Effects Of Leptin On Triglyceride Levers In Vitro Human L-02 Steatosis Hepatocyte

Objects: To observe the effects of leptin on triglyceride levels , and the mRNA expression of OB-Rb,STAT3 and MTP in vitro human L-02 steatosis hepatocyte,deducing the possible relationship among Lptin,OB-Rb,and the subsequent signal pathway on nonalcoholic fatty liver .To study the mechanism of leptin regulating the level of fat in steatosis hepatocyte,offering the evidence for next investigation.Methods: The investigation was conducted by means of randomized controlled trial. To establish a model of hepatocyte steatosis, we incubate the normal human L-02 hepatocyte in RPMI-1640 complete medium with 10% (V/V) fetal bovine serum plus 10% (V/V) medical fat emulsion injection for 24 hours. Then add recombinant human leptin of varies concentration into the medium and incubate for another 24 hours. The concentration of leptin was respectively 10-6mol/L,10-7mol/L and 10-8mol/L.And observe the formation of cellular morphology and lipid droplets in the cells under oil red O stain and determin the intracellular triglyceride levels through high performance liquid chromatography (HPLC). The semiquantitative RT-PCR was used to detect the mRNA levels of OB-Rb,STAT3 and MTP of the hepatocytes of each group. The best concentration of leptin that decreases the amount of intracellular triglyceride levels was decided and the dose-effect relationship curve was established. Then expose the cell to leptin at the optimal concentration for 0hr,6hr,12hr,24hr and 48h respectively, observe the above-mentioned parameters and determine the best duration for leptin to decrease intracellular triglyceride levels, and draw out the duration-effect relation curve. All the data was analyzed by SPSS13.0 statistical software.Results: After incubated in fat emulsion injection for 24 hours, numerous lipid droplets were scattered in the cytolymph of human L-02 hepatocyte by inverted microscope, which can be dyed to red with oil red O dying. And intracellular TG levels were found out to be increased dramatically through measurement of HPLC. Thus, the model of steatosis hepatocytes was established successfully. After exposed to leptin(10-6mol/L, 10-7mol/L and 10-8mol/L) for 24 hours, the intracellular lipid droplets of steatosis hepatocytes were much less than the model group by oil red O stain, the intracellular TG levels decreased remarkablely with dose-dependent pattern with measurement of HPLC. The semi quantitative RT-PCR was employed to detect the mRNA levels of OB-Rb and its target genes STAT3,MTP in the hepatocytes of each group. The expression of OB-Rb and target genes was found out to be much higher in the group treated with leptin than in the model ones, which is statistically significant(P<0.05). After exposed to the leptin of 10-7mol/l for0,6,12, 24 and 48 hours, the results show the decrease of TG levels and increase of OB-Rb expression in the foam cell was occured in a time-dependent manner.Conclusions:1. Leptin decreases the quantity of intracellular triglyceride in steatosis hepatocyte with a time and dose dependent manner.2. The treatment of leptin in fatty changed human L-02 hepatocyte could promote the mRNA expression of OB-Rb and its target genes STAT3,MTP by a dose-dependent manner .3. Leptin can decrease the intracellular TG levels in steatosis hepatocyte in a dose-dependent manner and the possible mechanisms are related to upregulation of the mRNA levels of OB-Rb and its target genes.4. There will be leptin resistance in vitro steatosis hepatocyte ,because of the OB-R quantities decreasing.