This study was performed on seven-week-old female Sprague–Dawley rats (200–230 g body weight) from Envigo RMS Srl (San Pietro al Natisone, Udine, Italy). Food and water were available ad libitum. The University of Messina Review Board for animal care (OPBA) approved the study. Animal care was in accordance with Italian regulations on protection of animals used for experimental and other scientific purposes (D.M.116192) as well as with EEC regulations (O.J. of E.C. L 358/1 12/18/1986), and in compliance with the requirements of the Italian Legislative Decree no. 26/2014 and subsequent guidelines issued by the Italian Ministry of Health on March 16, 2015. All animal experiments complied with the EU regulations (EU Directive 2010/63) and ARRIVE guidelines.

Given the nature of chronic FIC pathology that can afflict cats, we chose a validated chronic model of CYP-induced cystitis as shown above (16). Briefly, cyclophosphamide (CYP, 40 mg/kg) (Endoxan Baxter. Roma, Italy) was injected in the rat peritoneal cavity every 3 days (i.e., on days 0, 3 and 6) as shown in Figure 1. All rats were sacrificed by cervical dislocation at the end of the experimental protocol. A midline ventral abdominal incision was made to collect the bladder. Next, the Lumbar6-Sacral1 (L6-S1) area of the spinal cord was collected through a longitudinal incision along the midline of the back.

The animals were randomly allocated into the following groups, sample size was calculated by G-Power (n = 6 each):

All the oral treatments were administered by oral gavage. The study supplement is a 3:1 mixture of micronized PGA and HSP.

Nociceptive response was evaluated through an electronic von Frey system (Bioseb, Vitrolles, France), as previously described (16). Briefly, von Frey filaments of increasing forces were applied to previously shaved abdomen, close to the urinary bladder, before CYP or saline injection (basal, day 0), at day 7 and day 10 (Figure 1). The stimulation was applied three times at each timepoint, with the mean value being considered the mechanical threshold (expressed in grams), corresponding to the pressure that induced a behavioral response (i.e., retraction/licking of the lower abdomen, or jumping).

On day 10, under light anesthesia (3% sevoflurane in air) the bladder was non-invasively observed using an ultrasound imaging system (Esaote My Lab Vet 30 Gold, Genoa, Italy) equipped with an 18 MHz linear probe. The bladder thickness was measured on the acquired images with ImageJ software (National Institutes of Health, United States).

On day 10, rats were euthanized with anesthetic overdose (5% sevoflurane in air) and bladder and spinal cord (L6-S1) collected for further analysis. Wet weight of each bladder was recorded. The bladders were photographed, and examined macroscopically for bleeding and edema. Bladder damage was scored according to a 4-point scale, as previously reported (36): 0-normal bladder, 1-mild (little edema and no bleeding), 2-moderate (fluid limited to the internal mucosa and little bleeding), and 3-severe (fluid in the bladder wall and internal mucosa, with evident bleeding).

Bladder tissues were fixed for 24 h in 4% paraformaldehyde/0.1 M phosphate-buffered saline at room temperature, dehydrated through a graded series of ethanol and embedded in Paraplast (Bio-Optica Milan, Italy). Tissue sections (7 μm) were deparaffinized with xylene, stained with hematoxylin and eosin (H&E) and evaluated by light microscopy. The degree of bladder inflammation was scored on a 4-grade scale based on submucosa edema, epithelial thinning, petechial hemorrhage, cell infiltration, and desquamation of the uroepithelium, as reported by Zhang and collaborators (37). Briefly, Grade 1 = normal control, Grade 2 = simple edema, Grade 3 = edema combined with epithelial layer cleavage and thinning, with mucosal abrasion and polymorphonuclear leucocyte (PMN) infiltration (i.e., mild cystitis) and Grade 4 = complete cystitis, i.e., increased severity and spread of all the above signs plus petechial hemorrhage (37). Bladder sections (7 μm) were also stained by Masson’s Trichrome method for collagen detection according to manufacturer protocol (Bio-Optica, Milan, Italy), and observed under light microscopy. The degree of fibrosis was evaluated as % fibrotic area (blue staining) and quantified using image analysis ImageJ software. For mast cell count, the bladder and spinal cord sections were stained with toluidine blue (Bio-Optica, Milan, Italy) and mast cells were counted in 10 cross-sections at 40x magnification in the most infiltrated area. All histological evaluations were performed by two independent pathologists blinded to the grouping. All observations were performed on Leica DM6 microscope (Leica Microsystems SpA, Milan, Italy) associated with Leica LAS X Navigator software (Leica Microsystems SpA, Milan, Italy).

Myeloperoxidase (MPO) enzymatic activity, an index of neutrophilic granulocyte infiltration, was determined spectrophotometrically in the bladder tissues as previously described (26). MPO activity was defined as the quantity of enzyme degrading 1 μm of peroxide per minute at 37° C and was expressed in units per gram of wet tissue. Malondialdehyde (MDA) is a stable end product of lipid peroxidation and a marker of oxidative stress. MDA levels in the bladder samples were determined spectrophotometrically at 532 nm by Thiobarbituric Acid Assayas previously described (38), the levels of MDA were determined using a microplate reader at 532 nm and expressed as nmol/mg of tissue.

Immunohistochemical analyzes were performed as previously described (39). Briefly the sections were incubated with the following primary antibodies: anti-occludin (1:100, WH0004950M1), anti-claudin-1 (1:100, Novus Biologicals, NBP177036), anti-mast cell chymase (CC1) (1:50, SCB sc-59586), and anti-mast cell tryptase (1:100, SCB sc-33676). Then the sections were processed as previously described (40). Digital images were analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, United States) using the color deconvolution plug-in. When the Immunohistochemistry Profiler plugin is selected, it mechanically plots a histogram profile of the deconvoluted diaminobenzidine image, and a corresponding scoring log is exhibited. The histogram profile refers to the positive pixel intensity value obtained from a computer program.

Bladder tissue was collected and transferred to cold phosphate-buffered saline (pH 7.4). It was then cut into thin slices with a surgical scalpel, suspended in a cooled sucrose solution (0.25 M), and dried quickly on filter paper. Tissues were homogenized to release soluble proteins in a cooled tris hydrochloride buffer (10 mM, pH 7.4), and then centrifuged at 7000 rpm for 20 min as previously described (9).

After harvesting, the spinal cord tissues of the L6-S1 segment were separated and immediately stored at −80°C for further analysis. Samples were lysed, proteins were separated by SDS-PAGE electrophoresis and subsequently transferred onto polyvinylidene fluoride membranes at 300 mA, as previously described (41). The membranes were then incubated overnight at 4°C with the following primary antibodies: c-Fos (E-8, 1:1000; sc-166940; SantaCruz Biotechnology), ionized calcium-binding adapter molecule 1 (Iba-1, 1:1000 v\v; ab5076, Abcam), phosphor-p38 (Tyr180/182, 1:1000; #4511, Cell Signaling Technology), glial fibrillary acidic protein (GFAP, 1:1000; #3670, Cell Signaling Technology), β-actin (1:1000; sc-47778; SantaCruz Biotecnology). Then, the membranes were washed three times and incubated with the secondary antibodies conjugated to horseradish peroxidase (1 h at 20–25°C). Protein bands were visualized using the Chemiluminescent ECL assay (Bio-Rad, Hercules, CA, United States) and visualized with the Chemi Doc XRS (Bio-Rad, Hercules, CA, United States). Band density was quantified using ImageJ analysis software (National Institutes of Health, United States).

The expression levels of histamine and serotonin (5-hydroxytryptamine, 5-HT) in bladder tissue were measured with enzyme-linked immunosorbent assay (ELISA) kits (Elabscience Biotechnology Co., Ltd., Houston, Texas, 77,079, United States). The procedures were performed according to the manufacturer’s instructions, on bladder tissue homogenates obtained as described above. The results of histamine and 5-HT were expressed as pg./mL.

Total RNA was purified from bladder tissues using the RNeasy Kit according to the manufacturer’s protocol (Qiagen, Milan, Italy). RNA was then quantified using a Nanodrop Spectrometer. Reverse transcription of the extracted RNA was performed using RevertAid H Minus First Strand cDNA Synthesis Kit (ThermoFisher, Monza, Italy). cDNA samples were loaded in triplicate and were run according to the manufacturer’s settings on a BioRad CFX Connect RT-PCR machine using QuantiTect SYBR Green PCR Kits (Qiagen, Milan, Italy), and QuantiTech primers (Qiagen, Milan, Italy) according to specific manufacturer protocols. The 2^−ΔΔCq method was used for the calculation of the relative fold change, with gene expression being normalized to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

All values are shown as mean ± standard error of the mean (SEM) of N observations. Figures are representatives of three independent experiments. The time-course data (i.e., von Frey testing) were analyzed by two-way ANOVA, while one-way ANOVA was used for all the other comparisons, with Kruskal-Wallis test being used for non-parametric variables. Post hoc evaluations were performed using Bonferroni adjustment for multiple comparisons. A value of p of less than 0.05 was considered significant. All analysis were performed on GrapPad Prism 10 software (GraphPad Software.USA).

The repeated injection of CYP produced persistent visceral pain as shown by the significant reduction in mechanical threshold compared to saline-injected controls, assessed by von Frey test at day 7 and day 10 (Figure 2). Not only mesna but also PGA-HSP (higher dose) relieved CYP-induced visceral pain at both timepoints, as shown by the significant increase of mechanical threshold compared to the CYP group (Figure 2).

At ultrasonography, a significant although irregular thickening of the bladder wall secondary to cystitis was observed in the longitudinal images from CYP-injected rats (Figure 3B) compared to controls (Figure 3A). The alteration was significantly reversed by both mesna (Figure 3C) and PGA-HSP at the higher dose (Figure 3E), as confirmed by the analysis of sonographic thickness of the bladder wall (Figure 3F).

At the macroscopic examination, extensive edema and bleeding were observed in the urinary bladder of CYP-injected animals (Figures 4B,K), being consistent with severe cystitis (36, 42). Edema was observed both in the bladder wall and internal mucosa. The bladder-to-body weight ratio increased accordingly compared to control group (Figure 4L). At the higher dose, the study dietary intervention significantly reduced cystitis, approaching the efficacy of mesna, as shown both by the index of severity (Figure 4K) and bladder to body weight ratio (Figure 4L).

Histological characterization showed that CYP injection resulted in inflammation, characterized by vascular congestion, microhemorrhages, and extensive submucosa edema (Figure 5B) compared to controls (Figure 5A). Accordingly, the inflammatory severity score significantly increased in CYP-injected animals compared to the control group (Figure 5K). Although the net activity level was still very low (i.e., lower than 3 units per gram of wet tissue), a significant increase of MPO activity was also observed following CYP injection (Figure 5L). The level of MDA also increased significantly compared to the control group (Figure 5M). Both the inflammatory and oxidative changes were similarly counteracted by mesna (Figures 5C,K–M) or the higher dose of the study supplement (Figures 5D,K–M). At Masson’s trichrome staining, increased collagen density along the deeper layers of the lamina propria and interstitial fibrosis between smooth muscle bundles were evident in the bladder of CYP-injected rats (Figure 5G) compared to controls (Figure 5F). Mucosal sloughing and denuded urothelial mucosa with a thinner layer of epithelial cells were also observed. Both mesna (Figure 5H) and the study supplement (higher dose, Figure 5J) reverted collagen deposition and significantly reduced fibrosis as shown by the percentage of fibrotic area compared to CYP group (Figure 5N).

The bladder mRNA expression level of the inflammatory cytokines IL-1β, TNF-α, IL-6 and CCL2 was significantly increased in the CYP group compared to saline-injected controls (Figure 6). PGA-HSP at the higher dose was able to significantly downregulate the gene expression of the investigated cytokines (Figure 6), and similar results were observed in the mesna-treated group.

Metachromatic staining of bladder mucosal mast cells showed a significant three-fold increase in cell density in CYP-injected rats compared to controls (Figures 7A,B,P). Similarly, significantly increased numbers of chymase- (Figures 7F,G,Q) and tryptase-positive mast cells (Figures 7K,L,R) were found in the bladder of CYP-injected animals compared to controls. Not only mesna but also the higher-dose PGA-HSP significantly decreased mucosal mast cell density at toluidine blue (Figures 7C,E,P) and immunohistochemical chymase (Figures 7H,J,Q) and tryptase staining (Figures 7M,O,R). Interestingly, the lower-dose PGA-HSP was also significantly effective in reducing the CYP-induced increase of both chymase- (Figures 7I,Q) and tryptase-positive mast cells (Figures 7N,R).

Since bladder mast cells are the main and candidate sources of histamine and 5-HT respectively, (43, 44), the level of the two mediators was evaluated in bladder tissues, as an index of degranulation (Figure 8). CYP significantly increased the level of both biogenic amines, while treatment with PGA-HSP (high dose) significantly counteracted the increase, with the effect being similar to that of mesna (Figures 8A,B).

In order to investigate whether the bladder epithelial barrier was compromised following CYP injection, the expression of the transmembrane barrier proteins claudin-1 and occludin was studied. Compared to the control group (Figures 9A,F), CYP-injected rats had reduced bladder expression of claudin-1 (Figures 9B,K) and occludin (Figures 9G,L), which was significantly restored by mesna (Figures 9C,H,K,L) as well as PGA-HSP at higher dose (Figures 9E,J–L).

Since accumulating evidence suggest that neuroinflammation plays an important role in the pathogenesis of cystitis (45, 46), we also evaluated the effect of PGA-HSP on CYP-induced neuroinflammatory changes in the lumbosacral spinal cord. Western blot analysis showed a significant increase of Iba-1 and GFAP expression (indicative of microglia and astrocyte activation, respectively) in the CYP group compared to controls (Figure 10A). Moreover, a significant increase of c-fos and p38 protein expression (activation markers for spinal neurons and microglia, respectively) was also observed in response to CYP (Figure 10B). Finally, the spinal mRNA expression level of the inflammatory cytokines IL-1β, TNF-α, IL-6 and CCL2 was also significantly increased in the CYP group compared to saline-injected controls (Figure 10C). Treatment with PGA-HSP at the higher dose exerted a significant protective effect on CYP-induced neuroinflammatory changes, with the mixture being able to significantly counteract the increase in all spinal cell activation markers (Figures 10A,B), as well as cytokine expression levels (Figure 10C), at a similar extent as the reference drug mesna.

Mast cells play a key role in pain processes both in peripheral tissues and central nervous system, including the spinal cord (47). We thus evaluated spinal mast cell count from the different treatment groups. As shown in Figure 11, the number of mast cells in the spinal cord from CYP-injected group (Figure 11B) increased compared to control group (Figure 11A). The mast cell density significantly increased accordingly (Figure 11P). The increase was significantly reduced by PGA-HSP (higher dose) as well as mesna (Figure 11P). The number of chymase-and tryptase-positive spinal mast cells increased accordingly in response to CYP (Figures 11G,L). Moreover, PGA-HSP supplementation, either at the higher or lower dose, significantly blocked CYP-induced increase in the number of both chymase-and tryptase-positive cells, with similar results being observed with mesna treatment (Figures 11Q,R).