AMPK regulates murine hepatic ischemia-reperfusion injury via mTOR/Nix signaling pathway_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

DU Chenyang ¹ , WANG Zhen ¹ , SONG Hu ¹ , LI Shipeng ² , ZHANG Haiming ³ ,  ZHANG Jianjun ³

1. The First Central Clinical College, Tianjin Medical University, Tianjin 300192, P. R. China;
2. Department of General Surgery, The People’s Hospital of Jiaozuo City, Jiaozuo, Henan 454150, P. R. China;
3. Department of Organ Transplantation, Tianjin First Central Hospital, Tianjin 300192, P. R. China;

Corresponding author：

ZHANG Jianjun, Email: zhangjianjun99@medmail.com.cn

Keywords：

ischemia-reperfusion injury; mitophagy; AMP-activated protein kinase; mechanism; mouse

DOI：

10.7507/1007-9424.201704050

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Objective To investigate the mechanism of AMP-activated protein kinase (AMPK) in hepatic ischemia-reperfusion injury (HIRI). Methods ① Grouping. Forty-two mice were randomly divided into Sham group, 4 ischemia reperfusion (IR) group of different times (2, 6, 12, and 24 h), Compound C group, and Compound C+repamycin (Rapa) group, each group enrolled in 6 mice. Compound C group: mice were modeled at 1 h after intraperitoneal injection of Compound C (25 mg/kg). Compound C+Rapa group: mice were modeled at 1 h after intraperitoneal injection of rapamycin (1 mg/kg) and Compound C (25 mg/kg). Mice of 4 IR groups, Compound C group, and Compound C+Rapa group were used to prepare HIRI model. Mice of Sham group were treated only for laparotomy, freeing the first portal hepatis and closing peritoneal. ② To filter the best IR time. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of mice in Sham group and IR groups of 4 different reperfusion time points were measured. The pathological changes of liver tissues were observed by HE staining, and the expressions of related proteins in liver tissue of mice were detected by Western blot. Considering the results of blood biochemical test, HE staining, and Western blot together to determine the best IR point. ③ The exploration of signal pathway for AMPK. The expressions of proliferating cell nuclear antigen (PCNA) were observed by immunohistochemical staining in the liver tissues of IR-12 h group, Compound C group (12 h after IR) and compound C+Rapa group (12 h after IR). The mitochondrial damage was observed by rhodamine 123 staining, and the apoptotic status of liver cells was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay (TUNEL). Results ① The 12 h after IR was the best observation time point. Compared with IR-12 h group, the levels of ALT and AST in Sham group, IR-2, 6, and 24 h groups were lower (P<0.05). HE staining showed that liver tissue destruction in IR-12 h group was the most severe. Western blot showed that, expressions of AMPKα, phosphorylated adenylate activated protein kinase α (p-AMPKα), Nip3-like protein X (Nix), BCL-2 homologous water-soluble protein (Bax), as well as ratio of autophagy microtubule-associated protein light chain 3 (LC3)Ⅱto LC3Ⅰof Sham group, IR-2, 6, and 24 h group were all lower than those of IR-12 h group (P<0.05), but the expressions of phosphorylated mammalian target of Rapa (p-mTOR) of Sham group, IR-2, 6, and 24 h group were all higher (P<0.05). Therefore, 12 h after IR was the best time to observe. ② Compared with IR-12 h group, the expression level of PCNA protein in liver tissue of Compound C group was lower (P<0.05), the mitochondrial luminescence intensity was weaker and the apoptotic cells were more. Compared with Compound C group, the expression of PCNA protein in the liver tissue of the Compound C+Rapa group was higher (P<0.05), the mitochondrial intensity was stronger and the apoptotic cells were less. ③ Compared with IR-12 h group, the expressions of Nix and p-AMPKα, and ratio of LC3Ⅱ to LC3Ⅰ in liver tissue of Compound C group decreased (P<0.05), while the expressions of p-mTOR, Caspase-3, and Cleaved Caspase-3 increased (P<0.05). Compared with Compound C group, the expressions of p-AMPKα and Nix in the liver tissue of Compound C+Rapa group increased (P<0.05), while the expressions of p-mTOR, Caspase-3, and Cleaved Caspase-3 decreased (P<0.05). Conclusion During the HIRI in mouse, AMPK regulates mitophagy and apoptosis through the mTOR/Nix pathway.

Citation： DU Chenyang, WANG Zhen, SONG Hu, LI Shipeng, ZHANG Haiming, ZHANG Jianjun. AMPK regulates murine hepatic ischemia-reperfusion injury via mTOR/Nix signaling pathway. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2017, 24(10): 1191-1199. doi: 10.7507/1007-9424.201704050 Copy

1.	Fayed NA, Sayed EI, Saleh SM, et al. Effect of dexmedetomidine on hepatic ischemia-reperfusion injury in the setting of adult living donor liver transplantation. Clin Transplant, 2016, 30(4): 470-482.
2.	Li S, Zhang J, Wang Z, et al. MicroRNA-17 regulates autophagy to promote hepatic ischemia/reperfusion injury via suppression of signal transductions and activation of transcription-3 expression. Liver Transpl, 2016, 22(12): 1697-1709.
3.	Yang Y, Zhang S, Fan C, et al. Protective role of silent information regulator 1 against hepatic ischemia: effects on oxidative stress injury, inflammatory response, and AMPKs. Expert Opin Ther Targets, 2016, 20(5): 519-531.
4.	Lee HM, Jang HJ, Kim SS, et al. Protective Effect of Eupatilin Pretreatment Against Hepatic Ischemia-Reperfusion Injury in Mice. Transplant Proc, 2016, 48(4): 1226-1233.
5.	Czaja MJ, Ding WX, Donohue TM Jr, et al. Functions of autophagy in normal and diseased liver. Autophagy, 2013, 9(8): 1131-1158.
6.	Gupta NA, Kolachala VL, Jiang R, et al. Mitigation of autophagy ameliorates hepatocellular damage following ischemia-reperfusion injury in murine steatotic liver. Am J Physiol Gastrointest Liver Physiol, 2014, 307(11): G1088-G1099.
7.	Williams JA, Ding WX. A Mechanistic Review of Mitophagy and Its Role in Protection against Alcoholic Liver Disease. Biomolecules, 2015, 5(4): 2619-2642.
8.	Park MS, Joo SH, Kim BS, et al. Remote Preconditioning on Rat Hepatic Ischemia-Reperfusion Injury Downregulated Bax and Cleaved Caspase-3 Expression. Transplant Proc, 2016, 48(4): 1247-1250.
9.	Zhang J, Ney PA. Role of BNIP3 and NIX in cell death, autophagy, and mitophagy. Cell Death Differ, 2009, 16(7): 939-946.
10.	Li W, Zhang X, Zhuang H, et al. MicroRNA-137 is a novel hypoxia-responsive microRNA that inhibits mitophagy via regulation of two mitophagy receptors FUNDC1 and NIX. J Biol Chem, 2014, 289(15): 10691-10701.
11.	Viola G, Bortolozzi R, Hamel E, et al. MG-2477, a new tubulin inhibitor, induces autophagy through inhibition of the Akt/mTOR pathway and delayed apoptosis in A549 cells. Biochem Pharmacol, 2012, 83(1): 16-26.
12.	Vucicevic L, Misirkic M, Janjetovic K, et al. Compound C induces protective autophagy in cancer cells through AMPK inhibition-independent blockade of Akt/mTOR pathway. Autophagy, 2011, 7(1): 40-50.
13.	Guo Y, Zhang Y, Hong K, et al. AMPK inhibition blocks ROS-NFκB signaling and attenuates endotoxemia-induced liver injury. PLoS One, 2014, 9(1): e86881.
14.	Zhu J, Hua X, Li D, et al. Rapamycin Attenuates Mouse Liver Ischemia and Reperfusion Injury by Inhibiting Endoplasmic Reticulum Stress. Transplant Proc, 2015, 47(6): 1646-1652.
15.	Zhu J, Lu T, Yue S, et al. Rapamycin protection of livers from ischemia and reperfusion injury is dependent on both autophagy induction and mammalian target of rapamycin complex 2-Akt activation. Transplantation, 2015, 99(1): 48-55.
16.	Li CX, Ng KT, Shao Y, et al. The inhibition of aldose reductase attenuates hepatic ischemia-reperfusion injury through reducing inflammatory response. Ann Surg, 2014, 260(2): 317-328.
17.	关连越, 付佩尧, 李巍. 肝脏缺血再灌注损伤机制的研究进展. 中国实验诊断学, 2015, 19(9): 1602-1605.0.
18.	Saidi RF, Kenari SK. Liver ischemia/reperfusion injury: an overview. J Invest Surg, 2014, 27(6): 366-379.
19.	Zhang J, Ng S, Wang J, et al. Histone deacetylase inhibitors induce autophagy through FOXO1-dependent pathways. Autophagy, 2015, 11(4): 629-642.
20.	Lee SC, Kim KH, Kim OH, et al. Activation of Autophagy by Everolimus Confers Hepatoprotection Against Ischemia-Reperfusion Injury. Am J Transplant, 2016, 16(7): 2042-2054.
21.	Sun K, Xie X, Liu Y, et al. Autophagy lessens ischemic liver injury by reducing oxidative damage. Cell Biosci, 2013, 3(1): 26.
22.	Kim JS, Wang JH, Biel TG, et al. Carbamazepine suppresses calpain-mediated autophagy impairment after ischemia/reperfusion in mouse livers. Toxicol Appl Pharmacol, 2013, 273(3): 600-610.
23.	Go KL, Lee S, Zendejas I, et al. Mitochondrial Dysfunction and Autophagy in Hepatic Ischemia/Reperfusion Injury. Biomed Res Int, 2015, 2015: 183469.
24.	Long B, Yin C, Fan Q, et al. Global Liver Proteome Analysis Using iTRAQ Reveals AMPK-mTOR-Autophagy Signaling Is Altered by Intrauterine Growth Restriction in Newborn Piglets. J Proteome Res, 2016, 15(4): 1262-1273.
25.	Wang LT, Chen BL, Wu CT, et al. Protective role of AMP-activated protein kinase-evoked autophagy on an in vitro model of ischemia/reperfusion-induced renal tubular cell injury. PLoS One, 2013, 8(11): e79814.
26.	Seo K, Seo S, Ki SH, et al. Compound C Increases Sestrin2 Expression via Mitochondria-Dependent ROS Production. Biol Pharm Bull, 2016, 39(5): 799-806.
27.	Gong G, Hu L, Liu Y, et al. Upregulation of HIF-1α protein induces mitochondrial autophagy in primary cortical cell cultures through the inhibition of the mTOR pathway. Int J Mol Med, 2014, 34(4): 1133-1140.
28.	Gordeev SA, Bykova TV, Zubova SG, et al. mTOR kinase inhibitor pp242 causes mitophagy terminated by apoptotic cell death in E1A-Ras transformed cells. Oncotarget, 2015, 6(42): 44905-44926.
29.	Park CW, Hong SM, Kim ES, et al. BNIP3 is degraded by ULK1-dependent autophagy via MTORC1 and AMPK. Autophagy, 2013, 9(3): 345-360.
30.	Kanki T. Nix, a receptor protein for mitophagy in mammals. Autophagy, 2010, 6(3): 433-435.
31.	Novak I, Kirkin V, McEwan DG, et al. Nix is a selective autophagy receptor for mitochondrial clearance. EMBO Rep, 2010, 11(1): 45-51.
32.	Hamacher-Brady A, Brady NR, Logue SE, et al. Response to myocardial ischemia/reperfusion injury involves Bnip3 and autophagy. Cell Death Differ, 2007, 14(1): 146-157.
33.	王振, 李世朋, 于瑶, 等. 干扰素调节因子 1 调控线粒体自噬参与小鼠肝缺血再灌注损伤. 中华器官移植杂志, 2016, 37(10): 614-619.0.
34.	Mazure NM, Pouysségur J. Hypoxia-induced autophagy: cell death or cell survival? Curr Opin Cell Biol, 2010, 22(2): 177-180.
35.	李丽君, 唐圣松. BNIP3 调控肿瘤细胞自噬及凋亡的研究进展. 国际病理科学与临床杂志, 2014, 34(6): 779-785.0.
36.	Li SP, He JD, Wang Z, et al. miR-30b inhibits autophagy to alleviate hepatic ischemia-reperfusion injury via decreasing the Atg12-Atg5 conjugate. World J Gastroenterol, 2016, 22(18): 4501-4514.

1. Fayed NA, Sayed EI, Saleh SM, et al. Effect of dexmedetomidine on hepatic ischemia-reperfusion injury in the setting of adult living donor liver transplantation. Clin Transplant, 2016, 30(4): 470-482.
2. Li S, Zhang J, Wang Z, et al. MicroRNA-17 regulates autophagy to promote hepatic ischemia/reperfusion injury via suppression of signal transductions and activation of transcription-3 expression. Liver Transpl, 2016, 22(12): 1697-1709.
3. Yang Y, Zhang S, Fan C, et al. Protective role of silent information regulator 1 against hepatic ischemia: effects on oxidative stress injury, inflammatory response, and AMPKs. Expert Opin Ther Targets, 2016, 20(5): 519-531.
4. Lee HM, Jang HJ, Kim SS, et al. Protective Effect of Eupatilin Pretreatment Against Hepatic Ischemia-Reperfusion Injury in Mice. Transplant Proc, 2016, 48(4): 1226-1233.
5. Czaja MJ, Ding WX, Donohue TM Jr, et al. Functions of autophagy in normal and diseased liver. Autophagy, 2013, 9(8): 1131-1158.
6. Gupta NA, Kolachala VL, Jiang R, et al. Mitigation of autophagy ameliorates hepatocellular damage following ischemia-reperfusion injury in murine steatotic liver. Am J Physiol Gastrointest Liver Physiol, 2014, 307(11): G1088-G1099.
7. Williams JA, Ding WX. A Mechanistic Review of Mitophagy and Its Role in Protection against Alcoholic Liver Disease. Biomolecules, 2015, 5(4): 2619-2642.
8. Park MS, Joo SH, Kim BS, et al. Remote Preconditioning on Rat Hepatic Ischemia-Reperfusion Injury Downregulated Bax and Cleaved Caspase-3 Expression. Transplant Proc, 2016, 48(4): 1247-1250.
9. Zhang J, Ney PA. Role of BNIP3 and NIX in cell death, autophagy, and mitophagy. Cell Death Differ, 2009, 16(7): 939-946.
10. Li W, Zhang X, Zhuang H, et al. MicroRNA-137 is a novel hypoxia-responsive microRNA that inhibits mitophagy via regulation of two mitophagy receptors FUNDC1 and NIX. J Biol Chem, 2014, 289(15): 10691-10701.
11. Viola G, Bortolozzi R, Hamel E, et al. MG-2477, a new tubulin inhibitor, induces autophagy through inhibition of the Akt/mTOR pathway and delayed apoptosis in A549 cells. Biochem Pharmacol, 2012, 83(1): 16-26.
12. Vucicevic L, Misirkic M, Janjetovic K, et al. Compound C induces protective autophagy in cancer cells through AMPK inhibition-independent blockade of Akt/mTOR pathway. Autophagy, 2011, 7(1): 40-50.
13. Guo Y, Zhang Y, Hong K, et al. AMPK inhibition blocks ROS-NFκB signaling and attenuates endotoxemia-induced liver injury. PLoS One, 2014, 9(1): e86881.
14. Zhu J, Hua X, Li D, et al. Rapamycin Attenuates Mouse Liver Ischemia and Reperfusion Injury by Inhibiting Endoplasmic Reticulum Stress. Transplant Proc, 2015, 47(6): 1646-1652.
15. Zhu J, Lu T, Yue S, et al. Rapamycin protection of livers from ischemia and reperfusion injury is dependent on both autophagy induction and mammalian target of rapamycin complex 2-Akt activation. Transplantation, 2015, 99(1): 48-55.
16. Li CX, Ng KT, Shao Y, et al. The inhibition of aldose reductase attenuates hepatic ischemia-reperfusion injury through reducing inflammatory response. Ann Surg, 2014, 260(2): 317-328.
17. 关连越, 付佩尧, 李巍. 肝脏缺血再灌注损伤机制的研究进展. 中国实验诊断学, 2015, 19(9): 1602-1605.0.
18. Saidi RF, Kenari SK. Liver ischemia/reperfusion injury: an overview. J Invest Surg, 2014, 27(6): 366-379.
19. Zhang J, Ng S, Wang J, et al. Histone deacetylase inhibitors induce autophagy through FOXO1-dependent pathways. Autophagy, 2015, 11(4): 629-642.
20. Lee SC, Kim KH, Kim OH, et al. Activation of Autophagy by Everolimus Confers Hepatoprotection Against Ischemia-Reperfusion Injury. Am J Transplant, 2016, 16(7): 2042-2054.
21. Sun K, Xie X, Liu Y, et al. Autophagy lessens ischemic liver injury by reducing oxidative damage. Cell Biosci, 2013, 3(1): 26.
22. Kim JS, Wang JH, Biel TG, et al. Carbamazepine suppresses calpain-mediated autophagy impairment after ischemia/reperfusion in mouse livers. Toxicol Appl Pharmacol, 2013, 273(3): 600-610.
23. Go KL, Lee S, Zendejas I, et al. Mitochondrial Dysfunction and Autophagy in Hepatic Ischemia/Reperfusion Injury. Biomed Res Int, 2015, 2015: 183469.
24. Long B, Yin C, Fan Q, et al. Global Liver Proteome Analysis Using iTRAQ Reveals AMPK-mTOR-Autophagy Signaling Is Altered by Intrauterine Growth Restriction in Newborn Piglets. J Proteome Res, 2016, 15(4): 1262-1273.
25. Wang LT, Chen BL, Wu CT, et al. Protective role of AMP-activated protein kinase-evoked autophagy on an in vitro model of ischemia/reperfusion-induced renal tubular cell injury. PLoS One, 2013, 8(11): e79814.
26. Seo K, Seo S, Ki SH, et al. Compound C Increases Sestrin2 Expression via Mitochondria-Dependent ROS Production. Biol Pharm Bull, 2016, 39(5): 799-806.
27. Gong G, Hu L, Liu Y, et al. Upregulation of HIF-1α protein induces mitochondrial autophagy in primary cortical cell cultures through the inhibition of the mTOR pathway. Int J Mol Med, 2014, 34(4): 1133-1140.
28. Gordeev SA, Bykova TV, Zubova SG, et al. mTOR kinase inhibitor pp242 causes mitophagy terminated by apoptotic cell death in E1A-Ras transformed cells. Oncotarget, 2015, 6(42): 44905-44926.
29. Park CW, Hong SM, Kim ES, et al. BNIP3 is degraded by ULK1-dependent autophagy via MTORC1 and AMPK. Autophagy, 2013, 9(3): 345-360.
30. Kanki T. Nix, a receptor protein for mitophagy in mammals. Autophagy, 2010, 6(3): 433-435.
31. Novak I, Kirkin V, McEwan DG, et al. Nix is a selective autophagy receptor for mitochondrial clearance. EMBO Rep, 2010, 11(1): 45-51.
32. Hamacher-Brady A, Brady NR, Logue SE, et al. Response to myocardial ischemia/reperfusion injury involves Bnip3 and autophagy. Cell Death Differ, 2007, 14(1): 146-157.
33. 王振, 李世朋, 于瑶, 等. 干扰素调节因子 1 调控线粒体自噬参与小鼠肝缺血再灌注损伤. 中华器官移植杂志, 2016, 37(10): 614-619.0.
34. Mazure NM, Pouysségur J. Hypoxia-induced autophagy: cell death or cell survival? Curr Opin Cell Biol, 2010, 22(2): 177-180.
35. 李丽君, 唐圣松. BNIP3 调控肿瘤细胞自噬及凋亡的研究进展. 国际病理科学与临床杂志, 2014, 34(6): 779-785.0.
36. Li SP, He JD, Wang Z, et al. miR-30b inhibits autophagy to alleviate hepatic ischemia-reperfusion injury via decreasing the Atg12-Atg5 conjugate. World J Gastroenterol, 2016, 22(18): 4501-4514.

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AMPK regulates murine hepatic ischemia-reperfusion injury via mTOR/Nix signaling pathway

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