Cannabinoid receptor 2 activation decreases severity of cyclophosphamide‐induced cystitis via regulating autophagy

PURPOSE: Cannabinoids have been shown to exert analgesic and anti-inflammatory effects, and the effects of cannabinoids are mediated primarily by cannabinoid receptors 1 and 2 (CB1 and CB2). The objective of this study was to determine efficacy and mechanism of CB2 activation on cyclophosphamide (CYP)-induced cystitis in vivo. METHODS: Cystitis was induced by intraperitoneal (IP) injection of CYP in female C57BL/6J mice. Mice were pretreated with CB2 agonist JWH-133 (1 mg/kg, intraperitoneally), CB2 antagonist AM-630 (1 mg/kg, intraperitoneally) or autophagy inhibitor 3-methyladenine (3-MA) (50 mM, intraperitoneally) before IP injection of CYP. Peripheral nociception and spontaneous voiding were investigated in these mice. Bladders were collected, weighed, and processed for real-time polymerase chain reaction, immunoblotting analysis, histological and immunohistochemical analysis. RESULTS: Twenty-four hours after IP injection of CYP, the bladder of CYP-treated mice showed histological evidence of inflammation. The expression of CB2 in bladder was significantly increased in CYP-treated mice. Mechanical sensitivity was significantly increased in CYP-treated mice and CB2 agonist JWH-133 attenuated this effect (P < .05). The number of urine spots was significantly increased after CYP treatment and it was decreased in JWH-133 treated mice (P < .05). Activating CB2 with JWH-133 significantly alleviated bladder tissue inflammatory responses and oxidative stress induced by CYP. Activation of CB2 by JWH-133 increased the expression of LC3-II/LC3-I ratio, and decreased the expression of SQSTM1/p62 in the bladder of cystitis mice, whereas AM-630 induced inverse effects. Further study indicated that JWH-133 could promote autophagy and blocking autophagy by 3-MA dismissed the effort of CB2 in alleviating bladder tissue inflammatory responses and oxidative stress injury. Furthermore, treatment with 3-MA decreased the expression of p-AMPK and induced the phosphorylation of mTOR in the presence of JWH-133 stimulation in cystitis model. CONCLUSIONS: Activation of CB2 decreased severity of CYP-induced cystitis and ameliorated bladder inflammation. CB2 activation is protective in cystitis through the activation of autophagy and AMPK-mTOR pathway may be involved in the initiation of autophagy.