Sinomenine Alleviates Neuropathic Pain Via Inhibiting Hyperpolarization Activated Nucleotide-gated Channels In Spinal Dorsal Horn In Mice

Neuropathic pain（NeuP）,which is defined as pain owing to a disease or lesion of the somatosensory nervous system,has unique clinical manifestations such as spontaneous pain,hyperalgesia and allodynia.It is wildly recognized as a common and complex pain condition with serious impacts on patients’physiological and psychosocial function.Current first-line pharmacological strategy for Neu P includes antiepileptic drugs and antidepressants.Given that the outcomes for many patients are often unsatisfactory and are sometimes associated with side effects,improving treatment for Neu P remains a huge challenge in medical care and an important goal for the research community.Recently,converging preclinical works described that sinomenine has satisfactory analgesic effect in rodent models of Neu P.However,up to now,little is known about the central mechanisms responsible for the anti-nociceptive action leaded by this herbal medicine.Specifically,lamina II of the spinal cord,namely the substantia gelatinosa（SG）acts as the first relay center for controlling the modulation and transmission of nociceptive information from the periphery.Alteration of neuronal excitability in SG neurons is an indispensable mechanism underlying the initiation and maintenance of chronic pain.Hyperpolarization-activated cyclic nucleotide-gated（HCN）channels are unique candidates involved in modifying cellular excitability by carrying an inward mixed Na⁺-K⁺current（Ih）.Mounting data have linked abnormal HCN expression or kinetic property alteration to the pathogenesis of chronic pain.Here in this study,by using electrophysiological,molecular biological,immunofluorescence staining together with behavioral methods,we investigated the role of spinal HCN channels in the anti-Neu P effect of sinomenine in both in vivo and in vitro aspects,and thus provide novel theoretical foundation for its clinical application.In this study,through immunofluorescence method,we identified somatic expression of all four homologous isoforms of the HCN channel family（HCN1,HCN2,HCN3,HCN4）in SG neurons.Meanwhile,by using GAD67-GFP mouse in which GFP are expressed only in inhibitory neurons,we found HCN-immunoreactive SG neurons were mainly colocalized with GFP-negative neurons,while only a few neurons displayed colocalization of HCN-immunoreactivity with GFP-immunoreactivity,suggesting that the vast majority of HCN-immunoreactive neurons were excitatory neurons in the superficial spinal dorsal horn.To investigate the role of Ih,which is mediated by HCN channels,in regulating the excitability of SG neurons,whole-cell patch-clamp recordings were performed on acute spinal cord slices,and neurons were divided into two groups:with Ih and without Ih.We found that Ih-expressing neurons had smaller input resistance（Rin）,more hyperpolarized action potential（AP）thresholds,and shorter AP durations than neurons without Ih.Abolishment of Ih with ZD7288 significantly reduced Rin,decreased AP amplitudes and prolonged AP durations in SG neurons with Ih.These findings demonstrate that Ih is an essential ionic conductance contributing to modify SG neuronal electrophysiological properties.Next,we established the model of NeuP in mice with partial sciatic nerve ligation（PSNL）.We explored whether PSNL could affect the expression of HCN channels in mouse superficial spinal dorsal horn on protein level by using immunofluorescence and Western blot on day 14 postoperatively.While the expression levels of HCN channel subunits,including HCN1,HCN3,and HCN4,were not markedly altered;the expression of HCN2 subunit within the ipsilateral superficial spinal dorsal horn was significantly enhanced following PSNL modeling.To corroborate examination of HCN protein expression levels,we also tested HCN channel function by using whole-cell patch-clamp recordings.We noticed greatly raised Ih current amplitude,potentiated Ih current density,and increased firing rate in SG neurons resulted by PSNL,as compared to sham-operated mice.Profound reductions of both mechanical allodynia and thermal hypersensitivity on ipsilateral paw starting from day 1 to day 14 were observed in PSNL mice treated with consecutive sinomenine injection（40 mg/kg/d,i.p.）.Both immunofluorescent and Western blot results suggested that repeated sinomenine administration downregulated PSNL-caused overexpressed HCN2.Notably,enhanced HCN channel function and increased SG neuronal excitability induced by PSNL was reversed under repeated treatment of sinomenine.Having established that sinomenine has an antinociceptive action via modulating anomalous expression and function of HCN channels in vivo,we next detailed the efficacy and potency of sinomenine on inhibiting HCN channels in mouse SG neurons in vitro.Sinomenine suppressed Ih in a dose-dependent manner with IC₅₀ obtained as 317μM,and also inhibited the firing of SG neurons.In addition,we confirmed that sinomenine could bind against the HCN2 channel model via hydrogen bond directly by using computational docking experiment.In summary,HCN2 is the major HCN subtype involved in PSNL-induced pain hypersensitivity,and sinomenine may alleviated Neu P by modulating HCN2 expression and HCN channel function.