| Atherosclerosis is the key physiopathologic basis of cardiovascular disease, and elevatedLp(a) is an independent risk factor for atherosclerosis, but in the present which doesn’t havespecifically and aggressively methods to decreasing lipoprotein(a) of patients with highlipoprotein(a).apolipoprotein(a)[apo(a)] is the unique apolipoprotein of lipoprotein(a)[Lp(a)], a large number of studies show that the synthesis rates and size of apo(a) decided Lp(a)concentrations and pathogenic. Thus,how inhibited apo(a) synthesis is the main strategies forreduced Lp(a) in plasma.Recently, one study indicated that the human apo (a) promoter contains the Ets-1bindingmotif and DR-1motif, which can be bind to Elk1and HNF4α to regulated apo(a) transcription.Elk-1, which belongs to the family of Ets domain-containing transcription factors, is awell-characterized common nuclear substrate for activated ERK1/2; moreover, HNF4α havebeen indicated inihited by ERK1/2activation. FGF21can stimulate glycogen synthesis andlipid metabolism via ERK1/2in the liver, and its media by FGFR. Thus, The purpose of thisstudy is to assess the effects of FGF21on the expression of apo(a) in HepG2cells and toexplore its mechanism. For the drugs that lower plasma lipoprotein (a) provide newtheoretical basis.PartⅠ: Effect of FGF21on the expression of apo (a) in HepG2cellsObjectives: To indentify the effects of FGF21on the expression of apo (a) in HepG2cells.Methods: After reaching confluence, HepG2cells were maintained in fresh serum-freemedium for6hours to get synchronization growth. After that, the medium was replaced withfresh serum-free medium containing different concentrations of FGF21(0ng/ml,50ng/ml,100ng/ml,200ng/ml,400ng/ml)and incubated with cells for24h, or with200ng/ml FGF21fordifferent times(0h,6h,12h,24h，48h). Real Time-PCR, Western blotting and Immunohistochemical Staining were applied to determine the apo(a) expression in mRNA andprotein levels, respectively. Enzyme-linked Immunosorbent Assay (ELISA) detected apo (a)secretion.Results: Compare with control group, the mRNA and protein expression of apo (a) and thelevels of the apo(a) protein in the medium decreased with increasing FGF21concentration,with the strongest effect observed at a concentration of200ng/ml,400ng/ml;Compare with0hgroup, the mRNA and protein expression of apo(a) were significantly downregulated inHepG2cells after24h of incubation with200ng/ml FGF21. The expression of apo (a)decreased as the incubation time was prolonged, with the strongest effect observed in the24hgroup.Summary:FGF21decreases apo(a) expression in a dose-and time-dependent Manner.PartⅡ:The mechanism of FGF21influence apo(a) expression in HepG2cellObjectives: To investigate the molecular mechanism of FGF21down-regulate apo(a)expression in HepG2cell.Methods: Pretreatment with ERK1/2inhibitor PD98059or FGFR1inhibitor PD166866orElk-1siRNA for HepG2cells. And then added to FGF21(200ng/ml) for24h. Western-blottinganalyses for ERK1/2、p-ERK1/2、Elk-1、p-ERK1/2、FGFR1-4、HNF4α、FXR and apo(a)expression, and the expression of apo(a) and FGFR1-4Mrna detected by Real Time-PCR.Moreover, the apo(a) in the culture medium were extracted and subjected to ELISAResults: FGF21should be significant up-regulate ERK1/2activation, and the apo(a)expression should be inhibited when treatment with ERK1/2inhibitor; Treatment with siRNAfor Elk1siRNA or FGFR1inhibitor was attenuates FGF21-mediated inhibition of apo(a)expression. Moreover, the FGFR1inhibition should be attenuates ERK1/2and Elk-1activated.Futhermore, we also found that the expression of HNF4α was down-regulate by FGF21, butFXR did not change, and treatment with FGFR1inhibitor or ERK1/2inhibitor was attenuatesFGF21-mediated inhibition of HNF4α.Summary: FGF21down-regulation apo(a) expression via bothFGFR1-ERK1/2-Elk-1pathway and FGFR1-ERK1/2-HNF4α Conclution:①FGF21decreases apo(a) expression in a dose-and time-dependent Manner②FGF21down-regulation apo(a) expression via both FGFR1-ERK1/2-Elk-1pathway andFGFR1-ERK1/2inhibite HNF4α. |
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