The Role Of HCN4 Channels In Spinal Dorsal Horn In Diabetic Neuropathic Pain

As a common chronic complication,diabetic neuropathic pain(DNP)seriously affects the quality of life of patients with diabetes.Spinal dorsal horn plays a key role in nociceptive information transmission and regulation.It has been confirmed that spinal hyperpolarization-activated cyclic nucleotide gated(HCN)channels are involved in the occurrence and development of pathological pain.Of them,HCN4 channels is highly expressed in the inhibitory interneurons of spinal dorsal substantia gelatinosa.Recent research shows that decreased potassium-chloride cotransporter 2(KCC2)expression in spinal dorsal horn promoted the generation of neuropathic pain.KCC2 is also highly expressed in neurons within the superficial spinal dorsal horn.It remains to be clarified whether K~+efflux induced by HCN4 channels activation in diabetic neuropathic pain modulate KCC2 function and subsequently affect the role of GABA/GABAA receptors of neurons in spinal dorsal horn.In this study,streptozocin-induced diabetic rats were used to observe:1)the role of HCN4 channels in spinal dorsal horn in diabetic neuropathic pain;2)whether KCC2 is involved in the funcation of HCN4 channels in diabetic neuropathic pain.The research will clarity the role and relative mechanism of HCN4 channels in spinal dorsal horn in DNP,and provide some evidence for developing effective analgesics based on HCN4 channels as new therapeutic targets.Objective:1.To observe the role of HCN4 channels in spinal dorsal horn in diabetic neuropathic pain.2.To observe whether KCC2 is involvemented in the role of HCN4 channels.Methods:1.Healthy male SD rats(4~5 weeks old)were divided into 3 groups(n=12):(1)control group,(2)STZ group(intrathecal administration of saline),(3)ZD7288 + STZ group(intrathecal administration of HCN channels blocker ZD7288).Diabetes model was prepared by single intraperitoneal injection of 60 mg/kg streptozotocin(STZ).HCN channels blocker ZD7288 was intrathecally given to diabetic rats after injection of STZ for 2 w.Fasting blood glucose(FBG)and body weight of rats were measured before and 14,21,28 d after STZ injection.The mechanical withdrawal threshold(MWT)and thermal withdrawal latency(TWL)were detected before and 14,21,28 d after STZ injection by behavior test.HCN4 channels,glutamate decarboxylase 65(GAD65)and KCC2 protein expression in spinal dorsal horn were detected by Western Blot.GABA content in spinal dorsal horn of rats was detected by high performance liquid chromatography with pre-column derivatization of o-phthalaldehyde(OPA).2.Healthy male SD rats(4~5 weeks old)were divided into 5 groups(n=12):(1)control group,(2)STZ group(intrathecal administration of saline),(3)ZD7288 + STZ group(intrathecal administration of HCN channels blocker ZD7288),(4)DIOA + STZ group(intrathecal administration of KCC2 inhibitor DIOA),(5)DIOA + ZD7288 + STZ group(Pre-intrathecal administration of DIOA,10 min later,intrathecal administration of ZD7288).Diabetes model was prepared by single intraperitoneal injection of 60 mg/kg STZ.KCC2 inhibitor DIOA and HCN channels blocker ZD7288 were intrathecally given to diabetic rats after injection of STZ for 2 w.MWT and TWL were detected at 30,60,90,120 min after DIOA injection.HCN4 channels expression in spinal dorsal horn were detected by Western Blot.Results:1.Compared with the control group,the body weight of diabetic rats was significantly decreased(P < 0.01)and the FBG level was significantly increased(P < 0.01).Intrathecal injection of HCN channels blocker ZD7288 had no significant effect on the body weight and FBG of diabetic rats(P > 0.05).MWT and TWL in DNP group was significantly lower than that in control group(P < 0.01).Intrathecal administration of ZD7288 markedly suppressed the decrease of MWT and TWL after injection of STZ(P < 0.01).The expression of HCN4 channels significantly increased(P < 0.01),KCC2 expression decreased(P < 0.01),while the expression of GAD65 had no significant change in spinal dorsal horn of diabetic rats(P > 0.05).Intrathecal administration of HCN channels blocker ZD7288 significantly blocked the increasement of HCN4 channels expression(P < 0.01)and the decrease of KCC2 expression(P < 0.01)in spinal dorsal horn of DNP rats.The content of GABA in spinal dorsal horn of DNP rats significantly increased(P < 0.05).Intrathecal administration of ZD7288 significantly decreased the content of GABA(P < 0.05).2.Intrathecal administration of KCC2 inhibitor DIOA significantly reduced MWT and TWL in DNP rats(P < 0.01).Further more,intrathecal administration of DIOA significantly inhibited the analgesic effect of the HCN channels blocker ZD7288(P < 0.01).At the same time,intrathecal administration of KCC2 inhibitor DIOA significantly increased the expression of HCN4 channels(P < 0.01)in the spinal dorsal horn of DNP rats.Conclusion:1.Increased expression of HCN4 channels in spinal dorsal horn promotes the occurrence and development of diabetic neuropathic pain.2.The role of HCN4 channels in DNP may be related to down regulation of KCC2 expression and altered nature of GABA in spinal dorsal horn.