Effect of Heat-Treated Garlic (Allium sativum L.) on Growth Parameters, Plasma Lipid Profile and Histological Changes in the Ileum of Atherogenic Rats

Dietary supplementation with raw garlic has a preventive and healing effect in cardiovascular diseases, but it could also damage the intestinal mucosa, resulting in impairment of nutrient absorption. Garlic processing, including heat treatment, changes the content and biological activity of garlic, so it is crucial to find food-processing methods that will preserve the health-promoting properties of garlic while minimizing its negative impact on the digestive system. Therefore, in this study, the effect of garlic (Allium sativum L.) on growth parameters, plasma lipid profile, and morphological parameters in the ileum of Wistar rats subjected to various types of heat treatment (90 s blanching garlic, 10 min boiling in water, 10 min pan frying without fat, microwave heating fresh garlic, 90 s blanching and microwave heating garlic, 10 min boiling in water and microwave heating garlic, and 10 min pan frying without fat and microwave heating garlic) was determined in an atherogenic diet (containing 1% addition of cholesterol). In the conducted research, it was found that the diet supplemented with heat-treated garlic used in the atherogenic diet improved the consumption and growth parameters of rats, depending on the type and time of its use. The highest consumption was recorded in atherogenic groups supplemented with garlic subjected to a longer (10 min) heat treatment and was then heated in a microwave oven. Garlic subjected to the shortest heat treatment proved to be most effective, and a significant improvement in the lipid profiles of rats’ plasma with atherogenic was observed. Extending the time of heat treatment of garlic and, additionally, its microwaving significantly weakened the action of garlic in the body, but still retained its hypolipidemic effect. The greatest influence on the structural changes in the mucosa of the rats’ iliac intestine, manifested by degeneration of the mucosa, shortening the length of the intestinal villi, damage to the brush border, and thus impairment of the intestinal absorption, was exerted by supplementing the atherogenic diet with garlic subjected to short-term heat treatment. Among the processes used, blanching was the least favorable, and the long-lasting thermal processes (cooking, frying for 10 min) had a positive effect on the mucosa of the rats’ intestines. The results obtained in this study confirm that the selection of an appropriate method of thermal processing of garlic may allow for the maintenance of preventive and therapeutic efficacy of garlic in cardiovascular diseases, while ensuring the safety of its long-term use in the context of degenerative changes in the gastrointestinal tract.

Curcumin exerts a protective effect against obesity and liver injury induced by an atherogenic diet

Background: Curcumin (Cur) is a natural yellow polyphenol extracted from the turmeric rhizome (Curcuma longa). Cur is known for its potential therapeutic properties as an analgesic, anti-inflammatory, antioxidant, antimicrobial, hepatoprotective, and anti-mutagenic, although some of these biological activities remain unproven. Epidemiological studies have shown a positive relationship between high-fat diets and diet-related chronic diseases. We hypothesized that some adverse effects of consuming atherogenic or high-fat diets (AD) can be ameliorated by Cur supplementation. Using an experimental model of rats, this study investigated the significance of Cur when it is given as a supplement in an AD.Methods: Healthy adult Wistar rats were randomly assigned to one of three groups. Controls (C) received a standard diet and experimental rats were fed with AD or AD+Cur for 5 weeks. Cur (100 mg/kg body weight) was given orally daily, plus piperine (5 mg/kg body weight). The effect of Cur supplementation was studied on zoometrics, visceral fat content, serum lipids profile, hepatosteatosis, liver function and oxidative status. Results: Diets did not alter energy consumption. As compared to the other groups, AD+Cur group showed a lower total visceral fat content, percentage of perirenal, mesenteric, and pelvic fat, and body weight gain (P< 0.05). Serum total cholesterol (P<0.0001), non-HDL-C (P<0.0001) levels were significantly higher in AD groups as compared with C. Serum triglycerides and HDL-C levels remained similar among groups (P>0.05). AD induced a liver injury with macrovesicular steatosis and portal inflammation. AD+Cur rats presented microvesicular steatosis with no inflammation, achieving the lowest level of alanine aminotransferase (ALT; P<0.0001) and reductions of aspartate aminotransferase (AST; P<0.0001). Liver homogenates from AD+Cur showed that Cur supplementation reduced the dichlorofluorescein diacetate (DCFH-DA) oxidation rate induced by AD by 25 % and deferoxamine and superoxide dismutase inhibited DCFH-DA. Conclusion: Cur as a dietary supplement showed a protective effect against obesity and inflammation, but its cardioprotective ability remained unproved. Cur may develop as a promising therapeutic agent for liver diseases induced by oxidative stress. This study provides supporting evidence to confirm the beneficial effects of curcumin from the point of view of functional food science. Keywords: curcumin, liver injury, ROS, atherogenic diet, visceral fat, obesity

Link between aging and atheroprotection in Mif-deficient atherosclerotic mice

Atherosclerosis is a lipid-triggered chronic inflammatory condition of our arteries and the main underlying pathology of myocardial infarction and stroke. Pathogenesis is age-dependent, but the mechanistic links between disease progression, age, and atherogenic cytokines and chemokines are incompletely understood. Here, we studied the chemokine-like inflammatory cytokine macrophage migration inhibitory factor (MIF) in atherogenic Apoe-/- mice across different stages of aging and cholesterol-rich high-fat diet (HFD). MIF promotes atherosclerosis by mediating atherogenic monocyte and T-cell recruitment, amplifying lesional inflammation, and suppressing atheroprotective B-cell responses. However, age-related links between atherogenesis and MIF and its role in advanced atherosclerosis in aged mice have not been systematically explored. We compared effects of global Mif-gene deficiency in 30-, 42-, and 48-week-old Apoe-/- mice on HFD for 24, 36, or 42 weeks, respectively, and in 52-week-old mice on a 6-week HFD. While a regio-specific atheroprotective phenotype of Mif-deficiency was observed in the 30/24-week-old group, atheroprotection was not detected in the 48/42- and 52/6-week-old groups, suggesting that atheroprotection afforded by global Mif-gene deletion differs across aging stages and atherogenic diet duration. We identify a combination of mechanisms that could explain this phenotype: i) Mif-deficiency promotes lesional Trem2+ macrophage numbers in younger but not aged mice; ii) Mif-deficiency favors formation of lymphocyte-rich stage-I/II ATLOs in younger mice but ATLO numbers equalize with those in Apoe-/- controls in the older mice; and iii) plasma anti-oxLDL-IgM antibody levels are decreased in aged Mif-deficient mice. Of note, these three markers (Trem2+ macrophages, ATLOs, anti-oxLDL-IgM antibodies) have been previously linked to atheroprotection. Together, our study thus suggests that regio-specific atheroprotection due to global Mif-gene deficiency in atherogenic Apoe-/- mice is lost upon advanced aging and identifies mechanisms that could explain this phenotype shift. These observations may have implications for translational MIF-directed strategies.

SIRT1 Inhibition Impairs Ca2+ Buffering in Coronary Smooth Muscle Cells of Ossabaw Miniature Swine

Background: SIRT1 is a deacetylase that has diverse roles in intracellular Ca2+ signaling, metabolism, and cardiovascular disease. SIRT1 increases sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) activity that is essential to buffer the increase in Ca2+ induced by release from the sarcoplasmic reticulum (SR). Our lab has shown that metabolic syndrome (MetS) impairs SERCA activity in coronary smooth muscle cells and causes coronary artery disease in Ossabaw miniature swine. We hypothesized that SIRT1 inhibition and MetS would impair Ca2+ buffering. Methods: CRISPR/Cas9 methods delivered a leucine to proline point mutation in SIRT1 (SIRT1L100P) into the Ossabaw swine genome to compare to wild type (WT) and mimic the naturally occurring mutation in humans and decrease SIRT1 activity. Four treatment groups of juvenile swine were based on genotype and diet: WT Lean, SIRT1 Lean, WT MetS, and SIRT1 MetS. Lean swine were fed normal chow and MetS were fed a hypercaloric, atherogenic diet for 7 months. The left anterior descending coronary artery was harvested and enzymatically digested to obtain cells. Fluorescence microscopy measured the Ca2+ indicator fura-2 in single cells. The cells were exposed to 5 mM caffeine to maximally release stores of Ca2+ from the SR. Ca2+ buffering capacity of each cell was analyzed after the caffeine-induced peak increase to assess Ca2+ efflux and SERCA activity. Results: MetS was confirmed by increased body weight, impaired glucose tolerance, hyperinsulinemia, and hypercholesterolemia. Coronary atherosclerosis was shown by angiography, intravascular ultrasound, and gross imaging. The rapid phase of Ca2+ buffering due to Ca2+ efflux was not affected by SIRT1 mutation or MetS. The slower phase of Ca2+ buffering due to SERCA activity was impaired only by SIRT1 mutation (p<0.0005), not by MetS. Conclusion: SIRT1 mutation alone inhibited SERCA buffering of Ca2+ in coronary smooth muscle. (Support: NIH T35HL110854, DK120240, DK09751.)

Mangrove (Rhizophora sp) Fruit Extract Inhibits Tumor Growth Factor (TGF)-β1 Expression in High Cholesterol Diet-Fed Rats

Background: Atherosclerosis is a major finding in cardiovascular disease. One of the pro-fibrotic cytokines that play an important role in the atherosclerosis process is Tumor Growth Factor (TGF)-β1, where the presence of high TGF-β1 secretion due to hypercholesterolemia will trigger excessive collagen matrix formation. Objective: To analyze Rhizophora sp fruit extract effect on TGF-β1 expression in high cholesterol diet-fed rats. Material and Methods: Eighteen 10-week-old rats weighing 150-200 g were used in this study. These animals were grouped into 3 groups, each consisting of 6 rats. Group A (normal control) is a group of rats that received a normal diet. Group B (atherogenic control) received a diet that induces atherosclerosis (atherogenic diet). This diet contains 2% cholesterol, 5% goat fat, 0.2% cholic acid and standard diet up to 100%. Atherogenic diet was given for 3 days, and on the first day this group also received vitamin D3 700,000 IU/kg. Group C (treated), apart from receiving an atherogenic diet, was also given Rhizophora sp fruit extract 500 mg/kg body weight. The Rhizophora sp fruit extract effect on TGF-β1 expression was evaluated by immunohistochemical procedure. The area of ​​the expression is calculated using the ImageJ. Results: The results of this study indicate that the expression of TGF-β1 is higher in the group receiving the atherogenic diet than the normal control group (17.3 vs. 8.9; P=0.000). Rhizophora sp fruit extract reduced this expression remarkably (17.3 vs. 11.4; P=0.001). Conclusion: Rhizophora sp fruit extract inhibits the the expression of TGF-β1 in high cholesterol diet-fed rats.

Deficiency of ASGR1 in pigs recapitulates reduced risk factor for cardiovascular disease in humans

Genetic variants in the asialoglycoprotein receptor 1 (ASGR1) are associated with a reduced risk of cardiovascular disease (CVD) in humans. However, the underlying molecular mechanism remains elusive. Given the cardiovascular similarities between pigs and humans, we generated ASGR1-deficient pigs using the CRISPR/Cas9 system. These pigs show age-dependent low levels of non-HDL-C under standard diet. When received an atherogenic diet for 6 months, ASGR1-deficient pigs show lower levels of non-HDL-C and less atherosclerotic lesions than that of controls. Furthermore, by analysis of hepatic transcriptome and in vivo cholesterol metabolism, we show that ASGR1 deficiency reduces hepatic de novo cholesterol synthesis by downregulating 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), and increases cholesterol clearance by upregulating the hepatic low-density lipoprotein receptor (LDLR), which together contribute to the low levels of non-HDL-C. Despite the cardioprotective effect, we unexpectedly observed mild to moderate hepatic injury in ASGR1-deficient pigs, which has not been documented in humans with ASGR1 variants. Thus, targeting ASGR1 might be an effective strategy to reduce hypercholesterolemia and atherosclerosis, whereas further clinical evidence is required to assess its hepatic impact.

The anti-integrity activity of eugenol on inflammatory markers of chronic atherosclerosis

The putative anti-atherogenic, anti-oxidative, and anti-inflammatory potential of atherogenic diet-fed, eugenol-treated Wistar rats were studied. The levels of hematological parameters (ESR, WBC, and platelet count); the mean levels of nitric oxide (NO), and C-reactive protein (CRP) were investigated in serum samples. The mean ESR, platelet, and total WBC counts were all significantly higher in atherogenic diet-fed (Group II) rat when compared with normal (Group I), whereas the mean ESR, platelet, and total WBC counts were significantly lower in atherogenic diet-fed followed by eugenol treatment (Group III). The mean hepatic tissue mRNA transcript level of the gene responsible for CRP, iNOS, TNF-α, IL-1β, and NF-κB and expression of their protein levels were determined in liver tissue samples were found to be significantly higher in the atherogenic diet-fed followed saline treatment (Group II) rats than the normal fed (Group I) rats. However, in hepatic tissue samples from an atherogenic diet-fed followed by eugenol treated rats, the mean mRNA the transcript level of the gene encoding CRP, iNOS, TNF-α, IL-1β, NF-κB and their mean concentration of the protein translation were significantly lower than the values in Group II rat’s hepatic tissue, approaching near-normal levels. These results are exhibited anti-atherogenic potential in hypercholesterolemic Wistar rats due to their hypocholesterolemic, antioxidant, and anti-inflammatory effects.

The Influence of Plants from the Alliaceae Family on Morphological Parameters of the Intestine in Atherogenic Rats

Bulbs from the Alliaceae family have been well-known and valued spices for thousands of years, not only for their unique flavor and aroma features, but also for their high nutritional and health-promoting values. Long-term or excessive consumption of these vegetables, especially raw garlic, can have side effects in the body (including in the digestive tract), causing a number of pathological changes in the intestinal wall; these changes lead, in turn, to its damage, dysfunction, and disorder development. Therefore, the aim of this study was to investigate the effect of the addition of freeze-dried vegetables from the Alliaceae family, i.e., garlic (Allium sativum L.), white onion, and red onion (Allium cepa L.) on the morphometric parameters (intestinal villi length, crypt depth, thickness of tunica mucosa, and the thickness of tunica muscle) of the jejunum of rats fed a semi-synthetic atherogenic diet (1% dietary cholesterol). In freeze-dried vegetables administered to rats, the contents of selected bioactive ingredients and their antioxidant potentials were determined. The effect of the onion vegetable supplements on growth parameters, serum lipid profile, plasma antioxidant potential, and the intestinal morphological parameters of rats loaded with cholesterol was determined. In an animal experiment, 30 male Wistar rats were divided into 5 diet groups, diet consumption and FER were studied. Supplementation of the atherogenic diet with vegetables improved the blood plasma lipid profiles and atherogenic indices, in a manner that was dependent on the type of supplementation used, with the best hypolipidemic and anti-atherosclerotic effects found in garlic use. The atherogenic diet, as well as the supplementation of this diet with the tested vegetables from the Alliaceae family, influenced the histological changes in the epithelium of the jejunum of rats. The damage to the intestinal mucosa was the greatest in animals fed an atherogenic diet supplemented with garlic. Bearing in mind that the desired beneficial therapeutic or prophylactic effects of onion vegetables (in particular garlic) in the course of various metabolic ailments (including atherosclerosis) are achieved during long-term supplementation, it is important to remember their possible cytotoxic effects (e.g., on the digestive tract) in order to achieve real benefits related to the supplementation with vegetables from the Alliaceae family.

THE EFFECT OF EGGPLANT JUICE TO THE THICKNESS OF AORTIC WALL IN WHITE RAT

Atherosclerosis is a complex chronic disease characterized by the accumulation of lipids within arterial walls. Delphinidin-3-(p-coumaroylrutinoside)-5-glucoside (nasunin), an anthocyanin, was isolated as purple-colored crystals from eggplant peels. Nasunin protection against induced lipid peroxidation in rat. The aim of study was to determine the effect of eggplant juice to the thickness of aortic wall of white rat with atherogenic diet. This true experimental laboratoric study using control group post test design performed in white rat that placed in pharmacologic laboratory of Brawijaya University. Sampling was carried out by completely random sampling with 25 rats for the total sample. Data were processed and analyzed using SPSS 16. Statistical test using one-way ANOVA and continuing with post hoc Tukey. The result showed there was a significant effect between positive control of diet atherogenic with the dosage I (1,3 gr/3 ml), dosage II (2,6 gr/3 ml), and dosage III (5,2 gr/3 ml) of eggplant juice to aorta wall thickness (p=0,000;p<0,05) . We can conclude that the effective dosage of eggplant juice for reducing the progression of aortic wall thickening is dosage III (5,2 gr/3 ml).

The effect of Sorghum Tempeh (Sorghum bicolor L. Moench) on low-density lipoprotein (LDL) and malondialdehyde (MDA) levels in atherogenic diet-induced rats

An atherogenic diet induces oxidative stress leading to hypercholesterolemia. This condition causes atherosclerosis followed by increased LDL and MDA. Sorghum tempeh contains fiber and antioxidants that can protectively improve LDL and MDA levels. Therefore, this research aims to determine the effect of sorghum tempeh on LDL and MDA levels in atherogenic diet-induced rats compared to sorghum flour. It used a randomized pre-post test with a control group design. The test subjects were 30 male Sprague Dawley rats, consisting of 6 normal conditioned rats (C1), and 24 that were induced by an atherogenic diet (C2, T1, T2, T3) for 2 weeks. Sorghum flour was administered at a dose of 4.095 g (T1) and the sorghum tempeh at 3.041 g (T2) and 6.081 g (T3) for 4 weeks. Furthermore, C2 was constantly induced through an atherogenic diet. Total cholesterol and LDL levels were then analyzed using the CHOD-PAP method, and MDA levels, using the ELISA method. Meanwhile, statistical analysis for these variables was carried out using IBM SPSS Statistics 21 software. The results showed that the administration of sorghum flour and tempeh significantly reduced total cholesterol, LDL, MDA levels in each group (p = 0.001). Furthermore, it showed that there was a significantly strong correlation between LDL and MDA levels before and after treatment (r = 0.610, r = 0.805, and p = 0.001). The administration of sorghum tempeh at a dose of 6.081 g caused the greatest reduction (∆) in LDL levels at -44.19 ±2.58 mg.dL-1, although, it was not the same as normal control. Meanwhile, sorghum flour at a dose of 4.095 g was the most influential in reducing MDA levels to the same as normal control with delta (∆) at -7.67 ±0.37 ng.mL-1. In conclusion, sorghum tempeh and flour were the most effective at reducing LDL and MDA levels, respectively.