Cholesterol-lowering Effect And Mechanisms Of Sargassum Fucoidan On HepG2 Cell

This study was conducted to investigate the cholesterol-lowering effect and mechanisms of Sargassum fucoidan, to comprehensive evaluation of the cholesterol-lowering activity of six different crude sargassum fucoidan with chemical and cell experiment in vitro. The crude fucoidan from Sargassum zhangii was isolated and purified by DEAE-52 cellulose chromatograph and determined its chemical composition which cholesterol-lowering activity was best. Discussed the effect and mechanisms on cholesterol metabolism of the different components from Sargassum zhangii fucoidan by HepG2 cell. Finally provide the theory basis for the research on cholesterol-lowering activity of sargassum fucoidan. The main research content is as follows.(1) Comparative studied the cholesterol micellar inhibition rate,bile acid salt binding rate and the effect of the total cholesterol levels on HepG2 cell of crude fucoidan from six different sargassum(Sargassum wightii, Sargassum zhangii, Sargassum hemiphyllum, Sargassum henslowianum, Sargassum naozhouense, Sargassum integerrimum). The results showed that, crude fucoidan from Sargassum zhangii had a relative advantages of cholesterol-lowering effect which polysaccharide content was 33.37%, fucose content was 7.91% and the content of sulfate was 13.63%.(2) Using DEAE-52 cellulose chromatograph purified the crude focoidan of Sargassum zhangii, and there were three different components(named ZF1,ZF2,ZF3). Detecting chemical composition of each component, the polysaccharide content of ZF1, ZF2, ZF3 were 58.29%, 60.84%, 54.26%, fucose content were 4.94%, 17.81%, 26.01%, the content of sulfate were 3.29%, 19.39%, 18.89%. Using GC-MS to analyze monosaccharide composition of ZF1, ZF2, ZF3. The three components were composed of fucose, galactose, rhamnose, xylose, mannose and glucose, but the percentage of each monosaccharide were different.(3) Comparative study of the cholesterol-lowering effect of ZF1, ZF2, ZF3 on HepG2 cell were cultured for 24 h and 48 h. The results showed that three kinds of fucoidan can decrease the cholesterol level of HepG2 cell at the concentration of 200 μg/m L and 800 μg/m L(p﹤0.05), showing a better effect at 24 h than at 48 h, and the cholesterof-lowering effect of ZF2 was best. In addition, the levels of intracellular TC and the levels of TBA, ApoA1 and ApoB in culture medium were determined after the cells were cultured for 0, 12, 24, 36, 48 h with ZF2 at concentrations of 800 μg/mL or were cultured for 24 h in the presence of 50, 200, 800 μg/mL. When the concentrations of 200 μg/mL, 800 μg/mL, ZF2 could decreased the levels of intracellular TC and the secretion of ApoB as well as increased TBA and ApoA1 secretion obviously, but the effect of 50 μg/m L ZF2 was opposite. When HepG2 were cultured from 0 h to 48 h with 800 μg/m L ZF2, a more pronounced effect in intracellular TC and TBA, ApoA1, ApoB in culture medium was observed when culture time was 24 h(p﹤0.05). And the effect would weakened after 24 h until returned to normal level.(4) The gene expression levels of SREBP-2, LDLR, HMGCR, LXR-α, ABCA1, CYP7A1 were measured by real-time quantitative PCR after the cells were incubated with 50, 200, 800 μg/m L ZF2 for 24 h or were cultured for 0, 12, 24, 36, 48 h with 800 μg/m L ZF2. The results showed that, each of mRNA expression were a rising trend when the HepG2 cells were cultured with 50, 200, 800 μg/m L ZF2 for 24 h, but only the gene expression were significant with the concentration of 800 μg/m L(p﹤0.01). The highest mRNA expression of LDLR, HMGCR, LXR-α, ABCA1 were noted after 24 h, and the highest mRNA expression of CYP7A1 were noted after 48 h when HepG2 cells were incubated with 800 μg/mL for 0, 12, 24, 36, 48 h(p﹤0.05). These results suggested that the cholesterol-lowering mechanism of Sargassum zhangii fucoidan is by multiple ways to achieve and the cholesterol-lowering effect related with its pollysaccharide content, sulfate content and composition of monosaccharides. Firstly, it could promote the expression of bile acid synthesis enzyme CYP7A1 mRNA, liver receptor LXR-α mRNA, ABCA1 mRNA, thereby increased the discharge of cholesterol. Then it could reduce intracellular cholesterol synthesis by affecting HMG-CoA reductase activity.Finally, intracellular cholesterol levels activated expression of SREBP-2 mRNA with a feedback mechanism, and incresed expression of LDLR which was target gene of SREBP-2 mRNA, thereby accelerated the catabolism of LDL in plasma.