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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(P<0.05);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(P<0.05),且慢性酒精组升高幅度明显大于2月龄及9月龄对照组(P<0.05)。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(P<0.05)。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。
OBJECTIVE: To investigate the effect of supernatant recovered from lactobacillus bulgaricus culture MRS broth (LBG-S) on Toll-like receptor 4 (TLR4) -TANK binding in chronic alcohol-induced rat intestinal epithelial cells The role of TANK binding kinase-1 (TBK1) signaling pathway. Methods: Thirty male Wistar rats (2 months old and weighing 250-300 g) were divided into three groups: control group (2 months old) Free to eat the standard diet and drinking double distilled water in July; chronic alcohol group, 9 months old, drinking double distilled water containing 25% ethanol for 6 consecutive months. After the rats were sacrificed, Escherichia coli and Lactobacillus were isolated from the small intestine mucosa of rats in each group. Small intestine epithelial cells of rats in each group were isolated and cultured. After pretreatment with or without LBG-S (10μg / mL) After stimulation with lipopolysaccharide (LPS, 10 EU / m L), the levels of TLR4 and TBK1 in epithelial cells of small intestine of rats in each group were detected by Western blot. The expressions of TLR4 and TBK1 in epithelial cells of small intestine in each group were detected by enzyme-linked immunosorbent assay Tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in cell culture supernatants. Results: After chronic drinking, the number of Lactobacillus in the intestine of rats was significantly lower than that of the corresponding control group (P <0.05); the number of E. coli did not increase significantly. LPS significantly increased the levels of TLR4, TBK1 and TNF-α and IFN-γ in rat intestinal epithelial cells (P <0.05), and the increase in chronic alcohol group was significantly greater than that of 2-month-old and 9-month- Group (P <0.05). Pretreatment with LBG-S significantly inhibited the upregulation of TLR4, TBK1, TNF-α and IFN-γ induced by LPS in isolated rat intestinal epithelial cells (P <0.05). CONCLUSION: Chronic alcohol intake leads to high sensitivity of TLR4-TBK1 pathway to LPS in rat intestinal epithelium, and LBG-S can significantly inhibit this hypersensitivity.