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.
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.
摘要：
目的：探讨核因子E2相关因子2(Nrf2)对脓毒症肝损伤(SALI)小鼠铁凋亡的影响。
方法：雄性SD小鼠按随机数字表法分为6组，每组6只小鼠。采用盲肠结扎穿孔法(CLP)建立小鼠SALI模型,Sham组仅接受剖腹手术治疗。CLP+Fer-1组,CLP+Erastin组,CLPML385组和CLP姜黄素组腹腔注射铁死亡抑制剂Ferrostatin-1（Fer-1）10mg×kg-1×d-1，铁死亡激活剂Erastin20mg×kg-1×d-1，Nrf2抑制剂ML38530mg×kg-1×d-1和Nrf2激活剂姜黄素100mg×kg-1×d-1，CLP后Sham组和CLP组分别给予生理盐水10mg×kg-1×d-1，每组连续给药10天。手术后十天，取小鼠血清和肝组织检测血清中谷丙转氨酶(ALT)和谷草转氨酶(AST)水平,和丙二醛（MDA）的水平，谷胱甘肽（GSH）和Fe2；在肝匀浆中。苏木精-伊红(HE)染色后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。Westernblotting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。
结果：与Sham组相比，CLP组血清ALT和AST水平明显升高；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MDA和Fe2+含量显著升高,GSH含量显著降低;Nrf2和GPX4在肝组织中的蛋白表达降低,PTGS2蛋白表达明显升高。与CLP组相比，CLP+Fer-1组和CLP+姜黄素组血清ALT和AST水平明显下降[ALT（U/L）：80.65±19.44，103.45±20.52。283.50±37.12，AST（U/L）：103.33±11.90，127.33±15.79vs.288.67±36.82，均P<0.05]；显微镜下，肝索不规则，细胞轻微肿胀,线粒体长度显着增加（μm：1.42±0.09，1.43±0.21vs.1.07±0.25，均P<0.05）；肝组织中MDA和Fe2；GSH含量显著增加[MDA（mol/g）：0.87±0.23，1.85±0.43。4.47±0.95,Fe2+(μg/g):63.80±7.15,67.48±6.28vs.134.52±14.32，GSH（mol/g）：1.95±0.29，1.95±0.45vs.0.55±0.29,均P<0.05];Nrf2和GPX4在肝组织中的蛋白表达明显升高,PTGS2蛋白表达明显降低(Nrf2/GAPDH:1.80±0.28,2.10±0.43vs.0.70±0.24,GPX4/GAPDH:0.80±0.06,0.93±0.07vs.0.48±0.02，PTGS2/GAPDH：0.76±0.05，0.84±0.01vs.1.02±0.09，均P<0.05）。然而,CLP+Erastin组和CLP+ML385组的上述指标结果相反,血清ALT和AST水平明显升高[ALT（U/L）：344.52±40.79，321.70±21.10vs.283.50±37.12，AST（U/L）：333.50±27.90，333.00±16.67vs.288.67±36.82，均P<0.05]；显微镜下，肝索的排列紊乱，细胞明显肿胀坏死,线粒体长度明显缩短（μm：0.78±0.13，0.67±0.07vs.1.07±0.25,均P<0.05);肝组织MDA和Fe2+含量显著升高,GSH含量显着降低[MDA（mol/g）：5.92±1.06，5.62±0.56。4.47±0.95,Fe2+(μg/g):151.40±8.03,151.88±8.68vs.134.52±14.32，GSH（mol/g）：0.25±0.08，0.23±0.11vs.0.55±0.29,均P<0.05];Nrf2和GPX4在肝组织中的蛋白表达显著降低,PTGS2蛋白表达明显升高(Nrf2/GAPDH:0.46±0.09,0.46±0.11vs.0.70±0.24，GPX4/GAPDH：0.34±0.05，0.40±0.01vs.0.48±0.02,PTGS2/GAPDH:1.24±0.13,1.16±0.11vs.1.02±0.09，均P<0.05）。
结论：CLP诱导的SALI可导致小鼠肝细胞铁凋亡，肝组织中的Nrf2蛋白可以通过调节铁凋亡来介导SALI。
方法：雄性SD小鼠按随机数字表法分为6组，每组6只小鼠。采用盲肠结扎穿孔法(CLP)建立小鼠SALI模型,Sham组仅接受剖腹手术治疗。CLP+Fer-1组,CLP+Erastin组,CLPML385组和CLP姜黄素组腹腔注射铁死亡抑制剂Ferrostatin-1（Fer-1）10mg×kg-1×d-1，铁死亡激活剂Erastin20mg×kg-1×d-1，Nrf2抑制剂ML38530mg×kg-1×d-1和Nrf2激活剂姜黄素100mg×kg-1×d-1，CLP后Sham组和CLP组分别给予生理盐水10mg×kg-1×d-1，每组连续给药10天。手术后十天，取小鼠血清和肝组织检测血清中谷丙转氨酶(ALT)和谷草转氨酶(AST)水平,和丙二醛（MDA）的水平，谷胱甘肽（GSH）和Fe2；在肝匀浆中。苏木精-伊红(HE)染色后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。Westernblotting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。
结果：与Sham组相比，CLP组血清ALT和AST水平明显升高；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MDA和Fe2+含量显著升高,GSH含量显著降低;Nrf2和GPX4在肝组织中的蛋白表达降低,PTGS2蛋白表达明显升高。与CLP组相比，CLP+Fer-1组和CLP+姜黄素组血清ALT和AST水平明显下降[ALT（U/L）：80.65±19.44，103.45±20.52。283.50±37.12，AST（U/L）：103.33±11.90，127.33±15.79vs.288.67±36.82，均P<0.05]；显微镜下，肝索不规则，细胞轻微肿胀,线粒体长度显着增加（μm：1.42±0.09，1.43±0.21vs.1.07±0.25，均P<0.05）；肝组织中MDA和Fe2；GSH含量显著增加[MDA（mol/g）：0.87±0.23，1.85±0.43。4.47±0.95,Fe2+(μg/g):63.80±7.15,67.48±6.28vs.134.52±14.32，GSH（mol/g）：1.95±0.29，1.95±0.45vs.0.55±0.29,均P<0.05];Nrf2和GPX4在肝组织中的蛋白表达明显升高,PTGS2蛋白表达明显降低(Nrf2/GAPDH:1.80±0.28,2.10±0.43vs.0.70±0.24,GPX4/GAPDH:0.80±0.06,0.93±0.07vs.0.48±0.02，PTGS2/GAPDH：0.76±0.05，0.84±0.01vs.1.02±0.09，均P<0.05）。然而,CLP+Erastin组和CLP+ML385组的上述指标结果相反,血清ALT和AST水平明显升高[ALT（U/L）：344.52±40.79，321.70±21.10vs.283.50±37.12，AST（U/L）：333.50±27.90，333.00±16.67vs.288.67±36.82，均P<0.05]；显微镜下，肝索的排列紊乱，细胞明显肿胀坏死,线粒体长度明显缩短（μm：0.78±0.13，0.67±0.07vs.1.07±0.25,均P<0.05);肝组织MDA和Fe2+含量显著升高,GSH含量显着降低[MDA（mol/g）：5.92±1.06，5.62±0.56。4.47±0.95,Fe2+(μg/g):151.40±8.03,151.88±8.68vs.134.52±14.32，GSH（mol/g）：0.25±0.08，0.23±0.11vs.0.55±0.29,均P<0.05];Nrf2和GPX4在肝组织中的蛋白表达显著降低,PTGS2蛋白表达明显升高(Nrf2/GAPDH:0.46±0.09,0.46±0.11vs.0.70±0.24，GPX4/GAPDH：0.34±0.05，0.40±0.01vs.0.48±0.02,PTGS2/GAPDH:1.24±0.13,1.16±0.11vs.1.02±0.09，均P<0.05）。
结论：CLP诱导的SALI可导致小鼠肝细胞铁凋亡，肝组织中的Nrf2蛋白可以通过调节铁凋亡来介导SALI。
