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Experimental Cannabinoid 2 Receptor Activation by Phyto-Derived and Synthetic Cannabinoid Ligands in LPS-Induced Interstitial Cystitis in Mice

Affiliations

Experimental Cannabinoid 2 Receptor Activation by Phyto-Derived and Synthetic Cannabinoid Ligands in LPS-Induced Interstitial Cystitis in Mice

Geraint Berger et al. Molecules.

Abstract

Interstitial cystitis (IC) is a chronic bladder disorder with unclear etiology. The endocannabinoid system has been identified as a key regulator of immune function, with experimental evidence for the involvement of cannabinoid receptors in bladder inflammation. This study used intravital microscopy (IVM) and behavioral testing in lipopolysaccharide-induced IC, to investigate the anti-inflammatory analgesic effects of a natural dietary sesquiterpenoid, beta-caryophyllene (BCP), which is present in cannabis among other plants, and has reported agonist actions at the cannabinoid 2 receptor (CB2R). BCP's anti-inflammatory actions were compared to the synthetic CB2R-selective cannabinoid, HU308, and to an FDA-approved clinical treatment (dimethyl sulfoxide: DMSO). IVM data revealed that intravesical instillation of BCP and/or HU308 significantly reduces the number of adhering leukocytes in submucosal bladder venules and improves bladder capillary perfusion. The effects of BCP were found to be comparable to that of the selective CB2R synthetic cannabinoid, HU308, and superior to intravesical DMSO treatment. Oral treatment with BCP was also able to reduce bladder inflammation and significantly reduced mechanical allodynia in experimental IC. Based on our findings, we believe that CB2R activation may represent a viable therapeutic target for IC, and that drugs that activate CB2R, such as the generally regarded as safe (GRAS) dietary sesquiterpenoid, BCP, may serve as an adjunct and/or alternative treatment option for alleviating symptoms of inflammation and pain in the management of IC.

Keywords: allodynia; cannabinoid receptors; endocannabinoid system; inflammation; interstitial cystitis; microcirculation; sesquiterpenoid.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Chemical structures of (a) Beta-Caryophyllene and (b) HU308.
Figure 2
Figure 2
Still frame images from leukocyte trafficking (a) and capillary perfusion (b) videos obtained from intravital microscopy of mouse bladder (magnification = 200×). Rhodamine-6G and FITC were administered i.v into the tail vein to allow fluorescent visualization of leukocyte-endothelial interactions and capillary blood flow, respectively. White arrows indicate leukocytes interacting with the endothelium of the venule.
Figure 3
Figure 3
Leukocyte adhesion in submucosal bladder venules of female CD-1 mice for the following experimental groups: control (CON; n = 9), lipopolysaccharide (LPS)-induced interstitial cystitis (IC) (LPS; n = 9), LPS-induced IC treated with HU308 (5 mg/kg, LPS + HU308; n = 4), LPS-induced IC treated with beta-caryophyllene (BCP) (100 mg/kg, LPS + BCP; n = 7). LPS, BCP, and HU308 were all administered via i.p. injection. Data presented as mean ± SD. * p < 0.05 vs. CON. # p < 0.05 vs. LPS.
Figure 4
Figure 4
Capillary perfusion quantified through functional capillary density (FCD) within the bladder microcirculation of female CD-1 mice for the following experimental groups: control (CON; n = 9), lipopolysaccharide (LPS)-induced IC (LPS; n = 9), LPS-induced IC treated with HU308 (5 mg/kg, LPS + HU308; n = 4), LPS-induced IC treated with BCP (100 mg/kg, LPS + BCP; n = 7). Saline, LPS, BCP, and HU308 were all administered via intraperitoneal injection. Data are presented as percentage relative to the control group. * p < 0.05 vs. CON. # p < 0.05 vs. LPS.
Figure 5
Figure 5
Leukocyte adhesion in submucosal bladder venules of female BALB/c mice for the following experimental groups: control (CON; n = 6), LPS-induced IC (LPS; n = 5), LPS-induced IC treated with 50% DMSO (LPS + 50% DMSO; n = 5), and LPS-induced IC treated with BCP (100 mg/kg, LPS + BCP; n = 6). Saline, LPS, DMSO, and BCP were all administered via intravesical instillation. Data presented as mean ± SD. * p < 0.05 vs. CON. # p < 0.05 vs. LPS.
Figure 6
Figure 6
Capillary perfusion quantified through FCD within the bladder microcirculation of female BALB/c mice for the following experimental groups: control (CON; n = 6), LPS-induced IC (LPS; n = 5), LPS-induced IC treated with 50% DMSO, (LPS + 50% DMSO, n = 5), LPS-induced IC treated with BCP (100 mg/kg, LPS + BCP; n = 6). Saline, LPS, DMSO, and BCP were all administered via intravesical instillation. Data are presented as mean ± SD. * p < 0.05 vs. CON. # p < 0.05 vs. LPS.
Figure 7
Figure 7
Leukocyte adhesion in submucosal bladder venules of female BALB/c mice for the following experimental groups: control (CON; n = 5), LPS-induced IC (LPS; n = 5), LPS-induced treated with oral BCP (100 mg/kg, LPS + BCP; n = 5), healthy animals administered oral BCP (100 mg/kg, BCP; n = 5). All groups were pretreated with olive oil gavage, with the treatment groups receiving BCP dissolved in olive oil. Saline and LPS were administered via intravesical instillation. Data presented as mean ± SD. * p < 0.05 vs. Control, # p < 0.05 vs. LPS.
Figure 8
Figure 8
Capillary perfusion quantified through FCD within the bladder microcirculation of female BALB/c mice for the following groups: control (CON; n = 5), LPS-induced IC (LPS; n = 5), IC treated with oral BCP (100 mg/kg, LPS + BCP; n = 5), healthy animals administered oral BCP (100 mg/kg, BCP; n = 5). All groups were pretreated with olive oil gavage, with the treatment groups receiving BCP dissolved in olive oil. Saline and LPS were administered via intravesical instillation. Data presented as mean ± SD. * p < 0.05 vs. LPS.
Figure 9
Figure 9
Behavioral score for each category of female BALB/c mice; (a) breathing rate, (b) eye opening, (c) motor activity, (d) posture, before cystitis induction (T0) and after treatment (T1) for the following experimental groups: healthy control animals (Control, n = 5), LPS-induced IC (LPS; n = 5), LPS-induced IC treated with oral BCP (100 mg/kg, LPS + BCP; n = 5), and healthy animals administered BCP (100 mg/kg, BCP; n = 5) All groups were pretreated with olive oil gavage, with the treatment groups receiving BCP dissolved in olive oil. Saline and LPS were administered via intravesical instillation. Data presented as the mean score ± SD for each parameter. Individual data points are also shown. * p < 0.05 vs. Control, # p < 0.05 vs. LPS.
Figure 10
Figure 10
Composite score of behavioral results of female BALB/c mice before cystitis induction (T0) and after treatment (T1) for the following experimental groups: control (CON; n = 5), LPS-induced IC (LPS; n = 5), LPS-induced IC treated with BCP (100 mg/kg, LPS + BCP; n = 5), and BCP administered to healthy animals (100 mg/kg, BCP; n = 5). All groups were pretreated with olive oil gavage, with the treatment groups receiving BCP dissolved in olive oil. Saline and LPS were administered via intravesical instillation. Data presented as mean composite score ± SD. Individual data points are also shown. * p < 0.05 vs. Control, # p < 0.05 vs. LPS.
Figure 11
Figure 11
Force applied (g) by an electronic von Frey aesthesiometer before a response withdrawal was observed in female BALB/c mice for the following experimental groups: control (CON; n = 5), untreated LPS-induced IC (LPS; n = 5), LPS-induced IC treated with BCP (100 mg/kg, LPS + BCP; n = 5) and BCP administered to healthy animals (100 mg/kg, BCP; n = 5). All groups were pretreated with olive oil gavage, with the treat groups receiving BCP dissolved in olive oil. Saline and/or LPS was administered via intravesical instillation. Data presented as mean total force ± SD before cystitis induction (T0) and after the treatment period (T1). Individual data points are also shown. * p < 0.05 T1 LPS + BCP vs. T1 LPS.

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