Electroacupuncture Attenuates Visceral Hypersensitivity By Suppressing Spinal PAR-2 And CGRP Expressions In Animals With TNBS-induced Ileitis

Inflammatory bowel diseases(IBDs)including Crohn’s disease(CD)and ulcerative colitis(UC)are immune-mediated chronic and remittent-relapsing intestinal inflammation.Visceral hypersensitivity(VH),an important symptom of the IBDs,has received remarkable attention in the recent years.Because,the continuous release of inflammatory mediators result in sensitization of enteric nerve endings and abdominal pain in 70% of patients with IBDs,which is present not only during acute flares of the diseases but also during their remission.It is worth noticing that two thirds of CD manifest as a transmural inflammation in the distal ileum.Whether CD patients with ileitis suffer from visceral sensitivity disturbances seems somewhat controversial,and the evidence is likewise scarce.The controversies regarding VH and its underlying mechanism need to be addressed properly intervening ileal CD model as a choice to upgrade knowledge in this filed.Despite the several proposed factors including inflammation,psychosocial and aberrant sensory-motor function of the gut to contribute for peripheral and central sensitization,the exact mechanism underlying VH is not fully elucidated.The role of the co-expression of cell-membrane protease-activated receptor-2(PAR-2)with calcitonin gene-related peptide(CGRP)in the VH development could attract researchers to expand the knowledge in this field.There is no single line of therapy that attenuates VH in patients.Presently,therapeutic options for VH are mainly focused on suppressing the aberrant mucosal immune responses.Although therapies are reasonably successful in some patients,a great number cannot be efficacious particularly during remission,which requires the novel therapeutic option.Acupuncture is a traditional medicine treatment that has been employed empirically in China for several millennia.Compared with pharmacological therapeutics,electroacupuncture(EA)is safe and complication free.Although EA appears to be a promising treatment for VH in patients and experimental animals with gastrointestinal disorders,no data exist so far about the mechanism by which EA attenuates ileitis accompanied VH.The scientific evidences are necessary to validate the efficacy of EA through its intervention on PAR-2 and CGRP before it is being employed as a novel approach for the treatment of VH.Therefore,the experiments were conducted initially to unravel the existence of VH in TNBS-induced ileitis,and thereafter the intervention of EA stimulation on PAR-2 mediated CGRP release,and c-Fos expressions during VH were explored in the spinal cord.1.Visceral hypersensitivity is provoked by TNBS-induced ileitisObjective To investigate the existence of visceral hypersensitivity provoked by 2,4,6-trinitrobenzene sulfonic(TNBS)-induced ileitis in rats.Methods Ninety six Male Sprague-Dawley rats were anaesthetized and laparotomized for intraileal injection of TNBS(0.6 ml,80 mg/kg body weight in 30% ethanol,n=48),an equal volume of 30% Ethanol(n=24),and Saline(n=24),respectively.Visceral hypersensitivity was assessed by visceromotor responses(VMR)to 20,40,60,80,and 100 mmHg colorectal distension pressure(CRD)at day 1,3,7,14,21,and 28.Immediately after CRD test,the rats were euthanized for collecting the terminal ileal segment for histopathological examinations and ELISA of myleoperoxidase(MPO)and cytokines(TNF-α,IL-1β,IL-6),and dorsal root ganglia(T11)for determination of calcitonin gene-related peptide by immunohistochemistry,respectively.Results Among all groups,TNBS-treatment showed transmural inflammation initially at 3 days,reached maximum at 7 days and persisted up to 21 days.The rats with ileitis exhibited(P<0.05)VMR to CRD at day 7 to day 21.The calcitonin gene-related peptide-immunoreactive positive cells increased(P<0.05)in dorsal root ganglia at day 7 to 21,which was persistently consistent with this visceral hypersensitivity in the TNBS-treated rats.Conclusions and clinical relevancies TNBS injection into the ileum induced transmural ileitis including granuloma and visceral hypersensitivity.As this model mimics clinical manifestations of CD,it may provide a road map to probe the pathogenesis of gut inflammation and visceral hypersensitivity,as well as for establishing the therapeutic protocol for Crohn’s ileitis.2.Electroacupuncture intervention of visceral hypersensitivity is involved in activation of PAR-2 and CGRP in the spinal cordObjective To study the effects of electroacupuncture(EA)stimulation at bilateral Hou-San-Li(ST-36)acupoints on visceral hypersensitivity(VH)induced in TNBS-ileitis goats,and to confirm whether EA attenuates VH through PAR-2 mediating CGRP and c-Fos expression in the spinal cord.Methods After proper anaesthetization,the right flank laparotomy was performed.TNBS 30 mg mixed in 30% Ethanol in VH group(n=12),Sham group(n=6)and EA group(n=6),and an equal volume of 0.9% NaCl in Control group(n=12)was injected in the wall of ileum.Six goats representating each VH group and Control group were laparotomized at day 7 for the resection of terminal ileum segments for assessing the inflammatory status by histopathological examination and ELISA for MPO.Experimental goats were restrained in sternal recumbency.The EA(60Hz,3mA)was performed bilaterally at acupoints ST-36 for 0.5 h in goats of EA group firstly at 7 days,thereafter repeated on every 3 days and lasted for 22 days(total 6 times).Sham acupuncture was performed for 0.5 h by inserting needles bilaterally at the acupoints ST-36 with no electrical stimulation or manual manipulation.The goats in Control and VH groups were restrained for 0.5 h in the same manner as the EA-treated goats but without needling and electric stimulation.Subsequently,VMR to CRD were recorded quantitatively by electromyography(EMG).Soon after CRD test,the goats were euthanized and sampled the ileal segment for assessing the inflammatory status,and spinal cord(T10-L1)for topographic distribution and quantitative assessment of PAR-2,CGRP and c-Fos expression by immunohistochemistry,Western blotting and qPCR.Results TNBS administrated groups exhibited mucoid to bloody diarrhea,sluggishness,weight loss,and markedly increased macro-and microscopic scores,and MPO concentrations at day 7.Microscopically,the low grade of ileal inflammation persisted in goats of VH and Sham groups but not in EA group at day 22.The goats of EA group showed lower VMR with all CRD pressures as compared to goats of VH group.Compared with their initial values,the repetitive EA stimulations attenuated VMR to 80(P<0.05)and 100 mmHg(P< 0.01)distension pressures at day 16.Furthermore at day 19 and 22,the magnitude of VMR to 40,60,80 and 100 mmHg distension pressures decreased(P<0.01)with increased frequency of EA stimulations.The repetitive EA treatments significantly suppressed PAR-2,CGRP and c-Fos expressions in the spinal cord.Conclusions and clinical relevancies TNBS injection in the ileal wall successfully induced transmural ileitis.Electroacupuncture attenuates ileal inflammation and VH through down-regulation of PAR2-regulated pathways co-expressed CGRP and c-Fos involved in the spinal transmission of nociceptive stimuli in the brain-gut axis.The repeated EA applications show profound anti-hypersensitivity effects.The repetitive EA therapies could be effective for the treatment of VH provoked in IBDs,irritable bowel syndromes(IBS)and other functional bowel disorders.