乳酸杆菌培养上清抑制慢性酒精大鼠小肠上皮TLR4-TBK1通路

目的:探讨保加利亚乳酸杆菌培养上清(supernatant recovered from lactobacillus bulgaricus culture MRS broth,LBG-S)对慢性酒精摄入大鼠小肠上皮细胞Toll样受体4(Toll-like receptor 4,TLR4)-TANK结合激酶-1(TANK binding kinase-1,TBK1)信号通路的作用。方法:雄性健康Wistar大鼠30只(2月龄,体重250~300 g)分为三组:2月龄对照组(即基线对照组),顺应1周后即处死;9月龄对照组,自由取食标准鼠粮及饮用双蒸水7月;慢性酒精组,9月龄,饮用含25%乙醇的双蒸水连续6月。处死大鼠后,分离培养并计数各组大鼠小肠黏膜中的大肠杆菌和乳酸杆菌;分离并培养各组大鼠的小肠上皮细胞,在有或无LBG-S(10μg/m L)预处理的情况下,给予脂多糖(lipopolysaccharide,LPS,10 EU/m L)刺激后,Western blot检测各组大鼠小肠分离上皮细胞中的TLR4及TBK1水平,酶联免疫吸附法检测各组分离小肠上皮细胞培养上清中的肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和干扰素-γ(interferon-γ,IFN-γ)。结果:慢性饮酒后,大鼠肠腔内乳酸杆菌数量明显低于相应对照组(P0.05);大肠杆菌数量无明显增加。LPS能明显升高各组分离大鼠小肠上皮细胞TLR4、TBK1以及生成TNF-α和IFN-γ的水平(P0.05),且慢性酒精组升高幅度明显大于2月龄及9月龄对照组(P0.05)。LBG-S的预处理能明显抑制LPS对各组分离大鼠小肠上皮细胞TLR4、TBK1以及生成TNF-α和IFN-γ水平的上调作用(P0.05)。结论:慢性酒精摄入导致大鼠小肠上皮TLR4-TBK1通路对LPS的高敏感性,LBG-S能明显抑制这一高敏感性。

The Culture Supernatant of Lactobacilli Inhibits Intestinal EpithelialTLR4-TBK1 Pathway in Rats Chronically Exposed to Ethanol

Objective:To determine whether the supernatant recovered from Lactobacillus bulgaricus culture MRS broth (LBG-S)inhibits intestinal epithelial Toll-like receptor 4 (TLR4)-TANK binding kinase-1 (TBK1) pathway in rats chronically exposed to ethanol.Methods:Thirty healthy male Wistar rats, 2 months old, 250~300 g, were randomly divided into three groups: 10 rats killed immediatelyafter acclimation (baseline control), 10 rats treated with 25% (vol/vol) ethanol for 6 months (ethanol group), and 10 rats givendouble-distilled water and killed simultaneously with the ethanol group (9-month control). The amounts of intestinal mucosal E.coli and Lactobacilli were evaluated in each group. Isolated intestinal epithelia from each rat were used to examine lipopolysaccharide(LPS) responsiveness with or without LBG-S pretreatment by quantification of TLR4, TBK1, interferon-r(IFN-r) and tumor necrosisfactor-alpha(TNF-alpha).Results:Compared with both control groups, the amount of mucosal Escherichia coli in the ethanol group was notchanged, whereas the number of intestinal Lactobacilli in the ethanol group was significantly reduced (P<0.05). In all groups, LPStreatment significantly up-regulated the expression of TLR4, TBK1 and production of TNF-alpha and IFN-gama in isolated intestinal epithelia(P<0.05). The enhancements of expression of TLR4, TBK1 and production of TNF-alpha and IFN-gama by LPS treatment in isolated intestinalepithelia of the ethanol group were significantly higher than those in either the baseline control or 9-month control group (P<0.05). TheLPS-enhanced expression of TLR4, TBK1 and production of IFN-gama and TNF-alpha in isolated intestinal epithelia in all groups weredramatically inhibited by LBG-S (P<0.05).Conclusion:LBG-S inhibits the hypersensitivity of intestinal epithelia to LPS via up regulatedTLR4-TBK1 pathway in rats chronically exposed to ethanol.