Protective effect of palmitoylethanolamide, a naturally-occurring molecule, in a rat model of cystitis

Federica Pessina , Raffaele Capasso , Francesca Borrelli , Teresa Aveta , Lorena Buono , Giuseppe Valacchi , Paolo Fiorenzani , Vincenzo Di Marzo , Pierangelo Orlando , Angelo A. Izzo

PII: S0022-5347(14)04930-1
DOI: 10.1016/j.juro.2014.11.083
Reference: JURO 12017

To appear in: The Journal of Urology Accepted Date: 12 November 2014

Please cite this article as: Pessina F, Capasso R, Borrelli F, Aveta T, Buono L, Valacchi G, Fiorenzani P, Di Marzo V, Orlando P, Izzo AA, Protective effect of palmitoylethanolamide, a naturally-occurring molecule, in a rat model of cystitis, The Journal of Urology® (2014), doi: 10.1016/j.juro.2014.11.083.

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Protective effect of palmitoylethanolamide, a naturally-occurring molecule, in a rat model of cystitis
Running title: Palmitoylethanolamide and cystitis

Federica Pessina1, Raffaele Capasso2,6, Francesca Borrelli2,6, Teresa Aveta3,6, Lorena Buono4,6 Giuseppe Valacchi5, Paolo Fiorenzani1, Vincenzo Di Marzo3,6 Pierangelo Orlando4,6, Angelo A. Izzo2,6*
1Department of Molecular and Developmental Medicine, University of Siena, Via A. Moro, 2, 53100 Siena, Italy;
2Department of Pharmacy, University of Naples Federico II, via D Montesano 49, 80131 Naples, Italy;
3Institute of Biomolecular Chemistry, National Research Council, Pozzuoli (NA), Italy; 4,6Institute of Protein Biochemistry, National Research Council, Naples, Italy, Italy and guest researcher at National Institute of Optics – CNR, via C. Flegrei 34 Pozzuoli, Italy;
5Department of Life Science and Biotechnologies, University of Ferrara, Via Borsari, 46, 44100 Ferrara, Italy;
6Endocannabinoid Research Group

*Corresponding Author: Department of Pharmacy, University of Naples Federico II, via D Montesano 49, 80131 Naples, Italy; email: [email protected], tel 081.678439

Keywords

bladder, bladder pain syndrome; cancer, cystitis, palmitoylethanolamide.

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ABSTRACT

Purpose: Palmitoylethanolamide is both an endogenous mediator released together with the endocannabinoid anandamide from membrane phospholipids, and a plant-derived compound with analgesic and antinflammatory properties. Here, we verified if the pathophysiology of
experimental cystitis involves changes in the levels of palmitoylethanolamide and of some of its targets [i.e. cannabinoid (CB1 and CB2) receptors, and PPARα] and if exogenous-administered palmitoylethanolamide can be proposed as a preventive measure for cystitis.
Material and Methods: Cystitis was induced by cyclophosphamide in female rats. Nociceptive responses, voiding episodes, gross damage, myeloperoxidase activity, bladder weight, bladder palmitoylethanolamide and endocannabinoid levels (measured by liquid chromatography-mass spectrometry), and expression of palmitoylethanolamide targets (measured by quantitative reverse transcription-PCR) were recorded.
Results: Cyclophosphamide induced pain behaviour, bladder inflammation and voiding dysfunction associated to increased bladder levels of palmitoylethanolamide, up-regulation of CB1 receptors mRNA expression, down-regulation of PPARα mRNA and no changes in CB2 receptor mRNA expression. Exogenously-administered ultramicronized palmitoylethanolamide attenuated pain behaviour, voids and bladder gross damage. The CB1 antagonist rimonabant and the PPARα antagonist GW6471 counteracted the beneficial effect of palmitoylethanolamide on gross damage. Additionally, GW6471 further decreased voiding episodes in palmitoylethanolamide-treated rats.
Conclusions: The present study provides strong evidence for a protective role of palmitoylethanolamide as well as for alteration of bladder palmitoylethanolamide levels (and of some of its targets) in cyclophosphamide-induced cystitis.

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INTRODUCTION

Haemorrhagic cystitis is a frequent (up to 30%) and severe complication of treatment with the chemotherapeutic agent cyclophosphamide (CYP) [1]. It is a difficult clinical problem to treat, complicated by the fragility of patients, who may have metastatic disease and are often elderly [1]. Mesna, hyperhydration and bladder irrigation have been the most frequently used measures to prevent CYP-related cystitis, but the evidence for their efficacy is far from to be compelling [1,2]. The lack of robust clinical data on clinical efficacy and the side effects associated to preventive and treatment strategies highlights the need for a better understanding of the pathophysiology of the disease and for the identification of novel, safe and effective therapeutic options.
Palmitoylethanolamide (PEA) is both a food ingredient (contained, for example, egg yolk, peanuts and soy seeds) [3,4] and an endogenous lipid, chemically related to the endocannabinoid anandamide [5]. Animal studies have shown that PEA exerts therapeutically-relevant analgesic, anti-inflammatory and neuroprotective effects and is an endogenous mediator produced during pathophysiological states to attenuate inflammation, neuronal damage and pain [6]. Peroxisome proliferator-activated receptor α (PPARα) has been identified as one of the main pharmacological targets of PEA action [7]. Additionally, cannabinoid (CB1 and CB2) receptors may be indirectly activated by PEA via the so-called “entourage effect”, i.e. the augmentation of the endocannabinoid levels and/or actions at cannabinoid receptors [8,9].
The role and the effects of PEA in bladder function have been poorly investigated to date. Some papers published more than ten years ago showed that exogenous-administered PEA attenuated viscero-visceral hyper-reflexia induced by intra-vesical instillation of nerve growth factor or turpentine [10,11,12]. In these models of bladder inflammation, the pharmacological action of PEA was attributed to activation of CB2-like receptors, since it was counteracted by the selective CB2 receptor antagonist SR144528 [10,11].

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The objective of the present study was to address the potential use of PEA to alleviate experimental cystitis. We specifically evaluated bladder-pain-related behaviours, frequency of voids and gross damage of the bladder. Furthermore, in order to give novel insights into the role of endogenous PEA in experimental cystitis, we measured i) the levels of endogenous PEA, ii) the mRNA expression of N-acylethanolamine-hydrolyzing acid amidase (NAAA) – a key specific enzyme in PEA catabolism, as well as some of the pharmacological targets of PEA action, i.e. cannabinoid CB1 and CB2 receptors and PPARα following CYP administration. MATERIALS AND METHODS
Animals

Female Wistar rats (weight range: 200-240 g; Harlan Italy, S. Pietro al Natisone UD, Italy) were used. Rats were housed in groups of 3-4 per cage at room temperature (20-23 °C) and 50-70% relative humidity with an alternating 12h light/dark cycle. Food and water were freely given. At the end of the procedures, rats were euthanized by CO2 inhalation followed by cervical dislocation. The study was conducted in compliance with the Italian D.L. no.116 of 27 January 1992 and associated guidelines in the European Communities Council Directive of 24 November 1986 (86/609/ECC). A total of 132 rats were used in the experiments described here.
Induction of experimental cystitis

Acute cystitis was induced by a single i.p. injection of CYP at a final dose of 200 mg/kg (volume 5ml/kg) as previously described [13]. Control animals received the correspondent volume of saline.
Nociceptive response and voids following CYP-induced cystitis

Rats were allowed to acclimate, individually, in a transparent metabolic cage (20x20x30) 2 h before cystitis induction. The animals were observed and videotaped. Experiments were always performed between 8.00 and 12.00 am to minimize the potential variations in the behavioral responses. Behavioral parameters were used as pain indices and scored (the observer of the

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behavioural testing was blinded to the specific treatments) as described below. The time-course of the observation was 30-240 min after CYP injection. The behavioural parameters scoring scales were adapted from those described earlier [13]. A maximum value of 10 was used for each of the 2 parameters observed, eye closure and abnormal posture (rounded back and stretched posture) and an extra 10 for animals showing also other parameters (as licking the abdomen;
brief pain crisis) yielding a maximum score of 30 and a minimal score of 0 if no parameters were affected (no pain). For ‘brief pain crisis’ we mean tail hyperextension, abdominal retractions, backward withdrawal movements. For abnormal posture a score of 0 indicated a normal posture, a score of 5 occasionally rounded back or stretching, score of 7 almost rounded back or stretched position, a score of 10 both rounded back and stretching. For eye closure, score of 0 was given for completely open eyes, score of 5 for half closed eyes, score of 10 for eyes completely closed.
The final pain score was obtained as a composite of the scores derived from 14 measurements (each one scoring maximum of 30) given at 15 min intervals starting from 30 min after CYP injection.
Voiding frequency was assessed, from 60 to 180 min after CYP injection, by observing directly the animals (experiments were performed scoring two rats in parallel, each one in its own metabolic cage). Moreover all the experiments were video recorded, thus to verify again the data obtained. The total volume of urine was measured by collecting the urine of each rat from its own metabolic cage.
Gross evaluation of urinary bladder damage

After the observations, rats were sacrificed and the urinary bladder weighted and examined macroscopically for edema formation and bleeding. According to Gray et al. 1986, the following score was used: 3, for severe damage (fluid externally present in the wall of the bladder and evident bleeding), 2 for moderate (fluid found in the internal mucosa, little bleeding), 1 for mild (little edema, no bleeding), 0 for no effects in the bladder [14].

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Pharmacological treatment

PEA was administered (i.p.) at various doses (5-20 mg/kg) 30 min before CYP injection. Fifteen min after PEA injection, 30 ml/kg of H20 was administered by gavaging to rats in order to increase urine production. This water hydration procedure increased the number of voiding, and reduced the variation of bladder capacity. In some experiments, the effect of PEA (10 mg/kg) was evaluated in rats pre-treated (i.p., 15 min before PEA) with the CB1 antagonist rimonabant (0.3 mg/kg), the CB2 receptor antagonist AM630 (3 mg/kg) or the PPARα antagonist GW6471 (1 mg/kg). The doses of these antagonists were selected on the basis of previous work [15,16,17]. The observer of behavioural testing, voiding and bladder inflammation, was blinded to the treatments.
Identification and quantification of palmitoylethanolamide and endocannabinoids

PEA and endocannabinoids (anandamide and 2-AG) were analyzed in the bladder by isotope dilution liquid chromatography–atmospheric pressure–chemical ionization mass spectrometry as previously described [18] (see supplementary materials for details).
Quantitative (real-time) RT-PCR analysis

The bladder from rats treated with CYP or saline were removed (210 min after CYP administration), collected in RNA later (Invitrogen, Carlsbad, CA, USA) and homogenized by a rotorstator homogenizer in 1.5 mL of Trizol® (Invitrogen). Total RNA was purified, quantified, characterized and retro-transcribed as previously described [19] (see supplementary materials for details).
Materials

Ultramicronized palmitoylethanolamide was kindly provided by Epitech Group (Saccolongo, Italy), rimonabant and SR144528 by SANOFI Recherche, Montpellier, France. Cyclophosphamide was purchased from Sigma Aldrich S.r.l. (Milan, Italy), GW6471 and AM630 were obtained from Tocris (Bristol, UK ). All chemicals and reagents employed in this

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study were of analytical grade. PEA was dissolved in ethanol (amount injected: 0.4µl/g) GW6471 in saline, rimonabant and AM630 were dissolved in DMSO (amount injected: 0.4µl/g). CYP was dissolved in saline. All drugs solutions were freshly prepared at the desired final concentration. Control animals received the equal volume of vehicle through the respective route of administration of the drug treated animals. The vehicle for PEA, rimonabant, AM630 and GW6471, by themselves, had no significant effects on the responses under study.
Statistics

Data are expressed as the mean ± SEM of n experiments. To determine statistical significance, Student’s t test was used for comparing a single treatment mean with a control mean, and a one way analysis of variance followed by the Tukey-Kramer multiple comparisons test or by the Bonferroni’s test was used for analysis of multiple treatment means. Values of P less than 0.05 were considered significant.
RESULTS

PEA reduced CYP-induced nociceptive behaviour

CYP (200 mg/kg, i.p.) induced marked modifications in the behaviour of freely moving conscious rats, as revealed by the assessment of closing of the eyes, brief crises and occurrence of specific postures as previously characterized. During the time-course considered, PEA (5-10
mg/kg, i.p.) reduced the pain score, the effect being significant – and reaching a plateau – starting from the 10 mg/kg dose (pain score: control 72±22.83, CYP 346±23.19, CYP + PEA 10 mg/kg 236.4±11.66**, n=6-8 for each experimental group). (Figure 1). Detailed analysis of the time- course of pain behaviour revealed that rats exhibited abnormal behaviour starting from 15 min after CYP injection and PEA, at the doses of 10 and 15 mg/kg, reduced the pain score throughout the time-course considered (Figure 2).
PEA reduced CYP-induced voiding episodes

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The number of voiding episodes was measured from 60 to 180 min after CYP injection. As shown in Figure 3, rats treated with CYP had a significant increase of urinary frequency in comparison with the control rats. More importantly, rats receiving PEA before CYP injection, showed a significant reduction in the number of voiding episodes, with a maximal inhibitory effect at the 15 mg/kg dose (number of voids: control 2.75±0.854, CYP 9.0±0.982, CYP + PEA 5.0±0.837, n=6-8 for each experimental group). (Figure 3). No significant effect was observed at the 20 mg/kg dose of PEA.
CYP increased bladder PEA levels

Compared to control tissues, endogenous levels of PEA significantly increased in the bladder after CYP administration (PEA levels, pmol/mg lipid extract: control 15.12±5.6, CYP 49.2±11.5, n=5 for each experimental group, Figure 4). No significant changes were observed in N- acylethanolamine acid amidase (NAAA) mRNA expression between control and CYP-treated bladder (normalised fold expression: control 1.0±0.17, CYP 0.6±0.009, n=3-4 for each experimental group).
The mRNA expression of CB1 receptors, and PPARα are altered in the bladder of CYP- treated rats
In these experiments, we measured the mRNA expression of some of the pharmacological targets of PEA. CYP challenge up-regulated CB1 mRNA expression (normalised fold expression: control 1.0±0.2, CYP 6.5±0.8, n=3-4 for each experimental group), down-regulated PPARα (normalised fold expression: control 3.89±0.34, CYP 1.0±0.14 n=3-4 for each experimental group) and left unchanged the mRNA expression of CB2 (Figure 5). Endocannabinoid levels are altered in the bladder of CYP-treated rats
CYP treatment significantly decreased 2-AG bladder levels (pmol/mg lipid extract: control 260.8±25.40, CYP 172.8±17.5, n=5 for each experimental group) but it did not significantly

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change anandamide levels, although a strong tendency toward an increase was observed (Figure 6).
The beneficial effect of PEA on experimental cystitis involved CB1 receptors and PPARα In this set of experiments, we evaluated the effect of PEA on nociceptive behaviour, voiding
episodes and bladder gross damage in the presence of rimonabant (a CB1 receptor antagonist, 0.3 mg/kg), AM630 (a CB2 receptor antagonist, 3 mg/kg) and GW6471 (a PPARα antagonist, 1 mg/kg). Results, depicted in Figure 7, show that i) rimonabant, AM630 and GW6471 did not significantly change the effect of PEA on nociceptive behaviour (Figure 7A); ii) the PPARα antagonist, GW6471 further decreased voiding episodes in PEA-treated rats (Figure 7B), while the cannabinoid antagonists rimonabant and AM630 did not significantly modify the effect of PEA on voids; and iii) both GW6471 and rimonabant significantly counteracted the beneficial effect of PEA on gross damage (severity index) (Figure 7C). Rimonabant, AM630 and
GW6471, at the doses used in the present investigation, did not significantly modify, when administered alone (that is, in absence of PEA), the CYP-induced effects. At a higher dose, i.e. 1 mg/kg, rimonabant, given alone, caused severe pain crises when CYP was injected. DISCUSSION
We have shown that intraperitoneal PEA attenuated the nociceptive behaviour, voiding episodes and bladder inflammation associated to CYP administration to female rats. Our results, together with the previously reported ability of PEA to reduce viscero-visceral hyper-reflexia induced by intra-vesical nerve growth factor or turpentine [11,12], lends further support to the beneficial effect of this naturally-occurring acylethanolamide on bladder function. Others have shown that PEA exerts protective effects in a number of experimental models of inflammation and/or pain such as the formalin test [20], the carrageenan-induced paw edema [21] and the chronic constriction injury of the sciatic nerve [22].

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Endogenous PEA levels have been shown to change in a number of tissues in response to a variety of inflammatory stimuli [23]. In the present study we have shown that its levels are greatly increased in response to CYP administration. Consistent with our data, it has been shown that PEA increased in the rat bladder with experimental inflammation induced by acrolein, a CYP metabolite [24]. By contrast, the expression of the mRNA encoding for NAAA, a specific enzyme involved in PEA catabolism, did not significantly change in the bladder of CYP-treated rats. Because we have here observed that exogenously administered PEA attenuates CYP- induced gross damage (see above), we hypothesise that that endogenous PEA might be produced as a defence mechanism of the bladder to counteract local inflammation.
A further step of our study was to investigate the possible mode of action of PEA. We focused on the possible involvement of PPARα and cannabinoid receptors. This because: i) PPARα mediates the anti-inflammatory effect of PEA [25]; ii) cannabinoid receptors have been involved in bladder pathophysiology [26,27], including cystitis [28,29,30]; iii) PEA may indirectly activate cannabinoid receptors via the so-called “entourage effect”, i.e. the augmentation of the endocannabinoid levels and/or actions at cannabinoid receptors [8,9]; iv) in previous studies, the effect of PEA on bladder hyper-reflexia was believed to be mediated by CB2-like receptors [10,11]. Our results show that: i) the CB1 antagonist rimonabant significantly counteracted the effect of PEA on gross damage. Because PEA does not directly activate CB1 receptors, it is likely that the involvement of such receptors derives from the so-called “entourage effect”, i.e. via enhancement of the action of anandamide on CB1 receptors. Interestingly, CYP administration caused increased bladder expression of CB1 mRNA expression and the alteration in endocannabinoid levels. ii) the CB2 receptor antagonist AM630 did not significantly change the effect of PEA on bladder gross damage; iii) the PPARα antagonist GW6471 had a bidirectional influence on PEA effect: indeed, the antagonist significantly counteracted PEA action on bladder gross damage while it decreased the number of voids in rats with experimental

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cystitis treated with PEA. While the former effect of the antagonist is in agreement with the general anti-inflammatory role of the nuclear receptor, and suggests that PPARα is involved in the protective action of PEA, the effect on the number of voids is difficult to interpret given the paucity of data on the role of PPARα in bladder function, and, accordingly, remains to be investigated. To this point, it is worthy of note that PPARα mRNA expression is down-regulated in experimental cystitis.
CONCLUSIONS

The findings described in the present study indicate that the naturally occurring molecule PEA is elevated in the bladder in response to an inflammatory insult and exerts beneficial effects in experimental cystitis when given exogenously. Although we did not provide data on bladder function or capacity, we anticipate that the protective effect of exogenous PEA, which involves cannabinoid CB1 and PPARα receptors, might certainly be of translational interest, for the treatment of symptoms related to cystitis, notably in patients under chemotherapy with CYP. Abbreviations: CB, cannabinoid; PPARα, peroxisomal proliferator activated receptor α; PEA, palmitoylethanolamide; 2-AG, 2-arachydonylglycerol; NAAA; N-acylethanolamine-hydrolyzing acid amidase.
Conflict of interest

Vincenzo Di Marzo is co-inventor of patents claiming the use of palmitoylethanolamide against inflammatory conditions, and receives research support from Epitech Italia S.r.l. which markets palmitoylethanolamide. The other authors declare that they have no conflict of interest. Acknowledgements
This work was supported by a grant from Programma Operativo Nazionale “Research and Development of bioregulators active on epigenetic mechanisms of inflammatory processes in chronic and degenerative diseases (BIAM-EPI)”, nr. 01_02512.

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ACCEPTED

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Figure 1. Effect of palmitoylethanolamide PEA (PEA 5-20 mg/kg, i.p.) on cyclophosphamide (CYP)-induced pain-related behaviors in rats. PEA was given i.p. 30 min before CYP injection. Data are reported as a composite of the scores derived from 14 measurements (each one with a maximal score of 30) given at 15 min intervals for 210 min, as described in Methods section Data are means±SEM (n=6-8 rats for each experimental group). °°°P< 0.001 vs control, **P< 0.01 vs CYP.

Figure 2 Effect of palmitoylethanolamide PEA (PEA 5-20 mg/kg, i.p.) on cyclophosphamide (CYP)-induced pain-related behaviors in rats. PEA was given i.p. 30 min before CYP injection. Pain behavior score is plottet against time. Data are means±SEM (n=6-8 rats for each experimental group).

Figure 3. Effects of palmitoylethanolamide (PEA 5-20 mg/kg) on the number of voids in cyclophosphamide (CYP)-treated rats. PEA was given (i.p.) 30 min before CYP. The number of voiding episodes was measured during 2h time (from 60 to 180 min after CYP injection). Data are the mean±SEM of voids (n=6-8 rats for each experimental group). ; °P< 0.05 vs control; *P< 0.05; **P< 0.01 vs CYP.

Figure 4. Palmitoylethanolamide (PEA) levels in the bladder of control rats and rats treated with cyclophosphamide (CYP). Tissues were analysed 210 min after vehicle or CYP administration. Data are mean± SEM of five rats. *P<0.05 vs control.

Figure 5. Relative mRNA expression of cannabinoid CB1 receptors (A), cannabinoid CB2 receptors (B) and PPAR-α (C) in control and cyclophosphamide (CYP)-treated rats. Tissues were analysed 210 min after vehicle or CYP administration. Results are mean±SEM of 3-4 rats

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for each experimental group. RT-PCR analysis was performed as described in methods, for statistical significance analysis Cq data were also analysed by the RESTR 2009 software (Pfaffl M.W., Nucleic Acid Research 2002, 30, E-36). *P<0.05 vs control

Figure 6. Anandamide (AEA, Figure 5A) and 2-arachydonoylglycerol (2-AG, Figure 5B) levels in bladder of rats treated or not with cyclophosphamide (CYP). Tissues were analysed 210 min after vehicle or CYP administration. Data are mean± SEM of five rats. *P<0.05 vs control.

Figure 7. Effect of palmitoylethanolamide (PEA, 10 mg/kg, ip) alone or in presence of the cannabinoid CB1 receptor antagonist rimonabant (SR1, 0.3 mg kg-1, i.p.), the CB2 receptor antagonist AM630 (3 mg/kg, i.p.), or PPARα antagonist GW6471 (1 mg/kg) on total pain score (Figure 6A), number of voids (Figure 6B) and gross damage (Figure 6C) in rats treated with cyclophosphamide (CYP).The antagonists were given 15 min before PEA administration. PEA was given 30 min before CYP. Data are means ± SEM (n=6-8 rats for each experimental group). °°P<0.01 and °°°P<0.001 vs control (CTRL); *P< 0.05 and **P< 0.01 vs CYP; ##P<0.01 and ###P<0.001 vs PEA.

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GW6471

Supplementary dates Materials and methods
1.Myeloperoxidase (MPO) activity in the urinary bladder

MPO activity, a measure of neutrophil recruitment to the rat bladder, was measured as previously reported [1]. The bladders were removed four h after cyclophosphamide injection and homogenized in an appropriate lysis buffer (0.5% hexadecyltrimethylammonium bromide in MOPS 10 mM) in ratio 50 mg tissue/1 ml MOPS. The samples were then centrifuged for 20 minutes at 15,000 g at 4 °C. An aliquot of the supernatant was then incubated with NaPP (sodium phosphate buffer pH 5.5) and tetra-methylbenzidine 16 mM. After 5 minutes, H2O2 (9.8 M) in NaPP was added and the reaction stopped adding acetic acid. The rate of exchange in absorbance was measured by a spectrophotometer at 650 nm. Different dilutions of human MPO enzyme of known concentration were used to obtain a standard curve. MPO activity was expressed as unit (U)/mg of tissue.
2.Identification and quantification of palmitoylethanolamide and endocannabinoids Bladder from rats receiving CYP or saline were removed (210 min after CYP administration), immediately immersed into liquid nitrogen, and stored at -80°C until extraction of PEA, and endocannabinoids [anandamide and 2-arachidonoylglycerol (2-AG)]. Tissues were extracted with chloroform/methanol (2:1, by volume) containing each 10 pmol of d8-anandamide, 50 pmol
of d4-PEA and d5-2-AG, synthesized as described previously (for the former two compounds) or provided by Cayman Chemicals (for d5-2-AG, AnnArbor, MI). The lipid extracts were purified by silica column chromatography and the fractions containing anandamide, PEA, and 2-AG were analyzed by isotope dilution liquid chromatography–atmospheric pressure–chemical ionization mass spectrometry. Results were expressed as picomoles per milligram of lipid extract.
3.Quantitative (real-time) RT-PCR analysis

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For all samples tested, the RNA integrity number (Bionalyzer 2100, Agilent) was greater than 8 relative to a 0–10 scale. Quantitative real-time PCR was performed by an iCycler-iQ5® in a 20µL reaction mixture as described using 20 ng of cDNA. Assays were performed in quadruplicate (maximum ∆Ct of replicate samples <0.5), and a standard curve from consecutive five-fold dilutions (100 to 0.16 ng) of a cDNA pool representative of all samples was included for PCR efficiency determination. Optimized primers for SYBR-green analysis and optimum annealing temperatures were designed by the Allele-Id software version 7.0 (Biosoft International, Palo Alto, CA, USA) and were synthesized (HPLC-purification grade) by MWG- Biotech. For each target, all mRNA sequences at http://www.ncbi.nlm.nih.gov/gene/, were aligned and common primers were designed. Relative expression calculation, correct for PCR efficiency and normalized with respect to reference genes β-actin and HPRT was performed by the iQ5 software. Results are expressed as fold expression, compared with control (=1) [2]. Results
1. PEA reduced bladder gross damage, weight and MPO

Experimental cystitis results in increase in bladder weight, bladder gross damage and MPO activity, which were all significantly reduced by PEA (10 mg/kg) (bladder weight: control 50.15±3.621, CYP 129.7±10.22°°°, CYP + PEA 80.37±10.09*, n=3-4; bladder gross damage: control 0.0±0.0, CYP 3.0±0.410°°°, CYP + PEA 1.25±0.250**, n=6; MPO activity: control 0.2941±0.1153, CYP 1.679±0.3546°°°, CYP + PEA* 0.6342±0.1594, n=3-4; °°°P<0.001 vs control, *P<0.05 and **P<0.01 vs CYP)
References

1.Martins JP, Silva RB, Coutinho-Silva R, et al. The role of P2X7 purinergic receptors in inflammatory and nociceptive changes accompanying cyclophosphamide-induced haemorrhagic cystitis in mice. Br J Pharmacol 2012;165:183-96.

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2.Romano B, Borrelli F, Fasolino I, et al. The cannabinoid TRPA1 agonist colitis. Br J Pharmacol 2013;169:213-29.
cannabichromene inhibits nitric oxide production in macrophages and ameliorates murine

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Abbreviations: CB, cannabinoid; PPARα, peroxisomal proliferator activated receptor α; PEA, palmitoylethanolamide; 2-AG, 2-arachydonylglycerol; NAAA; N-acylethanolamine-hydrolyzing acid amidase.
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