Study On The Mechanism By Which Electroacupuncture Alleviates Inflammatory Intestinal Hypersensitivity Through Regulating ADORA1

The analgesic effect of electroacupuncture（EA）has been proved in the clinical application and scientific research on different diseases,especially in neuropathic pain and visceral pain.Studies have found that a variety of adenosine receptors play an important role in the alleviation effect of visceral hypersensitivity（VH）by EA.Adenosine A1receptor（ADORA1）which has been confirmed to alleviate VH symptoms by inhibiting the release of IL-1β and SP in the colon,but ADORA1 is involved in the alleviation of VH by EA at the central level remains unclear.The analgesic effect of EA in ruminants is better than that in other species.In this experiment,the VH model was established on goats and the potential therapeutic mechanism was explored through EA treatment.On this basis,mice were used as the model animal for in-depth exploration to further clarify the central mechanism of EA alleviating VH through ADORA1.1.Electroacupuncture alleviates visceral hypersensitivity through the ADORA1 signaling pathway in the descending pain modulatory system of goatsIn order to explore the central mechanism of alleviating VH by EA,TNBS was injected into the ileum wall to establish the ileum inflammatory VH model in goats.Starting from the 7th day of study,the goat"Zusanli"point was stimulated by EA for 30 minutes every2 days,a total of 7 times.Painful behavior and visceral motor response（VMR）changes induced by colorectal dilatation（CRD）were recorded after each EA stimuli.The ileum of goats was collected on the 7th and 25th day,and the inflammatory status was assessed by the changes of macroscopic and microscopic pathological and the concentration of inflammatory substances.On the 25th day,PAG,RVM and spinal cord（SC）T11 segments were collected from the goats to detect the protein expression levels of c-Fos and CGRP,for evaluating the central sensitization status of goats.The contents of adenosine（ADO）and Phosphatidylinositol diphosphate（PIP2）in PAG,RVM and SC were detected.The expression levels of ADORA1,PLCB3 and TRPV1 in PAG,RVM and SC and the distribution of ADORA1 were measured.The results showed that on the 7th day after modeling,TNBS could induce serious ileitis and significant increase of VMR in goats.And the expression levels of c-Fos and CGRP have been upregulated,resulting in central sensitization.On the 25th day after modeling,expression levels of ADORA1 and PLCB3 were downregulated,but expression of TRPV1 was upregulated which induced by TNBS in goats.Compared with the TNBS group,increase of VMR and inflammatory substance induced by TNBS were inhibited by EA significantly.The increased expressions of c-Fos and CGRP induced by TNBS were downregulated and the central sensitization of goats was inhibited.Meanwhile,concentration of ADO and expression levels of ADORA1 and PLCB3 were upregulated,expression of TRPV1 was downregulated,and content of PIP2was decreased by EA in PAG,RVM and SC of goats at same time.These results suggest that EA may activate the ADORA1-PLCB3 signaling pathway to reduce the content of PIP2 and inhibit activation of TRPV1 to alleviate visceral hypersensitivity in goats.2.Electroacupuncture alleviates visceral hypersensitivity through the ADORA1 signaling pathway in the spinal cord of miceRecent studies from ADO and ADORA1 suggest that they play a positive role in the treatment of various types of pain.The author has proved that EA may alleviate VH through ADORA1 signaling pathway in goats through experiments and established an ileal inflammatory VH model in mice successfully.However,EA alleviates VH in mice by the same or similar mechanism or not as in goats remains unclear.Therefore,on the basis of the results in goats,this study continued to explore the central mechanism of EA in alleviating VH with mice as the model animal.In this experiment,the mouse VH model was established firstly by injecting TNBS ethanol solution into the ileum cavity.After anesthesia,the ileum and cecum were removed surgically,and 100μL 40%ethanol solution containing 5 mg TNBS was injected into the ileum cavity 3 cm from the ileocecal ligament,while the control group was injected with the same amount of saline.The changes of disease activity index（DAI）and pain threshold were evaluated at 3,7 and 20 days,respectively.The ileum and T11 segment of spinal cord were collected to observe the macroscopic and microscopic pathological changes of the ileum.The protein expression levels of c-Fos and CGRP in the spinal cord were detected to evaluate the central sensitization status of mice.The results showed that TNBS could induce severe ileitis in mice,and the pathological changes were obvious on day 3 and 7.By day 20,the differences had disappeared in various inflammatory markers.The change of pain threshold of mice was significantly lower than that in the control group since the beginning of modeling.It was still significantly lower than that of the control group until the 20th day although it increased to a certain extent during the period.The protein expression levels of c-Fos and CGRP of TNBS were significantly higher than those in the control group in the spinal cord at the 7th and 20th day,suggesting that the central sensitization lasted until the 20th day.These results indicated that 100μL 40%ethanol solution containing 5 mg TNBS could induce ileitis for 7 days,and hypersensitivity of pain lasting at least 20 days injected in ileum cavity in mice.And then the VH mice were treated with EA stimuli.It was found that similar results in the mouse model as in the goat model:VH was alleviated by EA and the expression of ADORA1 was also upregulated in the spinal cord of mice by EA.On the basis of the above experiments,the VH mice were treated with EA stimuli after intrathecal injection of ADORA1 agonist or antagonist.The results showed that the antagonist of ADORA1 reversed the analgesic effect of EA,which proved that the analgesic effect of EA needs participation of ADORA1 indeed.The results also showed that single EA stimuli could increase content of ADO in the spinal cord of mice,and the pain threshold of mice was positively correlated with ADO content in the spinal cord.The content of ADO was increased and content of PIP2 decreased at the same time by EA.In this study,the ADO and ADORA1 downstream pathways were further explored in primary neurons of mouse:after ADORA1 activation,content of PIP2 and Ca²⁺was decreased in neurons significantly,which was consistent with the results of PLCB3activation alone.On the contrary,when PLCB3 was inhibited before ADORA1 was activated,PIP2 and Ca²⁺in neurons maintained high content,indicating that PIP2 and Ca²⁺were rapidly consumed through PLCB3 pathway after ADORA1 activation in neurons.These results indicate that EA could alleviate VH by increasing ADO in spinal cord to activate ADORA1-PLCB3 pathway in mice.The effect of EA has a cumulative effect,that is,the effect of multiple EA is better than that of single EA.Therefore,this experiment continued to explore the dose-effect relationship between EA and expression of ADORA1 or content of ADO,and the results showed that:with the increase of the number of EA stimuli（within 7 times）,expression level of ADORA1 gradually increased,but there was no such dose-effect relationship between EA and content of ADO.It is worth mentioned that EA also has dose-effect relationship with the expression level of TRPV1 in the spinal cord of mice:with the increase of the number of EA stimuli,the expression level of TRPV1gradually decreases.The above results prove jointly that EA could reduce the VH by increasing ADO content in the spinal cord and activating the ADORA1-PLCB3 signaling pathway to rapidly consume PIP2 and Ca²⁺in neurons.And the cumulative effect of alleviate by multiple EA on VH was achieved through upregulating expression of ADORA1 and downregulating expression of TRPV1.