肠道菌对肌萎缩侧索硬化症小鼠运动能力的影响及其机制研究

Effect of intestinal bacteria on motor function and its mechanism in amyotrophic lateral sclerosis mice

目的探讨肠道菌对肌萎缩侧索硬化症(ALS)小鼠运动能力的影响及其机制。方法以20只野生型(WT)C57BL/6J小鼠(WT组)和20只SOD1-G93A转基因ALS小鼠(ALS组)为实验对象。(1)按随机数字表法分别取WT组和ALS组小鼠各10只,其中每组取5只饲养于SPF环境下,剩余5只饲养于无菌环境下,分别定义为WT+SPF组、WT+无菌组及ALS+SPF组、ALS+无菌组。(2)按随机数字表法分别取WT组和ALS组小鼠各10只,饲养于无菌环境下,其中每组取5只进行ALS小鼠粪菌移植,剩余5只不予干预,分别定义为WT+移植组、WT+未移植组及ALS+移植组、ALS+未移植组。采用握力计量器检测小鼠的握力,采用滚筒跑步机、转棒实验检测小鼠的运动协调能力,采用Nissl染色检测小鼠脊髓前角运动神经元数量,采用免疫组化染色检测小鼠脊髓组织中小胶质细胞激活标志物离子钙接头蛋白(IBA-1)的表达,采用Western blotting检测小鼠脊髓组织中肿瘤坏死因子(TNF)-α、白介素(IL)-6的蛋白表达,采用高效液相色谱-串联质谱法检测小鼠脊髓组织中β-甲氨基-L-丙氨酸(BMAA)的表达。结果(1)ALS+无菌组小鼠的握力、掉落潜伏期、掉落时间均明显高于ALS+SPF组小鼠,Nissl染色阳性细胞数量明显高于ALS+SPF组小鼠,IBA-1阳性细胞数量明显低于ALS+SPF组小鼠,TNF-α、IL-6蛋白表达水平及BMAA浓度均明显低于ALS+SPF组小鼠,差异均有统计学意义(P<0.05)。(2)ALS+移植组小鼠的握力、掉落潜伏期、掉落时间均明显低于ALS+未移植组小鼠,Nissl染色阳性细胞数量明显低于ALS+未移植组小鼠,IBA-1阳性细胞数量明显高于ALS+未移植组小鼠,TNF-α、IL-6蛋白表达水平及BMAA浓度均明显高于ALS+未移植组小鼠,差异均有统计学意义(P<0.05)。结论肠道菌稳态失调可以促进ALS小鼠的运动能力降低,其机制与小胶质细胞激活有关。

Objective To study the effect of intestinal bacteria on motor ability of amyotrophic lateral sclerosis(ALS)mice models and its mechanism.Methods Twenty wild type C57BL/6J mice(WT group)and 20 SOD1-G93A transgenic ALS mice(ALS group)were selected as the research subjects.(1)Ten mice in both WT group and ALS group were selected,respectively;5 mice in each group were fed in SPF environment,and the remaining 5 mice were fed in aseptic environment;they were defined as WT+SPF group,WT+aseptic group,ALS+SPF group and ALS+aseptic group.(2)Ten mice in WT group and ALS group were fed in sterile environment;5 mice in each group were transplanted with fecal bacteria,and the remaining 5 mice were not interfered;they were defined as WT+transplantation group,WT+non-transplantation group,ALS+transplantation group and ALS+non transplantation group.The grip strength of mice was measured by grip force meter,the motor coordination ability of mice was tested by roller treadmill and rotating rod test,the number of motor neurons in the anterior horn of spinal cord was measured by Nissl staining,the expression of microglia activation marker ionic calcium junction protein(IBA-1)in spinal cord tissues was detected by immunohistochemical staining,and the expressions of tumor necrosis factor(TNF)-αand interleukin(IL)-6 in spinal cord tissues were detected by Western blotting;theβ-N-methylamino-L-alanine(BMAA)expression was detected by high performance liquid chromatography-tandem mass spectrometry.Results(1)The grip strength,drop latency and drop time of ALS+aseptic mice were significantly higher than those of ALS+SPF mice,the number of Nissl-stained positive cells was significantly larger than that of ALS+SPF mice,the number of IBA-1 positive cells was significantly smaller than that of ALS+SPF mice,the levels of TNF-αand IL-6 protein expressions and BMAA concentration were statistically lower than those of ALS+SPF mice(P<0.05).(2)The grip strength,drop latency and drop time of ALS+transplantion mice were significantly lower than those of ALS+non-transplantation mice,the number of Nissl-stained positive cells was significantly smaller than that of ALS+non-transplantation mice,the number of IBA-1 positive cells was significantly larger than that of ALS+non-transplantation mice,the TNF-αand IL-6 protein expressions and BMAA concentration were significantly higher than those of ALS+non-transplantation mice(P<0.05).Conclusion Imbalance of intestinal bacteria homeostasis can decrease the motor ability of ALS mice,which is related to the activation of microglia.