Effects Of Leptin On Triglyceride Levels And Expression Of PPARa,CPT-â… ,LPL In Human L-02 Hepatocyte

Objects: To observe the effects of leptin on triglyceride levels, the formation of lipid droplets and the mRNA expresion of PPARαand its target genes CPT-I,LPL in human L-02 steatosis hepatocyte. The aim is to study the pathogenesis of hepatocyte steatosis and to construct theoretical foundation for the prevention and cure earlier period NAFL in clinic.Methods: To establish a model of hepatocyte steatosis, we incubate the normal human L-02 hepatocyte in RPMI-1640 complete medium with 10% fetal bovine serum plus 10% medical fat emulsion injection for 24 hours. The experiment is divided into 7 groups: the first group is normal hepatocytes; the second one is fatty change hepatocytes; Group 3 to 5(different amounts of leptin is added to the fatty changed hepatocytes, to make the concentration of leptin to be 10-8mol/L, 10-7mol/L and 10-6mol/L, respectively); Group 6 is the positive control group, i.e Gemifibrozil is added to the hepatocyte with fatty change to make the concentration to be 1000mg/L. Incubate these human L-02 hepatocyte for another 24 hours. 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 PPARαand its target genes CPT-I,LPL in the hepatocytes of each group. According to the descendent level of intracellular triglyceride, the dose effect curve was made out. Then the cells were exposed to leptin at the 10-7mol/L concentration for 12hr, 24hr and 48h, respectively. The above-mentioned parameters were observed and the duration-effect curve was drawn out. All the data was analyzed by SPSS11.0 statistical software, P value of <0.05 is considered significantly important.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(10-8mol/L, 10-7mol/L and 10-6mol/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 PPARαand its target genes CPT-I,LPL in the hepatocytes of each group. The expression of PPARαand 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<0.01). After exposed to the leptin of 10-7mol/l for12, 24 and 48 hours, the results show the decrease of TG levels and increase of PPARαexpression 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 PPARαand its target genes CPT-I,LPL 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 PPARαand its target genes.