The Role And Preliminary Mechanism Of GPR18 In Cyclophosphamide-induced Cystitis

Backgrounds and purposes:Interstitial Cystitis/Bladder Pain Syndrome(IC/BPS)is a bladder disease whose pathological mechanisms are not fully understood,and its main clinical manifestations are lower urinary tract symptoms(such as frequent urination,urgency and nocturia)and pain in the pelvic region(such as perineal and suprapubic pain).Although the pathological mechanisms of IC/BPS are still not fully understood,more and more studies suggest that the sensitization of bladder afferent nerve is involved in the pathogenesis of IC/BPS.Bladder afferent sensitization is manifested as a decrease in the activation threshold of the bladder afferent nervous system,an increase in nerve electrical activity,and an enhanced response to various external noxious and non-noxious stimuli.According to this theory,clinical and animal experiments have proved that intravesical injection of resinous toxin,botulinum toxin and lidocaine to block nerve electrical activity or desensitize nerves can relieve symptoms of overactive bladder and pain to a certain extent.This indicates that the therapy targeting afferent nerve sensitization will be an important direction for the treatment of IC/BPS.Studies suggest that the endocannabinoid system is involved in the regulation of bladder function and pain.The symptoms of overactive bladder and pain were significantly improved by the use of classical cannabinoid receptor agonists,and the related mechanism was related to the regulation of activity of bladder afferent nerve.The G protein-coupled receptor GPR18 is a non-classical cannabinoid receptor that is also involved in the regulation of bladder function and pain.However,the expression,function and mechanism of GPR18 in IC/BPS have not been studied.Therefore,this study aims to explore the expression,role and mechanism of GPR18 in IC/BPS,and provide a new theoretical basis for the clinical treatment of IC/BPS.Methods:Part 1: Expression and localization of GPR18 in cyclophosphamide-induced cystitisFemale Sprague-Dawley rats were randomly divided into the cyclophosphamide group(CYP)and control group(Con).The CYP group was injected intraperitoneally with 200mg/kg cyclophosphamide to construct an animal model of IC/BPS,and the Con group was intraperitoneally injected with the same dose of normal saline.The rat body weight and bladder weight were weighed.HE staining was used to observe the histological changes of the bladder.Plantar mechanical pain threshold and bladder function assays were used to analyze changes in pain and bladder function.RT-PCR and Western blotting were used to analyze the expression of GPR18 in the bladder.Immunofluorescence was used to determine the localization of GPR18 in bladder afferent nerve fibers and dorsal root ganglia(DRG).Part Ⅱ: Effects and mechanisms of GPR18 on pain and bladder functionPlantar mechanical pain threshold and bladder function assays were used to observe the effects of GPR18 agonist(Resolvin D2,Rv D2)and GPR18 blocker(O-1918)on pain and bladder function.RT-PCR and Western blotting were used to analyze the expression of TRPV1 in the bladder.Immunofluorescence was used to determine the co-localization of GPR18 and TRPV1 in the DRG.Calcium imaging experiments were used to analyze the effect of Rv D2 and O-1918 on calcium influx induced by capsaicin-activated TRPV1.Results:1.In the CYP group,bladder weight increased;HE staining showed the damage of bladder epithelial,submucosal edema,submucosal hemorrhage,and infiltration of inflammatory cell;decreased plantar mechanical pain threshold,shortened voiding interval,but no significant changes in maximum intravesical pressure;The m RNA and protein levels of GPR18 were decreased in bladder;the m RNA level of GPR18 was decreased in DRG.Immunofluorescence showed that GPR18 was expressed in neurons of all sizes,and the expression of GPR18 was also decreased in DRGs from CYP group.2.In the CYP group,by intrathecal injection of Rv D2,the plantar mechanical pain threshold was increased,the voiding interval was prolonged,and the maximum intravesical pressure was not affected;O-1918 could block the therapeutic effect of Rv D2.The m RNA and protein expression levels of TRPV1 were up-regulated in the bladder of the CYP group.Immunofluorescence results suggested that GPR18 and TRPV1 co-localized in the DRG.Calcium imaging results suggest that in CYP group,capsaicin-activated TRPV1 can induce stronger calcium influx;in both Con and CYP groups,Rv D2 can inhibit capsaicin-induced calcium influx,and this effect of Rv D2 can be inhibited by O-1918.Conclusions:1.The animal model of IC/BPS was successfully established by intraperitoneal injection of cyclophosphamide,and its histological and functional changes were basically consistent with those of IC/BPS.2.In the CYP group,the expression of GPR18 was down-regulated in the bladder and DRG.GPR18 is expressed in bladder afferent nerve fibers and DRG.3.Activation of GPR18 relieves pain and improves bladder function by inhibiting TRPV1.