Elangovan Vellaichamy

Quantification of the overall REACTIVE OXYGEN SPECIES scavenging capacity of biological fluids and tissues

A method has been developed for measuring and evaluating the overall antioxidant activity derived from the low-molecular weight antioxidants (scavengers). The principle governing this method is based on a common chemical characteristic of the scavengers, their reducing properties. It was hypothesized and then demonstrated that an evaluation of the overall reducing power of a biological sample correlates with the overall scavenging activity of the sample. In order to quantify the total reducing power, the cyclic voltammetry methodology was applied. The resulting measurements correlated with the antioxidant activity of both hydrophilic and lipophilic scavengers. The method is suitable for use in biological fluids and in tissue homogenates, and can supply information concerning the type of antioxidants and their total concentration without having to determine specific compounds. A noninvasive procedure for determining skin overall scavenging activity is also described. This method is based on a well containing an extraction solution that is attached to the skins surface. Following incubation time the extraction solution is analyzed using the cyclic voltammeter instrument and other methods. We have found these methods suitable for evaluating the reducing capacity status in various clinical conditions such as diabetes, ionizing and nonionizing irradiation, brain degenerative diseases, head trauma, and inflammatory bowel diseases. This method is also an efficient tool for evaluating the overall antioxidant capacity of mixtures of antioxidant preparations in vitro. The measurements themselves are simple and rapid. Furthermore, they do not require manipulation of the samples.

Involvement of the NF-κB/Matrix Metalloproteinase Pathway in Cardiac Fibrosis of Mice Lacking Guanylyl Cyclase/Natriuretic Peptide Receptor A

Mice carrying a targeted disruption of the Npr1 gene (coding for guanylyl cyclase/natriuretic peptide receptor A (NPRA)) exhibit increased blood pressure, cardiac hypertrophy, and congestive heart failure, similar to untreated human hypertensive patients. The objective of this study was to determine whether permanent ablation of NPRA signaling in mice alters the expression of matrix metalloproteinase (MMP)-2 and MMP-9 and pro-inflammatory mediators such as tumor necrosis factor-α (TNF-α), leading to myocardial collagen remodeling. Here, we report that expression levels of the MMP-2 and MMP-9 genes were increased by 3-5-fold and that the expression of the TNF-α gene was enhanced by 8-fold in Npr1 homozygous null mutant (Npr1-/-) mouse hearts compared with wild-type (Npr1+/+) control mouse hearts. Myocardial fibrosis, total collagen, and the collagen type I/III ratio (p < 0.01) were dramatically increased in adult Npr1-/- mice compared with age-matched wild-type counterparts. Hypertrophic marker genes, including the β-myosin heavy chain and transforming growth factor-β1, were significantly up-regulated (3-5-fold) in both young and adult Npr1-/- mouse hearts. NF-κB binding activity in ventricular tissues was enhanced by 4-fold with increased translocation of the p65 subunit from the cytoplasmic to nuclear fraction in Npr1-/- mice. Our results show that reduced NPRA signaling activates MMP, transforming growth factor-β1, and TNF-α expression in Npr1-/- mouse hearts. The findings of this study demonstrate that disruption of NPRA/cGMP signaling promotes hypertrophic growth and extracellular matrix remodeling, leading to the development of cardiac hypertrophy, myocardial fibrosis, and congestive heart failure.

Role of Myocytes in Myocardial Collagen Production

Excessive collagen deposition may cause abnormal stiffness of the heart during hypertrophy and heart failure. The potent vasoconstrictor angiotensin II seems, via an unknown mechanism, to stimulate collagen production. This study describes the in vitro and ex vivo effects of [Sar1]Ang II on collagen production by fibroblasts in culture and in beating, nonworking heart preparations. The effects of [Sar1]Ang II on isolated rat hearts or rat heart fibroblasts were determined by quantifying transcript levels of collagen phenotypes I and III through videodensitometry after Northern blot analysis with specific cDNA probes (collagen [P &agr;2r2] rat &agr;2[I] probe for type I and human skin fibroblast &agr;1[III] probe for type III). When [Sar1]Ang II was added in vitro to neonatal or adult 28-week-old Wistar-Kyoto rat heart fibroblasts, questionable stimulation in the mRNAs of types I and III occurred. In contrast, when 10−8 mol/L [Sar1]Ang II was added to beating, nonworking Wistar-Kyoto rat heart preparation ex vivo, a 1.5- to 2.5-fold stimulation of collagen mRNAs of phenotypes I and III was observed. When neonatal fibroblasts were cocultured with neonatal myocytes in vitro, with 10−10 mol/L [Sar1]Ang II added, there was no stimulation of either phenotype. However, significant stimulation of both collagen transcripts was recorded when 10−10 mol/L [Sar1]Ang II was added to adult fibroblasts cocultured with either neonatal or adult myocytes. Our data suggest that factors produced by myocytes are necessary for upregulation of collagen genes in vitro and demonstrate that fibroblast-myocyte cross-talk is required for Ang II–induced collagen upregulation.

Rapid synthesis of biocompatible silver nanoparticles using aqueous extract of Rosa damascena petals and evaluation of their anticancer activity

OBJECTIVE To optimize the process parameters involved in the green synthesis of silver nanoparticles (G-SNPs) by aqueous extract of Rosa damascena petals and to evaluate the biocompatibility and anti cancer activity of the synthesized silver nanoparticles against human lung adenocarcinoma (A549). METHODS The process variables that include concentration of extract, mixing ratio of reactants, silver salt concentration and interaction time were analyzed. The compatibility of the G-SNPs was verified by incubating with erythrocytes and the anticancer property of the G-SNPs against A549 cells was performed by MTT assay. RESULTS Formation of G-SNPs was confirmed by the visual change in the colour of the reaction mixture from pale yellow to brown yellow. Surface plasmon resonance of synthesized G-SNPs was observed at 420 nm; the size of G-SNPs were analyzed by DLS and found to be in the range of (84.00±10.08) nm. Field emission scanning electron microscope and high resolution transmission electron microscopy analysis confirmed that the G-SNPs were fairly spherical. Fourier transform infrared spectroscopy spectroscopy and X-ray diffraction revealed the characteristic peaks of G-SNPs. Energy dispersive X-ray analysis showed a signal of silver around 3 keV. The synthesized G-SNPs exhibited anticancer activity as evidenced by the MTT assay. IC50 value of G-SNPs was found to be 80 μg/mL. CONCLUSION The results of the present study suggest that G-SNPs can be synthesized rapidly within first minute of the reaction; they are biocompatible and possess anticancer activity against human lung adenocarcinoma.

Enhanced activation of pro-inflammatory cytokines in mice lacking natriuretic peptide receptor-A.

Natriuretic peptide receptor-A (NPRA) is the principal receptor for the cardiac hormones ANP and BNP. Mice lacking NPRA develop progressive cardiac hypertrophy and congestive heart failure. However, the mechanisms responsible for hypertrophic growth in the absence of NPRA signaling are not yet known. In the present study, we determined whether deficiency of NPRA/cGMP signaling alters the cardiac pro-inflammatory cytokines gene expression in Npr1 (coding for NPRA) gene-knockout (Npr1(-/-)) mice exhibiting cardiac hypertrophy and fibrosis as compared with control wild-type (Npr1(+/+)) mice. A significant up-regulation of cytokine genes such as TNF-alpha (five-fold), IL-6 (three-fold) and TGF-beta1 (four-fold) were observed in mutant mice hearts lacking NPRA as compared with the age-matched wild-type mice. In parallel, NF-kappaB binding activity was almost five-fold greater in the nuclear extract of Npr1(-/-) mutant mice hearts as compared with wild-type Npr1(+/+) mice hearts. Guanylyl cyclase (GC) activity and cGMP levels were drastically reduced by 10- and 5-fold, respectively, in ventricular tissues of mutant mice hearts relative to wild-type controls. The present findings provide direct evidence that ablation of NPRA/cGMP signaling activates inflammatory cytokines, probably via NF-kappaB mediated signaling pathway, and is associated with hypertrophic growth of null mutant mice hearts.

Topical application of Gallic acid suppresses the 7,12-DMBA/Croton oil induced two-step skin carcinogenesis by modulating anti-oxidants and MMP-2/MMP-9 in Swiss albino mice

Gallic acid (GA - 3,4,5-trihydroxybenzoic acid), a dietary anti-oxidant has been shown to inhibit cancer cell growth in in vitro. Herein, we investigated the in vivo chemo preventive activity of GA on 7,12-Dimethylbenz[a]anthracene (DMBA)/Croton oil induced two-step skin carcinogenesis in Swiss albino mice. Skin tumor incidence and tumor volume were recorded during the 16 weeks of experimental period. In addition, LDH-isozyme shift, skin collagen content, activities of matrix metalloproteinases (MMP-2/MMP-9) enzymes and enzymatic and non-enzymatic antioxidant were studied in the skin and serum of experimental mice. Tumor incidence was significantly increased in the DMBA/Croton oil induced mice (100%; p<0.001) when compared to GA co-treated mice (60%; p<0.01) and 5-FU treated mice (50%; p<0.01). Skin collagen content, MMPs activities, LDH-isoenzymes and MMP-2/-9 expressions were increased in DMBA/Croton oil induced skin while decreased levels of enzymatic (GST, SOD, CAT & GPx) and non-enzymatic anti-oxidant (GSH) were noticed. On the other hand, GA co-treatment exhibited a significant protection by reverting back the altered levels of LDH-isoenzymes, antioxidants, collagen and MMP-2/MMP-9 activities. The results of this study indicate that topical application of GA inhibits DMBA/Croton oil induced two-stage skin carcinogenic process by modulating the antioxidants and MMPs (-2 & -9) in the mouse skin.

Evaluation of hemocompatibility and in vitro immersion on microwave-assisted hydroxyapatite–alumina nanocomposites

This study reports the microwave-assisted synthesis and characterization of nHAp (nano-hydroxyapatite)-alumina composites. The crystalline phase and interaction of alumina with nHAp was analyzed using X-ray diffraction (XRD) and Raman microscopy analysis, respectively. High resolution transmission electron microscopy (HRTEM) micrographs exhibit morphological changes of nHAp composites with increasing alumina concentrations. Microhardness studies reveal the enhanced mechanical strength of nHAp10 and nHAp20 nanocomposites than pure nHAp. In vitro bioactivity of the nanocomposites was studied by immersing samples in simulated body fluid (Hanks solution) for 21 days. The surface of biomineralized samples were analyzed using field emission scanning electron microscopy (FESEM) and energy dispersive X-ray spectroscopy (EDX). Hemolytic assay revealed acceptable compatibility for varying concentrations of all the samples. Cell proliferation assay was systematically investigated for 1 day and 3 days on Saos-2 osteoblast-like cell lines and it was found that nHAp nanocomposites improved the proliferation.

Genetically altered mutant mouse models of guanylyl cyclase/natriuretic peptide receptor-A exhibit the cardiac expression of proinflammatory mediators in a gene-dose-dependent manner

The objective of this study was to examine whether genetically determined differences in the guanylyl cyclase/natriuretic peptide receptor-A gene (Npr1) affect cardiac expression of proinflammatory cytokines, hypertrophic markers, nuclear factor-κB (NF-κB), and activating protein-1 (AP-1) in am Npr1 gene-dose-dependent manner. In the present studies, adult male Npr1 gene-disrupted (Npr1(-/-)), wild-type (Npr1(+/+)), and gene-duplicated (Npr1(++/++)) mice were used. The Npr1(-/-) mice showed 41 mm Hg higher systolic blood pressure and 60% greater heart weight to body weight (HW/BW) ratio; however, Npr1(++/++) mice exhibited 15 mm Hg lower systolic blood pressure and 12% reduced HW/BW ratio compared with Npr1(+/+) mice. Significant upregulation of gene expression of proinflammatory cytokines and hypertrophic markers along with enhanced NF-κB/AP-1 binding activities were observed in the Npr1(-/-) mouse hearts. Conversely, hypertrophic markers and proinflammatory cytokines gene expression as well as NF-κB/AP-1 binding activities were markedly decreased in Npr1(++/++) mouse hearts compared with wild-type mice. The ventricular guanylyl cyclase activity and cGMP levels were reduced by 96% and 87%, respectively, in Npr1(-/-) mice; however, these parameters were amplified by 2.8-fold and 3.8-fold, respectively, in Npr1(++/++) mice. Echocardiographic analysis revealed significantly increased fractional shortening in Npr1(++/++) mice (P < .05) but greatly decreased in Npr1(-/-) mice (P < .01) hearts compared with Npr1(+/+) mice. The present findings suggest that Npr1 represses the expression of cardiac proinflammatory mediators, hypertrophic markers, and NF-κB/AP-1-mediated mechanisms, which seem to be associated in an Npr1 gene-dose-dependent manner.

Atrial natriuretic peptide (ANP) inhibits DMBA/croton oil induced skin tumor growth by modulating NF-κB, MMPs, and infiltrating mast cells in swiss albino mice.

Cardiac hormone atrial natriuretic peptide (ANP) and its receptor, natriuretic peptide receptor-A (NPR-A) are implicated as a vital regulator of cancer cell growth and tumor progression. However, the underlying mechanism by which ANP opposes the cancer growth in in-vivo remains unknown. Herein, we investigated the anti-cancer activity of ANP on 7, 12-dimethyl benzanthracence (DMBA)/Croton oil- induced two-step skin carcinogenic mouse model. Skin tumor incidence and tumor volume were recorded during the experimental period of 16 weeks. ANP (1 μg/kg body weight/alternate days for 4 weeks) was injected subcutaneously from the 13th week of DMBA/Croton oil induction. ANP treatment markedly inhibited the skin tumor growth (P<0.001). A significant reduction in the level of NF-κB activation (P<0.001), infiltrating mast cell count (P<0.01) and MMP-2/-9 (P<0.001, respectively) were noticed in the ANP treated mice skin tissue. Further, ANP treatment revert back the altered levels of serum LDH-4, C-reactive protein (CRP), and enzymatic antioxidants (SOD and CAT activities) to near normal level. Taken together, the results of this study suggest that ANP opposes the skin carcinogenesis by suppressing the inflammatory response and MMPs.

RETRACTED: Anti-fibrotic cardio protective efficacy of aminoguanidine against streptozotocin induced cardiac fibrosis and high glucose induced collagen up regulation in cardiac fibroblasts

This study mainly focuses on cardio protective anti-fibrotic activity of aminoguanidine against streptozotocin induced cardiac fibrosis and high glucose induced collagen accumulation in cardiac fibroblasts. Dysregulation of matrix metalloproteinase especially 2 and 9 were considered to be responsible for the abnormal collagen deposition, which resulting improper cardiac contractile function in diabetic mice. Mice received a single dose of streptozotocin (100 mg/kg) through tail vein to induce diabetes. Normal and diabetic mice received aminoguanidine orally (100 mg/kg/day) throughout the study period of 8 weeks. Cardiac fibroblasts cultured and exposed to high glucose, aminoguanidine and both for 48 h. Collagen quantitatively estimated in both in vivo and in vitro models. Altered structural changes were studied using the Masson tri-chrome staining, TEM images of cardiac sections. Increased collagen and metalloproteinase activities were confirmed using gelatin zymography, western blotting and gene expression studies. The exact mechanism responsible for high glucose induced collagen up regulation in diabetic heart was incompletely understood. From this above in vivo and in vitro results, we conclude that, the cardio protective anti fibrotic activity of amino guanidine was mainly attributed by exhibiting the inhibitory efficacy against streptozotocin and high glucose induced collagen accumulation probably by inhibiting high glucose altered metalloproteinase-2 and -9 activities.