Duan’s group at Lund University, Sweden generously provided alk-SMase^-/- mice generated from mice on a C57BL/6 background[23]. The alk-SMase^+/+ mice and alk-SMase^-/- mice used in the experiments were crossbred from alk-SMase^+/- mice. The genotypes of the animals were confirmed by polymerase chain reaction (PCR). All mice were kept in the animal facilities under laboratory conditions (23 °C, 12 h/12 h light/dark, 50% humidity, commercial standard pellets and free access to drinking water). The animal protocol was designed to minimize pain or discomfort. All the experimental animals were anesthetized with isoflurane prior to the operation.

Twelve-week-old mice were provided 3% DSS (MW 36000-50000) (MP Biomedicals, Santa Ana, CA, United States) in their daily drinking water for 4 or 6 d and were fed a normal diet during the induction of acute colitis. The disease activity index (DAI) was measured by examining weight loss, stool consistency and blood in the stool during the experiments according to previous methods[20] (Table 1). After DSS was induced, the mice were anesthetized by isoflurane inhalation, and blood was harvested for subsequent analysis. The organs were removed, and the colon length was measured. Colon tissue sections (0.5 mm) were stained with hematoxylin-eosin, and histopathological examinations were performed by microscopy. Colonic mucosal tissues were scraped for subsequent experiments. For the colitis model treated with tertiary butylhydroquinone (t-BHQ) (Sigma-Aldrich, St Louis, MO, United States), KO mice in the t-BHQ group were intraperitoneally injected with 50 mg/kg/d t-BHQ for 6 d and subsequently sacrificed. The experiments were performed as described above.

A fluorescein isothiocyanate dextran (FITC-D) experiment was performed to examine intestinal permeability. Briefly, on days 0, 4 and 6 after 3% DSS treatment, the mice were administered 4 kDa FITC-D (50 mg/100 g body weight) by gavage and sacrificed after 4 h. Serum was obtained by centrifugation at 1000 × g for 15 min, after which the fluorescence of FITC-D was measured with a fluorescence microplate reader at excitation and emission wavelengths of 485 nm and 535 nm, respectively. The concentration of FITC-D in serum was calculated based on the standard curve.

Mouse serum lipopolysaccharide (LPS) concentrations were analyzed using an enzyme-linked immunosorbent assay (ELISA) kit (AndyGene Biotechnology, Beijing, China). An anti-mouse LPS antibody was added to the microporous plate. Serum samples and standards were pipetted into separate wells, followed by the addition of a biotin-conjugated LPS antibody. The LPS in the sample or standard was sandwiched between pairs of antibodies. After the wells were thoroughly washed, HRP-conjugated streptavidin was added. The solution turned blue after the addition of the TMB substrate (HRP catalyzes the enzyme-substrate reaction). The LPS concentration of each serum sample was calculated using the standard curve. Intestinal mucosal tissue was homogenized and centrifuged, after which the proteins were extracted. The supernatant was carefully collected. The level of secretory immunoglobulin A (sIgA) in the intestinal mucosa was assessed by an ELISA kit (AndyGene Biotechnology) according to the manufacturer’s instructions.

Colon sections (0.5 cm) were removed and fixed in 2% glutaraldehyde. After fixation, embedding and staining, 60 nm continuous sections were cut. These sections were stained with 1% uranyl acetate for 20 min and lead citrate for 7 min. The microvilli, cell structure, organelles and cell junctions of intestinal mucosal epithelial cells were observed by electron microscopy.

Colonic mucosal tissues were first lysed in RIPA buffer and subsequently homogenized on ice using a homogenizer. The supernatants of the homogenates were collected after centrifugation at 13500 × rpm at 4°C for 15 min. Total extracted proteins (50 μg) were separated by sodium-dodecyl sulfate gel electrophoresis, transferred to nitrocellulose membranes and blocked with 5% skim milk for 1 h at room temperature. Afterward, the membranes were incubated with primary antibodies overnight at 4°C. The primary antibodies used were claudin-3 (1:800 dilution) (Abcam, Cambridge, United Kingdom), claudin-5 (1:800 dilution) (Abcam), occludin (1:400 dilution) (Abcam), zonula occludens-1 (ZO-1) (1:500 dilution) (Abcam), nuclear factor erythroid 2-related factor 2 (Nrf2) (1:500 dilution) (Santa Cruz Biotechnology, Dallas, TX, United States) and β-actin (1:8000 dilution). The membranes were washed with TBST buffer three times for 10 min each. Then, the membranes were incubated with the appropriate peroxidase-conjugated secondary antibodies (1:20000 dilution), after which chemiluminescence was detected. ImageJ software was used to measure the optical density of the bands. The expression levels of the target proteins relative to β-actin were calculated.

Total RNA was isolated from colonic mucosal tissues using TRIzol reagent (Invitrogen of Thermo Fisher Scientific, Waltham, MA, United States). To remove the inhibitory effect of DSS on qPCR, the RNA was purified using lithium chloride[24]. RNA concentration and purity were determined by using a Nanodrop (Thermo Fisher Scientific), and RNA integrity was verified by 2% agarose gel electrophoresis. Briefly, the RNA was reverse-transcribed into cDNA using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan). Real-time PCR was performed according to the instructions of the SYBR Green Real-time PCR Master Mix Kit (Toyobo). The primers used are shown in Table 2. The relative expression levels of the target genes were normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH)[25,26] in each sample by the 2^-ΔΔCt method.

The concentration of malondialdehyde (MDA) and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were determined using commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. The assay for SOD activity was based on its ability to inhibit the oxidation of hydroxylamine by O₂^-· produced from the xanthine-xanthine oxidase system. One unit of SOD activity was defined as the amount that reduced the absorbance at 550 nm by 50%. GSH-Px activity was assayed by quantifying the rate of oxidation of reduced glutathione to oxidized glutathione by H₂O₂, as catalyzed by GSH-Px. MDA levels were measured according to the thiobarbituric acid (TBA) method (Nanjing Jiancheng Bioengineering Institute). The method was based on spectrophotometric measurements of the color produced during the reaction of TBA with MDA. MDA concentrations were calculated by measuring the absorbance of TBA reactive substances at 532 nm.

The data are presented as the mean ± standard error of the mean. Each experiment was performed in triplicate and independently repeated a minimum of three times, and every experiment was performed with a sample size of no less than 3 mice per group. Statistical significance was assessed using an unpaired Student’s t-test for 2-group comparisons. Multigroup comparisons were performed using one-way analysis of variance (ANOVA), followed by using the Least Significant Difference test. A P value < 0.05 was considered statistically significant. Data analysis was conducted using SPSS 20.0 software (IBM Corp., Armonk, NY, United States), while graphical representations were created using GraphPad Prism software, version 5 (GraphPad Software, Inc., La Jolla, CA, United States).

In this study, 3% DSS successfully induced acute colitis in both wild-type (WT) and KO mice. As shown in Figure 1A, the body weights of WT and KO mice were slightly decreased on day 4 but were substantially decreased from day 5 to day 6 of DSS treatment. On day 6, the weight of KO mice decreased by 23.1%, whereas that of WT mice decreased by 14.5%. Similarly, the changes in the DAI scores were consistent with the effects on body weight, and the scores were significantly higher in KO mice than in WT mice on day 6 (Figure 1B). Compared with those of WT mice, the colon lengths of KO mice were significantly shorter before DSS treatment or after DSS treatment for 6 d, as shown in Figure 1C. The changes in tumor necrosis factor-alpha and interleukin-6 levels were significantly higher in KO mice than in WT mice after DSS treatment for 4 d (Figure 1D).

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Figure 1 Alkaline sphingomyelinase deficiency exacerbates dextran sulfate sodium-induced colitis in mice. The mice were treated with 3% dextran sulfate sodium (DSS) in drinking water for 6 d. A: Body weight (BW) was measured, and the percentage of BW loss was calculated (n = 6 per group); B: The disease activity index (DAI) was calculated according to the stool bleeding score, BW loss, stool consistency, and disease signs (n = 4 per group); C: After 6 d of DSS treatment, the colon was removed, and the length of the colon was measured (before DSS, n = 3 per group; after DSS, n = 4 per group); D: The mRNA levels of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 in colonic mucosal tissue were analyzed (n = 4 per group); E: Histopathological characterization of the colon was performed. Arrows: Decreased goblet cells; dysplastic glands; dilated congested blood vessels; destroyed mucosal layer; severe damage to the intestinal epithelium; intensive inflammatory cell infiltration to the submucosa and muscularis. ^aP < 0.05, ^bP < 0.01, ^cP < 0.005 compared with wild-type (WT). KO: Gene knockout.

Histopathological changes were characterized, and there were different degrees of epithelial damage, inflammatory cell infiltration, goblet cell depletion and crypt damage (Figure 1E). Before DSS induction, the colonic mucosal epithelium of WT and KO mice was intact, and abundant goblet cells were observed, with a clear mucosal structure and no inflammatory cell infiltration. On day 4 after DSS induction, WT mice exhibited relatively minor epithelial cell damage, reduced goblet cells, mild glandular hyperplasia, and infiltrating inflammatory cells. In contrast, KO mice exhibited extensive infiltration of inflammatory cells in the submucosal layer of connective tissue and a small amount of necrosis and desquamation of mucosal epithelial cells. By day 6, WT mice exhibited disorderly arranged glands, with partial necrosis and loss and a significant decrease in goblet cells. KO mice exhibited disruption of the intestinal epithelial layer, marked vascular dilation, abnormal morphology of goblet cells with a large cellular cavity, and widespread inflammation and inflammatory cells extending to blood vessels, the submucosa, and even the muscle layers.

To explore why the KO mice had more severe colitis than the WT mice, intestinal permeability after DSS administration was examined by measuring changes in the serum concentration of FITC-D. As shown in Figure 2A, the intestinal permeability of WT mice and KO mice did not change before DSS induction. However, on day 4 (P < 0.005) and day 6 (P < 0.05) after DSS induction, the intestinal permeability of KO mice was significantly higher than that of WT mice. Therefore, we verified that alk-SMase deficiency enhanced intestinal epithelial permeability on day 4 of DSS-induced colitis.

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Figure 2 Alkaline sphingomyelinase deficiency increases intestinal permeability in dextran sulfate sodium-induced colitis. The mice were treated with 3% dextran sulfate sodium (DSS) water, and blood was collected on days 0, 4, and 6. A: The mice were administered 4 kDa fluorescein isothiocyanate dextran (FITC-D) by gavage 4 h before blood sampling, after which the fluorescence intensity of FITC-D in serum was measured (n = 5-9 per group); B: Serum lipopolysaccharide (LPS) concentrations were detected on days 0, 4, and 6 after DSS treatment (n = 3 per group). ^aP < 0.05, ^cP < 0.005 compared with wild-type (WT) mice. KO: Gene knockout.

To observe the extent of colonic epithelial cell damage, we compared the levels of the bacterial translocation marker LPS in the serum of WT and KO mice (Figure 2B). We found that the serum concentration of LPS was significantly increased in WT and KO mice after DSS induction, but there was no significant difference between WT and KO mice on day 4, while serum concentrations were significantly higher in KO mice than in WT mice on day 6. The damage to the colonic mucosal epithelial cells in the KO mice had occurred on day 4 and was more severe on day 6.

The expression of ZO-1, occludin, claudin-2, claudin-3, claudin-5 and claudin-8 in mouse colonic mucosal tissues before and after DSS induction was examined. Before DSS treatment (day 0), the mRNA levels of claudin-3 and claudin-8 were significantly lower in KO mice than in WT mice, but claudin-5 mRNA levels were significantly lower (Figure 3A). However, the mRNA levels in KO mice were significantly lower than those in the WT mice after DSS induction (day 4) (Figure 3B). Western blotting was subsequently performed to detect the protein expression of ZO-1, occludin, claudin-3 and claudin-5. Before DSS treatment (day 0), the expression levels of all the proteins in KO mice were lower than those in WT mice (P < 0.05) (Figure 3C). However, after DSS induction for 4 d, the decreases in proteins levels in KO mice were more significant than those in WT mice (P < 0.01) (Figure 3D). Furthermore, the level of mucin 2 (MUC2), which is an important secretory protein in the gut, was significantly lower in KO mice than in WT mice on days 0 and 4 after DSS induction. We also examined the levels of the intestinal secretory protein sIgA, which is an important component of the intestinal mucosal immune barrier. On day 4 after DSS induction, sIgA levels were lower in WT and KO mice than before DSS induction, but no significant difference was observed between the two groups. However, on day 6, KO mice exhibited significantly lower levels of sIgA than WT mice, suggesting greater impairment of the intestinal immune barrier in KO mice (Figure 3E and F).

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Figure 3 Changes in intestinal mucosal barrier proteins in mice after dextran sulfate sodium induction. The mice were given normal drinking water before dextran sulfate sodium (DSS) treatment, and then the mice were given 3% DSS water for 4 d. Quantitative real-time reverse transcription-polymerase chain reaction and western blot analysis of intestinal barrier proteins were performed on homogenates of colonic mucosa tissues. A and B: The relative mRNA levels were determined relative to those in the wild-type (WT) group; C and D: The densities of the bands were determined relative to those in the WT group; E: Mucin 2 (MUC2) mRNA levels were examined in the homogenates of colonic mucosa tissues; F: The animals were euthanized on days 0, 4, and 6 after receiving DSS water. The levels of secretory immunoglobulin A (sIgA) in the homogenates of colonic mucosa tissues were examined by enzyme-linked immunosorbent assay. n = 3 per group. ^aP < 0.05, ^bP < 0.01, ^cP < 0.005 compared with WT mice. KO: Gene knockout; ZO-1: Zonula occludens-1.

As shown in Figure 4, before DSS treatment (day 0), the ultrastructure of colonic mucosal epithelial cells was normal, as shown by electron microscopy. However, there was slight loss of microvilli, swollen mitochondria and impaired tight junctions (TJs) in WT mice and KO mice on day 4 of DSS induction. The damage to the intestinal mechanical barrier in KO mice was more severe than that in WT mice on day 4. Moreover, we found widespread mitochondrial swelling and degeneration in intestinal epithelial cells after DSS induction for 4 d. However, on day 6, compared with WT mice, KO mice exhibited more severe colonic epithelial damage, and the intestinal epithelial cells in KO mice showed abscission of microvilli, widening of intercellular spaces, severe disruption of TJs, nuclear pyknosis, and epithelial ablation, as shown by the arrows in Figure 4.

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Figure 4 Ultrastructure of colonic mucosal epithelial cells in mice after dextran sulfate sodium induction. Mice were treated with 3% dextran sulfate sodium (DSS) water. Colon sections (0.5 cm) were removed on days 0, 4, and 6 after DSS treatment and fixed in 2% glutaraldehyde. The microvilli, cell structure, organelles and cell junctions of intestinal mucosal epithelial cells were examined by electron microscopy. Before DSS induction, the junctions between epithelial cells in wild-type (WT) and gene knockout (KO) mice were normal. After DSS induction, WT mice exhibited a loss of microvilli, pyknotic nuclei and impaired tight junctions. In KO mice, severe colonic epithelial damage was observed, and the intestinal epithelial cells exhibited damaged microvilli, widening of intercellular spaces, nuclear pyknosis, and mitochondrial swelling and degeneration (green arrows show damaged microvilli, orange arrows show mitochondrial swelling, and blue arrows show the intercellular space).

To verify whether mitochondrial damage caused changes in oxidative stress levels, changes in the antioxidant capability of serum were measured after DSS induction for 4 d. There was no difference in the levels of the antioxidant enzymes GSH-Px and SOD before DSS induction, but these levels were significantly lower in KO mice than in WT mice after DSS induction (Figure 5A and B). In contrast, the MOD of KO mice was significantly higher than that of WT mice after DSS induction (Figure 5C).

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Figure 5 Changes in antioxidant enzyme activity in the serum of mice after dextran sulfate sodium induction. The mice were given normal drinking water before dextran sulfate sodium (DSS) treatment, and the mice were then given 3% DSS water for 4 d. The concentration of malondialdehyde (MDA) and the activities of superoxide (SOD) and glutathione peroxidase (GSH-Px) were determined before and after DSS induction for 4 d. A: Serum MDA levels; B: GSH-Px activity; C: SOD activity. n = 4 per group. ^aP < 0.05, ^bP < 0.01 compared with wild-type (WT) mice. KO: Gene knockout.

To explore the extent to which antioxidant capacity decreased, changes in the expression of the key transcription factor Nrf2 in colonic mucosal tissues were examined. As shown in Figure 6A and B, the expression of Nrf2 in KO mice was significantly lower than that in WT mice after DSS stimulation for 4 d. Heme oxygenase-1 (HO-1), which is an antioxidant enzyme regulated by Nrf2, was further examined, and the mRNA level in KO mice was significantly lower than that in WT mice. Similarly, on day 4 after DSS treatment, the mRNA levels of GSH-Px, glutamate-cysteine ligase catalytic subunit (GCLc), and SOD in KO mice were significantly lower than those in WT mice (Figure 6C).

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Figure 6 Changes in the expression of colonic nuclear factor erythroid 2-related factor 2 and antioxidant enzymes after dextran sulfate sodium induction. The mice were given normal drinking water before dextran sulfate sodium (DSS) treatment, and the mice were then given 3% DSS water for 4 d. A and B: Western blot analysis of nuclear factor erythroid 2-related factor 2 (Nrf2) was performed on homogenates of colonic mucosa tissues before and after DSS induction; C: Quantitative real-time reverse transcription-polymerase chain reaction was used to measure the mRNA levels of Nrf2 and its target antioxidant genes in colonic mucosa tissues. n = 4 per group. ^aP < 0.05, ^bP < 0.01 compared with wild-type (WT) mice. GCLc: Glutamate-cysteine ligase catalytic subunit; GSH-Px: Glutathione peroxidase; HO-1: Heme oxygenase-1; KO: Gene knockout; SOD: Superoxide.

To verify the effect of alk-SMase on the Nrf2 signaling pathway, KO mice were intraperitoneally injected with the Nrf2 activator t-BHQ every day during DSS induction. Body weight loss (Figure 7A), active state, stool consistency and blood in the stool were effectively alleviated by t-BHQ in KO mice. The increased DAI of KO mice after DSS treatment was significantly decreased by t-BHQ-mediated activation of Nrf2 (Figure 7B). Furthermore, the changes in the thymus, spleen, and liver weights caused by the knockout of alk-SMase were significantly reversed to almost the same levels as those in WT mice after t-BHQ treatment of experimental colitis (Figure 7C-E). The colons of the KO mice treated with t-BHQ were obviously longer than those of KO mice that were not treated with t-BHQ (Figure 7F).

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Figure 7 Changes in the incidence of experimental colitis after intraperitoneal injection of tertiary butylhydroquinone in mice. The mice were given 3% dextran sulfate sodium (DSS) drinking water for 6 d, and gene knockout (KO) mice in the tertiary butylhydroquinone (t-BHQ) group were intraperitoneally injected with 50 mg/kg t-BHQ every day. A: Body weight (BW) loss; B: The disease activity index (DAI), calculated according to BW loss, active state, stool consistency, and blood in the stool; C-E: On day 6 of DSS treatment, the mice were killed, the organs were removed and weighed, the ratios of organ weight to BW were calculated; F: The colon lengths were measured. n = 4 or 5 per group. ^aP < 0.05, ^bP < 0.01, ^cP < 0.005, compared to wild-type (WT) mice. ^dP < 0.05, ^eP < 0.01, ^fP < 0.005, compared to KO mice.

Ulcerative colitis (UC) is a chronic inflammatory condition of the colon with unknown causes. Alkaline sphingomyelinase (alk-SMase), expressed in intestinal epithelial cells, shows anti-inflammatory effects, but its mechanism remains to be clarified.

This study was motivated by the need to understand the mechanism behind the anti-inflammatory effects of alk-SMase, particularly in relation to intestinal barrier function and oxidative stress in dextran sulfate sodium (DSS)-induced colitis.

The primary objective was to explore how alk-SMase impacts intestinal barrier function and manages oxidative stress, contributing to its anti-inflammatory role in DSS-induced colitis.

Mice were given 3% DSS drinking water to induce colitis. The study assessed disease activity, intestinal permeability, bacterial translocation, and the ultrastructure of intestinal epithelial cells. Western blotting and quantitative real-time reverse transcription-polymerase chain reaction were employed to evaluate intestinal barrier proteins and mRNA, and serum oxidant and antioxidant levels were analyzed.

In gene knockout (KO) mice, inflammation and intestinal permeability were more severe compared to wild-type mice after DSS induction. There was a significant reduction in intestinal barrier proteins and an increase in serum malondialdehyde levels, indicating lower antioxidant capacity. Notably, the administration of the nuclear factor erythroid 2-related factor 2 (Nrf2) activator tertiary butylhydroquinone (t-BHQ) relieved colitis in KO mice.

This study introduces the model that alk-SMase regulates intestinal barrier stability and antioxidant activity via the Nrf2 pathway, offering a new perspective on managing colitis. We employed a novel approach by using alk-SMase KO mice and treating them with t-BHQ to isolate and understand the specific effects of alk-SMase and Nrf2 in colitis.

The findings underscore the potential of alk-SMase in maintaining intestinal barrier stability and increasing antioxidant capacity, offering insights into novel therapeutic approaches for colitis. Future research will delve into the mechanisms by which alk-SMase influences the Nrf2 pathway, further illuminating its therapeutic potential in colitis.