Anti-inflammatory effect of sea buckthorn in an HCl-induced cystitis rat model

Abstract

Purpose

Although the mechanism underlying interstitial cystitis/bladder pain syndrome (IC/BPS) remains unclear, oxidative stress is suggested to be implicated in IC/BPS development. Sea buckthorn (SB; Hippophae rhamnoides L.) contains several compounds with antioxidant properties. In addition, intravesical application of hydrochloric acid (HCl) in rats induces histological changes similar to those observed in humans with IC. Therefore, the aim of this study was to evaluate the anti-inflammatory effects of SB in an HCl-induced rat cystitis model.

Materials and Methods

Twenty 8-week-old female Sprague–Dawley rats were instilled with HCl in their bladders to create an IC/BPS model. The model rats were divided into three groups and orally administrated distilled water (control, n=4), concentrated SB (n=8), or pentosan polysulfate (PPS, n=8) daily. Pathologic inflammation grade (H&E staining), number of mast cells per square millimeter (toluidine blue staining), fibrotic changes (Masson’s trichrome staining), and apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling staining) of bladder tissue samples were compared among the groups.

Results

Compared to the control group, the SB and PPS groups showed reduced edema (5.25±0.96 vs. 2.25±0.46 vs. 2.50±0.54, p=0.004, p=0.005, respectively), number of mast cells (12.5±3.6 vs. 6.8±1.9 vs. 6.6±1.8, p=0.010, p=0.002, respectively), ratio of fibrotic submucosal tissue (63.9%±7.0% vs. 43.6%±9.9% vs. 40.5%±5.2%, p<0.001, p<0.001, respectively), and ratio of apoptotic nucleus (40.7%±11.7% vs. 7.7%±6.5% vs. 5.1%±4.9%, p<0.001, p<0.001, respectively).

Conclusions

SB exhibited anti-inflammatory effects comparable to those of PPS in the HCl-induced chemical cystitis model.

Graphical Abstract

INTRODUCTION

Interstitial cystitis/bladder pain syndrome (IC/BPS) is an intractable chronic urological condition characterized by complex symptoms, including urethral pain, severe oliguria, and urgency [1]. Although the etiology of IC/BPS is unclear, various factors have been implicated as possible causes, including inflammatory responses, mast cell activation, loss of the glycosaminoglycan layer of the bladder wall, urethral epithelial dysfunction, autoimmune mechanisms, infection, ischemia, and injury [2].

IC/BPS shows a poor response to treatment, even when various treatment methods are applied in combination. A meta-analysis has indicated that pentosan polysulfate (PPS)—the only U.S. Food and Drug Administration-approved oral drug for IC/BPS—exhibits only moderate effects [3]. Furthermore, intravesical drug injections, hydrodistension, bladder wall cauterization, and partial or total cystectomy are sometimes performed to relieve IC/BPS symptoms [4]. Many patients resort to complementary and alternative medicine after experiencing a lack of efficacy with conventional treatment [5].

Patients with IC/BPS reportedly exhibit reduced urinary bladder perfusion during the filling phase [6]. These dysfunctions originate from ischemia, hypoxia, and reperfusion-related oxidative damage and free radical generation [7]. Therefore, hypoxic damage to the bladder tissue is key to the IC/BPS pathogenesis [8]. Although high-pressure oxygen therapy and low-intensity shock wave therapy have been explored as treatments for hypoxia, the results have been inadequate. Therefore, the development of additional treatment options, especially natural therapies, is in high demand.

Hippophae rhamnoides L., commonly known as sea buckthorn (SB), is a thorny plant along with orange-colored berries. It has a long history of application in traditional medicine for treating cough, digestive diseases, and pain in several countries [9]. Its fruits contain highly concentrated anti-inflammatory and antioxidant substances [10], which could be effective in IC/BPS treatment. Rivas et al. [11] reported that intravesical application of hydrochloric acid (HCl) in rat results in detrusor mast cell accumulation and eosinophilia, mimicking changes observed in human IC. Notably, this reliable model has since been widely used in IC/BPS animal studies [12]. The aim of this study was to analyze and compare the anti-inflammatory effects of orally administered SB and PPS using an HCl-induced rat model of IC/BPS.

MATERIALS AND METHODS

1. Preparation of the SB

Fermented SB extract was provided by Vitamin Tree Labs Corp. The fruits harvested from the farm were transported, washed, and then shredded. The by-products, which were separated into skin, seeds, and pulp, were discarded, while the rest was subjected to high-pressure filtering twice with 200 mesh to remove large particles such as fiber. The filtered juice was sterilized at 110℃ for 40 seconds and then stored in a fermentation room for 3 months.

2. Animals

This was a pilot in vitro study. Twenty 8-week-old female Sprague–Dawley rats (Orient Bio) weighing 180–200 g were used for the experiment after 1 week of adaptation. The rats were maintained in sawdust-covered cages, with two to three rats per cage. Drinking water and dried feed were provided ad libitum, and the temperature and humidity of the rearing room were maintained at 21℃–24℃ and 50%–60%, respectively, under a 12-h/12-h light/dark cycle (on at 8 a.m. and off at 8 p.m.).

The procedures used and the care of animals were approved by the Animal/Preclinical Study Ethics Committee of Ewha Womans University Medical School (approval number: ESM 14-0272).

3. IC/BPS model creation

Gel-covered polyethylene catheters (24-gauge, 19 mm, Angiocath Plus; BD Biosciences) were inserted into the bladders of the rats under isoflurane anesthesia. The bladders were emptied by applying pressure to the abdomen. Subsequently, the bladders were washed with 0.2 mL phosphate-buffered saline (PBS) and then injected with 0.4 M HCl. HCl was removed after 90 seconds, and the bladder was washed with 0.2 mL PBS [13].

4. Oral administration

Four weeks after the induction of IC/BPS, the rats were divided into three groups. The positive control group received PPS at 25 mg/kg/d (n=8) [14], while the treatment group received 10 mL/kg SB (Vitamin Tree Labs Corp., n=8), and the negative control group received distilled water (DW, n=4), via oral gavage for 4 weeks.

5. Tissue collection and analysis

After the oral treatment, the rats were euthanized via CO₂ gas application. After skin disinfection with 70% ethanol, a midline incision was made, and the abdominal cavity was revealed. The bladders were identified, removed, and fixed in 10% formalin. Subsequently, the specimens were dehydrated with ethanol, rinsed with xylene, immersed in paraffin solution, embedded in paraffin blocks, and sliced to a thickness of 4 µm (VT 1000S; Leica Biosystems).

The tissue sections were stained with H&E to confirm the presence of interstitial edema in the lamina propria. The edema grade was scored as 0 (no edema), 1 (mild edema), 2 (edema of less than twice the thickness of the mucous membrane), or 3 (edema of more than twice the thickness of the mucous membrane) by a single, experienced urologist who was blinded to other test results [15]. Toluidine blue staining was performed to determine the number and density of mast cells in bladder wall tissue. The results were compared among the groups.

Masson’s trichrome (MT) staining was used to assess fibrosis and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was used to assess apoptosis (TUNEL Assay Kit-HRP-DAB, ab206386; Abcam).

Semi-quantitative image analysis was performed on selected areas of each section. To quantify fibrosis and apoptosis, MT and TUNEL staining-positive areas were determined using ImageJ version 1.54f (National Institutes of Health) [14].

6. Statistical analysis

Statistical analyses were performed using IBM SPSS Statistics version 29 (IBM Corp.). All data are expressed as the mean±standard deviation. Differences among the groups were analyzed and post-hoc analysis was performed using the Kruskal–Wallis test. Statistical significance was set at p<0.05.

RESULTS

H&E staining revealed severe inflammation in the DW group. Thicker lamina propria and more monocyte and lymphocyte infiltrations were observed in the DW group than in the other groups. Denudation of the mucosa and hemorrhage of the lamina propria were also observed in the DW group. The mean edema score of the PPS, SB, and DW groups was 2.50±0.54, 2.25±0.46, and 5.25±0.96, respectively. The PPS and SB groups had significantly lower edema scores than the DW group (p=0.005 and p=0.004, respectively) (Fig. 1).

Fig. 1. Edema score of HCl-induced cystitis rat bladder wall determined using H&E staining after oral administration of pentosan polysulfate (PPS), sea buckthorn (SB), and distilled water (DW). PPS and SB groups showed a significantly lower score than the DW group (p=0.005 and p=0.004, respectively).

Mast cell infiltration was more prominent in the lamina propria and muscle layers than in the bladder mucosa. The mean number of mast cells per mm² in the PPS, SB, and DW groups was 6.6±1.8 vs. 6.8±1.9 vs. 12.5±3.6, respectively. The PPS and SB groups had significantly fewer mast cells than the DW group (p=0.002 and p=0.010, respectively) (Fig. 2).

Fig. 2. Mast cell count per square millimeter of HCl-induced cystitis rat bladder wall determined using toluidine blue staining. The pentosan polysulfate (PPS) and sea buckthorn (SB) groups showed significantly less activation of mast cells than the distilled water (DW) group (p=0.002 and p=0.010, respectively).

Masson’s trichrome staining confirmed fibrosis in the bladder wall. The area between the mucosal and muscle layers was stained blue. The ratio of stained area among the groups was 40.5%±5.2% vs. 43.6%±9.9% vs. 63.9%±7.0%, respectively. The PPS and SB groups showed a significantly lower ratio of positively stained areas than the DW group (p<0.001 and p<0.001, respectively) (Fig. 3A–C, Fig. 4).

Fig. 3. HCl-induced cystitis rat bladder wall determined using Masson’s trichrome staining in the pentosan polysulfate (PPS) (A), sea buckthorn (SB) (B), distilled water (DW) (C) groups (×100, scale bars=500 µm). TUNEL staining in the PPS (D), SB (E), and DW (F) groups (×200, scale bars=500 µm). DW group showed thicker collagen tissue and apoptotic mucosa than the other two groups.

Fig. 4. Percent area of fibrotic tissue determined using Masson’s trichrome staining in the pentosan polysulfate (PPS), sea buckthorn (SB), and distilled water (DW) groups. PPS and SB groups showed a significantly smaller area than the DW group (p<0.001 and p<0.001, respectively).

The TUNEL assay revealed an increase in the number of apoptotic nuclei in the mucosal layer. The ratio of immunostaining of the PPS, SB, and DW groups was 5.1%±4.9%, 7.7%±6.5%, and 40.7%±11.7%, respectively. The PPS and SB groups showed a significantly lower ratio of TUNEL staining than the DW group (p<0.001 and p<0.001, respectively), whereas the PPS group showed a relatively lower ratio than the SB group (p=0.025) (Fig. 3D–F, Fig. 5).

Fig. 5. Percent area of apoptosis determined using TUNEL staining in the pentosan polysulfate (PPS), sea buckthorn (SB), and distilled water (DW) groups. PPS and SB groups showed a significantly reduced ratio than the DW group (p<0.001 and p<0.001, respectively). A few outliers (^*) were observed in the PPS and SB groups.

DISCUSSION

The treatment options of IC/BPS are inadequate as its etiology is unclear [16]. Recently, bladder hypoxia and inflammation have been suggested to play important roles in the pathogenesis of IC/BPS. Patients with IC/BPS have elevated levels of oxidative stress biomarkers, such as 8-hydroxy-2-deoxyguanosine and 9-isoprostane, in the urine, with these levels correlating with severe glomerulation grade and low maximal bladder capacity [17]. SB is an oral supplement effective for recovery from oxidative damage. Our study demonstrated a significant decrease in inflammation, fibrosis, and apoptosis in the bladder after SB administration, as determined using histological analysis. Moreover, we compared the effects of SB with those of PPS and found no significant differences in histological findings, except for TUNEL staining.

To the best of our knowledge, this is the first study on the application of SB in treating IC/BPS. Various supplementary oral agents have been studied for the treatment of IC/BPS. For example, the administration of red ginseng to an IC/BPS rat model reportedly decreased inflammation and fibrosis in the bladder tissue, with reduced levels of inflammatory cytokines and oxidative stress-related factors (malondialdehyde and superoxide dismutase) compared to those in the control group [18]. Moreover, urinary frequency, nociceptive behavior, and urodynamics improved, while mast cell count and inflammation-related cytokine concentrations decreased following intravesical administration of Houttuynia cordata extract in a cyclophosphamide-induced IC/BPS rat model [19]. Since ancient times, SB has been used in herbal medicines in Asia to treat diarrhea and gastrointestinal and dermatological disorders. Teng et al. [20] reported that polysaccharides extracted from SB exhibit anti-fatigue activity, hypoglycemic activity, immunoregulatory activity, antitumor activity, liver protection, intestinal protection, microbiota regulation, antiviral, anti-obesity, and antitumor effects. In addition, SB polysaccharides possess antioxidant and anti-inflammatory effects that are important in the treatment of IC/BPS. Wei et al. [21] reported that polysaccharides extracted from SB exhibit an antioxidant effect by decreasing malondialdehyde and protein carbonyls while increasing superoxide dismutase and glutathione. Bao et al. [22] reported that penicillin-binding protein-3 and calcium phospholipid-binding protein of SB have free radical scavenging effects. Zhao et al. [23] demonstrated that SB polysaccharides protect cells from inflammation, apoptosis, and barrier dysfunction by inhibiting the TLR4/NF-κB signaling pathway in vitro. Lan et al. [24] reported that oral administration of SB polysaccharide in high-fat diet mice improved neuroinflammation and synaptic dysfunction by regulating gut dysbiosis.

In the present study, although we did not examine cystometry or cytokine profiles, we analyzed fibrosis and apoptosis through additional staining. A systemic review of randomized controlled trials [3] demonstrated the effectiveness of oral PPS, the only oral medication that can restore the damaged glycosaminoglycan layer of the bladder wall, compared to the placebo. Similarly, a systemic review of patients with Hunner’s lesions or glomerulations, determined using cystoscopy, described PPS as an effective treatment for patients with IC/BPS [25]. In this study, oral PPS administration significantly reduced HCl-induced cystitis, and SB showed a similar effect. However, the anti-inflammatory effect of SB cannot be considered superior to PPS, because the two treatment groups did not show a significant difference; therefore, SB cannot be used as a replacement for PPS. However, Nickel et al. [26] reported that 13.3% of patients discontinued PPS use because of adverse effects. Although no comparison of the side effects of PPS and SB has been made, to the best of our knowledge, the SB fruit has no known side effects other than liver dysfunction and skin pigmentation due to overdose [27]. Therefore, SB may be considered as an alternative medication. Moreover, a human pilot study on the efficacy of combination therapy with PPS and other oral formulations [28] demonstrated that the oral administration of a formulation containing cranberry extract, chondroitin sulfate, and hyaluronic acid complex exhibits clinically positive effects against IC/BPS-related symptoms [28]. In particular, the improvement in bladder pain achieved with this formulation was superior to that with PPS treatment alone. These findings indicate the possibility of a synergistic effect when PPS is combined with various natural products, indicating that the combined therapy of PPS and SB could yield positive results.

In the present study, TUNEL staining revealed different apoptotic changes in bladder mucosa nuclei. We cannot rule out the possibility that PPS may have more effectively reduced apoptosis than SB; however, the possibility of test error should also be considered. TUNEL staining tends to be considerably affected by the experimental environment such as time and temperature during the staining process [29]. Immunoreactivity may decrease in some cases of immunostaining when samples are fixed in paraffin blocks and stored at room temperature, compared to when they are stored under cold conditions [30]. Additionally, the intensity of immunostaining significantly decreases with prolonged sample storage time, especially over 6 months. This reduction may account for the TUNEL staining results of the PPS and SB groups, which exhibited relatively dispersed staining. Nevertheless, the results of the two groups were significantly different from those of the DW group. Additionally, we consider that the effects of PPS and SB are not significantly different, as the edema score, mast cell count, and MT staining demonstrated consistent trends.

Our study had some limitations. First, the most effective concentration or volume of SB was not established. Furthermore, other parts (including leaves, fruits, seeds, and oils) or a mixture of SB products could be more effective in treating IC/BPS. Second, multidrug administration may reduce the therapeutic effects or exacerbate the adverse effects, although many physicians recommend lifestyle modifications and complementary medicine in addition to conventional medication. Although validating the effect of PPS and SB combination treatment is necessary, we did not include this combination group in this study. Nevertheless, to our knowledge, this is the first in vitro pilot study comparing natural products with conventional oral medication. Third, we did not compare the HCl-induced cystitis, the PPS, or the SBtreated groups with the normal control group. Nevertheless, we regarded that the drug was effective, as there was a significant difference between the cystitis group and the treatment groups.

CONCLUSIONS

The present study indicated that treatment with SB and PPS alleviated bladder inflammation, fibrosis, and apoptosis in an HCl-induced chemical cystitis model. The results of this study may help develop a supplementary treatment option for patients with IC/BPS. A clinical trial to compare the efficacy of SB with PPS will be conducted in the future.