Effects Of Sodium Sulfite On Very Low Density Lipoprotein Assembly Pathway Of HL-7702 Liver Cells

Objective: Animal experiments have shown that sodium sulfite exposure can cause fatdeposition in the liver cells.We observed the effects of food additives sodium sulfite （Na₂SO₃）on triglyceride contents and the factors associated with very low densitylipoprotein（VLDL）assembly in the levels of transcription and translation in vitro humanHL-7702 hepatocyte,and found the key elements and links that food additives sodium sulfiteeffect on triglyceride contents or secretion of very low density lipoprotein of hepatocytes,thendetermined whether the sulphite affect the very low density lipoprotein ultimate secretion effluxby affecting its packaging process.Methods:Human diploid liver cell line（L-02） is the object of sudy.L-02 cells were cultured inDMEM/HIGH GLUCOSE medium containing 10% fetal bovine serum.Been exposed todifferent concentrations of Na₂SO₃and a positive control group （1mM oleic acid） for 24 hoursand 48 hours,cell viability was determined by CCK-8 assay,lipid droplets in the hepatocyteswere observed with oil red O staining, and the TG contents in hepatocytes were measured withTriglyceride Glycerol phosphate oxidase GPO-POD assay Kit.Western Blotting was used todetect very low density lipoprotein（VLDL）secretion of extracellular supernatant.Thefluorescence quantitative PCR （Q-PCR） was used to detect the mRNA levels of Microsomaltriglyceride transfer protein（MTP）,Triglyceride hydrolase（TGH）,Diacylglycerol acyltrans-ferase 2（DGAT2） of the hepatocytes.Results:1. The effect of different concentrations of Na₂SO₃on the activity of the L-02 cells: after beenexposed to Na₂SO₃for 24 hours and 48 hours, Na₂SO₃inhibited L-02 liver cells viability in adose-dependent manner （r24h=-0.941,p<0.01;r48h=-0.915, p<0.01）. Compared with the normalcontrol group, 10mM,2.5mM,0.625mM,0.156mM Na₂SO₃treated groups had significantdifferences （P<0.05）.The results indicate that Na₂SO₃can directly cause the reduction of livercell activity.2. The effect of different concentrations of Na₂SO₃on fat deposition in the L-02 cells: Observedby oil red O staining,negative control group cells were linked closely together,which werepolygon and arranged in single layer without overlapping growth,cell boundaries were clear andcells were adherent well.With the increase in exposure dose,it could clearly be seen that Na₂SO₃ treated groups had decreased in the number of cells and increased in cell gap.After beenexposed for 24 hours and 48 hours,each of Na₂SO₃exposure groups didn’t see orange fat dyeparticles,only the positive control oleic acid group showed obvious red lipid droplets.Theresults indicate that Na₂SO₃fail to cause intracellular significant fat deposition in the presentexperimental conditions.3. The effect of different concentrations of Na₂SO₃on intracellular TG contents in the L-02cells: After been exposed for 24 hours and 48 hours, compared with the negative controlgroup,10mM Na₂SO₃treated group and the positive control 1mM oleic acid group significantlyincreased the intracellular TG contents（P<0.05）.The results indi- cate that Na₂SO₃of a certaindose can increase TG contents in hepatocytes.4. The effect of different concentrations of Na₂SO₃on the secretion of VLDL in the L-02 cells:After been exposed for 24 hours,the VLDL level of culture supernatant in 10mM Na₂SO₃treated group was higher than the negative control group,and this different was statisticallysignificant（P<0.05）.While after been exposed for 48 hours, the VLDL level of culturesupernatant in all treated groups had no significant change in comparison with the negativecontrol group（P>0.05）.5. The effect of different concentrations of Na₂SO₃on the mRNA levels of MTP, DGAT2,TGHin the L-02 cells: After been exposed for 24 hours and 48 hours,the mRNA levels of MTP andDGAT2 between Na₂SO₃treated groups and the negative control group had no significantlydifferences（P>0.05）.After been exposed for 24 hours,the mRNA level of TGH in 10mMNa₂SO₃and 0.5mM Na₂SO₃treated groups increased and the discrepancies were statisticallysignificant compared with the negative control group（P<0.05）.After been exposed for 24hours,the mRNA level of TGH in 10mM Na₂SO₃and 1mM oleic acid treated groups were lowerdistinctly,and these differences were statistically significant in comparison with the negativecontrol group（P<0.05）,however,it raised in 2.5mM Na₂SO₃and 0.1mM Na₂SO₃treated groups（P<0.05）.Conclusion：1. Na₂SO₃inhibit L-02 liver cells viability in a dose-depedent manner, indicating that Na₂SO₃has toxic effects of liver cells.2. High concentration Na₂SO₃can increase the TG contents in L-02 hepatocyte, and causelipometabolism disorders in hepatocyte.3. Na₂SO₃can induce acumulation of TG in L-02 hepatocyte and thus promote the secretion ofVLDL under certain conditions.4. After been exposed to Na₂SO₃for 24 hours, the increase secrection of VLDL may be relatedto upregulation of the mRNA expression of TGH gene. After been exposed to Na₂SO₃for 48 hours, the increase of TG contents in L-02 hepatocyte may be due to downregulation of themRNA expression of TGH gene.