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. 2024 Jan 4:10:1327102.
doi: 10.3389/fvets.2023.1327102. eCollection 2023.

Uroprotective and pain-relieving effect of dietary supplementation with micronized palmitoyl-glucosamine and hesperidin in a chronic model of cyclophosphamide-induced cystitis

Enrico Gugliandolo  1 , Gianluca Antonio Franco  1 , Ylenia Marino  2 , Alessio Filippo Peritore  1 , Daniela Impellizzeri  2 , Marika Cordaro  3 , Rosalba Siracusa  2 , Roberta Fusco  2 , Ramona D'Amico  2 , Francesco Macrì  1 , Rosanna Di Paola  1 , Salvatore Cuzzocrea  2   4 , Rosalia Crupi  1
Affiliations

Uroprotective and pain-relieving effect of dietary supplementation with micronized palmitoyl-glucosamine and hesperidin in a chronic model of cyclophosphamide-induced cystitis

Enrico Gugliandolo et al. Front Vet Sci. .

Abstract

Introduction: Feline idiopathic cystitis is a common, chronic-relapsing disorder of the lower urinary tract. In addition to environmental modification/enrichment, long-term and safe treatment targeting specific pathophysiological changes may be of help. In this context, effective dietary interventions hold clinical promise. Palmitoyl-glucosamine (PGA) and hesperidin (HSP) are safe and authorized feed ingredients for animal nutrition under European regulations.

Methods: The current study aimed to investigate whether a 3:1 mixture of micronized PGA and HSP could represent a novel mechanism-oriented approach to chronic cystitis management. A newly validated rat model of cyclophosphamide (CYP)-induced chronic cystitis was used (40 mg/kg, three intraperitoneal injections every 3rd day). Animals were randomized to orally receive either vehicle or PGA-HSP at a low (72 + 24 mg/kg) or high (doubled) dose for 13 days, starting 3 days before the chronic CYP protocol, with mesna (2-mercaptoethane-sulfonate) being used as a reference drug.

Results: Higher PGA-HSP dose was effective at relieving chronic visceral pain, as measured by mechanical allodynia test (von Frey test). The severity of cystitis was also significantly improved, as shown by the reduced sonographic thickening of the bladder wall, as well as the decrease in edema, bleeding and bladder to body weight ratio compared to the vehicle treated group. A significant decrease of MPO activity, MDA level and fibrosis at Masson's trichrome staining was also observed in animals administered PGA-HSP in comparison to vehicle treated ones. The CYP-induced increase in bladder mRNA expression of pro-inflammatory cytokines was also significantly counteracted by the study mixture. Moreover, CYP-induced bladder mast cell accumulation and releasability were significantly decreased by PGA-HSP (even at the low dose), as determined by metachromatic staining, chymase and tryptase immunostaining as well as enzyme-linked immunosorbent assay for histamine and 5-hydoxytriptamine.

Discussion: PGA-HSP is able to block CYP-induced decrease of tight junction proteins, claudin-1 and occludin, thus preserving the urothelial bladder function. Finally, neuroinflammatory changes were investigated, showing that dietary supplementation with PGA-HSP prevented the activation of neurons and non-neuronal cells (i.e., microglia, astrocytes and mast cells) at the spinal level, and counteracted CYP-induced increase of spinal mRNA encoding for pro-inflammatory cytokines. Altogether, the present findings confirm the uroprotective and pain-relieving effect of PGA-HSP and pave the way to potential and relevant clinical applications of the study supplement in feline idiopathic cystitis.

Keywords: ALIAmides; cystitis; hesperidin; mast cells; neuroinflammation; palmitoyl-glucosamine; urothelium; visceral pain.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer CI declared a shared affiliation with the authors to the handling editor at the time of review.

Figures

Figure 1
Figure 1
Timeline of the experimental protocol.
Figure 2
Figure 2
Von Frey test performed on the vicinity of the urinary bladder on baseline (day 0) and days 7 and 10 after three i.p. doses of CYP (40 mg/kg) or saline. Data are expressed as mean ± SEM (n = 6), figures are representative of three independent experiments. CYP, Cyclophosphamide; HD, high dose; LD, low dose; PGA-HSP, 3:1 mixture of micronized palmitoyl-glucosamine and hesperidin. ***p < 0.001 versus CYP; °°°p < 0.001 versus control.
Figure 3
Figure 3
Ultrasound longitudinal view images of the urinary bladder in the different treatment groups, moderately distended with anechoic saline solution (A–E). Bladder wall thickness in the different treatment groups is reported in the histogram (F). Data are expressed as mean ± SEM (n = 6), figures are representative of three independent experiments. CYP, Cyclophosphamide; HD, high dose; LD, low dose; PGA-HSP, 3:1 mixture of micronized palmitoyl-glucosamine and hesperidin. **p < 0.01 versus CYP; ***p < 0.001 versus CYP; °°°p < 0.001 versus control.
Figure 4
Figure 4
Macroscopic appearance of the whole urinary bladder (A–E) and its internal surface (F–J) from the different treatment groups. (K) Severity of cystitis-associated bladder damage according to the following scale (36): 3-severe (fluid in the bladder wall and internal mucosa, with evident bleeding), 2-moderate (fluid limited to the internal mucosa and little bleeding), 1-mild (little edema and no bleeding) and 0-normal bladder. (L) Bladder weight to body weight ratio reflecting mucosa edema. Data are expressed as mean ± SEM (n = 6), figures are representative of three independent experiments. CYP, Cyclophosphamide; HD, high dose; LD, low dose; PGA-HSP, 3:1 mixture of micronized palmitoyl-glucosamine and hesperidin. * p < 0.05 versus CYP, **p < 0.01 versus CYP; ***p < 0.001 versus CYP; °°°p < 0.001 versus control.
Figure 5
Figure 5
Representative photomicrographs of H&E staining of bladder tissue from the different experimental groups (A–E) and the relative inflammatory severity score (K). MPO activity (L) and MDA levels (M) were quantified in bladder tissues from the different experimental groups. Masson’s trichrome staining images of bladders from the different experimental groups (F–J), showing extensive increase of collagen deposition and hence fibrosis in the CYP group compared with controls (blue staining). The percentage of fibrotic area as quantified using ImageJ software is shown in the histogram (N). Data are expressed as mean ± SEM (n = 6), figures are representative of three independent experiments. CYP, Cyclophosphamide; HD, high dose; LD, low dose; PGA-HSP, 3:1 mixture of micronized palmitoyl-glucosamine and hesperidin. **p < 0.01 versus CYP; ***p < 0.001 versus CYP; °°°p < 0.001 versus control.
Figure 6
Figure 6
Bladder gene expression of IL-1β, TNF-α, IL-6 and CCL2 presented as mRNA fold change to control (set to 1). All values are expressed as mean ± SEM (n = 6). CYP, Cyclophosphamide; HD, high dose; LD, low dose; PGA-HSP, 3:1 mixture of micronized palmitoyl-glucosamine and hesperidin. ***p < 0.001 versus CYP; °°°p < 0.001 versus control.
Figure 7
Figure 7
Bladder mucosal mast cells from the different treatment groups (yellow circles) in sections stained with toluidine blue (A–E) and immunostained for chymase (F–J) or tryptase (K–O). The mast cell density (i.e., the number of mast cells per mm2) was calculated in 10 cross-sections at 40x magnification and shown in the histogram (P). The number of chymase (Q) or tryptase positive pixels (R) was calculated with ImageJ software. Data are expressed as mean ± SEM (n = 6), figures are representative of three independent experiments. CYP, Cyclophosphamide; HD, high dose; LD, low dose; PGA-HSP, 3:1 mixture of micronized palmitoyl-glucosamine and hesperidin. **p < 0.01 versus CYP; ***p < 0.001 versus CYP; °°°p < 0.001 versus control.
Figure 8
Figure 8
Histamine (A) and 5-HT levels (B) as determined by ELISA in bladder tissues. Data are expressed as mean ± SEM (n = 6), figures are representative of three independent experiments. CYP, Cyclophosphamide; HD, high dose; LD, low dose; PGA-HSP, 3:1 mixture of micronized palmitoyl-glucosamine and hesperidin. ***p < 0.001 versus CYP; °°°p < 0.001 versus control.
Figure 9
Figure 9
Immunohistochemical staining for claudin-1 (A–E) and occludin (F–J) in bladder tissue from the different treatment groups. The histograms (K,L) represent the number of positive pixels calculated with ImageJ software. Data are expressed as mean ± SEM (n = 6), figures are representative of three independent experiments. CYP, Cyclophosphamide; HD, high dose; LD, low dose; PGA-HSP, 3:1 mixture of micronized palmitoyl-glucosamine and hesperidin. ***p < 0.001 versus CYP; °°°p < 0.001 versus control.
Figure 10
Figure 10
Western blot analysis of the activation markers Iba-1, GFAP (A), c-fos and p38 (B). Quantitative analysis of western blots was performed and levels were normalized relative to β-actin. Real time PCR assay for IL-1β, TNF-α, IL-6 and CCL2 (C). All results are presented as fold change to control (set to 1). All values are expressed as mean ± SEM (n = 6). CYP, Cyclophosphamide; HD, high dose; LD, low dose; PGA-HSP, 3:1 mixture of micronized palmitoyl-glucosamine and hesperidin. ***p < 0.001 versus CYP; °°°p < 0.001 versus control.
Figure 11
Figure 11
Mast cells in the spinal cord of the different treatment groups (yellow circles) in sections stained with toluidine blue (A–E) and immunostained for chymase (F–J) or tryptase (K–O). The mast cell density (i.e., the number of mast cells per mm2) was calculated in 10 cross-sections at 40x magnification and shown in the histogram (P). The number of chymase- (Q) or tryptase-positive pixels (R) was calculated with ImageJ software. Data are expressed as mean ± SEM (n = 6), figures are representative of three independent experiments. CYP, Cyclophosphamide; HD, high dose; LD, low dose; PGA-HSP, 3:1 mixture of micronized palmitoyl-glucosamine and hesperidin. **p < 0.05 versus CYP; ***p < 0.001 versus CYP; °°°p < 0.001 versus control.
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