High Fat Cholesterol Research Articles

Gut microbiota dysbiosis augments atherosclerosis in LDLR-/- mice

Aim: In recent years the gut microbiome has been recognized as an important participant in the etiology of obesity and -associated comorbidities. Moreover, recent studies have implicated an involvement of the gut microbiota in both cardiovascular health and disease. Although the gut microbiome has been suggested as a contributor to atherosclerosis, firm evidence to support a causal role for the gut microbiota in atherosclerosis is limited. Methods: To investigate the effect of the gut microbiota in atherosclerosis, we performed fecal microbiota transplantation to transplant the gut microbiota of caspase1-/- mice, an established model for dysbiosis, into LDLR-/- mice (LDLR-/-[casp1-/-]). Fecal microbiota transplantation of the gut microbiota of LDLR-/- mice into LDLR-/- mice (LDLR-/-[LDLR-/-]) served as control. Mice were fed a chow or high-fat cholesterol diet (HFC, 0,21% cholesterol) for 13 weeks and fecal samples were collected to determine microbiota composition by 16S rRNA sequencing. Plaque size was analyzed in the aortic root and immune cell subsets were analyzed by flow cytometry. Results: 16S rRNA sequencing of fecal samples confirmed the induction of dysbiosis in LDLR-/-[casp1-/-] compared to LDLR-/-[LDLR-/-] mice. Body weight, plasma triglyceride and cholesterol levels were significantly increased by HFC-feeding in LDLR-/-[casp1-/-] and LDLR-/-[LDLR-/-] mice. However, dysbiosis did not affect these parameters. In contrast, plaque size was significantly increased in HFC-fed LDLR-/-[casp1-/-] compared to HFC-fed (LDLR-/-[LDLR-/-] mice. Furthermore, dysbiosis in LDLR-/- mice was associated with a moderate increase in circulatory levels of Ly6C-high monocytes. Conclusions: Our data shows that gut microbiota dysbiosis augments atherosclerosis, possibly by exacerbating low-grade systemic inflammation.

Myrciaria cauliflora extracts attenuating hyperlipidemia and obesity in vivo

Obesity, the major reason causing the metabolic syndrome (MetS), leads to abnormal lipid metabolism, and also causes hyperlipidemia, morbidity and mortality in type 2 diabetes, cardiovascular and other diseases. These diseases often have increased insulin levels resulting from an insulin resistance lead to blood lipid abnormalities. Previously studies indicate that the polyphenols and anthocyanins ingredients have antioxidant, anti-arteriosclerosis, hypertension and diabetes suppression, hypolipidemic and tumor suppressor role. Therefore, this experiment will explore the preventive effect of extract from Myrciaria cauliflora (MCE) for the high-fat diet-induced obesity and dyslipidemia. In this study, we investigated the chemopreventive potential of the water extract from Myrciaria cauliflora (MCE) on the obesity male Wistar rat induced by lard oil for 10 weeks. The Serum biochemical parameters, body weight, were monitored every two weeks. After 10 weeks, the rats were sacrificed and the liver was assayed by HE stain and western blot. The results showed that dietary administration of MCE (0.5, 1, 1.5%) reduced the body weight of rat, serum biochemical parameters such as TG, TC, HDL-C, LDL-C, AST, ALT and lipid droplets in liver sections. Furthermore, MCE can also decrease the expression of lipid synthesis-related proteins by western blotting. These findings demonstrated that MCE prevented high-fat diet-induced high cholesterol and excess body fat, suggesting that MCE has a potentially protective effect in obesity-related and cardiovascular diseases.

Beneficial effects of TQRF and TQ nano- and conventional emulsions on memory deficit, lipid peroxidation, total antioxidant status, antioxidants genes expression and soluble Aβ levels in high fat-cholesterol diet-induced rats

The study determined the effect of thymoquinone rich fraction (TQRF) and thymoquinone (TQ) in the forms of nano- and conventional emulsions on learning and memory, lipid peroxidation, total antioxidant status, antioxidants genes expression and soluble β-amyloid (Aβ) levels in rats fed with a high fat-cholesterol diet (HFCD). The TQRF was extracted from Nigella sativa seeds using a supercritical fluid extraction system and prepared into nanoemulsion, which later named as TQRF nanoemulsion (TQRFNE). Meanwhile, TQ was acquired commercially and prepared into thymoquinone nanoemulsion (TQNE). The TQRF and TQ conventional emulsions (CE), named as TQRFCE and TQCE, respectively were studied for comparison. Statin (simvastatin) and non-statin (probucol) cholesterol-lowering agents, and a mild-to-severe Alzheimer's disease drug (donepezil) were served as control drugs. The Sprague Dawley rats were fed with HFCD for 6 months, and treated with the intervention groups via oral gavage daily for the last 3 months. As a result, HFCD-fed rats exhibited hypercholesterolaemia, accompanied by memory deficit, increment of lipid peroxidation and soluble Aβ levels, decrement of total antioxidant status and down-regulation of antioxidants genes expression levels. TQRFNE demonstrated comparable effects to the other intervention groups and control drugs in serum biomarkers as well as in the learning and memory test. Somehow, TQRFNE was more prominent than those intervention groups and control drugs in brain biomarkers concomitant to gene and protein expression levels. Supplementation of TQRFNE into an HFCD thus could ameliorate memory deficit, lipid peroxidation and soluble Aβ levels as well as improving the total antioxidant status and antioxidants genes expression levels.

Sivelestat alleviates nonalcoholic steatohepatitis in mice through inhibiting activation of Kupffer cells

Objective: To investigate the role of neutrophil elastase inhibitor, sivelestat, in preventing and treating nonalcoholic steatohepatitis (NASH) and its underling mechanisms. Methods: A total of forty 4-week-old male C57BL/6J ApoE-/-mice were equally divided into the following four groups: standard chow (SC)+isotonic saline; SC+sivelestat; high-fat, high-cholesterol (HFHC) diet+isotonic saline; and HFHC+sivelestat. These mice were treated with above methods for 12 weeks. Blood and liver tissue samples were collected to measure biochemical parameters, hepatic steatosis and non-alcoholic fatty liver disease (NAFLD) activity score (inflammation) were evaluated by oil red O staining and HE staining, respectively. The mRNA and protein expression levels of hepatic inflammatory cytokines, CD68, and F4/80 were determined by quantitative RT-PCR and immunohistochemistry, respectively. Comparison of means between the four groups was made by one-way analysis of variance, and comparison between any two groups was made by the LSD or SNK method (for data with homogeneity of variance) or the Tamhane or Dunnett method (for data with heterogeneity of variance). Results: Mice fed with an HFHC diet for 12 weeks developed typical pathological features of NASH compared with those fed with SC. Compared with mice fed with HFHC diet without sivelestat, those treated with HFHC and sivelestat exhibited the following features: (1) significantly reduced fast blood glucose, blood cholesterol, and hepatic biochemical parameters, as well as increased insulin sensitivity; (2) significantly reduced NAFLD activity score (5.71±1.11 vs 3.16±1.16, P < 0.05); (3) reduced monocyte chemoattractant protein-1 and tumor necrosis factor -α; (4) significantly reduced mRNA levels of CD68 and F4/80; and (5) reduced expression of CD68 in the liver. Conclusion: Sivelestat alleviates the hepatic steatosis and inflammation of NASH in mice by inhibiting the activation of Kupffer cells.

Abstract 444: A Six-Microrna Panel Identified as a Potential Biomarker for Early-Stage Atherosclerotic Lesions

Introduction: Atherosclerosis is a progressive disease, and it’s the common cause of cardiovascular disease (CVD). CVD is the leading cause of morbidity and mortality in the US. It is important to detect early-stage asymptomatic atherosclerosis prior to progression to plaques. However, it is not feasible to obtain target tissues from humans with early-stage atherosclerosis. We tested the hypothesis that a panel of microRNAs (miRNAs) expressed in atherosclerotic lesions and expressed in plasma of the same baboons are potential biomarkers indicative of initiation and progression of atherosclerosis. Methods and Results: We used small RNA-Seq to identify miRNA expression profiles in atherosclerotic lesions and in plasma of baboons. We challenged adult baboons (n=24) with a high cholesterol, high fat (HCHF) diet for two years. After the diet challenge, common iliac arteries were harvested, fixed in 10% buffered formalin and stained with Sudan IV. We observed interesting fatty streak lesion variations, including early stage (EGES), flat (F) and raised (R) lesions, corresponding, respectively, to AHA lesion types I, II and V. We identified 45 miRNAs differentially expressed in fatty streak lesions (ES vs F=0; ES vs R =36; F vs R=9), and 43 miRNAs in plasma of the same animals (ES vs F=0; ES vs R =21; F vs R=22). Further, we observed that miR-30-5p, miR-340-5p, miR-548-5p and let-7-3p were expressed in flat lesions as well as in plasma, whereas miR-30-5p and miR-21-5p were expressed in raised lesions and in plasma. FDR &lt; 0.05. Conclusions: We conclude that a panel of six miRNAs differentially expressed in fatty streak lesions and differentially expressed in plasma of the same animals is a potential biomarker indicative of initiation and progression of atherosclerosis in baboons. Future studies will focus on translating baboon findings to humans.

Curcumin improves intestinal barrier function: modulation of intracellular signaling, and organization of tight junctions.

Association between circulating lipopolysaccharide (LPS) and metabolic diseases (such as type 2 diabetes and atherosclerosis) has shifted the focus from high-fat high-cholesterol containing Western-type diet (WD)-induced changes in gut microbiota per se to release of gut bacteria-derived products (e.g., LPS) into circulation due to intestinal barrier dysfunction as the possible mechanism for the chronic inflammatory state underlying the development of these diseases. We demonstrated earlier that oral supplementation with curcumin attenuates WD-induced development of type 2 diabetes and atherosclerosis. Poor bioavailability of curcumin has precluded the establishment of a causal relationship between oral supplementation and it is in vivo effects. We hypothesized that curcumin attenuates WD-induced chronic inflammation and associated metabolic diseases by modulating the function of intestinal epithelial cells (IECs) and the intestinal barrier function. The objective of the present study was to delineate the underlying mechanisms. The human IEC lines Caco-2 and HT-29 were used for these studies and modulation of direct as well as indirect effects of LPS on intracellular signaling as well as tight junctions were examined. Pretreatment with curcumin significantly attenuated LPS-induced secretion of master cytokine IL-1β from IECs and macrophages. Furthermore, curcumin also reduced IL-1β-induced activation of p38 MAPK in IECs and subsequent increase in expression of myosin light chain kinase involved in the phosphorylation of tight junction proteins and ensuing disruption of their normal arrangement. The major site of action of curcumin is, therefore, likely the IECs and the intestinal barrier, and by reducing intestinal barrier dysfunction, curcumin modulates chronic inflammatory diseases despite poor bioavailability.

Linarin Enriched Extract Attenuates Liver Injury and Inflammation Induced by High-Fat High-Cholesterol Diet in Rats.

The aim of this study was to explore the potential beneficial effects of linarin enriched Flos Chrysanthemi extract (Lin-extract) on nonalcoholic steatohepatitis (NASH) induced by high-fat high-cholesterol (HFHC) diet in rats. SD rats received normal diet, HFHC diet, or HFHC diet plus different doses of Lin-extract. The liver content of triglyceride and total cholesterol markedly increased in HFHC diet-fed model rats while middle and high dose of Lin-extract lowered liver cholesterol significantly. The expression of stearoyl-CoA desaturase (SCD1) was upregulated by HFHC diet and further elevated by high dose Lin-extract. High dose of Lin-extract also markedly lowered the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and inhibited the activation of c-Jun N-terminal kinase (JNK) induced by HFHC in livers. The HFHC-increased mRNA levels of hepatic inflammation cytokines, including monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), and chemokine (C-X-C motif) ligand 1 (CXCL1), were suppressed by Lin-extract dose-dependently. Furthermore, pathology evaluation showed that high dose Lin-extract greatly improved lobular inflammation. Our results suggest that Lin-extract could attenuate liver injury and inflammation induced by HFHC diet in rats. Its modulatory effect on lipid metabolism may partially contribute to this protective effect.

Pleiotropic Effect of Human ApoE4 on Cerebral Ceramide and Saturated Fatty Acid Levels.

Apolipoprotein E (ApoE) is known for its role in lipid trafficking and the ɛ4 allele is a risk factor for late onset Alzheimer's disease (AD). Recently, aberrant ceramide and fatty acid (FA) levels have been implicated in AD. To determine the specific effects of human ApoE4 (hE4) on cerebral ceramide and FA content during chow or a high fat/high cholesterol (HFHC) diet. Cerebral ceramide and FA profiles were determined by LC-MSMS in 15-month-old female wild-type (WT), ApoE-knockout (E0), and hE4-knockin mice fed chow or a HFHC diet for 3 months. mRNA levels of genes involved in ceramide and FA metabolism were determined by qPCR. Similar to E0, hE4 mice displayed lower cerebral total ceramide, Cer16 : 0, and Cer24 : 1 levels than WT mice on both diets. Akin to WT mice, hE4 mice had lower total and saturated FA levels on chow than E0 mice. The HFHC diet significantly increased total and saturated FA levels in hE4 mice. Chow-fed hE4 mice showed lower mRNA levels of ceramide synthase (CerS) 6, acid sphingomyelinase, and of most ceramide and FA transporters than WT and E0 mice. The HFHC diet downregulated the expression of CerSs in hE4 and WT mice, and of ceramide and FA transporters in WT mice, but not in E0 mice. hE4 reduced cerebral ceramide levels to levels observed in E0 mice independent of diet. The HFHC diet increased cerebral FA levels in hE4 mice. This was associated with alterations in the expression of ceramide and FA transporters specifically in hE4 mice.

Myeloid DLL4 Does Not Contribute to the Pathogenesis of Non-Alcoholic Steatohepatitis in Ldlr-/- Mice.

Non-alcoholic steatohepatitis (NASH) is characterized by liver steatosis and inflammation. Currently, the underlying mechanisms leading to hepatic inflammation are not fully understood and consequently, therapeutic options are poor. Non-alcoholic steatohepatitis (NASH) and atherosclerosis share the same etiology whereby macrophages play a key role in disease progression. Macrophage function can be modulated via activation of receptor-ligand binding of Notch signaling. Relevantly, global inhibition of Notch ligand Delta-Like Ligand-4 (DLL4) attenuates atherosclerosis by altering the macrophage-mediated inflammatory response. However, the specific contribution of macrophage DLL4 to hepatic inflammation is currently unknown. We hypothesized that myeloid DLL4 deficiency in low-density lipoprotein receptor knock-out (Ldlr-/-) mice reduces hepatic inflammation. Irradiated Ldlr-/- mice were transplanted (tp) with bone marrow from wild type (Wt) or DLL4f/fLysMCre+/0 (DLL4del) mice and fed either chow or high fat, high cholesterol (HFC) diet for 11 weeks. Additionally, gene expression was assessed in bone marrow-derived macrophages (BMDM) of DLL4f/fLysMCreWT and DLL4f/fLysMCre+/0 mice. In contrast to our hypothesis, inflammation was not decreased in HFC-fed DLL4del-transplanted mice. In line, in vitro, there was no difference in the expression of inflammatory genes between DLL4-deficient and wildtype bone marrow-derived macrophages. These results suggest that myeloid DLL4 deficiency does not contribute to hepatic inflammation in vivo. Since, macrophage-DLL4 expression in our model was not completely suppressed, it can’t be totally excluded that complete DLL4 deletion in macrophages might lead to different results. Nevertheless, the contribution of non-myeloid Kupffer cells to notch signaling with regard to the pathogenesis of steatohepatitis is unknown and as such it is possible that, DLL4 on Kupffer cells promote the pathogenesis of steatohepatitis.

Effect of Doenjang made with Lipoxygenase-free Genotypes Soybean on the Lipid Improvement and Antioxidant Enzyme Activity in Rat Fed a High Fat-cholesterol Diet

Effect of Doenjang made with Lipoxygenase-free Genotypes Soybean on the Lipid Improvement and Antioxidant Enzyme Activity in Rat Fed a High Fat-cholesterol Diet

Lipid-lowering effects of Zanthoxylum schinifolium Siebold & Zucc. seed oil (ZSO) in hyperlipidemic rats and lipolytic effects in 3T3-L1 adipocytes.

On the basis of the antiatherosclerotic effect of Zanthoxylum schinifolium, the therapeutic potential of Zanthoxylum schinifolium seed oil (ZSO) was tested in terms of the blood lipid profile and obesity in rats. The lipolytic effects of ZSO were determined in adipocytes and the total body and liver weight were decreased in rats. Compared with the high-cholesterol high-fat (HCHF) group, the rats in the HCHF+ZSO group showed improved levels of hyperlipidemia indicators. Furthermore, western blot analysis confirmed that the improvement of hyperlipidemia indicators was induced by stimulation of lipoprotein lipase expression. Additional results indicated that the reduction in body weight was likely caused by phosphorylation of hormone-sensitive lipase (HSL) via the protein kinase A pathway, ultimately leading to lipolysis. In conclusion, the results of the in vivo experiment showed that ZSO improved the lipid profiles in the blood, lowering cardiovascular disease and arteriosclerosis and degrading cellular lipids by activating HSL.

Macrophage Stimulating Protein Enhances Hepatic Inflammation in a NASH Model.

Non-alcoholic steatohepatitis (NASH) is a common liver disease characterized by hepatic lipid accumulation (steatosis) and inflammation. Currently, therapeutic options are poor and the long-term burden to society is constantly increasing. Previously, macrophage stimulating protein (MSP)—a serum protein mainly secreted by liver—was shown to inhibit oxidized low-density lipoprotein (OxLDL)/lipopolysaccharides (LPS)-induced inflammation in mouse macrophages. Additionally, MSP could reduce palmitic acid (PA)-induced lipid accumulation and lipogenesis in the HepG2 cell line. Altogether, these data suggest MSP as a suppressor for metabolic inflammation. However, so far the potential of MSP to be used as a treatment for NASH was not investigated. We hypothesized that MSP reduces lipid accumulation and hepatic inflammation. To investigate the effects of MSP in the early stage of NASH, low-density lipoprotein receptor (Ldlr-/-) mice were fed either a regular chow or a high fat, high cholesterol (HFC) diet for 7 days. Recombinant MSP or saline (control) was administrated to the mice by utilizing subcutaneously-implanted osmotic mini-pumps for the last 4 days. As expected, mice fed an HFC diet showed increased plasma and hepatic lipid accumulation, as well as enhanced hepatic inflammation, compared with chow-fed controls. Upon MSP administration, the rise in cholesterol and triglyceride levels after an HFC diet remained unaltered. Surprisingly, while hepatic macrophage and neutrophil infiltration was similar between the groups, MSP-treated mice showed increased gene expression of pro-inflammatory and pro-apoptotic mediators in the liver, compared with saline-treated controls. Contrary to our expectations, MSP did not ameliorate NASH. Observed changes in inflammatory gene expression suggest that further research is needed to clarify the long-term effects of MSP.

Anti-hyperlipidemic and Anti-hypercholesterolemic effect of aqueous extract of guava (Pisidum guajava Linn.) leaves on rats

The aim of the present study is to investigate the ameliorative effect of aqueous guava leaf extract (AGLE) with different doses (200, 350, 500 & 650 mg/kg b.wt.) taken p.o in rats fed high fat high cholesterol (HFC) diet for 8 weeks. The four doses of AGLE cause a significant decrease in food intake, final body weight, gain in body weight, serum TAG, VLDL-C, risk ratio as well as liver TAG and malondialdehyde level. Also doses of AGLE (350,500 and 650 mg/kg b.wt.) cause a significant decrease in relative liver weight, serum AST and LDL-C but a significant increase in HDL-C and leptin hormone. While doses (500 & 650 mg/kg b.wt.) cause a significant decrease in serum TC. The highest dose causes a significant decrease in serum ALT and adiponectin hormone. Total liver cholesterol was decreased significantly in groups given (200 & 350 mg/kg b.wt.) but no significant decrease in doses (500 & 650 mg/kg b.wt.). As well as ghrelin hormone does not show significant decrease in the four treated groups fed HFC diet and given AGLE compared to rats fed HFC diet only.

Detrimental effects of a high fat/high cholesterol diet on memory and hippocampal markers in aged rats

High fat diets have detrimental effects on cognitive performance, and can increase oxidative stress and inflammation in the brain. The aging brain provides a vulnerable environment to which a high fat diet could cause more damage. We investigated the effects of a high fat/high cholesterol (HFHC) diet on cognitive performance, neuroinflammation markers, and phosphorylated Tau (p-Tau) pathological markers in the hippocampus of Young (4-month old) versus Aged (14-month old) male rats. Young and Aged male Fisher 344 rats were fed a HFHC diet or a normal control diet for 6 months. All animals underwent cognitive testing for 12days in a water radial arm maze to assess spatial and working reference memory. Hippocampal tissue was analyzed by immunohistochemistry for structural changes and inflammation, and Western blot analysis. Young and Aged rats fed the HFHC diet exhibited worse performance on a spatial working memory task. They also exhibited significant reduction of NeuN and calbindin-D28k immunoreactivity as well as an increased activation of microglial cells in the hippocampal formation. Western blot analysis of the hippocampus showed higher levels of p-Tau S202/T205 and T231 in Aged HFHC rats, suggesting abnormal phosphorylation of Tau protein following the HFHC diet exposure. This work demonstrates HFHC diet-induced cognitive impairment with aging and a link between high fat diet consumption and pathological markers of Alzheimer’s disease.

Lipocalin-2 mediates non-alcoholic steatohepatitis by promoting neutrophil-macrophage crosstalk via the induction of CXCR2

Inflammatory cell infiltration in the liver is a hallmark of non-alcoholic steatohepatitis (NASH). However, the pathological events which trigger the infiltration of inflammatory cells to mediate NASH pathogenesis remains poorly understood. This study aims to investigate the role of neutrophil-derived lipocalin 2 (LCN2) in mediating the transition from simple steatosis to NASH. Animal models of NASH were induced by high fat high cholesterol (HFHC) diet and methionine- and choline-deficient (MCD) diet in LCN2 knockout mice and wild-type controls. Circulating levels of LCN2 and its hepatic expression were markedly increased in both murine models and human subjects with NASH, and these changes were associated with increased infiltration of neutrophils. In diet-induced NASH models, hepatic injury, necroinflammation and infiltration of neutrophils and macrophages were substantially attenuated by genetic depletion of LCN2. In contrast, chronic infusion of recombinant LCN2 exacerbated diet-induced liver injury, inflammation and macrophage accumulation in a neutrophil-dependent manner. Primary mouse neutrophils lacking LCN2 exhibited a defective migration capacity, which can be reversed by replenishment with recombinant LCN2. Mechanistically, LCN2 induced the expression of the chemokine (C-X-C motif) receptor 2 (CXCR2), thereby leading to activation of ERK1/2 and production of proinflammatory chemokines. LCN2-induced inflammation, infiltration of macrophages and liver injury was abrogated in CXCR2-deficient mice. These findings demonstrated that LCN2 acts as a central mediator to facilitate the crosstalk between neutrophils and hepatic macrophages via induction of the chemokine receptor CXCR2, thereby exacerbating steatohepatitis. Lipocalin-2 levels in blood and the liver were markedly increased in both mouse models and human subjects with NASH, and these changes were associated with increased infiltration of neutrophils in the liver. In diet-induced NASH models, hepatic injury, necroinflammation and infiltration of neutrophils and macrophages were substantially attenuated by genetic depletion of lipocalin-2, but was augmented by chronic infusion of recombinant lipocalin-2. Lipocalin-2 induced the expression of the chemokine receptor CXCR2, thereby leading to activation of the mitogen-activated protein (MAP) kinase ERK1/2 and production of proinflammatory chemokines. Lipocalin-2-induced inflammation, infiltration of macrophages and liver injury was abrogated in CXCR2-deficient mice.

배암차즈기 열수 추출물 첨가가 고지방-고콜레스테롤 식이를 급여한 흰쥐의 항산화 활성과 지질조성에 미치는 영향

This study examined the antioxidant activity and lipid composition of rats fed a high fat-high cholesterol diet containing Salvia plebeia water extract. Three levels (50 mg/kg, 200 mg/kg, 500 mg/kg) of Salvia plebeia water extract were administered to seven weeks old male Sprague Dawley rats for four weeks. The activity of glutathione peroxidase in serum was high in all groups that received the Salvia plebeia water extract (p<0.05). The glutathione reductase activity was high (p<0.05) in groups that received 50 mg/kg and 500 mg/kg of Salvia plebeia water extract. When rats received Salvia plebeia water extract, the content of MDA (malondialdehyde) in the serum and liver was low in all groups. In addition, the serum total lipid, triglyceride, total cholesterol and LDL-cholesterol contents decreased significantly (p<0.05). Total cholesterol and triglyceride levels in the liver were significantly lower in the group that received the Salvia plebeia water extract than in the control group (p<0.05). Salvia plebeia water extract improves lipid metabolism and it almost meets with the increase of antioxidant activity and peroxide formation reduction. Based on these results, supplemented of a high fat-hypercholesterol diet with Salvia plebeia can increase antioxidant activity and depress peroxide formation in serum, as well as liver cholesterol and triglycerides.

Abstract 647: Induction of Cardiovascular Calcification in Non-transgenic Mice via a Single Injection of Pcsk9 Adeno-associated Viral Vector

Background: Studying atherosclerotic calcification in vivo requires mouse models with genetic deletion of low-density lipoprotein receptor (Ldlr) or apolipoprotein E. A previous study showed a rapid induction of atherosclerosis by proprotein convertase subtilisin/kexin type 9 (PCSK9) in mice. Here, we hypothesize that this method is a useful in vivo tool to study cardiovascular calcification in non-genetically modified C57BL/6 mice. Results: 10 week old C57BL/6 mice received a single tail vein injection of recombinant adeno-associated viral vector (AAV) encoding PCSK9 (rAAV8/D377Y-mPCSK9). Ldlr -/- and saline injected C57BL/6 mice served as controls. Mice consumed a high-fat, high-cholesterol (HF/HC) diet for 15-20 weeks. PCSK9 and total cholesterol serum levels were significantly increased within one week after injection and maintained for 20 weeks (cholesterol: 82 mg/dL to 820 mg/dL, p&lt;0.01; PCSK9: 0.14 μg/ml to 20 μg/ml, p&lt;0.01). Total cholesterol levels remained 20-30% lower than those of of Ldlr -/- mice. Atherosclerotic lesion size was similar between PSCK9 and Ldlr -/- mice. Saline injected mice did not show any lesions. Plaque collagen content was 31.9%±6.6 in PCSK9 mice and 62.9%±16.6 in Ldlr -/- mice at 15 weeks of HF/HC diet (p=0.01). However, by 20 weeks, the PCSK9 mice had 57.9%±18.6 plaque collagen, suggesting a different stage of plaque progression. Fluorescence reflectance imaging of a near infrared calcium tracer in intact arteries detected 0.4%±0.4 aortic calcification in PCSK9 mice and 9.7%±1.6 in Ldlr -/- mice at 15 weeks of HF/HC diet (p=0.01); by 20 weeks, the PCSK9 mice had 5.3%±1.0 aortic calcification. Tissue non-specific alkaline phosphatase activity positive lesion area was 7.9%±4.0 and 8.3%±2.6 in PCSK9 mice and 10.8%±2.5 and 12.7%±1.7in Ldlr -/- mice at 15 and 20 weeks, respectively. Immunofluorescence analysis demonstrated accumulation of CD68 and RUNX2-positive cells in the plaques of PCSK9 mice similar to Ldlr -/- . Conclusion: While injection of recombinant AAV encoding PCSK9 into C57BL/6 mice induces atherosclerotic calcification with slower sclerotic plaque remodeling compared to Ldlr -/- mice, it may serve as a useful tool to study cardiovascular calcification in mice independent of their genetic background.

Hypolipidemic and Antioxidative Effects of Glossogyne tenuifolia in Hamsters Fed an Atherogenic Diet.

Glossogyne tenuifolia (GT) Cassini is a special herbal tea in the Penghu Islands, Taiwan, and has a long history of being used as an antipyretic, detoxifying, and anti-inflammatory remedy in folk medicine among local residents. The aim of this study was to investigate the effect of hot water extracts from GT on oxidative stress and lipid metabolism in animals. Five- to 6-week-old male Syrian hamsters were divided into four groups (n = 14) for different treatments, that is: control group (C), high-fat/cholesterol (HF) group, HF diet containing 0.5% (GT0.5) and 1.5% (GT1.5) GT extracts for 4 weeks. Hamsters fed with 0.5% GT powder as well as 1.5% GT powder exhibited reduced serum total cholesterol (TC), conjugated diene of low-density lipoprotein (LDL), and increased serum antioxidant capacity, but 1.5% GT powder was more potent at lowering serum LDL cholesterol and thiobarbituric acid reactive substance concentrations than 0.5% GT. GT extracts significantly lowered liver triacylglycerol (TG) concentration by diminishing activities of fatty acid synthase (FAS) and glucose-6-phosphate dehydrogenase (G-6-PDH). In addition, fecal excretion of cholesterol and bile acids were increased in GT extract groups. In conclusion, GT extracts increase the antioxidative capacity, decrease serum TC, inhibit the activities of FAS and G-6-PDH, and further reduce liver TG accumulation in hamster fed on atherogenic diets.

Development of Human-Like Advanced Coronary Plaques in Low-Density Lipoprotein Receptor Knockout Pigs and Justification for Statin Treatment Before Formation of Atherosclerotic Plaques.

BackgroundAlthough clinical trials have proved that statin can be used prophylactically against cardiovascular events, the direct effects of statin on plaque development are not well understood. We generated low‐density lipoprotein receptor knockout (LDLR −/−) pigs to study the effects of early statin administration on development of atherosclerotic plaques, especially advanced plaques.Methods and Results LDLR −/− pigs were generated by targeted deletion of exon 4 of the LDLR gene. Given a standard chow diet, LDLR −/− pigs showed atherosclerotic lesions starting at 6 months of age. When 3‐month‐old LDLR −/− pigs were fed a high‐cholesterol, high‐fat (HCHF) diet for 4 months (HCHF group), human‐like advanced coronary plaques developed. We also fed 3‐month‐old LDLR −/− pigs an HCHF diet with pitavastatin for 4 months (Statin Prophylaxis Group). Although serum cholesterol concentrations did not differ significantly between the 2 groups, intravascular ultrasound revealed 52% reduced plaque volume in statin‐treated pigs. Pathological examination revealed most lesions (87%) in the statin prophylaxis group were early‐stage lesions, versus 45% in the HCHF diet group (P<0.01). Thin‐cap fibroatheroma characterized 40% of the plaques in the HCHF diet group versus 8% in the statin prophylaxis group (P<0.01), intraplaque hemorrhage characterized 11% versus 1% (P<0.01), and calcification characterized 22% versus 1% (P<0.01).ConclusionsResults of our large animal experiment support statin prophylaxis before the occurrence of atherosclerosis. Early statin treatment appears to retard development of coronary artery atherosclerosis and ensure lesion stability. In addition, the LDLR −/− pigs we developed represent a large animal model of human‐like advanced coronary plaque suitable for translational research.

Effect of resveratrol on experimental non-alcoholic fatty liver disease depends on severity of pathology and timing of treatment.

Non-alcoholic steatohepatitis (NASH) is a leading cause of chronic liver disease with few therapeutic options. Resveratrol (RSV) prevents the development of steatosis in a number of experimental fatty liver (non-alcoholic fatty liver [NAFL]) models, but the preventive or therapeutic effects on experimental NASH are not yet clarified, and clinical results on non-alcoholic fatty liver disease are ambiguous. Thus, we aimed to compare the RSV-mediated preventive and therapeutic effects on experimental NAFL and NASH. We used a high-fat (HF) diet to generate a rat NAFL model and a high-fat, high-cholesterol (HFC) diet to generate a rat NASH model. The preventive and therapeutic potential of RSV was tested by adding RSV to the HF and HFC diet from study start or after 1 week of the diets. Animals were sacrificed after 8 weeks with appropriate controls. Blood and liver were harvested for analysis, including measurement of RSV metabolites. Resveratrol reduced the development of histological steatosis (P = 0.03) and partly triglyceride accumulation (fold change reduced from 3.6 to 2.4, P = 0.08) in the male NAFL model, although effects were moderate. In NASH prevention, RSV reduced the accumulation of triglyceride in hepatic tissue (P < 0.01), while there was no effect on biochemical, histopathological, or transcriptional NASH changes. Further, RSV had no therapeutic effect on established NASH. We found RSV metabolites but no parent RSV in serum or liver tissue, confirming low bioavailability. These experimental findings suggest that a weak hepatic benefit of RSV treatment is seen in prevention of steatosis only.