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

Abstract

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.

Pharmacological Properties, Therapeutic Potential and Molecular Mechanisms of JWH133, a CB2 Receptor-Selective Agonist

“…Furthermore, Liu et al (2020a) showed that JWH133 diminished mechanical hyperalgesia, reduced urine spot numbers, and enhanced the micturition frequency mediated by cyclophosphamide-induced cystitis in mice. They also observed a reduction in bladder tissue inflammation and oxidative damage as indicated by reduced levels of proinflammatory mediators, including IL-1β, TNF-α, and IL-8, and enhanced activities of cellular GSH and SOD, while lowering MDA levels.…”

Endocannabinoids in Bladder Sensory Mechanisms in Health and Diseases

“…No changes in the expression of CBR2 were reported in the mouse bladder in cyclophosphamide-induced cystitis (Liu et al, 2020). These discrepancies may be due to species differences and/or the method of induction of cystitis.…”

“…Whether this is due to reduced inflammation or direct effects on bladder afferents, or both is not clear. A growing body of evidence indicates that CBR2 activation decreases infiltration of inflammatory cells and markers such as leukocytes, interleukins, and tissues necrosis factors in cystitis (Rice et al, 2002;Tambaro et al, 2014;Wang et al, 2014;Liu et al, 2020). This suggests that a reduction in inflammation itself by CBR2 activation may be one of the main causes for a reduction in inflammatory hyperalgesia.…”

“…However, in acrolein-induced cystitis in mice, there was no changes in CBR2 expression ( Wang et al, 2014 ). No changes in the expression of CBR2 were reported in the mouse bladder in cyclophosphamide-induced cystitis ( Liu et al, 2020 ). These discrepancies may be due to species differences and/or the method of induction of cystitis.…”

“…Animal models suggest that CBR2 may be a potential target to ameliorate pain and bladder inflammation in cystitis. Selective CBR2 agonists (JWH015, JWH133 and GP1a) reduce the inflammation, pain behavior, and urinary frequency associated with cystitis ( Tambaro et al, 2014 ; Wang et al, 2014 ; Liu et al, 2020 ). Whether this is due to reduced inflammation or direct effects on bladder afferents, or both is not clear.…”