The Role of Sodium Phenylbutyrate and Suramin in the Prevention of Ulcerative Colitis Induced by Dextran Sulfate Sodium

doi:10.21203/rs.3.rs-4211305/v1

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The Role of Sodium Phenylbutyrate and Suramin in the Prevention of Ulcerative Colitis Induced by Dextran Sulfate Sodium

https://doi.org/10.21203/rs.3.rs-4211305/v1

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Ulcerative colitis is an inflammatory bowel disease characterized by inflammation and increased oxidative stress in the colon tissue. Sodium phenylbutyrate (PBA) and suramin are histone deacetylase inhibitors that alter gene expression keeping histones in the acetylated form. Our aim in this study was to determine the effects of PBA and suramin on colon tissue in an acute colitis model in mice and to clarify the mechanisms of their action. Ulcerative colitis was induced by 3% dextran sulfate sodium (DSS). After PBA and suramin injection, disease activity index (DAI) and colitis scoring were used to verify colitis damage. Alterations in proliferation, antioxidant enzyme activities and oxidative stress were shown by Ki-67 immunohistochemistry and spectrophotometry respectively. Active caspase-3 and COX-2 as well as cytokine levels and H3K9me3 histone modification were designated by Western blotting. The gene expression of STAT1, STAT3 and SIRT were analyzed by real-time PCR. DSS treatment increased DAI, MDA, MPO, TNF-α, IL-1β, IL-6, caspase-3 and COX-2 levels, and decreased Ki-67, IL-10, GSH levels and antioxidant enzyme activities in the colon tissue. Injection of PBA or suramin into DSS-treated animals significantly prevented colitis damage by improving the manifestations. Also, both of them induced significant changes in STAT and SIRT gene expressions and histone modifications compared to the DSS colitis group. Our study demonstrated that PBA and suramin have anti-apoptotic, anti-inflammatory, antioxidant and protective effects in DSS-induced ulcerative colitis. Considering these results, PBA and suramin can be potential agents to be used therapeutically in inflammatory bowel diseases.

Ulcerative colitis

dextran sulfate sodium

histone deacetylase inhibitors

sodium phenylbutyrate

suramin

mouse

Ulcerative colitis is an inflammatory bowel disease characterized by ulceration and inflammation of the colon. The disease can be triggered by genetic susceptibility and environmental factors such as stress, alcohol and smoking [1]. The pathogenesis of ulcerative colitis is not well understood. It has been suspected that increased production of pro-inflammatory cytokines and oxidative stress in the colon tissue as well as reduced levels of antioxidant enzymes are among the triggering factors playing a role in the development of the disease [2]. The drugs currently used to treat ulcerative colitis are not aimed at dispensing with the factors involved in causing colitis. Anti-inflammatory drugs suppressing the production of inflammatory mediators are often used to eliminate the symptoms of the disease. However, long-term use of these drugs also has some side effects that can lead to diabetes, hepatitis and liver toxicity. Therefore, new and effective treatments are needed to treat the disease [3]. For experimental purposes, various animal models are used to study the pathogenesis of the disease and prospect potential therapeutic approaches. The DSS-induced colitis model is often favored in experimental studies as it reflects many clinical and histopathological features similar to ulcerative colitis observed in humans [4].

Chemical compounds inhibiting histone deacetylase enzymes are called histone deacetylase inhibitors (HDACi). HDACis are molecules that play a role in many biological processes, such as altered gene expression, cell growth, proliferation, apoptosis, cell differentiation and inflammatory response. Due to these properties, they are considered potential therapeutics for many diseases. Sodium phenylbutyrate (PBA) is an HDACi that inhibits class I and IIA histone deacetylase (HDAC) enzymes. Studies in various experimental models show that PBA suppresses inflammation and oxidative damage by suppressing NF-kB activity and reducing the expression of pro-inflammatory cytokines and the production of reactive oxygen species (ROS) [5, 6]. Suramin is an HDACi that suppresses the deacetylase activity of sirtuins (SIRTs), which belong to class III of HDAC enzymes [7]. There are studies in the literature showing that suramin protects cells from inflammatory damage by inhibiting the binding of various proinflammatory cytokines to cell receptors or by reducing their expression [8, 9].

A review of the literature revealed that the mechanism of action of PBA in DSS-induced ulcerative colitis is not fully understood and the effects of suramin on this disease are unknown. The main objective of our study was to investigate the effects of PBA and suramin on colon morphology, cell proliferation and apoptosis, oxidative stress, histone modifications, antioxidant defense system, and inflammatory mediators in an acute DSS-induced colitis model in mice. By comparing the effects of PBA and suramin, which inhibit different classes of HDACs, on colitis, this study may provide insight into the utility of histone deacetylase inhibitors as novel therapeutic targets for the prevention and treatment of inflammatory bowel disease.

Animal model

This experimental study was approved by the Local Ethics Committee for Animal Experiments of Istanbul University, Aziz Sancar Research Institute of Experimental Medicine (ASDETAE) with the decision letter dated 25.02.2016 and number 07. In our study, 48 male C57BL/6 mice 8–10 weeks old and weighing 20–25 g were randomly selected and divided into 6 groups. Prior to euthanasia, the animals were fasted overnight. The animals were euthanized on day 8 of the experiment under anaesthesia with sodium pentobarbital. The colon tissue was removed for analysis. Dextran sulfate sodium (DSS) 3% (w/v) (MP Biomedicals, molecular weight: 36,000–50,000 Da), used to induce colitis model in mice, was freshly prepared each day with autoclaved sterile tap water. During the study, the mice were weighed at the same time of each day to determine weight changes, and the amount of water/DSS water they drank was recorded. The injections were administered to the mice at the same time of each day.

Experimental groups

Group I (control group) (n = 8): Mice were fed ad libitum for 7 days, given sterile tap water and were injected intraperitoneally (ip.) with phosphate buffer (PBS, pH 7.4).

Group II (control group given PBA) (n = 8): Mice were fed ad libitum for 7 days, given sterile tap water and were injected ip. with 150mg/kg dose of PBA dissolved in PBS once a day.

Group III (suramin treated control group) (n = 8): Mice were fed ad libitum for 7 days and given sterile tap water, injected ip. with suramin at a dose of 25mg/kg dissolved in PBS once daily.

Group IV (experimental group given DSS) (n = 8): Mice were given sterile tap water for the first 2 days of the experiment and then were given 3% DSS dissolved in sterile tap water for 5 days starting from the 3rd day and were injected ip. with PBS once a day for 7 days to ensure equal conditions with all experimental animals in the study.

Group V (experimental group given DSS and PBA) (n = 8): Mice were given sterile tap water for the first 2 days of the experiment, and then were given 3% DSS dissolved in sterile tap water for 5 days starting from the 3rd day and were injected ip. with 150mg/kg dose of PBA dissolved in PBS once a day.

Group VI (experimental group given DSS and suramin) (n = 8): Mice were given sterile tap water for the first 2 days of the experiment, and then were given 3% DSS dissolved in sterile tap water for 5 days starting from the 3rd day, and were injected ip. with suramin dissolved in PBS at a dose of 25mg/kg once a day.

All injections were performed at the same time every day, 0.1 ml/day. All animals were fasted the night before the experiment. Mice were anesthetized with i.p. injection of sodium pentobarbital (150 mg/kg dose) after 24 hours from the last treatment. Colon tissues were removed for analysis.

Determination of the percentage change in body weight and colon length in mice

The mice were weighed at the same time each day throughout the experiment and their weight was recorded. The percentage change in weight was calculated from the difference between the body weight of the mice on that day and the weight before the start of the experiment. On the last day of the experiment, the colon tissue of the anesthetized mice was completely removed through a midline incision and the fecal contents were carefully cleaned. The lengths of the entire colon of all mice were measured and the values of the lengths were statistically analyzed.

Determination of disease activity index

Clinical evaluation of colitis was performed by determining the disease activity index (DAI), a method developed by Cooper et al. (1993) [10]. The maximum score for an individual was accepted as 4. In our study, the DAI was calculated daily for each DSS-treated mouse, and mice that had an DAI score of ≥ 3 at the end of the experiment in the DSS-treated experimental groups were considered to have colitis.

Histopathologic evaluation

The tissues taken from the proximal part of the distal colon were fixed in Bouin's solution and embedded in paraffin for histologic examination. Sections 5 µm thick were taken from the paraffin-embedded colon tissues, stained with hematoxylin-eosin (H&E) and examined with Olympus BX53F light microscope and photographed with Olympus DP27-CU Camera. The colitis scoring system developed by Dieleaman et al. (1998) and shown in Table 1 was performed on the sections stained with H&E for microscopic determination of the colitis damage score [11]. According to this colitis scoring system, the sections of each mouse were scored microscopically according to criteria such as the severity of inflammation, spread of inflammation, and crypt damage. The score of each criterion was multiplied by the percentage of involvement of that criterion in the section, and the scores of all criteria were summed. The maximum colitis damage score was considered to be 40 for an individual.

Immunohistochemical analysis

To determine the cell proliferation index, colon sections were immunolabeled with the anti-Ki-67 antibody using the "streptavidin-biotin-peroxidase" method. The colon tissue was fixed in 10% buffered formalin and embedded in paraffin. 4µm thick sections were taken from the paraffin blocks. A specific primary antibody for Ki-67 was diluted 1:100 with PBS to detect proliferating cells. Sections were counterstained with Mayer hematoxylin. In each column section, 1000 cells were counted in different randomly selected areas and the cell proliferation index was calculated as a percentage.

Biochemical analyses

Mouse colonic tissue was reconstituted with 10 ml of 0.9% FTS per 1 g of tissue and homogenised using the MagNA lyser (Roche 4.0). The homogenates were centrifuged at 10,000 g for 20 minutes at + 40°C. The clear supernatant was used for spectrophotometric analysis. The total protein content in the column supernatant was calculated according to the Lowry method using bovine serum albumin (BSA) standards [12]. Malondialdehyde levels resulting from lipid peroxidation were determined by the Ledwozyw method [13]. The Beutler method was used to determine the amount of glutathione in the colon tissue [14]. Catalase (CAT) activity was determined spectrophotometrically according to the Aebi method [15], glutathione peroxidase (GPx) activity according to the Paglia-Valentine method [16] and superoxide dismutase (SOD) activity according to the Sun method [17].

ELISA

Colon tissues were reconstituted with 500 µl lysis buffer per 25 mg of tissue and homogenized with a MagNA lyser. Samples were centrifuged at 1500 g for 15 minutes. A mouse-specific MPO sandwich ELISA kit (Hycult biotech-HK210) was used to determine the level of myeloperoxidase (MPO) in the supernatants. The absorbance of the samples was read in an ELISA reader at a wavelength of 450 nm compared to the blank, and the MPO level in the samples were determined using the standard curve generated with the kit's standard solutions.

Western blotting

The levels of tumor necrosis factor-alpha (TNF-α), interleukin 1-beta (IL-1β), interleukin-6 (IL-6), interleukin 10 (IL-10), cyclooxygenase-2 (COX-2), caspase-3 and H3K9 were determined by Western blotting in colon tissue samples from mice. The tissues were homogenized in the MagNA lyser by adding RIPA buffer with EDTA and protease inhibitor. The protein levels in the samples were determined using the Bradford method [18]. Thermo Fisher mini gel tanks and 4–12% Bis-Tris gels were used for the gel run. The gels were run at 160 volts and 300 amps for 50 minutes, and a dry transfer was performed. A blocking solution of 5% BSA in TBST was prepared and applied to the membranes for 1 hour at room temperature. All antibodies were diluted with 1% BSA in TBST. The membranes were incubated with the primary antibodies overnight at + 4°C and then with the secondary antibodies for 60 minutes at room temperature. The TNF-α, IL-1β, COX-2 and caspase-3 concentrations in the tissues were calculated by dividing the intensity of the bands in the visualized membranes by the intensity of the GAPDH bands in the same samples.

Quantitative PCR

RT-PCR reactions were performed to assess gene expressions of STAT1, STAT3, SIRT1, SIRT2, SIRT3 and SIRT5 genes using BioLine SensiFast™ SYBR® No-Rox Kit (London, UK) on LightCycler® 480 Instrument II (Roche Life Science). Primer sequences and their annealing temperatures of genes were given in Table 2. The expression results of the selected genes were normalized using the internal control, β-actin, relative to a control group for every PCR assay. Controls for each PCR reaction included without a template in which 1 µL of water was added instead of the sample cDNA. Each of the samples used in RT-PCR experiments to determine mRNA expression values was studied in triplicate, and the average of the obtained CT values was calculated. The changes in mRNA expression in relation to control samples were calculated using the ΔΔCt method as described previously [19]. Data were presented as mean fold change relative to controls.

Statistical analysis

The data were analyzed using the statistical program SPSS 23.0. The data on the disease activity index were analyzed using the Kruskal-Wallis test and the Mann-Whitney U test. For all other parameters, a one-way ANOVA test followed by an independent t-test was used. The results were expressed as "mean ± standard error".

Changes of body weight and colon length in mice with acute colitis

On day 8 of the experiment, when the animals were sacrificed, the changes in percentage body weight in each group are shown in Fig. 1A. As a result of the measurements, a statistically significant decrease in the body weight of the mice in the DSS group compared to the control group was observed (p < 0.001). No significant difference was found in the percentage body weight of the mice in the PBA and Suramin groups compared to the control group. The percent body weights of the mice in the DSS + PBA and DSS + suramin groups were significantly increased compared to the mice in the DSS-treated colitis group (p < 0.001).

The observed changes in colon lengths are shown in Fig. 1B. When measuring the length of the colon tissue, a significant decrease was observed in the PBA group (p < 0.01) and the DSS colitis group (p < 0.001) compared to the control group. The decrease in the suramin group was not statistically significant compared to the control group. In the DSS + PBA group, the length of the colon decreased compared to both the control group (p < 0.001) and the PBA group (p < 0.01), but showed a significant increase (p < 0.05) compared to the DSS group. The colon lengths of the DSS + suramin group showed a significant decrease compared to the control group (p < 0.001) and the suramin group (p < 0.001); however, there was no significant change compared to the DSS group.

While DSS increases DAI, PBA and suramin decrease DAI from the fourth day of the experiment

The distribution of disease activity indices (DAI) obtained 5 days after DSS administration in each group is shown in Fig. 1C. It was found that the DAI was significantly higher in the colitis group than in the control group on all days after DSS administration (days 1, 2 and 3 p < 0.01; days 4 and 5 p < 0.001). Similarly, a significant increase in the DAI of the mice was observed in the DSS + PBA (day 1 and 2 p < 0.05; day 3 p < 0.01; day 4 and 5 p < 0.001) and DSS + suramin (day 1 p < 0.05; day 2 p < 0.01; day 3, 4 and 5 p < 0.001) groups compared to the control group. The DAI values of the mice in the DSS + PBA group were statistically significantly lower than the DAI values of the mice in the DSS group on day 4 and 5 of DSS administration (p < 0.001). Similarly, the DAI values of the mice in the DSS + suramine group were statistically significantly lower than the DAI values of the mice in the DSS group mice.

PBA and suramin reverse the histological damages induced by DSS treatment

The histological findings of the colon tissue, which were evaluated semi-quantitatively according to the colitis scoring system, are shown in Fig. 1D. Of all experimental groups, the highest histologic colitis score was observed in the colon tissues of the DSS-treated mice. There was a significant increase in this group compared to the control group (p < 0.01). Although a significant increase was observed in the PBA-only group compared to the control group (p < 0.001), there was no significant difference in the colitis score of the suramin-only group compared to the control group. The administration of PBA and suramin to the DSS group significantly reduced histologic damage compared to the DSS-treated colitis group (p < 0.05 and p < 0.01).

The histological sections of the control group showed a healthy and normal appearance (Fig. 1E). Sections from the distal colon tissue of mice in the PBA and suramin groups showed a normal histologic appearance, except for mild inflammatory cell infiltration and epithelial loss observed in a few animals. Examination of sections from the DSS-treated group revealed significant degenerative damage compared to mice in the control group. In this group, diffuse and severe infiltration of inflammatory cells in the mucosa and submucosa of some individuals, advanced inflammation, diffuse submucosal edema, focal mucosal disruption, and occasional loss of surface epithelium were observed. In addition, completely destroyed crypt structure and widespread crypt loss, especially vacuolar hydropic degeneration and necrosis in the crypt cells, were frequently observed in the distal colon sections of mice in this group. When comparing the sections of the DSS + PBA and DSS + suramin groups with the DSS group, it was found that the histologic damage was significantly less. Edema, severe lesions in the crypt, loss of goblet cells in large areas and widespread and severe inflammation were not observed in the histologic examinations of the sections of the animals in these groups.

PBA or suramin co-treatment promotes the proliferation of colon epithelial cells

Light micrographs of Ki-67 immunohistochemistry in distal colon sections of mice are shown in Fig. 1G, and the distribution of cell proliferation index (%) by group is shown in Fig. 1F. The cell proliferation indexes of the DSS colitis group (p < 0.001) and the PBA group (p < 0.05) showed a significant decrease compared to the control group, while no significant change was observed in the proliferation index of the suramin group compared to the control group. In the DSS + PBA group, the percentage of Ki-67-positive cells decreased compared to the control group (p < 0.01) and the PBA group (p < 0.001). However, there was a statistically significant increase compared to the DSS group (p < 0.001). Although the cell proliferation index in the DSS + suramin group showed a decrease compared to both the control group and the suramin group, it showed a significant increase compared to the DSS group (p < 0.001).

Changes in the glutathione levels and lipid peroxidation of experimental animals

The distribution of GSH and MDA levels obtained from the mouse intestinal tissue homogenates is shown in Fig. 2. A significant decrease in GSH levels was observed in the DSS colitis group compared to the control group (p < 0.001). There was a significant increase in GSH levels in the DSS + PBA and DSS + suramin groups compared to the DSS colitis group (p < 0.001). A decrease (p < 0.05) was observed in the DSS + suramin group compared to the control and suramin groups. When comparing all experimental groups, the highest MDA levels were observed in the DSS group. Injection of PBA and suramin into DSS-induced colitis resulted in a non-statistically significant decrease in the elevated MDA levels observed in the DSS colitis group. Although MDA levels changed between the experimental groups, these changes were not statistically significant.

Changes the activity of antioxidant enzymes GPx, CAT, SOD

The distribution of GPx, CAT and SOD activities obtained from the homogenates of colon tissue by group is shown in Fig. 2. The GPx activities of the mice in the DSS group showed a significant decrease compared to the control group (p < 0.001). The enzyme activity in the PBA group showed no significant change compared to the control group. In addition, GPx activity increased 2.6-fold in the suramin-treated group compared to the control group (p < 0.001). In the DSS + PBA (p < 0.01) and DSS + suramin (p < 0.001) groups, there was a significant increase in GPx activity compared to the DSS colitis group. In the DSS + suramin group, a decrease in GPx activity was observed in the mice compared to the mice in the suramin group (p < 0.001).

The CAT activities of the mice in the DSS colitis group showed a significant decrease compared to the mice in the control group (p < 0.01). CAT activities in the PBA group showed a insignificant increase compared to the control group. However, the CAT activity of the mice in the suramin group increased significantly compared to the control group (p < 0.01). CAT activity in both the DSS + PBA and DSS + suramin groups showed a significant increase compared to the DSS group (p < 0.01). CAT activity decreased in the DSS + PBA group compared to the PBA-only group (p < 0.01). A decrease in CAT activity was observed in the DSS + suramin group compared to the suramin group (p < 0.01). However, a significant increase in CAT activity was observed in this group compared to both the control group (p < 0.01) and the DSS group (p < 0.01).

There was a significant decrease in SOD activity in the DSS group compared to the control group (p < 0.01). The SOD activities of the PBA group and the suramin group were close to those of the control group. In the DSS + PBA and DSS + suramin groups, a significant increase in low SOD activities was observed in the DSS colitis group (p < 0.01). In addition, the increase in SOD activity was significant in the DSS + PBA group compared to the control group and the PBA group (p < 0.01).

ELISA analysis shows decreased levels of MPO in PBA and suramin injected mice with DSS-induced colitis

The distribution of MPO levels from the homogenates of the colon tissue of the mice by group is shown in Fig. 2. When considering all DSS-treated groups, the DSS colitis group showed the highest increase (approximately 18-fold) in MPO levels compared to the control group (p < 0.001). The MPO levels of individuals in the DSS + PBA (p < 0.001) and DSS + suramin (p < 0.01) groups showed a significant decrease compared to the DSS-colitis group. The MPO levels were higher in the DSS + suramin group than in the control group and in the suramin group (p < 0.001).

Western blot analysis demonstrates decreased caspase-3 activity and the expression of TNF-α, IL-1β, IL-6, COX-2 and increase IL-10 levels in PBA and suramin treated groups

The results and analysis of related parameters are shown in Fig. 3. An increase in the active caspase-3 level was observed in all experimental groups compared to the control group. However, the highest increase was observed in the colitis group treated with DSS at 2.2-fold (p < 0.001). However, separate injections of PBA or suramin into the DSS group clearly and significantly reduced the increased caspase-3 activity in the DSS group (p < 0.01 and p < 0.001, respectively). The concentrations of TNF-α in the colon tissue were significantly increased in the DSS-treated mice compared to the control group (p < 0.001). The highest TNF-α, IL-1β and IL-6 levels were found in the individuals with DSS-induced ulcerative colitis (p < 0.001 for TNF-α, IL-1β; p < 0.01 for IL-6). Their levels in the DSS + PBA and DSS + suramin groups showed a significant decrease compared to the DSS colitis group (p < 0.001). When comparing all groups with each other, the lowest IL-10 level in the colon tissue was found in the DSS colitis group. In the PBA and suramin groups, IL-10 expression was increased compared to the control group. Although the increase in the PBA group was not significant, the increase in the suramin group was significant (p < 0.05). Injection of PBA into DSS-treated mice resulted in a significant increase in IL-10 level compared to the DSS-only group (p < 0.001). However, its level was high than PBA group (p < 0.001). In the DSS + suramin group, IL-10 levels were significantly increased compared to both DSS-treated mice (p < 0.001) and control samples (p < 0.01). The highest IL-10 level among all experimental groups was found in the group in which suramin and DSS were administered together. In the DSS + suramin group, the IL-10 level increased two-fold compared to the DSS group and 1.5-fold compared to the control group. A significant increase was found in the COX-2 expression of each group compared to the control group (p < 0.001). However, the highest increase was observed in the colitis group treated with DSS at 4.3-fold (p < 0.001). However, separate injections of PBA or suramin into the DSS group significantly reduced the increased COX-2 expression (p < 0.001).

The global trimethyl-H3K9 modification increases PBA and suramin treated mice with DSS-induced colitis

Figure 4 represents the distribution of H3K9me3 expression levels in each group. The H3K9me3 level of colitis group was significantly decreased compared to the control group (p < 0.01). A significant increase in H3K9me3 level was observed in the PBA group compared to the control group (p < 0.05), but there was no change in H3K9me3 level of suramin group compared to the control group. The highest trimethylation level was observed in the DSS + PBA group and it was significant compared to the control, PBA and DSS groups (p < 0.01, p < 0.01 and p < 0.001, respectively). An increase in H3K9me3 level was also observed in the DSS + suramin group compared to the DSS group (p < 0.001).

PBA increases STAT1 but Suramin increase STAT3 mRNA levels in mice with acute colitis The results of the mRNA expression of the genes STAT1, STAT3, SIRT1, SIRT2, SIRT3 and SIRT5 according to the quantitative PCR analysis of the colon tissue of the mouse are shown in Figs. 5. A statistically significant decrease in STAT1 gene expression was observed in all groups compared to the control group, with the DSS group showing the lowest gene expression of all groups. While a significant decrease of 81% was observed in the DSS group compared to the control group, a significant decrease of 76% was observed in the group receiving suramin for therapeutic purposes. A 3.5-fold significant increase was observed in the therapeutic PBA group compared to the DSS group and a 1.5-fold significant increase compared to the PBA group.

When analyzing STAT3 gene expression levels, the highest levels were found in the PBA group. STAT3 gene expression increased significantly by 2.62-fold in the PBA group, 1.52-fold in the DSS group, 1.34-fold in the PBA-treated group and 2.05-fold in the suramin-treated group compared to the control group. For the same gene, a 1.31-fold significant increase was observed in the suramin-treated group compared to the DSS group and a 2.5-fold significant increase compared to the suramin group, while PBA caused a 49% significant decrease compared to the PBA group.

PBA and suramin decrease mRNA levels of SIRT1, SIRT2, SIRT3 and SIRT5 in mice with ulcerative colitis

The mRNA expression levels of the SIRT1 gene showed a significant decrease of 48% in the suramin group, a 2.67-fold significant increase in the DSS group, a 2.42-fold significant increase in the DSS + PBA group and a 1.96-fold significant increase in the DSS + suramin group compared to the control group. The DSS colitis group had the highest gene expression. The separate application of PBA and suramin in the DSS group resulted in a significant decrease in gene expression compared to the DSS colitis group. Suramin caused a significant decrease in SIRT1 gene expression by 26.6% compared to the DSS group; PBA caused a 2.42-fold significant increase compared to the PBA group; suramin caused a 3.77-fold significant increase compared to the suramin group (Fig. 5).

SIRT2 gene expression decreased significantly by 90% in the suramin group, by 4.22-fold in the DSS group, by 2.73-fold in the PBA group and by 1.89-fold in the suramin group compared to the control group. A significant decrease of 35.31% was observed in the DSS + PBA group and 55.22% in the DSS + suramin group compared to the DSS group. PBA and suramin caused a 2.37-fold and 18.9-fold significant increase in the SIRT2 gene compared to the PBA and suramin groups, respectively.

SIRT3 gene expression was significantly decreased by 44% and 58% in the PBA group and the suramin group, respectively, while it was significantly increased by 1.4-fold, 1.13-fold and 1.11-fold in the DSS group, the DSS + PBA and the DSS + suramin groups, respectively, compared to the control group. Compared to the DSS group, PBA treatment significantly decreased SIRT3 gene expression by 19.28% and suramin treatment by 21.43%. PBA treatment increased SIRT3 gene expression by 2.02-fold compared to the PBA group and suramin treatment increased SIRT3 gene expression by 2.64-fold compared to the suramin group.

SIRT5 gene expression increased significantly by 1.44-fold in the DSS group compared to the control group, while it decreased significantly by 55.55% and 48.61% in the DSS + PBA or suramin group compared to the DSS group, respectively. However, the expression in the DSS + PBA and DSS + suramin groups was also significantly reduced compared to the control group.

The DSS-induced colitis model has similarities to human ulcerative colitis, particularly with clinical findings such as weight loss, shortening of the colon, mucosal ulceration, diarrhea, softening of stool consistency, and rectal bleeding, as well as histopathological features such as crypt loss, increased infiltration of inflammatory cells, and necrotic damage, and is commonly used to induce an animal model of colitis [20, 21]. In our study, the experimental colitis induced by administration of 3% DSS (w/v) to C57BL/6 mice for 5 days resembled acute colitis models described in the literature in terms of bloody stools, diarrhea, weight loss, high DAI, decreased colon length, and histopathological findings [22].

There is evidence that HDACi exert anti-inflammatory effects in some inflammatory disease models [23, 24]. HDACi exert their anti-inflammatory effects by acting on T cells, macrophages and cytokines, which play an important role in colon inflammation. In a study in which various doses of PBA were administered intraperitoneally and orally in a DSS-induced colitis model in mice, PBA suppressed the development of colitis by reversing the increase in disease activity index (DAI), colonic shortening, increased inflammatory cytokine levels, and histopathologic changes in the colon as a result of colitis damage [6, 25]. In our study, we found that 150 mg/kg PBA administered to C57BL/6 mice for 7 days significantly suppressed the effects of DSS, significantly decreased DAI score, and increased colon length, which decreased in the colitis group. Suramin is a histone deacetylase inhibitor that inhibits the NAD+-dependent enzyme activities of SIRTs that form the HDAC-III class. Although there are several physiological studies in the literature investigating the effects of suramin administered as a purinergic receptor antagonist on nerve excitation and intestinal motility, there is no article investigating the biological effects of suramin in DSS-induced ulcerative colitis. In our study, we found that 25 mg/kg suramin significantly suppressed the effects of DSS and significantly decreased the DAI score.

In ulcerative colitis, the disruption of the balance between apoptosis and proliferation in the epithelial cells of the colon leads to damage to the epithelial barrier [26]. In our study, we found that the number of proliferative cells in the colonic mucosa of mice with acute colitis induced by DSS administration was significantly reduced compared to mice in the control group. However, in our study, it was also found that the apoptosis rate was significantly increased in animals with colitis compared to mice in the control group. Histone deacetylase inhibitors with anticarcinogenic activity have been reported to inhibit tumor growth by inhibiting proliferation in cancer cell lines [27, 28]. The anti-proliferative effects of PBA, an HDACi, on the cell cycle have been demonstrated in cancer cell lines and cancer patients [29, 30]. In contrast to the results of studies with PBA in cancer cell lines and experimental models, in our study the cell proliferation index in the colon was significantly increased when PBA was administered to mice in which colitis was induced by DSS compared to the colitis group. However, the expression of caspase-3 was also significantly decreased in the DSS group injected with PBA. All these findings suggest that PBA prevents apoptosis induced by DSS-induced colitis in the colon and ensures cell cycle continuity. Various doses of suramin have been applied to different human cancer cell lines, and it was reported that proliferation decreased with increasing suramin dose [31]. There are no studies in the literature investigating the effects of suramin on ulcerative colitis. In our study, injection of suramin into mice with DSS-induced colitis stimulated cell proliferation in the colon and inhibited colitis-induced apoptosis, and this inhibition was higher compared to PBA. In addition, the proliferation index, which decreased in colonic epithelial cells with colitis formation when the animals given suramin, increased more than in PBA.

Oxidative stress, which occurs when inflammatory cells, neutrophils and macrophages produce large amounts of ROS, plays an important role in the pathogenesis of inflammatory diseases. ROS has a major impact on the pathogenesis and progression of ulcerative colitis [32]. It is known that in DSS-induced colitis, the ROS concentration in the colon increases significantly and the concentration of antioxidant enzymes decreases [33]. Our study is fully consistent with these findings from the literature. In the DSS colitis group, there was a significant increase in MPO and MDA levels and a significant decrease in SOD, CAT, GPx and GSH levels, and oxidative damage to colon tissue was induced by DSS. Sodium phenylbutyrate inhibits oxidative damage and inflammation by suppressing ROS and pro-inflammatory cytokine levels [34]. However, the studies performing the effects of PBA and suramin on the markers of oxidative system are quite limited, and there are no reports investigating their effects on oxidative stress in case of ulcerative colitis. In our study, the elevated levels of MPO and MDA in the colon tissue of mice with DSS-induced colitis decreased with the administration of PBA and suramin. The GSH levels decreased but activities of SOD, GPx and CAT increased with the administration of PBA and suramin in the colitis group. Our results suggest that PBA and suramin injections protect colon tissue against oxidative damage in DSS-induced colitis by decreasing MDA and MPO levels, the markers of oxidative stress, and increasing SOD, GPx and CAT activities and GSH levels, the parameters of the antioxidant system. When comparing the effects of suramin and PBA on the antioxidant system and oxidative stress in the DSS-induced colitis model, it was found that suramin increased the levels of the enzymes CAT and GPx in the colon tissue more than PBA. When considering that both enzymes exert their antioxidant effects by reducing H₂O₂, it can be suggested that the antioxidant activity of suramin is mainly related to H₂O₂ detoxification. In addition, PBA was more effective than suramin in enhancing GSH and SOD levels. When colonic MDA levels were analyzed, although MDA levels of the DSS + PBA group decreased more than that of the DSS + Suramin group, no significant differences were observed in both groups compared to the colitis group. Similarly, colonic MPO levels showed a more significant decrease in the DSS + PBA group than in the DSS + Suramin group compared to the colitis group.

Cytokines are mediators that play a key role in the initiation, development and healing of inflammation in inflammatory diseases [35]. Many studies on biopsy samples from patients with ulcerative colitis and DSS colitis models have reported elevated levels of TNF-α, IL-1β, IL-6, IL-8 and IL-12 in colonic tissue [33, 36]. IL-10 is an anti-inflammatory cytokine that plays a crucial role in intestinal homeostasis, and its loss of function is thought to be associated with the early stages of inflammatory bowel disease. In our study, in parallel with these findings, a significant increase in pro-inflammatory cytokine levels such as TNF-α, IL-1β and IL-6 and a significant decrease in IL-10 levels were observed in the colonic tissue of mice with acute colitis induced by 3% DSS compared to mice in the control group. Sodium phenylbutyrate is known to exert anti-inflammatory effects by suppressing the expression of pro-inflammatory cytokines [5, 25] reported that PBA administered at various doses to ICR mice with DSS-induced colitis exhibited anti-inflammatory effects by significantly decreasing IL-1β, TNF-α and IL-6 cytokine levels in the colon at a dose of 150 mg/kg [12]. In our study, injection of PBA into C57BL/6 mice administered DSS resulted in a decrease in TNF-α, IL-1β and IL-6 cytokine levels and an increase in IL-10 levels in the colon, in parallel with the results in the literature. Suramin contains six sulfonate groups, or sodium sulfonates, which are thought to be responsible for its anti-inflammatory effects. It was found that suramin administration decreased serum levels of TNF-α, IL-6 and NF-kBp65 in an acute liver injury model in C57BL/6 mice [37]. In our study, it was observed that injection of suramin at a dose of 25 mg/kg/day for 7 days significantly decreased TNF-α, IL-1β and IL-6 levels in the colon of DSS-treated mice. In addition, suramin administration also increased IL-10 levels, which decreased with DSS. Considering all cytokine levels, suramin was more effective than PBA in lowering TNF-α, IL-1β and IL-6 levels and increased IL-10 levels. In view of our results, suramin showed a stronger anti-inflammatory effect in the DSS-induced colitis model compared to PBA.

Cyclooxygenase-2 (COX-2) is an enzyme that plays a role in the repair process following mucosal damage in the gastrointestinal tract and its expression can be stimulated by many cytokines such as IL-1, TNF-α and is responsible for the formation of prostanoids including thromboxanes and prostaglandins. It is known that ulcerative colitis not only increases the production of cytokines such as IL-1, IL-6 and TNF-α, but also increases COX-2 gene expression [38]. It has been reported in the literature that COX-2 levels increase in experimental animals with DSS-induced colitis compared to animals in the control group [39, 40]. In consistent with these studies, we also found that COX-2 expression was significantly increased in the colonic tissue of mice with DSS-induced colitis compared to mice in the control group. No reports were found in the literature investigating the effects of sodium phenylbutyrate and suramin on colonic tissue COX-2 levels in ulcerative colitis. We also found that elevated COX-2 expression decreased after the administration of both PBA and suramin and its level approached that of control samples. It is noteworthy that the effects of suramin and PBA, which cause inhibition of different classes of histone deacetylase enzymes, on DSS-induced COX-2 levels in the colon are quite similar.

Epigenetic mechanisms, in particular histone modifications, modulate inflammatory responses positively or negatively, depending on the type of modification. For example, trimethylation of H3 at Lys9 (H3K9me3) disrupts the interaction between transcription factors and promoters through the formation of heterochromatin, a tight genomic packaging. H3K9 methylation is thought to influence NF-κB access to the IL-6 promoter. Studies have shown that dynamic changes in H3K9 methylation are associated with proinflammatory genes [41]. No study was found that examined H3K9 trimethylation in DSS-induced colitis. In parallel to this effect on H3K9me3 and pro-inflammatory cytokines in the literature, our study found that H3K9 trimethylation decreased in colon tissue in DSS-induced colitis compared to the control group. Administration of the histone deacetylase inhibitors PBA and suramin led to an increase in H3 methylation, which decreased with colitis. It is conceivable that both agents individually increase H3K9 trimethylation and lead to the formation of heterochromatin, thereby suppressing the expression of proinflammatory cytokines through genomic repression. However, since the presence of an epigenetic modification alone does not guarantee that the transcription of the associated gene is affected, and it is difficult to associate specific epigenetic modifications with disease activity, many studies are needed to evaluate the effect of epigenetic modifications on colitis to be sure that they are associated with the disease and to develop targeted epigenetic therapies.

Several studies have highlighted that the analysis of STATs activation can be used as a biomarker in diseases characterized by intestinal inflammation and that the STAT1/3 pathway could be an important target in the development of future therapeutics for these diseases [42, 43]. Suzuki et al. (2001) demonstrated that STAT3 mRNA levels are elevated in ulcerative colitis and stated that STAT3 could be an important biomarker for characterizing T cell activation in inflammatory bowel disease [44]. According to the results of the gene expression analysis, STAT1 gene expression decreased in the group with DSS-induced ulcerative colitis compared to the control group, while STAT3 gene expression increased as expected. PBA, one of the histone deacetylase inhibitors administered for therapeutic purposes, significantly increased STAT1 gene expression compared to the DSS group, while it caused a non-significant decrease in STAT3 gene expression. Administration of suramin did not alter STAT1 gene expression, but significantly increased STAT3 gene expression levels compared to the DSS group. However, it is known that the STAT1 and STAT3 proteins are activated by increased phosphorylation levels in ulcerative colitis [42, 45]. It can be assumed that the phosphorylation levels of the STAT1 and STAT3 proteins may also have changed. Histone deacetylase inhibitors have been shown to have a positive effect on the prognosis of colitis, and the statuses that promote histone acetylation, such as the administration of some histone deacetylase inhibitors, may improve colitis through a mechanism involving the production of various anti-inflammatory cytokines [46]. Moreover, acetylation and deacetylation are also involved in regulatory mechanisms for the activation or inactivation of transcription factors such as STAT1 and STAT3, and the histone deacetylases that mediate this process can be modulated by histone deacetylase inhibitors. Preclinical and clinical studies have demonstrated reduced gene expression and activity of SIRT1 and SIRT5 in patients with ulcerative colitis and in various experimental animal models [20, 47]. In addition, it has been reported that reduced gene expression of SIRT1 may be responsible for the sustained production of proinflammatory cytokines and generation of oxidative stress in ulcerative colitis [48]. However, reduced gene expression of SIRT1 also protects the gastrointestinal barrier, suggesting a protective role of sirtuin in inflammatory bowel disease. Moreover, several studies have reported that exogenous administration of sirtuin ameliorates colitis in various models of inflammatory bowel disease [49, 50]. In this present study, SIRT1, SIRT2, SIRT3 and SIRT5 gene expressions were increased in C57BL/6 mice with DSS-induced ulcerative colitis compared to the control group, while PBA and suramin administration for therapeutic purposes caused a decrease in DSS-induced gene expression. It was also observed that suramin, one of the two histone deacetylase inhibitors used, was more effective in reducing the elevated sirtuin gene expressions compared to PBA.

In conclusion, this study is the first experimental study to investigate the biological role of suramin in a model of ulcerative colitis induced by DSS. Furthermore, PBA and suramin exhibited antioxidant activity by suppressing oxidative stress, and their relationship to epigenetic modifications was determined for the first time in a colitis model. The cytoprotective activity of PBA and suramin in colon tissue of the mouse with DSS-induced colitis, their ability to maintain the balance between cell proliferation and apoptosis and their activity to stimulate the endogenous antioxidant system by reducing oxidative stress and also their immunomodulatory role on inflammatory mediators by suppressing the expression of pro-inflammatory cytokines make them promising agents in the treatment of inflammatory diseases triggered by oxidative stress.

Funding

This work was supported by the The Scientific and Technological Research Council of Turkey (TUBITAK) (Project numbers: KBAG-118Z061).

Competing Interest

The authors have no relevant financial or non-financial interests to disclose.

Author Contributions

C.O.C. and P.A. conceived and designed research; C.O.C. and E.F.K. performed experiments; C.O.C., E.F.K., S.O., E.K. and P.A. analyzed data; S.O., E.K. and P.A. interpreted results of experiments; C.O.C. and E.F.K. prepared figures; P.A. drafted manuscript; C.O.C., E.F.K, S.O., E.K. and P.A. edited and revised manuscript; C.O.C., E.F.K., S.O., E.K. and P.A. approved final version of manuscript.

Ethics Approval

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Tables 1 and 2 are available in the Supplementary Files section.

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The Role of Sodium Phenylbutyrate and Suramin in the Prevention of Ulcerative Colitis Induced by Dextran Sulfate Sodium

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Abstract

Figures

Introduction

Materials and Methods

Animal model

Experimental groups

Determination of the percentage change in body weight and colon length in mice

Determination of disease activity index

Histopathologic evaluation

Immunohistochemical analysis

Biochemical analyses

ELISA

Western blotting

Quantitative PCR

Statistical analysis

Results

Changes of body weight and colon length in mice with acute colitis

PBA and suramin reverse the histological damages induced by DSS treatment

PBA or suramin co-treatment promotes the proliferation of colon epithelial cells

Changes in the glutathione levels and lipid peroxidation of experimental animals

Changes the activity of antioxidant enzymes GPx, CAT, SOD

The global trimethyl-H3K9 modification increases PBA and suramin treated mice with DSS-induced colitis

Discussion

Conclusion

Declarations

References

Tables

Additional Declarations

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