Effects Of Necroptosis And Autophagy In Sodium Sulfite-induced Hepatocyte Injury

Objective:To study the effects of necroptosis and autophagy in damage of liver cells caused by sodium sulfite(Na2SO3), and to provide a scientific basis for the possible mechanism of hepatotoxicity of sodium sulfite. Methods:Part Ⅰ: HL-7702 cells were respectively exposed to the sodium sulfite medium with the final concentrations of 0, 0.01, 0.1, 1, 10 mmol/L, and 50 mmol/L CCl4, 0.5% DMSO, then cultured for 2, 4, 12, 24 and 48 h respectively. The cells’ morphological changes were observed under an inverted optical microscope. The cell viability was assayed by CCK-8 kit, the ALT, AST and LDH activities were detected by Automatic biochemical analyzer, and the protein expression of RIP1 was tested by Western Blot.Part Ⅱ: HL-7702 cells were respectively exposed to the sodium sulfite medium with the final concentrations of 0.1, 1, 2.5, 5 mmol/L, and 20 mmol/L CCl4, 0.2% DMSO, then cultured for 2, 4, 12, 24 and 48 h respectively. The ATP of liver cells were detected by luciferase chemiluminescence. The expression of autophagy associated protein LC3 was detected by Immunofluorescence(IF). The protein expression of autophagy associated protein LC3, p62 and AMPK were tested by Western Blot. Results:Part Ⅰ:(1) Compared with the control group, after treated with 10 mmol/L Na2SO3 for 24 h and 48 h, the morphology of liver cells was changed, and the survival rates of HL-7702 cells of 1 and 10 mmol/L Na2SO3 treated groups for 2 h-48 h were significantly decreased(P<0.05).(2) Compared with the control group, the activities of ALT were significantly increased in 10 mmol/L Na2SO3 treated groups for 24 h and 48 h(P<0.05). The activities of AST were significantly decreased in 1 mmol/L Na2SO3 treated groups for 2 h and 24 h and in 10 mmol/L Na2SO3 treated groups for 2 h and 4 h,but these were significantly increased in 10 mmol/L Na2SO3 treated groups for 24 h and 48 h(P<0.05). The activities of LDH were significantly decreased in 10 mmol/L Na2SO3 treated groups for 2 h and 4 h, but these were significantly increased in 10 mmol/L Na2SO3 treated groups for 24 h and 48 h(P<0.05).(3) Compared with the control group,the protein expression of RIP1 in all Na2SO3 treated groups were increased(P<0.05).Part Ⅱ:(1) Compared with the control group, the ATP concentration of each group treated by Na2SO3 were significantly increased(P<0.05), and with the increase of Na2SO3 dose, the ATP concentration showed a increasing trend. The ATP concentration treated by 20 mmol/L CCl4(the positive control group) were significantly increased(P<0.05).(2) Compared with the control group, the intensity of green fluorescence which stand for the expression of LC3 protein in all Na2SO3 treated groups were increased significantly. And the green fluorescent intensity also significantly increased in the positive control group, especially in 24 h and 48 h. It suggesting that exposure to sodium sulfite may increase the expression of LC3 protein in HL-7702 cells.(3) Compared with the control group, the protein expression of LC3-Ⅱ treated by Na2SO3 for 2 h-24 h were significantly increased(P<0.05). With the increase of Na2SO3 dose for 48 h, the protein expression of LC3-Ⅱ showed a decreasing trend. Compared with the control group, the protein expression of p62 treated by Na2SO3 for 2 h-24 h were significantly increased(P<0.05).While with the increase of Na2SO3 dose for 48 h, the protein expression of p62 decreased gradually. Compared with the control group, the protein expression of AMPK in all Na2SO3 treated groups were not changed. Conclusion:(1) The time of increasing of RIP1 protein expression was earlier than morphological change induced by Na2SO3, and the exposure dose of increasing of RIP1 protein expression was also significantly lower than the cell viability change. Therefore, sodium sulfite may cause liver cell damage via necroptosis.(2) Sodium sulfite can cause significant increase of protein expression of LC3 and P62 in the early stages, and significant decrease in the later stages. It suggests that sodium sulfite may alleviate hepatocytes injury by promotion of autophagy.