Hydrogen sulphide protects against NSAID-enteropathy through modulation of bile and the microbiota

Abstract

Background and purpose: Hydrogen sulphide is an important mediator of gastrointestinal mucosal defence. The use of non-steroidal anti-inflammatory drugs (NSAIDs) is significantly limited by their toxicity in the gastrointestinal tract. Particularly concerning is the lack of effective preventative or curative treatments for NSAID-induced intestinal damage and bleeding. We evaluated the ability of a hydrogen sulphide donor to protect against NSAID-induced enteropathy.

Experimental approach: Intestinal ulceration and bleeding were induced in Wistar rats by oral administration of naproxen. The effects of suppression of endogenous hydrogen sulphide synthesis or administration of a hydrogen sulphide donor (diallyl disulphide) on naproxen-induced enteropathy was examined. Effects of diallyl disulphide on small intestinal inflammation and intestinal microbiota were also assessed. Bile collected after in vivo naproxen and diallyl disulphide administration was evaluated for cytotoxicity in vitro using cultured intestinal epithelial cells.

Key results: Suppression of endogenous hydrogen sulphide synthesis by β-cyano-L-alanine exacerbated naproxen-induced enteropathy. Diallyl disulphide co-administration dose-dependently reduced the severity of naproxen-induced small intestinal damage, inflammation and bleeding. Diallyl disulphide administration attenuated naproxen-induced increases in the cytotoxicity of bile on cultured enterocytes, and prevented or reversed naproxen-induced changes in the intestinal microbiota.

Conclusions and implications: Hydrogen sulphide protects against NSAID-enteropathy in rats, in part reducing the cytotoxicity of bile and preventing NSAID-induced dysbiosis.

Figure 1

Inhibition of H₂S synthesis by cystathionine γ-lyase. BCA exacerbated naproxen-induced intestinal damage and bleeding. Panel A: administration of naproxen (10 mg·kg⁻¹) twice daily over 4.5 days resulted in marginal intestinal damage. Co-treatment with BCA significantly worsened naproxen-induced intestinal erosions. Panel B: rats co-treated with BCA and naproxen had significantly reduced haematocrit compared with rats treated with vehicle and naproxen. Panel C: treatment with BCA twice a day did not significantly change intestinal MPO activity. Results are shown as mean ± SEM (n ≥ 6 per group). *P < 0.05, **P < 0.01, significantly different from vehicle; unpaired, two-tailed Student's t-test.

Figure 2

Dose-dependent reduction of naproxen-induced intestinal ulceration by DADS. Rats were co-treated, twice daily, with naproxen (20 mg·kg⁻¹) and vehicle or DADS (10, 30, or 60 mmol·kg⁻¹) for 4.5 days. Panel A: naproxen-induced small intestinal damage was significantly reduced by co-treatment with DADS at doses of 30 and 60 mmol·kg⁻¹kg⁻¹. Panel B: naproxen administration caused significant bleeding compared with vehicle treatment, but co-treatment with DADS at doses of 30 or 60 mmol·kg⁻¹ significantly reduced the naproxen-induced decrease in haematocrit. Panel C: weight loss caused by naproxen administration was significantly reduced by co-treatment with DADS at doses of 30 or 60 mmol·kg⁻¹kg⁻¹. Results are shown as mean ± SEM (n ≥ 6 per group). ***P < 0.001, significantly different from vehicle; ^ψP < 0.05, ^ψψP < 0.01, (^ψψψP < 0.001, significantly different from naproxen alone; one-way anova followed by Dunnett's and Bonferroni post hoc tests.

Figure 3

DADS did not prevent systemic COX inhibition by naproxen. Naproxen administration significantly suppressed (by 99%) whole-blood synthesis of TXB₂ (panel A), and this was not affected by co-administration of DADS (10, 30 or 60 mmol·kg⁻¹). Three hours after the final dose, naproxen administration also significantly inhibited (by 64%) intestinal PGE₂ synthesis compared with vehicle-treated rats (panel B). However, co-treatment with DADS at 30 and 60 mmol·kg⁻¹ increased intestinal PGE₂ synthesis in naproxen-treated rats to levels comparable with vehicle-treated rats. Results are shown as mean ± SEM (n ≥ 6 per group). *P < 0.05, **P < 0.01, significantly different from vehicle; one-way anova followed by Dunnett's and Bonferroni post hoc tests.

Figure 4

DADS dose-dependently prevented naproxen-induced mucosal inflammation and structural damage. Panel A: naproxen administration significantly increased intestinal MPO activity compared with vehicle-treated rats. However, co-treatment with DADS at doses of 30 or 60 mmol·kg⁻¹ significantly diminished the naproxen-induced increase in MPO activity. Panel B: loss of mucosal structure in the intestine after naproxen treatment. Mucosal structure remained intact when naproxen-treated rats were co-administered DADS (30 mmol·kg⁻¹) (panel D), with a similar appearance to tissue from vehicle-treated rats (panel C). Results are shown as mean ± SEM (n ≥ 6 per group). ***P < 0.001, significantly different from vehicle; ^ψψψP < 0.001, significantly different from naproxen alone; one-way anova followed by Dunnett's and Bonferroni post hoc tests. Scale bar, 100 μm (applicable to each panel).

Figure 5

DADS dose-dependently reduced naproxen-induced bile cytotoxicity. Bile collected from rats treated with naproxen (20 mg·kg⁻¹) twice daily for 4.5 days was significantly more cytotoxic to cultured rat IEC-6 cells than bile collected from vehicle-treated rats. Co-treatment with DADS at 30 mmol·kg⁻¹ significantly reduced the naproxen-induced increase in cytotoxicity of bile. Co-treatment with DADS at 60 mmol·kg⁻¹ further reduced naproxen-induced bile cytotoxicity, to a level similar to that of bile from vehicle-treated rats. Data shown are from the 1:6 dilutions of bile samples, and are expressed as the mean ± SEM of at least six rats per group. ***P < 0.001, significantly different from vehicle; ^ψP < 0.05, ^ψψψP < 0.001, significantly different from naproxen alone; one-way anova followed by Dunnett's and Bonferroni post hoc tests.

Figure 6

Co-treatment with DADS prevented naproxen-induced dysbiosis. Panel A: DGGE analysis revealed that naproxen (20 mg·kg⁻¹) administration to rats caused dysbiosis of the caecal microbiota, with distinct clustering from vehicle-treated rats. Co-treatment with DADS at 30 mmol·kg⁻¹ shifted the microbiota of naproxen-treated rats back to being similar to that of vehicle-treated rats. Using a resampling technique (majority UPGMA algorithm), the dendrogram clustering observed in Panel A was confirmed, indicating a robust difference in microbiota composition between groups (panel B). Panel C: The total number of aerobes in the jejunum did not significantly differ in rats treated with vehicle, naproxen, or naproxen plus DADS (10, 30 or 60 mmol·kg⁻¹) twice daily for 4.5 days. Results in panel C are from samples plated on CBA and shown as mean ± SEM (n ≤ 5 per group). The data were analysed by a one-way anova followed by Dunnett's multiple comparison test.