Electroacupuncture Treatment Of Visceral Hypersensitivity And Its Accompanying Depression By Activating CB2 Receptor And Downregulating P2Y12 Receptor Expression

Objective:Inflammatory bowel disease(IBD)is a chronic and recurrent intestinal inflammatory disease that can cause abdominal pain and emotional reactions such as depression.At present,there is still no effective treatment for IBD.Medial prefrontal cortex(m PFC)plays an important role in pain and depressive.The type 2 purinergic receptor Y12(P2Y12R),a metabolic purinergic receptor specifically expressed on microglia,which is closely related to neuropathic pain.Pain causes depressive behavior by activating P2Y12 R,and then promote the high expression of interleukin-1β(IL-1β),a key mediator of depression-like behavior.In previous studies,electroacupuncture(EA)has a therapeutic effect on pain and depression,in which cannabinoid receptor 2(CB2R)plays a key role.In addition,activation of CB2 R can inhibit the expression of P2Y12 R.The purpose of this study is to investigate whether P2Y12 R in the m PFC are involved in visceral pain and associated depressive behaviors in IBD,and whether EA can alleviate visceral pain and associated depressive behaviors in IBD through CB2R-P2Y12R-IL-1β pathway,and clarify its neurobiological mechanism.The above research can provide effective theoretical basis for electroacupuncture analgesia and antidepressant.Methods:(1)IBD model of mice were induced by colonic injection of 2,4,6-trinitrobenzenesulfonic acid(TNBS)in C57BL/6 mice.The mechanical pain threshold of IBD mice were measured by von Frey filament,the visceral hypersensitivity of IBD mice was tested by abdominal withdraw reflex score,and the depression-like behaviors of mice were assessed by sucrose preference test,tail suspension test and forced swimming test.(2)P2Y12R antagonist MRS2395 was injected intraperitoneally to block the function of P2Y12R;P2Y12R short hairpin RNA(P2Y12R sh RNA)was injected into the bilateral m PFC of C57BL/6 mice to knock down the expression of P2Y12 R in the m PFC.(3)The acupuncture points selected for EA treatment were the bilateral "Dachangshu",which is also named bladder meridian 25 acupoint(BL25).The EA intensity was 1 m A,wave width was 0.3 ms,frequency was 2 Hz,lasted 30 min,once a day,and lasted 7 days.And the sham EA was only superficial subcutaneously,with no electricity.(4)CB2R-Konck out(KO)mice were used to establish the IBD model and then treated with EA.CB2R-KO mice were stereotaxically injected with lentivirus overexpressing CB2R(LV-Cnr2)into the bilateral m PFC to up-regulate the expression of CB2 R in the m PFC.(5)Western blotting was used to detect the expression of P2Y12 R,IL-1β and CB2 R in the m PFC of mice.RT-PCR was used to detect the expression of P2Y12 R m RNA in the m PFC of mice,and ELISA was used to detect the expression of IL-1β in the m PFC of mice.(6)Immunofluorescence staining was used to detect the expression of P2Y12 R in the m PFC.IBA-1,CD68,GFAP and Neu N were used to label microglia,activated microglia,astrocytes and neurons in the m PFC,respectively.Results:(1)IBD model of mice were successfully constructed,which showed typical clinical symptoms of IBD,as well as significant visceral pain and depression-like behavior.At the same time,the expression of P2Y12 R in the m PFC of IBD model mice was up-regulated.(2)Systemic administration of P2Y12 R antagonist MRS2395 significantly alleviated mechanical allodynia and visceral hypersensitivity in IBD model mice,but didn’t affect the accompanying depression,and the expression of P2Y12 R and IL-1β in the m PFC.P2Y12 R sh RNA downregulated the P2Y12 R expression in the m PFC,inhibited the activation of microglia in the m PFC,downregulated the expression of IL-1β in the m PFC,thereby alleviating mechanical allodynia,visceral hypersensitivity and its accompanying depression in IBD model mice.(3)EA significantly downregulated the P2Y12 R expression,inhibit the activation of microglia in the m PFC,and further downregulate the expression of IL-1β in the m PFC.Moreover,weight loss and diarrhea in IBD model mice were alleviated,and their mechanical allodynia,visceral hypersensitivity and its accompanying depression were relieved.(4)EA significantly upregulated the expression of CB2 R in the m PFC.After CB2 R gene knockout,the effect of EA in relieving visceral hypersensitivity and its accompanying depression in IBD model mice was reversed.Meanwhile,EA no longer inhibited the upregulation of the P2Y12 R and IL-1β expression in the m PFC.Upregulation of CB2 R in the m PFC of CB2R-KO mice restored the effect of EA in relieving visceral hypersensitivity and its accompanying depression in IBD model mice,as well as the downregulation of P2Y12 R and IL-1β expression in the m PFC.Conclusion:(1)P2Y12R in the m PFC plays an important role in visceral pain and associated depression-like behaviors in IBD mice,suggesting that P2Y12 R may be a new target for the EA treating of visceral pain and associated depression-like behaviors in IBD.(2)EA upregulated the expression of CB2 R in the m PFC,downregulated the expression of P2Y12 R in the m PFC,and reduced the expression of inflammatory factor IL-1β,which may be involved in the treatment of IBD visceral pain and its accompanying depression-like behaviors.