Chinnasamy Thirunavukkarasu

The extra cellular synthesis of gold and silver nanoparticles and their free radical scavenging and antibacterial properties

The bio reduction of chloro auric acid (HAuCl(4)) and silver nitrate (AgNO(3)) is achieved extracellularly by using the aqueous extract of Solanum torvum (S. torvum) fruit. The nanoparticle formation was screened by UV-visible spectroscopy through color conversion due to surface plasma resonance bands at 560 nm and 430 nm for gold and silver nanoparticles respectively. The spherical shapes with smooth surface of gold and silver nanoparticles were analyzed through scanning electron microscope and its presence was confirmed by energy dispersive X-ray spectroscopy (SEM/EDX). The functional groups in the gold and silver salts and the bio interactive functional groups present in the S. torvum extract were characterized by employing Fourier transform infra-red spectroscopy (FTIR). The biomedical properties of gold and silver nanoparticles were premeditated as free radical scavenging activity and antibacterial static agents. Gold and silver nanoparticles serve as strong hydroxyl, superoxide, nitric oxide and DPPH radical scavengers in contrast to their corresponding metal oxides. The radical quenching properties of gold and silver nanoparticles were found to correlate with in vitro DNA protective effect. The silver nanoparticles show strong zone of inhibition against Escherichia coli, Pseudomonas and Bacillus whereas, gold nanoparticles exhibit fair zone of inhibition. To our knowledge this is the first report that S. torvum extract can reduce metal acids to nano materials.

Mechanisms of endotoxin‐induced NO, IL‐6, and TNF‐α production in activated rat hepatic stellate cells: Role of p38 MAPK

Compelling experimental evidence indicates that the interactions between endotoxin and hepatic stellate cells (HSCs) can play a significant role in the pathogenesis of liver disease. Endotoxin‐induced release of a multifunctional mediator NO (via inducible NO synthase) and the proinflammatory cytokines tumor necrosis factor α (TNF‐α) and interleukin (IL)‐6 by HSCs could be an important mechanism of pathological changes in the liver. However, the signaling mechanisms of these effects are poorly understood. In this study, we found that endotoxin causes activation of mitogen‐activated protein kinases (MAPKs) (extracellular signal‐regulated protein kinase [ERK] 1 and 2, p38, and c‐Jun NH2‐terminal kinase [JNK]) and nuclear factor κB (NF‐κB) and production of H2O2 in culture‐activated HSCs. However, only p38 and NF‐κB were found to be responsible for the synthesis of NO, IL‐6, and TNF‐α. Exogenous H2O2 caused modest stimulation of TNF‐α synthesis, did not affect the synthesis of NO or IL‐6, and did not activate NF‐κB or MAPKs. Inhibition of p38 and NF‐κB activation by SB203580 and pyrrolidine dithiocarbamate, respectively, blocked endotoxin‐induced H2O2, NO, TNF‐α, and IL‐6 synthesis. Inhibition of ERK1/2 and JNK phosphorylation did not alter these effects of endotoxin. Whereas SB203580 inhibited endotoxin‐induced NF‐κB activation, pyrrolidine dithiocarbamate did not affect p38 phosphorylation in endotoxin‐stimulated cells. In conclusion, endotoxin‐induced synthesis of NO, TNF‐α, and IL‐6 in HSCs is mediated by p38 and NF‐κB, with involvement of H2O2 in TNF‐α production. (HEPATOLOGY 2006;44:389–398.)

Augmenter of liver regeneration: An important intracellular survival factor for hepatocytes

BACKGROUND/AIMS Augmenter of liver regeneration (ALR), a protein synthesized and stored in hepatocytes, is associated with mitochondria, and possesses sulfhydryl oxidase and cytochrome c reductase activities. We sought to determine the effects of ALR depletion in hepatocytes by antisense oligonucleotide transfection. METHODS Rat hepatocytes in primary culture were transfected with antisense oligonucleotide for ALR mRNA (ALR-AS) or scrambled oligonucleotide. Various analyses were performed at times up to 24h after transfection. RESULTS Treatment with ALR-AS caused a decrease in ALR mRNA, cellular depletion of ALR protein primarily from mitochondria, and decreased viability. Flow cytometric analysis of ALR-AS-transfected hepatocytes stained with annexin-Vcy3 and 7-aminoactinomycin D revealed apoptosis as the predominant cause of death up to 6h; incubation beyond this time resulted in necrosis in addition to apoptosis. ALR-AS-transfection caused release of mitochondrial cytochrome c, activation of caspase-3, profound reduction in the ATP content, and cellular release of LDH. Inhibition of caspase-3 inhibited the early phase of ALR-AS-induced death but not the late phase that included ALR and LDH release. CONCLUSIONS These results suggest that ALR is critically important for the survival of hepatocytes by its association with mitochondria and regulation of ATP synthesis.

Normal rat hepatic stellate cells respond to endotoxin in LBP-independent manner to produce inhibitor(s) of DNA synthesis in hepatocytes†

Endotoxin is implicated in the pathology of acute liver failure. The mechanisms of its actions on quiescent hepatic stellate cells (qHSCs) and their implications in hepatocyte injury are incompletely understood. We investigated effects of endotoxin (bacterial lipopolysaccharide; LPS) on qHSCs and subsequently on hepatocytes. After overnight culture following their isolation, qHSCs were incubated with or without endotoxin for 24 h. The cells and the culture supernatant were analyzed for cytokines and nitric oxide (NO) synthesis. The effects of qHSC‐conditioned media on hepatocytes were then determined. LPS increased inducible NO synthase expression, stimulated NO synthesis, and inhibited DNA synthesis in qHSCs. qHSC‐conditioned medium inhibited DNA synthesis in hepatocytes without affecting NO synthesis, while LPS (1–1,000 ng/ml)‐conditioned qHSC medium stimulated NO synthesis and caused further inhibition of DNA synthesis and apoptosis. These effects of LPS were more pronounced when qHSCs were incubated with serum, but not with LPS‐binding protein (LBP) although CD14 (a receptor for LPS‐LBP complex) was found in qHSCs. LPS stimulated the synthesis of TNF‐α, interleukin (IL)‐6, and IL‐1β but not of TGF‐β in qHSCs. Individually or together, L‐NG‐monomethylarginine and antibodies to IL‐1β, IL‐6, and TNF‐α only partly reversed qHSC + LPS‐conditioned medium‐induced inhibition of DNA synthesis in hepatocytes. These results suggest that the effects of LPS on qHSCs are novel, occurring without the aid of LBP/CD14. They also indicate that other factors, in addition to NO, TGF‐β, TNF‐α, IL‐1β, and IL‐6 are involved in the mechanisms of the growth inhibitory effects of qHSCs on hepatocytes.

Crocin, a dietary colorant, mitigates cyclophosphamide-induced organ toxicity by modulating antioxidant status and inflammatory cytokines.

This study investigated the protective efficacy of crocin against hepatotoxicity induced by cyclophosphamide (CP) in Wistar rats.

p38-MAPK- and Caspase-3-Mediated Superoxide-Induced Apoptosis of Rat Hepatic Stellate Cells: Reversal by Retinoic Acid

Reactive oxygen species (ROS) activate retinoid‐containing quiescent hepatic stellate cells (qHSCs) to retinoid‐deficient fibrogenic myofibroblast‐like cells (aHSCs). However, ROS also cause apoptosis of aHSCs, and apoptotic aHSCs are observed in inflammatory fibrotic liver. Here, we investigated mechanisms of the effects of oxidative stress on the survival of qHSCs and aHSCs. HSCs from normal rat liver were used after overnight culture (qHSCs), or in 3–5 passages (aHSCs). For in vivo induction of oxidative stress, tert‐butylhydroperoxide was injected into control and CCl4‐induced cirrhotic rats. Spontaneous caspase‐3 activation and apoptosis, observed in cultured qHSCs, decreased with time and were unaffected by superoxide. In contrast, superoxide caused caspase‐3 and p38‐MAPK activation, reduction in Bcl‐xL expression, and apoptosis in aHSCs. Inhibition of caspase‐3 and p38‐MAPK did not affect the viability of qHSCs in the absence or presence of superoxide, but inhibited superoxide‐induced death of aHSCs. Glutathione (GSH) level and activities of superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) were lower in aHSCs than qHSCs. Superoxide increased GSH content, and activities of SOD, catalase and GPx in qHSCs but not in aHSCs. Incubation of 13‐cis‐retinoic acid (RA)‐treated aHSCs with superoxide increased their GSH content significantly, and prevented superoxide‐induced p38‐MAPK and caspase‐3 activation while dramatically reducing the extent of apoptosis. Finally, oxidative stress induced in vivo caused apoptosis of aHSCs in cirrhotic but not of qHSCs in control rats. These results suggest that the absence of retinoids render aHSCs susceptible to superoxide‐induced apoptosis via caspase‐3 and p38‐MAPK activation. J. Cell. Physiol. 218: 157–166, 2009.

Endothelin receptor antagonist TAK-044 arrests and reverses the development of carbon tetrachloride induced cirrhosis in rats

Background and aims: Hepatic concentrations of the powerful vasoconstrictor and fibrogen endothelin 1 (ET-1) and its receptors increase in human and experimental cirrhosis, suggesting a major role for ET-1 in the pathology of chronic liver disease. We investigated whether ET-1 receptor antagonism, after the development of fibrosis and cirrhosis, arrests/reverses the progression of chronic liver disease. Methods: Chronic liver injury was induced in rats by carbon tetrachloride (CCl4) treatment (0.15 ml/kg intraperitoneally twice a week) in conjunction with phenobarbital in drinking water (0.4 g/l) for four (group 1: fibrosis) and eight (group 2: cirrhosis) weeks. Rat were then treated concurrently with the ET-1 receptor antagonist TAK-044 (10 mg/kg/day) and CCl4/phenobarbital for a further four weeks. Results: Histopathological examination revealed significant arrest of progression to cirrhosis in group 1 and reversal of cirrhosis in group 2 rats. TAK-044 treatment caused significant amelioration of portal hypertension, systemic hypotension, and liver injury (reduced activities of serum aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase), and improved hepatic synthetic capacity (increased serum albumin concentration) in both groups of rats relative to vehicle treated rats. TAK-044 treatment reduced collagen synthesis, as evidenced by decreased hepatic hydroxyproline content, mRNA expression of collagen-α type I, and tissue inhibitors of matrix metalloproteinases 1 and 2, and mRNA and protein expression of a potent fibrogenic cytokine, transforming growth factor β1. Conclusions: The results emphasise the role of ET-1 in the development of cirrhosis and strongly suggest that blockade of its actions can be a rational therapy for chronic liver disease and its complications.

Spectroscopic and molecular docking studies on the interaction of troxerutin with DNA.

Troxerutin (TXER) is a derivative of naturally occurring bioflavonoid rutin. It possesses different biological activities in rising clinical world. The biological activity possessed by most of the drugs mainly targets on macromolecules. Hence, in the current study we have examined the interaction mechanism of TXER with calf thymus DNA (CT-DNA) by using various spectroscopic methods, isothermal titration calorimetry (ITC) and molecular docking studies. Further, DNA cleavage study was carried out to find the DNA protection activity of TXER. UV-absorption and emission spectroscopy showed low binding constant values via groove binding. Circular dichroism study indicates that TXER does not modify native B-form of DNA, and it retains the native B-conformation. Furthermore, no effective positive potential peak shift was observed in TXER-DNA complex during electrochemical analysis by which it represents an interaction of TXER with DNA through groove binding. Molecular docking study showed thymine guanine based interaction with docking score -7.09 kcal/mol. This result was compared to experimental ITC value. The DNA cleavage study illustrates that TXER does not cause any DNA damage as well as TXER showed DNA protection against hydroxyl radical induced DNA damage. From this study, we conclude that TXER interacts with DNA by fashion of groove binding.

Unconjugated bilirubin exerts pro-apoptotic effect on platelets via p38-MAPK activation

Thrombocytopenia is one of the most frequently observed secondary complications in many pathological conditions including liver diseases, where hyperbilirubinemia is very common. The present study sought to find the cause of thrombocytopenia in unconjugated hyperbilirubinemic conditions. Unconjugated bilirubin (UCB), an end-product of heme catabolism, is known to have pro-oxidative and cytotoxic effects at high serum concentration. We investigated the molecular mechanism underlying the pro-apoptotic effect of UCB on human platelets in vitro, and followed it up with studies in phenylhydrazine-induced hyperbilirubinemic rat model and hyperbilirubinemic human subjects. UCB is indeed found to significantly induce platelet apoptotic events including elevated endogenous reactive oxygen species generation, mitochondrial membrane depolarization, increased intracellular calcium levels, cardiolipin peroxidation and phosphatidylserine externalization (p < 0.001) as evident by FACS analysis. The immunoblots show the elevated levels of cytosolic cytochrome c and caspase activation in UCB-treated platelets. Further, UCB is found to induce mitochondrial ROS generation leading to p38 activation, followed by downstream activation of p53, ultimately resulting in altered expression of Bcl-2 and Bax proteins as evident from immunoblotting. All these parameters conclude that elevated unconjugated bilirubin causes thrombocytopenia by stimulating platelet apoptosis via mitochondrial ROS-induced p38 and p53 activation.

Synthesis and biological evaluation of isoindoloisoquinolinone, pyroloisoquinolinone and benzoquinazolinone derivatives as poly(ADP-ribose) polymerase-1 inhibitors.

A series of novel fused isoquinolinones with isoindoloisoquinolinone, pyroloisoquinolinone, and benzoquinalizinone skeletons were synthesized from corresponding phenethylimides. The isoquinolinone derivatives were evaluated for their protective effect on chicken erythrocytes subjected to oxidative damage. The effect of isoquinolinone derivatives were analysed by estimation of cell viability, antioxidant enzyme activities, DNA damage (comet assay), PARP-1 inhibition assay and molecular docking of the compounds with PARP-1 active site. The compounds CRR-271, CRR-288 and CRR-224+225 showed significant protective effect at 100 μM concentration. The PARP-1 inhibition assay revealed the IC50 values of CRR-271, CRR-288 and CRR-224+225 as <200 nM, further molecular docking studies shows higher binding energies with PARP-1 active site. Interesting findings in this study suggest that the novel isoquinolinone derivatives inhibit PARP-1 activity and protect cells against oxidative DNA damage, which could be implemented in the treatment of inflammatory diseases.