Objective: To investigate the effect of fecal microbiota transplantation (FMT) on acute lung injury (ALI) induced by lipopolysaccharide (LPS) and its regulatory mechanism. Methods: Fifteen rats were divided into control group, LPS group and LPS+FMT group by random number table method. LPS group and LPS+FMT group were intraperitoneally injected with LPS to generate rat ALI model. After 24 h of modeling, feces (10 ml/kg) were given to the LPS+FMT group twice a day, and the control group and LPS group were given the same amount of normal saline. The intervention lasted for 2 days. After 24 h of the last fecal microbiota transplantation, arterial blood gas analysis was performed in each group. Then rats were sacrificed and enzyme-linked immunosorbent (ELISA) method was used to detect intercellular adhesion molecule 1 (ICAM-1) content in the serum and bronchoalveolar lavage fluid (BALF). The lung wet-dry weight ratio (W/D) was evaluated; HE staining and lung tissue pathology scoring, immunohistochemical detection of nuclear factor-kappa B (NF-κB) predominate nuclear expression and expression of ICAM-1 of alveolar epithelial cells were conducted; Western blot was used to detect the expression of proteins related to the intracellular phosphatidylinositol kinase (PI3K)/protein kinase (AKT) signaling pathway. Samples of rat feces were collected and DNA was extracted. Polymerase chain reaction (PCR) products of the V3 and V4 regions of the 16S ribosomal RNA gene (16SrDNA) were sequenced at high throughput, and bioinformatics analysis was conducted on the microbial community based on the operational classification unit. Results: The lung W/D and lung histopathological score of the LPS group were significantly higher than those of the control group, while the arterial partial oxygen pressure (PaO(2)) of the LPS group was significantly lower than that of the control group [(79.2±5.89 vs 95.2±2.77) mmHg, 1 mmHg=0.133 kPa](all P<0.05). The results of intestinal flora sequencing revealed that the diversity index of LPS group was significantly higher than that of the control group, while the lactobacillus of LPS group rats was significantly lower than that of the control group. The content of ICAM-1 in serum, BALF and its relative expression on the cell membrane in the LPS group was significantly higher than that in the control group [(8.64±0.87) vs (7.40±0.32) ng/L; (0.941±0.035) vs (0.739±0.079) ng/L; (0.250±0.010) vs (0.076±0.010)] (all P<0.05). Moreover, the relative expression levels of phosphorylated P65 (p-P65), p-PI3K and p-AKT nucleoprotein in the LPS group were significantly higher than those in the control group (4.89±0.27 vs 3.28±0.13, 0.265±0.030 vs 0.036±0.013 and 0.444±0.040 vs 0.109±0.016) (all P<0.05). The above injury effect was reduced after fecal fungus transplantation. The lung W/D and lung pathological score of LPS+FMT group were significantly lower than those of LPS group, and PaO(2) of LPS+FMT group was significantly higher than that of LPS group [(88.0±3.53) mmHg]. The results of intestinal flora sequencing revealed that the diversity index of LPS+FMT group was significantly lower than that of LPS group, and the lactobacillus genus of LPS+FMT group was significantly higher than that of LPS group. ICAM-1 in the blood serum ((7.44±0.46) ng/L), BALF (0.834±0.040) ng/L) and its relative expression on alveolar epithelial cell membrane (0.173±0.030), the relative expression of p-P65, p-PI3K and p-AKT protein of NF-κB in alveolar epithelial cells was down-regulated ((2.99±0.28, 0.090±0.013 and 0.206±0.018) in LPS+FMT group than those of LPS group, the differences were statistically significant (all P<0.05). Conclusion: Fecal transplantation can alleviate lipopolysaccharide-induced acute lung injury in rats, and its regulatory effect may be related to inhibiting the activation of PI3K/AKT/NF-κB signaling pathway and reducing the expression of inflammatory factor ICAM-1.
目的： 探讨粪菌移植（FMT）对脂多糖（LPS）诱导的大鼠急性肺损伤（ALI）的影响及调控机制。 方法： 采用随机数字表法将15只大鼠分为对照组、LPS组和LPS+FMT组各5只，LPS组和LPS+FMT组分别腹腔内注射LPS复制大鼠ALI模型，于造模完成24 h后，LPS+FMT组每日给予粪便（10 ml/kg）灌胃2次，对照组和LPS组同时给予等量生理盐水灌胃。干预持续2 d，于最后一次FMT 24 h后各组分别行动脉血气分析，然后处死大鼠，用酶联免疫吸附法检测血清、支气管肺泡灌洗液（BALF）中细胞间黏附分子-1（ICAM-1）的含量，检测肺湿重/肺干重比值（W/D），行肺组织HE染色及病理学评分，免疫组化检测肺泡上皮细胞中的核因子（NF）-κB的核表达和ICAM-1的膜表达，Western印迹法检测胞内磷脂酰肌醇激酶（PI3K）/蛋白激酶（AKT）信号通路相关蛋白表达。收集大鼠粪便样品并提取DNA，对16S核糖体RNA基因（16SrDNA）的V3、V4区的聚合酶链式反应产物进行高通量测序，并对操作分类单元为分类基础的微生物群落进行生物信息学分析。 结果： LPS组W/D、肺组织病理评分均显著高于对照组，而LPS组动脉血氧分压（PaO(2)）显著低于对照组[（79.2±5.89）比（95.2±2.77）mmHg，1 mmHg=0.133 kPa]（均P<0.05）；肠道菌群测序LPS组多样性指数显著高于对照组，而LPS组乳酸杆菌属显著低于对照组；LPS组血清、BALF中ICAM-1含量及其胞膜的相对表达量均显著高于对照组[（8.64±0.87）比（7.40±0.32）ng/L、（0.941±0.035）比（0.739±0.079）ng/L、（0.250±0.010）比（0.076±0.010）]（均P<0.05），且LPS组NF-κB的p-P65、p-PI3K及p-AKT（p代表磷酸化）核蛋白相对表达量均显著高于对照组（4.89±0.27比3.28±0.13、0.265±0.030比0.036±0.013及0.444±0.040比0.109±0.016）（均P<0.05）。FMT后上述损伤效应减轻，LPS+FMT组肺W/D、肺组织病理评分均显著低于LPS组，LPS+FMT组PaO(2)[（88.0±3.53）mmHg]显著高于LPS组；肠道菌群测序LPS+FMT组多样性指数显著低于LPS组，LPS+FMT组乳酸杆菌属显著高于LPS组；血清、BALF中ICAM-1含量[（7.44±0.46）、（0.834±0.040）ng/L]及其胞膜的相对表达量（0.173±0.030）以及NF-κB的p-P65、p-PI3K及p-AKT核蛋白相对表达量（2.99±0.28、0.090±0.013、0.206±0.018）均显著低于LPS组（均P<0.05）。 结论： FMT可以改善LPS诱导的大鼠ALI，其可能与抑制PI3K/AKT/NF-κB信号通路活化、减少炎症因子ICAM-1等表达有关。.
Keywords: Acute lung injury; Fecal microbiota transplantation; Lipopolysaccharides; Rats.