| Cholesterol is an important component of mammalian cell membrane, which plays a key role in maintaining the structure and function of cell membrane, and also displays an important action in some physiological processes, such as the intracellular material transportation, cell differentiation and embryonic development. But the increased intracellular cholesterol level could destroy the cell membrane structure and promote the cell apoptosis. Besides, the overhigh cholesterol level could induce various diseases, such as atherosclerosis and coronary heart disease(CHD) etc. Researches indicated that the up-taking of excessive cholesterol by diet is one of the main causes of hypercholesterolemia, therefore, the study on cholesterol absorption is the hot point in the current area of lipid metabolism.SAK-HV protein is a multifunctional fusion-protein with MW 17 k D, constructed in our laboratory, whose molecular structure includes a SAK mutant, RGD sequence, and a thrombin-binding area, among them SAK mutant displayed fibrin-dissolving effect, RGD sequence inhibited the platelet aggregation, and the thrombin-binding area possessed anti-thrombin effect. The original intention of constructing SAK-HV fusion protein was expecting to achieve a multi-targeted anti-atherosclerotic effect with thrombolysis, anti-coagulation, and anti-inflammation. In the study on pharmacodynamics, it was found that the fusion protein SAK-HV can inhibit the development of atherosclerotic plaque and inflammatory reaction of hemoendothelial cells in Apo E-/-mice fed on high fat diet. Besides, we surprisingly found that SAK-HV also can decrease the plasma cholesterol level in Apo E-/-mice, but its mechanism is not yet clear.The internal cholesterol metabolism of mammal includes the absorption, synthesis, and excretion etc. Small intestine is the main site of cholesterol absorption, NPC1L1 expressed on the brush border membrane of small intestine epithelia is an important protein to mediate the absorption of cholesterol in small intestine, NPC1L1 protein can mediate the absorption of cholesterol by intestinal epithelial cells through its N-terminal cysteine-rich domain(NTD)binding cholesterol, further entering the cell by means of the endocytosis of sarcostyle.The researches indicated that the cholesterol absorption level in the small intestine of the NPC1L1 gene- knocked out mice was decreased by 70%, but Ezetimibe, the inhibitor of NPC1L1, can decrease the cholesterol level of the zebra fish fed on high fat diet. Besides, ABCG5/ABCG8 expressed on the brush border membrane of epithelial cells in small intestine can reversely transport the free cholesterol into the intestinal lumen, then being excreted together with feces. The researches demonstrated that the ABCG8 gene deletion in the mice significantly increased the absorption of cholesterol and sitosterol by the mice, but the over-expression of ABCG5 and ABCG8 in the mice could decrease the absorption of cholesterol and sitosterol. Liver X receptor(LXR) is a ligand-activated transcription factor, in this receptor superfamily, LXRɑ(Liver X receptor-α)and LXRβ(Liver X receptor-β)participate in the regulation of internal cholesterol metabolism. The up-regulation of LXRαand LXRβ expression in Caco-2 cells and mouse intestine can decrease NPC1L1 expression, and up-regulate the expression of ABCG5 and ABCG8.Liver is the main site for cholesterol synthesis, the rate-limiting enzyme of liver cholesterol synthesis is 3-hydroxy-3-methylglutaryl-Co A reductase( HMGCR),intrahepatic ABCG5 and ABCG8 protein can accelerate the excretion of hepatic ABCG5/ABCG8 by bile, and the expression of NPC1L1 protein in human and primate can reabsorb the cholesterol in bile.The previous experimental results suggested that the fusion protein SAK-HV could decrease significantly the plasma cholesterol level in the Apo E-/-micefed on high fat diet, but whether this effect was caused by inhibiting the absorption of cholesterol by small intestine or not, that need to be confirmed by our further research. Therefore, this study explored the influence of SAK-HV on absorption of cholesterol by intestine and its possible mechanism. As liver plays an important role in cholesterol metabolism, we also paid attention to the influence of liver on expression of the genes related with cholesterol synthesis and excretion. The main results were as follows:1. The influence of SAK-HV on absorption of cholesterol and its possible mechanismPrevious experiments demonstrated that the plasma cholesterol level was decreased in the SAK-HV-treated Apo E-/-mice fed on high fat diet, therefore, this study utilized the Apo E-/-mice as the test object to establish an high fat model, and in the same time the wild-type of C57 mice fed on the normal feed as the control group. At the time point after feeding on high fat diet for one week, the detection of blood fat level indicated that the plasma levels of total cholesterol(TC), triglyceride(TG), and low density lipoprotein(LDL) in the Apo E-/- mice fed on high fat diet were significantly higher than those in C57 miceof the control group, however, high density lipoprotein(HDL) levels were not significantly changed, confirming that the high lipid model was successfully constructed. Subsequently the Apo E-/-mice fed on high fat diet were randomly divided into one model group and two SAK-HV- administered groups(one with the dose of 0.125mg/kg, and the other one with the dose of 0.5mg/kg) each containing 12 mice. The mice were injected via the tail vein for continuous 14 days in SAK-HV-administered groups; and the equal volume of normal saline was injected into each animal in the model group. After finishing the administration, assaying the blood fat level demonstrated that the plasma levels of TC and TG in SAK-HV-administered group were significantly decreased in the SAK-HV group were significantly decreased in comparison with those in the model group, but those were not significantly different between two SAK-HV administered groups, and LDL and HDL were not notably changed, illustrating the recombinant protein SAK-HV can decrease the plasma cholesterol level of Apo E-/-mice. Afterwards, the Apo E-/-mice in administered group at the dose of 0.5mg/kg and the model group were taken as the study objects to explore the mechanism for decreasing the plasma cholesterol level in Apo E-/-mice by SAK-HV, q PCR and Westernblot were used respectively to monitor the m RNA and protein expression levels of the related genes, as a result, it was found that NPC1L1 expression level in the small intestine was decreased, both ABCG5/ABCG8 and LXRβ were increased in the SAK/HV-administered group, but the LXRα was slightly down-regulated. however, the expression levels of ABCG5/ABCG8 and LXRα/β in liver were not different between these two groups, but HMGCR expression level in the SAK-HV administered group was down-regulated in comparison with the model group. 2. The influence of SAK-HV on absorption of cholesterol at cellular level and its possible mechanismIn order to illustrate the influence of the protein SAK-HV on the absorption of cholesterol in the intestinal epithelial cells, Caco-2 cells were utilized as the study object and stimulated with various concentrations of SAK-HV for 24 h, then the absorption of cholesterol in Caco-2 cells was determined by NBD-cholesterol as a fluorescent probe, as a result, it was found that the absorption level of cholesterol in cacao cells started to be decreased at the concentration of 50μg/ml, but not to be further decreased at 100μg/ml, thus, dose-effect relationship was not so definite.q PCR and Westernblot were used to assay m RNA and protein expression levels of the related genes, as a result, it was found that the m RNA and protein expression levels of NPC1L1 was down-regulated at the concentration of 100μg/ml. ABCG5 and ABCG8 were up-regulated at the concentration of 20μg/ml and 50μg/ml. LXRα was up-regulated at the concentration of 20μg/ml. but LXRβ was up-regulated only at the concentration of 100μg/ml. Afterwards, 100μg/ml SAK-HV was used to stimulate the Caco-2 cells for different time and to analyze the influence of different stimulation time on the cholesterol absorption and the expression of related genes in the caco cells. As a result, the absorption level of cholesterol in Caco-2 cells was significantly decreased after the stimulation for 6h. The results from q PCR and Westernblot indicated that m RNA and protein expression levels of NPC1L1 was down-regulated after stimulation with SAK-HV for 6h. ABCG5/ABCG8 and LXRβ all were up-regulated after the stimulation with SAK-HV for 3h, however, LXRα was down-regulated after stimulation with SAK-HV for 12 h.As we found that the expression of the rate-limiting enzyme HMGCR for cholesterol synthesis was decreased in the Apo E-/-mice fed on high fat diet, in order to explore the influence of SAK-HV on the expression of cholesterol metabolism-related genes in the normal liver cells, Hep G2 cells were taken as the test object and stimulated by various concentrations of SAK-HV for 24 h, then q PCR and Westernblot were utilized to determine the test results, and it was found that the expression of ABCG5/ABCG8, LXRα/β and HMGCR in Hep G2 cell all were not influenced by SAK-HV stimulation.In summary, it was confirmed that the plasma cholesterol level in Apo E-/-micecan be decreased by the recombinant protein SAK-HV in the animal experiment of this study, and it was also found that NPC1L1 protein expression in small intestine was down-regulated, but that of ABCG5/ABCG8 and LXRβ was up-regulated, thus we hypothesized that SAK-HV inhibited the absorption of cholesterol by small intestine through activating the inhibition of LXRβ expression.In the cell experiment, we further demonstrated that SAK-HV can inhibit the absorption of cholesterol by Caco-2 cells, and revealed the mechanism identical to that in the animal experiment.Therefore, it was concluded that the recombinant protein SAK-HV can inhibit the absorption of cholesterol into the small intestine, its possible mechanism might regulate the expression of NPC1L1 and ABCG5/8 in the small intestine via activating the expression of transcription factor LXRα/β, finally inhibit the absorption of cholesterol in the small intestine. The above-mentioned results laid a firm basis for illustration of SAK-HV action mechanism and the further development. |
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