S. Niranjali Devaraj

Cardioprotective effect of the alcoholic extract of Terminalia arjuna bark in an in vivo model of myocardial ischemic reperfusion injury

The present study was designed to investigate the effects of chronic administration of the alcoholic extract of Terminalia arjuna (TAAE) bark on isoproterenol induced myocardial injury. The TAAE was administered orally to Wistar albino rats (150-200 g) in three different doses, by gastric gavage [3.4 mg/kg: (T1), 6.75 mg/kg: (T2) and 9.75 mg/kg: (T3)] 6 days/week for 4 weeks. At the end of this period, all the animals, except the normal untreated rats that served as the control group, were administered isoproterenol (ISO) 85 mg/kg, S.C., for two consecutive days to induce in vivo myocardial injury. After 48 hours rats were anaesthetized with anaesthetic ether, then sacrificed and the hearts were harvested for biochemical and histological studies. A significant rise in myocardial thiobarbituric acid reactive substances (TBARS) and loss of reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (suggestive of increased oxidative stress) occurred in the hearts subjected to in vivo myocardial ischemic reperfusion injury. The 6.75 mg/kg TAAE treatment group (baseline) shows a significant increase in myocardial TBARS as well as endogenous antioxidants (GSH, SOD, and catalase), but not in the other treatment groups. In in vivo ischemic reperfusion injury of the TAAE treated rats there was a significant decrease in TBARS in all the groups. In 6.75 mg/kg treatment group, a significant rise in the levels of GSH, SOD and catalase were observed, and it shows better recovery profile than the other groups subjected to in vivo ischemic reperfusion injury. In histological studies, all the groups, except the isoproterenol treated group, showed preserved myocardium. The present study demonstrates that the 6.75 mg/kg TAAE augments endogenous antioxidant compounds of the rat heart and also prevents the myocardium from isoproterenol induced myocardial ischemic reperfusion injury.

Lutein protects HT-29 cells against Deoxynivalenol-induced oxidative stress and apoptosis: Prevention of NF-κB nuclear localization and down regulation of NF-κB and Cyclo-Oxygenase - 2 expression

Increasing evidence suggests that oxidative stress is closely linked to toxic responses in cells. The tricothecene mycotoxin, Deoxynivalenol (DON), primarily affects cells of the immune system and the GI tract. DONs cytotoxicity is closely linked to intracellular ROS, and it exerts its toxic effect by a mechanism known as ribotoxic stress response, which drives both cytokine expressions at low dosages and apoptosis at high dosages. Studies to alleviate DONs toxicity are sparsely reported in literature. In the present study, the cytoprotective effect of lutein, was tested in HT-29 cells against DON-induced oxidative stress and cytotoxicity. MTT assay revealed IC(20) values of DON at 250 ng/ml. Pre-treatment of cells with 10 microM lutein resulted in 95% cell viability. Lutein combated DON-induced oxidative stress and downregulated expression of inflammatory genes, NF-kappaB and COX-2. Lutein also prevented DON-induced migration of NF-kappaB into the nucleus, as measured by immunofluorescence. Morphological studies by Electron microscopy and Cell cycle analysis by flow cytometry indicated that lutein prevented DON-induced apoptosis. The results of the present study demonstrate for the first time that lutein exerts a cytoprotective role in DON-induced toxicity.

Effect of tincture of Crataegus on the LDL-receptor activity of hepatic plasma membrane of rats fed an atherogenic diet

Tincture of Crataegus, (TCR), is a hypocholesterolemic and antiatherosclerotic drug made from berries of hawthorn, Crataegus oxyacantha. Its main constituents are flavonoids, triterpene saponins and a few cardioactive amines. TCR, when administered simultaneously to rats fed an atherogenic diet, significantly increased the binding of 125I-LDL to the liver plasma membranes, in vitro. Scatchard analysis of the specific binding data revealed that under the influence of TCR treatment the liver membranes bound to a greater number of 125I-LDL molecules indicating an enhancement in the LDL-receptor activity. TCR was also shown to increase bile acid excretion and to depress hepatic cholesterol synthesis in atherogenic diet fed rats. With these observations in view, the hypocholesterolemic action of TCR appears to be due to an upregulation of hepatic LDL-receptors resulting in greater influx of plasma cholesterol into the liver. TCR also prevents the accumulation of cholesterol in the liver by enhancing cholesterol degradation to bile acids and by simultaneously suppressing cholesterol biosynthesis. The various constituents of TCR may act synergistically to bring about the observed effects.

Morin regulates the expression of NF-κB-p65, COX-2 and matrix metalloproteinases in diethylnitrosamine induced rat hepatocellular carcinoma.

Morin--a bioflavonoid is a naturally available dietary agent believed to impede cancer promotion and progression. The present study was conducted to decipher, in vivo, the role of morin on the expression of matrix metalloproteinases, cyclooxygenase (COX)-2 and nuclear factor kappa B (NF-kappaB)-p65 during diethylnitrosamine (DEN)-induced hepatocarcinogenesis in Wistar albino rats. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that administration of DEN (200 mg/kg bodyweight in drinking water) to experimental animals caused inflammation of the liver due to up-regulation of NF-kappaB-p65 and COX-2. RT-PCR and immunoblot analysis also revealed that the oral supplementation of morin (500 ppm in diet) to DEN-induced hepatocellular carcinoma rats down-regulated the expression of COX-2 and NF-kappaB-p65, thereby preventing inflammation and angiogenesis mediated hepatocellular carcinogenesis. Further, immunohistological analysis for NF-kappaB-p65 nuclear localization confirms the above observations. Gelatin zymography was performed for matrix metalloproteinase MMP-2 and MMP-9 expression to confirm their role in angiogenesis in DEN induced hepatocellular carcinoma and its modulation by morin. Both MMP-2 and MMP-9 levels were found to be increased in DEN-induced animals when compared to control. MMP-2 and MMP-9 levels were down-regulated in morin post-treated animals when compared to DEN-induced animals favouring prevention of angiogenesis. In conclusion, our findings indicate that morin possessed anti-inflammatory and anti-cancer properties favouring suppression of DEN-induced hepatocellular carcinoma.

Pretreatment with alcoholic extract of Crataegus oxycantha (AEC) activates mitochondrial protection during isoproterenol - : induced myocardial infarction in rats

Crataegus oxycantha (hawthorn) is used in herbal and homeopathic medicine as a cardiotonic. The present study was done to investigate the effect of the alcoholic extract of Crataegus oxycantha (AEC) on mitochondrial function during experimentally induced myocardial infarction in rat. AEC was administered orally to male albino rats (150–200 g), at a dosage of 0.5 ml/100 g body weight/day, for 30 days. At the end of the experimental period, the animals were administered isoproterenol (85 mg/kg body weight, s.c) for 2 days at an interval of 24 h. After 48 h, the rats were anaesthetized and sacrificed. The hearts were homogenized for biochemical and electron microscopic analysis. AEC pretreatment maintained mitochondrial antioxidant status, prevented mitochondrial lipid peroxidative damage and decrease in Krebs cycle enzymes induced by isoproterenol in rat heart.

Co-overexpression of p53 and bcl-2 proteins in HPV-induced squamous cell carcinoma of the uterine cervix☆

OBJECTIVE The aim of this study was to analyze aberrant expression of both apoptotic protein p53 and antiapoptotic protein bcl-2 in squamous cell carcinoma (SCC) of the uterine cervix with HPV infection and its significance as a marker for progression of cervical lesions. METHODS One hundred and five cervical lesions and 20 normal (age matched) cervical epithelium from patients with complaints other than cervical lesions were investigated immunocytochemically for aberrant expression of p53 and bcl-2 using the streptavidin-biotin-peroxidase method with respective monoclonal antibodies. HPV status was also anlayzed using type-specific primers for HPV 16/18 and HPV 6/11 by polymerase chain reaction (PCR). The statistical correlation analysis was carried out using Spearmans correlation test and univariate analysis by the SPSS system. RESULTS An abnormal nuclear expression of tumor-suppressor protein p53 and cytoplasmic expression of bcl-2 were observed using immunocytochemistry in biopsies of cervical lesions but not in normal subjects. The intensity of immunoreactivity for both p53 and bcl-2 proteins varied between different histopathological grades of cervical lesions and the correlation analysis showed a highly significant positive correlation for their expression level with different stages from mild dysplasia to invasive cancer with r = 0.88842; P = 0.00001 and r = 0.86929; P = 0.00001, respectively. A highly significant positive correlation was also observed between the expression of both p53 and bcl-2 proteins and HPV infection. The current study indicates a very good significant direct correlation (r = 0.83925; P = 0.00001) between p53 expression and bcl-2 expression in the study population, suggesting the co-overexpression of these proteins in HPV-associated cervical cancer. CONCLUSIONS From the observations it is suggested that the immunodetection of both p53 and bcl-2 proteins in squamous cell carcinoma of the uterine cervix can be used as an independent diagnostic marker for cervical cancer associated with HPV infection. The highly significant association of these proteins with HPV infection suggests that the high-risk HPV infection may be responsible for the co-overexpression of p53 and bcl-2 in cervical cancer even though both of them are antagonistic in their function. This study thus helps to understand the molecular mechanism underlying cervical carcinogenesis and which in turn may improve the therapeutic approach.

Assessment of the no-observed-adverse-effect level (NOAEL) of gallic acid in mice

Gallic acid is a naturally occurring plant phenol obtained by the hydrolysis of tannins and is known to show some pharmacological activities. The purpose of this paper is to establish the safety of gallic acid in mice. In this study, acute administration of gallic acid even at a dose as high as 5 g/kg body weight did not produce any signs of toxicity or mortality. In the subacute study, gallic acid at a dose of 1000 mg/kg body weight did not significantly alter the hematological parameters. Further, no appreciable change was noted in the various biochemical parameters such as SGOT and SGPT, as well as many serum constituents such as protein, cholesterol, urea and bilirubin. Therefore, from this study, it may be concluded that gallic acid is non-toxic up to a level of 5000 mg/kg body weight, when given orally. In addition, the subacute study indicated the absence of cumulative toxicity, as reflected by the non-significant alterations in the parameters investigated. The NOAEL was 5000 mg/kg body weight, the highest dose tested.

Lactobacilli facilitate maintenance of intestinal membrane integrity during Shigella dysenteriae 1 infection in rats

OBJECTIVE Lactobacilli are used in various dairy products and fermented foods for their potential health beneficial effects. Recently we reported the protective role of Lactobacillus rhamnosus and Lactobacillus acidophilus during Shigella dysenteriae 1 infection. Nevertheless, investigations on the membrane-stabilizing effect of L. rhamnosus and L. acidophilus have not been done. Hence, the present study evaluated the effect of L. rhamnosus and L. acidophilus on the maintenance of intestinal membrane integrity during S. dysenteriae 1-induced diarrhea in rats. METHODS Rats were divided into eight groups (n = 6 in each group). Induced rats received single oral dose of S. dysenteriae (12 x 10(8) colony-forming units [cfu]/mL). Treated rats received L. rhamnosus (1 x 10(7)cfu/mL) or L. acidophilus (1 x 10(7)cfu/mL) orally for 4 d, alone or in combination, followed by Shigella administration. At the end of the experimental period, animals were sacrificed and the assay of membrane-bound adenosine triphosphatases (Na(+)/K(+)-ATPase, Ca(2+)-ATPase, and total ATPase), immunoblot analysis of tight junctional proteins (claudin-1 and occludin), and transmission electron microscopic studies were performed. RESULTS Induced rats showed a significant (P < 0.05) reduction in the membrane-bound ATPases and reduced expression of tight junction proteins in the membrane, coupled with their increased expression in the cytosol, indicating membrane damage. Transmission electron microscopic studies correlated with biochemical parameters. Pretreatment with combination of L. rhamnosus and L. acidophilus significantly prevented these changes. CONCLUSION Lactobacillus rhamnosus and L. acidophilus synergistically offered better protection to the intestinal membrane when compared with individual treatments with these strains during S. dysenteriae 1 infection.

Surfactant free rapid synthesis of hydroxyapatite nanorods by a microwave irradiation method for the treatment of bone infection

Mesoporous nanocrystalline hydroxyapatite (nHAp) rods of size 40-75 nm long and 25 nm wide (resembling bone mineral) were synthesized under microwave irradiation without using any surfactants or modifiers. The surface area and average pore size of the nHAp were found to be 32 m(2) g(-1) and 4 nm, respectively. Rifampicin (RIF) and ciprofloxacin (CPF) loaded nHAp displayed an initial burst followed by controlled release (zero order kinetics). Combination of CPF and RIF loaded nHAp showed enhanced bacterial growth inhibition against Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis) and Escherichia coli (E. coli) compared to individual agent loaded nHAp and pure nHAp. In addition, decreased bacterial adhesion (90%) was observed on the surface of CPF plus RIF loaded nHAp. The biocompatibility test toward MG63 cells infected with micro-organisms showed better cell viability and alkaline phosphatase activity (ALP) for the combination of CPF and RIF loaded nHAp. The influence on cell viability of infected MG63 cells was attributed to the simultaneous and controlled release of CPF and RIF from nHAp, which prevented the emergence of subpopulations that were resistant to each other. Hence, apart from the issue of the rapid synthesis of nHAp without surfactants or modifiers, the simultaneous and controlled release of dual drugs from nHAp would be a simple, non-toxic and cost-effective method to treat bone infections.

Methylated chrysin induces co-ordinated attenuation of the canonical Wnt and NF-kB signaling pathway and upregulates apoptotic gene expression in the early hepatocarcinogenesis rat model

Hepatocellular carcinoma (HCC), a highly aggressive form of solid tumor, has been increasing in South East Asia. The lack of effective therapy necessitates the introduction of novel chemopreventive strategies to counter the substantial morbidity and mortality associated with the disease. Recently, we reported that dimethoxy flavone (DMF), a methylated flavone derived from chrysin, significantly suppressed the development of preneoplastic lesions induced by N-nitrosodiethylamine (DEN) in rats, although the mechanism of action was not known. In the present study, we have investigated the effects of DMF administration on gene expression changes related to the inflammation-mediated NF-kB pathway, Wnt pathway and apoptotic mediators in DEN-induced preneoplastic nodules. There was a significant increase in inflammatory markers like cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) and a decrease in apoptotic mediators like p53, caspase-3 and bax in DEN-treated rats when compared to the control group. Activation of NF-kB was noticed by an elevated expression of nuclear protein expression of NF-kB and cytoplasmic phospho-IkBαSer(32/36) in the same animals. Likewise, upregulation of canonical Wnt pathway was noticed by elevated expression of nuclear protein levels of phospho-β-cateninThr(393) and cytoplasmic casein kinase-2 (CK2), Dvl2 and cyclin D1 levels, along with a simultaneous decrease in expression of phospho-GSK3β(Ser9). Dietary DMF (100mg/kg) administration inhibited liver nodule incidence and multiplicity by 82% and 78%, respectively. DMF also reversed the activation of NF-kB and Wnt pathway as shown by the decrease in protein expression of several proteins. Results of the present investigation provide evidence that attenuation of Wnt pathway and suppression of inflammatory response mediated by NF-kB could be implicated, in part, in the chemopreventive effects of methylated flavone. Therefore, the present findings hold great promise for the utilization of DMF as an effective chemotherapeutic agent in treating early stages of liver cancer.