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Prophylactic Treatment of Probiotic and Metformin Mitigates Ethanol-Induced Intestinal Barrier Injury: In Vitro, In Vivo, and In Silico Approaches.

ABSTRACT: Ethanol depletes intestinal integrity and promotes gut dysbiosis. Studies have suggested the individual role of probiotics and metformin Met in protecting intestinal barrier function from injuries induced by ethanol. The objective of the current study is to investigate the potential mechanism by which coadministration of probiotic Visbiome® (V) and Met blocks the ethanol-induced intestinal barrier dysfunction/gut leakiness utilizing Caco-2 monolayers, a rat model with chronic ethanol injury, and in silico docking interaction models. In Caco-2 monolayers, exposure to ethanol significantly disrupted tight junction (TJ) localization, elevated monolayer permeability, and oxidative stress compared with controls. However, cotreatment with probiotic V and Met largely ameliorated the ethanol-induced mucosal barrier dysfunction, TJ disruption, and gut oxidative stress compared with ethanol-exposed monolayers and individual treatment of either agent. Rats fed with ethanol-containing Lieber-DeCarli liquid diet showed decreased expression of TJ proteins, and increased intestinal barrier injury resulting in pro-inflammatory response and oxidative stress in the colon. We found that co-administration of probiotic V and Met improved the expression of intestinal TJ proteins (ZO-1 and occludin) and upregulated the anti-inflammatory response, leading to reduced ER stress. Moreover, co-administration of probiotic V and Met inhibited the CYP2E1 and NOX gene expression, and increase the translocation of Nrf-2 as well as anti-oxidative genes (SOD, catalase, Gpx, and HO-1), leading to reduced colonic ROS content and malondialdehyde levels. The combined treatment of probiotic V and Met also improved their binding affinities towards HO-1, Nrf-2, SLC5A8, and GPR109A, which could be attributed to their synergistic effect. Our findings based on in-vitro, in-vivo, and in-silico analyses suggest that the combination of probiotic V and Met potentially acts in synergism, attributable to their property of inhibition of inflammation and oxidative stress against ethanol-induced intestinal barrier injury.

SUBMITTER: Patel F

PROVIDER: S-EPMC8490080 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Alcohol liver disease (ALD) is a major cause of liver-related mortality globally, yet there remains an unmet demand for approved ALD drugs. The pathogenesis of ALD involves perturbations to the intestinal barrier and subsequent translocation of bacterial endotoxin that, acting through toll-like receptor 4 (TLR4), promotes hepatic inflammation and progression of ALD. In the present study we investigated the ability of fenretinide (Fen) [N-(4-hydroxyphenyl) retinamide], a synthetic retinoid with known anti-cancer and anti-inflammatory properties, to modulate intestinal permeability and clinical hallmarks of ALD in a mouse model of chronic ethanol (EtOH) exposure. Our results show that EtOH-treated mice had reductions in mRNA and protein expression of intestinal tight junction proteins, including claudin one and occludin, and increases in intestinal permeability and endotoxemia compared to pair-fed mice. Also, EtOH-treated mice had marked increases in hepatic steatosis, liver injury, and expression of pro-inflammatory mediators, including TNF-α, and TLR4-positive macrophages, Kupffer cells, and hepatocytes in the intestines and liver, respectively. In contrast, EtOH + Fen-treated mice were resistant to the effects of EtOH on promoting intestinal permeability and had higher intestinal protein levels of claudin one and occludin. Also, EtOH + Fen-treated mice had significantly lower plasma levels of endotoxin, and reductions in expression of TNF-α and TLR4 positive macrophages, Kupffer cells, and hepatocytes in the intestine and liver. Lastly, we found that EtOH + Fen-treated mice exhibited major reductions in hepatic triglycerides, steatosis, and liver injury compared to EtOH-treated mice. Our findings are the first to demonstrate that Fen possesses anti-ALD properties, potentially through modulation of the intestinal barrier function, endotoxemia, and TLR4-mediated inflammation. These data warrant further pre-clinical investigations of Fen as a potential anti-ALD drug.

Project description:BackgroundIntestinal barrier dysfunction, which is associated with reactive enteric glia cells (EGCs), is not only a result of early sepsis but also a cause of multiple organ dysfunction syndrome. Inhibition of platelet activation has been proposed as a potential treatment for septic patients because of its efficacy in ameliorating the organ damage and barrier dysfunction. During platelet activation, CD40L is translocated from α granules to the platelet surface, serving as a biomarker of platelet activation a reliable predictor of sepsis prognosis. Given that more than 95% of the circulating CD40L originate from activated platelets, the present study aimed to investigate if inhibiting platelet activation mitigates intestinal barrier dysfunction is associated with suppressing reactive EGCs and its underlying mechanism.MethodsCecal ligation and puncture (CLP) was performed to establish the sepsis model. 24 h after CLP, the proportion of activated platelets, the level of sCD40L, the expression of tight-junction proteins, the intestinal barrier function and histological damage of septic mice were analyzed. In vitro, primary cultured EGCs were stimulated by CD40L and LPS for 24 h and EGCs-conditioned medium were collected for Caco-2 cells treatment. The expression of tight-junction proteins and transepithelial electrical resistance of Caco-2 cell were evaluated.ResultsIn vivo, inhibiting platelet activation with cilostazol mitigated the intestinal barrier dysfunction, increased the expression of ZO-1 and occludin and improved the survival rate of septic mice. The efficacy was associated with reduced CD40L+ platelets proportion, decreased sCD40L concentration, and suppressed the activation of EGCs. Comparable results were observed upon treatment with compound 6,877,002, a blocker of CD40L-CD40-TRAF6 signaling pathway. Also, S-nitrosoglutathione supplement reduced intestinal damage both in vivo and in vitro. In addition, CD40L increased release of TNF-α and IL-1β while suppressed the release of S-nitrosoglutathione from EGCs. These EGCs-conditioned medium reduced the expression of ZO-1 and occludin on Caco-2 cells and their transepithelial electrical resistance, which could be reversed by CD40-siRNA and TRAF6-siRNA transfection on EGCs.ConclusionsThe inhibition of platelet activation is related to the suppression of CD40L-CD40-TRAF6 signaling pathway and the reduction of EGCs activation, which promotes intestinal barrier function and survival in sepsis mice. These results might provide a potential therapeutic strategy and a promising target for sepsis.

Project description:BackgroundIntestinal barrier dysfunction, which is associated with reactive enteric glia cells (EGCs), is not only a result of early sepsis but also a cause of multiple organ dysfunction syndrome. Inhibition of platelet activation has been proposed as a potential treatment for septic patients because of its efficacy in ameliorating the organ damage and barrier dysfunction. During platelet activation, CD40L is translocated from α granules to the platelet surface, serving as a biomarker of platelet activation a reliable predictor of sepsis prognosis. Given that more than 95% of the circulating CD40L originate from activated platelets, the present study aimed to investigate if inhibiting platelet activation mitigates intestinal barrier dysfunction is associated with suppressing reactive EGCs and its underlying mechanism.MethodsCecal ligation and puncture (CLP) was performed to establish the sepsis model. 24 h after CLP, the proportion of activated platelets, the level of sCD40L, the expression of tight-junction proteins, the intestinal barrier function and histological damage of septic mice were analyzed. In vitro, primary cultured EGCs were stimulated by CD40L and LPS for 24 h and EGCs-conditioned medium were collected for Caco-2 cells treatment. The expression of tight-junction proteins and transepithelial electrical resistance of Caco-2 cell were evaluated.ResultsIn vivo, inhibiting platelet activation with cilostazol mitigated the intestinal barrier dysfunction, increased the expression of ZO-1 and occludin and improved the survival rate of septic mice. The efficacy was associated with reduced CD40L+ platelets proportion, decreased sCD40L concentration, and suppressed the activation of EGCs. Comparable results were observed upon treatment with compound 6877002, a blocker of CD40L-CD40-TRAF6 signaling pathway. Also, S-nitrosoglutathione supplement reduced intestinal damage both in vivo and in vitro. In addition, CD40L increased release of TNF-α and IL-1β while suppressed the release of S-nitrosoglutathione from EGCs. These EGCs-conditioned medium reduced the expression of ZO-1 and occludin on Caco-2 cells and their transepithelial electrical resistance, which could be reversed by CD40-siRNA and TRAF6-siRNA transfection on EGCs.ConclusionsThe inhibition of platelet activation is related to the suppression of CD40L-CD40-TRAF6 signaling pathway and the reduction of EGCs activation, which promotes intestinal barrier function and survival in sepsis mice. These results might provide a potential therapeutic strategy and a promising target for sepsis.

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Prophylactic Treatment of Probiotic and Metformin Mitigates Ethanol-Induced Intestinal Barrier Injury: In Vitro, In Vivo, and In Silico Approaches.

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