Spinal Cord JAK2-STAT3 Pathway Is Involved In The Development Of Electroacupuncture Tolerance

Acupuncture is a traditional Chinese therapeutic method with a long history in treating diseases and relieving pain.Electroacupuncture(EA),evolving from traditional manual acupuncture,has begun to replace manual manipulation because of its better analgesia,lower side effects and objectively quantified and controlled properties in the stimulation references.However,like morphine tolerance the analgesic effect of EA would become attenuated,even finally disappear,after prolonged or repeated EA stimulations.This phenomenon is called as EA tolerance.The phenomenon of EA tolerance exsits in the clinical generally,arousing high attention from scholars.It has been confirmed that EA exerts the antinociceptive effect by releasing the endogenous opioid peptides,including endorphin,enkephalin.Meanwhile EA stimulation also mobilize some anti-analgesia substance,such as FQ orphanin,cholecystokinin-octopeptide-8 and antiotensin ?.It has been verified that there is a bidirectional cross-tolerance between EA and morphine,suggesting EA and morphine analgesia may share similarity underlying mechanisms.Studies demonstrated that chronic i.t.administration of morphine significantly upregulates the expression levels of pSTAT3 in rats' s spinal cord.Also many studies proved that spinal JAK2-STAT3 pathway is involved in the development and maintance of rats neuropathic pain,inhibiting the activation of JAK2-STAT3 pathway can attenuate allodynia and hyperanalgesia,indicating that JAK2-STAT3 pathway may play an important role in pain response.However,the roles of JAK2-STAT3 pathway in EA tolerance remains unknown.The objective of the present study was to investigate the role of spinal JAK2-STAT3 pathway involved in EA tolerance.Rats with more than 50% increase in tail flick latency(TFL)after single EA stimulation were selected as responders.Seventy responders were randomly divided into two groups: EA group and Sham group.EA tolerance model of rats was established by repeated EA stimulation,when the analgesic effect of EA become attenuated,even disappear means EA tolerance has developed.In the present study we adopted 2Hz frequency to stimulate rat's bilateral “Zusanli”and “Sanyinjiao”acupoints once a day,lasting 30 min,for consecutive 8 days in EA group.While rats in the Sham group only inserted needles into acupoints without electricity.Thermal radiation method was adopted to measure TFL.Recording TFL as thermal threshold before and after EA stimulation,the change rate in pain threshold was calculated and was presented as the EA analgesia effect.To investigate the role of spianl JAK2-STAT3 pathway in EA tolerance,rats were sacrificed at days 0,2,4,6 and 8d respectively,and their L4~6 spinal cord were taken as sample.The mRNA levels of JAK2 and STAT3 were detected with qPCR,the protein levels of spinal phosphorylated JAK2(pJAK2)and phosphorylated STAT3(pSTAT3)were detected with Western Blot.Immunohistochemistry was used to verify the distribution of pJAK2 and pSTAT3 in spinal cord.Intrathecal administrate WP1066,an inhibitor of JAK2-STAT3 pathway,to further determine the role of spinal JAK2-STAT3 pathway in EA tolerance.Forty-five rats were randomly divided into three groups: EA group,EA+ Dimethylsulphoxide(DMSO)group and EA+WP1066 group,fifteen rats per group.DMSO(solvent)and WP1066(inhibitor)was injected into lumbar enlargement before EA stimulation respectively.Recording TFL before and after EA stimulation and calculating the change rate in pain threshold.Detecting the expression level of pJAK2 and pSTAT3 with immunohistochemistry.Repeated EA show that the analgesic effect was declined and almost disappeared as the times of EA increased.The TFL change rate in Sham group have no significant difference in all time points.Compared with Sham group,the TFL change rate was higher(p < 0.05)in EA group from day 1 to day 5,but show no difference from day 6 to day 8(p > 0.05),indicating the development of EA tolerance.qPCR result show no significant difference in the mRNA level of JAK2 and STAT3 between two groups(p > 0.05).Compared with sham group,Western Blot show the protein level of pJAK2 in EA group was significant increased(p < 0.05)from day 2 to day 6,and have a peak on day 4,while have no difference on day 8(p > 0.05).pSTAT3 have a similar expression pattern like pJAK2.The results of immunohistochemistry were accordance with Western Blot and found that EA stimulation causing pJAK2 and pSTAT3 expression increased in laminae? in spinal cord dorsal horn(SCDH).The experiment of intrathecal injection show that analgesia effect between EA group and EA+DMSO group have no difference(p > 0.05),the effect of EA nearly disapper from day 6 to day 8.Athough the analgesia effect of EA+WP1066 group declined with repeated EA stiulation,it still significant higher than EA group and EA+DMSO group from day 4 to day 8(p < 0.05).Immunohistochemistry result show the expression tendance of pJAK2 and pSTAT3 were accordance with repeated EA experiment,its were upregulated from day 2 to day 6,and have a peak on day 4.The number of immunopositive cells of pJAK2 and pSTAT3 in EA+DMSO group and EA group have no difference(p > 0.05).The number of immunopositive cells of pJAK2 and pSTAT3 in EA+WP1066 group were increased after EA stimulation.Collectively,JAK2-STAT3 pathway play a role in EA tolerance,and intrathecal administrate WP1066 inhibitor activation of JAK2-STAT3 pathway can attenuate the development of EA tolerance.