间歇低氧对大鼠肠道细菌移位发生及肠系膜淋巴结结构的影响

Effects of intermittent hypoxia intestinal bacterial translocation on mesenteric lymph node injury

目的观察间歇低氧对肠道细菌移位及肠系膜淋巴结(MLN)结构的影响,探索其机制。方法将成年雄性Wister大鼠24只按照随机数字表法分为实验组及对照组各12只,实验期间两组以相同条件饲养,实验组模拟睡眠呼吸暂停给予间歇低氧处理。实验开始后第2、4周的最后1天,使用20%乌拉坦按照0.7 ml/100 g剂量麻醉大鼠,剖腹后无菌方式取MLN及距回盲瓣2 cm以上的小肠组织,HE染色观察组织微观结构,MLN无菌研磨液进行病原学培养,以ELISA法检测研磨液超氧化物歧化酶(SOD)、脂质过氧化物(MDA)及活性氧(ROS),评估氧化应激反应程度;采集中心静脉血液检测血清二胺氧化酶(DAO),评估肠道黏膜损伤程度。结果实验组大鼠MLN及其对应小肠肠管均有损伤,随着间歇低氧时间的延长,光镜下可见:MLN生发中心数目显著减少,体积减小,皮质髓质融合加重,面积比下降,肠管组织表现出肠上皮受损严重,通透性增高,黏膜水肿,肠绒毛高度、厚度、面积及隐窝深度明显下降等。第4周实验组大鼠MLN厌氧状态下培养出产气荚膜梭菌,证实出现肠道细菌移位。第2周对照组大鼠MLN组织的ROS、SOD及MDA含量分别为(177±9)U/ml、(5.20±0.43)U/ml及(0.95±0.22)nmol/ml,第4周对照组为(176±9)U/ml、(5.19±0.43)U/ml及(0.93±0.16)nmol/ml,组间比较差异无统计学意义(P>0.05);第4周实验组大鼠MLN组织ROS[(284±13)U/ml]、MDA[(2.30±0.34)nmol/ml]明显高于第2周[(217±21)U/ml、(1.17±0.24)nmol/ml],差异有统计学意义(P<0.05),但SOD变化情况不明显[(5.98±0.43)、(5.99±0.43)U/ml,P>0.05];与对照组相比,实验组大鼠MLN组织ROS、SOD及MDA含量均较同周期对照组大鼠有明显升高(P<0.05)。实验组大鼠在第2、4周血清DAO水平均高于对照组(P<0.05)。提示实验组大鼠肠道黏膜损伤程度较对照组严重。结论低氧暴露可加重大鼠机体氧化应激反应的程度,随着间歇低氧时间逐渐延长,大鼠肠道黏膜出现严重损伤,肠道菌群移位加重对肠系膜淋巴结结构损伤,氧化应激进一步加重,进而影响肠道自身免疫功能的完整。

Objective To observe the effect of intermittent hypoxia on intestinal bacterial translocation and mesenteric lymph node(MLN)structure and explore its mechanism.Methods Twenty-four adult male Wistar rats were randomly divided into an experimental group(HI group)and a control group(UC group),with 12 rats in each.During the experiment,both groups were fed under the same conditions,but the HI group received simulated sleep apnea with hypoxic treatment.On the last day of the 2nd and 4th week of the experiment,20%urethane(0.7 ml/100g)was used for anesthesia,and MLNs and corresponding small intestinal tissues were aseptically collected.HE staining was used to observe the microscopic changes of the tissues.The lymph node tissue was sent for pathogenic culture.The levels of oxide dismutase(SOD),lipid peroxide(MDA)and reactive oxygen species(ROS)were measured for the extent of oxidative stress.Serum diamine oxidase(DAO)was measured to assess the extent of intestinal mucosal damage.Result MLNs and their corresponding small intestines were damaged in the HI group as compared to the UC group.With the prolongation of intermittent hypoxic time,the number of germinal centers in MLNs was significantly reduced,with the volume reduced,cortical medullary fusion aggravated,and the area ratio increased.The intestinal tissue showed severe damage to the intestinal epithelium,increased permeability,mucosal edema,and changes of the crypts.At the 4th week,MLNs in the HI group grew Clostridium perfringens under anaerobic conditions,confirming intestinal bacterial translocation.The contents of ROS,SOD and MDA in MLNs of the HI group were significantly higher than those in the UC group(P<0.05).At the 2nd week and the 4th week,the contents of ROS,SOD and MDA were not significantly changed in the UC group(P>0.05).While the content of ROS and MDA in MLNs of the HI group at 4th week was significantly higher than that in the second week(P<0.05),but no change of SOD was observed(P>0.05).Serum DAO levels in the HI group were higher than those in the UC group at week 2 and week 4(P<0.05),suggesting that the degree of intestinal mucosal injury in the HI group was more serious than that in the UC group.Conclusion Hypoxic exposure aggravated the degree of oxidative stress in rats.With the prolongation of intermittent hypoxia,the intestinal mucosa of rats was seriously damaged.The intestinal flora shifted to damage the structure of mesenteric lymph nodes,and oxidative stress was further aggravated,which in turn affected the integrity of the intestinal autoimmune function.