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Melvin Amey
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Results and discussion: The oral administration of ESEG significantly lowered the levels of lipids in rabbits that were fed a CRD diet. This treatment also adjusted the protective system against oxidation in the arteries by decreasing the oxidation of lipids and proteins. Additionally, the levels of IL-1b, IL-6, sICAM-1, and sVCAM-1 in the bloodstream decreased significantly, and this was accompanied by a reduction of atherosclerotic lesions in all branches of the arteries. The findings suggest that EEPC may be a possible option for additional management of atherosclerosis.
Plinia cauliflora fruit peels present intense violet color from accumulating different polyphenolic pigments, including anthocyanins and ellagic acid derivatives (Neves et al., 2018). A study by Romo et al. (2019) identified 37 compounds in a preparation obtained from P. cauliflora fruit peels, including organic acids, phenolic acid derivatives, flavonoids, anthocyanins, and hydrolysable tannins (gallotannins and ellagitannins).
Isoflurane and potassium chloride were purchased from Cristlia (Itapira, SP, Brazil). Simvastatin and cholesterol were obtained from Sigma-Aldrich (St. Louis, MO, United States of America). All of the other reagents were acquired in analytical grade.
The animals were initially divided into five experimental groups, with each group consisting of six animals. They were then given a standard commercial diet (Nestl Purina PetCare, San Luis, Missouri, EUA), which was supplemented with 1% cholesterol. To prepare the CRD (cholesterol-rich diet), the commercial diet was crushed, and cholesterol dissolved in corn oil was added. After thorough mixing, the resulting mass was shaped into pellets and dried. For a period of 60 days, the CRD was freely available to the rabbit groups. Only animals with confirmed hypercholesterolemia were included in the study (Barboza et al., 2016). Thirty days after starting the diet, different experimental groups received orally, once a day, the EEPC (10, 30, and 100 mg/kg), vehicle (filtered water, 1 mL/kg; negative control), or simvastatin (SMV; 2.5 mg/kg; positive control) for 30 days. The nave group was fed a cholesterol-free diet and was treated only with the vehicle.
Body weight gain, behavioral changes, and mortality rate were monitored throughout the experimental period. On the morning of the sixty-first day, all animals were fasted for 6 hours and anesthetized with isoflurane. Blood samples were obtained from the jugular vein, and serum was obtained by centrifugation (1,500 x g for 5 min). The levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglyceride (TG) were measured using an automated biochemical analyzer (Roche Cobas Integra 400 plus). Malondialdehyde (MDA) levels were measured using an MDA assay kit (Cayman Chemical, Ann Arbor, MI, United States of America). Plasma nitrite levels were determined by the technique described by Schmidt et al. (1989). Interleukin-1β (IL-1β), IL-6, soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble intercellular adhesion molecule-1 (sICAM-1), nitrotyrosine (NT), and serum-oxidized low-density lipoprotein (ox-LDL) levels were measured by enzyme-linked immunosorbent assay (ELISA; BD Biosciences, San Jose, CA, United States of America). Subsequently, all animals were euthanized (35 mg/kg potassium chloride, i.v.). A part from the aorta segments, including the arch and iliac branches, was removed and fixed in 10% formalin. After 48 h, a part of each arterial branch was stained in Sudan-IV according to the previously described method (Gasparotto et al., 2019). The luminal surface was assessed for sudanophilic lesions by Motic Images Plus 2.0 software. The edited image was classified using an iterative algorithm for multiple threshold detection. A second part of the arterial branches was dehydrated, embedded in paraffin, and sectioned at 5 μm. Then, the samples were stained with hematoxylin/eosin and microscopically examined. The images were obtained, and the intima and media layers were measured by Motic Images Plus 2.0 software, according to Gasparotto et al. (2019). Finally, a third part of the arterial branches was sectioned and homogenized in K+ phosphate buffer (0.1 M, pH 6.5). The superoxide dismutase (SOD) (Gao et al., 1998) and catalase (CAT) (Aebi, 1984) levels were measured.
The body weight gain of the different experimental groups is presented in Figure 2. At the beginning of the treatments, the body weight of the rabbits of all experimental groups was homogeneous. The animals in the negative control and EEPC 30 mg/kg groups showed a significant reduction in weight gain at the end of the 60 days of treatment. On the sixty-first day, the animals treated with EEPC at 100 mg/kg and the rabbits treated with simvastatin presented a final body weight similar to that found in nave animals. During the experimental period, we did not identify deaths or significant behavioral changes, with appearance (skin, eyes, and appendages), reflexes, walking, and gastrointestinal function within the normal range for the species and gender.
World Health Organization points out that cardiovascular diseases (CVD) are the leading causes of morbidity and mortality worldwide, with values estimated at 17.9 million deaths annually (Nugroho et al., 2022). Among CVD, atherosclerosis contributes a large part to this outcome. Atherosclerosis is a complex inflammatory disease that affects medium and large-caliber arteries, especially in areas of more significant shear stress or bifurcations. The speed of its evolution depends on several factors, especially the intake of a diet rich in fats, hypertension, smoking, diabetes, and family history (Libby et al., 2019). Epidemiological studies indicate that high levels of LDL-C play a central role in the development of atherosclerosis (Whayne, 2017). Preclinical data and population studies show that endothelial dysfunction and increased LDL-C in its oxidized form (OxLDL) are indeed the primary cause of atherosclerosis. The morphological change in endothelial cells, followed by increased permeability to OxLDL particles in the subendothelial space, is the kickstart of atherosclerosis (Pirillo et al., 2013).
Reactive oxygen species (ROS) are produced by cellular metabolism. Under certain conditions, including dyslipidemia, cells will overproduce ROS, and several response mechanisms will be activated, including enzymatic antioxidants such as SOD and CAT. SOD primarily metabolizes superoxide anion into hydrogen peroxide and molecular oxygen, while CAT neutralizes excess hydrogen peroxide (Sies, 2015). Excessive production of ROS can alter cell homeostasis, leading to a chronic inflammatory response and contributing to the genesis of numerous chronic diseases, including atherosclerosis (Kattoor et al., 2017). It is already widely known that oxidative stress reduces nitric oxide (NO) production, impairs vasodilation, and causes endothelial dysfunction (Incalza et al., 2018). Moreover, oxidative stress triggers oxidative changes that can occur in the LDL-C molecule. After binding to proteoglycans of the extracellular matrix, the OxLDL induces the release of cytokines and the expression of cell adhesion molecules on the endothelial cells, recruiting monocytes and T lymphocytes to the inflamed arterial area. Differentiation of monocytes into macrophages express scavenger receptors to recognize OxLDL. The atherosclerotic plaque is mainly formed of foam cells, originating from the metamorphosis of swollen macrophages from OxLDL molecules. Finally, the plaque is covered by a fibrous cap, which can also be affected by oxidative stress. ROS can degrade the fibrous wall of plaque via the release of matrix metalloproteinases, causing thrombus formation, blockage of blood flow, and tissue infarction (Kattoor et al., 2017). We showed that EEPC could attenuate the oxidative stress induced by the atherogenic diet, reducing protein and lipid peroxidation and increasing nitrite levels, an indirect marker of nitric oxide bioavailability. This effect likely has an essential contribution from tissue antioxidant enzymes, especially SOD and CAT, as prolonged treatment with EEPC modulated the presence of these enzymes in different arterial branches. The evidence points to a synergistic effect between the reduction of serum lipids and antioxidant activity, reverberating in a significant decrease in LDL-C oxidation and the release of inflammatory mediators, including IL-1β, IL-6, sVCAM, and sICAM. As an endpoint, we found a significant reduction in lipid streaks and mature atherosclerotic plaques in all evaluated arterial branches.
The antioxidant potential of natural products is already well known, mainly due to the polyphenols (phenolic acids, flavonoids, anthocyanins, lignans, and stilbenes), carotenoids (xanthophylls and carotenes) and vitamins (vitamin E and C) (Lu et al., 2021). Generally, these natural antioxidants, especially polyphenols, exhibit a wide range of cardiovascular effects, such as in both animal models and clinical trials (Zhang et al., 2021). In recent years, several studies have explored the antiatherosclerotic effects of natural products in New Zealand rabbits and have associated polyphenolic compounds as potential agents responsible for the cardioprotective effects. In our study, we identified a large number of polyphenolic compounds in EEPC, including organic acids, phenolic acid derivatives, flavonoids, anthocyanins, and hydrolysable tannins. These findings indicate that these compounds are likely to contribute to the presented antiatherosclerotic response. However, it would be highly speculative to attribute the antiatherosclerotic activity of EEPC solely to one compound, like a flavonoid. We believe that the cardioprotective effects occur due to a synergistic and coordinated activity of different polyphenolic molecules, which contribute directly to the observed dose-response effect.
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