Peripheral Cannabinoid-1 Receptor Blockade Ameliorates Cystitis Severity

Abstract

Background: The endocannabinoid system (ECS) plays a key physiological role in bladder function and it has been suggested as a potential target for relieving lower urinary tract symptoms (LUTSs). Whereas most studies indicate that activating the ECS has some beneficial effects on the bladder, some studies imply the opposite. In this study, we investigated the therapeutic potential of peripheral cannabinoid-1 receptor (CB₁R) blockade in a mouse model for LUTSs. Materials and Methods: To this end, we used the cyclophosphamide (CYP; 300 mg/kg, intraperitoneal)-induced cystitis model of bladder dysfunction, in which 12-week-old, female C57BL/6 mice were treated with the peripherally restricted CB₁R antagonist, JD5037 (3 mg/kg), or vehicle for three consecutive days. Bladder dysfunction was assessed using the noninvasive voiding spot assay (VSA) as well as the bladder-to-body weight (BW) ratio and gene and protein expression levels; ECS tone was assessed at the end of the study. Results: Peripheral CB₁R blockade significantly ameliorated the severity of CYP-induced cystitis, manifested by reduced urination events measured in the VSA and an increased bladder-to-BW ratio. Moreover, JD5037 normalized CYP-mediated bladder ECS tone imbalance by affecting both the expression of CB₁R and the endocannabinoid levels. These effects were associated with the ability of JD5037 to reduce CYP-induced inflammatory response, manifested by a reduction in levels of the proinflammatory cytokine, tumor necrosis factor alpha (TNFα), in the bladder and serum. Conclusions: Collectively, our results highlight the therapeutic relevance of peripheral CB₁R blockade in ameliorating CYP-induced cystitis; they may further support the preclinical development and clinical use of peripherally restricted CB₁R antagonism for treatment of LUTSs.

Keywords: cannabinoid-1 receptor; cyclophosphamide; cystitis; endocannabinoid system; lower urinary tract symptoms.

FIG. 1.

CYP-induced cystitis exacerbates micturition events and bladder inflammation. CYP-induced cystitis—experimental design (see the Materials and Methods section; A). CYP-induced cystitis elevated micturition events (B, C), bladder-to-BW ratio (D), and bladder mRNA expression levels of inflammatory markers (E–I). The mRNA levels of fibrotic markers showing elevation in expression of Col3 (J), but not Col1 and Fn1 (K, L), following injection of CYP. Representative panoramic H&E staining of bladders from each group, original magnification×5, scale bar—500 μm (M). Data represent the mean±SEM. For (C, D), n=9 mice per group, and for (E–L), n=7 mice per group. **p<0.01, ***p<0.001, and ****p<0.0001. BW, body weight; CYP, cyclophosphamide; H&E, hematoxylin and eosin; mRNA, messenger RNA; SEM, standard error of mean; Veh, vehicle.

FIG. 2.

CYP-induced cystitis upregulates CB₁R and TRPV1 expression. No significant changes in the bladder mRNA expression levels of Cnr1 or Cnr2 in CYP-injected mice (A, B). Significant elevation in the bladder protein expression levels of CB₁R (C, D) and TRPV1 (E, F). Representative immunohistochemical staining of CB₁R in the bladder's urothelial cells (G) and detrusor muscle (H) and quantification of CB₁R-positive areas in these cells (I) for each group, magnification×40, scale bar—50 μm. Data represent the mean±SEM. For (A, B), n=7 mice per group, and for (D, F, I), n=5 mice per group. *p<0.05 and ***p<0.001. CB₁R, cannabinoid-1 receptor; TRPV1, transient receptor potential cation channel subfamily V member 1.

FIG. 3.

Peripherally restricted CB₁R antagonism attenuates CYP-induced cystitis severity and inflammation. A CYP-induced cystitis model with CB₁R antagonism—experimental design (see the Materials and Methods section; A). Treatment with the peripherally restricted CB₁R antagonist, JD5037 (3 mg/kg, IP), significantly reduced micturition events (B, C), the bladder-to-BW ratio (D), and submucosal edema and hemorrhage, as shown in the representative panoramic H&E staining of bladders from each group, (original magnification×5, scale bar—500 μm; and×40 scale bar—50 μm; E), as well as normalized the elevated bladder mRNA (F) and protein (G) levels of TNFα and its circulating levels (H). Data represent the mean±SEM. For (C), n=16 mice per group; for (D), n=27 mice per group; for (F), n=7 mice per group; for (G), n=7 mice for the Veh group and 17 mice for the CYP-treated groups; and for (H), n=18 mice for the Veh group and 22 mice for the CYP-treated groups. **p<0.01, ***p<0.001, and ****p<0.0001. CYP, cyclophosphamide; IP, intraperitoneal; JD, JD5037; TNFα, tumor necrosis factor alpha; Veh, vehicle.

FIG. 4.

Peripherally restricted CB₁R antagonism restores the ECS tone. JD5037 treatment restored bladder protein expression levels of CB₁R (A, B) in CYP-injected mice, but not the elevated protein levels of TRPV1 (A, C), as shown in the representative immunoblots. CYP did not induce any significant changes in the mRNA expression levels of Nape-pld (D); however, it did induce a significant reduction in Faah (E). Accordingly, the actual amount of AEA increased in the CYP-injected mice and was normalized by JD5037 (F). CYP induced a significant reduction in the bladder mRNA levels of Daglb (G) and significant upregulation of Magl (H). Accordingly, the actual amount of 2-AG was significantly reduced in the CYP-injected mice and normalized by JD5037 (I). Bladder protein concentrations (J). Data represent the mean±SEM. For (B–E and G, H), n=7 mice per group, and for (F, I, J), n=9 mice per group. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. 2-AG, 2-arachidonoylglycerol; AEA, anandamide; Daglb, diacylglycerol lipase b; ECS, endocannabinoid system; Faah, fatty acid amide hydrolase; Magl, monoacylglycerol lipase; Nape-pld, N-acyl phosphatidylethanolamine phospholipase D.