[Regulation of autophagy by GLT25D2 gene in acetaminophen-induced hepatotoxicity injury].

Objective: To investigate whether GLT25D2 gene regulates autophagy in acetaminophen (APAP)-induced hepatotoxicity injury.

Methods: GLT25D2 wild-type C57BL/6J mice and GLT25D2 C57BL/6J mice were selected as subjects. (1) In vivo experiment: 20 for wild-type mice and 20 for GLT25D2 mice were respectively divided into phosphate buffer (PBS) control group and APAP intervention group according to random number table, with 10 mice in each group. The hepatotoxicity injury model of mice was reproduced by intraperitoneal injection of 25 g/L APAP solution 500 mg/kg. The PBS control group was intraperitoneally injected with the same amount of PBS. The mice were sacrificed immediately after model reproduction, and the liver tissues were harvested. Western Blot was used to detect the expressions of autophagy-related proteins ATG5, ATG7, microtubule-associated protein 1 light chain 3 (LC3) and P62. The ultrastructural changes in liver tissue were observed under electron microscope to observe the level of autophagy. (2) In vitro experiment: primary hepatocytes extracted by two-step collagen perfusion from one GLT25D2 wild-type mouse and one GLT25D2 mouse were divided into two parts respectively. One part was treated with 5 mmol/L APAP solution. The cells were harvested at 0, 8, and 12 hours, and the expressions of autophagy-related proteins ATG5, ATG7, LC3, and P62 were determined by Western Blot. The other part was transfected with the green fluorescent protein-LC3 plasmid (GFP-LC3) for 24 hours. The cells were cultured with PBS (PBS control group) or 5 mmol/L APAP (APAP intervention group) for 12 hours, and the positive expression of GFP-LC3 was observed under the fluorescence microscope, thereby reflecting the expression of autophagosomes.

Results: (1) In vivo experiment: compared with the corresponding PBS control group, the expressions of the positive-associated proteins ATG5, ATG7 and LC3-II in liver tissue of the APAP intervention group were down-regulated in the wild-type and GLT25D2 mice, while the expression of the negative correlation protein P62 was up-regulated, indicating that the overall level of autophagy decreased after treatment with APAP. Compared with wild-type mice, the expressions of autophagy positive correlation proteins ATG5 and ATG7 were up-regulated in GLT25D2 mice (ATG5/β-actin: 1.21±0.29 vs. 0.84±0.19, ATG7/β-actin: 1.29±0.14 vs. 1.54±0.40, both P > 0.05), LC3-II expression was slightly down-regulated (LC3-II/β-actin: 0.52±0.06 vs. 0.58±0.06, P > 0.05), while negative correlation protein P62 was down-regulated (P62/β-actin: 1.13±0.94 vs. 1.54±0.40, P > 0.05), indicating that the expression of autophagy in GLT25D2 mice was higher than that in wild-type mice. Ultrastructural observation under electron microscope showed that the number of autophagosomes in the liver tissue of wild-type mice did not change significantly after APAP intervention as compared with that in PBS control group, but the number of autophagosomes in GLT25D2 mice was increased. (2) In vitro experiment: with the prolongation of APAP intervention, the expressions of ATG5 and ATG7 in the primary hepatocytes of wild-type and GLT25D2 mice were up-regulated, LC3 was slightly fluctuated, and the expression of negative-related protein P62 was gradually down-regulated. The peak value or the trough value reached at 12 hours. It was indicated that the expression of autophagy in APAP-stimulated cells was enhanced with a time-dependent manner. Compared with wild-type mice, the expressions of autophagy correlation proteins ATG5, ATG7, LC3-II and P62 were up-regulated in GLT25D2 mice at 12 hours (ATG5/β-actin: 0.93±0.09 vs. 0.74±0.06, ATG7/β-actin: 0.80±0.09 vs. 0.65±0.10, LC3-II/β-actin: 1.35±0.30 vs. 1.15±0.20, P62/β-actin: 0.36±0.02 vs. 0.31±0.03, all P > 0.05), indicating that the expression of autophagy was enhanced after gene knockout. Fluorescence microscopy showed that GFP-LC3 positive cells in both wild-type and GLT25D2 mice hepatocytes were significantly increased after APAP intervention as compared with those of PBS control group, and the proportion of GFP-LC3 positive cells in GLT25D2 mice was significantly higher than that in wild-type mice (0.64±0.08 vs. 0.36±0.05, P > 0.05).

Conclusions: GLT25D2 is a negative regulator of autophagy. Knockout of GLT25D2 gene can enhance the autophagy level of APAP-induced hepatotoxicity injury in mice.