Halagowder Devaraj

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.

Expression profile of mucins (MUC2, MUC5AC and MUC6) in Helicobacter pylori infected pre-neoplastic and neoplastic human gastric epithelium.

BackgroundHelicobacter pylori (H. pylori) causes gastritis and intestinal metaplasia (IM) that may evolve to gastric carcinoma. The objective of this study was to compare the profile of mucins in the progressive stages of H. pylori infected pre-neoplastic and neoplastic human gastric epithelium. We used a panel of monoclonal antibodies with well-defined specificities of MUC2, MUC5AC and MUC6 to characterize the expression pattern of mucins by immunohistochemistry.MethodsRUT and ELISA were down for H. pylori confirmation. Human gastric biopsy sections were stained using immunohistochemistry with MUC2, MUC5AC and MUC6 antibodies.ResultsMUC5AC was expressed in the superficial epithelium and the upper part of the gastric pits. MUC6 expression was detected in the lower part of the gastric glands. MUC2 was expressed in intestinal metaplasia, mostly in goblet cells. The mucin expression profile in the progressive stages of H. pylori infected human gastric epithelium allows the identification of intestinal metaplasia, which is characterized by a decreased expression of the gastric mucins (MUC5AC and MUC6) and de novo expression of MUC2.ConclusionIn conclusion, our results suggest that there is altered expression of MUC5AC and MUC6 together with the aberrant expression of MUC2 in intestinal metaplasia, during the process of gastric carcinogenesis. The present study indicates that the MUC2 mucin expression pattern is a reliable marker of intestinal metaplasia, which appears in the context of H. pylori infected individuals.

Interaction of MUC1 with β-catenin modulates the Wnt target Gene cyclinD1 in H. pylori-induced gastric cancer

β‐catenin can function as an oncogene when it is translocated to the nucleus, binds to T‐cell factor (TCF) or lymphoid enhance factor and transactivate its target gene. The mechanism responsible for the activation of Wnt signaling pathway in the Cytotoxin‐associated antigen A (CagA) Helicobacter pylori (H. pylori)‐infected gastric carcinoma has not been elucidated. We hypothesize that whether interaction of MUC1 with β‐catenin modulates the Wnt signaling and its target gene cyclinD1 in CagA H. pylori‐infected gastric carcinoma. The result demonstrate that binding of MUC1 CT with Protein Kinase C δ (PKC δ), tyrosine phosphorylation of MUC1 CT, and CagA are strongly associated with the interaction of MUC1 with β‐catenin in CagA H. pylori‐infected gastric carcinoma. A statistically significant difference (χ2 = 24.49; P < 0.001) was found when the binding of MUC1 CT and β‐catenin was compared to subcellular localization of β‐catenin. We also observed significant statistical correlation (χ2 = 14.885; P < 0.001) between the cyclinD1 overexpression and the subcellular localization of β‐catenin. The overexpression of cyclinD1 was significantly higher (χ2 = 13.785; P < 0.002) in advanced gastric carcinoma with CagA H. pylori infection. In addition cyclinD1 overexpression was significantly higher (χ2 = 37.267; P < 0.001) with the interaction of MUC1 CT with β‐catenin in advanced gastric cancer. These findings indicate that MUC1 CT plays a role in the intracellular signaling through its interaction with β‐catenin and upregulate the Wnt target gene cyclinD1 in CagA H. pylori‐infected gastric carcinoma.

Chrysin abrogates early hepatocarcinogenesis and induces apoptosis in N-nitrosodiethylamine-induced preneoplastic nodules in rats

Flavonoids possess strong anti-oxidant and cancer chemopreventive activities. Chrysin (5,7-dihydroxyflavone) occurs naturally in many plants, honey, and propolis. In vitro, chrysin acts as a general anti-oxidant, causes cell cycle arrest and promotes cell death. However, the mechanism by which chrysin inhibits cancer cell growth and the subcellular pathways activated remains poorly understood. Effect of dietary supplementation with chrysin on proliferation and apoptosis during diethylnitrosamine (DEN)-induced early hepatocarcinogenesis was investigated in male Wistar rats. To induce hepatocarcinogenesis, rats were given DEN injections (i.p., 200 mg/kg) three times at a 15 day interval. An oral dose of chrysin (250 mg/kg bodyweight) was given three times weekly for 3 weeks, commencing 1 week after the last dose of DEN. Changes in the mRNA expression of COX-2, NFkB p65, p53, Bcl-xL and β-arrestin-2 were assessed by quantitative real-time PCR. Changes in the protein levels were measured by western blotting. Chrysin administration significantly (P<0.001) reduced the number and size of nodules formed. Also, a significant (P<0.01) reduction in serum activities of AST, ALT, ALP, LDH and γGT was noticed. Expression of COX-2 and NFkB p65 was significantly reduced whereas that of p53, Bax and caspase 3 increased at the mRNA and protein levels. Likewise, a decrease in levels of β-arrestin and the anti-apoptotic marker Bcl-xL was also noted. These findings suggest that chrysin exerts global hepato-protective effect and its chemopreventive activity is associated with p53-mediated apoptosis during early hepatocarcinogenesis.

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.

Buthionine sulfoximine-induced glutathione depletion: Its effect on antioxidants, lipid peroxidation and calcium homeostasis in the lung

The administration of buthionine sulfoximine (BSO), an irreversible inhibitor of gamma-glutamylcysteine synthetase, produces glutathione (GSH) depletion in tumors, making them sensitive to drugs and radiation. During the process, it also depletes GSH from normal tissues. Certain tumors require frequent doses of BSO for several days to produce GSH depletion. In this study, we determined that this chronic GSH-deficient condition lowers the antioxidant defense of the lung by diminishing the activities of superoxide dismutase, catalase, and glutathione peroxidase and the levels of ascorbic acid and alpha-tocopherol. Impaired antioxidant defense leads to enhanced lipid peroxidation, as indicated by increased levels of thiobarbituric acid reactive substances and conjugated dienes. The alteration of protein thiols by lipid peroxidation, is responsible for altered Ca2+ homeostasis, which, in turn, leads to cell injury. Cell injury was confirmed by elevated activities of angiotensin converting enzyme and lactate dehydrogenase, increased levels of protein and lactate, and histopathological changes.

Adherence of Shigella dysenteriae 1 to Human Colonic Mucin

The pathogenic potential of Shigella is correlated with the ability of the organism to invade and multiply within the cells of colonic epithelium. Although invasion is the ultimate event, a preceding step is adherence. Shigella dysenteriae 1 preferentially adhered to colonic mucin and not to small intestinal mucin. The pathogen showed a very strong adherence pattern to human colonic mucin when compared with guinea pig and rat mucin. The adherence pattern of S. dysenteriae 1 was not altered on preincubation with monosaccharides present in mucins, suggesting that the receptor for the pathogen is not a simple sugar. Binding of S. dysenteriae 1 to human colonic mucin was not by weak hydrophobic forces. The bacterium also adhered to glycolipids, emphasizing the role of glycoconjugates as receptors for S. dysenteriae 1.

Synergistic effect of tincture of Crataegus and Mangifera indica L. extract on hyperlipidemic and antioxidant status in atherogenic rats

This study was designed to address the synergistic effect of tincture of Crataegus (TCR) and Mangifera indica L. (MNG) extracts on the lipid and antioxidant parameters in the development of aortic lesions in diet-induced atherosclerosis in rats. TCR, is an alcoholic extract made from the berries of Hawthorn, Crataegus oxyacantha with flavanoids as the main constituent. MNG, is an alcoholic extract made from the stem bark of Mangifera indica L. with polyphenols as the main constituent. Simultaneous oral administration of these two extracts (0.5 ml/100 g body weight) to rats fed with an atherogenic (4% cholesterol, 1% cholic acid, 0.5% thiouracil) diet prevented the elevation of lipids in the serum and heart and also caused a significant decrease in lipid accumulation in the liver and aorta reverting the hyperlipidaemic condition of these rats. These extracts significantly restored the activity of antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase, and glutathione, thereby restoring the antioxidant status of the organism to almost normal levels. This effect could be attributed to the synergistic activity of flavonoids in TCR and polyphenols of MNG.

Morin ameliorates chemically induced liver fibrosis in vivo and inhibits stellate cell proliferation in vitro by suppressing Wnt/β-catenin signaling.

The anti-fibrotic effect of morin was examined in LX-2 cells (culture-activated human hepatic stellate cells) and in diethylnitrosamine induced rat model of liver fibrosis. The in vitro study was designed to determine whether morin affects the survival of cultured LX-2 cells, while the in vivo study was designed to evaluate the antioxidant and anti-fibrotic efficacy of morin on diethylnitrosamine induced liver fibrosis in male albino Wistar rat. The activities of liver function enzymes in serum, liver lipid peroxide levels, activities of serum antioxidant enzymes and liver architecture were monitored to cast light on the antioxidant and hepatoprotective nature of morin. To establish the anti-fibrotic effects of morin, the levels of key Wnt signaling molecules which are strongly associated with the signal transduction pathway of HSC activation were measured. Overall, from the in vitro results, it was observed that morin at 50 μM concentration inhibited the proliferation of cultured LX-2 cells, inhibited Wnt signaling and induced G1 cell cycle arrest. The in vivo results further confirmed that morin by downregulating the expressions of GSK-3β, β-catenin and cyclin D1 ameliorated DEN-induced liver fibrosis. Hence morin could be employed as a promising chemopreventive natural supplement for liver fibrosis.