pre-incubate 10 min）+ ATP（100 μmol/L）group.Changes of [Ca²⁺]i in neurons were detected by laser scanning confocal microscopy.Results:1.PWTL and PWMT of CCI rats were significantly lower than that in the sham group after operation（P < 0.05）.Intrathecal administration of CP55940（0.05 mg/kg）,cannabinoid receptors agonist,markedly inhibited the decrease of PWTL and PWMT after nerve injury（P < 0.05）.Intrathecal administration of AM251（CB1R antagonist,0.05 mg/kg）,and AM630（CB2R antagonist,0.05 mg/kg）,markedly blocked the analgetic effect of CP55940.The Western blot assay showed that the expression of CB1 R,CB2R,P2X2 R and P2X3 R was markedly enhanced in the ipsilateral dorsal root ganglions on days 7d（P < 0.05）and 14d（P < 0.05）after CCI.Intrathecal administration of CP55940 markedly inhibited the increased expression of CB1 R,CB2R,P2X2 R and P2X3 R after CCI.The inhibitory effect of CP55940 was blocked by AM251 and AM630,respectively（P < 0.05）.2.ATP（100 μmol /L）caused the increase of [Ca²⁺] i in DRG neurons via activation of P2 X receptors.A 10 min incubation with CP55940（0.001-1 μmol/L）inhibited ATP-induced [Ca²⁺]i increase in DRG neurons in a concentration-dependent manner（P < 0.05）.The inhibitory effect of CP55940 on ATP-induced [Ca²⁺]i increase was blocked by CB1 receptor antagonist AM251（10 μmol/L）and CB2 receptor antagonist AM630（10 μmol/L）,respectively（P < 0.05）.Forskolin（adenosine cyclase agonist,10 μmol/L）markedly suppressed the inhibitory effect of CP55940（P < 0.05）.Conclusion:1.Activation of cannabinoid receptors have a significant analgesic effect on neuropathic pain induced by CCI.Cannabinoid receptors activation can inhibits the expression of purine P2 X receptors in DRG in neuropathic pain.2.Cannabinoid receptors agonist CP55940 can inhibit ATP-induced [Ca²⁺]i increase in DRG neurons.The inhibitory effect of CP55940 is mediated by CB1 and CB2 receptors,which may be through inhibiting the activity of PKA.