Ischemic Postconditioning Alleviates Intestinal Ischemia-Reperfusion Injury by Enhancing Autophagy and Suppressing Oxidative Stress through the Akt/GSK-3 β/Nrf2 Pathway in Mice

Abstract

Aims: Ischemic postconditioning (IPO) has a strong protective effect against intestinal ischemia-reperfusion (IIR) injury that is partly related to autophagy. However, the precise mechanisms involved are unknown.

Methods: C57BL/6J mice were subjected to unilateral IIR with or without IPO. After 45 min ischemia and 120 min reperfusion, intestinal tissues and blood were collected for examination. HE staining and Chiu's score were used to evaluate pathologic injury. We test markers of intestinal barrier function and oxidative stress. Finally, we used WB to detect the expression of key proteins of autophagy and the Akt/GSK-3β/Nrf2 pathway.

Results: IPO significantly attenuated IIR injury. Expression levels of LC3 II/I, Beclin-1, and p62 were altered during IIR, indicating that IPO enhanced autophagy. IPO also activated Akt, inhibited GSK-3β/Nrf2 pathway.

Conclusion: Our study indicates that IPO can ameliorate IIR injury by evoking autophagy, activating Akt, inactivating GSK-3β, and activating Nrf2. These findings may provide novel insights for the alleviation of IIR injury.β/Nrf2 pathway.

Figure 1

IPO alleviates IIR-induced intestinal injury in mice. (a) Histopathologic changes of the intestine under a light microscope (hematoxylin-eosin staining, ×200, scale bar 50 μm). Intestinal mucosal injury was graded by Chiu's score. (b) Intestinal water content was used to assess gut permeability. Serum concentrations of DAO (c), I-FABP (d), and D-LA (e) were detected to determine intestinal epithelial function. Data are presented as the mean ± SD (n = 8). ^∗p < 0.05 versus S; ^#p < 0.05 versus IIR.

Figure 2

IPO suppresses oxidative stress and evokes intestinal autophagy during IIR-induced intestinal injury in mice. MDA levels (a), SOD activity (b), and the GSH/GSSG ratio (c) were determined using colorimetric assays. (d) Expression of key molecules involved in autophagy in the gut was detected by western blot analysis in each group. (e) Autophagosomes were observed under an electron microscope (red arrows point to autophagosomes, blue arrows point to autolysosomes, scale bar 1.0 μm). Histogram shows the average number of autophagosome structures per view (371 μm²) obtained by examining at least 50 images per testing sample. (f) Representative immunoblots for the LC3 II/I ratio and GAPDH with and without CQ. Data are presented as the mean ± SD (n = 8). ^∗p < 0.05 versus S; ^#p < 0.05 versus IIR.

Figure 3

Defective autophagy abrogates the protective effects of IPO. Mice were pretreated with 3-MA or RAP prior to ischemia. (a) Autophagic protein LC3 in the ischemic gut was examined by western blot analysis. Levels of serum DAO (b), I-FABP (c), and D-LA (d) were analyzed as a measure of tissue injury. MDA levels (e), SOD activity (f), and the GSH/GSSG ratio (g) were determined using colorimetric assays. Data are presented as the mean ± SD (n = 8). ^∗p < 0.05 versus S; ^#p < 0.05 versus IIR; ^•p < 0.05 versus vehicle.

Figure 4

IPO activates Akt, inhibits GSK-3β, induces Nrf2 nuclear translocation, and upregulates HO-1 and NQO1 expression. (a) Expression of p-Akt (Ser⁴⁷³), Akt, p-GSK-3β (Ser⁹), and GSK-3β. Expression of Nrf2 (b) (in the nucleus and cytoplasm) and its downstream targets HO-1 and NQO1 (c) was detected by western blot analysis in each group. Data are presented as the mean ± SD (n = 8). ^∗p < 0.05 versus S; ^#p < 0.05 versus IIR.

Figure 5

Protective effects of IPO depend on Akt/GSK-3β-mediated Nrf2 activation. Mice were pretreated with SC66 or brusatol prior to ischemia. (a) Expression of p-Akt (Ser⁴⁷³), Akt, p-GSK-3β (Ser⁹), and GSK-3β. Expression of Nrf2 (b) (in the nucleus and cytoplasm) and its downstream targets HO-1 and NQO1 (c) was detected by western blotting in each group. Data are presented as the mean ± SD (n = 8). ^∗p < 0.05 versus IPO.

Figure 6

Protective effects of IPO depend on the activation of Akt/GSK-3β/Nrf2-mediated autophagy. Mice were pretreated with SC66 or brusatol prior to ischemia. Levels of serum DAO (a), I-FABP (b), and D-LA (c) were analyzed as a measure of tissue injury. (d) Expression of LC3, Beclin-1, and p62 was detected by western blot analysis in each group. Data are presented as the mean ± SD (n = 8). ^∗p < 0.05 versus S; ^#p < 0.05 versus IIR; ^•p < 0.05 versus IPO.