Attenuation of cadmium-induced vascular toxicity by pro-angiogenic nanorods.

Cadmium (Cd) is a common heavy metal that causes major environmental pollution with adverse effects on human health and well-being. Exposure to Cd is known to exhibit detrimental consequences on all the vital organ systems of the body, especially the vascular system. Certain approaches using anti-oxidants and chelating agents have been demonstrated previously to mitigate Cd-induced toxicity. However, these approaches are associated with their own limitations. In this context, there is a critical need for the development of alternative treatment strategies to address the conditions associated with Cd-poisoning. One such novel approach is the application of nanomedicine which is well-known to resolve several health complications by improving disease therapy. Recently, our group demonstrated the role of europium hydroxide nanorods (EHN) in promoting vascular growth using in vitro and in vivo assay systems. Therefore, in the present study, we have evaluated the effect of EHN on health of endothelial cells (EA.hy926) and fibroblasts (NIH 3T3) intoxicated by Cd. The results revealed that EHN significantly improved the viability of EA.hy926 and NIH 3T3 cells, reduced apoptotic cell population, increased nitric oxide (NO) production and promoted blood vasculature development in the chick embryo model, which were hampered due to Cd insult. Molecular studies demonstrated the reduced expression of tumor suppressor (p53) and elevated anti-apoptotic protein (Bcl-xL) levels along with enhanced NO production through endothelial nitric oxide synthase (eNOS) activation as the plausible mechanisms underlying protective role of EHN against Cd-induced vascular toxicity. Considering the above observations, we strongly believe that EHN could be a potential nanomedicine approach for overcoming Cd-induced toxicity by improving vascular health and functioning.

Gymnema montanum improves endothelial function via inhibition of endoplasmic reticulum stress by activating Nrf2 signaling

Objective: To investigate the effects of Gymnema montanum leaf extract against endoplasmic reticulum (ER) stress-induced toxicity in endothelial cells.Methods: The immortalized endothelial hybrid cell, EA.hy926 was treated with different concentrations of Gymnema montanum leaf extract (0-100 μg/mL) and the ER stress inducer, tunicamycin. The cytotoxicity was assessed by MTT as well as lactate dehydrogenase and malondialdehyde levels were determined. The levels of ER stress markers, GRP78 and CHOP were analysed by Western blot assay. The Gymnema montanum leaf extract-mediated activation of nuclear factor erythroid 2-related factor 2 (Nrf2) was assessed by cell-based luciferase enzyme fragment complementation assay and antioxidant responsive element driven luciferase reporter assay. The levels of phosphoproteins of the MAPK pathway were analyzed using the Bioplex system. Results: A dose-dependent cytoprotective effect of Gymnema montanum leaf extract was observed in tunicamycin-induced toxicity. Gymnema montanum leaf extract significantly reduced lactate dehydrogenase activity and malondialdehyde levels in ER stress-induced endothelial cells. It also suppressed ER stress markers dose dependently and inhibited the phosphorylation of JNK, ERK, MEK and p38 MAPK in tunicamycin-induced endothelial cells. Moreover, Gymnema montanum leaf extract increased the expression of Nrf2 and its downstream targets in endothelial cells. Conclusions: Gymnema montanum leaf extract attenuates ER stress by increasing the expression of Nrf2 and its downstream genes.

Green synthesis of copper oxide nanoparticles using sinapic acid: an underpinning step towards antiangiogenic therapy for breast cancer.

Synthesis of copper oxide nanoparticles without any chemical reductant is always a challenging methodology for biological studies. In this study, sinapic acid, a phytochemical, is used for the synthesis of stable copper oxide nanoparticles. The as-synthesized nanoparticles were characterized thoroughly using UV-Visible, IR spectroscopy, Transmission Electron Microscopy (TEM) and X-ray photoelectron spectroscopy (XPS). Nanoparticles collected during different time intervals of synthesis (60,120 and 180 min) were subjected for analysis, where the occurrence of copper oxide nanoparticles with substantial morphology was seen at 180 min. Further, nanoparticles synthesized at 120 and 180 min were studied for their potential biological applications. These copper oxide nanoparticles evinced potential cytotoxic effects on breast cancer cells, MCF7 and MDA-MB231. Supplementarily, it also exhibited anti-angiogenic effect on endothelial cells (EA.hy926), thus confirming its potential to inhibit angiogenesis in cancer.

Shielding the Negative Impact of Cadmium in Avian Embryos by Supplementing with Beetroot Juice.

The increased correlation between maternal cadmium (Cd) exposure and restricted fetal growth has become a matter of global concern. Because dietary nitrate has been demonstrated to offer a range of beneficial vascular effects, we attempt to unveil the effect of one such nitrate-rich food, beetroot, on recovering Cd-induced fetal growth retardation. Using chick embryos as a model, our results confirm that retarded growth is a result of exposure to Cd at an early stage of development and that Cd hinders vasculogenesis and angiogenesis. We find that beetroot juice (BRJ) recovers fetal growth retardation effects of Cd via its vasodilatory effect and promotes embryonic angiogenesis. In conclusion, this study confirms that BRJ reduces the rate of congenital malformations with Cd exposure in utero and suggests the importance of dietary supplements for pregnant women.

Establishment of pancreatic microenvironment model of ER stress: Quercetin attenuates ß-cell apoptosis by invoking nitric oxide-cGMP signaling in endothelial cells.

The involvement of endoplasmic reticulum (ER) stress in endothelial dysfunction and diabetes-associated complications has been well documented. Inhibition of ER stress represents a promising therapeutic strategy to attenuate endothelial dysfunction in diabetes. Recent attention has focused on the development of small molecule inhibitors of ER stress to maintain endothelial homeostasis in diabetes. Here we have developed a reliable, robust co-culture system that allows a study on the endothelial cells and pancreatic ß-cells crosstalk under ER stress and validated using a known ER stress modulator, quercetin. Furthermore, sensitizing of endothelial cells by quercetin (25 µM) confers protection of pancreatic ß-cells against ER stress through nitric oxide (NO∙) signaling. In addition, increased intracellular insulin and NO∙-mediated cyclic 3',5'-guanosine monophosphate (cGMP) levels in pancreatic ß-cells further confirmed the mechanism of protection under co-culture system. In addition, the potential protein targets of quercetin against ER stress in the endothelial cells were investigated through proteomic profiling and its phosphoprotein targets through Bioplex analysis. On the whole, the developed in vitro co-culture set up can serve as a platform to study the signaling network between the endothelial and pancreatic ß-cells as well as provides a mechanistic insight for the validation of novel ER stress modulators.

Quercetin improves endothelial function in diabetic rats through inhibition of endoplasmic reticulum stress-mediated oxidative stress.

Endoplasmic reticulum (ER) stress attributes a crucial role in diabetes-induced endothelial dysfunction. The present study investigated the effects of quercetin, a potent antioxidant on the attenuation of ER stress-modulated endothelial dysfunction in streptozotocin (STZ)-induced diabetic rats. Oral administration of quercetin for six weeks to diabetic rats dose-dependently reduced the blood glucose levels and improved insulin secretion. Histopathological examination of pancreatic tissues in diabetic rats showed pathological changes such as shrunken islets, reduction in islet area and distorted ß-cells, which were found to be restored by quercetin treatment. In addition, quercetin reduced the pancreatic ER stress-induced endothelial dysfunction as assessed by immunohistochemical analysis of C/ERB homologous protein (CHOP) and endothelin-1 (ET-1). Moreover, quercetin administration progressively increased the expression of vascular endothelial growth factor (VEGF) and its receptor, VEGFR2 in diabetes rats. Quercetin-mediated decrease in the nitric oxide (NO∙) and cyclic 3',5'- guanosine monophosphate (cGMP) levels were also observed in the diabetic rats. Quercetin treatment reduced the lipid peroxidation in the diabetic rats, meanwhile increased the total antioxidant capacity in the pancreas from diabetic rats. Altogether, these results demonstrated the vasoprotective effect of quercetin against STZ-induced ER stress in the pancreas of diabetic rats.

Carvacrol induces mitochondria-mediated apoptosis in HL-60 promyelocytic and Jurkat T lymphoma cells.

The aim of the present study was to investigate the effect of carvacrol, a phenolic monoterpenoid on the induction of apoptosis in HL-60 (Human acute promyelocytic leukemia cells) and Jurkat (human T lymphocyte cells) cells. Carvacrol showed a potent cytotoxic effect on both cells with dose-dependent increase in the level of free radical formation as measured by an oxidation sensitive fluorescent dye, 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA) levels. The reduction in the level of antioxidants such as catalase (CAT) and superoxide dismutase (SOD) (P<0.05) was observed in carvacrol-treated cells. The major cytotoxic effect appears to be intervened by the induction of apoptotic cell death as assessed by annexin-V labeling assay using flow cytometry. Western blot analysis showed that Bax expression was increased, whereas Bcl-2 expression was significantly decreased in carvacrol exposed HL-60 cells and Jurkat cells. Further studies revealed that the dissipation of mitochondrial membrane potential of intact cells was accompanied by the activation of caspase-3. Our results found that the potential mechanism of cellular apoptosis induced by carvacrol is mediated by caspase-3 and is associated with the collapse of mitochondrial membrane potential, generation of free radicals, and depletion of the intracellular antioxidant pool.

Sinapic acid protects heart against ischemia/reperfusion injury and H9c2 cardiomyoblast cells against oxidative stress.

The present study was designed to evaluate antioxidant and cardioprotective potential of sinapic acid (SA) against ischemia/reperfusion (I/R) injury. Cardiac functional recovery after I/R was evaluated by percentage rate pressure product (%RPP) and percentage coronary flow (%CF). Myocardial injury was evaluated by 2,3,5-triphenyltetrazolium chloride (TTC) staining and LDH enzyme leakage. Oxidative stress was estimated by lipid peroxidation level. eNOS protein expression in reperfused heart was assessed using Western blot method. Finally, in order to support the antioxidant effect of SA, in vitro protective potential of SA was assessed on H2O2-induced oxidative stress in H9c2 cardiomyoblast cells. The overall results demonstrated that I/R induced cardiac dysfunction, injury and oxidative stress was attenuated by SA treatment. Moreover, in vitro results also shown that, SA protects H9c2 cells from oxidative stress and modulates mitochondrial membrane permeability transition (MPT). In conclusion, coupled results from both in vivo and in vitro experiments have confirmed that SA with antioxidant role protects cardiac cells and its functions from I/R induced oxidative stress.

Sinapic acid prevents hypertension and cardiovascular remodeling in pharmacological model of nitric oxide inhibited rats.

OBJECTIVES: Hypertensive heart disease is a constellation of abnormalities that includes cardiac fibrosis in response to elevated blood pressure, systolic and diastolic dysfunction. The present study was undertaken to examine the effect of sinapic acid on high blood pressure and cardiovascular remodeling. METHODS: An experimental hypertensive animal model was induced by L-NAME intake on rats. Sinapic acid (SA) was orally administered at a dose of 10, 20 and 40 mg/kg body weight (b.w.). Blood pressure was measured by tail cuff plethysmography system. Cardiac and vascular function was evaluated by Langendorff isolated heart system and organ bath studies, respectively. Fibrotic remodeling of heart and aorta was assessed by histopathologic analyses. Oxidative stress was measured by biochemical assays. mRNA and protein expressions were assessed by RT-qPCR and western blot, respectively. In order to confirm the protective role of SA on endothelial cells through its antioxidant property, we have utilized the in vitro model of H2O2-induced oxidative stress in EA.hy926 endothelial cells. RESULTS: Rats with hypertension showed elevated blood pressure, declined myocardial performance associated with myocardial hypertrophy and fibrosis, diminished vascular response, nitric oxide (NO) metabolites level, elevated markers of oxidative stress (TBARS, LOOH), ACE activity, depleted antioxidant system (SOD, CAT, GPx, reduced GSH), aberrant expression of TGF-ß, ß-MHC, eNOS mRNAs and eNOS protein. Remarkably, SA attenuated high blood pressure, myocardial, vascular dysfunction, cardiac fibrosis, oxidative stress and ACE activity. Level of NO metabolites, antioxidant system, and altered gene expression were also repaired by SA treatment. Results of in vitro study showed that, SA protects endothelial cells from oxidative stress and enhance the production of NO in a concentration dependent manner. CONCLUSIONS: Taken together, these results suggest that SA may have beneficial role in the treatment of hypertensive heart disease by attenuating fibrosis and oxidative stress through its antioxidant potential.

In vitro evaluation of free radical scavenging activity of Codariocalyx motorius root extract

OBJECTIVE@#To determine the phenolic content in Codariocalyx motorius root extract and to evaluate its antioxidant properties using various in vitro assay systems.@*METHODS@#The antioxidant activity was evaluated based on scavenging of 1,1-diphenyl-2-picrylhydrazyl, hydroxyl radicals, superoxide anions, nitric oxide, hydrogen peroxide, peroxynitrite, reducing power and by inhibition of lipid peroxidation which was estimated in terms of thiobarbituric acid reactive substances.@*RESULTS@#The root extract of the Codariocalyx motorius (C. motorius) exhibited potent total antioxidant activity that increased with increasing amount of extract concentration, which was compared with standard drug such as quercetin, butylated hydroxytoluene, tocopherol at different concentrations. The different concentrations of the extracts showed inhibition on lipid peroxidation. In addition, the extracts had effective reducing power, free radical scavenging, super oxide anion scavenging, nitric oxide scavenging, lipid peroxidation, and total phenolic content depending on concentration. High correlation between total phenolic contents and scavenging potential of different reactive oxygen species (r(2)=0.831-0.978) indicated the polyphenols as the main antioxidants.@*CONCLUSIONS@#Codariocalyx motorius (C. motorius) root possess the highly active antioxidant substance which can be used for the treatment of oxidative stress-related diseases.

Antidiabetic activity of alcoholic stem extract of Gymnema montanum in streptozotocin-induced diabetic rats.

In the present study, the effect of alcoholic stem extract of Gymnema montanum (GMSt) on blood glucose, plasma insulin, and carbohydrate metabolic enzymes were studied in experimental diabetes. Diabetes mellitus was induced by a single intraperitoneal injection of STZ (60 mg/kg bw). Five days after STZ induction, diabetic rats received GMSt orally at the doses of 25, 50, 100 and 200mg/kg daily for 3 weeks. Graded doses of stem extract showed a significant reduction in blood glucose levels and improvement in plasma insulin levels. The effect was more pronounced in 100 and 200mg/kg than 50mg/kg. GMSt showed significant increase in hexokinase, Glucose-6-phosphate dehydrogenase and glycogen content in liver of diabetic rats while there was significant reduction in the levels of glucose-6-phosphatase and fructose-1,6-bisphosphatase. The present study clearly indicated significant antidiabetic effect with the stem extract of G. montanum and lends support for its traditional usage.