Zhong Zhen, Ouyang Xunyan, Guo Qicai, Xiong Li, Liu Xianfa
Department of Emergency, First Affiliated Hospital of Gannan Medical University, Ganzhou 341000, Jiangxi, China.
Department of Orthopedics, First Affiliated Hospital of Gannan Medical University, Ganzhou 341000, Jiangxi, China. Corresponding author: Liu Xianfa, Email:
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2024 May;36(5):491-495. doi: 10.3760/cma.j.cn121430-20231105-00941.
To investigate the effect of nuclear factor E2-related factor 2 (Nrf2) protein on ferroptosis in mice with sepsis-associated liver injury (SALI).
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×d, iron death activator Erastin 20 mg×kg×d, Nrf2 inhibitor ML385 30 mg×kg×d and Nrf2 activator Curcumin 100 mg×kg×d after CLP, respectively; Sham group and CLP group were given normal saline 10 mg×kg×d, 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 Fe; 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.
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 Fe 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 Fe; 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, Fe (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 Fe 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, Fe (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).
CLP-induced SALI can lead to ferroptosis in mice hepatocytes, and Nrf2 protein in liver tissue can mediate SALI by regulating ferroptosis.
探讨核因子E2相关因子2（Nrf2）蛋白对脓毒症相关性肝损伤（SALI）小鼠铁死亡的影响。
将雄性Sprague-Dawley（SD）小鼠按随机数字表法分为6组，每组6只。采用盲肠结扎穿孔术（CLP）建立小鼠SALI模型，假手术组仅行剖腹术。CLP+Fer-1组、CLP+Erastin组、CLP+ML385组和CLP+姜黄素组在CLP术后分别腹腔注射铁死亡抑制剂Ferrostatin-1（Fer-1）10mg×kg×d、铁死亡激活剂Erastin 20mg×kg×d、Nrf2抑制剂ML385 30mg×kg×d和Nrf2激活剂姜黄素100mg×kg×d；假手术组和CLP组给予生理盐水10mg×kg×d，每组连续给药10天。术后10天，收集小鼠血清和肝组织，检测血清中丙氨酸氨基转移酶（ALT）和天冬氨酸氨基转移酶（AST）水平，以及肝匀浆中丙二醛（MDA）、谷胱甘肽（GSH）和铁（Fe）水平。苏木精-伊红（HE）染色后在光镜下观察肝组织病理变化。透射电镜下观察肝细胞线粒体的形态和长度。采用蛋白质印迹法检测肝组织中Nrf2、谷胱甘肽过氧化物酶4（GPX4）和前列腺素内过氧化物合酶2（PTGS2）的蛋白表达。
与假手术组比较，CLP组血清ALT和AST水平显著升高；组织学上，肝索紊乱，细胞肿胀坏死，线粒体长度显著缩短；肝组织中MDA和Fe水平显著升高，GSH含量显著降低；肝组织中Nrf2和GPX4蛋白表达降低，PTGS2蛋白表达显著升高。与CLP组比较，CLP+Fer-1组和CLP+姜黄素组血清ALT和AST水平显著降低[ALT（U/L）：80.65±19.44，103.45±20.52比283.50±37.12，AST（U/L）：103.33±11.90，127.33±15.79比288.67±36.82，均P<0.05]；镜下肝索不规则，细胞轻度肿胀，线粒体长度显著增加（μm：1.42±0.09，1.43±0.21比1.07±0.25，均P<0.05）；肝组织中MDA和Fe水平显著降低，GSH含量显著增加[MDA（mol/g）：0.87±0.23，1.85±0.43比4.47±0.95，Fe（μg/g）：63.80±7.15，67.48±6.28比134.52±14.32，GSH（mol/g）：1.95±0.29，1.95±0.45比0.55±0.29，均P<0.05]；肝组织中Nrf2和GPX4蛋白表达显著增加，PTGS2蛋白表达显著降低（Nrf2/GAPDH：1.80±0.28，2.10±0.43比0.70±0.24，GPX4/GAPDH：0.80±0.06，0.93±0.07比0.48±0.02，PTGS2/GAPDH：0.76±0.05，0.84±0.01比1.02±0.09，均P<0.05）。然而，CLP+Erastin组和CLP+ML385组上述指标结果相反，血清ALT和AST水平显著升高[ALT（U/L）：344.52±40.79，321.70±21.10比283.50±37.12，AST（U/L）：333.50±27.90，333.00±16.67比288.67±36.82，均P<0.05]；镜下肝索排列紊乱，细胞明显肿胀坏死，线粒体长度显著缩短（μm：0.78±0.13，0.67±0.07比1.07±0.25，均P<0.05）；肝组织中MDA和Fe水平显著升高，GSH含量显著降低[MDA（mol/g）：5.92±1.06，5.62±0.56比4.47±0.95，Fe（μg/g）：151.40±8.03，151.88±8.68比134.52±14.32，GSH（mol/g）：0.25±0.08,0.23±0.11比0.55±0.29，均P<0.05]；肝组织中Nrf2和GPX4蛋白表达显著降低，PTGS2蛋白表达显著升高（Nrf2/GAPDH：0.46±0.09，0.46±0.11比0.70±0.24，GPX4/GAPDH：0.34±0.05，0.40±0.01比0.48±0.02，PTGS2/GAPDH：1.24±0.13，1.16±0.11比1.02±0.09，均P<0.05）。
CLP诱导的SALI可导致小鼠肝细胞铁死亡，肝组织中的Nrf2蛋白可通过调节铁死亡介导SALI。
