Effects Of Alfalfa Saponins On LDLR,LXR,FXR MRNA Expression In BRL Liver Cells As Well As The Construction And Expression Of PNTAP-LDLR

In recent years, with the improvement of people’s living standards, high cholesterol, atherosclerosis, myocardial infarction and other cardiovascular diseases have become the top harmful factor to human health. Dyslipidemia, especially high blood cholesterol level is one of the main factors causing these diseases. Therefore, the control of hyperlipidemia is of great significance for the prevention of cardiovascular disease. Alfalfa saponins have regulating lipid metabolism, cholesterol-lowering effect and other biological activity, but there is a paucity of information on the mechanism of its cholesterol-lowering effect, especially from the cellular level. In order to study the effect of alfalfa saponins on cholesterol metabolism at the cellular level, in trial 1, we used rat BRL liver cells to study the effect of alfalfa saponins on some genes related to cholesterol metabolism; in trial 2, we constructed a key gene involved in cholesterol metabolism-low density lipoprotein receptor (LDLR) tandem affinity purification expression vector and further expressed in the HEK-293T cell. The details are as follows:Exp.l Effects of alfalfa saponins on genes involved in cholesterol metabolism in rat BRL liver cells.The normal BRL cells cultured for 2 d were divided into four groups:normal cells, normal cells with 50 mg/L,100 mg/L,150 mg/L alfalfa saponins. After cultured for 24 h, four groups were treated with different levels of alfalfa saponins for 24 h. Then the mRNA expression of LDLR, liver X receptor (LXR) and bile acid receptor (FXR) in treated cells were detected by qRT-PCR.Hyperlipidemic BRL cell model was established after treated with 50% fetal bovine serum for 48 h. After detection, hyperlipidemic BRL cells were divided into four groups: hyperlipidemic cells, hyperlipidemic cells with 50 mg/L,100 mg/L,150 mg/L alfalfa saponins. After cultured for 24 h, cells were collected and dected the mRNA expression of LDLR, LXR and FXR by qRT-PCR.The results showed that:(1) alfalfa saponins upregulated the mRNA expression of LDLR in normal cells. (2) after treated with alfalfa saponins, the mRNA expression of LXR in normal cells and hyperlipidemic BRL cells were downregulated. (3) after treated with alfalfa saponins, the mRNA expression of FXR in normal cells was significantly reduced, and extremely significantly decreased in hyperlipidemic BRL cells.Conclusion:alfalfa saponins might ameliorate hepatic steatosis by regulation of some genes involved in cholesterol metabolism, including upregulation of LDLR as well as downregulation of LXR and FXR.Exp.2 Construction and expression of pNTAP-LDLR.A pair of primers were designed according to multiple clone sites in pNTAP-B and LDLR mRNA sequence in Genbank. The total RNA was extracted from SD rat liver, and the cDNA encoding LDLR was obtained by qRT-PCR, which was confirmed by sequencing and BLAST, then it was cloned into T vertor and subsequently inserted in the expression vertor pNTAP-B. The expressed plasmids were transfected into eukaryotic cells, the expression product of pNTAP-LDLR was identified by Western blotting. The results showed that the coding region of LDLR was successfully amplified, and pNTAP-LDLR was successfully constructed. Western blotting analysis showed that LDLR was successfully expressed in transfected 293T cells. Conclusion:in the present study, LDLR was cloned, pNTAP-LDLR was successfully construsted and expressed in 293T cells, which lay a foundation for studying the function of LDLR and its interacting proteins in the further.