The Effects Of QO-58on Na~+Currents In DRG Neurons And Pain Behavior Of Adult Rats

Abnormal spontaneous activity within primary sensory neurons such asdorsal root ganglion (DRG) neurons, trigeminal neuronsis is involved in thegenesis of pain. Up to now, at least five functional voltage-gated sodiumchannel (VGSC) α-subunit isoforms have been identified in DRG neurons ofadult rat, including the TTX-sensitive (TTX-S) NaV1.1, NaV1.6and NaV1.7,and the TTX-resistant (TTX-R) NaV1.8and NaV1.9. NaV1.8is preferentiallyexpressed in small sensory neurons and produces a slowly activating andinactivating Na+current, which is essential for the process of nociception. Theprimary afferent neurons containing NaV1.8contribute to the hyperalgesia andallodynia following chronic pain. Mutations of NaV1.7may lead to PrimaryErythermalgia and Congenital Indifference to Pain (CIP). NaV1.6mayparticipate in the diabetic neuropathic pain. Multiple clinical and experimentalstudies show that sodium channel blockers have potentials in treatment ofpain-related disorder, which is the focus of the studies in the field ofneuroscience and new drug development.QO-58is a representative of novel serial compounds of pyrazolo[1,5-a]pyrimidin-7(4H)-ones (PPOs), and is a opener of Kv7K+channels. Previousstudy found that QO-58shows considerable anticonvulsant activity both inPTZ and MEM epileptic models of mice, and shows the analgesic effects inthe Chronic Constriction Injury (CCI) of the sciatic nerve models. Theseresults indicate that QO-58has potential to be developed to treat the diseasesrelated to the neuronal hyperexcitability. Studies have shown that epilepsy andpain are closely related to abnormal activity of sodium channel in sensorysystem. Since both activation of K+channels and inhibition of Na+channelswould dampen the excitability of neurons, the demonstrated antiepileptic andanalgesic effects of QO-58mentioned above could well arise form the inhibition of Na+channles besides the activation of K+channels. Thus thisstudy is intented first to study the effects of QO-58on Na+currents in rat DRGneurons. This study will also investigate the effects of QO-58on pain behaviorresponding to thermal and mechanical stimulation of rat inflammatory painmodel produced by CCL2(Monocyte chemoattractant protein-1) and CFA(Freund’s complete adjuvant), which has been suggested to produceneuropathic pain by modulation of Na+channel functions.Part1Effects of QO-58on the Na+currents in DRG neuronsObjective: To identify the effects of QO-58on the amplitude andbiophysical kinetics of total, TTX-R, NaV1.8and TTX-S sodium currents inDRG neurons.Methods: Whole cell Patch clamp technique was used to study theeffects of QO-58on the sodium channel.Results:(1) QO-58produced concentration-dependent reduction of totalsoidum currents recorded at0mV. Experimental data were fitted to the Hillequation.The fit gave a IC50of9.8±1.1μM, slope factor of1.4±0.12(n=7).(2)QO-58caused a concentration-dependent inhibition of total, TTX-R, NaV1.8and TTX-S soidum channel current from DRG neurons recorded at0mV. Twopopulations of TTX-S currents were identified: those with high sensitivity toQO-58inhibition and those with lower sensitivity to QO-58inhibition; QO-58inhibited two population of Na+currents by95.1±3.7%and28.7±5.2%(P<0.01), respectively.(3) QO-58(30μΜ) shifted the voltage-dependentactivation of total Na+and TTX-S currents to the more depolarized direction.However, the voltage-dependent activation of TTX-R and NaV1.8was notaffectd.(4) QO-58(30μΜ) shifted the voltage-dependent inactivation of total,TTX-R, NaV1.8and TTX-S soidum channel current to the hyperpolarizeddirection. Among the fast-and the slow-inactivated channel state of TTX-RNa+channel, QO-58shifted the slow-inactivation channel state to morehyperpolarized direction.Conclusions:(1) QO-58caused a concentration-dependent inhibition oftotal Na+current, NaV1.8and TTX-S sodium current in DRG neurons, Within the concentration of10μΜ to30μΜ, QO-58was more effective in reducingTTX-S than TTX-R Na+currents.(2) QO-58induced a right shift of thevoltage-dependent activation curve of total and TTX-S channels, and alsoproduced a leftward shift of the voltage-dependent inactivation curve of total,TTX-R, NaV1.8and TTX-S Na+currents; Among the fast-and theslow-inactivated channel state of TTX-R Na+channel, QO-58shifted theslow-inactivation channel state to more hyperpolarized direction.Part2The effects of QO-58on the pain behavior of rat pain modelinduced by CCL2and CFAObjective: To study the effects of QO-58on pain behavior of ratsinduced by CCL2/CFA and the underlying mechanism.Methods: CCL2/CFA-induced inflammatory pain model of rats wasused. Mechanical allodynia and thermal hyperalgesia was assessed using thevon Frey filaments and radiate heat tests, respectively.Results:(1) In the control rats,withdrawal latency to thermal stimulus ofCCL2/CFA-induced rats was11.7±1.6s,12.1±1.1s,11.4±1.2s and12.9±1.0s,respectively at the time points of3,4,5,6hour after i.p. injection of solvent(saline). After treatment with QO-58(30mg/kg, i.p.) the withdrawal latencywas significantly increased to16.9±1.0s,16.3±1.7s,21.0±1.9s,17.8±1.6s（P<0.001; P<0.05; P<0.001; P<0.01) at the same time points after theinjection. XE991did block the effects of QO-58.(2) QO-58(30mg/kg, i.p.)did not affect the threshold for the painful responses to the mechanicalstimulation of CCL2/CFA induced rats.Conclusions: QO-58significantly increased the threshold for the painbehavior induced by thermal stimulation of CCL2/CFA rats pain model, andthis analgesic effect of QO-58is possibly not related to the opening of Kv7K+channels but rather realted to the inhibition of Na+channels. QO-58didnot affect the threshold for the painful responses to the mechanical stimulationof CCL2/CFA rats pain model.