ADT-OH improves intestinal barrier function and remodels the gut microbiota in DSS-induced colitis

Abstract

Owing to the increasing incidence and prevalence of inflammatory bowel disease (IBD) worldwide, effective and safe treatments for IBD are urgently needed. Hydrogen sulfide (H₂S) is an endogenous gasotransmitter and plays an important role in inflammation. To date, H₂S-releasing agents are viewed as potential anti-inflammatory drugs. The slow-releasing H₂S donor 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADT-OH), known as a potent therapeutic with chemopreventive and cytoprotective properties, has received attention recently. Here, we reported its anti-inflammatory effects on dextran sodium sulfate (DSS)-induced acute (7 days) and chronic (30 days) colitis. We found that ADT-OH effectively reduced the DSS-colitis clinical score and reversed the inflammation-induced shortening of colon length. Moreover, ADT-OH reduced intestinal inflammation by suppressing the nuclear factor kappa-B pathway. In vivo and in vitro results showed that ADT-OH decreased intestinal permeability by increasing the expression of zonula occludens-1 and occludin and blocking increases in myosin II regulatory light chain phosphorylation and epithelial myosin light chain kinase protein expression levels. In addition, ADT-OH restored intestinal microbiota dysbiosis characterized by the significantly increased abundance of Muribaculaceae and Alistipes and markedly decreased abundance of Helicobacter, Mucispirillum, Parasutterella, and Desulfovibrio. Transplanting ADT-OH-modulated microbiota can alleviate DSS-induced colitis and negatively regulate the expression of local and systemic proinflammatory cytokines. Collectively, ADT-OH is safe without any short-term (5 days) or long-term (30 days) toxicological adverse effects and can be used as an alternative therapeutic agent for IBD treatment.

Keywords

inflammatory bowel disease / ADT-OH / intestinal permeability / gut microbiota

1 Introduction

Inflammatory bowel disease (IBD) is a global disease in the 21st century and has sharply increasing prevalence among all populations [1]. Clinically, IBD, including Crohn’s disease and ulcerative colitis, is characterized by chronic and recurrent gastrointestinal inflammation [2]. Although the incidence of IBD is increasing, its cause is yet to be understood. Recent studies indicate that genetic susceptibility, immune response dysregulation, and environmental factors (life-style, diet, and intestinal flora) play important roles in the pathogenesis of IBD [3–5]. Owing to the complex interplay among these factors, the efficacy of several clinical drugs for IBD, such as aminosalicylates, steroids, and antitumor necrosis factor-α antibodies, are limited and have significant side effects [6,7]. Therefore, developing novel, safe, and efficacious therapies for IBD is crucial.

Research into the molecular mechanisms of IBD has focused on the underlying immune cell-mediated inflammatory response regulation [8]. However, intestinal epithelium barrier dysregulation might play an important role in the development, perpetuation, and severity of IBD. For instance, an increase in intestinal permeability improves the recurrence rate of Crohn’s disease, indicating that it is a great risk factor for IBD [9]. Under normal physiologic conditions, the intestinal epithelium barrier exists as a semipermeable physical barrier, which allows the selective absorption of nutrients and prevents invasion by pathogens [10–12]. A defective intestinal barrier cannot respond to environmental toxins and physical stress, and damage to the intestinal epithelial barrier increases gut permeability and leads to the translocation of harmful microbiota into the body. These harmful bacteria produce ammonia and intestinal toxins, which can further exacerbate intestinal barrier disorders [13]. Significant changes in the composition of the gut microbiota have been reported in patients with IBD [14–17]. Thus, restoring intestinal barrier integrity and manipulating the gut microbiota might be developed as therapeutic strategies for patients with IBD.

Hydrogen sulfide (H₂S), the third endogenous gaseous molecule after nitric oxide (NO) and carbon monoxide (CO), is involved in the regulation of diverse biological effects, including the pathogenesis of IBD and colorectal cancer [18,19]. H₂S has anti-inflammatory and proinflammatory effects [20–22]. Inflammation might upregulate the expression of cystathionine β-lyase (CBS) and cystathionine-γ-lyase (CSE), which contribute to H₂S production [23]. High H₂S concentrations may promote inflammatory vasodilation and impair mitochondrial function by inhibiting complex IV in the electron transport chain, whereas low concentrations of mitochondrial H₂S actually promote oxidative phosphorylation and reduce oxidative stress [24,25]. Slow-release sulfide donors are superior to inorganic sulfide sources. Typical sulfide salts, including sodium sulfide (Na₂S) and sodium hydrogen sulfide (NaHS), rapidly but transiently increase sulfide concentrations; this effect may not be a characteristic of physiologic conditions [26]. Slow-release sulfide donors can mimic endogenous conditions. One of the most commonly used slow-release H₂S donors is 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADT-OH) [27], which has the special structure of the 3H-1,2-dithiole-3-thione (D3T) group (Fig.1). Members of the dithiolethione class, including olitipraz and ADT-OCH3, have antitumor and antioxidant properties [28,29]. For example, as a choleretic drug marketed as Sulfarlem, ADT-OCH3 offers good prospects for the chemoprevention of human lung cancer [30] and colon cancer [31]. As the main metabolite of anethole trithione (ADT, Sulfarlem), the derivative ADT-OH enhances the efficacy of the compound in providing hepatoprotective and antioxidant effects [32]. Moreover, ADT-OH suppresses LPS-induced neuroinflammatory responses and may be a candidate for treating inflammation-related neurodegenerative disorders [33,34]. However, little research has been conducted to assess the therapeutic effect of ADT-OH on IBD.

Here, we reported that low-dose ADT-OH alleviated dextran sodium sulfate (DSS)-induced inflammatory bowel disease in mice. Next, we used the well-established intestinal epithelial cell line Caco-2 to study the mechanism of the anti-inflammatory effect of ADT-OH. Our results suggested that ADT-OH protected cell monolayer integrity and maintained intestinal barrier functions by changing gut microbiota diversity and composition. This study expanded our understanding of the anti-inflammatory effect of ADT-OH and provided a potentially effective strategy for IBD therapy.

2 Materials and methods

2.1 Experimental animals

All C57BL/6J mice used in the study were purchased from Shanghai Laboratory of Animal Center (Shanghai, China) and housed in a temperature-controlled sterile room where humidity and light were carefully monitored. Animal welfare and experimental procedures were performed strictly in accordance with high-standard animal welfare practices and other related ethical regulations approved by the Nanjing University Animal Care and Use Committee.

2.2 Dextran sodium sulfate-induced acute colitis model

Eight-week-old C57BL/6J mice were randomly divided into six groups (n = 6–8): normal group (control), 3% DSS group (model), low-dose ADT-OH group (ADT-OH, 25 μmol/kg body weight), medium-dose ADT-OH group (ADT-OH, 50 μmol/kg body weight), high-dose ADT-OH group (ADT-OH, 100 μmol/kg body weight), and positive control group (NaHS, 40 μmol/kg body weight). All mice in the normal group were housed as usual and received neither DSS nor ADT-OH. Other groups were fed with sterile water containing 3.0% (w/v) DSS (36–50 kDa, MP Biomedicals) for 7 days. After 2 days, the mice were injected intraperitoneally with different concentrations of ADT-OH (100 μL per mouse), NaHS (40 μmol/kg, 100 μL per mouse), or vehicle (100 μL per mouse) per day.

2.3 Dextran sodium sulfate-induced chronic colitis model

Eight-week-old C57BL/6J mice received three cycles of DSS treatment consisting of 5 days with 2.5% DSS in sterile water and then underwent a 5-day recovery phase with sterile water (Fig.2). The control group received neither DSS nor ADT-OH and received sterile water. Daily intraperitoneal injections with ADT-OH were performed during the 2.5% (w/v) DSS treatment, whereas only one injection was applied during the regular water treatment. Other experimental treatments and groupings were the same as those used in the acute colitis model.

2.4 Evaluation of disease activity

In the DSS-induced colitis models, the life status, fecal condition (fecal morphology and occult blood phenomenon), and body weight of mice from each group were assessed every day. The disease activity index (DAI) was determined by scoring changes in body weight, blood in stool, and stool consistency with previously described methods [35]. Briefly, DAI was calculated by scoring weight loss (no loss, 0; 1%–5%, 1; 5%–10%, 2; 10%–15%, 3; > 15%, 4), stool consistency (normal = 0, mild looseness = 1, looseness = 2, diarrhea = 3, bloody stool = 4), and rectal bleeding (negative = 0, positive = 2, bloody stools visible to the naked eye = 4). Final DAI scores were obtained by dividing the combined scores by 3.

2.5 In vivo toxicity assessment

Eight-week-old C57BL/6J mice were maintained under standard feeding, light, and temperature conditions and had free access to food and water. Mice were divided randomly into four groups and intraperitoneally injected with vehicle or 25 μmol/kg, 50 μmol/kg, or 100 μmol/kg ADT-OH for five consecutive days. The body weights of the mice were monitored every day. On the sixth day, the mice were sacrificed, and serum was collected for liver and kidney function tests. Alanine aminotransferase (ALT), alkaline phosphatase (ALP), total blood bilirubin (TBIL) activity, blood urea nitrogen (BUN), and serum creatinine were measured using a serum multiple automatic biochemical analyzer (Chemray 800, China). The weights of the liver, kidney, and spleen were measured, and tissues were collected and fixed in formalin for hematoxylin and eosin (H&E) staining.

2.6 Hematoxylin and eosin staining and histological score

Formalin-fixed and paraffin-embedded colorectal tissues were cut into 4 μm sections and stained according to the standard protocol of the H&E staining assay. The histological diagnosis and scoring of all the samples were performed by two independent pathologists, and the scoring system used was described previously [36]. Briefly, we assessed the severity of inflammation (none = 0, mild = 1, moderate = 2, severe = 3), depth of injury (no mucosal injury = 0, mucosal injury = 1, mucosa and submucosal injury = 2, injury across the intestinal wall = 3), and inflammatory cell infiltration (no or very few inflammatory cells in the lamina propria of the mucosa = 0, more inflammatory cells in the lamina propria of the mucosa = 1, inflammatory cells spread to the submucosa = 2, exudation of inflammatory cells throughout the layer = 3). The colorectal histology score was obtained by combining the scores, and the total scoring range was 0–9 per mouse.

2.7 Determination of endogenous hydrogen sulfide content

The content of H₂S in colorectal tissues of mice or Caco-2 cells was detected with an endogenous hydrogen sulfide assay kit (Jiancheng Bioengineering Institute, Nanjing, China). Briefly, according to the manufacturer’s instructions, we extracted H₂S from colonic tissues or Caco-2 cells and then detected its content with the methylene blue method. A microplate reader (TECAN, Switzerland) was used to measure the absorbance of each sample at 665 nm.

2.8 Flow cytometry

Flow cytometry was used to detect cell apoptosis and Treg cell expression during the experiment. Cell apoptosis was detected with enhanced green fluorescent protein-conjugated annexin V (BD Pharmingen, San Diego, CA, USA) according to the manufacturer’s instructions. In the in vivo experiment, the mice were sacrificed to obtain single-cell suspensions from the intestine. Then, the cells were incubated with the corresponding antibodies for the detection of positive Treg cells. The following antibodies were used: CD45-APC-Cy7, CD3-PE/Cy7, CD4-FITC, CD25-PE, and CD127-APC (eBioscience, BioLegend, and BD Biosciences). The cells were analyzed with a FACSCanto flow cytometry (BD) and FlowJo software (TreeStar).

2.9 Cells and cell culture

Human colon adenocarcinoma cells (Caco-2), human colon adenocarcinoma cells (HCT-116), murine macrophage-like cells (RAW 264.7), and human umbilical vein endothelial cells (HUVECs) were purchased from the American Type Culture Collection (ATCC, Philadelphia, PA, USA). Caco-2 cells were cultured in minimum essential medium (Gibco, Shanghai, China) supplemented with 20% (v/v) fetal bovine serum (FBS, Gibco, USA), 100 U/mL penicillin, 100 μg/mL streptomycin, and 100 μg/mL sodium pyruvate. HCT-116 and RAW264.7 cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Shanghai, China) with 10% (v/v) FBS. HUVECs were cultured in endothelial cell medium (Gibco, Shanghai, China). All cells were cultured at 37 °C in a humidified chamber of 5% CO₂.

2.10 RNA extraction and quantitative real-time PCR

Total RNA of cells or colonic tissues was extracted using TRIzol reagent (Vazyme, Nanjing, China) according to the manufacturer’s instructions. RNA concentration and quality were determined for each sample in a 260/280 nm ratio with a microplate reader. RNA was reverse transcribed into cDNA by using a ReverTra Ace quantitative real-time polymerase chain reaction (qRT‒PCR) kit (TOYOBO, Japan) according to the manufacturer’s instructions. qRT‒PCR was performed using AceQ qPCR SYBR Green Master Mix (Vazyme, Nanjing, China) with an Applied Biosystems real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). The primer sequences are listed in Tables S1 and S2. Each qRT‒PCR was repeated at least three times, and β-actin was used as an internal control.

2.11 Trans-epithelial electrical resistance assay

Trans-epithelial electrical resistance (TEER) was monitored as an indication of epithelial monolayer integrity as previously described [37]. Transwell chambers (6.5 mm diameter, 0.4 μm pore size; Corning, NY, USA) were pre-activated with a serum-free medium in a cell culture incubator overnight. Caco-2 cells were seeded into the upper chamber of the Transwell plate at a density of 2 × 10⁵ cells/mL. A culture medium (600 μL) was added to the lower chamber, and the plate was gently shaken for the uniform distribution of cells. The culture was continued for 21 days, the TEER of the cells was measured regularly with an RE1600 epithelial cell voltage resistance meter (KingTech Technology Co., Ltd, Beijing, China), and the integrity of the cell monolayer was evaluated. When the cells were cultured to the 21st day, the TEER reached about 250 Ω·cm², indicating the formation of a complete and dense monolayer of cells. After TNF-α was added to stimulate the cells, the TEER of the cells was monitored every 1 h to 24 h and calculated using Formula (1). Three replicates were set for each experimental group.

(1)\mathrm{T}\mathrm{E}\mathrm{E}\mathrm{R}(\mathrm{\Omega }\cdot {\mathrm{c}\mathrm{m}}^2)=({\mathrm{R}}_{\mathrm{t}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{p}}-{\mathrm{R}}_{\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{p}})\times 0.33

where 0.33 is the membrane area in the upper chamber of the Transwell plate.

2.12 Epithelial monolayer permeability assay

First, Caco-2 cells were cultured in Transwell plates for 21 days to form a complete cell monolayer. Then, the cell monolayer was treated with different concentrations of ADT-OH for 24 h and stimulated by TNF-α for 24 h. Three replicates were set for each experimental group. The upper and lower chambers of the Transwell plate were washed 3 times with Hank’s balanced salt solution (Beijing Solarbio Science & Technology Co., Ltd, China). After that, 100 μL of fluorescein isothiocyanate-labeled dextran (FITC-dextran, 4-kDa, Sigma‒Aldrich) solution (10 mg/mL) was added to the upper chamber, and 600 μL of Hank’s balanced salt solution was added to the lower chamber, which was further incubated for 2 h at 37 °C in the dark. After 100 μL of the solution in the lower chamber was removed, its fluorescence value (excitation wavelength 485 nm, emission wavelength 528 nm) was detected with a microplate reader, and the apparent permeability coefficient (P_app) of transmembrane transport was calculated using Formula (2).

(2){\mathrm{P}}_{\mathrm{a}\mathrm{p}\mathrm{p}}(\mathrm{c}\mathrm{m}/\mathrm{s})=\frac{\mathrm{\Delta }\mathrm{Q}}{\mathrm{\Delta }\mathrm{t}\times 0.33\times C_0}

where ΔQ is the throughput of FITC-dextran within Δt, 0.33 is the membrane area in the upper chamber of the Transwell plate, and C₀ is the initial concentration of FITC-dextran added to the upper chamber of the Transwell plate (10 mg/mL).

2.13 Adhesion assays between macrophages and vascular endothelial cells

HUVECs were treated with different concentrations of ADT-OH in a 12-well plate for 24 h until confluence. RAW 264.7 cells that were uniformly seeded on the HUVEC monolayer were stimulated using TNF-α for 24 h. After TNF-α stimulation, the RAW 264.7 cells that underwent different treatments were harvested and then incubated with HUVECs at a density of 10⁵ cells per well. After 2 h, unattached RAW 264.7 cells were removed by washing with PBS buffer, and the remaining attached cells were observed with a microscope and counted with ImageJ software. The results were expressed as the mean number of RAW 264.7 cells attached to the HUVEC monolayer per square millimeter.

2.14 In vivo intestinal permeability assay

In vivo intestinal permeability was evaluated by the FITC-dextran permeability assay [38]. Briefly, mice fasted for 3 h were given 400 μL of FITC-dextran (FD4, Sigma‒Aldrich) solution by gavage. After 4 h, blood was collected from each mouse from the retro-orbital vein, left to stand in the dark at room temperature for 2–3 h, and centrifuged at 12 000 rpm for 30 min at 4 °C. Serum was analyzed for FITC-dextran concentration with a fluorescence spectrophotometer (excitation wavelength: 485 nm, emission wavelength: 528 nm). Standard curves for calculating the FITC-dextran concentration (0, 125, 250, 500, 1000, 2000, 6000, and 8000 ng/mL) in the samples were obtained by diluting the FITC-dextran solution in PBS.

2.15 Fecal microbiota analysis by 16S rRNA sequencing

The fecal samples were collected in 1.5 mL tubes and immediately frozen in liquid nitrogen. In this study, the number of mice used was 10 per group. Before sequencing, we mixed mouse fecal samples from two mice per group and then extracted DNA. 16S rRNA (rRNA) gene sequencing was performed as described previously [39]. Briefly, genomic DNA was extracted using a QIAamp DNA stool mini kit (51504, QIAGEN) according to the manufacturer’s instructions. The V3–V4 variable regions of the bacterial 16S rRNA gene were amplified by PCR with the specific primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The PCR conditions were as follows: initial denaturation at 94 °C for 4 min, followed by 25 cycles of 94 °C denaturation for 30 s, 50 °C annealing for 45 s, and 72 °C extension for 30 s; final extension at 72 °C for 5 min. PCR products were cleaned and subsequently sequenced by an Illumina MiSeq PE300 system (OEbiotech Co, Ltd.) according to standard protocols. The resultant sequences were screened for quality by using the QIIME software package (version 1.9.1), and the effective sequences were further clustered into operational taxonomic units (OTUs) of ≥97% similarity. The relative abundance of each OTU, other taxonomic levels (from phylum to species), beta diversity (between-sample dissimilarity), and principal component analysis were determined for each sample by using the MOTHUR program.

2.16 Fecal microbiota transplantation (FMT)

The experimental design is shown in Fig.3. After 1 week of acclimation, donor mice were divided randomly into two groups: (1) Control group (n = 12): mice were treated with PBS for 3 weeks; (2) ADT-OH group (n = 12): mice were treated with ADT-OH (25 μmol/kg body weight) by intraperitoneal injection daily. After 3 weeks of administration, fresh fecal samples were collected daily and transplanted into fecal microbiota transplantation (FMT)-recipient mice. All FMT-recipient mice were treated with 3% DSS in drinking water for 7 days, and fecal samples from the control group (Con-FMT) or ADT-OH group donor mice (ADT-OH-FMT) were transplanted. For the fecal transplants, fresh feces from each group were pooled and homogenized and diluted in sterile saline with a final concentration of 1 mg of feces/10 μL. Pooled samples were centrifuged at 3000 rpm for 5 min. The supernatant was collected through 70 μm filters and gavaged orally to each mouse (10 mL/kg) for five consecutive days.

2.17 Statistical analysis

All in vivo experiments were repeated three times, each group had six to eight mice, and the total sample size was over 18. For in vitro experiments, each sample had three replicates (biological), and the experiments were repeated three times. Statistical analysis was conducted by GraphPad Prism 8.0. All data are presented as the mean ± standard deviation (SD). One-way analysis of variance was used to analyze differences between groups, and a two-tailed Student’s t-test was performed for the analysis of the significance of two groups. The P values indicated in the figures are < 0.05 (*), < 0.01 (**), < 0.005 (***), and < 0.001 (****).

3 Results

3.1 ADT-OH alleviates DSS-induced acute inflammatory bowel disease in mice

In the current study, we first established an acute colitis model by treating mice with 3% DSS. One day after treatment, intraperitoneal injection of ADT-OH or the positive control drug NaHS into mice was conducted. During this process, body weight, stool consistency, and fecal occult blood were monitored daily. As shown in Fig.1, 3% DSS induced significant body weight loss, but ADT-OH treatment significantly slowed the decrease in body weight, especially in the low-dose (25 μmol/kg) ADT-OH group. In addition, the ADT-OH treatment groups showed significantly lower disease activity index (DAI) scores than the model group (P < 0.001), indicating that ADT-OH relieved DSS-induced acute colitis (Fig.1). In agreement with the reduced inflammation, colon length increased in the low-dose ADT-OH group compared with the model group (P < 0.01; Fig.1, 1E). Furthermore, H&E staining of the colonic tissue showed less disrupted tissue architecture and fewer signs of inflammation in the low-dose ADT-OH group compared with the model group (Fig.1). The data were further confirmed by histological score analysis, showing a lower score in the low-dose ADT-OH group (Fig.1). Intriguingly, the low-dose ADT-OH exhibited a better anti-inflammatory effect than the medium-dose ADT-OH (50 μmol/kg) and high-dose ADT-OH (100 μmol/kg). According to these results, we detected the production capacity of H₂S in mouse colonic tissues. As shown in Fig. S1A, 3% DSS induced a significant reduction in H₂S levels, but treatment with a low dose of ADT-OH reversed this outcome. Consistently, low-dose ADT-OH increased H₂S levels by increasing the protein expression levels of CBS and CSE in mouse colonic tissues (Fig. S1B and S1C). Notably, medium-dose or high-dose ADT-OH and NaHS cannot increase endogenous H₂S levels. Thus, we speculated that the H₂S level stimulated by ADT-OH at a relatively low concentration has a practical anti-inflammatory effect in mice.

3.2 ADT-OH is safe without any short- or long-term toxicological adverse effects in mice

To evaluate the safety of ADT-OH in vivo, six mice in each group were injected intraperitoneally with 0, 25, 50, and 100 μmol/kg ADT-OH, and body weight and relative organ weight were monitored for toxicity assessment (Fig.4). Continuous treatment with ADT-OH for 5 days did not affect body weight, and no significant difference was found among the treated groups (Fig.4). Meanwhile, no significant differences were observed in the liver, kidney, and spleen indexes of all groups (Fig.4). Potential cytotoxic and adverse effects were evaluated according to ALT, ALP, BUN, and TBIL activity and CREA levels. The markers were not elevated after treatment with different concentrations of ADT-OH (Fig.4). For the precise evaluation of the toxic effects of ADT-OH on major organs, H&E staining was performed (Fig.4). No obvious pathologic changes were found in the liver and kidney. These results suggested that ADT-OH has good biosafety and has no acute toxicity or severe side effects.

Furthermore, we conducted a long-term treatment study (one month) with ADT-OH in DSS-induced chronic colitis (Fig.2). Long-term prevention with ADT-OH blunted DSS-induced weight loss and decreased the disease activity index (Fig.2 and 3C). The shortening of the colon, which is a typical pathological feature of colitis, was substantially inhibited (Fig.2). Histological analysis showed the inhibitory effect of ADT-OH on colonic inflammation, which exhibited a reduced level of crypt damage and severity of inflammation (Fig. S2A). Consistent with the 5-day intervention study, long-term treatment with ADT-OH did not lead to any significant side effects. As shown in Fig.2, no significant differences in the weight indexes of the heart, liver, spleen, lung and kidney were found among the treatment groups. No tissue-specific toxicity in the major organs was indicated in the H&E staining results (Fig. S2B–S2D). Additionally, we evaluated organ toxicity by measuring biochemical parameters in the serum. As shown in Fig.2, all biochemical parameters (ALT, ALP, TBIL, BUN, and CREA) tested in this study were within the normal ranges after treatment with ADT-OH, suggesting that long-term treatment with ADT-OH is safe and does not induce toxicity in the liver and kidney.

3.3 ADT-OH regulates inflammatory factors and cytokines in colon tissues

As DSS-induced inflammation is associated with the release of an array of proinflammatory cytokines [36], we assessed whether ADT-OH treatment affects proinflammatory cytokine production. As expected, the mRNA expression levels of the proinflammatory cytokines TNF-α, interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) significantly increased in the DSS-treated mice. ADT-OH treatment decreased the expression levels of TNF-α, IL-1β, and IL-6 (Fig.5–4C). Myeloperoxidase (MPO) is a crucial indicator of inflammatory cell accumulation. Consistent with an attenuated inflammatory phenotype, ADT-OH decreased MPO levels in colon tissues (Fig.5). Furthermore, DSS increased CD45-positive immune regulatory cells and F4/80-positive macrophages in the colons of mice, indicating that DSS-treated mice displayed more severe colitis-associated symptoms. After ADT-OH (25 μmol/kg) administration, CD45-positive immune regulatory cells and F4/80-positive macrophages decreased by 63.58% and 66.40%, respectively (Fig.5 and 4F). In addition, ADT-OH significantly increased the number of regulatory T (Treg) cells in intestinal intraepithelial lymphocytes and lamina propria lymphocytes (Fig. S3A–S3C).

3.4 ADT-OH suppresses inflammatory response by blocking the activation of nuclear factor kappa-B (NF-κB) p65 signaling

To determine whether ADT-OH can attenuate inflammation in vitro, we used different concentrations of TNF-α to induce inflammation in Caco-2 and HCT-116 cells. As TNF-α concentrations increased, the relative mRNA levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), IL-1β, and IL-6 showed concentration-dependent increases (Figs. S4A and S5A). Cell counting kit-8 assay results showed that TNF-α (50 ng/mL) or ADT-OH (25 and 50 μmol/L) treatment did not affect cell viability; however, high-concentration ADT-OH (> 50 μmol/L) significantly inhibited cell viability (P < 0.0001; Figs. S4B and S5B). To reduce the cytotoxicity and enhance the anti-inflammatory effect in vitro, we used 50 ng/mL TNF-α to stimulate the cells, and 25 or 50 μmol/L ADT-OH was used to treat cells for further study. Flow cytometry analysis indicated that TNF-α and ADT-OH did not cause apparent cell apoptosis in vitro (Figs. S4C and S5C). In contrast to the positive control treated with the apoptosis inducer staurosporine (2 μmol/L), cleaved caspase-3 was not observed in other groups (Figs. S4D and S5D). Then, H₂S production level was determined in vitro. As shown in Fig. S4E, inflammation induced by TNF-α caused a significant reduction in H₂S levels; however, ADT-OH treatment completely restored the level of H₂S. Thus, we concluded that low-dose ADT-OH did not cause cell damage, and its ability to increase H₂S levels might endow cells with a certain anti-inflammatory ability in vitro and in vivo.

The potential anti-inflammatory effect of ADT-OH was further studied by qRT-PCR analysis in vitro. The results showed that ADT-OH inhibited increases in the mRNA expression levels of several proinflammatory factors, including iNOS, COX-2, IL-1β, and IL-6, induced by TNF-α stimulation (Fig.5). The nuclear factor kappa-B (NF-κB) signaling pathway is closely related to inflammation and inflammation-associated colorectal cancer [40]. Western blot results showed that TNF-α significantly increased the expression level of NF-κB p65 in the nuclei of Caco-2 cells, whereas ADT-OH co-treatment suppressed NF-κB p65 translocation into the nuclei (Fig.5). To confirm NF-κB activation and nuclear translocation, we performed an immunofluorescence staining assay. Clearly, the nuclear translocation of NF-κB p65 upon TNF-α treatment exhibited an increase from the cytoplasm to the nucleus, indicating that TNF-α effectively activated NF-κB. ADT-OH supplementation in the TNF-α treatment group decreased the nuclear translocation of NF-κB (Fig. S4F). Collectively, these results showed that ADT-OH treatment decreased the activation of NF-κB and its subsequent nuclear translocation.

3.5 ADT-OH inhibits adhesion between macrophages and endothelial cells in an inflammatory environment

The modulation of adhesion molecule expression by endothelial cells may be one mechanism of inflammation production. To investigate the impact of ADT-OH treatment on cell adhesion, we used a model of endothelial cell (HUVEC)–macrophage (RAW264.7) interactions in vitro. HUVECs are spindle-like in shape and are indicated by blue arrows, whereas RAW264.7 cells are small round cells and are indicated by red arrows (Fig.6). RAW 264.7 cells did not randomly adhere to endothelial cells in the control group. After TNF-α stimulation, RAW 264.7 cells showed a significantly greater ability to adhere and invade endothelial cells. As expected, ADT-OH co-treatment decreased the number of RAW264.7 macrophages that adhered to HUVECs (Fig.6). Cell adhesion mainly relies on the expression of adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), and endothelium selectin (E-selectin) [41]. The qRT-PCR results showed that TNF-α upregulated the expression of ICAM-1, VCAM-1, and E-selectin, whereas ADT-OH treatment significantly suppressed their upregulation (P < 0.001; Fig.6–5E). These data indicated that ADT-OH suppresses inflammatory responses induced by TNF-α by inhibiting cell adhesion function.

3.6 ADT-OH decreases intestinal permeability in an inflammatory environment

Inflammatory factors disrupt intestinal barrier integrity, leading to increased intestinal permeability in patients with IBD [42]. To assess whether ADT-OH can alleviate intestinal barrier integrity damage, we monitored the TEER value in a Caco-2 cell monolayer model. As shown in Fig.7, TNF-α induced a significant reduction in TEER to 0.61 ± 0.12-fold of baseline values after 5 h, whereas ADT-OH intervention blocked TNF-α-induced loss of TEER. Evidently, ADT-OH inhibited cell monolayer integrity damage in a dose-dependent manner. The TEER value of the 50 μM ADT-OH treatment group was similar to that of the blank control group 24 h after treatment (Fig.7). In the Caco-2 cell monolayer model, relative permeability was calculated as the ratio between the FITC signals in the lower chamber and the initial upper chamber. As expected, the application of ADT-OH blocked the TNF-α-induced increase in epithelial permeability of Caco-2 monolayers (Fig.7). Consistent with the in vitro results, ADT-OH treatment significantly inhibited the increase in intestinal permeability in mice (Fig.7). Collectively, these data indicated that ADT-OH plays a vital role in the regulation of intestinal epithelial barrier function in an inflammatory environment.

3.7 ADT-OH restores intestinal epithelial barrier dysfunction in vitro and in vivo

Given that tight junction (TJ) complexes are the basic elements that maintain the integrity of the intestinal barrier, we observed changes in their ultrastructures with a transmission electron microscope. In the control group, intestinal epithelial cell surface microvilli were arranged in neat rows, and the TJ (blue arrows) and desmosome (green arrows) structures were regular and clear (Fig.8). When acute colitis was induced in mice by 3% DSS treatment, the TJ structure was disordered, and desmosomes disappeared. However, after ADT-OH treatment, abnormalities in the structural integrity of TJs and desmosomes were not observed. The expression of the TJ proteins zonula occludens-1 (ZO-1) and occludin in the colon tissues of mice was further tested. As shown in Fig.8, the expression levels of ZO-1 and occludin significantly decreased in the DSS-induced group, whereas ADT-OH treatment effectively alleviated these changes. To further investigate the integrity of tight junctions, we performed ZO-1 and occludin immunofluorescence staining. As shown in Fig.8, ZO-1 and occludin expression levels significantly increased in the ADT-OH-treated group compared with those in the DSS group. Interestingly, Western blot results indicated that whether the cells were treated with TNF-α alone or in combination with ADT-OH, the protein expression levels of ZO-1 and occludin did not change in vitro (Fig. S4G). Consistent with the in vivo results, immunofluorescence staining results showed that ADT-OH restored the protein levels of ZO-1 and occludin in vitro (Fig.8–7G, Fig. S5E and S5F).

To better understand the mechanism by which ADT-OH regulates the intestinal barrier, we examined the dynamic remodeling of cytoskeletal filamentous actin (F-actin) in Caco-2 cells. After TNF-α stimulation, F-actin was obviously reorganized, and its distribution profiles became irregular and discontinuous. By contrast, ADT-OH treatment attenuated the TNF-α-induced abnormal remodeling of F-actin (Fig. S5G). Accumulating studies have shown that myosin II regulatory light chain (MLC) phosphorylation mediated by myosin light chain kinase (MLCK) plays a crucial role in proinflammatory cytokine-induced intestinal barrier disruption. MLCK-dependent MLC phosphorylation can result in the cellular redistribution of cell–cell junction proteins and further affect cytoskeleton remodeling [43]. Thus, the effect of ADT-OH on the MLCK-P-MLC signaling pathway was further investigated. After the Caco-2 monolayers were treated with ADT-OH alone, no significant change in the expression level of MLCK was observed (Fig.8). However, TNF-α treatment led to a great increase in MLCK protein levels and MLC phosphorylation levels (P < 0.0001). Compared with the TNF-α-alone treatment group, ADT-OH treatment blocked the TNF-α-induced elevation of MLC phosphorylation and MLCK protein expression. Taken together, we concluded that ADT-OH can attenuate TNF-α-induced intestinal barrier dysfunction by suppressing MLCK-mediated MLC phosphorylation.

3.8 ADT-OH improves gut microbiota diversity and restores gut microbiota composition in DSS-induced colitis mice

The gut flora is essential to protection against inflammation [13]. To investigate whether ADT-OH treatment can result in gut microbiota changes in vivo, we performed 16S rRNA sequencing and assessed alterations in the gut microbial community of mouse feces. After treatment with ADT-OH, the α-diversity of the gut microbiome was significantly increased compared with the DSS model group, as reflected by Chao1 index (403.02 vs. 252.78, P < 0.01), observed species (400.84 vs. 252.24, P < 0.01), Simpson’s diversity index (0.98 vs. 0.94, P < 0.01), and Shannon diversity index (7.12 vs. 5.43, P < 0.01; Fig. S6A–S6D). Principal coordinate analysis (PCoA) was used to examine global differences in microbial community structure. The PCoA successfully separated samples from the DSS and ADT-OH groups but failed to separate samples from the control and ADT-OH groups, in line with the NMDS analysis (Fig. S6E and S6F). In addition to using PCoA, samples were clustered using the unweighted pair group method with arithmetic mean. The results showed that the ADT-OH treatment group was closer to the control in terms of similarity of microbial communities than the DSS group (Fig. S6G).

Furthermore, clustering analysis of the bacterial community structure at different levels was performed (Fig. S7). The results showed that samples within the same group tended to cluster together, and all samples showed good concordance of duplicates and clustering according to the different groups. Notably, the control group showed the best sample clustering and was clearly separated from the DSS and ADT-OH groups. At the genus level, the ADT-OH treatment group clustered closer to the control group than the DSS alone group. The relative abundance of the gut microbiota was further analyzed. Eight bacterial phyla were detected in the DSS group, whereas the number of total phyla increased to 15 in the ADT-OH group (Fig.9). The relative abundance values of Campilobacterota, Deferribacterota, and Desulfobacterota in the DSS group (18.40%, 11.08%, and 8.98%, respectively) were higher than those in the control group (3.71%, 1.38%, and 3.49%, respectively), whereas the relative abundance of Bacteroidota was lower in the DSS group (30.84%) than in the control group (71.9%; Fig. S8A). Firmicutes and Bacteroidetes were the most dominant phyla, and DSS significantly changed the Firmicutes/Bacteroidetes (F/B) ratio (P < 0.0001; Fig. S8B). ADT-OH administration significantly reversed these changes in microbiota composition at the phylum level and decreased the F/B ratio (Fig.9 and 8C). Consistently, linear discriminant analysis effect size (LEfSe) indicated that bacteria belonging to Campilobacterota and Deferribacterota were enriched in the DSS-alone treatment group, whereas Actinobacteriota and Proteobacteria were enriched in the ADT-OH-treated DSS group (Fig.9; Fig. S8C and S8D). At the genus level, ADT-OH administration restored the gut microbiota composition in DSS-induced colitis mice. This effect was characterized by a significant increase in the abundance of Muribaculaceae, Alistipes, and Clostridia_UCG-014 (P < 0.05; Fig.9, Fig. S8E) and a marked decrease in the abundance of Helicobacter, Mucispirillum, Parasutterella, and Desulfovibrio (P < 0.01; Fig.9, Fig. S8F).

The possible correlations between the gut microbiota and proinflammatory cytokines were examined with Spearman’s correlation analyses. As shown in Fig.9, three bacteria were negatively correlated with proinflammatory cytokines (P < 0.05), including Muribaculaceae, Lactobacillus, and Alistipes. Notably, these three bacteria, especially Muribaculaceae and Alistipes, were separately enriched in the control and ADT-OH groups but not in the DSS group (Fig.9, Fig. S8E). Furthermore, Helicobacter, Mucispirillum, Lachnospiraceae_NK4A136_group, and Parasutterella were positively correlated with proinflammatory cytokines (P < 0.05). Intriguingly, except for Lachnospiraceae_NK4A136_group, these bacteria were all enriched in the DSS-alone treatment, whereas their relative abundance decreased significantly after ADT-OH treatment (Fig.9, Fig. S8F). These results confirmed that ADT-OH treatment can restore gut microbiota composition in DSS-induced colitis. Its anti-inflammatory effects might be correlated with the increased abundance of beneficial bacteria (Muribaculaceae, Alistipes) and decreased abundance of pathogenic bacteria (Helicobacter, Mucispirillum) in the gut flora of mice.

3.9 Transplantation of the gut microbiota of ADT-OH-treated mice attenuated DSS-induced colitis

To verify ADT-OH-mediated interactions between gut microbiota and inflammatory factors, we performed a fecal microbiome transplant experiment. The experimental design is shown in Fig.3. After 1 week of acclimation, donor mice were divided randomly into two groups: (1) Control group (n = 12): mice were treated with PBS for 3 weeks; (2) ADT-OH group (n = 12): mice were treated with ADT-OH (25 μmol/kg body weight) by intraperitoneal injection daily. After 3 weeks of administration, fresh fecal samples were collected daily and transplanted into FMT recipient mice. Our results showed that ADT-OH-modulated microbiota effectively alleviated DSS-induced weight loss (Fig.3). ADT-OH-FMT mice showed better stool consistency and a lower disease activity index than Con-FMT mice (Fig.3). Furthermore, macroscopic examination of the colons at day 13 revealed longer colons in the mice that received ADT-OH-FMT (Fig.3 and 9E). These results showed that the transplantation of feces from mice treated with ADT-OH can improve DSS-induced colitis in mice. Furthermore, we assessed the expression levels of proinflammatory cytokines in the serum and colonic tissues. The ELISA results showed that ADT-OH-FMT significantly reduced the levels of the proinflammatory factors TNF-α, IL-1β, and IL-6 in the sera (Fig.3). Meanwhile, the mRNA expression of colonic TNF-α, IL-1β, and IL-6 decreased in the ADT-OH-FMT mice (Fig.3). Overall, the above results demonstrated that transplanting ADT-OH-modulated microbiota can alleviate DSS-induced colitis and negatively regulate the expression of local and systemic proinflammatory cytokines.

4 Discussion

IBD is a worldwide health problem with a rapidly increasing incidence in all populations. Conventional anti-inflammatory drugs have been used for over four decades; however, these therapies generally cause significant side effects [44]. Increasing studies have reported physiologic and pathological effects of H₂S on the gastrointestinal tract, indicating that H₂S plays an important role in IBD and CRC [18,45]. Several novel derivatives released by H₂S donors have been developed and tested extensively in preclinical models. For instance, H₂S-releasing NSAIDs can overcome the gastric side effects and toxicity of traditional NSAIDs, showing good antioxidant effects [46]. In addition, H₂S-releasing aspirin prevents aspirin-induced gastric injury by reducing oxidative stress [47]. Thus, we believe that H₂S sustained-release donors are potential anti-inflammatory agents.

Currently, ADT-OH is considered the most widely used slow-releasing organic H₂S donor, and its derivatives release H₂S in a controlled manner in vivo and in vitro [48]. Indeed, ADT-OH plays a critical role in many inflammatory and autoimmune diseases. In the ischemic brain model, ADT-OH inhibited the expression of the proinflammatory markers iNOS and IL-1β while enhancing the expression of the anti-inflammatory markers arginase 1 and interleukin-10 [33]. In this study, we further broadened the potential anti-inflammatory effect of ADT-OH by demonstrating its ability to regulate intestinal barrier function in IBD. Our results showed that ADT-OH reduced the expression of proinflammatory cytokines, including TNF-α, IL-1β, and IL-6, and alleviated DSS-induced inflammatory bowel disease in vivo. The alleviation of intestinal inflammation was reflected by reduced MPO activity in the colonic tissues. Previous studies have demonstrated that transcription factors of the NF-κB family play a central role in the inducible expression of inflammatory genes during an immune response [49]. In the in vitro experiment, we confirmed that TNF-α treatment indeed promoted NF-κB p65 translocation into the nucleus, whereas ADT-OH significantly inhibited the activation of NF-κB p65 and its nuclear translocation. Notably, in the DSS-induced acute colitis model, we found that mice treated with a low concentration of ADT-OH had high endogenous hydrogen sulfide production in colonic tissues. Although researchers are actively developing methods for detecting H₂S, establishing an effective and convenient method remains a challenge [50,51]. Specifically, we cannot satisfactorily measure H₂S concentration accurately in the colon tissues of mice in real time. Meanwhile, ADT-OH is a slow-releasing organic H₂S donor, which might not directly determine the H₂S level of colon tissues in a dose-dependent manner. H₂S can be classified into exogenous or endogenous forms, and it cannot be clearly differentiated in vivo. Actually, the H₂S referred to in this study may be a mixture. We found that H₂S did not show a dose dependence during ADT-OH treatment in vivo. Thus, we supposed that the hydrogen sulfide detected by the kit might mainly belong to endogenous hydrogen sulfide, which is mainly produced by CBS, CSE and 3-mercaptopyruvate sulfurtransferase [52,53]. For validation, we examined the protein expression levels of CBS and CSE in the colon tissues of mice. As expected, Western blot results showed that the protein expression levels of CBS and CSE in colon tissues of mice treated with low concentrations of ADT-OH were significantly higher than those in the other groups. This is a very interesting and valuable point that deserves investigation into the underlying mechanism in our future study. In addition, we found that ADT-OH could not induce cell apoptosis within an inflammatory environment. As a potential anti-inflammatory drug, ADT-OH did not show toxicity toward the mouse model in the acute toxicity test [54], and long-term treatment with ADT-OH did not lead to any significant side effects.

In the last decade, cell adhesion molecules have been shown to play an essential role in immune regulation and the inflammatory response. Adhesion molecules, including ICAM-1, VCAM-1, and E-selectin, are mainly involved in leukocyte trafficking and interaction with their receptors on the surface of endothelial cells [41,55,56]. The upregulation of adhesion molecules resulted in a significant adhesion increase between macrophages and vascular endothelial cells, an early inflammation indicator [57]. Lin et al. found that exogenous H₂S suppressed the adhesion of macrophages to endothelial cells; this process is a crucial step in the inhibition of inflammation generation and progression [58]. In our study, we found that ADT-OH is involved in macrophage adhesion regulation. TNF-α successfully stimulated RAW264.7 cells, increasing adhesion molecules, including ICAM-1, VCAM-1, and E-selectin, whereas ADT-OH cotreatment significantly suppressed their upregulation. Junctional integrity within the intestine is mediated by several multiprotein adhesion complexes, including tight and adherence junctions [59]. Defects in intestinal epithelial barrier function, including alterations in TJ structure and epithelial permeability, have been observed in IBD [60]. ZO-1 and occludin are important tight junction proteins that are downregulated in various inflammatory diseases [11]. Our study showed that ADT-OH inhibited the increase in intestinal permeability by increasing the protein expression of the tight junction proteins ZO-1 and occludin and blocking the increase in MLC phosphorylation and MLCK protein expression.

Many studies have clearly indicated that increased intestinal permeability results in bacterial translocation and changes the gut microbial composition [9,13]. Coincidentally, gut microbiota disturbance can serve as a primary factor that augments proinflammatory cytokines and T helper cells, increasing intestinal barrier permeability [61]. Dysbiosis of the gut microbiome is considered a crucial pathogenesis factor of IBD and is characterized by decreased microbiota diversity in the gut [14]. We found that DSS treatment reduced gut microbiota richness and altered the Firmicutes to Bacteroidetes ratio, whereas ADT-OH administration restored the composition of the gut microbiota. In patients with IBD, decreased beneficial Lactobacillus and increased potential harmful Enterobacteria are closely related to IBD severity and treatment effectiveness [62]. Here, our results showed that ADT-OH heightened the colonization of beneficial bacteria in the gut, as evidenced by the significant increase in the relative abundance of Muribaculaceae and Alistipes in the ADT-OH group. A decrease in the abundance of Muribaculaceae is a crucial factor in the development of colitis, and Alistipes showed a significant protective effect against inflammatory diseases [63,64]. Moreover, ADT-OH treatment decreased the abundance of potentially pathogenic bacteria in DSS-induced mice, including Helicobacter, Mucispirillum, Parasutterella, and Desulfovibrio. Notably, our study indicated that Helicobacter and Mucispirillum are positively related to inflammatory cytokines (TNF-α, IL-β, and IL-6). In contrast, Muribaculaceae and Alistipes were negatively correlated with the three cytokines. Altered gut microbiota abundance correlates with immune gene expression in inflammatory diseases [65–67]. To verify whether the beneficial effects of ADT-OH on inflammation are mediated by the gut microbiota, we transplanted the fecal microbiota modulated by ADT-OH into DSS-induced mice. Interestingly, acute colitis was successfully alleviated by ADT-OH-FMT. Consistent with the results of intraperitoneal injection of ADT-OH, ADT-OH-FMT was more effective in alleviating the symptoms of colitis than Con-FMT. Transplanting ADT-OH-modulated microbiota can negatively regulate the expression of local and systemic proinflammatory cytokines. Considering these results, we believe that the ADT-OH-mediated gut microbiota can inhibit the production of proinflammatory factors and potentially exert beneficial effects on intestinal homeostasis.

5 Conclusions

Our research demonstrated that the slow-releasing H₂S donor ADT-OH is a potent anti-inflammatory agent that alleviates intestinal inflammation by inhibiting the expression of proinflammatory factors, reducing intestinal permeability, and restoring the gut microbiota (Fig.10). We believe that targeting intestinal barrier function with ADT-OH treatment is a novel therapeutic option for IBD, although further studies are needed to evaluate its clinical application.

5.0.0.0.1 Acknowledgements

This study was supported by grants from the National Natural Science Foundation of China (Nos. 82130106 and 32250016), Nanjing Special Fund for Life and Health Science and Technology (No. 202110016), and Changzhou Municipal Department of Science and Technology (Nos. CZ20210010, CJ20210024, and CJ20220019).

References

[1] Rubin SJS, Bai L, Haileselassie Y, Garay G, Yun C, Becker L, Streett SE, Sinha SR, Habtezion A. Mass cytometry reveals systemic and local immune signatures that distinguish inflammatory bowel diseases. Nat Commun 2019; 10(1): 2686–2699

[2] Zhou J, Huang S, Wang Z, Huang J, Xu L, Tang X, Wan YY, Li QJ, Symonds ALJ, Long H, Zhu B. Targeting EZH2 histone methyltransferase activity alleviates experimental intestinal inflammation. Nat Commun 2019; 10(1): 2427–2437

[3] Liu TC, Stappenbeck TS. Genetics and pathogenesis of inflammatory bowel disease. Annu Rev Pathol 2016; 11(1): 127–148

[4] Danese S, Fiocchi C. Etiopathogenesis of inflammatory bowel diseases. World J Gastroenterol 2006; 12(30): 4807–4812

[5] Zhang YZ, Li YY. Inflammatory bowel disease: pathogenesis. World J Gastroenterol 2014; 20(1): 91–99

[6] Bernstein CN. Treatment of IBD: where we are and where we are going. Am J Gastroenterol 2015; 110(1): 114–126

[7] TargownikLEBernsteinCN. Infectious and malignant complications of TNF inhibitor therapy in IBD. Am J Gastroenterol 2013; 108(12): 1835–1842, quiz 1843 doi:10.1038/ajg.2013.294

[8] Stappenbeck TS, Rioux JD, Mizoguchi A, Saitoh T, Huett A, Darfeuille-Michaud A, Wileman T, Mizushima N, Carding S, Akira S, Parkes M, Xavier RJ. Crohn disease: a current perspective on genetics, autophagy and immunity. Autophagy 2011; 7(4): 355–374

[9] Meddings JB, Sutherland LR, May GR. Intestinal permeability in patients with Crohn’s disease. Gut 1994; 35(11): 1675–1676

[10] Nusrat A, Parkos CA, Verkade P, Foley CS, Liang TW, Innis-Whitehouse W, Eastburn KK, Madara JL. Tight junctions are membrane microdomains. J Cell Sci 2000; 113(10): 1771–1781

[11] GroschwitzKRHoganSP. Intestinal barrier function: molecular regulation and disease pathogenesis. J Allergy Clin Immunol 2009; 124(1): 3–20, quiz 21–22 doi:10.1016/j.jaci.2009.05.038

[12] Turner JR, Rill BK, Carlson SL, Carnes D, Kerner R, Mrsny RJ, Madara JL. Physiological regulation of epithelial tight junctions is associated with myosin light-chain phosphorylation. Am J Physiol 1997; 273(4): C1378–C1385

[13] Garrett WS, Gordon JI, Glimcher LH. Homeostasis and inflammation in the intestine. Cell 2010; 140(6): 859–870

[14] Morgan XC, Tickle TL, Sokol H, Gevers D, Devaney KL, Ward DV, Reyes JA, Shah SA, LeLeiko N, Snapper SB, Bousvaros A, Korzenik J, Sands BE, Xavier RJ, Huttenhower C. Dysfunction of the intestinal microbiome in inflammatory bowel disease and treatment. Genome Biol 2012; 13(9): R79

[15] Mukhopadhya I, Hansen R, El-Omar EM, Hold GL. IBD-what role do Proteobacteria play? Nat Rev Gastroenterol Hepatol 2012; 9(4): 219–230 doi:10.1038/nrgastro.2012.14

[16] Lavelle A, Lennon G, O'Sullivan O, Docherty N, Balfe A, Maguire A, Mulcahy HE, Doherty G, O'Donoghue D, Hyland J, Ross RP, Coffey JC, Sheahan K, Cotter PD, Shanahan F, Winter DC, O'Connell PR. Spatial variation of the colonic microbiota in patients with ulcerative colitis and control volunteers. Gut 2015; 64(10): 1553–1561

[17] Wang M, Molin G, Ahrné S, Adawi D, Jeppsson B. High proportions of proinflammatory bacteria on the colonic mucosa in a young patient with ulcerative colitis as revealed by cloning and sequencing of 16S rRNA genes. Dig Dis Sci 2007; 52(3): 620–627

[18] Singh SB, Lin HC. Hydrogen sulfide in physiology and diseases of the digestive tract. Microorganisms 2015; 3(4): 866–889

[19] Szabo C, Hellmich MR. Endogenously produced hydrogen sulfide supports tumor cell growth and proliferation. Cell Cycle 2013; 12(18): 2915–2916

[20] Burguera EF, Meijide-Failde R, Blanco FJ. Hydrogen sulfide and inflammatory joint diseases. Curr Drug Targets 2017; 18(14): 1641–1652

[21] Fiorucci S, Orlandi S, Mencarelli A, Caliendo G, Santagada V, Distrutti E, Santucci L, Cirino G, Wallace JL. Enhanced activity of a hydrogen sulphide-releasing derivative of mesalamine (ATB-429) in a mouse model of colitis. Br J Pharmacol 2007; 150(8): 996–1002

[22] Muniraj N, Stamp LK, Badiei A, Hegde A, Cameron V, Bhatia M. Hydrogen sulfide acts as a pro-inflammatory mediator in rheumatic disease. Int J Rheum Dis 2017; 20(2): 182–189

[23] Miao X, Meng X, Wu G, Ju Z, Zhang HH, Hu S, Xu GY. Upregulation of cystathionine-β-synthetase expression contributes to inflammatory pain in rat temporomandibular joint. Mol Pain 2014; 10: 9

[24] AhmadASzaboC. Both the H₂S biosynthesis inhibitor aminooxyacetic acid and the mitochondrially targeted H₂S donor AP39 exert protective effects in a mouse model of burn injury. Pharmacol Res 2016; 113(Pt A):348–355

[25] Zhang HX, Liu SJ, Tang XL, Duan GL, Ni X, Zhu XY, Liu YJ, Wang CNH. H₂S attenuates LPS-induced acute lung injury by reducing oxidative/nitrative stress and inflammation. Cell Physiol Biochem 2016; 40(6): 1603–1612

[26] Bátai IZ, Sár CP, Horváth Á, Borbély É, Bölcskei K, Kemény Á, Sándor Z, Nemes B, Helyes Z, Perkecz A, Mócsai A, Pozsgai G, Pintér E. TRPA1 ion channel determines beneficial and detrimental effects of GYY4137 in murine serum-transfer arthritis. Front Pharmacol 2019; 10(10): 964

[27] Cai F, Xu H, Cao N, Zhang X, Liu J, Lu Y, Chen J, Yang Y, Cheng J, Hua ZC, Zhuang H. ADT-OH, a hydrogen sulfide-releasing donor, induces apoptosis and inhibits the development of melanoma in vivo by upregulating FADD. Cell Death Dis 2020; 11(1): 33–47

[28] De Long MJ, Dolan P, Santamaria AB, Bueding E. 1,2-Dithiol-3-thione analogs: effects on NAD(P)H: quinone reductase and glutathione levels in murine hepatoma cells. Carcinogenesis 1986; 7(6): 977–980

[29] Zhang Y, Munday R. Dithiolethiones for cancer chemoprevention: where do we stand? Mol Cancer Ther 2008; 7(11): 3470–3479 doi:10.1158/1535-7163.MCT-08-0625

[30] Lam S, MacAulay C, Le Riche JC, Dyachkova Y, Coldman A, Guillaud M, Hawk E, Christen MO, Gazdar AF. A randomized phase IIb trial of anethole dithiolethione in smokers with bronchial dysplasia. J Natl Cancer Inst 2002; 94(13): 1001–1009

[31] Reddy BS, Rao CV, Rivenson A, Kelloff G. Chemoprevention of colon carcinogenesis by organosulfur compounds. Cancer Res 1993; 53(15): 3493–3498

[32] Chegaev K, Rolando B, Cortese D, Gazzano E, Buondonno I, Lazzarato L, Fanelli M, Hattinger CM, Serra M, Riganti C, Fruttero R, Ghigo D, Gasco A. H₂S-donating doxorubicins may overcome cardiotoxicity and multidrug resistance. J Med Chem 2016; 59(10): 4881–4889

[33] Wang Y, Jia J, Ao G, Hu L, Liu H, Xiao Y, Du H, Alkayed NJ, Liu CF, Cheng J. Hydrogen sulfide protects blood-brain barrier integrity following cerebral ischemia. J Neurochem 2014; 129(5): 827–838

[34] Zhou X, Cao Y, Ao G, Hu L, Liu H, Wu J, Wang X, Jin M, Zheng S, Zhen X, Alkayed NJ, Jia J, Cheng J. CaMKKβ-dependent activation of AMP-activated protein kinase is critical to suppressive effects of hydrogen sulfide on neuroinflammation. Antioxid Redox Signal 2014; 21(12): 1741–1758

[35] Hirata I, Naito Y, Takagi T, Mizushima K, Suzuki T, Omatsu T, Handa O, Ichikawa H, Ueda H, Yoshikawa T. Endogenous hydrogen sulfide is an anti-inflammatory molecule in dextran sodium sulfate-induced colitis in mice. Dig Dis Sci 2011; 56(5): 1379–1386

[36] Meir M, Burkard N, Ungewiß H, Diefenbacher M, Flemming S, Kannapin F, Germer CT, Schweinlin M, Metzger M, Waschke J, Schlegel N. Neurotrophic factor GDNF regulates intestinal barrier function in inflammatory bowel disease. J Clin Invest 2019; 129(7): 2824–2840

[37] Leonard M, Creed E, Brayden D, Baird AW. Evaluation of the Caco-2 monolayer as a model epithelium for iontophoretic transport. Pharm Res 2000; 17(10): 1181–1188

[38] Rahman K, Desai C, Iyer SS, Thorn NE, Kumar P, Liu Y, Smith T, Neish AS, Li H, Tan S, Wu P, Liu X, Yu Y, Farris AB, Nusrat A, Parkos CA, Anania FA. Loss of junctional adhesion molecule a promotes severe steatohepatitis in mice on a diet high in saturated fat, fructose, and cholesterol. Gastroenterology 2016; 151(4): 733–746.e12

[39] Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Lozupone CA, Turnbaugh PJ, Fierer N, Knight R. Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc Natl Acad Sci USA 2011; 108(Suppl 1): 4516–4522

[40] Fordham RP, Sansom OJ. Colon contradictions: NF-κB signaling in intestinal tumorigenesis. J Exp Med 2015; 212(13): 2185

[41] Blankenberg S, Barbaux S, Tiret L. Adhesion molecules and atherosclerosis. Atherosclerosis 2003; 170(2): 191–203

[42] Howden CW, Gillanders I, Morris AJ, Duncan A, Danesh B, Russell RI. Intestinal permeability in patients with Crohn’s disease and their first-degree relatives. Am J Gastroenterol 1994; 89(8): 1175–1176

[43] Cao M, Wang P, Sun C, He W, Wang F. Amelioration of IFN-γ and TNF-α-induced intestinal epithelial barrier dysfunction by berberine via suppression of MLCK-MLC phosphorylation signaling pathway. PLoS One 2013; 8(5): e61944

[44] Li C, Zhao Y, Cheng J, Guo J, Zhang Q, Zhang X, Ren J, Wang F, Huang J, Hu H, Wang R, Zhang J. A proresolving peptide nanotherapy for site-specific treatment of inflammatory bowel disease by regulating proinflammatory microenvironment and gut microbiota. Adv Sci (Weinh) 2019; 6(18): 1900610

[45] Guo FF, Yu TC, Hong J, Fang JY. Emerging roles of hydrogen sulfide in inflammatory and neoplastic colonic diseases. Front Physiol 2016; 7: 156

[46] Wallace JL, Caliendo G, Santagada V, Cirino G, Fiorucci S. Gastrointestinal safety and anti-inflammatory effects of a hydrogen sulfide-releasing diclofenac derivative in the rat. Gastroenterology 2007; 132(1): 261–271

[47] Liu L, Cui J, Song CJ, Bian JS, Sparatore A, Soldato PD, Wang XY, Yan CDH. H₂S-releasing aspirin protects against aspirin-induced gastric injury via reducing oxidative stress. PLoS One 2012; 7(9): e46301

[48] Marutani E, Kosugi S, Tokuda K, Khatri A, Nguyen R, Atochin DN, Kida K, Van Leyen K, Arai K, Ichinose F. A novel hydrogen sulfide-releasing N-methyl-D-aspartate receptor antagonist prevents ischemic neuronal death. J Biol Chem 2012; 287(38): 32124–32135

[49] Sen N, Paul BD, Gadalla MM, Mustafa AK, Sen T, Xu R, Kim S, Snyder SH. Hydrogen sulfide-linked sulfhydration of NF-κB mediates its antiapoptotic actions. Mol Cell 2012; 45(1): 13–24

[50] Chen Y, Zhu C, Yang Z, Chen J, He Y, Jiao Y, He W, Qiu L, Cen J, Guo Z. A ratiometric fluorescent probe for rapid detection of hydrogen sulfide in mitochondria. Angew Chem Int Ed Engl 2013; 52(6): 1688–1691

[51] Nam B, Lee W, Sarkar S, Kim JH, Bhise A, Park H, Kim JY, Huynh PT, Rajkumar S, Lee K, Ha YS, Cho SH, Lim JE, Kim KW, Lee KC, Suk K, Yoo J. In vivo detection of hydrogen sulfide in the brain of live mouse: application in neuroinflammation models. Eur J Nucl Med Mol Imaging 2022; 49(12): 4073–4087

[52] Renga B. Hydrogen sulfide generation in mammals: the molecular biology of cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). Inflamm Allergy Drug Targets 2011; 10(2): 85–91

[53] Shatalin K, Shatalina E, Mironov A, Nudler E. H₂S: a universal defense against antibiotics in bacteria. Science 2011; 334(6058): 986–990

[54] Zhang J, Zhang Q, Wang Y, Li J, Bai Z, Zhao Q, Wang Z, He D, Zhang J, Chen Y. Toxicities and beneficial protection of H₂S donors based on nonsteroidal anti-inflammatory drugs. MedChemComm 2019; 10(5): 742–756

[55] Ghosh S, Panaccione R. Anti-adhesion molecule therapy for inflammatory bowel disease. Therap Adv Gastroenterol 2010; 3(4): 239–258

[56] Velikova G, Banks RE, Gearing A, Hemingway I, Forbes MA, Preston SR, Jones M, Wyatt J, Miller K, Ward U, Al-Maskatti J, Singh SM, Ambrose NS, Primrose JN, Selby PJ. Circulating soluble adhesion molecules E-cadherin, E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in patients with gastric cancer. Br J Cancer 1997; 76(11): 1398–1404

[57] Wang XH, Wang F, You SJ, Cao YJ, Cao LD, Han Q, Liu CF, Hu LF. Dysregulation of cystathionine γ-lyase (CSE)/hydrogen sulfide pathway contributes to ox-LDL-induced inflammation in macrophage. Cell Signal 2013; 25(11): 2255–2262

[58] Lin WC, Pan WY, Liu CK, Huang WX, Song HL, Chang KS, Li MJ, Sung HW. In situ self-spray coating system that can uniformly disperse a poorly water-soluble H₂S donor on the colorectal surface to treat inflammatory bowel diseases. Biomaterials 2018; 182: 289–298

[59] Egge N, Arneaud SLB, Wales P, Mihelakis M, McClendon J, Fonseca RS, Savelle C, Gonzalez I, Ghorashi A, Yadavalli S, Lehman WJ, Mirzaei H, Douglas PM. Age-onset phosphorylation of a minor actin variant promotes intestinal barrier dysfunction. Dev Cell 2019; 51(5): 587–601.e7

[60] Clayburgh DR, Shen L, Turner JR. A porous defense: the leaky epithelial barrier in intestinal disease. Lab Invest 2004; 84(3): 282–291

[61] Shi H, Yu Y, Lin D, Zheng P, Zhang P, Hu M, Wang Q, Pan W, Yang X, Hu T, Li Q, Tang R, Zhou F, Zheng K, Huang XF. β-glucan attenuates cognitive impairment via the gut-brain axis in diet-induced obese mice. Microbiome 2020; 8(1): 143

[62] Zhou Y, Xu ZZ, He Y, Yang Y, Liu L, Lin Q, Nie Y, Li M, Zhi F, Liu S, Amir A, González A, Tripathi A, Chen M, Wu GD, Knight R, Zhou H, Chen Y. Gut microbiota offers universal biomarkers across ethnicity in inflammatory bowel disease diagnosis and infliximab response prediction. mSystems 2018; 3(1): e00188–17

[63] Shang L, Liu H, Yu H, Chen M, Yang T, Zeng X, Qiao S. Core altered microorganisms in colitis mouse model: a comprehensive time-point and fecal microbiota transplantation analysis. Antibiotics (Basel) 2021; 10(6): 643

[64] Parker BJ, Wearsch PA, Veloo ACM, Rodriguez-Palacios A. The genus Alistipes: gut bacteria with emerging implications to inflammation, cancer, and mental health. Front Immunol 2020; 11: 906

[65] Li AL, Ni WW, Zhang QM, Li Y, Zhang X, Wu HY, Du P, Hou JC, Zhang Y. Effect of cinnamon essential oil on gut microbiota in the mouse model of dextran sodium sulfate-induced colitis. Microbiol Immunol 2020; 64(1): 23–32

[66] Hu Y, Liu JP, Zhu Y, Lu NH. The importance of Toll-like receptors in NF-κB signaling pathway activation by Helicobacter pylori infection and the regulators of this response. Helicobacter 2016; 21(5): 428–440

[67] Wang H, Huang J, Ding Y, Zhou J, Gao G, Han H, Zhou J, Ke L, Rao P, Chen T, Zhang L. Nanoparticles isolated from porcine bone soup ameliorated dextran sulfate sodium-induced colitis and regulated gut microbiota in mice. Front Nutr 2022; 9: 821404

RIGHTS & PERMISSIONS

Higher Education Press