Effects Of Sodium Sulfite On Lipid Metabolism Of Hepatocytes Co-cultured With Fat Cells

Objective:To study the effects of sodium sulfite on the content of triglyceride(TG)and cholesterol in liver cells by co-culturing with fat cells,and to provide a reference for the mechanism of hepatic toxicity of sodium sulfite.Methods:3T3-L3 preadipocytes were induced to differentiate into mature adipocytes,and verified by oil red O staining.With different concentrations(0.02,0.1,0.5,2.5mmol/L)of Na2SO3 and 1 mmol/L oleic acid(OA)exposure to fat cells for 12 h,fat cells supernatant were collected,the collected fat cells supernatant in each group toxicity in hepatic cells for 24 h,which were co-culture group.Then with the same concentration(0.02,0.1,0.5,2.5 mmol/L)of Na2SO3 and 1 mmol/L oleic acid direct exposure to hepatic cells for 24 h,namely separate culture group.Free fatty acid(FFA)quantitation kit was used to determine the content of FFA in fat cell supernatant.Adiponectin in fat cell supernatant was detected by ELISA.GPO-POD enzyme method was used to determine the content of TG in fat cell supernatant.TG,total cholesterol(TC)and free cholesterol(FC)in liver cells were extracted by organic extraction method,then determined with POD enzyme method.Using Western blot to detect the protein expression of adenosine 5?-monophosphate-activated protein kinase(AMPK?),P-AMPK?,acetyl-Co A carboxylase(ACC),P-ACC,sterol regulatory element-binding protern-l c(SREBP-1c),the fatty acid synthase(FASN),carnitine palmitoyl transferase 1(CPT-1),3-hydroxy-3-methylglutaryl coenzyme A reductase(HMGR),low density lipoprotein receptor(LDLR),acyl-coenzyme A:cholesterolacyltransferase(ACAT).Results:1.Compared with the negative control,exposure to Na2SO3 the FFA and adiponectin in fat cell supernatant were significantly reduced(P<0.05),TG showed no significant difference(P>0.05).2.In separate culture group the content of TG in liver cells exposed to Na2SO3 had no significant change(P>0.05)compared with the negative control group.While in co-culture group the 2.5 mmol/L Na2SO3 caused TG increased(P<0.05).3.Western blot results showed that the separate culture group,SREBP-1c,AMPK?,P-ACC and CPT-1 compared with the negative control group had no significant change(P>0.05).ACC except for 0.5 mmol/L Na2SO3,the other groups were significantly higher than the negative group(P<0.05).The protein level of FASN in 2.5 mmol/L Na2SO3 and 1 mmol/L OA groups were significantly increased(P<0.05).The co-culture group,P-ACC,SREBP-1c,FASN and CPT-1 had no significant change(P>0.05).P-AMPK? was not expressed in Hep G2 cells.The protein level of AMPK? and ACC were increased(P<0.05).4.In separate culture group the content of TC and FC in liver cells exposed to Na2SO3 had no significant change(P>0.05).While in co-culture group the 0.5mmol/L Na2SO3,2.5 mmol/L Na2SO3 and 1mmol/L OA caused TC decreased(P<0.05).And in co-culture group the 2.5 mmol/l Na2SO3 and 1 mmol/l OA can cause FC increased(P<0.05).5.Western blot results showed that the separate culture group,HMGCR compared with the negative control group had no significant change(P>0.05),ACAT protein expression increased(P<0.05),2.5 mmol/L Na2SO3 caused LDLR expression increased(P<0.05).The co-culture group,HMGCR and LDLR had no significant change(P>0.05).The protein level of ACAT were significantly increased(P<0.05).Conclusion:When people are exposed to lower doses of Na2SO3 under the environment,it should not cause the increase of hepatic cholesterol and TG.But when people are exposed to higher concentrations of 2.5 mmol/L Na2SO3 environment,they may show significant increase in TG and free cholesterol and decrease of total cholesterol in the liver which may derive form the cofunction of fat cells.