锰和高脂饮食联合暴露小鼠神经行为与肠道菌群相关性

Correlation between gut microbiota and neurological behavior in mice co-exposed to manganese and high-fat diet

目的探讨锰和高脂饮食(HFD)联合暴露对小鼠神经行为和肠道菌群结构的影响以及两者的相关性。方法将无特定病原体级雄性成年C57BL/6小鼠随机分为4组。对照组和锰暴露组小鼠予普通维持饲料饲养,HFD组和联合暴露组小鼠予高脂饲料饲养。采用腹腔注射法,锰暴露组和联合暴露组小鼠同时予剂量为10 mg/(kg·d)的氯化锰染毒,对照组和HFD组小鼠予等体积0.9%氯化钠溶液,1次/d,共60 d。染毒结束后,对小鼠进行神经行为学实验。其后处死小鼠,采集肠道粪便,采用高通量测序检测肠道菌群门水平和属水平菌群(相对丰度>1.000%)的相对丰度。结果染毒结束后,与对照组比较,HFD组和联合暴露组小鼠体质量均增加(P<0.05),锰暴露组小鼠体质量下降(P<0.05)。与对照组比较,锰暴露组小鼠掉落潜伏期、中央格停留时间、跨格次数、总路程和开臂停留时间百分比(OT%)均下降(P<0.05),闭臂停留时间百分比(CT%)升高(P<0.05)。与对照组和HFD组比较,联合暴露组小鼠掉落潜伏期、站立次数、中央格停留时间、跨格次数、总路程、OT%和进入开臂次数百分比(OE%)均下降(P<0.05),CT%升高(P<0.05)。联合暴露组小鼠总路程低于锰暴露组(P<0.05)。在门水平肠道菌群相对丰度上,与对照组比较,HFD组小鼠厚壁菌门(Firmicutes)升高(P<0.05),拟杆菌门(Bacteroidetes)和放线菌门(Actinobacteria)均下降(P<0.05);锰暴露组小鼠Firmicutes、变形菌门(Proteobacteria)和蓝藻菌门(Cyanobacteria)均升高(P<0.05),Bacteroidetes和Actinobacteria均下降(P<0.05)。在属水平肠道菌群相对丰度上,与对照组比较,HFD组小鼠颤螺菌属(Oscillospira)、拟杆菌属(Bacteroides)、普雷沃氏菌属(Prevotella)均下降(P<0.05);锰暴露组小鼠乳杆菌属(Lactobacillus)升高(P<0.05),Oscillospira、Bacteroides、Prevotella均下降(P<0.05)。与其余3组比较,联合暴露组小鼠Firmicutes、Cyanobacteria、Lactobacillus相对丰度均升高(P<0.05),其余6种细菌相对丰度均下降(P<0.05)。对于联合暴露组小鼠肠道菌群,掉落潜伏期与Bacteroidetes呈正相关(P<0.05);站立次数与Firmicutes呈正相关(P<0.05),与Actinobacteria呈负相关(P<0.01);OE%与Firmicutes呈负相关(P<0.05),与Actinobacteria呈正相关(P<0.05);跨格次数与Prevotella呈正相关(P<0.05)。结论锰联合HFD对小鼠神经行为异常具有协同作用;有关神经行为异常与小鼠肠道菌群稳态失衡具有一定相关性。

Objective To investigate the effects of manganese(Mn)and high fat diet(HFD)co-exposure on the neurological behavior and gut microbiota in mice,and to observe the correlation between them.Methods Specific pathogen free adult male C57 BL/6 mice were randomly divided into 4 groups.Mice in control group and Mn exposure group were fed with normal diet,while the HFD group and co-exposure group were fed with HFD.Both the Mn exposure group and the co-exposure group were exposed to 10 mg/(kg·d)manganese chloride by intraperitoneal injection,while the control group and HFD group were treated with 0.9%sodium chloride solution of the same volume,once per day for 60 consecutive days.At the end of exposure,the mice were subjected to experiments of neurological behaviors.Then,the mice were sacrificed and intestinal feces were collected.The relative abundance of gut microbiota(relative abundance>1.000%)was detected by high-throughput sequencing.Results After exposure,the body weight of the HFD group and the co-exposure group increased significantly(P<0.05),while that of the Mn exposure group decreased(P<0.05),compared with the control group.The latency,time in central,crossing,total distance and open arm time(OT%)of mice in the Mn exposure group were lower than that of the control group(P<0.05),and close arm time(CT%)prolonged(P<0.05).Compared with the control group and the HFD group,the latency,rearing,time in central,crossing,total distance,OT%and open arm entry(OE%)of mice in the co-exposure group decreased(P<0.05),and CT%increased(P<0.05).The total distance of mice in the co-exposure group was lower than that of the Mn exposure group(P<0.05).The relative abundance of Firmicutes increased(P<0.05),those of Bacteroidetes and Actinobacteria decreased in mice in the HFD group at the phylum level(P<0.05)compared with mice in the control group.The relative abundance of Firmicutes,Proteobacteria and Cyanobacteria increased(P<0.05),and Bacteroidetes and Actinobacteria decreased(P<0.05)in mice in the Mn exposure group.The relative abundance of Oscillospira,Bacteroides and Prevotella of mice in the HFD group reduced at the genus level(P<0.05)compared with the control group.The relative abundance of Lactobacillus increased in Mn exposure group(P<0.05),and Oscillospira,Bacteroides and Prevotella decreased(P<0.05).The relative abundance of Firmicutes,Cyanobacteria and Lactobacillus of mice in the co-exposure group increased(P<0.05),and those of the remaining 6 bacteria were lower(P<0.05)compared with mice in the other 3 groups.Among the mice of co-exposure group,the latency was positively correlated with Bacteroidetes(P<0.05).The rearing was positively correlated with Firmicutes(P<0.05)and negatively correlated with Actinobacteria(P<0.01).The OE%was negatively correlated with Firmicutes(P<0.05)and positively correlated with Actinobacteria(P<0.05).The crossing was positively correlated with Prevotella(P<0.05).Conclusion Manganese combined with HFD had a synergistic effect on the abnormality of neurological behavior of mice.There are some correlation between the abnormality of neurological behavior and the homeostatic imbalance of intestinal flora in mice.