Effects Of Procaine On Neuropathic Pain And The Underlying Mechanisms

[Background]Neuropathic pain(NPP)is a special type of pain,which is defined as "Pain initiated or caused by a primary lesion or dysfunction in the nervous system" by the International association for the study of pain(IASP).Neuropathic pain has become the top killer in chronic pain due to its long course,poor curative effect and seriously affecting the quality of life.Several mediators directly or indirectly induce NPP including proinflammatory cytokines and there is evidence that the Janus tyrosine kinase(JAK)and signal transducer and activator of transcription(STAT)pathway is involved in the formation of pronociceptive cytokines.The JAK/STAT signaling pathway is one of the most important cascades for the cellular transduction of signals in response to many pain modulators.It was reported that The JAK/STAT signaling pathways and MAPK cascades are vital signal transduction pathways for neuropoietic cytokines such as interleukin-6,among which the JAK2/STAT3 signaling can be activated by almost all the cytokines acting on JAKs.Peripheral nerve injury evokes the activation of JAK2/STAT3 with increased phosphorylation of STAT3,contributing to NPP.The regulation of JAK/STAT signaling pathway is mainly a feedback regulation mechanism.It is known that the negative regulatory factors are mainly protein tyrosine phosphatases(PTPs),cytokine signaling inhibitors(Protein tyrosine phosphatases,SOCS)and Protein inhibitors activated states(PIAS).However,it has been found that IFN-y and IFN gene induction can enhance the signal transduction of JAK/STAT pathway.The pathogenesis of neuropathic pain is complex,and multiple mechanisms have been found at present.But the effect is still disappointing if only mechanically treated for its mechanism.It has been reported that only 40-60%of patients receive partial pain relief in the current treatment.It is promoted that jointing variety of drugs and means were used for neuropathic pain treatment at present.Therefore,it is very important to find new drugs and methods.Procaine is a kind of fat-soluble local anaesthetics with low toxicity and good effects.Procaine acts as a kind of local anesthetic by blocking sodium channels.Studies have found that the pharmacological basis of local anesthesia also includes blocking calcium channels.Procaine can be used as a local anesthetic as well as intravenous anesthetics,which has been widely used in clinical anesthesia,although it is gradually replaced by more effective anesthetics.Furthermore,its effect in treating diseases has been revealed in recent years.For example,injection of 4 ml 1%procaine HC1 in median nerves improves the clinical and electrophysiological evaluations of carpal tunnel syndrome patients.Procaine represses the DNA-methylation level and promotes the proliferation arrest and apoptosis of gastriccancer cells.Procaine inhibited cell proliferation,migration but promoted apoptosis as well as inactivated ERK/MAPK/FAK pathways.It also facilitates the treatment of skin eruption.The role of procaine in relieving pains inspires us to investigate its effect on NPP.However,its role in modulating NPP has not been reported yet.[Objective]Iin this study,rats with procaine injected intrathecally received bilateral chronic constriction(bCCI)surgery were used as the model for NPP study.Then pain behaviors and changes of JAK2 and STAT3 were tested to investigate the role of procaine in modulating NPP.To investigate the possible mechanism of procaine in the treatment of neuropathic pain,we overexpress JAK2 in rat spinal cord and detect changes of JAK2 and STAT3 in spinal dorsal horn of rats.This study would offer fundamental information for further application of procaine in alleviating NPP.[Methods]1.To verify the effects of intrathecal procaine on neuropathic pain following chronic constrction injury in rats18 SD rats(180～210g)were divided into three groups randomly,namely,sham surgery group,CCI group and CCI+procaine group.The rats in CCI group and CCI+procaine group all received the whole procedure of bCCI surgery,while the sham surgery group underwent the surgery without ligation of sciatic nerve.The CCI+procaine group were injected of 2%procaine in DMSO(10 μl/kg)at three time points(3d before model construction,Id before model construction and just before model construction).While the other two groups were injected of the same volume of DMSO.The back heels of both sides of rats were all stimulated by mechanical stimulation,thermal stimulation and acetone cold stimulation before the pretreatment of procaine.Detection on the same rat was repeated for 3 times,the mean value was taken as mechanical stimulation threshold(MWT),Paw withdrawal thermal latency(PWTL)and total number of withdrawal upon cold stimulation.Behavior tests were also performed at night on 0,5,10,15 and 20 d post CCI surgery.After behavior tests at 20 d post surgery,The L4-L6 spinal dorsal horn of rats was sampled and quickly frozen in liquid nitrogen for RT-PCR and Western blot to investigate both mRNA and protein levels of JAK2 and STAT3.2.To verify the mechanism of procaine in alleviating NPP.In this study,The JAK2 overexpression vector was constructed via ligating the coding sequence of rat JAK2 into pcDNA3.I.The rats were injected intrathecally with JAK2 overexpression vector 1 d before CCl,and JAK2 was expressed in the spinal dorsal horn of rats for exploring the relationship between the therapeutic effect of procaine on neuropathic pain and the JAK/STAT signal transduction pathway.24 SD rats(180～210g)were divided into three groups randomly,namely,control group,procaine group,procaine+pcDNA3.1 group and procaine+pcDNA3.1-JAK2 group.The rats in all groups were stimulated by mechanical stimulation,thermal stimulation and acetone cold stimulation before the pretreatment of procaine and were measured.The procaine treated groups were injected with 2%procaine in DMSO(10 pl/kg)and the control group with the same volume of DMSO.To overexpress JAK2 in rats,we injected 100 μl of JAK2 overexpression vector(300 ng/ml)through the intrathecal catheter to rats of procaine+pcDNA3.1-JAK2 group on 1 d before model construction,while the procaine+pcDNA3.1 group was injected of the same amount of blank vector.Pain behaviors were performed at night on 15 d post CCI surgery.and then the L4-L6 spinal dorsal horn of rats was sampled and quickly frozen in liquid nitrogen for Western blot to detect the protein levels of JAK2 and STAT3.[Results]1.To verify the effects of intrathecal procaine on neuropathic pain following chronic constrction injury in ratsResults showed that there was no significant difference in MWT,PWTL and the acetone cold stimulation threshold before procaine pretreatment(P>0.05).Therefore,the mean value of bilateral hind foot was selected for statistical analysis.At 10,15 and 20 d post surgery,significant differences were found in MWT between CCI group and sham or CCI+ procaine group.CCI significantly decreased MWT compared to sham group(p<0.05),and procaine significantly increased MWT compared to CCI group(p<0.05).CCI could decrease PWTL when detected at 5 d post surgery(p<0.05).Compared to sham group,the obvious effect of procaine in recovering PWTL was detected at 15 and 20 d post surgery(p<0.05).As to cold stimulation,the number of withdrawal was increased significantly by CCI when detected at 5 d post surgery(p<0.05),and was significantly decreased by procaine pretreatment at 5 and 15 d post surgery(p<0.05).Taken together,the pain behaviors of CCI-induced rats were improved by procaine treatment,with the most obvious effect observed at the 15 th day post surgery.qRT-PCR showed that both JAK2 and STAT3 mRNA levels were elevated by CCI surgery(p<0.05),the NPP model rats with procaine pretreatment possessed obviously lower JAK2 and STAT3 levels(p<0.05).Western blot results showed the similar changing patterns of JAK2 and STAT3 proteins,and significant differences were detected between groups(p<0.05).2.To verify the mechanism of procaine in alleviating NPP.Results showed that there was no significant difference in MWT,PWTL and the acetone cold stimulation threshold before procaine pretreatment(p>0.05).Therefore,the mean value of bilateral hind foot was selected for statistical analysis.At 15 d post surgery,Behavior test results showed that procaine pretreatment in NPP model rats increased MWT and PWTL,and decreased the number of withdrawal upon cold stimulation compared to control group(p<0.05).JAK2 overexpression in model rats with procaine pretreatment significantly reduced MWT and PWTL(p<0.05),and significantly increased the number of withdrawal upon cold treatment(p<0.05),The expression level of JAK2 and STAT3 was further detected by western blot and results showed that both JAK2 and STAT3 protein levels were reduced by procaine pretreatment in model rats,but were elevated by JAK2 overexpression,with significant differences between control and procaine groups(p<0.05),as well as procaine+blank control and procaine+JAK2 groups(p<0.05).[Conclusions]JAK2/STAT3 signaling pathway is activated in rat CCI neuropathic pain model.Intrathecal procaine improved the pain behavior score of CCI rats,suggesting that procaine can relieve neuropathic pain in CCI rats.JAK2 overexpression can reverse the role of procaine in the treatment of neuropathic pain,suggesting that procaine relieves neuropathic pain in CCI-induced model rats and the mechanism may be associated with the inhibition of JAK2/STAT3 signaling.