Resveratrol attenuates non-steroidal anti-inflammatory drug-induced intestinal injury in rats in a high-altitude hypoxic environment by modulating the TLR4/NFκB/IκB pathway and gut microbiota composition

Abstract

Introduction: Non-steroidal anti-inflammatory drugs (NSAIDs) are currently the most widely used anti-inflammatory medications, but their long-term use can cause damage to the gastrointestinal tract(GIT). One of the risk factors for GIT injury is exposure to a high-altitude hypoxic environment, which can lead to damage to the intestinal mucosal barrier. Taking NSAIDs in a high-altitude hypoxic environment can exacerbate GIT injury and impact gut microbiota. The aim of this study is to investigate the mechanisms by which resveratrol (RSV) intervention alleviates NSAID-induced intestinal injury in a high-altitude hypoxic environment, as well as its role in regulating gut microbiota.

Methods: Aspirin was administered orally to rats to construct a rat model of intestinal injury induced by NSAIDs. Following the induction of intestinal injury, rats were administered RSV by gavage, and the expression levels of TLR4, NF-κB,IκB as well as Zonula Occludens-1 (ZO-1) and Occludin proteins in the different treatment groups were assessed via Western blot. Furthermore, the expression of the inflammatory factors IL-10, IL-1β, and TNF-α was evaluated using Elisa.16sRNA sequencing was employed to investigate alterations in the gut microbiota.

Results: The HCk group showed elevated expression of TLR4/NF-κB/IκB pathway proteins, increased expression of pro-inflammatory factors IL-1β and TNF-α, decreased expression of the anti-inflammatory factor IL-10, and expression of intestinal mucosal barrier proteins ZO-1 and Occludin. The administration of NSAIDs drugs in the plateau hypoxic environment exacerbates intestinal inflammation and damage to the intestinal mucosal barrier. After treatment with RSV intervention, the expression of TLR4/NF-κB/IκB signaling pathway proteins would be reduced, thereby lowering the expression of inflammatory factors in the HAsp group. The results of HE staining directly show the damage to the intestines and the repair of intestinal mucosa after RSV intervention. 16sRNA sequencing results show significant differences (P<0.05) in Ruminococcus, Facklamia, Parasutterella, Jeotgalicoccus, Coprococcus, and Psychrobacter between the HCk group and the Ck group. Compared to the HCk group, the HAsp group shows significant differences (P<0.05) in Facklamia, Jeotgalicoccus, Roseburia, Psychrobacter, and Alloprevotella. After RSV intervention, Clostridium_sensu_stricto bacteria significantly increase compared to the HAsp group.

Conclusion: Resveratrol can attenuate intestinal damage caused by the administration of NSAIDs at high altitude in hypoxic environments by modulating the TLR4/NF-κB/IκB signaling pathway and gut microbiota composition.

Fig 1. Rat jejunal tissue damage score.

Comparison with plain blank group:*P<0.05, **P<0.01, ***P<0.001; Comparison with plateau NSAIDs: #P<0.05, ##P<0.01, ###P<0.001.

Fig 2. Histopathological section results of rat jejunum.

(A) plain blank group (Ck); (B) plain aspirin-treated group (PAsp); (C) plateau blank group (HCk); (D) plateau aspirin-treated group (HAsp); (E) low-dose resveratrol-treated group (RSVL); (F) medium-dose resveratrol-treated group (RSVM); (G) high-dose resveratrol-treated group (RSVH); (H) Chiu’s of the jejunal tissue score.

Fig 3. Indicators of oxidative stress.

(A) Changes in myeloperoxidase in different treatment groups; (B) Changes in superoxide dismutase in different treatment groups.

Fig 4. Expression of inflammatory factors in jejunum and blood.

(A) Blood IL-1β; (B) Blood IL-10; (C) Blood TNF-α; (D) Jejunal IL-1β; (E) Jejunal IL-10; (F) Jejunal TNF-α.

Fig 5. Resveratrol intervention inhibits TLR4/NF-κB/IκB-mediated inflammation.

(A) WB blotting results of Occludin, ZO-1, TLR4, IκB, NF-κB, and β-actin; (B) Illustration of quantitative Western blotting of Occludin; (C) ZO-1 level; (D) IκB level; (E) TLR4 level; (F) NF- κB level. Band intensities were analyzed with Image J software and normalized with β-Actin.

Fig 6. Venn diagram of OTUs of gut microorganisms with different treatments and Alpha diversity.

(A) Venn diagram of OTUs of gut microorganisms with different treatments; (B) Observed ASVs; (C) Chao1 index; (D) Shannon diversity index; (E) Simpson index.

Fig 7. Bacterial community structure of each group at the phylum level and genus level.

(A) phylum level; (B) genus level.

Fig 8. LEfSe evolutionary branching diagram and the structure of bacterial community of each group at genus level.

(A) LEfSe evolutionary branching diagram, different colors represent different treatment groups:Ck(red), HCk(green), HAsp(blue), PAsp(purple), RSV_H(cyan), RSV_L(dark grey), RSV_M(light grey); (B) Relative abundance of bacterial communities at genus level of different treatment groups.

Fig 9. Correlation analysis between differential gut microbiota and jejunal immune factors in rats.