| Objects:To.study the effects of emodin on expression of SREBP-1c mRNA in steatotic hepatocytes and to investigate the mechanism of emodin regulating lipid metabolism.Methods:(1) To establish a model of hepatocyte steatosis,the normal human L02 hepatocytes were incubated in RPMI-1640 medium with 50%(V/V)calf serum for 24 hours,48 hours and 72 hours respectively to determine the best duration for establishing steatotic hepatocytes model.(2)The MTT assay was used to choose the appropriate concentration of emodin which would be used to treat steatotic hepatocytes.(3)After the model was established steatotic hepatocytes were incubated in different groups,such as normal group,model group, spontaneous recovery group,emodin group with the concentration of 5μM and 10μM for 24 hours and 48 hours respectively.Then the formation of cellular morphology and lipid droplets in the cells would be observed under oil red O stain and the intracellular triglyceride(TG) levels would be determined through automatic biochemical analyzer.Then the best concentration and duration of emodin were determined for ameliorating hepatocyte steatosis. (4)The semi quantitative reverse transcription polymerase chain reaction(RT-PCR) was employed to detect the expression level of SREBP-1c mRNA in hepatocytes in each group.Results:1.After incubated in RPMI-1640 medium with 50%(V/V)calf serum for 48 hours and 72 hours,numerous lipid droplets were scattered in the cytolymph of human L02 hepatocyte by inverted microscope,which can be dyed to orange-red or red with oil red O stain.And extracellular serum ALT,AST,ALP,LDH and intracellular TG levels were found out to be increased remarkably than normal group through automatic biochemical analyzer(P＜0.01).Thus,the model of steatotic hepatocytes was established successfully.2.MTT assay showed out the appropriate concentration of emodin on human L02 hepatocytes:1μM,2.5μM,5μM,10μM separately.This study used 5μM and 10μM as the concentration of emodin on human L02 hepatecytes.3.24 hours later after emodin treated,cellular level of TG in spontaneous recovery group was decreased remarkably than that in model group(P＜0.01);cellular level of TG in emodin 10μM group was decreased remarkably than spontaneous recovery group,which was statistically significant(P＜0.05).Oil red O stain presented numerous orange-red or red lipid droplets in cytoplasm of human L02 hepatocytes of model group under inverted microscope,the number of lipid droplets in cytoplasm and its color in normal group,spontaneous recovery group and emodin groups were decreased dramatically,and the emodin 10μM group was the most one.4.48 hours later after emodin treated,cellular level of TG in spontaneous recovery group was decreased remarkably than that in model group(P＜0.01);cellular level of TG in emodin treatment groups presented no significant difference with spontaneous recovery group(P＞0.05).Oil red O stain presented numerous orange-red or red lipid droplets in cytoplasm of human L02 hepatocytes of model group under inverted microscope,the number of lipid droplets in cytoplasm and its color in normal group,spontaneous recovery group and emodin groups were decreased dramatically,but it presented no difference between the emodin treatment groups and spontaneous recovery group under inverted microscope.5.24 hours later after emodin treated,the expression level of SREBP-1c mRNA of model group was much higher than that of normal group which was statistically significant (p＜0.01);the expression level of SREBP-1c mRNA in spontaneous recovery group was much lower than that of model group but presented no statistically significant(p＞0.05); the expression level of SREBP-1c mRNA presented no significant difference between different concentration emodin group and spontaneous recovery group(P＞0.05).Conclusions:1.The steatotic hepatocytes model was established successfully by incubating human L02 hepatocyte in RPMI-1640 medium with 50%(VN) calf serum for 48 hours and 72 hours.2.Emodin decreased the quantity of intracellular TG in steatotic hepatocytes;emodin of 10μM concentration has obviouser effect.3.The deposition of lipid in hepatocytes might be relative with the upregulate of SREBP-1c mRNA.The function of decreasing cellular TG by emodin might have the participance of other genes correlated with lipid metabolism.
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