The Role Of Cannabinoid Receptor-1 In Regulating The Function Of HCN Channels In Dorsal Root Ganglion Neurons In Neuropathic Pain

Neuropathic pain is defined as a chronic or persistent pain state that results from an injury or dysfunction of the nervous system.As a neurotransmitter,cannabinoid is involved in a variety of physiological and pathological processes.Cannabinoid receptors mainly include cannabinoid receptor-1(CB1R)and cannabinoid receptor-2(CB2R).CB1 R and CB2 R are widely distributed in the central and peripheral nervous systems of mammals.Recent studies have found that CB1 R and/or CB2 R activation have significant analgesic effects on various inflammatory and neuropathic pain.In addition,recent studies have shown that HCN channels play an important role in the neuropathic pain.Among them,HCN2 channel are mainly distributed in small and medium-sized DRG neurons related to nociceptive reception.As primary sensory neurons,increased excitability of dorsal root ganglion(DRG)neurons is the key to the production of pathological pain.Both CB1 R and HCN channels are expressed in DRG neurons.Whether CB1 R activation modulates the function of HCN channels in DRG neurons remains unclear.The aim of this study is to observe: 1)the role of CB1 R in chronic constriction injury of sciatic nerve(CCI)induced neuropathic pain and the expression of HCN channels in DRG;2)whether CB1 R activation modulate the function of HCN channels in cultured DRG neurons.Objective:1.To explore the role of CB1 R in CCI induced neuropathic pain and the expression of HCN channels in DRG.2.To explore the role of CB1 R on the function of HCN channels in cultured DRG neurons.Methods:1.In vivo: healthy male SD rats(7~8 weeks)were divided into 4 groups:(1)sham group;(2)CCI group(intrathecal injection of DMSO in saline);(3)CP55940 + CCI group(intrathecal injection of 0.05 mg/kg CP55940);(4)AM251 + CP55940 + CCI group(intrathecal injection of 0.05 mg/kg AM251,followed by 0.05 mg/kg CP55940 in 15 min later);PWMT were measured.The expression of HCN1,HCN2 channel was detected by Western Blot.2.In vitro: DRG neurons of 2~3 w SD rats were cultured and divided into 4 groups:(1)hyperpolarized stimulation group(control group);(2)CP55940 + hyperpolarized stimulation(1 μmol/L CP55940 pre-incubated for 5 min,then given hyperpolarized stimulation);(3)AM251 + CP55940 + hyperpolarized stimulation(1 μmol/L AM251pre-incubated for 5 min,followed by 1 μmol/L CP55940 for 5 min,then given hyperpolarized stimulation);(4)8-Br-c AMP + CP55940 + hyperpolarized stimulation(100 μmol/L 8-Br-c AMP pre-incubated for 5 min,followed by 1 μmol/L CP55940 for 5min,then given hyperpolarized stimulation).Ih currents in DRG neurons were recorded using whole cell patch clamp technique.Results:1.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 PWMT after nerve injury(P < 0.05).Intrathecal administration of AM251(CB1R antagonist,0.05 mg/kg),markedly blocked the analgetic effect of CP55940.The Western Blot assay showed that the expression of CB1 R,HCN1 and HCN2 channel proteins was markedly enhanced in the ipsilateral dorsal root ganglions on days 7 d(P < 0.05)and 14 d(P < 0.05)after CCI.Intrathecal administration of CP55940 markedly inhibited the increased expression of HCN1 and HCN2 channel after CCI.The inhibitory effect of CP55940 was blocked by AM251(P <0.05).2.The hyperpolarization-activated currents(Ih)were recorded in about 63% DRG neurons,which can be compeletly blocked by HCN channel antagonist ZD7288(10 μmol/L).CP55940(1 μmol/L),cannabinoid receptors agonist,markedly decreased Ih in DRG neurons(P < 0.05).The inhibitory effect of CP55940 on Ih was blocked by CB1 receptor antagonist AM251(1 μmol/L)(P < 0.05).8-Br-c AMP(c AMP analogue,100 μmol/L) markedly suppressed the inhibitory effect of CP55940(P < 0.05).Conclusion:1.Activation of CB1 R has a significant analgesic effect on CCI induced neuropathic pain,and inhibits the expression of HCN channels in DRG in CCI rats.2.CB1 R activation can inhibit the function of HCN channels in DRG neurons,which may be through inhibiting the activity of c AMP-PKA signal pathway.