粪菌移植通过肠道菌群调节脓毒症大鼠脑皮质胆碱能抗炎通路

Fecal microbiota transplantation regulates the cholinergic anti-inflammatory pathway in cerebral cortex of septic rats through intestinal microbiota

目的探讨粪菌移植对脓毒症大鼠肠道菌群及胆碱能抗炎通路的影响。方法将60只清洁级雄性SD大鼠按随机数字表法分为生理盐水(NS)对照组、脓毒症模型组和粪菌移植组,每组20只。经尾静脉注射脂多糖(LPS)10 mg/kg制备脓毒症大鼠模型;NS对照组同途径给予等量NS。粪菌移植组于制模后1 d将正常大鼠粪便稀释后灌胃,每次2 mL,每日3次;其余两组给予等量NS灌胃。于制模后7 d采集大鼠粪便,采用16SrDNA基因测序技术检测肠道菌群的变化;监测脑电图变化,计算脑电图异常波形的发生率;处死大鼠取脑组织,采用蛋白质免疫印迹试验(Western Blot)测定脑皮质α7烟碱型乙酰胆碱受体(α7nAChR)蛋白表达,采用免疫组化法检测脑皮质离子钙接头分子-1(Iba-1)阳性表达,采用酶联免疫吸附试验(ELISA)测定脑皮质白细胞介素(IL-6、IL-1β)和肿瘤坏死因子-α(TNF-α)含量。结果制模后7 d,NS对照组大鼠全部存活;脓毒症模型组和粪菌移植组分别死亡10只、8只,死亡率分别为50%、40%。最终,NS对照组n=20,脓毒症模型组n=10,粪菌移植组n=12。与NS对照组比较,模型组大鼠肠道菌群多样性及结构均发生改变,异常脑电图发生率明显升高,脑皮质α7nAchR蛋白表达明显下降,Iba-1、TNF-α、IL-6、IL-1β表达明显升高,提示脓毒症大鼠肠道菌群失衡,脑皮质内发生严重的炎症反应,脑功能受损。与模型组比较,粪菌移植组大鼠肠道菌群α多样性指数明显升高(物种指数:510.24±58.76比282.50±47.42,Chao1指数:852.75±25.24比705.50±46.50,均P<0.05),由LPS诱导的基于门、科、属水平的肠道菌群结构失衡也得到明显逆转;随着肠道菌群的改善,粪菌移植组大鼠异常脑电波形的发生率较模型组明显降低〔25.0%(3/12)比80.0%(8/10),P<0.05〕,脑皮质α7nAChR蛋白表达明显升高(α7nAChR/β-actin:1.56±0.05比0.82±0.07,P<0.05),免疫组化显示Iba-1阳性表达的小胶质细胞明显减少,且脑皮质TNF-α、IL-6、IL-1β含量均明显下降〔TNF-α(ng/L):6.28±0.61比12.02±0.54,IL-6(ng/L):28.26±3.15比60.58±4.62,IL-1β(ng/L):33.63±3.48比72.56±2.25,均P<0.05〕。结论通过粪菌移植可改善脓毒症大鼠肠道菌群失衡,同时可激活胆碱能抗炎通路,减轻脓毒症相关性脑病。

Objective To investigate the effects of fecal microbiota transplantation on septic gut flora and the cortex cholinergic anti-inflammatory pathway in rats.Methods Sixty clean grade male Sprague-Dawley(SD)rats were divided into normal saline(NS)control group,sepsis model group and fecal microbiota transplantation group by random number table,with 20 rats in each group.The rat model of sepsis was reproduced by injection of 10 mg/kg lipopolysaccharide(LPS)via tail vein,the rats in the NS control group was given the same amount of NS.The rats in the fecal microbiota transplantation group received nasogastric infusion of feces from healthy donor on the 1st day,2 mL each time,for 3 times a day,the other two groups were given equal dose of NS by gavage.Fecal samples were collected on the 7th day after modeling,the levels of intestinal microbiota composition was determined using the 16SrDNA gene sequencing technology.The brain function was evaluated by electroencephalogram(EEG),and the proportion of each waveform in EEG was calculated.After sacrifice of rats,the brain tissues were harvested,the levels of protein expression ofα7 nicotinic acetylcholine receptor(α7nAChR)were determined by Western Blot,and positive cells of Iba-1 in brain tissue were detected by immunohistochemistry method.The levels of interleukins(IL-6 and IL-1β)and tumor necrosis factor-α(TNF-α)were determined by enzyme-linked immunosorbent assay(ELISA).Results Seven days after the reproduction of the model,all rats in the NS control group survived,while 10 rats and 8 rats died in the sepsis model group and fecal microbiota transplantation group,respectively,with mortality rates of 50%and 40%respectively.Finally,there were 20 rats in the NS control group,10 in the sepsis model group and 12 in the fecal microbiota transplantation group.Compared with the NS control group,the diversity and composition of intestinal flora were changed,the incidence of abnormal EEG increased significantly,the expression ofα7nAchR in the cortex decreased significantly,and the levels of Iba-1,TNF-α,IL-6 and IL-1βwere significantly increased in the model group,suggested that the intestinal flora was dysbiosis,and severe inflammatory reaction occurred in the cerebral cortex,and brain function was impaired.Compared with the model group,the diversity of intestinal flora in the fecal microbiota transplantation group was significantly increased(species index:510.24±58.76 vs.282.50±47.42,Chao1 index:852.75±25.24 vs.705.50±46.50,both P<0.05),the dysbiosis of intestinal flora at phylum,family,genus level induced by LPS were also significantly reversed,and with the improvement of intestinal flora,the incidence of abnormal EEG waveforms was lower in the fecal microbiota transplantation group compared with that in the model group[25.0%(3/12)vs.80.0%(8/10),P<0.05],and the expression ofα7nAChR protein in the cerebral cortex was significantly increased(α7nAChR/β-actin:1.56±0.05 vs.0.82±0.07,P<0.05),immunohistochemistry analysis showed that Iba-1 positive expression of microglia decreased significantly,and cerebral cortex TNF-α,IL-6,IL-1βlevels were significantly decreased[TNF-α(ng/L):6.28±0.61 vs.12.02±0.54,IL-6(ng/L):28.26±3.15 vs.60.58±4.62,IL-1β(ng/L):33.63±3.48 vs.72.56±2.25,all P<0.05].Conclusion The results reveal that fecal microbiota transplantation has remarkably modulated the dysbiosis of intestinal microbiota and activated cholinergic anti-inflammatory pathway,and ameliorate the brain dysfunction in septic rats.