Inhibition Of Cholesterol Uptake Research Articles

Evaluation of the role of metabolizing enzymes and transporter variants in ezetimibe pharmacokinetics.

Ezetimibe inhibits cholesterol uptake by modulation of intestinal sterol absorption. Currently, although some studies have shown alterations in ezetimibe levels caused by alterations in the ABCG5, ABCG8, NPC1L1 or UGT1A1 genes, there are no pharmacogenetic guidelines to confirm these biomarkers. The aim of this work was to evaluate the effect of 49 variants in 22 pharmacogenes related to metabolism and transport. A total of 96 healthy volunteers from four bioequivalence clinical trials of ezetimibe as monotherapy or in combination with simvastatin were studied. Blood samples were extracted for unconjugated ezetimibe plasma quantification and genotyping. No association of metabolizing enzyme variants with ezetimibe pharmacokinetic parameters was found. The results show some trends in the univariate analysis for ABCB1 rs2032582 or ABCC2 rs2273697 and Cmax (p univariate (p uv ) = 0.056 and 0.087, respectively), which finally reach significance in the multivariate analysis (p multivariate (p mv ) = 0.049 and 0.048, respectively). Nevertheless, these results need to be validated in future studies.

Totum-070, a Polyphenol-Rich Plant Extract, Prevents Hypercholesterolemia in High-Fat Diet-Fed Hamsters by Inhibiting Intestinal Cholesterol Absorption.

Atherosclerotic cardiovascular disease is the leading cause of mortality worldwide, and hypercholesterolemia is a central risk factor for atherosclerosis. This study evaluated the effects of Totum-070, a plant-based polyphenol-rich supplement, in hamsters with high-fat diet (HFD)-induced dyslipidemia. The molecular mechanisms of action were explored using human Caco2 enterocytes. Totum-070 supplementation reduced the total cholesterol (-41%), non-HDL cholesterol (-47%), and triglycerides (-46%) in a dose-dependent manner, compared with HFD. HFD-induced hepatic steatosis was also significantly decreased by Totum-070, an effect associated with the reduction in various lipid and inflammatory gene expression. Upon challenging with olive oil gavage, the post-prandial triglyceride levels were strongly reduced. The sterol excretion in the feces was increased in the HFD-Totum-070 groups compared with the HFD group and associated with reduction of intestinal cholesterol absorption. These effects were confirmed in the Caco2 cells, where incubation with Totum-070 inhibited cholesterol uptake and apolipoprotein B secretion. Furthermore, a microbiota composition analysis revealed a strong effect of Totum-070 on the alpha and beta diversity of bacterial species and a significant decrease in the Firmicutes to Bacteroidetes ratio. Altogether, our findings indicate that Totum-070 lowers hypercholesterolemia by reducing intestinal cholesterol absorption, suggesting that its use as dietary supplement may be explored as a new preventive strategy for cardiovascular diseases.

Laminarin Reduces Cholesterol Uptake and NPC1L1 Protein Expression in High-Fat Diet (HFD)-Fed Mice.

Aberrantly high dietary cholesterol intake and intestinal cholesterol uptake lead to dyslipidemia, one of the risk factors for cardiovascular diseases (CVDs). Based on previous studies, laminarin, a polysaccharide found in brown algae, has hypolipidemic activity, but its underlying mechanism has not been elucidated. In this study, we investigated the effect of laminarin on intestinal cholesterol uptake in vitro, as well as the lipid and morphological parameters in an in vivo model of high-fat diet (HFD)-fed mice, and addressed the question of whether Niemann-Pick C1-like 1 protein (NPC1L1), a key transporter mediating dietary cholesterol uptake, is involved in the mechanistic action of laminarin. In in vitro studies, BODIPY-cholesterol-labeled Caco-2 cells were examined using confocal microscopy and a fluorescence reader. The results demonstrated that laminarin inhibited cholesterol uptake into Caco-2 cells in a concentration-dependent manner (EC50 = 20.69 μM). In HFD-fed C57BL/6J mice, laminarin significantly reduced the serum levels of total cholesterol (TC), total triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C). It also decreased hepatic levels of TC, TG, and total bile acids (TBA) while promoting the excretion of fecal cholesterol. Furthermore, laminarin significantly reduced local villous damage in the jejunum of HFD mice. Mechanistic studies revealed that laminarin significantly downregulated NPC1L1 protein expression in the jejunum of HFD-fed mice. The siRNA-mediated knockdown of NPC1L1 attenuated the laminarin-mediated inhibition of cholesterol uptake in Caco-2 cells. This study suggests that laminarin significantly improves dyslipidemia in HFD-fed mice, likely by reducing cholesterol uptake through a mechanism that involves the downregulation of NPC1L1 expression.

Novel application potential of cinaciguat in the treatment of mixed hyperlipidemia through targeting PTL/NPC1L1 and alleviating intestinal microbiota dysbiosis and metabolic disorders

Mixed hyperlipidemia, characterized by high levels of triglycerides and cholesterol, is a key risk factor leading to atherosclerosis and other cardiovascular diseases. Existing clinical drugs usually only work on a single indicator, decreasing either triglyceride or cholesterol levels. Developing dual-acting agents that reduce both triglycerides and cholesterol remains a great challenge. Pancreatic triglyceride lipase (PTL) and Niemann–Pick C1-like 1 (NPC1L1) have been identified as crucial proteins in the transport of triglycerides and cholesterol. Here, cinaciguat, a known agent used in the treatment of acute decompensated heart failure, was identified as a potent dual inhibitor targeting PTL and NPC1L1. We presented in vitro evidence from surface plasmon resonance analysis that cinaciguat interacted with PTL and NPC1L1. Furthermore, cinaciguat exhibited potent PTL-inhibition activity. Fluorescence-labeled cholesterol uptake analysis and confocal imaging showed that cinaciguat effectively inhibited cholesterol uptake. In vivo evaluation showed that cinaciguat significantly reduced the plasma levels of triglycerides and cholesterol, and effectively alleviated high-fat diet–induced intestinal microbiota dysbiosis and metabolic disorders. These results collectively suggest that cinaciguat has the potential to be further developed for the therapy of mixed hyperlipidemia.

Quantitative Proteomic Analysis Reveals the Mechanisms of Sinapine Alleviate Macrophage Foaming.

Rapeseed polyphenols have cardiovascular protective effects. Sinapine, one main rapeseed polyphenol, possesses antioxidative, anti-inflammatory, and antitumor properties. However, no research has been published about the role of sinapine in alleviating macrophage foaming. This study aimed to reveal the macrophage foaming alleviation mechanism of sinapine by applying quantitative proteomics and bioinformatics analyses. A new approach was developed to retrieve sinapine from rapeseed meals by using hot-alcohol-reflux-assisted sonication combined with anti-solvent precipitation. The sinapine yield of the new approach was significantly higher than in traditional methods. Proteomics was performed to investigate the effects of sinapine on foam cells, and it showed that sinapine can alleviate foam cell formation. Moreover, sinapine suppressed CD36 expression, enhanced the CDC42 expression, and activated the JAK2 and the STAT3 in the foam cells. These findings suggest that the action of sinapine on foam cells inhibits cholesterol uptake, activates cholesterol efflux, and converts macrophages from pro-inflammatory M1 to anti-inflammatory M2. This study confirms the abundance of sinapine in rapeseed oil by-products and elucidates the biochemical mechanisms of sinapine that alleviates macrophage foaming, which may provide new perspectives for reprocessing rapeseed oil by-products.

Totum-070 prevents diet-induced hypercholesterolemia in western diet fed mice and inhibits cholesterol uptake in human enterocyte caco2 cell line

Abstract Introduction It is well known that hypercholesterolemia is an important modifiable risk factor in the development of cardiovascular diseases. Lowering blood cholesterol level reduces the incidence of atherosclerotic disease. Totum-070 (T070) is a clinical stage polyphenol-rich active substance composed by the association of 5 plant extracts. Purpose We assessed the hypothesis that administration of T070 prevents hypercholesterolemia in western diet fed mice and explored the potential mechanisms involved in vitro in human enterocytes. Methods C57BL/6 mice were fed either a normal diet (ND) or western diet (WD) for 6 weeks and received either vehicle or T070 (1mg/kg or 3mg/kg) daily by gavage (n=14 mice per group). Food intake, body weight and plasma lipid were monitored along study duration. Liver and feces lipid content were quantified at the end of the study. For in vitro studies, human Caco2 cells were differentiated in transwells for 21 days. To perform cholesterol uptake assay, [1,2-3H(N)]-cholesterol was incubated in apical medium for 1 hour in presence or not of T070 prior to quantification of radioactivity in the cells. Results In mice, supplementation with T070 had no effect on food intake during the study. At the end of the in vivo study, body weight in WD-fed mice was increased to 28.3±0.8 g compared to the ND group (24.5±0.4 g, p<0.001). Interestingly, a significant (p<0.01) 29% reduction in body weight gain was observed in the T070 3mg/kg group compared to the WD group at the end of the study. Compared to WD group, total-cholesterol in mice supplemented by T070 was reduced in a dose dependent manner (298±8 mg/dl WD vs 276±9 mg/dl T070 1mg/kg, p=0.19, and 265±8 mg/dl T070 3mg/kg, p<0.05). Furthermore, hepatic steatosis induced by the WD was reduced by 73% (p<0.001) in the T070 3mg/kg group. Hepatic and intestinal gene expression were drastically altered by WD feeding compared to ND, while supplementation with T070 tended to restore normal expression levels for many genes implicated in lipid and lipoprotein metabolism. A significant increase in fecal cholesterol excretion was observed in mice supplemented with T070 3 mg/g compared to the mice fed with WD alone (442.3±151.7 nmol/day WD + T070 3mg/g vs 75.4±5.9 nmol/day WD, p<0.05). Studies in human enterocytes demonstrated that 1 g/l T070 reduced cholesterol uptake by 35% (p<0.001), suggesting that T070 inhibits intestinal cholesterol absorption. Conclusion Our results show the beneficial effect of supplementation with Totum-070 to prevent induction of hypercholesterolemia in a nutritional mouse model. Investigation of mechanisms of action in enterocytes demonstrate the role of Totum-070 to inhibit intestinal cholesterol absorption. These results highlight the interest of using Totum-070 for the management of hypercholesterolemia. Funding Acknowledgement Type of funding sources: None.

Emodin lows NPC1L1-mediated cholesterol absorption as an uncompetitive inhibitor

Emodin (EM) is one of the active components of the traditional Chinese medicine rhubarb, and there is evidence of its hypolipidemic activity, though the exact mechanism is unknown. NPC1L1 is a key protein in human cholesterol uptake that is primarily expressed in hepatocytes and gastrointestinal epithelial cells. Our findings suggest that rhodopsin inhibits cellular cholesterol uptake by influencing NPC1L1 cholesterol transport. The results showed that NBD-cholesterol uptake in human HepG2 cells was 27 %, 31.3 %, 33.6 %, 41.6 %, and 52.6 % of control after treatment with 100, 75, 50, 25, and 12.5 % M EM, respectively, compared to 50 % for 100 M Ezetimibe. Kinetic studies revealed that EM inhibited cellular uptake of cholesterol through anti-competitive inhibition. Furthermore, using confocal fluorescence quantification, we discovered that after cholesterol deprivation treatment reintroduced cholesterol supply, cholesterol uptake was significantly higher in HepG2 cells highly expressing NPC1L1 than in U2OS cells with low NPC1L1 expression. As a result, we hypothesize that EM may inhibit cholesterol uptake via NPC1L1 in human hepatocytes in an anti-competitive manner. Overall, as a dietary supplement or lipid-modifying drug, EM has the potential to lower cholesterol.

Cholesterol-Lowering Effects of Asperidine B, a Pyrrolidine Derivative from the Soil-Derived Fungus Aspergillus sclerotiorum PSU-RSPG178: A Potential Cholesterol Absorption Inhibitor.

Isolated secondary metabolites asperidine B (preussin) and asperidine C, produced by the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178, were found to exhibit inhibitory effects against 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase and oxidative stress in an in vitro assay. Whether or not the known pyrrolidine asperidine B and the recently isolated piperidine asperidine C have lipid-lowering effects remains unknown. Thus, this study aimed to investigate the hypocholesterolemic effects of asperidines B and C and identify the mechanisms involved in using in vitro, ex vivo, and in vivo models. The results show that both compounds interfered with cholesterol micelle formation by increasing bile acid binding capacity, similar to the action of the bile acid sequestrant drug cholestyramine. However, only asperidine B, but not asperidine C, was found to inhibit cholesterol uptake in Caco-2 cells by up-regulating LXRα without changing cholesterol transporter NPC1L1 protein expression. Likewise, reduced cholesterol absorption via asperidine-B-mediated activation of LXRα was also observed in isolated rat jejunal loops. Asperidine B consistently decreases plasma cholesterol absorption, similar to the effect of ezetimibe in rats. Therefore, asperidine B, the pyrrolidine derivative, has therapeutic potential to be developed into a type of cholesterol absorption inhibitor for the treatment of hypercholesterolemia.

Larvicidal activity of the pericarp extract of Garcinia mangostana against dengue vector Aedes aegypti in Sri Lanka

Objective: To assess the larvicidal activity of mangosteen (Garcinia mangostana) against larval stages of Aedes aegypti mosquitoes. Methods: A crude extract was prepared in ethanol from powdered mangosteen pericarps. A concentration gradient (0.01-4.92 g/ L) was prepared from the stock solution. Seven batches of 25 third instar larvae of Aedes aegypti were used for larval bioassays. Larval mortality rates were observed after one and 24 hours. Cholesterol and total lipid contents in 20 randomly selected dead larvae at each trial were assessed by colorimetric method. The experimental setup was repeated five times. The General Linear Model and Probit analysis were used to evaluate the relationship of mortality with cholesterol level, total lipid level and cholesterol to total lipid ratio. Results: The percentage mortalities significantly varied with different concentrations (F732=385.737; P<0.001). The LC50 and LC99 values were (0.041 ± 0.006) g/L and (10.616 ± 1.758) g/L, respectively after 24-hours. There was no mortality recorded within the one-hour exposure time. Only the cholesterol content (F524=173.245; P<0.001) in larvae exposed to different concentrations denoted a significantly decreasing trend within 24hour exposure. Larvae that were exposed to the lowest concentration (0.55 g/L) showed a higher cholesterol level (22.67 ± 1.33) μg. Conclusions: The Garcinia mangostana extract acts as an effective sterol carrier protein inhibitor that inhibits cholesterol uptake in Aedes aegypti mosquitoes. Hence, it could be explored for use as a key source for the development of an environment-friendly plant-based larvicide.

Isolation and Characterization of Bioactive Proteoglycan–Lipid Nanoparticles from Freshwater Clam (Corbicula fluminea Muller) Soup

Nanoparticles can be prepared by several sophisticated processes but until now, it cannot be prepared by simple home cooking. Here, we report that two incidental food nanoparticles (iFNPs) consisting of proteoglycans and phytosterols were isolated from soup made from freshwater clam (Corbicula fluminea Muller), a renowned folk remedy for liver problems in China and other parts of East Asia. These two bioactive iFNPs were obtained and characterized by anionic exchange chromatography coupled with multi-angle laser light scattering measurement. Their hydrodynamic diameters and ζ-potentials were 50 ± 0.2 nm and -28.0 mV and 67 ± 0.4 nm and -9.96 mV, respectively. FT-IR revealed that the proteoglycans in the particles contained α-type heteropolysaccharides. Both iFNPs were resistant to pH changes and separation by mechanical force but responsive to temperature changes. They effectively inhibited cholesterol uptake in vitro, which resonates with the traditional belief that freshwater clam soup provides hepatoprotective benefits. This study suggests that these two proteoglycan-lipid iFNPs are the active moieties and offers a supramolecular structure-based approach to study the function of such complex matrices derived from food.

Abstract PO-11: Targeting scavenger receptor type B1 in cholesterol-addicted lymphomas abolishes glutathione peroxidase 4 expression and results in ferroptosis

Abstract Introduction: In B-cell lymphomas, disparate molecular aberrations often produce common onco-metabolic phenotypes, including a dependency upon cholesterol uptake from serum lipoproteins rather than de novo synthesis. Recent reports have linked cancer cell cholesterol to ferroptosis, a newly appreciated form of programmed cell death characterized by the accumulation of oxidized membrane polyunsaturated fatty acids (PUFAs), facilitated by reduced glutathione peroxidase 4 (GPX4) activity. Diffuse large B-cell lymphoma (DLBCL) and anaplastic large T-cell lymphoma (ALCL) were identified as potentially sensitive to ferroptosis. We have developed high-density biomimetic lipoprotein nanoparticles (HDL NPs), a first-in-class platform that specifically targets scavenger receptor type B1 (SCARB1) and inhibits cholesterol uptake. In DLBCL, HDL NPs potently induces cell death. Accordingly, we hypothesized that HDL NPs induce ferroptosis in DLBCL and would be potent therapy for other cholesterol-addicted malignancies. Methods: Burkitt's lymphoma (Ramos), DLBCL (SUDHL4), ALCL (SR-786, SUDHL1), and histiocytic lymphoma (U937) cell lines were used. RNA, protein, and cholesterol were isolated from cells treated with HDL NPs and analyzed using RT-qPCR, Western blot, and total cholesterol assays, respectively. Oxidized PUFA accumulation was quantified using the C11-BODIPY dye and flow cytometry. The lipophilic antioxidant ferrostatin-1 (Fer-1) and iron chelator deferoxamine (DFO) were used to inhibit ferroptosis. Following informed consent, primary B-cell lymphoma samples were isolated from patients and assayed for SCARB1 expression and viability following HDL NP treatment. Results: HDL NPs reduced cholesterol levels in Ramos and SUDHL4 cells, leading to upregulation of cholesterol biosynthesis genes and downregulation of GPX4. This correlated with an increase in accumulation of oxidized PUFAs, and eventually cell death by ferroptosis. Fer-1 and DFO rescued Ramos and SUDHL4 cells from HDL NP-induced cell death. Oxidized PUFA accumulation and GPX4 downregulation were confirmed in vivo using HDL NP-treated SUDHL4 xenografts, which resulted in reduced tumor burden compared with controls. These results were replicated in other ferroptosis-sensitive malignancies, including SCARB1+ ALCL (SUDHL1, SR-786) and histiocytic lymphoma (U937) cell lines. Finally, primary B-cell lymphoma cells isolated from patients with follicular lymphoma (n=4) and DLBCL (n=2) demonstrated SCARB1 expression and were sensitive to HDL NP-induced cell death. Conclusion: HDL NP binding to SCARB1 results in inhibition of cholesterol uptake and triggers a metabolic compensatory response, namely upregulation of cholesterol biosynthesis genes and downregulation of GPX4, obligating cells to ferroptosis. As this onco-metabolic profile is observed across a range of cancers, HDL NPs represent a unique platform for metabolic therapy for cholesterol- and GPX4-dependent malignancies. Citation Format: Jonathan S. Rink, Adam Lin, Kaylin M. McMahon, Andrea E. Calvert, Shuo Yang, Amir Behdad, Reem Karmali, Colby S. Thaxton, Leo I. Gordon. Targeting scavenger receptor type B1 in cholesterol-addicted lymphomas abolishes glutathione peroxidase 4 expression and results in ferroptosis [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-11.

Phenolic acids from Coffea arabica L. suppress intestinal uptake of glucose and cholesterol.

The This study assessed the hypoglycaemic and hypocholesterolaemic effects of coffee fruit extracts and their active compounds involved in cholesterol and glucose uptake, as well as the inhibitory activity of α-glucosidase and α-amylase, the physiochemical changes of cholesterol micelles, and the uptake of glucose and cholesterol in Caco-2 cells. The results revealed that coffee green dry (CGD) coffee yellow dry (CYD), and coffee red bean dry (CRBD) extracts inhibited α-amylase activity by approximately 49% whereas caffeine and caffeic acid, showed high potency to inhibit α-amylase activity by 94.49% and 93.35%, respectively. In addition, coffee red pulps dry (CRPD) and citric acid inhibited αglucosidase by 29% and 46%, respectively. The extracts (1 mg/mL) and their active compounds (200 µM) inhibited glucose uptake in Caco-2 cells. All coffee fruit extracts inhibited cholesterol uptake in Caco-2 cells by approximately 40%, and they decreased the micellar solubility of cholesterol, except for coffee red fresh (CRF) and coffee red dry (CRD). Moreover, CYD strongly increased the size of cholesterol micelles. These findings demonstrate the potential effect of coffee extracts on anti-diabetic activity and their cholesterol-lowering activity according to the inhibitory activity of α-glucosidase and α-amylase and the uptake of glucose and cholesterol in Caco-2 cells.

Xanthohumol Suppresses NPC1L1 Gene Expression through Downregulation of HNF-4α and Inhibits Cholesterol Uptake in Caco-2 Cells.

Xanthohumol (Xan) is a prenylated chalcone mainly found in hops; it has been demonstrated to function against hypercholesterolemia, hyperlipidemia, and atherosclerosis. In this study, we focused on the hypocholesterolemic effect of Xan on cholesterol uptake and the underlying molecular mechanisms of Xan in human intestinal Caco-2 cells. The microarray data showed that Niemann-Pick C1-like 1 (NPC1L1), an essential transporter for dietary cholesterol absorption, was significantly downregulated in Xan-treated Caco-2 cells. We demonstrated that Xan (10 and 20 μM) suppressed the mRNA and protein expression of NPC1L1 by 0.65 ± 0.12-fold and 0.54 ± 0.15-fold and 0.72 ± 0.04-fold and 0.44 ± 0.12-fold, respectively, compared to that of the vehicle-treated Caco-2 cells. Moreover, Xan (10 and 20 μM) significantly inhibited cholesterol uptake by approximately 12 and 32% in Caco-2 cells. NPC1L1 promoter activity was significantly suppressed by Xan, and a DNA element within the NPC1L1 promoter involved in Xan-mediated NPC1L1 reduction located between the -120 and -20 positions was identified. Moreover, Xan markedly decreased the mRNA and protein levels of hepatocyte nuclear factor 4α (HNF-4α), a critical activator of NPC1L1 transcription, and subsequently attenuated HNF-4α/NPC1L1 promoter complex formation, resulting in the suppression of NPC1L1 gene expression. Finally, we demonstrated that Xan markedly abolished lovastatin-induced NPC1L1 overexpression in Caco-2 cells. These findings reveal that Xan suppresses NPC1L1 expression via downregulation of HNF-4α and exerts inhibitory effects on cholesterol uptake in the intestinal Caco-2 cells. Our findings suggest Xan could serve as a potential cholesterol-lowering agent and supplement for statin therapy.

Active polypeptides from Hirudo inhibit endothelial cell inflammation and macrophage foam cell formation by regulating the LOX-1/LXR-α/ABCA1 pathway

Background and aimsHirudo is an important Chinese medicine that has been widely used in patients with thrombosis-related diseases. We aimed to evaluate the protective effect and potential mechanism of Hirudo extract (HE) on the process of atherosclerosis (AS) as well as identify its active components in the lipopolysaccharide (LPS) - or oxidized low-density lipoprotein (ox-LDL)-induced cell models. MethodsAfter treatment, adhesion molecules and pro-inflammatory cytokines induced by LPS were examined by qPCR and ELISA. ROS production, cell apoptosis, and lipid accumulation in ox-LDL-induced cells were analyzed by flow cytometry, qPCR, western blotting, and immunofluorescence staining. In addition, the main active components of HE were identified and analyzed for preventing the progression of AS. ResultsIn this study, we found that HE pretreatment for 48 h significantly inhibited monocyte adhesion and reduced the levels of adhesion factors (ICAM-1 and VCAM-1) and pro-inflammatory factors (IL-6 and TNF-α) in LPS-induced endothelial cells. Moreover, HE attenuated ox-LDL-induced ROS accumulation and apoptosis in macrophage cells via mitochondrial apoptotic pathways. Additionally, HE pretreatment effectively inhibited cholesterol uptake and increased cholesterol efflux by regulating the LOX-1/LXR-α/ABCA1 pathway. Importantly, the polypeptides from HE (PP) with a molecular weight < 10,000 Da accounted for about 62.9% of the total amount of polypeptides, which in turn may be active components of HE that are responsible for inhibiting inflammation, foam cell formation and apoptosis. ConclusionPP from HE potently inhibits endothelial cell inflammatory injury and macrophage foam cell formation and apoptosis by regulating the LOX-1/LXR-α/ABCA1 pathway, thereby providing additional support to the beneficial effects of HE in preventing AS.

Synthesis and Evaluation of 2-Azetidinone and 1H-Pyrrole-2,5-dione Derivatives as Cholesterol Absorption Inhibitors for Reducing Inflammation Response and Oxidative Stress.

Excess lipid accumulation can initiate the development and progression of atherosclerotic lesions, thus eventually leading to cardiovascular disease. Lipid-lowering medication therapy is one of the cornerstones of cardiovascular disease therapy. On the basis of the cholesterol absorption inhibitor ezetimibe, we successfully synthesized seven 2-azetidinone derivatives and eighteen 1H-pyrrole-2,5-dione derivatives. Most of the new compounds significantly inhibited cholesterol uptake in vitro. In addition, one of the most active inhibitors, 3-(4-fluorophenyl)-1-[(3S)-3-hydroxy-3-(4-hydroxyphenyl)propyl]-4-(4-hydroxyphenyl)-1H-pyrrole-2,5-dione (14q), showed no cytotoxicity in L02 and HEK293T cell lines. Further evaluation indicated that 14q inhibited considerably the amount of TNF-α, ROS, MDA, and LDH in vitro. Therefore, 14q might be a novel cholesterol absorption inhibitor.

Endogenous cholesterol ester hydroperoxides modulate cholesterol levels and inhibit cholesterol uptake in hepatocytes and macrophages

Dysregulation of cholesterol metabolism represents one of the major risk factors for atherosclerotic cardiovascular disease (CVD). Oxidized cholesterol esters (oxCE) in low-density lipoprotein (LDL) have been implicated in CVD but the underlying mechanisms remain poorly defined. We use a targeted lipidomic approach to demonstrate that levels of oxCEs in human plasma are associated with different types of CVD and significantly elevated in patients with myocardial infarction. We synthesized a major endogenous cholesterol ester hydroperoxide (CEOOH), cholesteryl-13(cis, trans)-hydroperoxy-octadecadienoate (ch-13(c,t)-HpODE) and show that this endogenous compound significantly increases plasma cholesterol level in mice while decrease cholesterol levels in mouse liver and peritoneal macrophages, which is primarily due to the inhibition of cholesterol uptake in macrophages and liver. Further studies indicate that inhibition of cholesterol uptake by ch-13(c,t)-HpODE in macrophages is dependent on LXRα-IDOL-LDLR pathway, whereas inhibition on cholesterol levels in hepatocytes is dependent on LXRα and LDLR. Consistently, these effects on cholesterol levels by ch-13(c,t)-HpODE are diminished in LDLR or LXRα knockout mice. Together, our study provides evidence that elevated plasma cholesterol levels by CEOOHs are primarily due to the inhibition of cholesterol uptake in the liver and macrophages, which may play an important role in the pathogenesis of CVD.

Polydatin Attenuates Atherosclerosis in ApoE Mice through PBEF Mediated Reduction of Cholesterol Deposition.

Cholesterol metabolism becomes imbalanced during the formation of macrophage-derived foam cells. Pre-B-cell colony-enhancing factor (PBEF) has recently been found to affect lipid deposition and inflammation in atherosclerosis. Here, we aimed to study the effects and molecular mechanism of Polydatin on atherosclerosis in ApoE-knockout (ApoE ) mice. Thirty ApoE mice were fed a high-fat diet (HFD) for 12 weeks, and then treated with Polydatin for another 12 weeks. Whole aortas and cryosections were stained with oil red O. Blood lipid, PBEF and cytokine levels were measured by ELISA. The mRNAs of cholesterol metabolism-related genes were determined by qRT-PCR and protein levels by Western blotting. Cell cholesterol content and viability were determined in macrophages and RAW 264.7 cells. PBEF siRNA was used to study the effect of Polydatin on cholesterol metabolism in macrophages incubated with ox-LDL. Polydatin lowered blood lipids and decreased atherosclerotic lesions in ApoE mice. The expression of cytokines and the mRNA of cholesterol metabolism-related genes were markedly regulated by Polydatin. Meanwhile, PBEF mRNA and protein were both greatly down-regulated by Polydatin. In vitro, Polydatin protected RAW 264.7 cells treated by ox-LDL and inhibited cholesterol uptake by macrophages. The PBEF siRNA result indicates that Polydatin can modulate cholesterol metabolism in macrophages, partly through down-regulation of PBEF. In conclusion, Polydatin relieves atherosclerosis injury in ApoE mice, mainly through down-regulation of PBEF and inhibition of PBEF-inducing cholesterol deposits in macrophages.

Effect of Synthetic High Density Lipoproteins Modification with Polyethylene Glycol on Pharmacokinetics and Pharmacodynamics.

Synthetic high density lipoprotein nanoparticles (sHDLs) capable of mobilizing excess cholesterol from atherosclerotic arteries and delivering it to the liver for elimination have been shown to reduce plaque burden in patients. Unfortunately, sHDLs have a narrow therapeutic index and relative to the endogenous HDL shorter circulation half-life. Surface modification with polyethylene glycol (PEG) was investigated for its potential to extend sHDL circulation in vivo. Various amounts (2.5, 5, and 10%) and different chain lengths (2 and 5 kDa) of PEG-modified lipids were incorporated in sHDL's lipid membrane. Incorporating PEG did not reduce the ability of sHDL to facilitate cholesterol efflux, nor did it inhibit cholesterol uptake by the liver cells. By either adding more PEG or using PEG of longer chain lengths, the circulation half-life was extended. Addition of PEG also increased the area under the curve for the phospholipid component of sHDL (p < 0.05), but not for the apolipoprotein A-I peptide component of sHDL, suggesting sHDL is remodeled by endogenous lipoproteins in vivo. The extended phospholipid circulation led to a higher mobilization of plasma free cholesterol, a biomarker for facilitation of reverse cholesterol transport. The area under the cholesterol mobilization increased about 2-4-fold (p < 0.05), with greater increases observed for longer PEG chains and higher molar percentages of incorporated PEGylated lipids. Mobilized cholesterol was associated primarily with the HDL fraction, led to a transient increase in VLDL cholesterol, and returned to baseline 24 h postdose. Overall, PEGylation of sHDL led to beneficial changes in sHDL particle pharmacokinetic and pharmacodynamic behaviors.

Hypocholesterolemic effect of sericin-derived oligopeptides in high-cholesterol fed rats.

The beneficial effect of cholesterol-lowering proteins and/or peptides derived from various dietary sources is continuously reported. A non-dietary protein from silk cocoon, sericin, has also demonstrated cholesterol-lowering activity. A sericin hydrolysate prepared by enzymatic hydrolysis was also expected to posses this effect. The present study was aimed at investigating the cholesterol-lowering effect of sericin peptides, so called "sericin-derived oligopeptides" (SDO) both in vivo and in vitro. The results showed that SDO at all three doses tested (10mgkg-1day-1, 50mgkg-1day-1, and 200mgkg-1day-1) suppressed serum total and non-HDL cholesterol levels in rats fed a high-cholesterol diet. Triglyceride and HDL-cholesterol levels were not significantly changed among all groups. The fecal contents of bile acids and cholesterol did not differ among high-cholesterol fed rats. SDO dose-dependently reduced cholesterol solubility in lipid micelles, and inhibited cholesterol uptake in monolayer Caco-2 cells. SDO also effectively bound to all three types of bile salts including taurocholate, deoxytaurocholate, and glycodeoxycholate. Direct interaction with bile acids of SDO may disrupt micellar cholesterol solubility, and subsequently reduce the absorption of dietary cholesterol in intestines. Taking all data together, SDO or sericin peptides exhibit a beneficial effect on blood cholesterol levels and could be potentially used as a health-promoting dietary supplement or nutraceutical product.

Hypolipidemic activities of xanthorrhizol purified from centrifugal TLC

Hyperlipidemia is defined as the presence of either hypertriglyceridemia or hypercholesterolemia, which could cause atherosclerosis. Although hyperlipidemia can be treated by hypolipidemic drugs, they are limited due to lack of effectiveness and safety. Previous studies demonstrated that xanthorrhizol (XNT) isolated from Curcuma xanthorrhizza Roxb. reduced the levels of free fatty acid and triglyceride in vivo. However, its ability to inhibit cholesterol uptake in HT29 colon cells and adipogenesis in 3T3-L1 cells are yet to be reported. In this study, XNT purified from centrifugal TLC demonstrated 98.3% purity, indicating it could be an alternative purification method. The IC50 values of XNT were 30.81 ± 0.78 μg/mL in HT29 cells and 35.07 ± 0.24 μg/mL in 3T3-L1 adipocytes, respectively. Cholesterol uptake inhibition study using HT29 colon cells showed that XNT (15 μg/mL) significantly inhibited the fluorescent cholesterol analogue NBD uptake by up to 27 ± 3.1% relative to control. On the other hand, higher concentration of XNT (50 μg/mL) significantly suppressed the growth of 3T3-L1 adipocytes (5.9 ± 0.58%) compared to 3T3-L1 preadipocytes (81.31 ± 0.55%). XNT was found to impede adipogenesis of 3T3-L1 adipocytes in a dose-dependent manner from 3.125 to 12.5 μg/mL, where 12.5 μg/mL significantly suppressed 36.13 ± 2.1% of lipid accumulation. We postulate that inhibition of cholesterol uptake, adipogenesis, preadipocyte and adipocyte number may be utilized as treatment modalities to reduce the prevalence of lipidemia. To conclude, XNT could be a potential hypolipidemic agent to improve cardiovascular health in the future.