The anti-inflammatory effect of dimethyl trisulfide in experimental acute pancreatitis

Abstract

Various organosulfur compounds, such as dimethyl trisulfide (DMTS), display anti-inflammatory properties. We aimed to examine the effects of DMTS on acute pancreatitis (AP) and its mechanism of action in both in vivo and in vitro studies. AP was induced in FVB/n mice or Wistar rats by caerulein, ethanol-palmitoleic acid, or L-ornithine-HCl. DMTS treatments were administered subcutaneously. AP severity was assessed by pancreatic histological scoring, pancreatic water content, and myeloperoxidase activity measurements. The behaviour of animals was followed. Pancreatic heat shock protein 72 (HSP72) expression, sulfide, and protein persulfidation were measured. In vitro acinar viability, intracellular Ca²⁺ concentration, and reactive oxygen species production were determined. DMTS dose-dependently decreased the severity of AP. It declined the pancreatic infiltration of leukocytes and cellular damage in mice. DMTS upregulated the HSP72 expression during AP and elevated serum sulfide and low molecular weight persulfide levels. DMTS exhibited cytoprotection against hydrogen peroxide and AP-inducing agents. It has antioxidant properties and modulates physiological but not pathophysiological Ca²⁺ signalling. Generally, DMTS ameliorated AP severity and protected pancreatic acinar cells. Our findings indicate that DMTS is a sulfur donor with anti-inflammatory and antioxidant effects, and organosulfur compounds require further investigation into this potentially lethal disease.

Figure 1

Dimethyl trisulfide (DMTS) administration lowers the severity of caerulein (Caer)-induced necrotizing acute pancreatitis (AP). Mice were treated subcutaneously with 2 × 50, 2 × 75 or 2 × 100 mg/kg DMTS, whereas intraperitoneal injection with 10 × 50 µg/kg Caer was used to induce AP. Control animals received physiological saline rather than Caer, or vehicle instead of DMTS. Then, at 12 h after the first Caer or physiological saline injection, animals were sacrificed. (a) Representative histopathological images of pancreatic tissues of the treatment groups. Bar charts demonstrate the extent of pancreatic (b) water content (as measured by the dry–wet weight ratio) and oedema (evaluation of histological sections), (c) leukocyte infiltration, (d) myeloperoxidase (MPO) activity, and (e) cellular damage. Values represent means with standard deviation (SD). The total number of animals was 42, for details of exact means, SDs, and animals per group please view Supplementary Table 1. (b–d) One-way ANOVA was carried out followed by Dunnett’s post-hoc test where all of the groups were compared to the Caer-only group, *p < 0.05; **p < 0.01; ***p < 0.001. (e) Kruskal–Wallis test was performed followed by Dunn’s post-hoc test, the groups were compared to the Caer-only group, *p < 0.05; **p < 0.01; ***p < 0.001. Leukoc. inf., leukocyte infiltration.

Figure 2

Dimethyl trisulfide (DMTS) administration reduces the severity of ethanol and palmitoleic acid (EtOH-POA)-induced necrotizing acute pancreatitis (AP). Mice were treated subcutaneously with 3 × 75 or 3 × 100 mg/kg DMTS, whereas intraperitoneal injection with 2 × 1.35 g/kg EtOH and 2 × 150 mg/kg POA were used to induce AP. Control animals were given physiological saline instead of EtOH-POA, or vehicle rather than DMTS. Animals were sacrificed 24 h after the first injection of EtOH-POA or physiological saline injection. (a) Representative histopathological images of pancreatic tissues of the treatment groups. Bar charts illustrate the extent of pancreatic (b) water content (as measured by the dry–wet weight ratio) and oedema (evaluation of histological sections), (c) leukocyte infiltration, (d) myeloperoxidase (MPO) activity, and (e) cellular damage. Values represent means with standard deviation (SD). The total number of animals was 25, for details of exact means, SDs, and animals per group please view Supplementary Table 1. (b, e) One-way ANOVA was carried out followed by Dunnett’s post-hoc test where all of the groups were compared to the EtOH-POA only group, **p < 0.01; ***p < 0.001. (c, d) Kruskal–Wallis test was conducted followed by Dunn’s post-hoc test, the groups were compared to the EtOH-POA only group, *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3

Dimethyl trisulfide (DMTS) treatment lowers the severity of L-ornithine-HCl (LO)-induced necrotising acute pancreatitis (AP) in rats. Rats were treated with 2 × 50 or 4 × 25 mg/kg DMTS subcutaneously, whereas intraperitoneal injection with 3 g/kg LO was employed to induce AP. Control animals received physiological saline rather than LO, or vehicle instead of DMTS. Animals were sacrificed at 24 h after the first LO or physiological saline injection. (a) Representative histopathological images of pancreatic tissues of the treatment groups. Bar charts display the extent of pancreatic (b) water content (as measured by the dry–wet weight ratio) and oedema (evaluation of histological sections), (c) leukocyte infiltration, and (d) cellular damage. Values represent means with standard deviation (SD). The total number of animals was 27, for details of exact means, SDs, and animals per group please view Supplementary Table 1. (c–d) One-way ANOVA was performed followed by Dunnett’s post-hoc test where all of the groups were compared to LO only group, *p < 0.05; ***p < 0.001. (b) Kruskal–Wallis test was performed followed by Dunn’s post-hoc test, the groups were compared to the LO only group, ***p < 0.001.

Figure 4

High-dose dimethyl trisulfide (DMTS) treatment lowers spontaneous motor activity in caerulein (Caer)-induced acute pancreatitis (AP) in mice. Open field test was used for the measurements, where the animals were placed into a 60 × 60 × 60 cm open area. (a) Moving duration and (b) total distance covered by animals with Caer-induced AP after various doses of DMTS treatment (2 × 75 mg/kg and 2 × 100 mg/kg subcutaneously (s.c.)). The larger dose of DMTS (2 × 100 mg/kg s.c.) had no effect on motor activity in mice without pancreatitis (c). Values represent means with standard deviation (SD). The total number of animals was 39, for details of exact means, SDs, and animals per group please view Supplementary Table 1. One-way ANOVA was performed in the case of (a) and (b) followed by Dunnett’s post-hoc test, and in panel (c) Student’s t-test was done. Statistically significant difference was marked with *** p < 0.001.

Figure 5

Alterations in heat shock protein 72 (HSP72) levels in acute pancreatitis (AP) in mice treated with dimethyl trisulfide (DMTS). Representative Western blot image of pancreatic HSP72 expression is depicted in the bottom, and the bar chart shows the densitometry of Western Blot images for pancreatic HSP72 level. Band intensities were assessed by using ImageJ software and HSP72 expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels. Fig. S12 presents full blot images. Values represent means with standard deviation (SD). The total number of animals was 16, for details of exact means, SDs, and animals per group please view Supplementary Table 1. One-way ANOVA was performed followed by Dunnett’s post-hoc test. Statistically significant differences were marked with: *p < 0.05; **p < 0.01. Abbreviations: Caer, caerulein.

Figure 6

Dimethyl trisulfide (DMTS) exerts cytoprotective effects against reactive oxygen species (ROS) and acute pancreatitis (AP) inducing agents in isolated mouse pancreatic acinar cells. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method, indicated as acinar viability, was performed after 8 h treatment; and the propidium iodide (PI) method, indicated as toxicity, was used after 4, 6, and 8 h treatments. (a, b) The effect of 30 µg/ml DMTS and its vehicle (90 µg/ml Polysorbate 80) on acinar cells was determined. The effect of 500 µM H₂O₂ and its combination with 30 µg/ml DMTS on acinar cells was measured by (c) MTT and (d) PI methods. Investigation of how (e) 1 nM caerulein (Caer), or (f) 60 mM L-arginine-HCl (L-arg) and their combination with 30 µg/ml DMTS cause cellular toxicity was measured by the PI method. Triton X-100 (TX) was used as a positive control causing 100% toxicity. (g) The effect of sodium chenodeoxycholate (CDC; 0, 0.1, 0.3, 0.5 mM) and its combination with 30 µg/ml DMTS on acinar cells was measured by the MTT method at 8 h. (h) The PI method was applied after 4, 6, and 8 h to test the effect of 0.5 mM CDC and/or 30 µg/ml DMTS on acinar cells. Values represent means with standard deviation (SD). For MTT measurements 4–6 wells per group were used (a total of 68 wells), and the cells were derived from 2 animals. For the PI method 6 parallels were used (a total of 270 wells), and the cells were derived from 3 animals. For details of exact means, SDs, and animals per group please view Supplementary Table 1. One- or two-way ANOVA was performed followed by Dunnett’s or Tukey’s post-hoc tests (indicated in each plot). Statistically significant differences were marked with: **p < 0.01; *** p < 0.001.

Figure 7

The effect of dimethyl trisulfide (DMTS) on intracellular Ca²⁺ signalling and its antioxidant property in mouse pancreatic acinar cells. (a) Representative trace of intracellular Ca²⁺ concentration (ic[Ca²⁺]) in response to treatment with 0.1 nM caerulein (Caer) with or without 30 µg/ml DMTS. At the end of the observation, acinar cells were subjected to 100 µM carbachol. The number (shown as frequency) and height (shown as calcium response) of individual spikes recorded between 480 and 960 s (8 min) were counted and plotted on panels b and c, respectively. (d-g) Treatment of cells with 1 nM Caer with or without 30 µg/ml DMTS. At the end of the experiment, cells were subjected to 100 µM carbachol. When the ic[Ca²⁺] reached the maximum (8 ± 0.5 min) the values were plotted on panel (e). The area under the curve was determined in case of 1 nM Caer treatment (f), and the slope (decreasing part) of the response on Caer treatment (g). (h) Menadione (Menad) treatment at 10, 30, and 50 µM with or without 30 µg/ml DMTS. Values represent means with standard deviation (SD). For the reactive oxygen species (ROS) method 5 parallels were used (a total of 40 wells), and the cells were derived from 2 different animals. In the case of ic[Ca²⁺] a total of 20 measurements were performed, and the cells were derived from 4 different animals. For details of exact means, SDs, and animals per group please view Supplementary Table 1. Statistics: (h) One-way ANOVA was performed followed by Tukey’s post-hoc test; and (b, c, e–g) Student’s t-tests were applied. Statistically significant differences were marked in the following manner: *** p < 0.001; ‘a’, vs. control (p < 0.05); ‘b’, vs. H₂O₂ (p < 0.05).

Figure 8

Dimethyl trisulfide (DMTS) elevates serum sulfide and persulfide levels but does not affect pancreatic persulfidation and sulfide. The bar charts show cysteine (Cys-SH) and glutathione (GSH) persulfidation levels in serum (a, b) and pancreatic tissue samples (d, e), respectively. Sulfur metabolome analyses of sulfide (H₂S) levels in serum (c) and tissue samples (f). Values represent means with standard deviation (SD). The total number of animals was 22, for details of exact means, SDs and animals per group please view Supplementary Table 1. (b, d–f) One-way ANOVA was performed followed by Dunnett’s post-hoc test where all of the groups were compared to the Caer-only group, *p < 0.05; **p < 0.01. (a, c) Kruskal–Wallis test was performed followed by Dunn’s post-hoc test, the groups were compared to the Caer-only group, *p < 0.05; **p < 0.01; ***p < 0.001. Cys-SSH, cysteine persulfide; GSSH, glutathione persulfide.

Figure 9

Schematic view of the in vivo experimental setup. Treatment arrangements for acute pancreatitis induction and administration of dimethyl trisulfide (DMTS) are depicted. Arrows above or below the timeline show the injections. Control animals received only vehicles. i.p. intraperitoneal; s.c. subcutaneous.