The Role Of HCN Channels In Regulating The Function Of P2X Receptors In Dorsal Root Ganglion Neurons In Neuropathic Pain

Dorsal root ganglion?DRG?neurons,the primary sensory neurons,receive sensory information and transfer it to the spinal cord.Increased excitability of DRG neurons is the key to the development of neuropathic pain.Previous studies have shown that activation of hyperpolarization-activated cyclic nucleotide-gated cation?HCN?channels in DRG neurons promotes neuropathic pain.In addition,P2X receptors,including P2X_1–6,are also expressed in DRG neurons.Among them,P2X₃ receptors are selectively expressed in small and medium-sized DRG neurons closely related to nociceptive reception.Previous research showed that activation of P2X receptors expressed in DRG neurons promotes the development of neuralgia.HCN channels and P2X receptors are all expressed in DRG neurons.It is not clear whether there is a mutual regulation between the HCN channel and P2X receptor distributed in DRG neurons.The aim of this study is to observe:1)the role of HCN channels in chronic constriction injury of sciatic nerve?CCI?induced neuropathic pain and P2X receptor expression in DRG.2)whether HCN channels activation modulate the function of P2X receptors in cultured DRG neurons.The research work will clarify the role of HCN channels in regulating the function of P2X receptors in DRG neurons in neuropathic pain,and provide some evidence for developing effective analgesics bases on HCN channels as new therapeutic targets.Objective:1.To investigate the role of HCN channels in the generation and development of neuropathic pain and its effect on the expression of P2X receptors in DRG.2.To observe whether HCN channels activation modulate the function of P2X receptors in cultured DRG neurons.Methods:1.In vivo:Forty healthy male SD rats?7⁸ weeks?were divided into 5 groups?n=8?:?1?sham group;?2?CCI group?intrathecal injection of saline?;?3?⁶CCI+ZD7288 group?intrathecal injection of 10,30,50?g ZD7288?.In the sham-operated rats,the sciatic nerve was visualized and left intact.SD rats in other groups were intrathecally cathetered at 5d before CCI operation.Mechanical withdrawal threshold?MWT?were measured.The expression of P2X₂R,P2X₃R was detected by western blot.2.In vitro:DRG neurons of 2³w SD rats were cultured and divided into 4 groups:?1?ATP?100?mol/L ATP?;?2?ZD7288+ATP?10,100,1000?mol/L ZD7288 pre-incubated for 10min,then administration of 100?mol/L ATP?;?3?8-Br-cAMP+ATP?100?mol/L 8-Br-cAMP pre-incubated for 5min,then administration of 100?mol/L ATP?;?4?ZD7288+8-Br-cAMP+ATP?100?mol/L ZD7288 pre-incubated for 5min,followed by 100?mol/L 8-Br-cAMP pre-incubated for 5min,then administration of 100?mol/L ATP?.ATP?100?mol/L,2s?was given by MPS-2 multichannel fast extracellular micro dosing system.ATP induced current in DRG neurons was recorded using whole cell patch clamp technique.Results:1.MWT of CCI rats was significantly lower than that in the sham group after operation?P<0.05?.Intrathecal administration of ZD7288?30?g?,HCN channels antagonist,markedly inhibited the decrease of MWT after nerve injury?P<0.05?.The Western blot assay showed that the expression of P2X₂R and P2X₃R was markedly enhanced in the ipsilateral L_4-6 DRG on days 7d?P<0.05?and 14d?P<0.05?after CCI.Intrathecal administration of ZD7288 markedly inhibited the increased expression of P2X₂R and P2X₃R after CCI?P<0.05?.2.Three types of currents?transient,sustained and biphasic?were evoked by ATP?100?mol/L?in cultured DRG neurons.The HCN channel blocker ZD7288?100?mol/L,10min?can significantly inhibit the amplitude of ATP induced transient,sustained and biphasic current?P<0.05?.The inhibitory effect of ZD7288 on the ATP induced current of DRG neurons was dose-dependent.8-Br-cAMP?100?mol/L,c AMP analogue?enhanced the ATP induced current of DRG neurons.ZD7288?100?mol/L?incubation for 10min can significantly inhibit the effect of 8-Br-cAMP.Conclusion:1.Activation of HCN channels in DRG can promote the occurrence and maintenance of neuropathic pain caused by CCI.HCN channels activation can promote the expression of the P2X₂ and P2X₃ receptors in DRG at neuropathic pain state.2.Activation of HCN channels can promote the role of P2X receptors in DRG neurons,probably through the cAMP-PKA signal pathway.