摘要
目的探讨大豆来源的蛋白酶抑制剂(BBI)是否能阻断脂多糖(LPS)对肠道上皮细胞间紧密连接蛋白的下调作用及其机制。方法用CCK8试剂盒检测LPS和BBI对HT-29细胞的毒性作用。用BBI预处理HT-29细胞6 h,再用LPS刺激,分别用实时荧光定量PCR和蛋白质印迹法(Western Blot)检测细胞紧密连接蛋白(ZO-1,Occludin)、TLR4及MyDD8的表达;通过Western Blot检测NF-κB的激活。结果LPS 1 000 ng/mL和BBI1 000μg/mL对HT-29细胞均无毒性作用。LPS可显著地上调HT-29细胞TLR4表达,且上调作用具有时间与剂量效应;能明显下调紧密连接蛋白的表达,其下调作用与LPS浓度成正比;能明显激活NF-κB,且具有剂量效应;LPS对HT-29细胞的这种作用可被BBI显著地抑制。结论通过抑制LPS诱导的肠道上皮细胞TLR4的表达和NF-κB的活化,BBI能显著地阻断LPS对肠道上皮细胞间紧密连接蛋白的抑制作用。
Objective To evaluate the blocking effect and mechanism of Soybean-derived Bowman-Birk inhibitor(BBI)on LPS-mediated downregulation for tight junction protein(HT-29 cells)in intestinal epithelial cells(IECs).Methods The toxic effect of LPS and BBI on HT-29 cells was detected by CCK8 Kit.HT-29 cells were pretreated by BBI for 6 hours prior to LPS stimulation,the expression of tight junction protein(ZO-1 and Occludin),TLR4,and MyDD8 was detected by the quantitative real-time polymerase chain reaction(PCR)and Western Blot;activation of NF-κB was measured by Western Blot.Results LPS(1 000ng/mL)and BBI(1 000μg/mL)showed no cytotoxicity on HT-29 cells.LPS could significantly upregulate the expression of TLR4 in HT-29 cells,the up-regulation had time-dose effect,and could significantly downregulate the expression of tight junction protein,the down-regulation effect was directly proportional to the concentration of LPS,could activate NF-κB,and had dose effect,effect of LPS on HT-29 cells could be significantly inhibited by BBI.Conclusion By inhibiting the expression of TLR4 and activation of NF-κB in IECs induced by LPS,BBI can significantly block the LPS-mediated inhibitory effect on tight junction protein in intestinal epithelial cells.
作者
古俊
刘金彪
霍文哲
GU Jun;LIU Jin-biao;HUO Wen-zhe(School of Basic Medical Science,Wuhan University,Wuhan 430071,China)
出处
《中国感染控制杂志》
CAS
北大核心
2018年第3期185-190,共6页
Chinese Journal of Infection Control
基金
国家自然科学基金(81271334
81571962)
国家传染病防治重大科技专项(2012ZX10004501-001-004)
