肠道菌群在体外循环心脏手术后围手术期神经认知障碍中的作用:基于菌群人源化大鼠模型

Role of gut microbiota in perioperative neurocognitive disorders after cardiopulmonary bypass surgery in rats with humanized gut flora

目的通过肠道菌群人源化大鼠模型探讨体外循环(CPB)后肠道菌群紊乱可否通过激发炎症反应参与围手术期神经认知障碍(PND)的发生。方法SPF级成年雄性SD大鼠30只随机分为3组(n=10):伪无菌大鼠(S)组、伪无菌大鼠+接种健康人源粪菌滤液(NP)组和伪无菌大鼠+接种PND患者粪菌滤液(P)组。造模成功后对所有组别大鼠粪便进行宏基因组学检测,Elisa法测定血清炎症因子IL-1β、IL-6及TNF-α的含量,Western-blot法检测海马组织中GFAP、p-Tau蛋白的表达水平,Morris水迷宫实验评估造模后学习和记忆等认知功能。结果宏基因组学测序结果显示粪菌移植后3组大鼠肠道菌群各自聚集,分区良好;S组克雷伯氏菌属(Klebsiella)(P<0.005)、P组阿克曼菌属(Akkermansia)(P<0.005)、NP组拟杆菌属(Bacteroides)(P<0.005)相对丰度显著增高,差异有统计学意义;与S组相比,NP组和P组IL-1β(P<0.05)、IL-6(P<0.05)和TNF-α(P<0.05)的浓度显著降低,GFAP(P<0.05)、p-Tau(P<0.05)蛋白表达水平均下降,NP组逃生平台穿越次数(P<0.05)和兴趣象限停留时间(P<0.05)均明显增加,P组逃生平台穿越次数(P>0.05)和兴趣象限停留时间(P>0.05)均增加,但无统计学差异;与P组相比,NP组IL-1β(P<0.05)、IL-6(P<0.05)和TNF-α(P<0.05)的浓度显著降低,GFAP(P<0.05)、p-Tau(P<0.05)蛋白表达水平均下降,逃生平台穿越次数(P<0.05)和兴趣象限停留时间(P<0.05)均明显增加。结论肠道菌群的差异性组成参与了CPB心脏手术后PND的发生,其机制可能与炎症反应的发生相关。

Objective To investigate whether gut microbiota disturbance after cardiopulmonary bypass(CPB)contributes to the development of perioperative neurocognitive disorders(PND).Methods Fecal samples were collected from healthy individuals and patients with PND after CPB to prepare suspensions of fecal bacteria,which were transplanted into the colorectum of two groups of pseudo-germ-free adult male SD rats(group NP and group P,respectively),with the rats without transplantation as the control group(n=10).The feces of the rats were collected for macrogenomic sequencing analysis,and serum levels of IL-1β,IL-6 and TNF-αwere measured with ELISA.The expression levels of GFAP and p-Tau protein in the hippocampus of the rats were detected using Western blotting,and the cognitive function changes of the rats were assessed with Morris water maze test.Results In all the 3 groups,macrogenomic sequencing analysis showed clustering and clear partitions of the gut microbiota after the transplantation.The relative abundances of Klebsiella in the control group(P<0.005),Akkermansia in group P(P<0.005)and Bacteroides in group NP(P<0.005)were significantly increased after the transplantation.Compared with those in the control group,the rats in group NP and group P showed significantly decreased serum levels of IL-1β,IL-6 and TNF-αand lowered expression levels of GFAP and p-Tau proteins(all P<0.05).Escape platform crossings and swimming duration in the interest quadrant increased significantly in group NP(P<0.05),but the increase was not statistically significant in group N.Compared with those in group P,the rats in group NP had significantly lower serum levels of IL-1β,IL-6 and TNF-αand protein expressions of GFAP and p-Tau(all P<0.05)with better performance in water maze test(P<0.05).Conclusion In patients receiving CPB,disturbances in gut mirobiota contributes to the development of PND possibly in relation with inflammatory response.