Cannabinoid Receptor 2 Activation Decreases Severity Of Cyclophosphamide-induced Cystitis Via Regulating Autophagy

BackgroundThe role of the cannabinoid system in the regulation of inflammation and pain has attracted considerable interest in recent years,Cannabinoids function primarily by cannabinoid receptors 1 and 2(CB1 and CB2),which belong to G-protein coupled receptor family.Previous studies have shown that CB2 play an important role in early inflammatory events in both the central nervous system and peripheral tissues.CB2 has been shown to be involved in the regulation of immunity.CB2 is expressed in different types of leukocytes mediating cannabinoid anti-inflammatory effects and immunomodulation.Since CB2 was found in the bladder tissue of various species,including humans,rodents,and monkeys,there were several studies showing that CB2 play a functional role in bladder.Treatment with a selective CB2 agonist restored bladder function in rats with partial urethral obstruction and increased volume and micturition interval in normal rats.Activation of cannabinoid receptor 2 decreased severity of acrolein-induced cystitis.However,the specific role and downstream signaling pathway of CB2 in cystitis is still unknownInterstitial cystitis/Bladder pain syndrome(IC/BPS)is a chronic,pelvic pain condition,with no known etiology.IC/BPS is characterized by a series of unpleasant sensations,including pain,discomfort,and pressure throughout the pelvic region.Furthermore,patients experience an increased frequency of voiding and urinary urgency,without the presence of infection or other secondary causes.The diagnosis of this condition is based on the symptoms,followed by an exclusion of other pathologies,the risk factors for the development of IC/BPS have not yet been determined.Given the prevalence and healthcare costs associated with IC/BPS,the identification of treatment strategies to alleviate symptoms in IC/PBS patients has garnered considerable interest.Autophagy is a process by which cytoplasmic components,including organelles and macromolecular aggregates,are delivered to lysosomes for degradation.Autophagosomes(double-membrane vesicles)sequester cytoplasmic material such as organelles and macromolecular aggregates,and then,fuse with lysosomes to form autolysosomes for bulk degradation.More than 30 autophagy-related genes(ATG)have been identified,and the most investigated genes include microtubule-associated protein 1 light chain 3 beta(MAP1LC3B/LC3B),SQSTM1/p62,ATG5,and BECN1/Beclin1.MAP1LC3B/LC3B is a mammalian ortholog of Atg8 and plays an essential role in the formation of autophagosomes;thus,the expression level of LC3B-Ⅱ is a critical marker for monitoring the autophagy process in mammalian cells.Autophagy has always been recognized as a stress response to nutrient deprivation for the maintenance of cellular energy homeostasis.Recent studies demonstrated that autophagy play an important role in a variety of human inflammatory diseases and autoimmune diseases.However,whether the cystitis was associated with autophagy and whether the role of CB2 in the pathogenesis of cystitis was mediated by autophagy was still unknown.In the present study,we explored the protective role of CB2 activation in cyclophosphamide(CYP)-induced cystitis and investigated the mechanism underlying the function of the CB2 against CYP-induced bladder injury.MethodsFemale C57BL/6J mice(age range,10-13 weeks)were used in this study.The animals were housed in a temperature-controlled room with a 12:12-hour day-night cycle at～25℃ and had free access to food and water.At least six mice were randomly assigned to each group.Experimental design:(a)To verify the differential expression of CB2 in mice with CYP-induced cystitis.Mice were randomly divided into two groups(n=12 per group):control group for normal mice treated with saline,CYP group for cystitis mice treated with CYP.Twenty-four hours after CYP administration,six animals in each group were used for bladder weight,hematoxylin/eosin(H&E)staining or immunohistochemical staining.Another six mice were prepared and bladder tissues were used for immunoblotting and reverse transcription polymerase chain reaction(RT-PCR).(b)To explore the effects of CB2 on the mechanical sensitivity and voiding function of CYP-treated mice,mice were randomly divided into four groups(n=10 per group):control group for normal mice treated with saline,CYP group for cystitis mice treated with vehicle before the injection of the same dosage of CYP,CYP+AM-630 group for cystitis rats treated with AM-630 before the injection of CYP,and CYP+JWH-133 group for cystitis rats treated with JWH-133 before the injection of CYP.(c)To evaluate the effects of CB2 on the bladder inflammation,oxidative stress of CYP-treated mice.Mice were randomly divided into four groups(n=12 per group):control group,CYP group,CYP+AM-630 group,and CYP+JWH-133 group.Six animals in each group were used for measurement of oxidative products and antioxidant enzyme activity.Another six mice were prepared for RNA isolation and RT-PCR.(d)To evaluate the effects of CB2 on the bladder autophagy and AMPK-mTOR pathway of CYP-treated mice.Mice were randomly divided into five groups(n=12 per group):control group,CYP group,CYP+AM-630 group,CYP+JWH-133 group,and CYP+JWH-133+3-MA group.Six animals in each group were prepared for immunoblotting and six mice were used for transmission electron microscopy(TEM)measurement.(e)To verify whether activation of autophagy by CB2 underlies the anti-inflammatory and antioxidative stress activities.Mice were randomly divided into five groups(n=12 per group):control group,CYP group,CYP+AM-630 group,CYP+JWH-133 group,and CYP+JWH-133+3-MA group.Six animals in each group were used for measurement of oxidative products and antioxidant enzyme activity Another six mice were prepared for RNA isolation and RT-PCR.Results(a)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.(b)Mechanical sensitivity was significantly increased in CYP-treated mice and CB2 agonist JWH-133 attenuated this effect(P<0.05).The number of urine spots was significantly increased after CYP treatment and it was decreased in JWH-133 treated mice(P<0.05).(c)Activating CB2 with JWH-133 significantly alleviated bladder tissue inflammatory responses and oxidative stress induced by CYP.(d)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.(e)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.Conclusion 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.