Study On The Roles Of JAK2/STAT3 And Thymosin ?4 In Electroacupuncture-induced Visceral Analgesia And Tolerance Respectively

Electroacupuncture(EA)has shown to be effective for various painful disorders(including body pain and visceral pain).Studies on EA-induced body analgesia have verified that EA can cause the release of a variety of analgesic substances(such as endogenous opioid peptides: enkephalins,endorphins,dynorphin,etc.)and anti-analgesic substances(such as anti-opioid peptides: cholecystokinin octapeptide,orphanin FQ,etc.).EA analgesia is the result of combination of various active substances in the central nerve system.The research of EA in relieving visceral pain developed slowly,mainly because of no suitable model of visceral pain.The purpose of this study was to establish an appropriate visceral hypersensitivity(VH)model and to study the central molecular mechanism of EA in attenuating VH.EA show the best analgesic effect in ruminants(goats).Due to the high cost of goat experiment,the visceral hypersensitivity model was firstly established in rats,and then ileitis-induced visceral hypersensitivity model was established in goats.Repeated or continuous EA will lead to the decrease of its analgesic effect and the phenomenon of "EA tolerance"(EAT),which is the negative effect of EA treatment and one of the main obstacles in its clinical application.Studies have shown that anti-analgesic substances play an important role in EAT.A recent study have shown that thymosin 4(T?4)has an anti-electroacupuncture analgesic(EAA)effect,and its role in EAT is worthy of attention.The effect of T?4 on classical active substances(opioid peptides and anti-opioid peptides)in electroacupuncture tolerance was further explored to elucidate the mechanism of EAT.1.Establishment of visceral hypersensitivity model with 2,4,6-trinitrobenzenesulfonic acid in ratsInflammatory bowel disease(IBDs)are immune-mediated chronic,intermittent recurrent intestinal inflammation.VH is an important pathophysiological mechanism of IBDs and has attracted more and more attentions in recent years.In 70% IBDs,the inflammatory mediators constantly were released during the acute and convalescent phase of disease progression,leading to the sensitization of the nerve endings of intestinal wall,finally causing abdominal pain.Two-thirds of patients with IBDs present with transmural ileitis.The existing studies on VH resulting from IBDs is based on colitis,which are obviously not suitable.Whether VH presents in IBDs is controversial and lacks evidence.To address this controversy,this trial aimed to establish ileitis with 2,4,6-trinitrobenzenesulfonic acid(TNBS)in rats and to observe the presence of VH.Male Sprague-Dawley rats were anesthetized,the abdominal cavity was opened,TNBS(0.6 mL,80 mg/kg,dissolved in 30% ethanol)was injected into the ileum lumen at 15 cm from the ileocecal ligament,and the control group was injected with an equal volume of 30% ethanol or saline.The visceral motor response(VMR)to 20,40,60,80 and 100 mmHg colorectal distension pressure(CRD)was assessed at day 1,3,7,14,21 and 28,respectively.Immediately after CRD test,the rats were euthanized,and the end of intestine were collected.The concentrations of myleoperoxidase and cytokines(TNF-?,IL-1? and IL-6)were detected with ELISA.The calcitonin gene-related peptide(CGRP)levels in eleventh thoracic vertebrae(T11)dorsal root ganglia were determined with immunohistochemistry.The results showed that rats in TNBS group began to show transmural inflammation at day 3,which was most obvious at day 7,and then the inflammation gradually recovered,and no lesions were observed at day 21.The VMR to CRD in TNBS group was higher(P < 0.05)than that in ethanol or saline group at day 7 to 21.The CGRP immunoreactive cells in the dorsal root ganglion increased(P < 0.05)at day 7 to 21,and positively correlated with VMR.These results indicated that TNBS injecting into the ileum induced transmural inflammation and VH.On the basis of rat model,30 mg TNBS-40% ethanol solution(1.2 mL)was injected into the ileum wall at 15 cm from the ileocecal ligament in goats,and the control group was injected with the same amount of normal saline.Ileum inflammation and visceral motor response were evaluated at day 3,7,14,21 and 28.The results showed that TNBS induced obvious ileal inflammation in goats at day 3 to 7,and visceral hypersensitivity began to appear at day 7 and lasted until day 28 in goats.This model simulates the clinical manifestations of IBDs,providing ideas for the exploration of the pathogenesis of intestinal inflammation and visceral hypersensitivity,and contributing to the further treatment of visceral hypersensitivity.This model mimics the clinical manifestations of IBDs,provides insights into the pathogenesis of intestinal inflammation and VH,and provides a basis for further treating VH.2.Electroacupuncture attenuates visceral hypersensitivity via inhibiting JAK2/STAT3 signaling pathway in the descending pain modulation system in goatsIt is currently believed that inflammation,psychological and intestinal abnormalities and motor dysfunction can lead to peripheral and central sensitization,and further induced VH,but the inside mechanism is not fully understood.The cytokine(IL-6)-activated JAK2/STAT3 signaling pathway is a key signaling pathway for central sensitization and promotes the development of hypersensitivity.Therefore,JAK2/STAT3 signaling pathway may be a potential target for treating hypersensitivity in humans and animals.EA is a traditional treatment method,it is safe and without complications,and may be an effective supplementary treatment for VH,but the specific mechanism is still unclear.EA induces analgesia by activating(or inhibiting)a descending inhibitory(or facilitatory)system,mainly including the periaqueductal gray(PAG)-the rostral ventral medial region(RVM)-the spinal cord dorsal horn(SCDH).Whether EA regulates VH through JAK2/STAT3 signaling pathway in PAG-RVM-SCDH axis remains to be confirmed.Goats were anaesthetized and laparotomized for injecting TNBS-ethanol solution(30 mg TNBS dissolved in 40% ethanol)into the ileal wall to induce VH.EA was treated for 30 min from day 7,then every three days for six times.VH was assessed by visceromotor response(VMR)and pain behavior response to 20,40,60,80 and 100 mmHg colorectal distension pressures at day 7,10,13,16,19 and 22.The spinal cord in the eleventh thoracic vertebra and the brain were collected at day 22.The protein and mRNA levels of IL-6,JAK2 and STAT3 in the SCDH were detected with western blot and qPCR,respectively.The distribution of these substances was observed with immunohistochemistry in the ventrolateral PAG(vlPAG),RVM(mainly the nucleus raphe magnus,NRM),SCDH,the nucleus tractus solitaries(NTS)and the dorsal motor nucleus of vagi(DMV).Results: Goats administered with TNBS-ethanol solution showed diarrhea,enhanced VMR and pain behavior response,and increased IL-6,phosphorylated JAK2 and STAT3(pJAK2 and pSTAT3)in the vlPAG,NRM,NTS and DMV,and their protein and mRNA levels in the SCDH.EA relieved diarrhea,VMR and pain behavior response,decreased IL-6,pJAK2 and pSTAT3 levels in the vlPAG,NRM,SCDH,NTS and DMV except for pSTAT3 in the DMV,but did not affect mRNA level of these three substances in the SCDH.Conclusion: EA attenuates VH probably through inhibiting JAK2/STAT3 signaling pathway in the PAG-RVM-SCDH axis.3.Thymosin beta 4 is involved in the development of electroacupuncture toleranceEA with appropriate intervals can produce good analgesic effects.However,prolonged or repeated EA stimulations would attenuate and finally nullify analgesic effect,which is termed EAT.EAT is a negative effect of acupuncture treatment,which has attracted more and more attentions from clinicians and researchers.A large number of studies have shown that EA induces the release of opioid peptides,including enkephalin(ENK),endorphin(END)and dynorphin(DYN),but also anti-opioid peptides,including cholecystokinin(CCK-8)and orphanin(OFQ).These studies have elucidated the role of these active substances and their relationship in EAA,but the specific mechanism of EAT remains unclear.Recently,T?4 was reported to play a role in anti-EAA,the potential mechanism is worthy attention.Whether it is involved in EAT,and its relationship with classical EAA related substances(including opioid peptides and anti-opioid peptides and their related receptors)need to further study.Rats were treated with EA once daily for eight consecutive days to establish EAT,effect of T?4 on the development of EAT was determined through microinjection of T?4 antibody and siRNA into the cerebroventricle.The mRNA and protein expression profiles of T?4,opioid peptides(enkephalin,dynorphin and endorphin),and anti-opioid peptides(cholecystokinin octapeptide,CCK-8 and orphanin FQ,OFQ),and mu opioid receptor(MOR)and CCK B receptor(CCKBR)in the brain areas(hypothalamus,thalamus,cortex,midbrain and medulla)were characterized after T?4 siRNA was administered.Results: T?4 levels were increased at day 1,4 and 8 and negatively correlated with the changes of tail flick latency in all areas.T?4 antibody and siRNA postponed EAT.T?4 siRNA caused decreased T?4 levels in all areas,which resulted in increased enkephalin,dynorphin,endorphin and MOR levels in most measured areas during repeated EA,but unchanged OFQ,CCK-8 and CCKBR levels in most measured areas.T?4 levels were negatively correlated with enkephalin,dynorphin,endorphin or MOR levels in all areas except medulla,while positively correlated with OFQ and CCK-8 levels in some areas.These results confirmed T?4 facilitates EAT probably through negatively changing endogenous opioid peptides and their receptors and positively influencing anti-opioid peptides in the central nervous system.