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Aging and disease    2019, Vol. 10 Issue (1) : 82-93     DOI: 10.14336/AD.2018.0210
Orginal Article |
DRAM is Involved in Hypoxia/Ischemia-Induced Autophagic Apoptosis in Hepatocytes
Jianji Xu1,2,4, Yunjin Zang1,3, Dongjie Liu1,2,4, Tongwang Yang1,2,4, Jieling Wang1,2,4, Yanjun Wang1,2,4, Xiaoni Liu1,2,4,*, Dexi Chen1,2,4,*
1Beijing You’an Hospital Affiliated with Capital Medical University, Beijing 100069, China
2Beijing Institute of Hepatology, Capital Medical University, Beijing 100069, China
3Organ Transplantation Center, The Affiliated Hospital of Qingdao University, Qingdao 266000, China
4The Beijing Precision Medicine and Transformation Engineering Technology Research Center of Hepatitis and Liver Cancer, Beijing 100069, China
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Abstract  

Liver hypoxia/ischemia injury leads to acute liver injury, delayed graft dysfunction, and failure during liver transplantation. Previous studies showed that autophagy is involved in liver hypoxia/ischemia injury. Our and others’ studies have found that the damage-regulated autophagy modulator (DRAM) could induce the autophagic apoptosis. However, the role of DRAM regulating autophagy in liver hypoxia/ischemia injury remains unclear. The aim of this study was to determine whether DRAM is involved in oxygen-glucose deprivation (OGD)-induced hepatocyte autophagic apoptosis. Normal hepatocytes (HL-7702) were treated with OGD while Balb/c mice underwent surgery to induce 70% liver ischemia. To evaluate the role of DRAM in hypoxia/ischemia-induced hepatic injury, DRAM siRNA was used to knockdown DRAM expression in cultured hepatocytes and a recombinant adenovirus vector expressing DRAM was used to overexpress DRAM in cultured hepatocytes in vitro and in the liver in vivo. Hepatic injury was analyzed by histopathological methods and measurement of hepatocyte enzyme release. Cell apoptosis was analyzed by flow cytometry and TUNEL staining. Several autophagic biomarkers were observed by western blot analysis. OGD and 70% hepatic ischemia significantly induced cell autophagy, apoptosis and DRAM expression in hepatocytes in vitro and in vivo. OGD-induced autophagic apoptosis was inhibited by 3-Methyladenine (3-MA). OGD-induced injury and autophagy in HL-7702 cells were significantly attenuated by DRAM knockdown but aggravated by DRAM overexpression in vitro. Similarly, DRAM overexpression increased ischemia-induced liver injury and hepatic apoptosis in vivo. Our data demonstrate that hypoxia/ischemia induces hepatic injury through a DRAM-dependent autophagic apoptosis pathway. These data also suggest that DRAM plays an important role in ischemia-induced liver injury and hepatocyte apoptosis.

Keywords DRAM      autophagy      apoptosis      hypoxia/ischemia      hepatocyte     
Corresponding Authors: Liu Xiaoni,Chen Dexi   
About author:

These authors equally contributed to the work.

Issue Date: 17 December 2017
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Xu Jianji
Zang Yunjin
Liu Dongjie
Yang Tongwang
Wang Jieling
Wang Yanjun
Liu Xiaoni
Chen Dexi
Cite this article:   
Xu Jianji,Zang Yunjin,Liu Dongjie, et al. DRAM is Involved in Hypoxia/Ischemia-Induced Autophagic Apoptosis in Hepatocytes[J]. Aging and disease, 2019, 10(1): 82-93.
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http://www.aginganddisease.org/EN/10.14336/AD.2018.0210     OR     http://www.aginganddisease.org/EN/Y2019/V10/I1/82
Figure 1.  Experimental protocol for the study.
Figure 2.  ODG treatment induced activation of autophagy and apoptosis in HL-7702 cells. The cells were treated with oxygen-glucose deprivation for 20 min, 40 min, 60 min, and 80 min, and the supernatant was collected for indicated experiments thereafter. (A) Western blot analysis was performed with antibodies against LC3 and p62 to detect autophagy. GAPDH was used as a loading control. (B and C) Quantitative densitometry scan results from (A). (D) Cells were stained with Annexin V/PI and analyzed by flow cytometry. (E) Apoptosis ratio of cells from (D). (F) Cell death was assessed by LDH release into the supernatant. Data in graph A,C,E and F were presented as mean ± SD from three independent experiments. *p<0.05.
Figure 3.  OGD treatment induced autophagy activation in HL-7702 cells. HL-7702 cells underwent OGD for 40 minutes with or without pre-treatment of 5 mM 3-MA for 3 hours. (A) Western blot analysis with the indicated antibody (LC3 and p62 antibody for detecting autophagy; GAPDH was used as a loading control). (B) Confocal microscopy was used to detect the formation of GFP-LC3 puncta. Original magnification, 400×. (C) Quantification of cells with >5 GFP-LC3 puncta. The data were presented as the mean ±SD from three independent experiments. (D) Apoptosis was assessed by flow cytometry with Annexin V/PI stain. (E) Cell death was assessed by LDH release into the supernatant. *p<0.05.
Figure 4.  The effects of DRAM knockdown in response to OGD-induced autophagy and apoptosis in HL-7702 cells. Cells were transfected with siRNA-DRAM and siRNA-control and then treated with OGD for 40 minutes. (A) DRAM protein levels were detected by western blot. (B and C) Western blot analysis was used to detect autophagy levels with indicated antibody (LC3 and p62). GAPDH was used as a loading control. (D) Quantification of cells with >5 GFP-LC3 puncta. The data were presented as mean ± SD of three independent experiments. (E) Cell death was assessed by LDH release assay into the supernatant. (F) Apoptosis was assessed by flow cytometry with Annexin V/PI scan. * p<0.05.
Figure 5.  Overexpression of DRAM enhanced OGD-induced autophagy and aggravated cell apoptosis. HL-7702 cells were transfected with rAd-DRAM or rAd-con for 48 hours before OGD treatment. (A) The efficiency of adenovirus-mediated DRAM expression was evaluated by western blot analysis. (B) Western blot analysis was used to detect autophagy levels with the indicated antibody (LC3 and p62 antibody for detecting autophagy; GAPDH was used as a loading control). (C) Quantification of cells with >5 GFP-LC3 puncta. The data were presented as mean ± SD of three independent experiments. (D) Cell death was assessed by LDH release assay. (E) Apoptosis was assessed by Flow Cytometry with Annexin V/PI stain. *p < 0.05.
Figure 6.  Effects of DRAM overexpression on liver function and histopathology in a mouse liver ischemia model. Balb/c mice were injected with rAd-DRAM or rAd-control through the tail vein 72 hours before the onset of 70% hepatic ischemia. (A) Representative histopathology of liver sections (hematoxylin-eosin) in each group. Magnification, 400×. The black arrows in the figures represent hepatocytes vacuolization. (B) The severity of liver injury assessed by the modified Suzuki classification. (c and d) Serology tests of AST and ALT in each group. Data were presented as mean ± SD. *p < 0.05.
Figure 7.  Effect of DRAM overexpression on liver ischemia-induced cell apoptosis. Balb/c mice were injected with rAd-DRAM or rAd-control through the tail vein 72 hours before the onset of 70% hepatic ischemia. (A) TUNEL staining results (200×), the red arrow indicates an apoptotic nucleus. (B) Statistical analysis of (A). Data were presented as mean ± SD. *p < 0.05.
Figure 8.  Effects of DRAM overexpression on autophagy and apoptosis in a mouse model with 70% hepatic ischemia. Balb/c mice were injected with rAd-DRAM or rAd-control through the tail vein 72 hours before the onset of 70% hepatic ischemia. Western blot analysis was used to detect protein levels of LC3 and p62 (markers for autophagy) and PARP and p53 (markers for apoptosis). GAPDH was used as a loading control.
[1] Meng X, Tan J, Li M, Song S, Miao Y, Zhang Q (2017). Sirt1: Role Under the Condition of Ischemia/Hypoxia. Cell Mol Neurobiol, 37: 17-28
[2] Cursio R, Colosetti P, Gugenheim J (2015). Autophagy and liver ischemia-reperfusion injury. Biomed Res Int, 2015: 417590
[3] Clavien PA (2006). How far can we go with marginal donors? J Hepatol, 45: 483-484
[4] Selzner N, Rudiger H, Graf R, Clavien PA (2003). Protective strategies against ischemic injury of the liver. Gastroenterology, 125: 917-936
[5] Li Y, Mei Z, Liu S, Wang T, Li H, Li XX, et al. (2017). Galanin Protects from Caspase-8/12-initiated Neuronal Apoptosis in the Ischemic Mouse Brain via GalR1. Aging Dis, 8: 85-100
[6] Meng X, Chu G, Yang Z, Qiu P, Hu Y, Chen X, et al. (2016). Metformin Protects Neurons against Oxygen-Glucose Deprivation/Reoxygenation -Induced Injury by Down-Regulating MAD2B. Cell Physiol Biochem, 40: 477-485
[7] Wang J, Cao B, Han D, Sun M, Feng J (2017). Long Non-coding RNA H19 Induces Cerebral Ischemia Reperfusion Injury via Activation of Autophagy. Aging Dis, 8: 71-84
[8] Mizushima N, Levine B (2010). Autophagy in mammalian development and differentiation. Nat Cell Biol, 12: 823-830
[9] Rubinsztein DC, Marino G, Kroemer G (2011). Autophagy and aging. Cell, 146: 682-695
[10] Deretic V, Levine B (2009). Autophagy, immunity, and microbial adaptations. Cell Host Microbe, 5: 527-549
[11] Adams JM (2003). Ways of dying: multiple pathways to apoptosis. Genes Dev, 17: 2481-2495
[12] Crighton D, Wilkinson S, O’Prey J, Syed N, Smith P, Harrison PR, et al. (2006). DRAM, a p53-induced modulator of autophagy, is critical for apoptosis. Cell, 126: 121-134
[13] Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, et al. (2000). LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J, 19: 5720-5728
[14] Komatsu M, Waguri S, Koike M, Sou YS, Ueno T, Hara T, et al. (2007). Homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice. Cell, 131: 1149-1163
[15] Liu WJ, Ye L, Huang WF, Guo LJ, Xu ZG, Wu HL, et al. (2016). p62 links the autophagy pathway and the ubiqutin-proteasome system upon ubiquitinated protein degradation. Cell Mol Biol Lett, 21: 29
[16] Liu K, Shi Y, Guo XH, Ouyang YB, Wang SS, Liu DJ, et al. (2014). Phosphorylated AKT inhibits the apoptosis induced by DRAM-mediated mitophagy in hepatocellular carcinoma by preventing the translocation of DRAM to mitochondria. Cell Death Dis, 5: e1078
[17] Gupta NA, Kolachala VL, Jiang R, Abramowsky C, Shenoi A, Kosters A, et al. (2014). Mitigation of autophagy ameliorates hepatocellular damage following ischemia-reperfusion injury in murine steatotic liver. Am J Physiol Gastrointest Liver Physiol, 307: G1088-1099
[18] Abe Y, Hines IN, Zibari G, Pavlick K, Gray L, Kitagawa Y, et al. (2009). Mouse model of liver ischemia and reperfusion injury: method for studying reactive oxygen and nitrogen metabolites in vivo. Free Radic Biol Med, 46: 1-7
[19] Zhang C, Huang J, An W (2017). Hepatic stimulator substance resists hepatic ischemia/reperfusion injury by regulating Drp1 translocation and activation. Hepatology, 66: 1989-2001
[20] Suzuki S, Toledo-Pereyra LH, Rodriguez FJ, Cejalvo D (1993). Neutrophil infiltration as an important factor in liver ischemia and reperfusion injury. Modulating effects of FK506 and cyclosporine. Transplantation, 55: 1265-1272
[21] Deretic V (2009). Links between autophagy, innate immunity, inflammation and Crohn’s disease. Dig Dis, 27: 246-251
[22] Liu K, Lou J, Wen T, Yin J, Xu B, Ding W, et al. (2013). Depending on the stage of hepatosteatosis, p53 causes apoptosis primarily through either DRAM-induced autophagy or BAX. Liver Int, 33: 1566-1574
[23] Qian L, Yuanshao L, Wensi H, Yulei Z, Xiaoli C, Brian W, et al. (2016). Serum IL-33 Is a Novel Diagnostic and Prognostic Biomarker in Acute Ischemic Stroke. Aging Dis, 7: 614-622
[24] Vavilis T, Delivanoglou N, Aggelidou E, Stamoula E, Mellidis K, Kaidoglou A, et al. (2016). Oxygen-Glucose Deprivation (OGD) Modulates the Unfolded Protein Response (UPR) and Inflicts Autophagy in a PC12 Hypoxia Cell Line Model. Cell Mol Neurobiol, 36: 701-712
[25] Fan J, Liu Y, Yin J, Li Q, Li Y, Gu J, et al. (2016). Oxygen-Glucose-Deprivation/Reoxygenation-Induced Autophagic Cell Death Depends on JNK-Mediated Phosphorylation of Bcl-2. Cell Physiol Biochem, 38: 1063-1074
[26] Xiao Q, Yang Y, Qin Y, He YH, Chen KX, Zhu JW, et al. (2015). AMP-activated protein kinase-dependent autophagy mediated the protective effect of sonic hedgehog pathway on oxygen glucose deprivation-induced injury of cardiomyocytes. Biochem Biophys Res Commun, 457: 419-425
[27] Liu J, Weaver J, Jin X, Zhang Y, Xu J, Liu KJ, et al. (2016). Nitric Oxide Interacts with Caveolin-1 to Facilitate Autophagy-Lysosome-Mediated Claudin-5 Degradation in Oxygen-Glucose Deprivation-Treated Endothelial Cells. Mol Neurobiol, 53: 5935-5947
[28] Xue LX, Xu ZH, Wang JQ, Cui Y, Liu HY, Liang WZ, et al. (2016). Activin A/Smads signaling pathway negatively regulates Oxygen Glucose Deprivation-induced autophagy via suppression of JNK and p38 MAPK pathways in neuronal PC12 cells. Biochem Biophys Res Commun, 480: 355-361
[29] Mo ZT, Li WN, Zhai YR, Gong QH (2016). Icariin Attenuates OGD/R-Induced Autophagy via Bcl-2-Dependent Cross Talk between Apoptosis and Autophagy in PC12 Cells. Evid Based Complement Alternat Med, 2016: 4343084
[30] Urbanek T, Kuczmik W, Basta-Kaim A, Gabryel B (2014). Rapamycin induces of protective autophagy in vascular endothelial cells exposed to oxygen-glucose deprivation. Brain Res, 1553: 1-11
[31] Ju W, Li S, Wang Z, Liu Y, Wang D (2015). Decorin protects human hepatoma HepG2 cells against oxygen-glucose deprivation via modulating autophagy. Int J Clin Exp Med, 8: 13347-13352
[32] Shi R, Weng J, Zhao L, Li XM, Gao TM, Kong J (2012). Excessive autophagy contributes to neuron death in cerebral ischemia. CNS Neurosci Ther, 18: 250-260
[33] Crighton D, Wilkinson S, Ryan KM (2007). DRAM links autophagy to p53 and programmed cell death. Autophagy, 3: 72-74
[34] Liu K, Zang Y, Guo X, Wei F, Yin J, Pang L, et al. (2017). The Delta133p53 Isoform Reduces Wtp53-induced Stimulation of DNA Pol gamma Activity in the Presence and Absence of D4T. Aging Dis, 8: 228-239
[35] Thorburn A (2008). Apoptosis and autophagy: regulatory connections between two supposedly different processes. Apoptosis, 13: 1-9
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