Inhibition of enteropathogenic Escherichia coli adhesion on host epithelial cells by Holarrhena antidysenterica (L.) WALL.

Bacterial adhesion is the first step in the sequence of events leading to infection. Previous data are available on the effect of Holarrhena antidysenterica on antidiarrhoeal and antibacterial action, but there is little information on the mechanism of action of the various aspects of EPEC-induced diarrhoea, namely adherence and translocation of the effector molecule to intestinal epithelial cells. The aim of the present study was to investigate the effects of alkaloids of Holarrhena antidysenterica (AHA) on interference in the mechanism of enteropathogenic Escherichia coli (EPEC) adhesion on host epithelial cells (INT 407 and HEp2). To determine the impact of AHA on epithelial cells, cytotoxicity (LDH), adherence, apoptotic and ultrastructural studies were performed. To analyse the effect of AHA on EPEC secreted proteins, especially EspD, INT 407 monolayers were infected with EPEC and AHA-treated EPEC, followed by immunoblotting, probed with anti EspD antisera. The maximum percentage of LDH leakage was reduced in AHA-treated EPEC (400 microg/mL) in both cell lines. Reduced bacterial adherence was observed under light microscopy and altered apoptotic changes were visualized using propidium iodide staining in conjunction with fluorescence microscopy, in both cell lines infected with AHA-treated EPEC and these results were confirmed with transmission electron microscope images. The suppression of type III secretory proteins (TTSPs), EspD ( approximately 40 kDa), was detected in INT 407 cells infected with AHA-treated EPEC. In conclusion, AHA reduces initial bacterial adhesion to intact epithelial cells and it may exert an antiadherence effect against the pathogenesis of EPEC in host epithelial cells. Thus, the investigations provide a rational basis for the treatment of EPEC-mediated diarrhoea with AHA.

Cardioprotective effect of the Hibiscus rosa sinensis flowers in an oxidative stress model of myocardial ischemic reperfusion injury in rat.

BACKGROUND: The present study investigates the cardioprotective effects of Hibiscus rosa sinensis in myocardial ischemic reperfusion injury, particularly in terms of its antioxidant effects. METHODS: The medicinal values of the flowers of Hibiscus rosa sinensis (Chinese rose) have been mentioned in ancient literature as useful in disorders of the heart. Dried pulverized flower of Hibiscus rosa sinensis was administered orally to Wistar albino rats (150-200 gms) in three different doses [125, 250 and 500 mg/kg in 2% carboxy methyl cellulose (CMC)], 6 days per week for 4 weeks. Thereafter, rats were sacrificed; either for the determination of baseline changes in cardiac endogenous antioxidants [superoxide dismutase, reduced glutathione and catalase] or the hearts were subjected to isoproterenol induced myocardial necrosis. RESULTS: There was significant increase in the baseline contents of thiobarbituric acid reactive substances (TBARS) [a measure of lipid per oxidation] with both doses of Hibiscus Rosa sinensis. In the 250 mg/kg treated group, there was significant increase in superoxide dismutase, reduced glutathione, and catalase levels but not in the 125 and 500 mg/kg treated groups. Significant rise in myocardial thiobarbituric acid reactive substances and loss of superoxide dismutase, catalase and reduced glutathione (suggestive of increased oxidative stress) occurred in the vehicle treated hearts subjected to in vivo myocardial ischemic reperfusion injury. CONCLUSION: It may be concluded that flower of Hibiscus rosa sinensis (250 mg/kg) augments endogenous antioxidant compounds of rat heart and also prevents the myocardium from isoproterenol induced myocardial injury.

Chronic depletion of glutathione (GSH) and minimal modification of LDL in vivo: its prevention by glutathione mono ester (GME) therapy.

A decline in reduced glutathione (GSH) level is associated with aging and free radical mediated diseases. The objective of this study was to determine whether the chronic depletion of extra cellular GSH causes oxidative damage to the circulating macromolecules such as lipoproteins. Decreased concentrations of plasma glutathione, vitamin E and ascorbic acid were recorded in the rats treated with buthionine sulfoximine (BSO), a selective GSH inhibitor. In LDL isolated from BSO-treated animals, the concentration of malondialdehyde (MDA) and conjugated dienes were significantly increased (P<0.01), whereas the levels of vitamin E were decreased (P<0.01). The analysis of total and LDL cholesterol revealed significant changes between the control and experimental groups. Of interest, altered concentrations of lyso-phosphatidyl choline (Lyso-PC) and phosphatidyl choline (PC) were recorded from the BSO mediated minimally modified LDL. A negative correlation between LDL-BDC/MDA and its antioxidant capacity was noted. Upon in vitro oxidation with CuSO(4), the electrophoretic behavior of purified LDL-apoprotein-B on agarose gel showed an increased mobility in BSO-treated rats, indicative of in vivo modification of LDL to become susceptible for in vitro oxidation. The increased mobility of LDL (after in vitro oxidation) isolated from the BSO-treated animals correlates with a decrease in its amino groups, as determined by the trinitrobenzene sulfonic acid (TNBS) reactants. However, the mobility of LDL molecule was not altered due to BSO treatment in vivo. Interestingly, the minimal modification on LDL does not lead to any vascular damage in the dorsal aorta of the rats injected with BSO. The administration of glutathione monoester (GME), at a dose of 5 mmol/kg body weight, twice a day, for 30 days, to animals treated with l-buthionine-SR-sulfoximine (BSO, 4 mmol/kg body weight, twice a day, for 30 days) normalized the antioxidant status and prevented the minimal modifications on LDL. Thus, increasing the cellular GSH levels may trigger beneficial effects against oxidative stress.

Modulation of rat erythrocyte antioxidant defense system by buthionine sulfoximine and its reversal by glutathione monoester therapy.

The protective effects of glutathione monoester (GME) on buthionine sulfoximine (BSO)-induced glutathione (GSH) depletion and its sequel were evaluated in rat erythrocyte/erythrocyte membrane. Animals were divided into three groups (n=6 in each): control, BSO and BSO+GME group. Administration of BSO, at a concentration of 4 mmol/kg bw, to the albino rats resulted in depletion of blood GSH level to about 59%. GSH was elevated several folds in the GME group as compared to the control (P<0.05) and BSO (P<0.001) groups. Decreased concentration of vitamin E was found in the erythrocyte membrane isolated from BSO-administered animals. Antioxidant enzymes, catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPX) were also found to be altered due to BSO-induced GSH depletion in blood erythrocytes. The SOD and CAT activities in BSO group were significantly lower (P<0.001) than the other groups. Lipid peroxidation index and malondialdehyde (MDA) levels in erythrocytes and their membranes were increased to about 45% and 40%, respectively. The activities of Ca2+ ATPase, Mg2+ ATPase and Na+K+ ATPase were lower than those of control group (P<0.05), whereas the activities of these enzymes were found to be restored to normal followed by GME therapy (P<0.05). Cholesterol, phospholipid and C/P ratio and some of the phospholipid classes like phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and sphingomyelin were significantly (P<0.05) altered in the erythrocyte membranes of BSO-administered rats compared with those of control group. These parameters were restored to control group levels in GME-treated group. Oxidative stress may play a major role in the BSO-mediated gamma glutamyl cysteine synthetase (gamma-GCS) inhibition and hence the depletion of GSH. In conclusion, our findings have shown that antioxidant status decreased and lipid peroxidation increased in BSO-treated rats. GME potentiates the RBC and blood antioxidant defense mechanisms and decreases lipid peroxidation.

Vitamin E prevents buthionine sulfoximine-induced biochemical disorders in the rat.

Antioxidant therapy can improve the protection and metabolic activity of cells and tissues. In this study, the effect of vitamin E administration on buthionine sulfoximine (BSO)-induced glutathione (GSH) depletion in the rat lung and liver was investigated. Hepatic GSH was depleted by intraperitoneal administration of BSO (4 mmol kg(-1)), twice a day, for 30 days to rats. We also investigated whether the lung and liver mitochondrial GSH contents were influenced by BSO administration and whether an extracellular supply of vitamin E could prevent the changes caused by BSO-mediated GSH depletion. Glutathione levels in lung and liver tissues were depleted by 47% and 60%, respectively. Depletion of hepatic and pulmonary GSH in turn causes decline in the levels of mitochondrial GSH, leading to impaired antioxidant defence function of mitochondria. Both the cytosolic and mitochondrial glutathione disulfides (GSSG) were altered during BSO treatment, and led to drastic increase in GSSG/GSH redox status. One of the experimental groups was given vitamin E (65 mg (kg diet)(-1)) mixed with rat feed. The rats fed with vitamin E were found to have partially restored GSH levels in liver and lung, diminished levels of TBARS and minimized tissue damage. The current findings suggest that the impaired glutathione and glutathione-dependent enzyme status may be correlated with the elevated lipid peroxidation and mitochondrial membrane damage and that vitamin E therapy to the BSO-administered rats prevents the above changes. However, vitamin E did not have any effect on the activity of gamma-glutamyl cysteine synthetase (gamma-GCS).

Lipid peroxidation as a possible secondary mechanism of sterigmatocystin toxicity.

Sterigmatocystin (Stg), a major secondary metabolite of Aspergillus versicolor and A. nidulans, is the precursor of aflatoxin B1. In this study, male albino rats were treated with Stg-contaminated diet for 30 days, resulting in reduced levels of glutathione, ascorbic acid, and alpha-tocopherol. The activity of catalase in liver was reduced, whereas an increase in the activities of superoxide dismutase and glutathione peroxidase was observed. The levels of cytochrome P450, cytochrome b5, cytochrome b5 reductase, cytochrome c reductase, hydroxyl radical, and hydrogen peroxide formation significantly increased in the Stg- treated rat liver microsomes. Hepatic parenchymal cell injury, necrosis, and Kupffer cells proliferation were noticed in histological sections of liver from animals treated with Stg. Overall results suggest that generation of free radicals imposes depletion of antioxidants. This led to enhanced lipid peroxidation. The observed elevation of hepatic thiobarbituric acid reactive substances appears to originate mainly from the damaged Kupffer cells. As a result, elevated levels of serum marker enzymes were also observed.

Binding of Shigella to rat and human intestinal mucin.

Invasion of epithelial cells by Shigella is an early step in their pathogenesis. Adherence is generally presumed to be a prerequisite for invasion. This study examined the possibility of intestinal mucins serving as initial binding sites for clinical isolates of S. boydii and S. sonnei. The interactions of Shigella with rat and human small intestinal and colonic mucin were investigated. In solid phase binding assays, [35S] labelled Shigella did not show any preferential binding to rat/human small intestinal mucin or to rat colonic mucin. On the other hand, Shigella bound specifically to human colonic mucin in a concentration-dependent manner. This specific binding to human colonic mucin was not by weak hydrophobic interactions and could not be attributed to the presence of contaminating glycolipids in the mucin preparation. The human colonic mucin receptor was sensitive to periodate treatment suggesting the involvement of the carbohydrate portion of the mucin. Reduction and alkylation of mucin enhanced adherence probably by exposing buried binding sites. The monosaccharides present in mucins were ineffective as hapten inhibitors as was the lectin wheat germ agglutinin suggesting that the mucin receptor is a more complex one. This study identifies, for the first time, the presence of a specific Shigella-binding site on the carbohydrate portion of human colonic mucin, which is not present in rat colonic mucin or in rat/human small intestinal mucin.

Pulmonary phospholipid changes induced by butylated hydroxy toluene, an antioxidant, in rats.

Butylated hydroxy toluene (BHT), 800 mg/kg body weight, dissolved in corn oil and administered (ip) in a single injection to male rats, damaged the lung as indicated by an increase in lavage ACE, protein and LDH and caused a significant increase in phospholipid, particularly, phosphatidyl choline (PC) in lung lavage and extracellular surfactant. The plasma lecithin cholesterol acyl transferase (LCAT) activity was inhibited leading to an increase in serum phospholipids and phosphatidyl choline. The results indicate that BHT-induced lung phospholipidosis may be attributed to an increase in surfactant phospholipids and/or due to the leakage of plasma phospholipids through damaged capillary membrane.

Lung injury by amiodarone, an antiarrhythmic drug, in male rats.

Administration of single dose (175 mg/kg body wt) of amiodarone dissolved in water through gavage for 3 weeks damaged the lung and changed the content of lung lavage. Activities of bronchoalveolar lavage (BAL) angiotensin converting enzyme (ACE) and lactate dehydrogenase (LDH) increased significantly. Also, the protein and lactate content of the lavage fluid increased significantly over the control. The drug also produced marked changes in morphology of the lung of experimental animals.