The Effect And Mechanism Of Ellagic Acid On Adipogenesis In3T3-L1Preadipocytes

Objective:To investigate prevalence of central obestiy in Uighur and kazak Population and to determine the effect of Ellagic Acid on sub-confluent proliferation and mitotic clonal expansion and adipogenesis in3T3-L1preadipocyte, try to combine the clinic phenomena with mechanism study and to provide promising target (s) for prevention of metabolic disease, finally to provide possible mechanism for developing pomegranat peel as natural anti-obesity agent. Methods:Survey contains samples of adults aged≥30years. Totally,1,379Uighur and1,123Kazak volunteers were enrolled according to inclusive and exclusive criteria. Questionnaires, blood pressure, anthropometric measurement, and blood serum lipoprotein were monitored. Data were processed by Chi-square test or t-test in SPSS17.0. To clarified the effect of EA on adipogenesis:Typan blue dye exclusion test was used to check the cell viability; For cell proliferation, WST-1staining and cellcounting were used for sub-and post-confluent preadipocytes; PI and ANNEXIN-V staining were used to check the effect of EA on sub-and post-confluent preadipocyte apoptosis; Staining neutral lipid in the cytoplasm with Oil Red O to check the effect of ellagic acid on adipogenesis, then we check the time period of EA action; The cell cycle related protein expression (cyclinA, CDK2, phospho-Rb/total-Rb, total C/EBPβ and C/EBPβ Thr188phosphorylation) and adipocyte specific protein expression were checked by Western Blott to testify thr role of EA; To know the cell cycle progression with EA, PI was used to stain DNA, which was checked by flow cytometry; EdU incoporation and Hoechst33342staining were used to show the cell entering S phase; Nucleus protein was extract to investigate the effect of EA on C/EBPβ and C/EBPa DNA binding acitivity by TRANS Active Motif ELISA kit; Luciferase reporter gene assay was choosed to describe the effect of EA on C/EBPβ transcriptional activation; Real time PCR and western blott were used to show the effect of EA on C/EBPβ target gene and protein expression. Results:The prevalence of central obesity in Uighur and Kazak population is51.6%and50.8%, respectively. And the pervalence of HTG is53.6%and24.1%, low HDL-c is49.1%and12.1%, respectively after age-and gender-adjusted. EA exert no impact on cell viability in3T3-L1preadipocytes but inhibit sub-and post-confluent preadipocytes with time-and concentration dependent manner; In another word, EA fail to induce apoptosis in sub-and post-confluent preadipocytes; EA inhibit adipogenesis in3T3-L1preadipocytes and the inhibitory effect on lipid accumulation was diminished when addition of EA was delayed to day2of differentiation; The cell cycle related protein (cyclinA, phospho-Rb/total-Rb and C/EBPβ Thr188phosphorylation) was impaired by EA, so did the adipocyte specific protein expression, but EA fait to influence on CDK and total protein expression. Then the data show that EA blocked the cell cycle progression, and restrained the cell from entering S phase possiblly through attenuating C/EBPβ a DNA binding acitivity; finally the results show that EA inhibit the expression of C/EBPβ target gene and protein. Conclusion:There is high prevalence of central obesity among Uighur and Kazak population, accompanying high prevalence of HTG and low HDL-c. EA reduce adipogenesis through inhibition of DNA binding activity and transcriptional factor activity of C/EBPβ and of preadipocytes proliferation and MCE, also EA attenuate late adipogenic marker in late stage of differentiation to promote the inhibition of adipogenesis. These data tend to provide laboratory basis to develop natural anti-obesity drug with less side effect and more efficacy.