Lipoxin A4 Preconditioning Attenuates Intestinal Ischemia Reperfusion Injury through Keap1/Nrf2 Pathway in a Lipoxin A4 Receptor Independent Manner

Abstract

Oxidative stress plays a critical role in the pathogenesis of intestinal ischemia reperfusion (IIR) injury. Enhancement in endogenous Lipoxin A4 (LXA4), a potent antioxidant and mediator, is associated with attenuation of IIR. However, the effects of LXA4 on IIR injury and the potential mechanisms are unknown. In a rat IIR (ischemia 45 minutes and subsequent reperfusion 6 hours) model, IIR caused intestinal injury, evidenced by increased serum diamine oxidase, D-lactic acid, intestinal-type fatty acid-binding protein, and the oxidative stress marker 15-F2t-Isoprostane. LXA4 treatment significantly attenuated IIR injury by reducing mucosal 15-F2t-Isoprostane and elevating endogenous antioxidant superoxide dismutase activity, accompanied with Keap1/Nrf2 pathway activation. Meanwhile, LXA4 receptor antagonist Boc-2 reversed the protective effects of LXA4 on intestinal injury but failed to affect the oxidative stress and the related Nrf2 pathway. Furthermore, Nrf2 antagonist brusatol reversed the antioxidant effects conferred by LXA4 and led to exacerbation of intestinal epithelium cells oxidative stress and apoptosis, finally resulting in a decrease of survival rate of rat. Meanwhile, LXA4 pretreatment upregulated nuclear Nrf2 level and reduced hypoxia/reoxygenation-induced IEC-6 cell damage and Nrf2 siRNA reversed this protective effect of LXA4 in vitro. In conclusion, these findings suggest that LXA4 ameliorates IIR injury by activating Keap1/Nrf2 pathway in a LXA4 receptor independent manner.

Figure 1

Chemical structure of Lipoxin A4 (C₂₀H₃₂O₅), Boc-2 (C₄₄H₅₉N₅O₈), and brusatol (C₂₆H₃₂O₁₁).

Figure 2

Effects of Lipoxin A4 on intestine ischemia reperfusion (IIR) injury. Representative photomicrographs (400x) showing H&E staining of intestine (a) and Chiu's score (b) was carried out to evaluate the injury degree; quantitative analysis using ELISA method was taken to assay the concentration of diamine oxidase (DAO) (c), D-lactic acid (DLA) (d), and intestinal-type fatty acid-binding protein (FABP2) (e) in serum. Each bar represents the mean ± SEM (n = 6 per group). ^∗ p < 0.05, ^∗∗ p < 0.01, one-way ANOVA with Tukey test.

Figure 3

Effects of Lipoxin A4 on Keap1/Nrf2 pathway. Quantitative analysis using ELISA method was taken to assay the concentration of oxidative marker 15-F2t-Isoprostane (a) and SOD activity (b) in intestine mucosa. Representative Western blots and quantitative analyses showing total Keap1 (c) and HO-1 (d) protein and nuclear Nrf2 (e) protein expressions in intestine mucosa. Each bar represents the mean ± SEM (n = 6 per group). ^∗ p < 0.05, ^∗∗ p < 0.01, one-way ANOVA with Tukey test. NS means no significant difference.

Figure 4

Brusatol reversed the protective effects conferred by Lipoxin A4. Representative photomicrographs (400x) showing H&E staining of intestine (a) and Chiu's score (b) was carried out to evaluate the injury degree; quantitative analysis using ELISA method was taken to assay the concentration of oxidative marker 15-F2t-Isoprostane (c) and SOD activity (d) in intestine mucosa. Representative Western blots and quantitative analyses showing total Keap1 (e) and HO-1 (g) protein and nuclear Nrf2 (f) protein expressions in intestine mucosa. Each bar represents the mean ± SEM (n = 6 per group). ^∗ p < 0.05, ^∗∗ p < 0.01, one-way ANOVA with Tukey test.

Figure 5

Lipoxin A4 attenuated intestinal epithelium cells apoptosis and finally increased rat survival rate. Representative photomicrographs showing fluorescent staining (a, 400x) of cytochrome C (green) or DAPI (blue) in intestinal epithelium cells and fluorescence intensity were measured (b). Cell apoptosis was also measured by using TUNEL assay (c, 400x) and TUNEL positive cells were counted (d). Each bar represents the mean ± SEM (n = 6 per group); the survival rate (e) was determined during 72 hours after reperfusion; n = 18 per group. ^∗ p < 0.05, ^∗∗ p < 0.01, one-way ANOVA with Tukey test.

Figure 6

Effects of Lipoxin A4 and knockdown of Nrf2 on H/R-induced cell damage in IEC-6 intestinal epithelium cells. (a) Effects of gene knockdown by Nrf2 siRNA. (b) Relative lactate dehydrogenase (LDH) release of IEC-6 cells. (c) Determining nuclear Nrf2 expression by western blot. Bars are mean ± standard deviation from four independent experiments. ^∗ p < 0.05, ^∗∗ p < 0.01, one-way ANOVA with Tukey test.

Figure 7

Proposed signaling mechanisms mediated by Lipoxin A4 in preventing intestinal epithelium cells from intestine ischemia reperfusion (IIR) injury. Lipoxin A4 promotes Keap1 dissociation from Nrf2 and leads to Nrf2 translocation from cytoplasm to nucleus, resulting in enhancing downstream HO-1 gene expression without binding to the Lipoxin A4 receptor (ALXR). Antioxidant enzyme HO-1 could possibly reduce cytochrome C release from mitochondria and decrease lipid peroxidative product 15-F2t-Isoprostane and elevate cell antioxidant ability evidenced by increase of superoxide dismutase (SOD) and finally decrease cells apoptosis. In addition, Lipoxin A4 binding to ALXR could also reduce damage of diamine oxidase (DAO) products, D-lactic acid (DLA), and intestinal-type fatty acid-binding protein (FABP2) release with other mechanisms.