| Neuropathic pain is defined as pain caused by a lesion or disease of the somatosensory system,which is characterized by persistent spontaneous pain,allodynia and hyperalgesia.Increased excitability of the nervous system hyperactivity pathway is often considered to be the basis of neuropathic pain,including peripheral sensitization and central sensitization.Ectopic discharges in primary sensory neurons is the underlying cause of peripheral sensitization.M channel plays a key role in regulating neuronal excitability,which is a non-activating subthreshold potassium current.It can suppress repeated diacharges of neurons,stable membrane potential and control neuronal excitability.Opening M channel(encoded by KCNQ genes,or Kv7 channel)will induce cell membrane hyperpolarization and thus suppress neuronal excitability.Therefore,M channel openers can treat diseases sociated with neuronal excitability,such as epilepsy,anxiety,schizophrenia,migraine,neuropathic pain,inflammatory pain and so on.QO-58 is a new compound with proprietary intellectual property rights discovered by high-throughput screening systems and its therapeutic target is Kv7/M channel.QO-58 has varying degrees of open effect on Kv7.1-Kv7.5 subunits,and has the strongest effect on Kv7.2/7.3 subunit.Previous pharmacodynamic observations confirmed that QO-58 lysine has a certain antiepileptic effect and analgesic effect.We intend to detect the mechanism and analgesic effects of QO-58 lysine in present studies,including neuropathic pain induced by peripheral sciatic nerve injury,trigeminal neuralgia and diabetic neuropathic pain,and to provide evidence for further drug researches and development.Objective: To analyse the concentration of QO-58 lysine in DRG and TG tissues of rats by HPLC-MS.To investigate the effect of QO-58 lysine on M currents recorded from DRG neurons and TG neurons by patch clamp.To evaluate the antinociceptive effects of QO-58 lysine on neuropathic pain models.Methods:(1)After 7 days of intragastric administration of 100 mg/kg QO-58 lysine,DRG and TG tissues was isolated,then HPLC-MS was performed to analyse the concentration of QO-58 lysine.(2)Perforated wholl cell patch clamp technique was used to record the amplitude of M currents from medium and small diameter cultured SD rats DRG and TG neurons.Then,we applied different concentrations of QO-58 lysine to observe its effect on M currents.(3)CCI model was established by ligating the peripheral sciatic nerve to induce neuropathic pain.Then,we evaluated the therapeutic effect of different doses of QO-58 lysine.MWT and TWL values were measured after repeated administration of 25 mg/kg、50 mg/kg、100 mg/kg QO-58 lysine.(4)ION-CCI model was established by ligating the infraorbital nerve to simulate trigeminal neuralgia.Then,we evaluated the therapeutic effect of different doses of QO-58 lysine.MWT values were measured after repeated administration of 25 mg/kg、50 mg/kg QO-58 lysine.(5)60 mg/kg intraperitoneal injection of 2% STZ to establish STZ-induced diabetic neuropathic pain model.Then,we evaluated the therapeutic effect of different doses of QO-58 lysine.50%MWT values were measured after repeated administration of 12.5 mg/kg、25 mg/kg、50 mg/kg QO-58 lysine.(6)Detecting the expression of KCNQ3 protein levels in DRG and TG by immunohistochemical staining.Results:(1)The content of QO-58 lysine in DRG tissues was 13.16 ± 7.76 μg/mg;the content of QO-58 lysine in TG tissues was 8.89 ± 1.48 μg/mg.(2)QO-58 lysine can activate M channel.In DRG neurons,1,3,10,100 μM QO-58 lysine increased the folds of M current to 0.43 ± 0.03,0.65 ± 0.11,1.59 ± 0.63 and 2.17 ± 0.14(n = 3-6),respectively,the EC50 value was 7.1μM.Compared with the M current before application of QO-58 lysine,P < 0.001.In TG neurons,0.01,0.1,1,3,10,30,100 μM QO-58 lysine increased the folds of M current to 0.09 ± 0.005,0.24 ± 0.09,0.36 ± 0.16,0.73 ± 0.16,1.46 ± 0.24,1.92 ± 0.20,2.30 ± 0.05(n = 3-8),respectively,the EC50 value was 9.3 ± 3.3 μM.Compared with the M current before application of QO-58 lysine,P < 0.001.(3)The CCI model was established successfully and the success rate was 85%,drugs were administrated on the 8th day after operation.The MWT and TWL values were the lowest in the model group on the 13 th day(14.9 ± 2.3 g,8.8 ± 0.6 s,respectively).On the 13th、15th and 17 th day,the MWT values(23.9 ± 4.6 g、25.3 ± 4.2 g、26.2 ± 3.9 g,respectively)and TWL values(11.6 ± 1.6 s、11.8 ± 0.8 s、11.5 ± 1.1 s,respectively)of PGB group were significantly increased compared with model group(P < 0.001 or P < 0.05).The MWT values of 25 mg/kg QO-58 lysine group were 22 ± 7 g and 24.4 ± 4.5 g respectively on the 13 th and 15 th day after operation.The TWL values were 10.9 ± 1.6 s、10.7 ± 1.2 s and 10.5 ± 1 s on the 13 th,15th and 17 th day after operation,which were significantly increased compared with model group(P < 0.01 or P < 0.05).The MWT value of 50 mg/kg QO-58 lysine was 19.8 ± 4.9 g on the 13 th day after operation.The TWL values on the 13 th and 17 th day were 9.7 ± 1.1 s and 10.1 ± 1.3 s respectively,which were significantly higher than those in model group(P < 0.05).The MWT value of 100 mg/kg QO-58 lysine was 24.2 g ± 3.7 g on the 15 th day after operation.The TWL values on the 13 th,15th and 17 th day were 10.1 ± 0.7 s,10.3 ± 0.7 s and 10.2 ± 0.7 s respectively,which were significantly increased compared with model group(P < 0.01 or P < 0.05).(4)The ION-CCI model was established successfully,drugs were administrated on the 10 th day after operation.On the 14 th,17th,21 th and 28 th day after operation,MWT of carbamazepine(1.43 ± 0.64 g,3.50 ± 1.91 g,3.6 ± 2.19 g,11.2 ± 3.6 g,respectively),25 mg/kg QO-58lysine(1.03 ± 0.64 g,2.8 ± 1.10 g,2.16 ± 1.07 g,7.33 ± 2.3 g,respectively),50 mg/kg QO-58lysine(1.45 ± 0.74 g,1.83 ± 1.06 g,1.70 ± 0.33 g,8.8 ± 1.8 g,respectively)were significantly increased compared with model group(P < 0.01 or P < 0.05).(5)STZ-induced diabetic neuropathic pain model was established successfully and the success rate was 68%.Drugs were administrated on day 22 after operation.On the 28 th and 31 th day after injection,50%MWT values of the model group were 3.24 ± 0.62 g and 3.43 ± 0.44 g,respectively.Compared with the model group,50%MWT values of PGB(13.72 ± 4.92 g,11.54 ± 3.82 g,respectively),12.5 mg/kg QO-58lysine(8.25 ± 3.83 g,9.36 ± 4.83 g,respectively)were significantly increased(P < 0.05 or P < 0.01).On the 28 th day after injection,50%MWT of 25 mg/kg QO-58lysine(8.05 ± 4.76 g)was significantly improved(P < 0.05).On the 31 st day after injection,50%MWT of 50 mg/kg QO-58lysine(6.89 ± 3.41 g)was significantly improved(P < 0.05).(6)Immunohistochemical staining results show that KCNQ3 protein was expressed in cell membrane of neurons.Large diameter neurons had weak or no expression,while medium and small diameter neurons were strongly expressed.The mean optical density of the model group was 0.180 ± 0.04,compared with the control group,the protein expression of the model group(0.161 ± 0.03)was significantly decreased,(P < 0.05).Compared with the model group,the mean optical density of PGB group,12.5 mg/kg QO-58 lysine and 50 mg/kg QO-58 lysine increased(0.216 ± 0.01,0.188 ± 0.01,0.184 ± 0.005,respectively),the protein expression was significantly increased,P < 0.001 or P < 0.05.Conclusions: QO-58 lysine has a certain concentration distribution in rats DRG and TG neurons after intragastric administration and it can increase the amplitude of M currents recorded from DRG and TG neurons.QO-58 lysine shows explicit antinociceptive effects on rats with CCI-induced sciatica、trigeminal neuralgia、diabetic neuropathic pain and increases the KCNQ expression in DRG and TG.QO-58 lysine plays a role in the treatment of neuropathic pain by regulating M channel. |
PDF Full Text Request