The Influences Of Food Additive Sodium Sulfite On The Triglyceride Contents And The Expressions Of Arf-1 And COPI Of Hepatocytes

Objective: Previous in vivo studies showed that sulfite may cause significant deposition of lipiddroplets in hepatocytcs. This experiment is to explore the possible liver injury ways of sulfite byobserving the changes of triglyceride content and the mRNA and protein levels of very lowdensity lipoprotein （VLDL） secretion related proteins in liver cells treated by sulfite by in vitrotest.Methods: The human hepatoma cell line （HepG2） were cultured in DMEM medium containing10% calf serum until the cells grow to the logarithmic growth phase, then the HepG2 cells weredigested into a single cell suspension with 0.25% trypsin solution and seeded into the cell cultureplates.After growing to a certain extent, the cells were treated with different concentration ofsodium sulfite （ 0 to 10 mmol/L） for 24 h, 48 h and 72 h, and 10 mmol/L carbon tetrachloride（CCL4） was used as a positive control, complete medium group as a negative control. Lactatedehydrogenase（LDH） activity assay of cultural supernatant was used to detect liver cellsmembrane permeability changes; CCK-8 method was used to detect cell viability changes andOil red O staining for fatty deposition detection and Glycerol phosphate oxidase （GPO）-PODassay for detection of intracellular triglyceride （ TG ） content;The Western blotting analysis wereused to detect the protein expression levels of the ARF-1, COPI, the APOE with theircorresponding antibody; and the quantity real-time PCR method was used to detect the mRNAlevels of the ARF-1, COPI and APOE.Results:1. The detection of LDH activity showed that 10 mmol/L sodium sulfite exposure for 24 hincreased significantly the LDH activity of HepG2 cells cultural supernatant compared withnegative control group （P<0.05）,and no other dosages of sodium sulfite exposure for 24 h and alldosages exposure for 48 h and 72 h changed significantly the LDH activity of culturalsupernatant.2. CCK-8 trials showed that exposure of Na₂SO₃for 24 h and 48 h, the cell survival rates ofHepG2 cells were reduced with increasing doses of Na₂SO₃, and the differences were significantcompared with medium control group （p <0.05）; Compared with medium control group,exposing for 72 h, the survival rates of Na₂SO₃treated groups （except 0.039 mmol/L group）were significantly reduced （p <0.05）.3. When treated for 24 and 48 h, we found few amount of lipid droplets in some sodiumsulfite exposure groups but no droplets could be found in the negative control group by Oil Red O staining; exposure for 72 h, 10 mmol/L sodium sulfite exposure group showed obvious lipiddroplets in many cells.4. Exposure of certain dose of sodium sulfite can cause the changes of intracellulartriglyceride （TG） content in HepG2 cells. The TG content in HepG2 cells exposed to 2. 5mmol/L Na₂SO₃for 24 h was statistically significant as compared with the control group（P<0.05）,there were no obvious differences between the other Na₂SO₃exposed groups andcontrol group. While duration of exposure reaching to 48 h, 10mmol/L Na₂SO₃and 0.039mmol/L Na₂SO₃increased TG content in HepG2 cells significantly compared with the controlgroup; When duration of exposure lasting for 72 h, there were no significant differences betweenthe treated groups and the control group except for 10 mmol/L Na₂SO₃group and 2.5 mmol/LNa₂SO₃group （P<0.05）.5. The protein expressions of ARF-1 and COPI changes in a time-dependent manner. For 24h exposure there were no significant changes between Na₂SO₃exposure groups and control whentreated. When treated for 48 h and 72 h, the ARF-1 and COPI protein expressions increased withthe increase of Na₂SO₃doses, protein levels at 2.5 10 mmol/L groups were significantly higherthan control.6. Compared with the negative control group, we found that the mRNA levels of ARF-1 at allNa₂SO₃treated groups and COPI at 0.039 mmol/L, 2.5 mmol/L, 10 mmol/L Na₂SO₃treatedgroups and APOE gene at 0.156 mmol/L, 2.5 mmol/L Na₂SO₃treated groups for 24 h weredecreased significantly（P<0.05）; There were no statistical differences between Na₂SO₃treatedgroups and control group for 48 h and 72 h incubation for all these genes except for APOE genelevels at 0.039 mmol/L Na₂SO₃were decreased significantly（P<0.05）.Conclusion:1. Na₂SO₃can inhibit cell viability in a dose-related manner in HepG2 cells.2. Certain dose of sodium sulfite exposure can increase cell membrane permeability andcause intracellular triglyceride accumulation in HepG2 cells.3. Certain dose of the Na₂SO₃exposure can increase the ARF-1 and COPI proteinexpressions of the HepG2 liver cells.4. At a certain extent, Na₂SO₃may inhibit the gene expressions of ARF-1, COPI and APOEin hepatocytes.