Mechanism Of Adenosine Receptor Involved In EA Treatment Of Trinitro-benzene-sulfonic Acid Induced Inflammatory Bowel Disease

Inflammatory bowel disease is a kind of chronic recurrent gastrointestinal disorder including ulcerative colitis and Crohn’s disease.The main symptoms of inflammatory bowel disease include abdominal pain,diarrhea,chronic intestinal inflammation,visceral hyperalgesia and anxiety,seriously affecting the quality of life and work efficiency of patients.As one of the traditional medical treatment in China,EA has significant therapeutic effect on inflammatory bowel disease.EA can not only alleviate symptoms such as visceral pain,diarrhea and weight loss,but also significantly improve pain-related anxiety.However,the underlying mechanism of EA in the treatment of inflammatory bowel disease is still unclear,which seriously hinders the further application of EA in clinical practice.Adenosine is an endogenous purine nucleoside with multiple physiological functions.Recent reports have shown that adenosine can modulate a variety of inflammatory immune responses,as well as many neural and mental activities such as emotion and cognition.There are four adenosine receptors: A1 receptors(A1R),A2 a receptors(A2a R),A2 b receptors(A2b R)and A3 receptors(A3R).Adenosine receptors are distributed in the colon,basal ganglia,striatum,amygdala,hippocampus,prefrontal cortex and other brain regions related to emotion.Adenosine and its receptors have been reported to be involved in the pathogenesis of inflammatory bowel disease.Activation of A1 R,A2a R and A3 R can increase the visceral pain threshold,relieve colonic mucosal inflammation,protect the inflammatory gastrointestinal mucosa and decrease colonic peristalsis,while A2 b R activation can increase the activity of pro-inflammatory factors in the intestinal epithelium,thereby aggravating intestinal inflammation.In addition,activation of A1 R in central nervous system can enhance the effect of anti-anxiety drugs,while inhibition of A2 aR can lead to antianxiety.However,whether adenosine receptors participate in the effect of EA on visceral pain,inflammation and anxiety in inflammatory bowel disease remains unknown.It has been reported that the peripheral substance P(SP),acting as the pain transmitter and regulator,and the proinflammatory factor interleukin-1β(IL-1β)contribute to the visceral inflammatory pain in inflammatory bowel disease by amplifying pain signals and maintaining the inflammatory state of the inflammatory bowel.Another important fact,brain-derived neurotrophic factor(BDNF)have the effect of antianxiety by regulating the release of other neurotransmitters.Whether EA could regulate these neurotransmitters or inflammatory factors to alleviate the visceral inflammatory pain or anxiety of inflammatory bowel disease remains unknown.In this study,we used trinitro-benzene-sulfonic acid(TNBS)to induce a mouse model of inflammatory bowel disease.Firstly,we investigated whether EA could regulate the expression of A1 R,A2a R,A2 b R and A3 R in colon tissues to inhibit the expression of SP and IL-1β,which can alleviate the visceral pain and inflammatory response of colon tissues induced by TNBS.Then we investigated whether EA could regulate the expression of A1 R and A2 a R in the medial prefrontal cortex(m PFC)and ventral hippocampus(v HPC)to affect the expression of BDNF and IL-1β,thereby improving the mechanical allodynia,visceral hyperalgesia and anxiety in mice with inflammatory bowel disease.Finally,we investigated the regulation effect of EA on the c-Fos expression of brain regions for inflammatory bowel disease mice induced by TNBS.Then chemical genetic technology was used to excite or inhibit the vHPC-mPFC neural circuit to antagonize or simulate the anti-anxiety and analgesia effect of EA.PART Ⅰ Peripheral mechanism of adenosine receptors involved in TNBS-induced visceral inflammatory pain in mice with inflammatory bowel diseaseOBJECTIVE(1)TNBS-induced inflammatory bowel disease mice was used to investigate the specific anti-inflammatory and analgesic effect and the regulation on the expression of SP and IL-1β in colon tissues of EA.(2)Adenosine A1,A2 a,A2b and A3 R antagonists were used before EA to discuss the mechanism of peripheral adenosine receptor involved in the EA treatment by observing the effect of EA on the expression of peripheral adenosine receptors and anti-inflammatory and analgesic effect.METHODS:(1)TNBS-induced inflammatory bowel disease model was established by single anorectal administration of 50μl TNBS(5% w/v)mixed with 50μl anhydrous ethanol per mouse.(2)EA or sham EA treatment: EA or sham EA treatment was given one day after TNBS injection daily for 7 consecutive days.In the EA group,a filiform needle for about 3mm was directly pierced the bilateral dachangshu(acupoint BL25),and connected with two electrodes with the same output of han’s acupoint nerve stimulator,current of 1m A,wave width of 0.3ms,frequency of 2Hz,and 30 minutes.The sham EA group pricks subcutaneously,leaving the needle for 30 minutes without needle stimulation or current stimulation.(3)Adenosine receptor antagonist injection: Adenosine receptor antagonist was pretreated in TNBS+ EA + antagonist group 1 day after modeling and before 7 EA treatments.Antagonist was intraperitoneal injected half an hour before each EA and continuously injected for 7 days.The dose of A1 R antagonist DPCPX was 3mg/kg,A2 a R antagonist ZM241385 at 1 mg/kg,A2 b R antagonist PSB603 at 3mg/kg,and A3 R antagonist MRS3777 at 5 mg/kg.(4)Behavioral test: Mechanical pain threshold of mice in each group was detected by Von Frey filament.The abdominal withdraw reflex score(AWRs)was recorded by colorectal distension(CRD)experiment.Weight and diarrhea scores were recorded daily.Intestinal propulsive rate was measured before sampling,and colonic length was measured after sampling.(5)Expression levels of A1,A2 a,A2b and A3 Rs as well as SP and IL-1β in colon tissues were determined by Western blot.RESULTS:(1)Behavioral tests showed that TNBS-induced inflammatory bowel disease mice developed mechanical allodynia and visceral hyperalgesia,weight loss,diarrhea,and shortened colon length.EA significantly alleviated mechanical allodynia,visceral hyperalgesia,weight loss,diarrhea,and shortened colon length in TNBS-induced inflammatory bowel disease mice.The effect of sham EA was not obvious.(2)Protein expression levels of pain factor SP and pro-inflammatory factor IL-1 in colonic tissues were determined by Western blot.The results showed that TNBS-induced expression of SP and IL-1β in colon tissues of mice with inflammatory bowel disease was significantly increased.EA significantly down-regulated the expression of SP and IL-1β in colon tissues of mice.The effect of sham EA was not obvious.(3)The role of A1 R in the EA treatment of visceral inflammatory pain in TNBS-induced inflammatory bowel disease mice: the expression of adenosine A1 R in colon tissues of TNBS-induced inflammatory bowel disease mice was significantly decreased.The expression of A1 R was significantly up-regulated by EA,but no significant effect was found in sham EA group.A1 R antagonist DPCPX significantly diminished the effect of EA on relieving mechanical allodynia and down-regulating the expression of SP and IL-1β in colon tissues.(4)The role of A2 a R in the EA treatment of visceral inflammatory pain in TNBS-induced inflammatory bowel disease mice: the expression of adenosine A2 a R in colon tissues of TNBS-induced inflammatory bowel disease mice was significantly decreased.The expression of A2 a R in colon tissues of mice with inflammatory bowel disease was significantly up-regulated by EA,but no significant effect was observed in sham EA group.A2 a R antagonist ZM241385 significantly diminished the effect of EA on relieving mechanical allodynia and down-regulating the expression of SP and IL-1β in colon tissues.(5)The role of A3 R in the EA treatment of visceral inflammatory pain in TNBS-induced inflammatory bowel disease mice: the expression of adenosine A3 R in colon tissues of TNBS-induced inflammatory bowel disease mice was significantly decreased.EA significantly up-regulated the expression of A3 R in colon tissues of mice with inflammatory bowel disease.The expression of A3 R in colon tissues of mice with inflammatory bowel disease was slightly up-regulated by sham EA,but it was still lower than that of EA group.The A3 R antagonist MRS3777 significantly diminished the effect of EA on relieving mechanical allodynia and down-regulating the expression of SP and IL-1β in colon tissues.(6)The role of A2 b R in the EA treatment of visceral inflammatory pain in TNBS-induced inflammatory bowel disease mice: the expression of adenosine A2 b R in the colon of TNBS-induced inflammatory bowel disease mice increased significantly.EA significantly down-regulated the expression of A2 b R in the colon of mice with inflammatory bowel disease.The expression of A2 b R in colon tissues of mice with inflammatory bowel disease was slightly down-regulated by sham EA,but it was still higher than that of EA group.A2 b R antagonist PSB603 had no significant effect on the effect of EA on relieving mechanical allodynia,but further enhanced the effect of EA on down-regulating the expression of SP and IL-1β in colon tissues(P < 0.05).CONCLUSION:(1)EA significantly alleviated mechanical allodynia and inflammation in TNBS-induced inflammatory bowel disease mice,while sham EA had no significant effect.EA also significantly decreased the expression of pain factor SP and pro-inflammatory factor IL-1β in colon tissues.The research proved that EA had specific anti-inflammatory and analgesic effect.(2)EA up-regulated the expression of A1,A2 a and A3 Rs in the colon tissues of mice with inflammatory bowel disease induced by TNBS,and down-regulate the expression of A2 b Rs.The upregulation of A1,A2 a and A3 Rs may be involved in the anti-inflammatory and analgesic effects of EA on TNBS-induced inflammatory bowel disease in mice.The downregulation of A2 b R may be involved in the pathophysiological mechanism of inhibition of visceral inflammation by EA.These results suggested that EA improved visceral inflammatory pain in TNBS-induced inflammatory bowel disease mice by up-regulating peripheral A1,A2 a and A3 Rs,down-regulating A2 b Rs,and inhibiting the expression of inflammatory cytokines SP and IL-1β.Part Ⅱ The central mechanism of adenosine A1 and A2 a Rs involved in EA improvement on pain sensation and pain emotion in TNBS-induced inflammatory bowel disease miceOBJECTIVE:(1)Firstly,determine the effects of EA on mechanical allodynia,visceral hyperalgesia,anxiety behavior,BDNF and IL-1β expression levels of m PFC and v HPC in mice with inflammatory bowel disease,so as to confirm the central effects of EA on analgesic and anti-anxiety.(2)Secondly,investigate the effects of EA on A1 and A2 a R expression of m PFC and v HPC in TNBS-induced inflammatory bowel disease mice,and the A1 and A2 a receptors of m PFC and v HPC were interfered with by sh RNA to observe their effects on analgesia and anti-anxiety effect of EA,in order to explore whether the central adenosine receptor is involved in the mechanism of relieving pain sensation and pain emotion of mice with inflammatory bowel disease by EA.METHODS:(1)Pain sensation measurement: Von Frey Filament was used to detect the mechanical pain threshold of mice in each group to evaluate the hypersensitivity of mechanical pain sensation.CRD test record AWRs evaluation of visceral hyperalgesia.(2)Anxiety behavior measurement: open field test(OFT)and elevated plus maze(EPM).(3)Expression levels of A1,A2 a,BDNF and IL-1β in m PFC and v HPC were detected by Western blot.(4)Stereotactic injection of the brain : Three weeks before modeling,virus were injected to the control + A1 / A2 a R empty virus group,TNBS+ A1 / A2 a R empty virus group,TNBS+ EA +A1 / A2 a R empty virus,control + A1 / A2 a R sh RNA group,TNBS + A1 / A2 a R sh RNA group,TNBS+ EA + A1 / A2 a R sh RNA group.Dose: empty virus/sh RNA 500 nl per mouse(Shanghai Ji Kai Ji due to buy).Bilateral m PFC injection location:(AP: + 2.5 mm,ML: + /-0.4 mm,DV: 2.5mm).Or bilateral v HPC(AP: 3.5 mm,ML: + /-2.8 mm,DV: 3.8 mm).Injection time is about 10 minutes,the injection speed is 30 nl/min,retaining needle for about 10 minutes.(5)EA and sham EA treatment: the EA group was treated by direct acupuncture at both sides of dachangshu(acupoint BL25)for about 3mm,and connected to the two electrodes of the same output of han’s acupoint nerve stimulator,1m A,wave width of 0.3ms,frequency of 2Hz,30 minutes.The sham EA group pricks subcutaneously,leaving the needle for 30 minutes without needle stimulation or current stimulation.RESULTS:(1)TNBS-induced inflammatory bowel disease mice developed mechanical allodynia and visceral hyperalgesia,accompanied by anxious behavior.EA significantly alleviated mechanical allodynia,visceral allodynia and anxiety in TNBS-induced inflammatory bowel disease mice.But sham EA had no apparent effect.(2)Protein expression levels of anti-anxiety factor BDNF and pain factor IL-1β in m PFC were determined by Western blot.The results showed that the expression of BDNF in m PFC and IL-1β in TNBS-induced inflammatory bowel disease was significantly decreased.Therefore,EA significantly up-regulated the BDNF expression of m PFC and down-regulated the expression of IL-1β receptor.Sham EA had no obvious effect.(3)Protein expression of A1 R and A2 a R of m PFC were determined by Western blot.The results showed that the expression of A1 R of m PFC in TNBS-induced inflammatory bowel disease was significantly decreased,while the expression of A2 a R was significantly increased.Therefore,EA significantly up-regulated A1 R expression of m PFC and down-regulated A2 a R expression.Sham EA had no obvious effect.(4)Protein expression levels of A1 R and A2 a R of v HPC were determined by Western blot.The results showed that A1 R expression of v HPC was significantly decreased and A2 a R expression increased in TNBS-induced inflammatory bowel disease mice.Therefore,EA significantly up-regulated A1 R expression of v HPC and down-regulated A2 a R expression.Sham EA had no obvious effect.(5)Interference effects of A1 sh RNA and A2 a sh RNA were detected by Western blot.The results showed that the expression of A1 and A2 a Rs in m PFC injected with A1 sh RNA and A2 a sh RNA was significantly decreased.(6)The role of A1 Rs of m PFC in improving pain sensation,pain emotion and regulating the expression of BDNF and IL-1β in mice with inflammatory bowel disease treated by EA.Injecting A1 sh RNA into m PFC had no significant effect on the improvement of mechanical allodynia and visceral hyperalgesia by EA,but diminished the effect of EA on the improvement of anxiety,and inhibited the effect of EA on the up-regulation of BDNF expression and down-regulation of IL-1β expression.(7)The role of m PFC A2 a R in improving pain sensation,pain emotion and regulating the expression of BDNF and IL-1β in mice with inflammatory bowel disease treated by EA.Injecting A2 a sh RNA into m PFC had no significant effect on the improvement of mechanical allodynia and visceral hyperalgesia by EA,but enhanced the effect of EA on the improvement of anxiety,and the effect of EA on the up-regulation of BDNF expression and down-regulation of IL-1β expression.(8)The role of A1 R of v HPC in improving pain sensation and emotion and regulating the expression of BDNF and IL-1β in mice with inflammatory bowel disease treated by EA.Injecting A1 sh RNA into v HPC had no significant effect on the improvement of mechanical allodynia and visceral hyperalgesia by EA,but diminished the effect of EA on the improvement of anxiety,and inhibited the up-regulation of BDNF expression and down-regulation of IL-1β expression by EA.(9)The role of v HPC A2 a R in improving pain sensation,pain emotion and regulating the expression of BDNF and IL-1β in mice with inflammatory bowel disease treated by EA.Injecting A2 a sh RNA into v HPC had no significant effect on the improvement of mechanical allodynia and visceral hyperalgesia by EA,but enhanced the effect of EA on the improvement of anxiety,and the effect of EA on the up-regulation of BDNF expression and down-regulation of IL-1β expression.CONCLUSION:(1)EA significantly alleviated the symptoms of TNBS-induced mechanical allodynia,visceral hyperalgesia and anxiety in mice with inflammatory bowel disease,and regulated the expression of anti-anxiety factor BDNF and pain factor IL-1β in m PFC and v HPC.It indicated that EA improve pain sensation and pain emotion by regulating pain and anxiety related factors in the center.(2)EA up-regulated A1 R expression and down-regulated A2 a R expression of m PFC and v HPC in TNBS-induced mice with inflammatory bowel disease.Interference with A1 R expression of m PFC and v HPC weakened the anti-anxiety effect and regulation of BDNF and IL-1β of EA,while interference with A2 a R expression of m PFC and vHPC enhanced the above effects of EA.However,interference with A1 R and A2 a R of m PFC and v HPC had no significant effect on EA analgesia.These results suggested that EA regulate the expression of pain-anxiety-related factors BDNF and IL-1β by up-regulating central A1 R and down-regulating central A2 a R,thereby improving the pain sensation and pain mood of TNBS-induced inflammatory bowel disease mice.PART Ⅲ Involvment of ventral hippocampal-medial prefrontal cortex projection in EA improvement on pain sensation and pain emotion in TNBS-induced inflammatory bowel disease mice OBJECTIVE:(1)In our study,we attempted to detect the expression of c-Fos in HPC,m PFC,insular cortex(IC),periaqueductal gray(PAG),supplementary motor cortex(M2),thalamic paraventricular nucleus(PVT)and anterior cingulated(ACC)in order to determine what brain regions of TNBS-induced inflammatory bowel disease mice will be activated and what effect of EA on these brain regions.(2)Then,we attempted to observe the changes of mechanical allodynia,visceral hyperalgesia and anxiety in TNBS-induced inflammatory bowel disease mice and the impact on mechanical allodynia and visceral hyperalgesia improvement effect of EA by activating or inhibiting the vHPC-mPFC projection pathway with chemical genetic technique.METHODS:(1)Pain sensation test: Mechanical pain threshold of mice in each group was detected by Von Frey filament.The abdominal withdraw reflex score(AWRs)was recorded by colorectal distension(CRD)experiment to determine visceral pain threshold.(2)Anxiety behavior measurement: open field test(OFT)and elevated plus maze(EPM).(3)Immunofluorescence technique was used to detect the c-Fos expression levels of HPC,m PFC,IC,PAG,M2,PVT and ACC in mice.(4)Virus construction for chemical genetic techniques: DIO virus r AAV-EF1α-dio-h M3Dq-m Cherry-WPRE-p A or r AAV-EF1α-dio-h M4Di-m Cherry-WPRE-p A was injected into v HPC.Retrograde Cre virus r AAV-h Syn-Cre-WPRE-p A(AAV2/R)was injected into m PFC basing on Cre-DIO strategy.HM3 Dq virus combined with Cre virus can activate vHPC-mPFC projection pathway,while HM4 Di virus combined with Cre virus can inhibit vHPC-mPFC projection pathway.(5)Stereotactic injection of the brain: Virus was injected three weeks before TNBS injection.Dose: 200 nl per mouse.Injection location: bilateral m PFC(AP: + 2.5mm,ML: ±0.4 mm,DV: 2.5 mm)or bilateral v HPC(AP: 3.5 mm,ML: ± 2.8 mm,DV: 3.8 mm).Injection time was about 7 min.The injection speed was 30 nl/min,retaining needle for about 10 min.(6)Chemogenetic manipulation: Clozapine N-oxide(CNO)was intraperitoneal injected 60 min before behavioral test or EA therapy to activate h M3 Dq or h M4 Di virus in mice(7)EA and sham EA treatment: the EA group was treated by direct acupuncture at both sides of dachangshu(acupoint BL25)for about 3mm,Which was connected to the two electrodes of the same output of han’s acupoint nerve stimulator,1m A,wave width of 0.3ms,frequency of 2Hz,lasting 30 minutes.The sham EA group pricked subcutaneously,and the needle was left for 30 minutes without stimulation of needle or current.RESULTS:(1)IC,PAG,ACC,M2,PVT,HPC and m PHC barin regions were activated in TNBS-induced inflammatory bowel disease mice.EA regulated the neuronal activity in IC,PAG,ACC,M2,PVT and HPC brain regions of TNBS-induced inflammatory bowel disease mice,but had no significant effect on the m PFC brain region.(2)Inhibition of vHPC-mPFC pathway significantly alleviated mechanical allodynia,visceral allodynia and anxiety in TNBS-induced inflammatory bowel disease mice.(3)Activation of vHPC-mPFC pathway significantly antagonized the EA improvement effect of mechanical allodynia,visceral allodynia in TNBS-induced inflammatory bowel disease mice,but had no impact on anxiety.CONCLUSION:(1)The corresponding brain regions can be activated in TNBS-induced inflammatory bowel disease mice,some of which can be regulated by EA.(2)Activation or Inhibition of vHPC-mPFC projection pathway can regulate pain sensation and emotion in TNBS-induced inflammatory bowel disease mice.The EA analgesic process can also be regulated by vHPC-mPFC projection pathway in TNBS-induced inflammatory bowel disease mice.