Study on the Effect and Mechanism of Ganoderic Acid C 2 on Lipid Metabolism of Hepatocytes by Regulating S6K/SREBPs Signaling Pathway / 中国药房

ABSTRACT

OBJECTIVE：To stu dy in vitro lipid-lowering effect of ganoderic acid C 2（GAC2），and to investigate its potential mechanism on the basis of S 6K/SREBPs signaling pathway. METHODS ：Using human liver cells HL- 7702 as objects ，MTT assay was used to test relative cell viability after treated with low ，medium and high doses （5，10，20 μmol/L，hereinafter）of GAC 2. Using lovastatin as positive control ，ELISA method was used to detect the contents of TC and TG in cells after treated with low ， medium and high doses of GAC 2. Nile red staining was used to observe the accumulation of lipids in cells. After transfected SREBPs report gene plasmid ，using 25-HC as positive control ，relative viability of SREBPs luciferase in cells were determined by luciferase assay after treated with low ，medium and high doses of GAC 2. Using 25-HC as positive control ，real-time fluorescent quantitative PCR was used to measure the mRNA expression of SREBPs and their downstream genes in cells after treated with medium and high doses of GAC 2. Using SREBPs inhibitor （25-HC）and S 6K inhibitor （rapamycin）as control ，Western blotting assay was adopted to determine the expression of SREBP- 1 and SREBP- 2（in the case of n-SREBPs ），relative expression ratio of phosphorylated S 6K to S 6K（p-S6K/S6K ratio ）. AutoDock 4.0 and other softwares were used for molecular docking of S 6K and GAC2. RESULTS ：There was no significant effect of low ， 0.05）. Compared with blank control group ，the content of TC qq.com in lovastatin group and GAC 2 high-dose group as well as thecontent of TG in lovastatin group ， GAC2 medium- and 床应用。电话：0371-65962746。E-mail：whui3697@126.com high-dose groups were decreased significantly （P＜0.05 or P＜ 0.01）；the number of lipid droplets in the cells of all medication groups decreased. Compared with blank control group ，relative viability of SREBPs luciferase in 25-HC group ，GAC2 low-，medium- and high-dose groups were decreased significantly ；mRNA expression of HMGCS1，MVK，SCD，HMGCR gene in 25-HC group and GAC 2 medium-，high-dose groups ，mRNA expression of DHCR7 gene in 25-HC group ，mRNA expression of SREBP-2 gene in GAC- 2 high-dose group as well as mRNA expression of DHCR24 and MSMO2 gene in 25-HC group and GAC 2 high-dose group were all decreased significantly ；relative protein expression of n-SREBP- 1 in 25-HC group ，GAC2 low-，medium- and high-dose groups ，relative protein expression of n-SREBP- 2 in 25-HC group and GAC 2 high-dose group as well as p-S 6K/S6K ratio in rapamycin group and GAC 2 groups were decreased significantly （P＜0.05 or P＜0.01）. The molecular docking results showed that GAC 2 could bound to amino acid residues Arg 335，Arg330 and Ala332 of S 6K via hydrogen bond. CONCLUSIONS ：GAC2 can reduce the lipid level of HL- 7702 cells，which may be associated with inhibiting the expression of S 6K/SREBPs signaling pathway.