Catechin suppresses an array of signalling molecules and modulates alcohol-induced endotoxin mediated liver injury in a rat model.

Induction of nuclear factor kappa B (NF-κB)-mediated gene expression has been implicated in the pathogenesis of alcoholic liver disease through enhanced production of reactive oxygen species and pro-inflammatory mediators. The present study was carried out to investigate the role of catechin as a chain breaking inhibitor against experimental alcoholic liver injury. Rats were administered 35% v/v ethanol orally at a dose of 10 g/Kg/day for two weeks, followed by 14 g/Kg/day for 10 weeks. Catechin (50 mg/Kg) was co-supplemented after 4 weeks of alcohol treatment till the end of the dosing period. Following chronic alcohol exposure, rats developed endotoxemia and severe pathological changes in the liver such as pronounced fatty change, vacuolar degeneration and inflammation. These changes were accompanied by activation of NF-κB and induction of inflammatory and cytotoxic mediators leading to increased level of tumor necrosis factor-alpha, enhanced formation of malondialdehyde in the liver followed by drastic alterations in the hepatic antioxidant defense systems. Additionally, nitrite levels and lactate dehydrogenase activities were also significantly elevated on chronic alcohol consumption. Alcohol exposure also increased the number of micronucleated cells indicating that alcohol abuse may again be associated with the nuclear changes. Supplementation with catechin ameliorated the alcohol-induced liver injury by downregulating the endotoxin-mediated activation of initial signalling molecule NF-κB and further going downstream the signalling cascade including tumor necrosis factor-alpha, nitric oxide and reactive oxygen species and by enhancing the antioxidant profile. These observations correlated well with the histological findings. Moreover, a remarkable decrease in the percentage of micronucleated cells was observed with catechin supplementation indicating an apparent protection against alcohol-induced toxicity. These findings suggest that catechin may alleviate experimental alcoholic liver disease by suppressing induction of NF-κB, a key component of signalling pathway, thus forming a pharmacological basis for designing novel therapeutic agents against alcohol induced endotoxin-mediated liver injury

In Vitro and In Vivo Synergistic Effects of Cryptdin 2 and Ampicillin against Salmonella▿

In view of the emergence of multidrug-resistant Salmonella strains, there is a need for therapeutic alternatives. To reduce the dose of antibiotic required in order to decrease the associated side effects, the present study was aimed at evaluating the synergism between cryptdin 2 (a Paneth cell antimicrobial peptide) and ampicillin (Amp) against Salmonella enterica serovar Typhimurium. The synergy was evaluated in terms of the fractional bactericidal concentration (FBC) index, time-kill assay results (in vitro), macrophage functions, i.e., intracellular killing, lipid peroxidation, superoxide dismutase activity, and generation of nitrite (ex vivo), and decreases in CFU of salmonellae in livers, spleens, and small intestines of infected mice treated with cryptdin 2 and/or Amp (in vivo). In vitro synergism between the two agents was observed on the basis of the FBC index and time-kill assays. When the agents were used in combination, ex vivo studies revealed an enhanced effect on macrophage functions, particularly exhibiting a synergetic effect in terms of SOD levels. In vivo synergy was indicated by larger log unit decreases in all target organs of mice treated with the combination than those for the drugs used alone. These results point toward the possible use of cryptdin 2 as an adjunct to ampicillin and may help in developing alternate strategies to combat Salmonella infections

Micronuclei analysis in the hepatocytes of alcohol-fed rats.

<p><b>A</b>, <b>B</b>) Dividing cells showing normal nuclei and micronuclei in hepatocytes of alcohol-fed rats. <b>BN</b>: Binucleated; <b>MN</b>: Mononucleus; <b>MNi</b>: Micronucleus; <b>C</b>) Effect of catechin on the extent of micronuclei formation in hepatocytes of alcohol- administered rats. Values are expressed as percentage of micronucleated cells. *p<0.001 vs. control; ^#p<0.05 vs. Alcohol (Alc).</p

Effect of catechin on LDH activity in alcohol-administered rats.

<p><b>A</b>) Serum LDH activity and <b>B</b>) hepatic LDH activity. Values are expressed as mean ± S.D. of eight different observations. *p<0.001 vs. control and catechin (CT) <i>per se</i>; ^#p<0.001 vs. Alcohol (Alc).</p

Effect of catechin on A) serum nitrite levels and B) hepatic nitrite levels in alcohol-fed rats.

<p>Values are expressed as mean ± S.D. of eight different observations. *p<0.001 vs. control and catechin (CT) <i>per se</i>; ^#p<0.001 vs. Alcohol (Alc).</p

Effect of catechin on alcohol-induced activation of NF-κB in liver.

<p>Values are expressed as mean ± S.D. of five different observations. *p<0.001 vs. control and catechin (CT) <i>per se</i>; ^#p<0.01 vs. Alcohol (Alc). Positive control <b>(+control)</b> refers to the TNF-α treated Hela whole cell extract; Negative control <b>(-control)</b> refers to the biotinylated double stranded non-specific competitor oligonucleotide probe which does not contain the NF-κB consensus sequence.</p

Effect of catechin on hepatic TNF-α levels in alcohol-fed rats.

<p>Values are expressed as mean ± S.D. of eight different observations. *p<0.001 vs. control and catechin (CT) <i>per se</i>; ^#p<0.001 vs. Alcohol (Alc).</p

Photomicrographs of hematoxylin-eosin stained rat liver sections after alcohol administration.

<p><b>A</b>) Photomicrograph of the normal/control rat liver showing normal liver morphology; <b>B</b>) Photomicrograph of rat liver of catechin <i>per se</i> group showing normal liver morphology; <b>C</b>, <b>D</b>) Photomicrograph of the liver from alcohol-administered rat showing vacuolar degeneration, micro- and macrovesicular fatty change; <b>E</b>) Photomicrograph of the liver from alcohol-administered rat showing portal triaditis with thin fibrous bridges radiating from the portal tract. Liver cells show vacuolar degeneration and microvesicular fatty change; <b>F</b>) Photomicrograph of the liver from alcohol-administered rat supplemented with catechin (Alc + CT) showing mild cytoplasmic vacuolation with no fatty change; <b>G</b>) Photomicrograph of the liver from alcohol-administered rat supplemented with catechin (Alc + CT) showing normal liver morphology.</p

Effect of catechin on liver MDA levels in alcohol-administered rats.

<p>Values are expressed as mean ± S.D. of eight different observations. *p<0.001 vs. control and catechin (CT) <i>per se</i>; ^#p<0.001 vs. Alcohol (Alc).</p

Effect of catechin on hepatic antioxidant profile in control and alcohol-administered rats.

<p>Values are expressed as mean ± S.D. of eight different observations.</p><p>*p<0.001 vs. control and catechin <i>per se</i>;</p>#<p>p<0.001 vs. Alc. Units for: <b>GSH-</b> micromoles of GSH/mg protein; <b>SOD-</b> Units/mg protein; <b>Catalase-</b> millimoles of catalase/mg protein; <b>GR-</b> micromoles of NADPH oxidized/min/mg protein; <b>GPx-</b> micromoles of NADPH oxidized/min/mg protein.</p