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
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.
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.
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