Sinomenine Alleviates Pain Behaviors Of Neuropathic Pain Through IL-17A/CaMK?/CREB Signaling Pathway

Background:Neuropathic pain is a special type of chronic pain that caused by injury or disease of nervous system.Neuropathic pain affects up to 10%of the total population and has become a worldwide public health problem.In addition to the persistent pain symptoms,neuropathic pain is usually accompanied by anxiety,depression and disordered sleep which severely affect the life quality of patients.Previous studies found that inflammation induced by nerve injury is an important reason for the development and maintenance of neuropathic pain.Sinomenine has anti-inflammatory,immunosuppressive,analgesic,antioxidant and other pharmacological effects.At present,sinomenine is widely used in clinical to treat rheumatoid arthritis,rheumatic diseases,nephritis,arrhythmia,etc.Sinomenine has been reported to alleviate neuropathic pain various animal models.However,the underlying molecular mechanism is unclear.Previous studies show that activation of IL-17A/CaMK?/CREB pathway may contribute to the development of neuropathic pain.This study will combine the animal behavioral models,cell culture and molecular biology techniques to confirm that sinomenine can alleviate neuropathy pain symptoms via inhibiting the activation of IL-17A/CaMK?/CREB pathway.This study can provide theoretical basis for sinomenine in the treatment of neuropathic pain,and expand the clinical application scope of sinomenine.Objective:To investigate the effect of sinomenine on neuropathic pain and its possible underlying mechanism by establishing neuropathic pain model in vivo and in vitro.Methods:Part 1Animal behavior tests and molecular biological techniques were used to determine the therapeutic effects of sinomenine on neuropathic pain and the underlying molecular mechanism.Spinal nerve ligation(SNL)was performed in rats to establish the neuropathic pain model.After measuring the basal pain threshold(mechanical allodynia and thermal hyperalgesia),rats were randomly assigned into the following groups(n=8):the sham group(operation without the spinal nerve ligation),SNL group(spinal nerve ligation),sinomenine group(sinomenine+SNL),IL-17A antibody group(IL-17A antibody+SNL),KN93(the CaMK?inhibitor)group(KN93+SNL),666-15(the CREB antagonist)group(666-15+SNL).The sham group and SNL group received intrathecal injection of the same volume of normal saline for consecutive 14 days.The sinomenine group,IL-17A antibody group,the KN93 group and the CREB group received intrathecal injection of the drugs for consecutive 14 days.Pain threshold was measured at day 1(P1),day 3(P3),day 5(P5),day 7(P7)and day 14(P14)after SNL.Western blot was used to measure IL-17A,p-CaMK?and p-CREB protein levels of the spinal cord at P14 after SNL(n=6).To clarify the role of IL-17A in the treatment of neuropathic pain by sinomenine,rats were further divided into the following groups(n=8):Sham group,SNL group,sinomenine group(SNL+sinomenine),sinomenine+IL-17A group,and sinomenine suspend group.The sham group and the SNL group received intrathecal injection with the same volume of normal saline,while the sinomenine group received intrathecal injection with sinomenine for 14 consecutive days.In the sinomenine+IL-17A group,rats received intrathecal injection of sinomenine for 7 days and combination with IL-17A for the remaining 7 days.In the sinomenine suspend group,the rats received intrathecal injection with sinomenine for 7 days,and then suspend the treatment.Mechanical pain threshold and thermal pain threshold were measured on day 7(P7)and day 14(P14)after SNL in rats.Then Western blot was used to measure IL-17A in spinal cord tissue of each group on the 14th day after SNL.Part 2In vitro,cell culture of DRG neurons were used to determine the effects of sinomenine on the IL–17A/p-CaMK?/p-CREB pathway activated by TNF-?.After induction of DRG neurons by TNF-?,protein(n=3)and m RNA(n=6)expression levels of TRPV1 and TRPA1,which are related to pain signal transduction,were detected by Western blot and RT-PCR,respectively.The changes in Ca²⁺concentration in DRG neurons were detected by Flu O-3/AM probe(n=6).MTT was used to detect the effect of sinomenine on the proliferation activity of DRG neurons after TNF-?treatment.The expression levels of LDH and ROS were also determined.Then,DRG neurons were randomly assigned into the following groups(n=6):the control group(no treatment),TNF-?group(TNF-a treatment),Sinomenine group(TNF-?+sinonine treatment),IL-17A antibody group(TNF-?+IL-17A antibody),and KN93 group(TNF-?+KN93 treatment).Western blot and immunofluorescence were used to detect the levels of IL-17A,p-CaMK?,p-CREB protein.RT-PCR was used to detect the TRPV1 and TRPA1 m RNA in spinal cord of rats in these groups.Part 3The therapeutic effects of sinomenine on neuropathic pain were compared with current methods in clinic.Spinal nerve ligation(SNL)was performed in rats to establish the neuropathic pain model.After measuring the basal pain threshold(mechanical allodynia and thermal hyperalgesia),rats were randomly assigned into the following groups(n=6):the Sham group(operation without the spinal nerve ligation),SNL(spinal nerve ligation),sinomenine group(sinomenine+SNL),oxycodone group(oxycodone+SNL)and pulsed radiofrequency group(pulsed radiofrequency+SNL),combined sinomenine with pulsed radiofrequency group(sinomenine+pulsed radiofrequency+SNL).The therapeutic effects of these methods on neuropathic pain were evaluated by measuring the mechanical and thermal pain thresholds at P1,P3,P5,P7 and P14 in each group.Results:Part 1Compared with the Sham group,the threshold of mechanical allodynia and thermal hyperalgesia of the SNL group were significantly reduced at the beginning of P1 and lasted for at least 14 days(P<0.05),and the levels of IL-17A,p-CaMK?and p-CREB protein of the spinal cord at P14 after SNL were significantly increased(P<0.05).Compared with SNL group,intrathecal injection of IL-17A antibody can improve the mechanical allodynia and thermal hyperalgesia induced by SNL in rats(P<0.05),and decrease the levels of IL-17A,p-CaMK?and p-CREB of the spinal cord at P14 after SNL(P<0.05).Compared with SNL group,intrathecal injection of CaMK?antagonist KN93 can improve the mechanical allodynia and thermal hyperalgesia induced by SNL in rats(P<0.05),and decrease the levels of p-CaMK?and p-CREB of the spinal cord at P14 after SNL(P<0.05).Compared with the SNL group,intrathecal administration of CREB antagonist can improve the mechanical allodynia and thermal hyperalgesia induced by SNL in rats(P<0.05),and decrease the levels of p-CREB of the spinal cord at P14 after SNL(P<0.05).Compared with SNL group,intrathecal administration of sinomenine can improve the mechanical allodynia and thermal hyperalgesia induced by SNL in rats(P<0.05)and decrease the levels of p-CREB of the spinal cord at P14 after SNL(P<0.05).In sinomenine+IL-17A group,the therapeutic effect of sinomenine could be reversed by IL-17A,and the expression level of IL-17A in spinal cord was significantly increased14 days after SNL(P<0.05).In the sinomenine suspend group,the pain of rats was improved on the 7th day after surgery,but decreased again on the 14th day after SNL.The expression level of IL-17A in the spinal cord was significantly increased on the14th day after SNL(P<0.05).Part 2Compared to the control group,TNF-?significantly up regulated TRPV1 and TRPA1 ion channel protein and m RNA expression levels in DRG neurons(P<0.05),as well as increased intracellular Ca²⁺concentration(P<0.05).Compared with 0?mol/L group,sinomenine at 100,200,400 and 800?mol/L significantly improved the cell viability induced by TNF-?.In addition,we also investigated the effects of different concentrations of sinomenine on intracellular reactive oxygen species and lactate dehydrogenase induced by TNF-?.The results showed that sinomenine,at the concentration?200?mol/L,significantly inhibited the expression of reactive oxygen species and lactate dehydrogenase in DRG.Therefore,the concentration of800?mol/L was selected for sinomenine in this part.Compared with the control group,the levels of IL-17A,p-CaMK?and p-CREB were significantly increased in the TNF-?group.Compared with the TNF-?group,the levels of IL-17A,p-CaMK?and p-CREB were significantly reduced in the IL-17A antibody group.Compared with the TNF-?group,the levels of p-CaMK?and p-CREB were significantly reduced in the KN93 group.Compared with the TNF-?group,the levels of IL-17A,p-CaMK?and p-CREB were significantly reduced in the sinomenine group.Compared with TNF-?group,the m RNA expressions of TRPV1 and TRPA1 were decreased in sinomenine group,IL-17A antibody group,and KN93 group.Part 3Compared with the sham group,the mechanical and thermal pain thresholds of the rats in the SNL group were significantly decreased.Compared with the SNL group,sinomenine,oxycodone,pulsed radiofrequency and sinomine/radiofrequency combined therapy all can significantly relieve the mechanical and thermal pain caused by the SNL model(P<0.05).Sinomenine,oxycodone and pulsed radiofrequency therapy had the same therapeutic effects on neuropathic pain.Sinomenine combined with pulsed radiofrequency therapy had better therapeutic effects than the single treatment group.Conclusions:Sinomenine can alleviate the neuropathy pain via inhibition of the activation of IL-17A/p-CaMK?/p-CREB pathway;the therapeutic effects of sinomenine,oxycodone and pulsed radiofrequency treatment were similar.sinomenine/radiofrequency combination treatment showed the best therapeutic effects in this research.