| Tight junctions (TJ) create a highly selective barrier that normally prevents the passage of harmful molecules, such as toxins and pathogens across the intestinal mucosa and into the interstitial tissue and circulation. The disruption of TJ and the loss of epithelial barrier function increase the intestinal permeability to injurious factors play a crucial role in the pathogenesis of a variety of gastrointestinal disorders, such as inflammatory bowel disease (IBD), obstructive jaundice (OJ), acute pancreatitis (AP), alcohol-induced liver injury, infectious enterocolitis and necrotizing enterocolitis (NEC) and so on. The mechanism involved in the barrier dysfunction in different diseases is not well understood. However, a growing body of evidence indicated that oxidative stress was an important factor induced disruption of TJ and barrier function.Hydrogen peroxide (H2O2) is constantly being generated within all cell types during the normal aerobic metabolism, and must be quickly detoxified by antioxidant defense enzymes, such as catalase in peroxisomes and glutathione peroxidase in mitochondria. If H2O2 is not detoxified quickly, amount of H2O2 would induce cells injure. H2O2 at low micro molar levels is poorly reactive with biological systems. However, at higher concentrations, H2O2 can inactivate glyceraldehyde-3-phosphate dehydrogenase, a glycolytic enzyme. A large number of studies have now indicated that H2O2 disrupted intestinal epithelial barrier function leading to elevate paracellular permeability. In Caco-2 cell monolayers, it was demonstrated that disruption of barrier function by oxidative stress was mediated by H2O2.Heme oxygenase-1 (HO-1), a ubiquitous and redox-sensitive inducible stress protein that degrades heme to CO, iron and biliverdin, it can be induced by various oxidative-inducing agents, including H2O2, heme, heavy metals, cytokines and endotoxin. Recently, numerous in vitro and in vivo studies have shown that the induction of HO-1 was an important cellular protective mechanism against oxidative injury. Curcumin (Cur) is a major active component of the food flavor turmeric, isolated from the powdered dry rhizome of Cur longa Linn. The component possesses a variety of pharmacological activities, including anti-oxidative, anti-inflammatory, anti-carcinogenic and anti-HIV and so on. Cur is a naturally occurring compound that has been identified as potent inducers of HO-1.Objective: To explore the protective effects of Cur on oxidative stress-induced disruption of TJ and barrier dysfunctions.Methods: Caco-2 cell monolayers were incubated without or with H2O2 (500μM) in the absence or presence of 5μM, 20μM, 80μM of Cur (administered 30min prior to H2O2) for 6h. In some experiments, cells were pretreated with Znpp (specific inhibitors of the HO-1) for 12h prior to Cur. DMSO group was incubated with DMEM without H2O2. In order to examine if the protective effect of Cur on TJ is time-dependent, Caco-2 cells were treated with Cur at 20μM (30 min prior to H2O2 administration) for 1h, 3h, 6h, but H2O2 was washed after cell monolayers were treated with H2O2 for 1h. The medium was harvested to measure LDH activity, MDA and SOD activity for cytotoxic and oxidative stress effects; The trans-epithelial electrical resistance (TEER) and the flux of sodium fluorescein of the filter-grown Caco-2 cell monolayers were measured to examine intestinal barrier functions; The expression and localization of TJ protein occludin and ZO-1 were measured by western blot and immunofluorescence microscopy; The protein and mRNA levels of HO-1 were analyzed by western blot and real-time PCR.Results:1 TEER and the flux of sodium fluorescein established in vitro intestinal epithelial barrier models with Caco-2 cells.2 Cur attenuated H2O2-induced cytotoxicity and oxidative stress in Caco-2 cells.①Incupation of Caco-2 cells with 500μM H2O2 for 6h resulted in dramatic increase in LDH (P<0.001). Pretreatment with Cur (5, 20 and 80μM) afforded cytoprotection against H2O2-induced cytotoxicity (P<0.05). However, the protective effect of each concentration was similar (P>0.05).②Incupation of Caco-2 cells with H2O2 resulted in dramatic increase in MDA (P<0.001). Pretreatment with Cur (5, 20 and 80μM) afforded cytoprotection against H2O2-induced increase in MDA (P<0.001). However, the protective effect of each concentration was similar (P>0.05). The SOD activity was contrary to MDA changes.3 Cur attenuated H2O2-induced disruption of paracellular permeability in Caco-2 cells.①H2O2 increased the flux of sodium fluorescein permeability compared to DMSO group (32.00±4.97% vs 6.10±1.43%, P<0.001). Pretreatment with Cur (5, 20 and 80μM) afforded cytoprotection against H2O2-induced monolayer hyper-permeability of Caco-2 cell monolayer (20.25±2.63%, 12.41±2.19%, 19.25±2.81% vs 32.00±4.97%, P<0.05).②H2O2 decreased TEER% compared to DMSO group (22.71±3.11% vs 99.60±1.30%, P<0.001). Pretreatment with Cur (5, 20 and 80μM) afforded cytoprotection against H2O2-induced decrease (49.05±13.99%, 52.02±10.15%, 43.24±12.26% vs 22.71±3.11%, P<0.05). The protective effect of Cur was dose-dependent and the best was seen at 20μM (P<0.05).4 Cur ameliorated H2O2-induced TJs distruption in Caco-2 cells.①H2O2 induced occludin protein decrease compared to DMSO group (53.67±24.19% vs 178.00±29.51%, P<0.001), but pretreatment with Cur (5, 20 and 80μM) prevented H2O2-induced decrease in occluding expression (115.00±18.03%, 175.67±29.50%, 120.67±32.72% vs 53.67±24.19%, P<0.05). The changes of ZO-1 protein expression were similar to occludin. H2O2 induced ZO-1 protein decrease compared to DMSO group (36.00±15.88% vs 133.00±26.46%, P<0.001), but pretreatment with Cur (5, 20 and 80μM) prevented H2O2-induced decrease in ZO-1 expression (90.33±22.50%, 139.67±33.71%, 92.00±24.33% vs 36.00±15.88%, P<0.05). The effect of Cur was dose-dependent and the best was seen at 20μM (P<0.05). Znpp could block the effect of Cur, the expressin of occludin (80.33±13.05%) and ZO-1 (50.00±15.13%) were similar to H2O2 induced changes (P>0.05). Cur at 20 μM was administed (30min prior to H2O2 administration) for 1h, 3h, 6h and H2O2 was washed after cells were treated with H2O2 for 1h. The control cells were treated with H2O2 for 1h. The occludin protein expression at indicated times were increased compared to control cells (185.00±51.64%, 300.67±27.10%, 424.33±69.66% vs 84.00±19.00%, P<0.05), the results of ZO-1 were similar to occludin expression. The ZO-1 protein expression at indicated times were increased compared to control cells (48.67±20.01%, 82.00±12.12%, 114.67±20.50% vs 15.00±12.52%, P<0.05). The protein levels of occludin and ZO-1were time-dependent manner and the highest levels were at 6h.②The localization of TJ proteins occludin and ZO-1 were measured by immunofluorescence microscopy assay. In the DMSO group, the expression of occludin and ZO-1 are integrity, and the fluorescence intensity was high. Loss of occludin was evident when H2O2 was administed for 6h, and the fluorescence intensity was lower. Pretreated with Cur at 20μM significantly prevented the disturbance of the TJ proteins. ZnPP could block the protective effect of Cur on H2O2-induced redistribution of the TJ proteins. The same as treated by H2O2. The changes of ZO-1 were similar to occludin. 5 Cur induced HO-1 of protein and mRNA expression in Caco-2 cells.①H2O2 induced HO-1 protein increase compared to control cells (99.00±10.00% vs 4.67±2.00%, P<0.001), but pretreatment with Cur (5, 20 and 80μM) significantly increased HO-1 protein expression compared to H2O2-induced increase (198.67±87.96%, 291.00±9.17%, 191.33±37.11% vs 99.00±10.00%, P<0.05). The effect of Cur was dose-dependent and the best was seen at 20μM (P<0.05). 20μM Cur was administed (30min prior to H2O2 administration) for 1h, 3h, 6h and H2O2 was washed after cells were treated with H2O2 for 1h. The HO-1 protein expression at indicated times were increase compared to control cells (149.33±6.81%, 263.67±16.50%, 372.33±102.34% vs 43.67±10.60%, P<0.05). The value was highest at 6h (P<0.05).②The HO-1 mRNA levels of HO-1 were measured by real time PCR assay. H2O2 induced HO-1 mRNA increase compared to control cells (P<0.05), but pretreatment with Cur (5, 20 and 80μM) significantly increased HO-1 mRNA compared to H2O2-induced increase (5.98±0.91, 7.58±1.55, 6.66±1.48 vs 3.84±0.90, P<0.05). The protective effect was similar at indicated concentrations (P>0.05). Znpp group (4.41±1.21) could block the effect of Cur (P<0.05). 20μM Cur was administed (30min prior to H2O2 administration) for 1h, 3h, 6h and H2O2 was washed after cells were treated with H2O2 for 1h. The HO-1 mRNA levels changes were similar to protein expression. The levels of HO-1 mRNA induced by Cur was observed at 1h and increased with time up to 6h of treatment(3.92±0.79, 4.34±1.03, 4.95±1.14 vs 1.00±0.00, P<0.05). Which were dramatic increased compared to cells treated with H2O2 for 1h.Conclusions: H2O2 could induce cytotoxic and oxidative stress effects. Cur attenuated H2O2-induced oxidative stress and cytotoxicity at low concentrations. Furthermore, Cur ameliorated H2O2-induced disruption of TJ and barrier dysfunctions in human intestinal epithelial cells via HO-1 pathway.
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