粪菌移植对大鼠脓毒症相关性脑病的影响及机制

Impact and mechanisms of fecal microbiota transplantation on sepsis associated encephalopathy via intestinal microbiota

目的观察粪菌移植对脓毒症相关性脑病(SAE)大鼠肠道菌群及脑功能的影响.方法选择60只成年雄性SD大鼠,按随机数字表法分为假手术组、模型组、粪菌移植组,每组20只.采用尾静脉注射脂多糖(LPS)10mg/kg的方法复制脓毒症大鼠模型,粪菌移植组通过灌胃移植正常大鼠粪菌.于制模后6d采集大鼠粪便检测肠道菌群水平;并监测脑功能变化,计算脑电图各波形比例;采用蛋白质免疫印迹试验(WesternBlot)和免疫组化法检测各组大鼠脑皮质Iba-1的蛋白和阳性细胞表达.结果①肠道菌群:模型组大鼠肠道菌群α多样性指数物种指数、Chao1指数较假手术组明显降低(物种指数:282±40比473±37,Chao1指数:730±21比837±27,均P<0.05);粪菌移植组较模型组升高(物种指数:461±20比282±40,Chao1指数:840±16比730±21,P<0.05).门、科和属水平分析显示,模型组大鼠肠道菌群中厚壁菌门和梭杆菌属比例较假手术组显著降低〔厚壁菌门:(22.12±1.34)%比(78.01±1.23)%,梭杆菌属:(2.03±0.17)%比(5.03±0.19)%,均P<0.05〕,变形杆菌门、拟杆菌门和氨基酸球菌科、梭杆菌科、肠杆菌科及另枝菌属比例则明显升高〔变形杆菌门:(70.21±2.35)%比(19.45±2.17)%,拟杆菌门:(4.12±0.19)%比(2.50±0.64)%;氨基酸球菌科:(12.51±0.87)%比(1.01±0.12)%,梭杆菌科:(13.62±1.27)%比(2.31±0.19)%,肠杆菌科:(18.24±2.13)%比(4.15±1.51)%;另枝菌属:(4.53±0.27)%比(1.47±0.33)%,均P<0.05〕;而粪菌移植组厚壁菌门和梭杆菌属水平比例则显著高于模型组〔厚壁菌门:(72.14±2.31)%比(22.12±1.34)%,梭杆菌属:(5.01±0.27)%比(2.03±0.17)%,均P<0.05〕,变形杆菌门、拟杆菌门和氨基酸球菌科、梭杆菌科、肠杆菌科比例则明显低于模型组〔变形杆菌门:(14.23±1.98)%比(70.21±2.35)%,拟杆菌门:(3.15±0.18)%比(4.12±0.19)%;氨基酸球菌科:(0.91±0.11)%比(12.51±0.87)%,梭杆菌科:(1.25±0.15)%比(13.62±1.27)%,肠杆菌科:(3.50±0.21)%比(18.24±2.13)%,均P<0.05〕.②脑电图:模型组大鼠脑电图δ波比例较假手术组明显增加〔(16.86±0.50)%比(10.67±0.65)%,P<0.05〕,粪菌移植组脑电图δ波比例则较模型组明显减少〔(12.87±0.60)%比(17.35±0.41)%,P<0.05〕.假手术组出现异常脑电图的比例均为0,模型组制模后出现异常脑电图的比例明显增加〔δ优势波、θ优势波、低电压比例分别为66.7%(6/9)、66.7%(6/9)、77.8%(7/9)〕,粪菌移植组上述指标均较模型组明显降低〔粪菌移植组上述指标比例分别为9.1%(1/11)、9.1%(1/11)、18.2%(2/11)〕.③WesternBlot结果显示:模型组大鼠脑皮质中Iba-1蛋白表达水平较假手术组明显增高(Iba-1/β-actin:1.39±0.16比0.67±0.18,P<0.05);粪菌移植组大鼠脑皮质中Iba-1蛋白表达则较模型组明显下降(Iba-1/β-actin:0.51±0.14比1.39±0.16,P<0.05).④免疫组化显示:假手术组脑皮质无Iba-1阳性细胞;模型组脑皮质可见Iba-1阳性细胞;粪菌移植组脑皮质Iba-1阳性细胞数量较模型组少.结论通过肠道菌群移植,可改善脓毒症大鼠的肠道菌群结构,减轻SAE.

Objective To observe the effects of fecal microbiota transplantation on intestinal microbiota and brain function in sepsis rats. Methods Sixty male Sprague Dawley (SD) rats were divided into sham operation group, model group and fecal microbiota transplantation (FMT) group by random number table, each group 20 rats. The rat model of sepsis was established by injection of lipopolysaccharide (LPS) 10 mg/kg in tail vein. FMT group received nasogastric infusion of feces from healthy donor. Fecal samples were collected on the 6th day after the modeling to detect the levels of intestinal microbiota composition;the brain function was also evaluated by electroencephalogram (EEG), and the proportion of each waveform in EEG was calculated. After sacrifice of rats in different groups, the brain tissues were taken, the levels of protein expression and positive cells of Iba-1 in brain tissue were detected by Western Blot and immunohistochemistry method. Results ① Intestinal flora analysis showed that: the diversity index and Chaol index of the intestinal microbiota in model group were significantly lower than that in sham operation group (observed species:282±40 vs. 473±37, Chao1 index: 730±21 vs. 837±27, both P < 0.05);compared with the model group, the diversity index and Chaol index in FMT group were obviously higher (observed species: 461±20 vs. 282±40, Chao1 index:840±16 vs. 730±21, both P < 0.05). At phylum, family, genus level analysis showed that the proportion of Firmicutes phylum and Fusobacterium were obviously lower than those of sham operation group [Firmicutes phylum (22.12±1.34)% vs.(78.01±1.23)%, Fusobacterium:(2.03±0.17)% vs.(5.03±0.19)%, both P < 0.05], and the proportions of Proteobacteria, Bacteroidetes phyla and Acidaminococcaceae, Fusobacteriaceae, Enterbacteriacecae, Alistipes were markedly higher in model group [Proteobacteria:(70.21±2.35)% vs.(19.45±2.17)%, Bacteroidetes phyla:(4.12±0.19)% vs.(2.50±0.64)%, Acidaminococcaceae:(12.51±0.87)% vs.(1.01±0.12)%, Fusobacteriaceae:(13.62±1.27)% vs.(2.31±0.19)%, Enterbacteriacecae:(18.24±2.13)% vs.(4.15±1.51)%, Alistipes:(4.53±0.27)% vs.(1.47±0.33)%, all P < 0.05];compared with the model group, the proportion of Firmicutes phylum and Faecalibacterium in FMT group were significantly higher [Firmicutes phylum:(72.14±2.31)% vs.(22.12±1.34)%, Faecalibacterium:(5.01±0.27)% vs.(2.03±0.17)%, both P < 0.05], and Proteobacteria, Bacteroidetes phyla and Acidaminococcaceae, Fusobacteriaceae, Enterbacteriacecae in FMT group were obviously lower [Proteobacteria:(14.23±1.98)% vs.(70.21±2.35)%, Bacteroidetes phyla:(3.15±0.18)% vs.(4.12±0.19)%, Acidaminococcaceae:(0.91±0.11)% vs.(12.51±0.87)%, Fusobacteriaceae:(1.25±0.15)% vs.(13.62±1.27)%, Enterbacteriacecae:(3.50±0.21)% vs.(18.24±2.13)%, all P < 0.05].② EEG analysis showed that the percentages of δ wave in EEG in model group was significantly higher after modeling than that in sham operation group [(16.86±0.50)% vs.(10.67±0.65)%, P < 0.05];the ratios of δ wave in EEG was significantly lower in FMT group than that in the model group [(12.87±0.60)% vs.(17.35±0.41)%, P <0.05]. The incidence of abnormal EEG in sham operation group was 0, the incidence of abnormal EEG in model group was significantly increased [the ratios of δpredominant wave,θpredominant wave, low-voltage were 66.7%(6/9), 66.7%(6/9), 77.8%(7/9) respectively], the ratios of above abnormal waves in EEG in FMT group were obviously lower than those in model group [the ratios of above abnormal waves in FMT group were respectively 9.1%(1/11), 9.1%(1/11), 18.2%(2/11)].③ Western Blot analysis showed that the protein expression of Iba-1 in cortex in model group obviously was higher than that in sham operation group (Iba-1/β-actin: 1.39±0.16 vs. 0.67±0.18, P < 0.05);the expression of Iba-1 in cortex tissue of FMT group was markedly lower than that in model group (Iba-1/β-actin: 0.51±0.14 vs. 1.39±0.16, P < 0.05).④ Immunohistochemistry of Iba-1 in cortex analysis showed that there were no Iba-1 positive cells in the cortex in sham operation group;Iba-1 positive cells were found in the cortex in model group;the number of Iba-1 positive cells in FMT group was less than that in model group. Conclusion FMT can improve the construction of intestinal microbiota, and ameliorate the brain dysfunction in SAE.