Electroacupuncture Elieves Visceral Hypersensitivity Through Modulating The Endogenous Cannabinoid System In Goats

Visceral hypersensitivity(VH)is a complex and common symptom in many gastrointestinal disorders,such inflammatory bowel diseases(IBDs)and irritable bowel syndrome(IBS),it exists in about 50%~70% IBDs patients and 20%~90% IBS patients.VH is defined as low thresholds of stimuli perception arising from the gut.It could lead to hyperalgesia,allodynia,altered bowel movement and increased mucosal secretion.VH is related to central sensitivity,peripheral sensitization,brain-gut axis disorder and so on.It’s drug treatment,that have severe side effects,which has became severe medical problem that puzzled people for a long time.Recently,acupuncture treatment of visceral hypersensitivity has achieved good effects in clinical practice.However,the mechanism of acupuncture for visceral hypersensitivity is unclear.Researches confirmed that the opioid system plays an important role in acupuncture analgesia.However,in the inflammatory induced chronic pain model,blocking the opioid system didn’t affect the effect of acupuncture analgesia,but blocking endocannabinoid system(ECS)successfully inhibited this effect.Those results suggest that ECS plays a unique role in the treatment of electroacupuncture to relieve pain in inflammatory conditions.The ECS is mainly made up of cannabinoid receptors,the endogenous ligands at these receptors and enzymes for the regulation of endocannabinoid levels,and are located primarily in central and peripheral neurons system,particularly in cingulate gyrus,medulla,spinal cord and enteric nervous system.Endocannabinoid,such as AEA and 2-AG,that produced by biosynthetic enzymes and released from post-synaptic neurons can active the CB1 in the pre-synaptic neurons.The activation of CB1 can re-establish an excessive activity of the post-synaptic neurons.Studies have shown that electroacupuncture can significantly increase the expression of CB1 and inhibit hyperalgesia in arthritic rats.The dominant enzyme responsible for the hydrolysis of AEA is fatty acid amide hydrolase(FAAH),and monoacylglycerol lipase(MAGL)is the primary hydrolase for 2-AG.In addition,The antagonists of FAAH and MAGL significantly increased the expression of AEA and 2-AG in rats,and could also alleviate the pain sensitivity in rats.However,whether CB1,FAAH and MAGL were concurrently involved in the inhibition of hypersensitivity were not reported.In present study,a ileitis-induced goats model of visceral hypersensitivity was used to examine the effect and mechanism of EA.A visceral hypersensitivity model was built after the injection of 2,4,6-trinitrobenzenesulfonic acid(TNBS)into terminal ileum wall in eighteen healthy hybrid male goats.A further six goats,that were treated with an equal volume of isotonic saline,were included as a control group.The visceral hypersensitivity goats were divided into three groups(n=6 each): the TNBS group,not treated with EA;the EA+TNBS group,EA was applied at day 7 after the establishment of model,the needle were inserted into bilateral “housanli”(ST-36)acupoints and stimulated by electrical current at 60 HZ in frequency for 30 min every three days,a total of 6 times;the Sham+TNBS group,the needle was inserted into ST-36 without electrical stimulation or manual manipulation,the other treatments were the same as those in EA group.At 7,10,13,16,19 and 22 days after operation,the goats were performed with EA and then submitted to colorectal distension(CRD),and electromyography(EMG)was used to measure the visceral motor response(VMR)to CRD(20~100mg)of goats.Once the EMG was recorded after the last time of EA,the samples of brain,spinal cord T11 and ileum were taken.The levels of CB1,FAAH and MAGL were tested with the method of SABC immunohistochemisty in the anterior cingulated cortex(ACC),periaqueductal gray(PAG),nucleus raphe magnus(NRM),gigantocellular reticular nucleus(GI),rostral ventrolateral medulla(rVLM),nucleus tractus solitarius(NTS),dorsal motor vagal nucleus(DMV),spinal cord dorsal horn(SDH)and ileum.The results showed that the colorectal distension-induced VMR for TNBS-treated goats significantly increased(P<0.05)compared to those in the saline-treated goats at day 7 and maintained such a high level to 22 day.There was no significant difference(P>0.05)in VMR between TNBS group and Sham+TNBS group.the VMR of goats in EA+TNBS group was no obvious difference(P>0.05)with the TNBS group at 7 day,but obviously higher(P<0.05)than the TNBS group at days 10,13,16,19 and 22.Those data suggested that the visceral hypersensitivity was observe in the goats with ileitis,meanwhile EA can alleviate the visceral hypersensitivity in chronic ileitis goats and have cumulative effects.In present study,the levels of CB1 in ACC,PAG,NRM,GI,RVLM,NTS,DMV,SDH and ileum obviously decreased(P<0.05)in TNBS group,while the levels of FAAH and MAGL significantly increased(P<0.05),compared with the contral group.After EA treatment,the protein level of CB1 significantly increased(P<0.05)in TNBS-induced ileitis goats,the protein level of FAAH and MAGL obviously reduced(P<0.05).Those data demonstrated that EA can contribute to the up-regulation of the CB1 and down-regulation the FAAH,MAGL in the ileitis-induced VH goats.The present study established visceral hypersensitivity model by the injection of TNBS into terminal ileum wall of goats and demonstrated that EA can effectively relive the ileitis-induced VH through up-regulated of CB1 and the down-regulated the FAAH,MAGL in the central and peripheral nervous system of goats,which is beneficial to discover the neural mechanism of EA on alleviating ileitis-induced visceral hypersensitivity of goats,and promote the development of intestinal disease treatment.