[Nuclear factor E2-related factor 2 protein mediates sepsis-associated liver injury by regulating ferroptosis].

Abstract

Objective: To investigate the effect of nuclear factor E2-related factor 2 (Nrf2) protein on ferroptosis in mice with sepsis-associated liver injury (SALI).

Methods: he male Sprague-Dawley (SD) mice were divided into 6 groups according to the random number table method, with 6 mice in each group. The SALI model of mice was established by cecal ligation and puncture (CLP), and the Sham group was only treated with laparotomy. CLP+Fer-1 group, CLP+Erastin group, CLP+ML385 group and CLP+Curcumin group were intraperitoneally injected with iron death inhibitor Ferrostatin-1 (Fer-1) 10 mg×kg^-1×d^-1, iron death activator Erastin 20 mg×kg^-1×d^-1, Nrf2 inhibitor ML385 30 mg×kg^-1×d^-1 and Nrf2 activator Curcumin 100 mg×kg^-1×d^-1 after CLP, respectively; Sham group and CLP group were given normal saline 10 mg×kg^-1×d^-1, each group was administered continuously for 10 days. Ten days after operation, the serum and liver tissues of mice were collected to detect the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum, and the levels of malondialdehyde (MDA), glutathione (GSH) and Fe²⁺; in liver homogenate. The pathological changes of liver tissue were observed under light microscope after hematoxylin-eosin (HE) staining. The shape and length of mitochondria in liver cells were observed under transmission electron microscope. The protein expressions of Nrf2, glutathione peroxidase 4 (GPX4) and prostaglandin-endoperoxide synthase 2 (PTGS2) in liver tissue were detected by Western blotting.

Results: Compared with Sham group, the serum levels of ALT and AST in the CLP group were significantly increased; histologically, the hepatic cord was disordered, the cells were swollen and necrotic, and the length of mitochondria was significantly shortened; the levels of MDA and Fe²⁺ in liver tissue increased significantly, and the content of GSH decreased significantly; the protein expressions of Nrf2 and GPX4 in liver tissue decreased, and the protein expression of PTGS2 increased significantly. Compared with CLP group, the serum levels of ALT and AST in CLP+Fer-1 group and CLP+Curcumin group were significantly decreased [ALT (U/L): 80.65±19.44, 103.45±20.52 vs. 283.50±37.12, AST (U/L): 103.33±11.90, 127.33±15.79 vs. 288.67±36.82, all P < 0.05]; microscopically, the hepatic cord was irregular, the cells were slightly swollen, and the mitochondrial length was significantly increased (μm: 1.42±0.09, 1.43±0.21 vs. 1.07±0.25, both P < 0.05); the levels of MDA and Fe²⁺; in liver tissue decreased significantly, and the content of GSH increased significantly [MDA (mol/g): 0.87±0.23, 1.85±0.43 vs. 4.47±0.95, Fe²⁺ (μg/g): 63.80±7.15, 67.48±6.28 vs. 134.52±14.32, GSH (mol/g): 1.95±0.29, 1.95±0.45 vs. 0.55±0.29, all P < 0.05]; the protein expressions of Nrf2 and GPX4 in liver tissue were significantly increased, and the protein expression of PTGS2 was significantly decreased (Nrf2/GAPDH: 1.80±0.28, 2.10±0.43 vs. 0.70±0.24, GPX4/GAPDH: 0.80±0.06, 0.93±0.07 vs. 0.48±0.02, PTGS2/GAPDH: 0.76±0.05, 0.84±0.01 vs. 1.02±0.09, all P < 0.05). However, the results of the above indexes in the CLP+Erastin group and CLP+ML385 group were opposite, and the serum levels of ALT and AST were significantly increased [ALT (U/L): 344.52±40.79, 321.70±21.10 vs. 283.50±37.12, AST (U/L): 333.50±27.90, 333.00±16.67 vs. 288.67±36.82, all P < 0.05]; microscopically, the arrangement of hepatic cords was disordered, the cells were obviously swollen and necrotic, and the length of mitochondria was significantly shortened (μm: 0.78±0.13, 0.67±0.07 vs. 1.07±0.25, both P < 0.05); the levels of MDA and Fe²⁺ in liver tissue increased significantly, and the content of GSH decreased significantly [MDA (mol/g): 5.92±1.06, 5.62±0.56 vs. 4.47±0.95, Fe²⁺ (μg/g): 151.40±8.03, 151.88±8.68 vs. 134.52±14.32, GSH (mol/g): 0.25±0.08, 0.23±0.11 vs. 0.55±0.29, all P < 0.05]; the protein expressions of Nrf2 and GPX4 in liver tissue were significantly decreased, and the protein expression of PTGS2 was significantly increased (Nrf2/GAPDH: 0.46±0.09, 0.46±0.11 vs. 0.70±0.24, GPX4/GAPDH: 0.34±0.05, 0.40±0.01 vs. 0.48±0.02, PTGS2/GAPDH: 1.24±0.13, 1.16±0.11 vs. 1.02±0.09, all P < 0.05).

Conclusions: CLP-induced SALI can lead to ferroptosis in mice hepatocytes, and Nrf2 protein in liver tissue can mediate SALI by regulating ferroptosis.