Rapid disruption of intestinal epithelial tight junction and barrier dysfunction by ionizing radiation in mouse colon in vivo: protection by N-acetyl-l-cysteine.

The goals of this study were to evaluate the effects of ionizing radiation on apical junctions in colonic epithelium and mucosal barrier function in mice in vivo. Adult mice were subjected to total body irradiation (4 Gy) with or without N-acetyl-l-cysteine (NAC) feeding for 5 days before irradiation. At 2-24 h postirradiation, the integrity of colonic epithelial tight junctions (TJ), adherens junctions (AJ), and the actin cytoskeleton was assessed by immunofluorescence microscopy and immunoblot analysis of detergent-insoluble fractions for TJ and AJ proteins. The barrier function was evaluated by measuring vascular-to-luminal flux of fluorescein isothiocyanate (FITC)-inulin in vivo and luminal-to-mucosal flux in vitro. Oxidative stress was evaluated by measuring protein thiol oxidation. Confocal microscopy showed that radiation caused redistribution of occludin, zona occludens-1, claudin-3, E-cadherin, and ß-catenin, as well as the actin cytoskeleton as early as 2 h postirradiation, and this effect was sustained for at least 24 h. Feeding NAC before irradiation blocked radiation-induced disruption of TJ, AJ, and the actin cytoskeleton. Radiation increased mucosal permeability to inulin in colon, which was blocked by NAC feeding. The level of reduced-protein thiols in colon was depleted by radiation with a concomitant increase in the level of oxidized-protein thiol. NAC feeding blocked the radiation-induced protein thiol oxidation. These data demonstrate that radiation rapidly disrupts TJ, AJ, and the actin cytoskeleton by an oxidative stress-dependent mechanism that can be prevented by NAC feeding.

Glutamine supplementation attenuates ethanol-induced disruption of apical junctional complexes in colonic epithelium and ameliorates gut barrier dysfunction and fatty liver in mice.

Previous in vitro studies showed that glutamine (Gln) prevents acetaldehyde-induced disruption of tight junctions and adherens junctions in Caco-2 cell monolayers and human colonic mucosa. In the present study, we evaluated the effect of Gln supplementation on ethanol-induced gut barrier dysfunction and liver injury in mice in vivo. Ethanol feeding caused a significant increase in inulin permeability in distal colon. Elevated permeability was associated with a redistribution of tight junction and adherens junction proteins and depletion of detergent-insoluble fractions of these proteins, suggesting that ethanol disrupts apical junctional complexes in colonic epithelium and increases paracellular permeability. Ethanol-induced increase in colonic mucosal permeability and disruption of junctional complexes were most severe in mice fed Gln-free diet. Gln supplementation attenuated ethanol-induced mucosal permeability and disruption of tight junctions and adherens junctions in a dose-dependent manner, indicating the potential role of Gln in nutritional intervention to alcoholic tissue injury. Gln supplementation dose-dependently elevated reduced-protein thiols in colon without affecting the level of oxidized-protein thiols. Ethanol feeding depleted reduced protein thiols and elevated oxidized protein thiols. Ethanol-induced protein thiol oxidation was most severe in mice fed with Gln-free diet and absent in mice fed with Gln-supplemented diet, suggesting that antioxidant effect is one of the likely mechanisms involved in Gln-mediated amelioration of ethanol-induced gut barrier dysfunction. Ethanol feeding elevated plasma transaminase and liver triglyceride, which was accompanied by histopathologic lesions in the liver; ethanol-induced liver damage was attenuated by Gln supplementation. These results indicate that Gln supplementation ameliorates alcohol-induced gut and liver injury.

Host-mediated gene silencing of a single effector gene from the potato pathogen Phytophthora infestans imparts partial resistance to late blight disease.

RNA interference (RNAi) has proved a powerful genetic tool for silencing genes in plants. Host-induced gene silencing of pathogen genes has provided a gene knockout strategy for a wide range of biotechnological applications. The RXLR effector Avr3a gene is largely responsible for virulence of oomycete plant pathogen Phytophthora infestans. In this study, we attempted to silence the Avr3a gene of P. infestans through RNAi technology. The P. infestans inoculation resulted in lower disease progression and a reduction in pathogen load, as demonstrated by disease scoring and quantification of pathogen biomass in terms of Pi08 repetitive elements, respectively. Transgenic plants induced moderate silencing of Avr3a, and the presence and/or expression of small interfering RNAs, as determined through Northern hybridization, indicated siRNA targeted against Avr3a conferred moderate resistance to P. infestans. The single effector gene did not provide complete resistance against P. infestans. Although the Avr3a effector gene could confer moderate resistance, for complete resistance, the cumulative effect of effector genes in addition to Avr3a needs to be considered. In this study, we demonstrated that host-induced RNAi is an effective strategy for functional genomics in oomycetes.

Neuroprotective efficacy of curcumin in arsenic induced cholinergic dysfunctions in rats.

Our recent studies have shown that curcumin protects arsenic induced neurotoxicity by modulating oxidative stress, neurotransmitter levels and dopaminergic system in rats. As chronic exposure to arsenic has been associated with cognitive deficits in humans, the present study has been carried out to implore the neuroprotective potential of curcumin in arsenic induced cholinergic dysfunctions in rats. Rats treated with arsenic (sodium arsenite, 20mg/kg body weight, p.o., 28 days) exhibited a significant decrease in the learning activity, assessed by passive avoidance response associated with decreased binding of (3)H-QNB, known to label muscarinic-cholinergic receptors in hippocampus (54%) and frontal cortex (27%) as compared to controls. Decrease in the activity of acetylcholinesterase in hippocampus (46%) and frontal cortex (33%), staining of Nissl body, immunoreactivity of choline acetyltransferase (ChAT) and expression of ChAT protein in hippocampal region was also observed in arsenic treated rats as compared to controls. Simultaneous treatment with arsenic and curcumin (100mg/kg body weight, p.o., 28 days) increased learning and memory performance associated with increased binding of (3)H-QNB in hippocampus (54%), frontal cortex (25%) and activity of acetylcholinesterase in hippocampus (41%) and frontal cortex (29%) as compared to arsenic treated rats. Increase in the expression of ChAT protein, immunoreactivity of ChAT and staining of Nissl body in hippocampal region was also observed in rats simultaneously treated with arsenic and curcumin as compared to those treated with arsenic alone. The results of the present study suggest that curcumin significantly modulates arsenic induced cholinergic dysfunctions in brain and also exhibits neuroprotective efficacy of curcumin.

Neuroprotective effect of Acorus calamus against middle cerebral artery occlusion-induced ischaemia in rat.

The neuroprotective potential of ethanol:water (1:1) extract of rhizomes of Acorus calamus (AC-002) has been investigated in middle cerebral artery occlusion (MCAO)-induced ischaemia in rats. A significant behavioural impairment in Rota-Rod performance and grid walking was observed in rats, 72 hours after MCAO as compared to sham-operated animals. These rats also exhibited an increase in lipid peroxidation (cortex -157%, corpus striatum - 58%) and a decrease in glutathione levels (cortex - 59%, corpus striatum - 34%) and superoxide dismutase (SOD) activity (cortex - 64%, corpus striatum - 32%) as compared to sham-operated animals. Ischaemic rats treated with AC-002 (25 mg/kg, p.o.) exhibited a significant improvement in neurobehavioural performance viz. Rota-Rod performance and grid walking as compared to the MCAO group. Interestingly, treatment with AC-002 in MCAO rats significantly decreased malonaldialdehyde levels in cortex as compared to ischaemic rats. A significant increase in reduced glutathione levels and SOD activity was also observed both in cortex and corpus striatum in MCAO rats treated with AC-002 in comparison to MCAO rats. Treatment with AC-002 in MCAO rats also reduced the contralateral cortical infarct area (19%) as compared to MCAO rats (33%). Neurological function score was improved in the AC-002-treated rats as compared to the MCAO group. The results of the present study indicate the neuroprotective efficacy of A. calamus in the rat model of ischaemia.

Protective effect of curcumin against lead neurotoxicity in rat.

Curcumin (diferuloylmethane), an active ingredient of turmeric, is known to have multiple activities, including an antioxidant property, and has been suggested to be of use in treatment of several diseases. The present study has been undertaken to investigate the protective effect of curcumin against lead-induced neurotoxicity in rats. Exposure of rats to lead (50 mg/kg po) for 45 days caused an increase in lipid peroxidation (LPO) and a decrease in reduced glutathione (GSH) levels in cerebellum, corpus striatum, hippocampus and frontal cortex as compared with controls. Lead levels were significantly increased in these rats. Activity of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) decreased in all the brain regions following lead exposure. Interestingly, cotreatment with curcumin (100 mg/kg po) and lead (50 mg/kg po) for 45 days caused a significant decrease in LPO with concomitant decrease in lead levels in all the brain regions as compared with those treated with lead alone. A significant increase in reduced glutathione (GSH) levels, SOD and CAT activities was also observed in all the four brain regions in rats simultaneously treated with curcumin and lead. The results suggest that curcumin may prevent lead-induced neurotoxicity.

Protective effect of acorus calamus against acrylamide induced neurotoxicity.

Exposure of rats to acrylamide (ACR) caused hind limb paralysis in 58% of the animals on day 10 and decreased behavioural parameters, namely distance travelled, ambulatory time, stereotypic time and basal stereotypic movements compared with the control group. These rats also had a decrease in the reduced glutathione (GSH) content and glutathione-S-transferase (GST) activity in the corpus striatum and an increase in striatal dopamine receptors, as evident by an increase in the binding of 3H-spiperone to striatal membranes. Treatment with the ethanol:water (1:1) extract of the rhizomes of Acorus calamus (AC-002) increased the GSH content and GST activity in the corpus striatum while insignificant changes were observed in other parameters. Rats treated with ACR and AC-002 in combination had a lower incidence of paralysis (18%) compared with those treated with ACR alone on day 10 of the experiment. The rats also showed a partial recovery in other behavioural parameters. The levels of GSH content and GST activity increased in the corpus striatum, while the dopamine receptors decreased compared with the ACR treated rats. The results suggest that the neurobehavioural changes produced by ACR may be prevented following treatment with Acorus calamus rhizomes.