肠道菌群紊乱在老龄小鼠围术期神经认知障碍中的作用

Role of intestinal flora disturbance in perioperative neurocognitive disorders in aged mice

目的评价肠道菌群紊乱在老龄小鼠围术期神经认知障碍中的作用。方法SPF级健康雄性C57BL/6J小鼠60只,18月龄,采用随机数字表法分为4组(n=15):对照组(C组)、手术组(O组)、手术+鼠李糖乳杆菌组(OL组)和手术+粪菌移植组(OF组)。O组、OL组和OF组行剖腹探查术。PL组术毕立即灌胃给予鼠李糖乳杆菌200μl,1次/d,连续10 d,小鼠每天约可获得0.2×10^9 CFU益生菌。OF组术前7周时,将广谱抗生素混合液(氨苄西林舒巴坦1.5 g/L、万古霉素500 mg/L、环丙沙星200 mg/L、亚胺培南西司他丁250 mg/L和甲硝唑1 g/L)加入小鼠饮用水中,并于术前72 h时更换为无菌自来水;术毕立即灌胃给予粪菌滤液200μl,1次/d,连续10 d。术后10 d时,每组处死5只小鼠,采用伊文思蓝渗漏实验检测空肠和回肠的血管通透性。术后10 d时,每组处死5只小鼠,取小肠、海马组织和眼眶静脉血标本,采用ELISA法检测IL-6、IL-17、TNF-α、IFN-γ、IL-4和IL-10的水平。术后10 d时,每组取5只小鼠,采用Morris水迷宫实验测定小鼠认知功能。结果与C组比较,O组空肠和回肠血管通透性升高,小肠、外周血和海马IL-6、IL-17、TNF-α和IFN-γ的水平升高,IL-4和IL-10的水平降低,游泳距离和逃避潜伏期延长,目标象限停留时间缩短(P<0.05或0.01),OL组和OF组上述各指标差异无统计学意义(P>0.05);与O组比较,OL组和OF组空肠和回肠血管通透性降低,小肠、外周血和海马IL-6、IL-17、TNF-α和IFN-γ的水平降低,IL-4和IL-10的水平升高,游泳距离和逃避潜伏期缩短,目标象限停留时间延长(P<0.05)。结论肠道菌群紊乱可介导海马炎症反应,导致老龄小鼠围术期神经认知障碍。

Objective To evaluate the role of intestinal flora disturbance in perioperative neurocognitive disorders in aged mice.Methods Sixty SPF healthy male C57BL/6J mice,aged 18 months,were divided into 4 groups(n=15 each)by a random number table method:control group(group C),operation group(group O),operation plus lactobacillus rhamnosus group(group OL)and operation plus fecal microbiota transplantation group(group OF).Exploratory laparotomy was performed in O,OL and OF groups.In group PL,lactobacillus rhamnosus 200μl(1×10^9 CFU/ml,200μl/day)was given by gavage once a day for 10 days starting from the end of surgery,and mice received about 0.2×10^9 CFU probiotics per day.In group OF,broad-spectrum antibiotic mixture(ampicillin and sulbactam 1.5 g/L,vancomycin 500 mg/L,ciprofloxacin 200 mg/L,imipenem cilastatin 250 mg/L and metronidazole 1 g/L)was added to the drinking water at 7 weeks prior to operation and replaced with sterile tap water at 72 h before operation,and fecal filtrates 200μl was given by gavage once a day for 10 days starting the end of operation.Five mice were sacrificed at day 10 after operation in each group,and Evans blue extravasation test was used to measure the vascular permeability of jejunum and ileum.Five mice were sacrificed at day 10 after operation in each group,and the small intestinal and hippocampal tissues and orbital venous blood samples were obtained for determination of interleukin-6(IL-6),IL-17,tumor necrosis factor-alpha(TNF-α),interferon gamma(IFN-γ),IL-4 and IL-10 levels by enzyme-linked immunosorbent assay.Five mice were selected at day 10 after operation in each group,and the Morris water maze test was used to assess the cognitive function.Results Compared with group C,the vascular permeability of jejunum and ileum was significantly increased,the levels of IL-6,IL-17,TNF-αand IFN-γin the small intestine,peripheral blood and hippocampus were increased,the levels of IL-4 and IL-10 were decreased,the swimming distance and escape latency were prolonged,and the time spent in the target quadrant was shortened in group O(P<0.05 or 0.01),and no significant change was found in the parameters mentioned above in OL and OF groups(P>0.05).Compared with group O,the vascular permeability of jejunum and ileum was significantly decreased,the levels of IL-6,IL-17,TNF-αand IFN-γin the small intestine,peripheral blood and hippocampus were decreased,the levels of IL-4 and IL-10 were increased,the swimming distance and escape latency were shortened,and the time spent in the target quadrant was prolonged in OL and OF groups(P<0.05).Conclusion Intestinal flora disturbance can mediate inflammatory responses in the hippocampus and cause perioperative neurocognitive disorders in aged mice.