干酪乳杆菌补充通过LPS-TLR4-NFκB信号通路对抗结核药物所致肝损伤作用的机制研究

The incidence of antituberculosis drug-induced liver injury (ATDILI) was high. There were still a lack of additional measures to protect liver without adverse reactions. In our preliminary animal experiments, it was found that lactobacillus casei could improve ATDILI to a certain extent, but the mechanism was not clear. The activated TLR4 can initiate the inflammatory cytokines dependent on NFκB, which was the membrane receptor of LPS in hepatic cell. The intestinal microecology that can be destroyed by isoniazide and rifampicin produced a large number of LPS to cause or aggravate liver injury. Lactobacillus casei can reduce LPS by improving intestinal disorder. Also it might affect the expression of negative regulatory factors in the LPS-TLR4-NFκB pathway to regulate activation of signals. Therefore, there were 72 male Wistar rats for 8 weeks intervention in this study, which were divided into blank control group, model group, positive control group, low, middle and high dose of lactobacillus casei groups. The indexes of liver function and colon intestinal barrier were detected. The changes of intestinal flora were analyzed by collecting rat feces. The expressions of activation or inhibition proteins and mRNA of LPS-TLR4-NFκB pathway in the liver and spleen were detected to explore whether the intervention of lactobacillus casei can play a protective role in ATDILI through this pathway, and propose a new way to repair ATDILI.

抗结核药物所致肝损伤（ATDILI）的发生率较高，但目前仍缺乏无不良反应的辅助保肝措施。我们的前期动物实验发现，干酪乳杆菌能在一定程度上改善ATDILI程度，可相关机制尚不明确。TLR4是LPS的肝细胞膜受体，激活后能启动NFκB依赖的炎性细胞因子，导致肝损伤。异烟肼和利福平联用可破坏肠道微生态，产生大量LPS而引发或加重肝损伤。而干酪乳杆菌可通过改善肠道紊乱减少LPS产生；还可能影响LPS-TLR4-NFκB通路的负性调节因子表达来调控信号活化。故拟选取SPF级雄性Wistar大鼠72只，分为空白对照组、模型组、阳性对照组、低、中和高剂量乳杆菌组，干预8周。检测肝功能和结肠肠道屏障功能指标；收集大鼠粪便分析肠道菌群变化；测定肝脾LPS-TLR4-NFκB通路激活或抑制的相关蛋白及mRNA表达。探讨干酪乳杆菌干预是否通过该通路对ATDILI发挥保护作用，提出辅助修复ATDILI的新思路。

我国抗结核药物所致肝损伤（ATDILI）的发生率较高，是导致抗结核治疗中断或失败、产生耐药性的重要原因。从营养途径探寻对ATDILI 起保护作用的辅助方案及机制对结核病患者治疗和康复具有临床和社会意义。.本项动物研究建立了抗结核药物（异烟肼和利福平）所致肝损伤的大鼠和小鼠模型，雄性Wistar大鼠60只随机分为5组（12只/组），干预6周：正常对照组、模型组、阳性对照组、低和高剂量干酪乳杆菌组；雄性C57BL/6J小鼠72只随机分为6组（12只/组），干酪乳杆菌分为高、中、低3个剂量组，其他三组同大鼠实验，干预8周。.观察抗结核药物对肝脏及肠道功能的影响，结果发现，抗结核药物可造成大鼠和小鼠不同程度肝损伤（细胞水肿和部分区域脂肪变形为主）及肠道细胞轻微受损；小鼠结肠紧密连接蛋白Claudin 1表达量、MUC2和sIgA水平、LZM活性较正常对照组均明显下降；肠道菌群多样性分析认为干预2周时菌群变化最为明显，α及β多样性结果均显示抗结核药物使肠道菌群多样性降低，微生态发生紊乱。分析干酪乳杆菌补充对肠道屏障及对肝损伤的改善效果，认为干酪乳杆菌干预后，高剂量组可使肝脏和肠道组织病理结构部分改善，而低剂量改善效果不明显；还可使Claudin 1蛋白表达量和MUC2水平显著升高；2周时肠道微生态轻微恢复，而6周时各组肠道菌群均恢复稳态。.测定LPS-TLR4-NFκB通路的相关蛋白及mRNA表达，各组间的血清LPS水平均无显著差异，但发现模型组的LPS水平较正常对照组有升高趋势，干酪乳杆菌干预后则下降。抗结核药物使肝脏MyD88蛋白表达量显著升高，干酪乳杆菌则使其显著降低；其他蛋白表达量在各组间虽没有显著差异，但模型组TLR4、NFκB蛋白表达量较对照组有升高趋势，干酪乳杆菌干预后则有降低趋势。模型组脾脏IκBα蛋白表达量明显降低，中、高剂量组的P-IκBα和IκBα蛋白表达量均显著高于模型组。负性调节因子的mRNA表达量在各组间无显著差异。.本研究结果认为抗结核药物应用可对肝功能和肠道屏障功能产生损伤，不同剂量的干酪乳杆菌具有不同程度改善效果。LPS-TLR4-NFκB通路可能是干酪乳杆菌干预的调节通路，但是效果并不明显，仍需进一步加大样本并从其他通路深入探讨。总之，本实验仍为通过肠道功能修复改善肝损伤的辅助机制提供科学依据，并开拓了辅助修复ATDILI的新思路。