Sulforaphane alleviates intestinal ischemia-reperfusion injury by inhibiting ferroptosis via activation of the Nrf2/HO-1 pathway

Intestinal ischemia-reperfusion (IR) injury is a common and critical pathological process in clinical practice. Ferroptosis, a form of cell death characterized by iron-dependent accumulation of uncontrolled lipid peroxidation, has been confirmed to be involved in intestinal IR injury. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key molecule in regulating ferroptosis, and its activation can inhibit this form of cell death, thereby alleviating tissue injury. Sulforaphane (SFN), a classical Nrf2 activator, exerts antioxidant protective effects. This study aims to investigate whether SFN alleviates intestinal IR injury by activating the Nrf2 pathway to inhibit ferroptosis.

① SFN-related target genes, ferroptosis-related genes, and intestinal IR injury-related genes were retrieved and integrated from online databases, including TCMSP, GeneCards and OMIM, and the intersection genes were identified and presented in a Venn diagram. Then the intersection genes were imported into STRING to construct a protein-protein interaction (PPI) network, and topological analysis was performed using Cytoscape (v3.9.1) to screen core targets. Molecular docking was employed to predict the binding affinity between SFN and the key target Nrf2. ② Twenty-four SPF-grade male C57BL/6 mice (6 to 8 weeks old, weighing 22±2g) were randomly divided into 4 groups (n=6): Sham group, IR group, IR+SFN group, and IR+SFN+ML385 (a specific Nrf2 inhibitor) group. Except for the Sham group, the other groups were inflicted with clamping the superior mesenteric artery for 45 min followed by reperfusion for 30 min to establish an IR injury model. The IR+SFN and IR+SFN+ML385 groups were given an intraperitoneal injection of SFN (5 mg/kg) 1 h before clamping, while the IR+SFN+ML385 group additionally received intraperitoneal injection of ML385 (30 mg/kg) 2 h before clamping. After 30 min of reperfusion, small intestinal tissues were harvested. Histopathological changes were observed by light microscopy after HE staining, and the severity of intestinal injury was evaluated using Chiu’s score. Levels of malondialdehyde (MDA), superoxide dismutase (SOD), and reduced glutathione (GSH) were measured using biochemical reagent kits. Western blotting was performed to detect the protein expression of acyl-CoA synthetase long-chain family member 4 (ACSL4), glutathione peroxidase 4 (GPX4), Nrf2, and heme oxygenase-1 (HO-1).

① Network pharmacology analysis identified 162 SFN target genes, 2772 ferroptosis-related genes, and 2100 intestinal IR injury-related genes, with 43 intersection genes. PPI network topological analysis revealed 36 core genes, including Nrf2. Molecular docking showed that SFN could form a binding conformation with Nrf2, with a binding energy of -3.3 kcal/mol. ② In vivo results demonstrated that compared with the Sham group, the IR group exhibited significantly aggravated intestinal mucosal injury, as evidenced by an increased Chiu’s score (P<0.05), elevated MDA level, and decreased SOD and GSH activities (P<0.05). Meanwhile, the expression of GPX4 was downregulated while that of ACSL4 was upregulated (P<0.05), suggesting the occurrence of ferroptosis in intestinal IR. SFN pretreatment ameliorated the above pathological and biochemical changes, upregulated Nrf2 and HO-1 expression (P<0.05), restored GPX4 expression and downregulated ACSL4 expression (P<0.05). The protective effects of SFN and its regulatory effects on the Nrf2/HO-1 axis and ferroptosis-related proteins were partially reversed by the addition of ML385 (P<0.05).

SFN can alleviate intestinal IR injury, and its effect may be related to enhancing the antioxidant response associated with the Nrf2/HO-1 signaling axis and ameliorating ferroptosis-related molecular changes.