Remote ischemic post‑conditioning alleviates ischemia/reperfusion‑induced intestinal injury via the ERK signaling pathway‑mediated RAGE/HMGB axis

Abstract

Intestinal ischemia reperfusion (I/R) injury is a tissue and organ injury that frequently occurs during surgery and significantly contributes to the pathological processes of severe infection, injury, shock, cardiopulmonary insufficiency and other diseases. However, the mechanism of intestinal I/R injury remains to be elucidated. A mouse model of intestinal I/R injury was successfully established and the model mice were treated with remote ischemic post‑conditioning (RIPOC) and/or an ERK inhibitor (CC‑90003), respectively. Histopathological changes of the intestinal mucosa were determined by hematoxylin and eosin staining. In addition, the levels of high‑mobility group box 1 (HMGB1) and receptor for advanced glycation end products (RAGE) expression were confirmed by reverse transcription‑quantitative polymerase chain reaction, western blotting and immunohistochemistry assays. The levels of antioxidants, oxidative stress markers (8‑OHdG) and interleukin 1 family members were evaluated by ELISA assays and the levels of NF‑κB pathway proteins were analyzed by western blotting. The data demonstrated that RIPOC could attenuate the histopathological features of intestinal mucosa in the intestinal I/R‑injury mouse models via the ERK pathway. It was also revealed that HMGB1 and RAGE expression in the mouse models could be markedly reduced by RIPOC (P<0.05) and that these reductions were associated with inhibition of the ERK pathway. Furthermore, it was demonstrated that RIPOC produced significant antioxidant and anti‑inflammatory effects following an intestinal I/R injury and that these effects were mediated via the ERK pathway (P<0.05). In addition, RIPOC was demonstrated to suppress the NF‑κB (p65)/NLR family pyrin domain containing 3 (NLRP3) inflammatory pathways in the intestinal I/R injury mouse models via the ERK pathway. The findings of the present study demonstrated that RIPOC helped to protect mice with an intestinal I/R injury by downregulating the ERK pathway.

Keywords: antioxidants; extracellular signal‑regulated kinase; inflammation; ischemia‑reperfusion.

Figure 1.

RIPOC ameliorates the histopathological features of intestinal mucosa in mice with intestinal I/R injury via the ERK pathway. I/R injury mouse models were treated with RIPOC and/or CC-90003 and histopathological changes in intestinal mucosa were evaluated by (A) hematoxylin and eosin staining in the sham group and treated model group and (B) evaluated according to Chiu's score. **P<0.01 vs. Sham; ^#P<0.05 vs. I/R; ^$P<0.05 vs. the I/R+RIPOC group. RIPOC, remote ischemic post-conditioning; I/R, ischemia reperfusion.

Figure 2.

RIPOC downregulates HMGB1 and RAGE expression in the intestinal I/R injury mouse models via the ERK pathway. RIPOC and/or CC-90003 were used to treat the I/R injury mouse models. The levels of HMGB1 and RAGE were confirmed by (A) reverse transcription-quantitative polymerase chain and (B) western blot analysis, respectively. (C) The effects of RIPOC and CC-90003 on HMGB1 expression in the intestinal I/R mouse models were determined by immunohistochemistry. Magnification, ×400; Scale bar, 20 µm. **P<0.01 vs. the sham group; ^#P<0.05 vs. the I/R group; ^$P<0.05 vs. the I/R+RIPOC group. RIPOC, remote ischemic post-conditioning; HMGB1, high-mobility group box 1; RAGE, receptor for advanced glycation end products; I/R, ischemia reperfusion.

Figure 3.

RIPOC decreases ROS, MDA and Trx levels and increases SOD and GSH-Px levels in intestinal I/R injury mouse models via the ERK pathway. (A) Following treatment of the I/R injury mouse models with RIPOC and/or CC-90003, the levels of ROS were identified using a flow cytometer equipped with a dihydroethidium fluorescent probe. The concentrations of (B) MDA, (C) SOD, (D) Trx, (E) GSH-Px and (F) 8-OHdG in each group were examined using the corresponding commercial ELISA kits. **P<0.01 vs. the sham group; ^#P<0.05 vs. the I/R group; ^$P<0.05 vs. the I/R+RIPOC group. RIPOC, remote ischemic post-conditioning; ROS, reactive oxygen species; MDA, malondialdehyde; Trx, thioredoxin; SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; I/R, ischemia reperfusion.

Figure 4.

RIPOC reduces the levels of IL-1β, IL-18, IL-33 and NLRP3 in mice with intestinal I/R injury via the ERK pathway. Intestinal I/R injury mouse models were treated with RIPOC or/and CC-90003, respectively. The levels of (A) IL-1β, (B) IL-18 and (C) IL-33 were analyzed by ELISA. (D) Western blot assays were performed to evaluate p-ERK1/2, ERK1/2, p-p65, p65, p-IKBα, IKBα and NLRP3 expression. The relative levels of p-ERK1/2 and p-IKBα were defined as the ratio of phosphorylated protein to total protein. **P<0.01 vs. the sham group; ^#P<0.05 vs. the I/R group; ^$P<0.05 vs. the I/R+RIPOC group. RIPOC, remote ischemic post-conditioning; NLRP3, NLR family pyrin domain containing 3; I/R, ischemia reperfusion; p-, phosphorylated.