Hidrox® and Chronic Cystitis: Biochemical Evaluation of Inflammation, Oxidative Stress, and Pain

Interstitial cystitis/painful bladder syndrome (IC/PBS) is a chronic bladder condition characterized by frequent urination, inflammation, oxidative stress, and pain. The aim of the study was to evaluate the anti-inflammatory and antioxidant effects of an oral administration of Hidrox® (10 mg/kg) in the bladder and spinal cord in a rodent model of IC/BPS. The chronic animal model of cystitis was induced by repeated intraperitoneal injections of cyclophosphamide (CYP) for five consecutive days. Treatment with Hidrox® began on the third day of the CYP injection and continued until the 10th day. CYP administration caused macroscopic and histological bladder changes, inflammatory infiltrates, increased mast cell numbers, oxidative stress, decreased expression of the tight endothelial junction (e.g., zonula occludens-1 (ZO-1) and occludin), and bladder pain. Treatment with Hidrox® was able to improve CYP-induced inflammation and oxidative stress via the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) pathway. It was also able to reduce bladder pain which was aggravated by the activation of neuroinflammation in the central nervous system. In particular, Hidrox® reduced the brain-derived neurotrophic factor (BDNF), as well as the activation of astrocytes and microglia, consequently reducing mechanical allodynia. These results indicate that nutritional consumption of Hidrox® can be considered as a new therapeutic approach for human cystitis, increasing the conceivable potential of a significant improvement in the quality of life associated with a lowering of symptom intensity in patients with IC/BPS.


Introduction
Interstitial cystitis/painful bladder syndrome (IC/BPS) is a chronic inflammatory condition of the bladder characterized by bladder pain and frequent urination [1]. The symptom of chronic pain seriously affects the quality of life of patients. IC/BPS affects all age groups of both sexes, although a 14-fold higher prevalence in females was observed compared to males [2]. To date, the etiology and the pathophysiology of this disease still remains to be clarified. Several theories have been proposed, including urothelial defects of the bladder or dysfunction, activation of mast cells, and autoimmunity [3]. The dose of Hidrox ® was based on previous experiments [25,27,28].

Macroscopic Analysis of Bladder Damage
After completing the experiment, the animals were sacrificed by cervical dislocation. The bladders were weighed and analyzed macroscopically to observe the formation of bleeding and edema. The score was established as follows, according to the previous work of Gray et al.: 3-severe damage (fluid externally present in the bladder wall and evident bleeding), 2-moderate damage (fluid in the internal mucosa and little bleeding), 1-mild damage (little edema and no bleeding), and 0-no bladder effect [27].

Assessment of Mechanical Hypersensitivity in Chronic Cystitis Model
Mechanical hypersensitivity in mice was measured at different time points using an electronic von Frey hair esthesiometer (Ugo Basile, Comerio, Italy) consisting of a portable force transducer equipped with a plastic tip [28]. Briefly, mice were placed in plastic boxes under which there was a metal mesh floor, and they were allowed to acclimatize for 15 min before starting the test. Thereafter, the transducer tip was placed perpendicular to the pelvic region until a sharp retraction of the abdomen, immediate licking or scratching of the area of stimulation, and jumping were observed. The mechanical threshold (expressed in grams) corresponding to the pressure that stimulated a behavioral reaction (withdrawal of the abdomen) was recorded automatically by the electronic device. The stimulation was applied three times, and the mean value was calculated as the mechanical threshold for each mouse.

Histological Evaluation
For histopathological examination, the bladders were taken 7 days after the third injection of CYP. The tissues were fixed at room temperature (RT) in buffered formaldehyde solution (10% in PBS); histological sections (7 µm) were stained with hematoxylin and eosin (H&E) and evaluated using a Leica DM6 microscope (Leica Microsystems SpA, Milan, Italy) equipped with a motorized stage and associated with Leica LAS X Navigator software (Leica Microsystems SpA, Milan, Italy) [29]. The stained sections were scored by two investigators in a blind fashion, and the level of inflammation was evaluated on a scale of 0-5 as follows: 0 = no inflammation, 1 = mild inflammation, 2 = mild/moderate inflammation, 3 = moderate inflammation, 4 = moderate/severe inflammation, and 5 = severe inflammation. The degree of fibrosis was evaluated by Masson's trichrome method according to the manufacturer's protocol (Bio-Optica, Milan, Italy) [30,31]. The quantity of fibrosis was evaluated as the percentage fibrotic area (blue staining) and quantified using image analysis software (Image J 1.8.0). Mast cell analysis was performed by toluidine blue staining [32].

Immunohistochemical Analysis
Immunohistochemical localization of anti-occludin (1:100, Santa Cruz Biotechnology, sc-133256) or anti-ZO-1 (1:100, Santa Cruz Biotechnology, sc-33725) was performed in the bladder, while localization of anti-BDNF (1:100, Abcam, Cambridge, MA, USA, ab108319), anti-GFAP (1:200, Santa Cruz Biotechnology, sc-33673), or anti-Iba-1 (1:200, Santa Cruz Biotechnology, sc-32725) was investigated in the spinal cord. The sections were incubated O/N with primary antibodies, and then all sections were washed with PBS and treated as previously reported [36]. Stained sections were observed using a Leica DM6 microscope following a typical procedure. The histogram profile was related to the positive pixel intensity value obtained by the computer program [37]. Immunohistochemical analyses were performed by experienced people who were unfamiliar with the treatment.

Preparation of the Bladder Homogenate for the Evaluation of Oxidative Stress
Bladder tissue was collected and transferred to very 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 the homogenate was centrifuged at 7000 rpm for 20 min to obtain the supernatant, which was used for the estimation of oxidative stress parameters.

Estimation of Lipid Peroxidation
Malondialdehyde (MDA) levels in the bladder samples were determined as an indicator of lipid peroxidation as previously described [38,39]. Briefly, 200 µL of homogenate was mixed with 200 µL of 8.1% sodium dodecyl sulfate, 1.5 mL of 30% acetic acid, and 1.5 mL of 0.8% of thiobarbituric acid. The mixture was heated for 60 min at 95 • C and subsequently rapidly cooled on ice. After cooling, 1.0 mL of distilled water and 5.0 mL of an n-butanol/pyridine solution (15:1 v/v) were added to each tube, before immediately centrifuging at 5000 rpm for 20 min. The levels of MDA were determined using a microplate reader at 532 nm and expressed as µg/mg of protein.

Estimation of Superoxide Dismutase (SOD)
Bladder tissue determination of SOD activity was performed according to a previously described method [40]. Briefly, to 100 µL of tissue supernatant, 2.85 mL of phosphate buffer (0.1 M, pH 8.4) and 50 µL of pyrogallol (7.5 mM) were added. Enzyme levels were expressed as SOD activity in units per mg protein and were determined using a microplate reader at 420 nm.

Estimation of Glutathione
The levels of GSH and GSSG in the bladder tissue were measured using GSH and GSSG Assay Kits (Beyotime, China) based on the method described by Tietze [41], according to the kit manufacturer's instructions. GSH/GSSG ratio levels were expressed in µg/mg protein.

Estimation of Catalase (CAT)
The bladder tissue determination of CAT activity was performed according to a previously described method [42]. Briefly, 50 µL of tissue supernatant was withdrawn, and 1.0 mL of phosphate buffer (50 mM, pH 7) and 100 µBL of hydrogen peroxide (30 mM) were added to it. Enzyme levels were expressed as CAT activity in units per mg protein and were determined using a microplate reader at 240 nm.

Statistical Analysis
All values in the figures and text are expressed as the mean ± standard deviation (SD) of N observations. For the in vivo studies, N represents the number of animals studied. In experiments involving histology, the figures shown are representative of at least three experiments performed on different days on tissue sections collected from all animals in each group. The results were analyzed by one-way ANOVA followed by a Bonferroni post hoc test for multiple comparisons. A p-value < 0.05 was considered significant.

Effects of HD on Bladder Damage and Fibrosis after Repeated CYP Injections
Edema and bleeding in the bladders were observed in mice administered CYP ( Figure 1C,E), compared with the Sham and Sham + HD groups ( Figure 1A,B,E). The oral administration of HD reduced this bladder damage ( Figure 1D,E). In the Supplementary Materials, we show the data of the macroscopic analysis carried out on all the animals (Table S1). Furthermore, repeated injections of CYP increased bladder weight compared to control animals. HD reported a ratio of bladder weight to body weight closer to the Sham group ( Figure 1F). Histopathological examination of the bladder showed alterations after CYP injection. Severe signs of cystitis including inflammatory cell infiltrate and submucosal edema were evident 7 days after the third injection of CYP ( Figure 1I,I'; see histological score K). In control mice and in Sham + HD mice, an intact urothelium and normal muscularis were evident with no signs of edema ( Figure 1G,G' and H,H'; see histological score K). HD treatment significantly reduced histological damage ( Figure 1J,J'; see histological score K). Masson's trichrome stain revealed a noticeable increase in fibrosis in the inflamed bladders ( Figure 1N) compared to the Sham and Sham + HD groups ( Figure 1L,M). The degree of fibrosis (blue-colored fibrotic area) was reduced by HD treatment ( Figure 1O). tological score K). In control mice and in Sham + HD mice, an intact urothelium and no mal muscularis were evident with no signs of edema ( Figure 1G,G' and H,H'; see hist logical score K). HD treatment significantly reduced histological damage ( Figure 1J,J'; s histological score K). Masson's trichrome stain revealed a noticeable increase in fibrosis the inflamed bladders ( Figure 1N) compared to the Sham and Sham + HD groups (Figu 1L,M). The degree of fibrosis (blue-colored fibrotic area) was reduced by HD treatme ( Figure 1O).

Effects of HD on Oxidative Stress after Repeated CYP Injections
To understand the effect of HD on cellular stress response, we evaluated the actio of this compound on Nrf2 and HO-1. The results show a reduction in the expression Nrf2 and HO-1 in the bladders of mice treated with CYP, compared to control mice th instead showed basal levels of these proteins. Instead, the HD treatment induced an i crease in the expression of both Nrf2 and HO-1 ( Figure 2A,A' and 2B,B', respectively).

Effects of HD on Oxidative Stress after Repeated CYP Injections
To understand the effect of HD on cellular stress response, we evaluated the action of this compound on Nrf2 and HO-1. The results show a reduction in the expression of Nrf2 and HO-1 in the bladders of mice treated with CYP, compared to control mice that instead showed basal levels of these proteins. Instead, the HD treatment induced an increase in the expression of both Nrf2 and HO-1 (Figure 2A,A' and 2B,B', respectively).
A state of marked oxidative stress in the CYP group was also observed by measuring the levels of MDA as a marker of lipid peroxidation, as well as the levels of reduced glutathione/oxidized glutathione ratio (GSH/GSSG) and the activity of SOD and CAT as markers of antioxidant defense systems. In particular, a significant increase in MDA levels and a consistent reduction in GSH levels and in the activity of SOD and CAT in the bladders taken from mice treated with CYP were highlighted. Treatment with HD alleviated the effect both on MDA and GSH/GSSG, and on SOD and CAT activity ( Figure 3A-D). A state of marked oxidative stress in the CYP group was also observed by measuring the levels of MDA as a marker of lipid peroxidation, as well as the levels of reduced glutathione/oxidized glutathione ratio (GSH/GSSG) and the activity of SOD and CAT as markers of antioxidant defense systems. In particular, a significant increase in MDA levels and a consistent reduction in GSH levels and in the activity of SOD and CAT in the bladders taken from mice treated with CYP were highlighted. Treatment with HD alleviated the effect both on MDA and GSH/GSSG, and on SOD and CAT activity ( Figure 3A-D).  A state of marked oxidative stress in the CYP group was also observed by measuring the levels of MDA as a marker of lipid peroxidation, as well as the levels of reduced glutathione/oxidized glutathione ratio (GSH/GSSG) and the activity of SOD and CAT as markers of antioxidant defense systems. In particular, a significant increase in MDA levels and a consistent reduction in GSH levels and in the activity of SOD and CAT in the bladders taken from mice treated with CYP were highlighted. Treatment with HD alleviated the effect both on MDA and GSH/GSSG, and on SOD and CAT activity ( Figure 3A-D).

Effects of HD on Bladder Inflammation and on Tight Junction (TJ) after Repeated CYP Injections
To investigate whether HD was capable of acting on the inflammatory processes that are activated during chronic cystitis, we evaluated the release of inflammatory cytokines by Western blot analysis and the activation of mast cells (MCs) using toluidine blue staining. We observed a marked increase in the expression of cytokines IL-1β, IL-6, and TNF-α in the bladder tissue in the group treated with CYP compared to the Sham group. The HD-treated group showed decreased levels of IL-1β, IL-6, and TNF-α, demonstrating an inhibitory effect on the release of proinflammatory cytokines ( Figure 4A,A', 4B,B', and 4C,C', respectively). An increase in the number and activation of MCs was found in the bladders of mice treated with CYP ( Figure 4F,H), compared to control mice ( Figure 4E,H). HD significantly reduced the number of MCs ( Figure 4G,H).

Effects of HD on Bladder Inflammation and on Tight Junction (TJ) after Repeated CYP Injections
To investigate whether HD was capable of acting on the inflammatory processes that are activated during chronic cystitis, we evaluated the release of inflammatory cytokines by Western blot analysis and the activation of mast cells (MCs) using toluidine blue staining. We observed a marked increase in the expression of cytokines IL-1β, IL-6, and TNFα in the bladder tissue in the group treated with CYP compared to the Sham group. The HD-treated group showed decreased levels of IL-1β, IL-6, and TNF-α, demonstrating an inhibitory effect on the release of proinflammatory cytokines ( Figure 4A,A', 4B,B', and 4C,C', respectively). An increase in the number and activation of MCs was found in the bladders of mice treated with CYP ( Figure 4F,H), compared to control mice ( Figure 4E,H). HD significantly reduced the number of MCs ( Figure 4G,H).  There is a substantial body of evidence showing changes in the expression of TJassociated proteins in biopsies from patients with IC/BPS [43]. A decrease in the expression of ZO-1 and occludin was observed in the bladders of mice injected with CYP m, respectively), compared to the control groups ( Figure 5A,E and 5D,H, respectively). Treatment with HD significantly reduced this TJ alteration ( Figure 5C,G and 5D,H, respectively).
There is a substantial body of evidence showing changes in the expression of TJ-associated proteins in biopsies from patients with IC/BPS [43]. A decrease in the expression of ZO-1 and occludin was observed in the bladders of mice injected with CYP ( Figure 5B,F and 5D,H, respectively), compared to the control groups ( Figure 5A,E and 5D,H, respectively). Treatment with HD significantly reduced this TJ alteration ( Figure 5C,G and 5D,H, respectively).

Effects of HD on Neuroinflammation after Repeated CYP Injections
To demonstrate that CYP-induced alterations in the bladder also cause changes in the central nervous system, we evaluated the activation of MCs, the expression of BDNF, GFAP, and Iba-1, and the release of proinflammatory cytokines such as IL-1β and TNF-α in the L6-S1 area of the spinal cord. MC infiltration and degranulation were assessed by toluidine blue staining. In the spinal cord, a significant upregulation of the number of MCs, which play a key role in the inflammatory process, was observed in the mice treated with CYP ( Figure 6B,D), compared to the control group ( Figure 6A,D). Treatment with HD reduced the number of MCs in the L6-S1 area of the spinal cord ( Figure 6C,D). Immunohistochemical analysis of BDNF showed an upregulation of this protein in the group treated with CYP ( Figure 6F,H), compared to the basal levels of the Sham group ( Figure  6E,H). Treatment with HD significantly reduced BDNF levels in the spinal cord ( Figure  6G,H).

Effects of HD on Neuroinflammation after Repeated CYP Injections
To demonstrate that CYP-induced alterations in the bladder also cause changes in the central nervous system, we evaluated the activation of MCs, the expression of BDNF, GFAP, and Iba-1, and the release of proinflammatory cytokines such as IL-1β and TNF-α in the L6-S1 area of the spinal cord. MC infiltration and degranulation were assessed by toluidine blue staining. In the spinal cord, a significant upregulation of the number of MCs, which play a key role in the inflammatory process, was observed in the mice treated with CYP ( Figure 6B,D), compared to the control group ( Figure 6A,D). Treatment with HD reduced the number of MCs in the L6-S1 area of the spinal cord ( Figure 6C,D). Immunohistochemical analysis of BDNF showed an upregulation of this protein in the group treated with CYP ( Figure 6F,H), compared to the basal levels of the Sham group ( Figure 6E,H). Treatment with HD significantly reduced BDNF levels in the spinal cord ( Figure 6G,H).
In addition, significant positive staining of GFAP and Iba-1 was observed in the spinal cord of the CYP group mice ( Figure 7B,F and 7D,H, respectively), compared to the control mice ( Figure 7A,E and 7D,H, respectively). Treatment with HD decreased the activation of both astrocytes and microglia ( Figure 7C,G and 7D,H, respectively). Lastly, through Western blot analysis, we demonstrated that repeated CYP injections induced the release of IL-1β and TNF-α, whereas, in the Sham group, the levels of these inflammatory cytokines were low. Treatment with HD significantly prevented the release of IL-1β and TNF-α ( Figure 7I,J and 7I',J', respectively). In addition, significant positive staining of GFAP and Iba-1 was observed in the spinal cord of the CYP group mice ( Figure 7B,F and 7D,H, respectively), compared to the control mice ( Figure 7A,E and 7D,H, respectively). Treatment with HD decreased the activation of both astrocytes and microglia ( Figure 7C,G and 7D,H, respectively). Lastly, through Western blot analysis, we demonstrated that repeated CYP injections induced the release of IL-1β and TNF-α, whereas, in the Sham group, the levels of these inflammatory cytokines were low. Treatment with HD significantly prevented the release of IL-1β and TNF-α ( Figure 7I,J and 7I',J', respectively).

Effects of HD on Mechanical Allodynia in Chronic Cystitis
We investigated the effects of HD on pain associated with chronic cystitis using the von Frey test. Repeated injections of CYP gradually reduced the withdrawal threshold in response to abdominal stimulation, reaching its lowest value around day 7 and persisting until day 10. Treatment with HD resulted in a significant improvement in pain-related responses (Figure 8).

Effects of HD on Mechanical Allodynia in Chronic Cystitis
We investigated the effects of HD on pain associated with chronic cystitis using the von Frey test. Repeated injections of CYP gradually reduced the withdrawal threshold in response to abdominal stimulation, reaching its lowest value around day 7 and persisting until day 10. Treatment with HD resulted in a significant improvement in pain-related responses ( Figure 8A).

Discussion
Hydroxytyrosol is a polyphenyl that commonly induces hormetic dose responses in a wide range of biological models. Hydrox ® , which contains 40-50% hydroxytyrosol, has been shown in our recent study to have hormetic properties and to induce the activation of the phase II enzyme responsible for antioxidant responses through the activation of the Nrf2/HO-1 pathway [25,44]. This pathway represents one of the antioxidant systems most involved in the maintenance of the intracellular redox state and, therefore, is widely studied for the prevention of numerous diseases caused by oxidative stress [45,46]. It is welldocumented that CYP-induced cystitis induces an increase in oxidative stress and inflammation in the bladder [47,48]. Our study showed that treatment with HD through the overexpression of Nrf2 and HO-1 restored the levels of GSH, SOD, CAT, and reduced lipid myeloperoxidation. All these results indicate that HD is capable of reducing the oxidative stress induced by CYP treatment. Connected to oxidative stress is the activation of mast cells and the inflammatory response with the release of pro-inflammatory cytokines and chemokines in the bladder [49][50][51][52][53]. HD, in addition to having high antioxidant power, is able to act on inflammatory processes [25]. In the present study, we demonstrated the ability of HD to significantly reduce the number of activated MCs and the consequent release of IL-1β, IL-6 and TNF-α. The effect of HD on oxidative stress and bladder inflammation led to a significant reduction in histopathological damage and loss of tight junctions whose function is important as they prevent pathogens from entering the bladder which would lead to urinary disease [54]. Several reports have indicated that inflammation and in particular the increased number of MCs in the bladder increase afferent sensitivity and nociception, contributing to symptoms of urinary frequency, urgency and pelvic pain in patients with IC/BPS [55]. This is due to the interaction between the immune system and the neural system, known as the neuroimmune interface [56][57][58][59]. Our study demonstrated the presence of activated MCs in the L6-S1 region of the spinal cord of mice

Discussion
Hydroxytyrosol is a polyphenyl that commonly induces hormetic dose responses in a wide range of biological models. Hydrox ® , which contains 40-50% hydroxytyrosol, has been shown in our recent study to have hormetic properties and to induce the activation of the phase II enzyme responsible for antioxidant responses through the activation of the Nrf2/HO-1 pathway [25,44]. This pathway represents one of the antioxidant systems most involved in the maintenance of the intracellular redox state and, therefore, is widely studied for the prevention of numerous diseases caused by oxidative stress [45,46]. It is well-documented that CYP-induced cystitis induces an increase in oxidative stress and inflammation in the bladder [47,48]. Our study showed that treatment with HD through the overexpression of Nrf2 and HO-1 restored the levels of GSH, SOD, CAT, and reduced lipid myeloperoxidation. All these results indicate that HD is capable of reducing the oxidative stress induced by CYP treatment. Connected to oxidative stress is the activation of mast cells and the inflammatory response with the release of pro-inflammatory cytokines and chemokines in the bladder [49][50][51][52][53]. HD, in addition to having high antioxidant power, is able to act on inflammatory processes [25]. In the present study, we demonstrated the ability of HD to significantly reduce the number of activated MCs and the consequent release of IL-1β, IL-6 and TNF-α. The effect of HD on oxidative stress and bladder inflammation led to a significant reduction in histopathological damage and loss of tight junctions whose function is important as they prevent pathogens from entering the bladder which would lead to urinary disease [54]. Several reports have indicated that inflammation and in particular the increased number of MCs in the bladder increase afferent sensitivity and nociception, contributing to symptoms of urinary frequency, urgency and pelvic pain in patients with IC/BPS [55]. This is due to the interaction between the immune system and the neural system, known as the neuroimmune interface [56][57][58][59]. Our study demonstrated the presence of activated MCs in the L6-S1 region of the spinal cord of mice treated with CYP. Migration of MCs induced the release of proinflammatory cytokines such as IL-1β and TNF-α also in the spinal cord where they are responsible for pelvic pain [60][61][62]. Another factor that together with MCs seems to play a crucial role in increasing urinary frequency and inducing the neuroinflammation responsible for pelvic pain perceived in patients with IC/BPS is the neurotrophic factor BDNF [12,59,62]. Our results on molecular changes in the spinal cord demonstrated that HD is able to reduce the activation of MCs, as well as the levels of BDNF, GFAP, Iba-1, IL-1β, and TNF-α also in the spinal cord and consequently reduce CYP-induced mechanical allodynia.

Conclusions
HD, by modulating oxidative stress, reduces bladder inflammation by suppressing mast cell proliferation and inhibiting the release of proinflammatory mediators. This prevents mast cell migration into the spinal cord and central hypersensitivity, leading to neuroinflammation and increased mechanical allodynia. Therefore, Hidrox ® could be used as a dietary supplement to counter IC/BPS and improve the quality of life of patients.

Data Availability Statement:
The data presented in this study are available upon request of the corresponding author. The data is not publicly available due to the internal rules of the research group.