Tilianin improves lipid profile and alleviates atherosclerosis in ApoE-/- mice through up-regulation of SREBP2-mediated LDLR expression.

BACKGROUND: The huge global burden of atherosclerotic cardiovascular diseases (CVDs) represents an urgent unmet need for the development of novel therapeutics. Dracocephalum moldavica L. has been used as a traditional Uygur medicine to treat various CVDs for centuries. Tilianin is a major flavonoid component of D. moldavica L. and has potential for preventing atherosclerosis. However, the molecular mechanisms that tilianin attenuate atherosclerosis are far from fully understood. PURPOSES: The purpose of this study is to investigate the efficiency and underlying mechanisms of tilianin in controlling lipid profile and preventing atherogenesis. METHODS: The lipid-lowering effect of tilianin was evaluated in C57BL/6 and ApoE-/- mice by systematically determining serum biochemical parameters. The effects of tilianin on the atherosclerotic lesion were observed in aortic roots and whole aortas of ApoE-/- mice with oil red O staining. Caecal content from ApoE-/- mice were collected for 16S rRNA gene sequence analysis to assess the structure of the gut microbiota. The inhibition of hepatosteatosis was verified by histological examination, and a liver transcriptome analysis was performed to elucidate the tilianin-induced hepatic transcriptional alterations. Effects of tilianin on the expression and function of LDLR were examined in HepG2 cells and ApoE-/- mice. Further mechanisms underlying the efficacy of tilianin were investigated in HepG2 cells. RESULTS: Tilianin treatment improved lipid profiles in C57BL/6 and dyslipidemic ApoE-/- mice, especially reducing the serum LDL-cholesterol (LDL-C) level. Significant reductions of atherosclerotic lesion area and hepatosteatosis were observed in tilianin-treated ApoE-/- mice. The altered gut microbial composition in tilianin groups was associated with lipid metabolism and atherosclerosis. The liver transcriptome revealed that tilianin regulated the transcription of lipid metabolism-related genes. Then both in vitro and in vivo analyses revealed the potent effect of tilianin to enhance hepatic LDLR expression and its mediated LDL-C uptake. Further studies confirmed a critical role of SREBP2 in hepatic LDLR up-regulation by tilianin via increasing precursor and thus mature nuclear SREBP2 level. CONCLUSION: This study demonstrated the lipid-lowering effect of tilianin through SREBP2-mediated transcriptional activation of LDLR. Our findings reveal a novel anti-atherosclerotic mechanism of tilianin and underlie its potential clinical use in modulating CVDs with good availability and affordability.

NMR, LC-MS Characterization of Rydingia michauxii Extracts, Identification of Natural Products Acting as Modulators of LDLR and PCSK9.

Rydingia michauxii (Briq.) Scheen and V.A.Albert (Lamiaceae) is used in Iranian traditional medicine to treat malaria, diabetes, hyperlipidemia, rheumatism and cardiovascular diseases. NMR and LC-DAD-MSn analyses were used to establish extract composition and phenylethanoid, flavonoid glycosides, lignans, labdane diterpenes and iridoids were identified and quantified. The main constituents were isolated, and structures were elucidated based on NMR, polarimetric and MS measurements. A new natural compound, ent-labda-8(17),13-dien-18-glucopyranosyl ester-15,16-olide is described here. The effects of ent-labda-8(17),13-dien-18-oic acid-15,16-olide (1), ent-labda-8(17),13-dien-18-glucopyranosyl es-ter-15,16-olide (2), antirrhinoside (3), echinacoside (4), verbascoside (5), and apigenin 6,8-di-C-glucoside (6), on the low-density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9), were studied in the human hepatocarcinoma cell line Huh7. Among the six constituents, (3) showed the strongest induction of the LDLR (3.7 ± 2.2 fold vs. control) and PCSK9 (3.2 ± 1.5 fold vs. control) at a concentration of 50 µM. The in vitro observations indicated a potential lipid lowering activity of (3) with a statin-like mechanism of action.

Sexual dimorphism: increased sterol excretion leads to hypocholesterolaemia in female hyperbilirubinaemic Gunn rats.

Circulating bilirubin is associated with reduced serum cholesterol concentrations in humans and in hyperbilirubinaemic Gunn rats. However, mechanisms contributing to hypocholesterolaemia remain unknown. Therefore, this study aimed to investigate cholesterol synthesis, transport and excretion in mutant Gunn rats. Adult Gunn and control rats were assessed for daily faecal sterol excretion using metabolic cages, and water was supplemented with [1-13 C]-acetate to determine cholesterol synthesis. Bile was collected to measure biliary lipid secretion. Serum and liver were collected for biochemical analysis and for gene/protein expression using RT-qPCR and western blot, respectively. Additionally, serum was collected and analysed from juvenile rats. A significant interaction of sex, age and phenotype on circulating lipids was found with adult female Gunn rats reporting significantly lower cholesterol and phospholipids. Female Gunn rats also demonstrated elevated cholesterol synthesis, greater biliary lipid secretion and increased total faecal cholesterol and bile acid excretion. Furthermore, they possessed increased hepatic low-density lipoprotein (LDL) receptor and SREBP2 expression. In contrast, there were no changes to sterol metabolism in adult male Gunn rats. This is the first study to demonstrate elevated faecal sterol excretion in female hyperbilirubinaemic Gunn rats. Increased sterol excretion creates a negative intestinal sterol balance that is compensated for by increased cholesterol synthesis and LDL receptor expression. Therefore, reduced circulating cholesterol is potentially caused by increased hepatic uptake via the LDL receptor. Future studies are required to further evaluate the sexual dimorphism of this response and whether similar findings occur in females with benign unconjugated hyperbilirubinaemia (Gilbert's syndrome). KEY POINTS: Female adult hyperbilirubinaemic (Gunn) rats demonstrated lower circulating cholesterol, corroborating human studies that report a negative association between bilirubin and cholesterol concentrations. Furthermore, female Gunn rats had elevated sterol excretion creating a negative intestinal sterol balance that was compensated for by elevated cholesterol synthesis and increased hepatic low-density lipoprotein (LDL) receptor expression. Therefore, elevated LDL receptor expression potentially leads to reduced circulating cholesterol levels in female Gunn rats providing an explanation for the hypocholesterolaemia observed in humans with elevated bilirubin levels. This study also reports a novel interaction of sex with the hyperbilirubinaemic phenotype on sterol metabolism because changes were only reported in females and not in male Gunn rats. Future studies are required to further evaluate the sexual dimorphism of this response and whether similar findings occur in females with benign unconjugated hyperbilirubinaemia (Gilbert's syndrome).

Clinical efficacies, underlying mechanisms and molecular targets of Chinese medicines for diabetic nephropathy treatment and management.

Diabetic nephropathy (DN) has been recognized as a severe complication of diabetes mellitus and a dominant pathogeny of end-stage kidney disease, which causes serious health problems and great financial burden to human society worldwide. Conventional strategies, such as renin-angiotensin-aldosterone system blockade, blood glucose level control, and bodyweight reduction, may not achieve satisfactory outcomes in many clinical practices for DN management. Notably, due to the multi-target function, Chinese medicine possesses promising clinical benefits as primary or alternative therapies for DN treatment. Increasing studies have emphasized identifying bioactive compounds and molecular mechanisms of reno-protective effects of Chinese medicines. Signaling pathways involved in glucose/lipid metabolism regulation, antioxidation, anti-inflammation, anti-fibrosis, and podocyte protection have been identified as crucial mechanisms of action. Herein, we summarize the clinical efficacies of Chinese medicines and their bioactive components in treating and managing DN after reviewing the results demonstrated in clinical trials, systematic reviews, and meta-analyses, with a thorough discussion on the relative underlying mechanisms and molecular targets reported in animal and cellular experiments. We aim to provide comprehensive insights into the protective effects of Chinese medicines against DN.

Dietary Fruit and Vegetable Supplementation Suppresses Diet-Induced Atherosclerosis in LDL Receptor Knockout Mice.

BACKGROUND: Epidemiologic studies suggest that fruit and vegetable (F&V) consumption is inversely associated with incidence of cardiovascular disease (CVD). However, evidence for causality is lacking, and the underlying mechanisms are not well understood. OBJECTIVES: We aimed to determine whether there is a causal relation between consuming high levels of F&V and prevention of atherosclerosis, the hallmark of CVD pathogenesis. Furthermore, the underlying mechanisms were determined. METHODS: Six-week-old male LDL receptor-knockout mice were randomly assigned to 3 diet groups (12 mice/group) for 20 wk: control (CON, 10% kcal fat, 0.20 g/kg cholesterol), atherogenic (Ath, 27% kcal fat, 0.55 g/kg cholesterol), and Ath supplemented with 15% F&V (Ath + FV) (equivalent to 8-9 servings/d in humans). F&V was added as a freeze-dried powder that was prepared from the 24 most commonly consumed F&Vs in the United States. Body weight, aortic atherosclerotic lesion area, hepatic steatosis area, serum lipid profile and proinflammatory cytokine TNF-α concentrations, gut microbiota, and liver TNF-α and fatty acid synthase (Fasn) mRNA concentrations were assessed. RESULTS: F&V supplementation did not affect weight gain. Mice fed the Ath + FV diet had a smaller aortic atherosclerotic lesion area (71.7% less) and hepatic steatosis area (80.7% less) than those fed the Ath diet (both P < 0.001) independent of impact on weight, whereas no difference was found between Ath + FV and CON groups in these 2 pathologic markers. Furthermore, F&V supplementation prevented Ath diet-induced dyslipidemia (high concentrations of serum TG and VLDL cholesterol and lower concentrations of HDL cholesterol), reduced serum TNF-α concentration (by 21.5%), suppressed mRNA expression of liver TNF-α and Fasn, and ameliorated Ath-induced gut microbiota dysbiosis. CONCLUSIONS: Our results indicate that consuming a large quantity and variety of F&Vs causally attenuates diet-induced atherosclerosis and hepatic steatosis in mice. These effects of F&Vs are associated with, and may be mediated through, improved atherogenic dyslipidemia, alleviated gut dysbiosis, and suppressed inflammation.

Lecithin coating as universal stabilization and functionalization strategy for nanosized drug carriers to overcome the blood-brain barrier.

Lecithin coated cholesteryl oleate (ChOl) based nanoparticles (NPs) imitating natural lipoproteins represent a new and promising drug carrier strategy to cross the blood-brain barrier (BBB). In such systems lecithin serves as stabilizing as well as functionalizing agent and enables the adsorptive binding of apolipoprotein E3 (ApoE) as potential drug targeting ligand. The present work is focused on the effect of size reduction on the lecithin coating and ApoE binding. Furthermore, the transferability of this lecithin coating strategy to other NP cores, namely polylactic-co-glycolic acid (PLGA) and polylactic acid (PLA), is investigated in order to provide a universal strategy for a wide range of cores to overcome the BBB. The ChOl NPs' size was successfully reduced from 100 nm to 70 nm. Varying the core size of ChOl NPs illustrated, that the at least needed lecithin amount for sufficient stabilization could be calculated surface area dependently. However, the size reduction led to reduced dye loading per NP and increased ApoE need per NP mass. These effects turned out as huge disadvantages of smaller NPs by weakening the observed ApoE mediated effects. Nevertheless, the extended understanding of the lecithin coating could be used to transfer the concept to other core materials. PLGA and PLA NPs were investigated as alternative core materials for lecithin coating. PLGA was found to be unsuitable, whereas in the case of PLA sufficient stabilization and 100% adsorptive binding efficiency to ApoE could be achieved. The ApoE mediated effects of transcytosis at an in vitro BBB model by bypassing lysosomes were reproduced in even stronger quantities than with a ChOl core, proving lecithin coating as transferable strategy to disguise various NPs with a certain lipophilicity as lipoproteins.

Effect of the polyphenol-rich extract from Allium cepa on hyperlipidemic sprague-dawley rats.

Allium cepa is used for the prevention and treatment of hyperlipidemia-related diseases such as atherosclerosis in the folk. This study was mainly aimed at investigating the effects of A. cepa extract (ACE) enriched in polyphenols on hyperlipidemia Sprague-Dawley (SD) experiment rat models. The levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), malondialdehyde (MDA), and superoxide dismutase (SOD) activity in serum and liver were measured using ELISA kits. In addition, hematoxylin-eosin (HE) technique was used to observe the liver and the aortic arch pathology. Moreover, western blotting (WB) method was applied to analyze LDL receptor (LDLR) and 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase (HMGCR) in liver. As a result, quercetin (2.42 mg/g DW) and isoquercitrin (4.60 mg/g DW) were the main constituents of ACE using HPLC analysis. Furthermore, ACE reduced the levels of TC, TG, LDL-C, and MDA, and increased HDL levels and elevated SOD activity both in serum and liver in hyperlipidemic SD rats (p < .05). HE results showed that liver fat drops of the rats in ACE group were obviously decreased, and the lipid and foam cells of the aortic arch of the rats in ACE group were markedly ameliorated. WB results showed that ACE promoted the degradation of HMGCR and increased LDLR expression in liver (p < .05). In conclusion, ACE alleviated hyperlipidemia with downregulation of HMGCR and upregulation of LDLR. PRACTICAL APPLICATIONS: Atherosclerosis, a major cardiovascular disease, is the leading cause of mortality and morbidity in the developed countries. Moreover, accumulating data indicate that, during atherosclerosis development, hyperlipidemia is an important risk factor. To date, hyperlipidemia is mainly treated with hyperlipidemic agents including statins, in spite of the side effects and poor tolerance in some patients. In addition, Allium cepa is a medicinal and edible plant. Furthermore, A. cepa is used for the prevention and treatment of hyperlipidemia-related diseases such as atherosclerosis in the folk. But the underlying mechanism is still unclear. In fact, this research showed that A. cepa extract (ACE) alleviated hyperlipidemia with downregulation of HMGCR and upregulation of LDLR, suggesting that ACE might be a potential option for hyperlipidemia as non-statin lipid-lowering agent.

A polyoxyethylene sorbitan oleate modified hollow gold nanoparticle system to escape macrophage phagocytosis designed for triple combination lung cancer therapy via LDL-R mediated endocytosis.

Presently, a combination of chemotherapy, radiotherapy, thermotherapy, and other treatments has become a hot topic of research for the treatment of cancer, especially lung cancer. In this study, novel hollow gold nanoparticles (HGNPs) were used as drug carriers, and in order to improve the targeting ability of HGNPs to a lung tumor site, polyoxyethylene sorbitol oleate (PSO) was chosen here as a target ligand since it can be specifically recognized by the low-density lipoprotein (LDL) receptor which is usually over expressed on A549 lung cancer cells. In this way, a PSO-modified doxorubicin-loaded HGNP drug delivery system (PSO-HGNPs-DOX) was constructed and its physicochemical properties, photothermal conversion ability, and drug release of PSO-HGNPs-DOX was investigated. Further, the effects of triple combination therapy, the intracellular uptake, and the ability to escape macrophage phagocytosis of PSO-HGNPs-DOX were also studied using A549 cells in vitro. In addition, an in vivo mouse model was also used to study the targeting of PSO-HGNPs-DOX to lung cancer. PSO-HGNPs-DOX demonstrated a good triple therapeutic effect for lung cancer (A549 cell viability was only 10% at 500 µM) by LDL receptor mediated endocytosis and was able to escape macrophage phagocytosis to enhance its accumulation at the target site. Therefore, PSO-HGNPs-DOX is a novel, safe, promising, and targeted drug carrier designed for triple combination lung cancer therapy which should be further studied for such applications.

Lipoprotein imitating nanoparticles: Lecithin coating binds ApoE and mediates non-lysosomal uptake leading to transcytosis over the blood-brain barrier.

Lipoproteins are naturally occurring nano sized transport vehicles in the human body. Therefore, lipoproteins could be applied as a drug carrier system. Additionally, several reports of apolipoprotein mediated blood-brain barrier (BBB) crossing suggest lipoprotein mimicking nanoparticles (NPs) as possible drug delivery vehicles to the brain. This could extend the therapy opportunities of various diseases of the central nervous system. A lipoprotein imitating NP system, consisting of a lecithin coated lipophilic cholesteryl oleate core with embedded fluorescent dye and adsorbed apolipoprotein E3 (ApoE) has been established using a two-step solvent injection method. Lecithin coating was proven to stabilize the NPs in isotonic saline solution and to bind ApoE in a highly efficient way. Fluorescent dye load (as model drug) and ApoE amount were varied, obtaining 100 nm sized, monodisperse NPs. The NPs' interaction with the BBB formed by primary porcine brain capillary endothelial cells (PBCEC) was investigated by fluorescence microscopy observing that ApoE mediated a lysosome bypassing uptake mechanism. Using this in vitro BBB model, ApoE concentration dependent permeation over the cell layer could be proven in both directions. An ApoE mediated transcytosis could be achieved, as it had been observed earlier for low-density lipoproteins. These results show that the newly developed NP system successfully mimics endogenous lipoproteins. An ApoE dependent penetration of the BBB was confirmed and provided an indication of apolipoprotein mediated transcytosis, avoiding lysosomal degradation.

Extra Virgin Olive Oil Phenol Extracts Exert Hypocholesterolemic Effects through the Modulation of the LDLR Pathway: In Vitro and Cellular Mechanism of Action Elucidation.

This study was aimed at investigating the hypocholesterolemic effects of extra virgin olive oil (EVOO) phenols and the mechanisms behind the effect. Two phenolic extracts were prepared from EVOO of different cultivars and analyzed using the International Olive Council (IOC) official method for total phenols, a recently validated hydrolytic procedure for total hydroxytyrosol and tyrosol, and 1H-NMR analysis in order to assess their secoiridoid profiles. Both of the extracts inhibited in vitro the 3-hydroxy-3-methylglutaryl co-enzyme A reductase (HMGCoAR) activity in a dose-dependent manner. After the treatment of human hepatic HepG2 cells (25 µg/mL), they increased the low-density lipoprotein (LDL) receptor protein levels through the activation of the sterol regulatory element binding proteins (SREBP)-2 transcription factor, leading to a better ability of HepG2 cells to uptake extracellular LDL molecules with a final hypocholesterolemic effect. Moreover, both of the extracts regulated the intracellular HMGCoAR activity through the increase of its phosphorylation by the activation of AMP-activated protein kinase (AMPK)-pathways. Unlike pravastatin, they did not produce any unfavorable effect on proprotein convertase subtilisin/kexin 9 (PCSK9) protein level. Finally, the fact that extracts with different secoiridoid profiles induce practically the same biological effects suggests that the hydroxytyrosol and tyrosol derivatives may have similar roles in hypocholesterolemic activity.

Aloe-emodin exerts cholesterol-lowering effects by inhibiting proprotein convertase subtilisin/kexin type 9 in hyperlipidemic rats.

Hyperlipidemia (HPL) characterized by metabolic disorder of lipids and cholesterol is one of the important risk factors for cardiovascular diseases. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a potent circulating regulator of LDL through its ability to induce degradation of the low-density lipoprotein cholesterol receptor (LDLR) in the lysosome of hepatocytes. Aloe-emodin (AE) is one of potentially bioactive components of Chinese traditional medicine Daming capsule. In this study we evaluated the HPL-lowering efficacy of AE in both in vivo and in vitro HPL models. High-fat diet-induced rats were treated with AE (100 mg/kg per day, ig) for 6 weeks. We found that AE administration significantly decreased the levels of total cholesterol (TC) and LDL in the serum and liver tissues. Moreover, AE administration ameliorated HPL-induced hepatic lipid aggregation. But AE administration did not significantly inhibit HMG-CoA reductase activity in the liver of HPL rats. A cellular model of HPL was established in human hepatoma (HepG2) cells treated with cholesterol (20 µg/mL) and 25-hydroxycholesterol (2 µg/mL), which exhibited markedly elevated cholesterol levels. The increased cholesterol levels could be reversed by subsequent treatment with AE (30 µM). In both the in vivo and in vitro HPL models, we revealed that AE selectively suppressed the sterol-regulatory element-binding protein-2 (SREBP-2) and hepatocyte nuclear factor (HNF)1α-mediated PCSK9 signaling, which in turn upregulated LDL receptor (LDLR) and promoted LDL uptake. This study demonstrates that AE reduces cholesterol content in HPL rats by inhibiting the hepatic PCSK9/LDLR pathway.

Could PCSK9 be a new therapeutic target of Eugenol? In vitro and in silico evaluation of hypothesis.

PCSK9 (Proprotein convertase Subtilisin/Kexin Type 9), an important regulator of lipid metabolism, has been shown to play a role in hepatocellular carcinoma by promoting metastasis. PCSK9 interferes with LDL metabolism and causes dyslipidemias in hematological malignancies particularly acute lymphoblastic leukemia. Nutraceuticals like berberine, curcumin and polydatin have been found effective in modulating PCSK9 expression by lowering LDL levels. Eugenol, a nutraceutical has shown a promising role in cancer due to its antioxidant and antihypercholesterolemic effects. In the present study, PCSK9 expression was measured in acute lymphoblastic leukemia (ALL) patients and was found to be significantly induced. Based on the results of expression analysis, a plausible hypothesis was made. Eugenol being an antioxidant will prevent oxidation of LDL. In the absence of ox-LDL, LOX1 scavenger receptor, which regulates PCSK9 expression, will not be activated. As the circulating LDL is reduced, it will no longer be able to support leukemia cell growth. The hypothesis was validated by an in silico and in vitro study. Molecular docking revealed hydrophobic interactions between ligand eugenol and macromolecules PCSK9 and LOX1. Expression of both PCSK9 and LOX1 were significantly reduced by eugenol in Jurkat cells. To conclude, PCSK9 could therapeutically be targeted by eugenol in leukemia cells.

Comparing the serum TAG response to high-dose supplementation of either DHA or EPA among individuals with increased cardiovascular risk: the ComparED study.

Studies have shown that the reduction in serum TAG concentrations with long-chain n-3 fatty acid supplementation is highly variable among individuals. The objectives of the present study were to compare the proportions of individuals whose TAG concentrations lowered after high-dose DHA and EPA, and to identify the predictors of response to both modalities. In a double-blind, controlled, crossover study, 154 men and women were randomised to three supplemented phases of 10 weeks each: (1) 2·7 g/d of DHA, (2) 2·7 g/d of EPA and (3) 3 g/d of maize oil, separated by 9-week washouts. As secondary analyses, the mean intra-individual variation in TAG was calculated using the standard deviation from the mean of four off-treatment samples. The response remained within the intra-individual variation (±0·25 mmol/l) in 47 and 57 % of participants after DHA and EPA, respectively. Although there was a greater proportion of participants with a reduction >0·25 mmol/l after DHA than after EPA (45 υ. 32 %; P 0·25 mmol/l after both DHA and EPA had higher non-HDL-cholesterol, TAG and insulin concentrations compared with other responders at baseline (all P < 0·05). In conclusion, supplementation with 2·7 g/d DHA or EPA had no meaningful effect on TAG concentrations in a large proportion of individuals with normal mean TAG concentrations at baseline. Although DHA lowered TAG in a greater proportion of individuals compared with EPA, the magnitude of TAG lowering among them was similar.

Can Carob-Fruit-Extract-Enriched Meat Improve the Lipoprotein Profile, VLDL-Oxidation, and LDL Receptor Levels Induced by an Atherogenic Diet in STZ-NAD-Diabetic Rats?

Carob fruit extract (CFE) has shown remarkable in vitro antioxidant properties and reduces postprandial hyperglycemia and hyperlipidemia in healthy animals. Development of functional meat products that contain bioactive components are presented as a great nutritional strategy. Until now, the effect of the consumption of restructured meat enriched with CFE in a murine model of diabetes has not been investigated. The objective of this study was to evaluate the effect on glycemia, lipemia, lipoprotein profile, Ldlr, arylesterase (AE), and very low-density lipoproteins (VLDL) and liver oxidation in streptozotocin-nicotinamide (STZ-NAD) growing Wistar diabetic rats fed restructured meat in the frame of a high cholesterol/high saturated-fat diet. In the present study, three groups (D, ED and DE) were fed cholesterol-enriched (1.4% cholesterol and 0.2% cholic acid) and high saturated-fat diets (50% of total energy from fats and 20.4% from saturated fatty acids). Rats were subjected to a STZ-NAD administration at the 3rd week. Group D did not receive CFE, while ED and DE rat groups received CFE before and after the diabetic induction, respectively. After eight weeks, D rats showed hyperglycemia and hypercholesterolemia, an increased amount cholesterol-enriched VLDL (ß-VLDL), IDL and LDL particles and triglyceride-enriched HDL. ED and DE partially blocked the hypercholesterolemic induction with respect to D group (p < 0.001) and improved glycemia, cholesterol levels, lipoprotein profile, Ldlr, plasma AE activity and liver oxidation (p < 0.001). Fecal fat, moisture and excretion were higher while dietary digestibility was lower in ED and DE vs. D counterparts (p < 0.001). In conclusion, CFE-enriched meat shows, for the first time, hypoglycemic and hypolipidemic effects in STZ-NAD animals fed high cholesterol/high saturated-fat diets. Likewise, it manages to reverse possible diabetes lipoprotein alterations if CFE-enriched meat is consumed before pathology development or improves said modifications if Type 2 Diabetes Mellitus is already established.

Coenzyme Q10 or Creatine Counteract Pravastatin-Induced Liver Redox Changes in Hypercholesterolemic Mice.

Statins are the preferred therapy to treat hypercholesterolemia. Their main action consists of inhibiting the cholesterol biosynthesis pathway. Previous studies report mitochondrial oxidative stress and membrane permeability transition (MPT) of several experimental models submitted to diverse statins treatments. The aim of the present study was to investigate whether chronic treatment with the hydrophilic pravastatin induces hepatotoxicity in LDL receptor knockout mice (LDLr-/-), a model for human familial hypercholesterolemia. We evaluated respiration and reactive oxygen production rates, cyclosporine-A sensitive mitochondrial calcium release, antioxidant enzyme activities in liver mitochondria or homogenates obtained from LDLr-/- mice treated with pravastatin for 3 months. We observed that pravastatin induced higher H2O2 production rate (40%), decreased activity of aconitase (28%), a superoxide-sensitive Krebs cycle enzyme, and increased susceptibility to Ca2+-induced MPT (32%) in liver mitochondria. Among several antioxidant enzymes, only glucose-6-phosphate dehydrogenase (G6PD) activity was increased (44%) in the liver of treated mice. Reduced glutathione content and reduced to oxidized glutathione ratio were increased in livers of pravastatin treated mice (1.5- and 2-fold, respectively). The presence of oxidized lipid species were detected in pravastatin group but protein oxidation markers (carbonyl and SH- groups) were not altered. Diet supplementation with the antioxidants CoQ10 or creatine fully reversed all pravastatin effects (reduced H2O2 generation, susceptibility to MPT and normalized aconitase and G6PD activity). Taken together, these results suggest that 1- pravastatin induces liver mitochondrial redox imbalance that may explain the hepatic side effects reported in a small number of patients, and 2- the co-treatment with safe antioxidants neutralize these side effects.

"Benifuuki" Extract Reduces Serum Levels of Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1 Ligands Containing Apolipoprotein B: A Double-Blind Placebo-Controlled Randomized Trial.

(1) Background: Arteriosclerosis is associated with high levels of low-density lipoprotein (LDL) cholesterol. O-methylated catechins in "Benifuuki" green tea are expected to reduce cholesterol levels, although there is limited research regarding this topic; (2) Methods: This trial evaluated 159 healthy volunteers who were randomized to receive ice cream containing a high-dose of "Benifuuki" extract including 676 mg of catechins (group H), a low-dose of "Benifuuki" extract including 322 mg of catechins (group L), or no "Benifuuki" extract (group C). Each group consumed ice cream (with or without extract) daily for 12 weeks, and their lipid-related parameters were compared; (3) Results: A significant reduction in the level of lectin-like oxidized LDL receptor-1 ligand containing ApoB (LAB) was detected in group H, compared to groups L and C. No significant differences between the three groups were detected in their levels of total cholesterol, triglycerides, and LDL cholesterol; (4) Conclusions: "Benifuuki" extract containing O-methylated catechins may help prevent arteriosclerosis.

Jatrophane diterpenoids from Euphorbia helioscopia and their lipid-lowering activities.

The phytochemical study of Euphorbia helioscopia led to the isolation of 33 jatrophane diterpenoids (1-33), of which six (1-6) were new. This small jatrophane library was established to screen for potential lipid modulators. LDL-Uptake screening assay demonstrated that most of them improved LDL-Uptake rate in HepG2 cells, with compounds 16, 21 and 26 exhibiting more outstanding effects. Further exploration found that these three compounds could increase LDLR protein level in HepG2 cells dose-dependently. SAR studies suggested that substituted patterns of C-9, steric hindrance between C-14 and C-15, and the long conjugated fragment from C-5 to the carbonyl (C-9) were essential for the activity. Moreover, compound 21, a relatively abundant chemical in E. helioscopia, showed remarkable lipid-lowering effect in vivo, which makes it a promising lead for development of new lipid-lowering agents.

Blackcurrant anthocyanins stimulated cholesterol transport via post-transcriptional induction of LDL receptor in Caco-2 cells.

PURPOSES: We previously showed that polyphenol-rich blackcurrant extract (BCE) showed a hypocholesterolemic effect in mice fed a high fat diet. As direct cholesterol removal from the body via the intestine has been recently appreciated, we investigated the effect of BCE on the modulation of genes involved in intestinal cholesterol transport using Caco-2 cells as an in vitro model. METHODS: Caco-2 cells were treated with BCE to determine its effects on mRNA and protein expression of genes important for intestinal cholesterol transport, low-density lipoprotein (LDL) uptake, cellular cholesterol content, and cholesterol transport from basolateral to apical membrane of Caco-2 cell monolayers. Cells were also treated with anthocyanin-rich or -poor fraction of BCE to determine the role of anthocyanin on BCE effects. RESULTS: BCE significantly increased protein levels of LDL receptor (LDLR) without altering its mRNA, which consequently increased LDL uptake into Caco-2 cells. This post-transcriptional induction of LDLR by BCE was markedly attenuated in the presence of rapamycin, an inhibitor of mechanistic target of rapamycin complex 1 (mTORC1). In addition, BCE altered genes involved in cholesterol transport in the enterocytes, including apical and basolateral cholesterol transporters, in such a way that could enhance cholesterol flux from the basolateral to apical side of the enterocytes. Indeed, BCE significantly increased the flux of LDL-derived cholesterol from the basolateral to the apical chamber of Caco-2 monolayer. LDLR protein levels were markedly increased by anthocyanin-rich fraction, but not by anthocyanin-free fraction. CONCLUSION: mTORC1-dependent post-transcriptional induction of LDLR by BCE anthocyanins drove the transport of LDL-derived cholesterol to the apical side of the enterocytes. This may represent a potential mechanism for the hypocholesterolemic effect of BCE.

Multifunctional gold nanorods and docetaxel-encapsulated liposomes for combined thermo- and chemotherapy.

Personalized and precise nanomedicines are highly demanded for today's medical needs. Liposomes are ideal candidates for the construction of multifunctional drug delivery systems. In this study, a liposome was used to improve the clinical issues of docetaxel (Doc), a potent antimitotic chemotherapy for prostate cancer (PC). RLT, a low-density lipoprotein receptor (LDLR)-binding peptide, and PEG were conjugated to the liposomes, and gold nanorods (GNRs) were also incorporated into the liposomes. The GNRs/Doc-liposome-RLT (GNRs/DocL-R) was tested in PC-3 cells and in PC-3 tumor-bearing nude mice. Results showed that GNRs/DocL-R possessed a diameter approximately 163.15±1.83 nm and a zeta potential approximately -32.8±2.16 mV. GNRs/DocL-R showed enhanced intracellular entrance, increased accumulation in the implanted tumor region, and the highest tumor inhibition in vitro and in vivo. Therefore, the multifunctional GNRs/DocL-R was a potential cancer treatment via combined chemo- and thermotherapy.

Atorvastatin and lovastatin, but not pravastatin, increased cellular complex formation between PCSK9 and the LDL receptor in human hepatocyte-like C3A cells.

Statins are the first-line treatment for hypercholesterolemic patients. Herein, the effects of three statins on complex formation between proprotein convertase subtilisin-kexin 9 (PCSK9) and the low density lipoprotein receptor (LDLR), a critical step for the PCSK9-dependent degradation of LDLR in the lysosome, were examined. Human hepatocyte-like C3A cells grown in control (containing 10% fetal bovine serum) or MITO+ (supplemented with BD™ MITO + serum extender) medium were also treated with atorvastatin (Atorv), lovastatin (Lov), or pravastatin (Prav) for 24 h. RNA and protein expression studies and determinations of PCSK9/LDLR complex formation were performed. As expected, the statins increased the expression of PCSK9 and LDLR independently of the medium employed. Interestingly, Atov and Lov caused increases in PCSK9/LDLR complex formation, whereas Prav decreased complex formation when compared to cells treated without drugs. These results may explain why Prav works better for statin intolerant patients than other statins such as Atorv and Lov.