H₂S Inhibits APO（a） Expression Through PKCα/FXR And AKT/HNF4α Pathway In HepG2 Cell

Lipoprotein(a) [Lp(a)] is considered to be the strongest genetic risk factor for coronary heart disease(CHD) and Atherosclerosis(AS), however the metabolism of Lp(a) is poorly understood. The drug that have been shown to can lower Lp(a) also have effects on other CHD risk factors, but there is lacking efficient and secure drugs to lower high level of plasma Lp(a) concentrations recently. In the scientific research now, FXR(also known as NR1H4) was found to can compete with the HNF4α-mediated( HNF4α is also known as NR2A1) activation of APOA transcription for binding to repeat 1 element between nucleotides-826 and-814 motifs. And the activation of FXR was responsible for the lower Lp(a) levels in plasma of mice and cholestatic patients, hence, it is through regulating a variety of metabolic enzymes and transporters to relative to the maintenance of cholesterol homeostasis.Hydrogen sulfide(H2S), it is a member of the gastransmitter family, and it plays many important physiological roles in the human body, for instance it can protection against cardiovascular disease via PKCα-FXR or Akt-HNF4α signaling pathway to regulate lipid and glucose metabolism in hepatocytes and adipocytes. In this study, we tried to research the possible molecular mechanisms that may be influence apo(a) biosynthesis. We also probe into the effects of H2 S on Hep G2 cell apo(a) expression and secretion, and the possible mechanism of H2 S in these effects. For the drugs that lower plasma lipoprotein(a) provide new theoretical basis.PartⅠ: Effect of H2 S on the expression of apo(a) in Hep G2 cells[Objectives]To indentify the effects of H2 S on the expression of apo(a) in Hep G2 cells.[Methods]Hep G2 cells were cultured in High glucose medium containing 10% fetal bovine serum. When the cells were grown to 70% in cell culture flasks, the medium was replaced with fresh fetal bovine serum-free medium containing different concentrations of Na HS(0μmol/L、25μmol/L、50μmol/L、100μmol/L、200μmol/L) and then incubate for 24 h, or with 200μmol/L Na HS for different times(0、6h、12h、24h). We apply Western blot and RT-PCR to detect the protein and m RNA levels of apo(a) expression. And Enzyme-linked Immunosorbent Assay(ELISA) detected apo(a) secretion levels.[Results]The protein and m RNA expression of apo(a), and the secretion levels of apo(a) in the medium decreased with increasing Na HS concentration compare with control group, with a concentration of 100μmol/L、200μmol/L play the strongest effect. The protein and m RNA expression of apo(a) were significant decreased in Hep G2 cells after 24 h incubation with 200μmol/L Na HS compare with 0h group. As the incubation time prolong the expression of apo(a) decreased, and in the 24 h group play the strongest effect.[Summary]Na HS decreases expression of apo(a) in a dose- and time- dependent Manner.PartⅡ:The mechanism of Na HS influence expression of apo(a) in Hep G2 cell[Objectives]To explore the molecular mechanism of Na HS down-regulate expression of apo(a) in Hep G2 cell. [ Methods ] Pretreatment with FXR si RNA, HNF4α si RNA, PKCα inhibitor Calphostin C or AKT inhibitor LY294002 for Hep G2 cells. And then add in 200μmol/L Na HS for 24 h. Western-blot analyses for expression of FXR、HNF4α、PKCα、M-PKCα、AKT、p-AKT and apo(a), and the apo(a) m RNA expression detected by RT-PCR. Moreover, the secretion levels of apo(a) in the culture medium were extracted and subjected to ELISA.[Results]Na HS can be significantly up-regulate FXR、M-PKCα and p-AKT activation, and down-regulate HNF4α expression; The expression of apo(a) should be inhibited when treatment with PKCα or AKT inhibitor; Treatment with si RNA for FXR si RNA was attenuates Na HS-mediated inhibition of apo(a) expression. After treatment with HNF4α si RNA for 24 h, the Na HS-induced inhibition of apo(a) expression and secretion was not attenuated. Partially because HNF4α can compete with FXR for binds to a control element located at nucleotides-826 and-814 of the human APOA promoter, and Positive regulation Na HS-induced down-regulation of apo(a) expression.[ Summary ] H2 S down-regulation apo(a) expression via both PKCα-FXR and Akt-HNF4α pathways.[Conclution]1. H2 S decreases apo(a) expression in a dose- and time- dependent Manner; 2.H2 S down-regulation apo(a) expression via up-regulate PKCα-FXR pathway;3.H2 S down-regulation apo(a) expression through AKT inhibit HNF4 α...