微生物学报  2019, Vol. 58 Issue (9): 1636-1650   DOI: 10.13343/j.cnki.wsxb.20180390.
http://dx.doi.org/10.13343/j.cnki.wsxb.20180390
中国科学院微生物研究所,中国微生物学会,中国菌物学会
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文章信息

安云英, 吴敏娜, 李璞泽, 靖昕瑞, 张剑锋, 薛红飞, 邓保国, 钟根深. 2019
Yunying An, Minna Wu, Puze Li, Xinrui Jing, Jianfeng Zhang, Hongfei Xue, Baoguo Deng, Genshen Zhong. 2019
口服硫酸链霉素对帕金森小鼠症状的改善及其对肠道菌群的影响
Effect of oral administration of streptomycin sulfate on symptoms and gut microbiota of Parkinson's disease mice
微生物学报, 58(9): 1636-1650
Acta Microbiologica Sinica, 58(9): 1636-1650

文章历史

收稿日期:2018-09-06
修回日期:2018-12-29
网络出版日期:2019-02-13
口服硫酸链霉素对帕金森小鼠症状的改善及其对肠道菌群的影响
安云英1 , 吴敏娜1 , 李璞泽1 , 靖昕瑞1 , 张剑锋1 , 薛红飞1 , 邓保国1 , 钟根深2     
1. 新乡医学院基础医学院, 河南 新乡 453003;
2. 新乡医学院医学检验学院, 河南 新乡 453003
摘要[目的] 探讨硫酸链霉素对慢性帕金森病(Parkinson's disease,PD)小鼠症状的改善及肠道菌群的影响。[方法] 将40只C57BL/6小鼠分为正常对照组、硫酸链霉素对照组、帕金森病模型组和硫酸链霉素干预帕金森病模型组。帕金森病模型组在实验的前5周采用1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,25 mg/kg)和丙磺舒(250 mg/kg)联合诱导帕金森病模型;硫酸链霉素干预帕金森病模型组在模型构建的同时连续饮用链霉素水溶液(500μg/mL)至实验第8周末。综合运用转棒实验、爬杆实验、免疫组织化学、荧光定量PCR、高通量测序等多种实验方法,检测各组实验小鼠相关症状与指标。[结果] 慢性PD模型小鼠与正常对照小鼠相比,表现出极显著的运动障碍(P<0.01),脑黑质纹状体中多巴胺(Dopamine,DA)能神经元及其纤维极显著减少(P<0.01),肠道出现显著的功能紊乱和炎症,同时肠道菌群结构发生了显著的变化:厚壁菌门/拟杆菌门(Firmicutes/Bacteroidetes,F/B)比值升高,疣微菌科(Ruminococcaceae)丰度极显著增加(P<0.01),普雷沃氏菌科(Prevotellaceae)及Prevotellaceae_UCG-001丰度极显著降低(P<0.01)。硫酸链霉素干预显著提高慢性PD小鼠的运动能力(P<0.05),缓解脑黑质纹状体系统中DA能神经元及其纤维的减少(P<0.05),改善肠道功能障碍和肠道炎症,同时可降低F/B的比值,显著降低Ruminococcaceae、理研菌科(Rikenellaceae)和乳杆菌科(Lactobacillaceae)的丰度,增加PrevotellaceaePrevotellaceae_UCG-001的丰度。[结论] 硫酸链霉素可改善PD小鼠相关症状,且影响了PD小鼠的肠道菌群结构。
关键词肠道菌群    硫酸链霉素    帕金森病    炎症    
Effect of oral administration of streptomycin sulfate on symptoms and gut microbiota of Parkinson's disease mice
Yunying An1 , Minna Wu1 , Puze Li1 , Xinrui Jing1 , Jianfeng Zhang1 , Hongfei Xue1 , Baoguo Deng1 , Genshen Zhong2     
1. School of Basic Medical Sciences, Xinxiang Medical University, Xinxiang 453003, Henan Province, China;
2. School of Laboratory Medicine, Xinxiang Medical University, Xinxiang 453003, Henan Province, China
Abstract: [Objective] To investigate the effect of streptomycin sulfate on the symptoms and gut microbiota of chronic Parkinson's disease mice. [Methods] Forty C57BL/6 mice were randomly divided into the control group, streptomycin sulfate control group, Parkinson's disease (PD) model group and streptomycin sulfate treated PD model group. PD mice were induced by injecting 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (25 mg/kg) combined with probenecid (250 mg/kg) at the first 5 weeks of the experiment; the streptomycin sulfate treated PD mice began to drink the streptomycin aqueous solution (500 μg/mL) on the first day of the experiment until the end of the experiment. A variety of experimental methods, such as rotarod test, pole test, immunohistochemistry, real-time PCR, and high-throughput sequencing were used to detect the related symptoms and indicators of each group. [Results] Compared with the control, chronic PD mice showed motor impairment (P < 0.01), dopaminergic neurons in the nigrostria and their fibers in the striatum reduced significantly (P < 0.01), intestinal dysfunction and inflammation, along with the structure of gut microbiota changed significantly:the ratio of Firmicutes/Bacteroides and the abundance of Family Ruminococcaceae increased (P < 0.01), whereas the abundance of Family Prevotellaceae and Genus Prevotellaceae_UCG-001 decreased (P < 0.01). The intervention of streptomycin sulfate significantly improved the exercise capacity of chronic PD mice (P < 0.05), relieved the decrease of dopaminergic neurons and fibers in the nigrostriatal system (P < 0.05), and improved the intestinal dysfunction and inflammation, meanwhile reduced the ratio of the Firmicutes/Bacteroides and the abundance of Family Ruminococcaceae, Rikenellaceae and Lactobacillaceae and increased the abundance of Family Prevotellaceae and Genus Prevotellaceae_UCG-001. [Conclusion] Streptomycin sulfate can relieve the symptoms associated with PD mice and affect the gut microbiota of PD mice.
Keywords: gut microbiota    streptomycin sulfate    Parkinson's disease    inflammation    

帕金森病(Parkinson’s disease,PD)是一种多发于中老年人的神经退行性疾病,且患病率呈现随年龄增长而升高的趋势[1-2]。PD的病理特征为黑质多巴胺(dopamine,DA)能神经元的选择性丧失和纹状体DA能神经纤维的减少[3]。PD发病机制尚未完全明了,目前认为氧化应激是其主要的发病机制。近来越来越多的研究表明肠道菌群参与了PD的发生发展。Scheperjans等研究发现PD患者的普雷沃氏菌科(Prevotellaceae)丰度水平较健康对照下降了77.6%,且肠杆菌科(Enterobacteriaceae)的丰度与PD的运动症状相关联[4];Qian等探讨了中国PD患者的粪便微生物组成,发现PD患者粪便中富集了梭菌属IV (Clostridium IV)、水生杆菌属(Aquabacterium)、霍乱嗜热菌属(Holdemania)等,此外还发现埃希菌属/志贺菌属比值(Escherichia/Shigella)与疾病持续时间呈负相关,Butyricoccus和梭菌属XIVb (Clostridium XIVb)与认知障碍有关[5]。DA对调节运动和认知功能至关重要,而负责将酪氨酸转化为DA的酪氨酸羟化酶和多巴脱羧酶的合成由微生物-脑-肠轴控制[6],亦表明肠道菌群与PD的发生发展密切相关。

口服抗生素可通过微生物-脑-肠轴作用于中枢神经系统。溶血性链球菌M抗原可导致中枢多巴胺能系统的功能障碍,氨苄青霉素可防治暴露于该M抗原动物的运动和行为障碍,而这种神经保护潜力可能是通过调节肠道菌群发挥作用的[7]。米诺环素在PD中具有神经保护作用[810],且其具有通过降低Firmicutes/Bacteroidetes比值来重新平衡肠道菌群的能力[11],因此米诺环素对PD的保护作用亦可能是与其对肠道菌群的调节有关。然而,目前关于抗生素是否可通过肠道菌群调节中枢神经系统的研究较少,有待于进一步深入探讨。

高浓度的抗生素混合物(氨苄青霉素1 g/L,万古霉素0.5 g/L,新霉素0.5 g/L,庆大霉素100 mg/L,红霉素10 mg/L,自由饮用)对PD小鼠进行治疗(从5–6周龄至12–13周龄),小鼠的病情得到了缓解;该治疗主要是通过影响肠道菌群发挥作用的,但长期、高强度的抗生素治疗显然是不明智的,会导致其他免疫器官和代谢功能的紊乱[12]。硫酸链霉素(streptomycin sulfate,STR)属氨基糖苷类抗生素,口服吸收不良,故常肌内注射;利用其在肠道吸收极少的特性,硫酸链霉素常被用于干扰肠道菌群进行其他研究,如金葡菌在胃肠道定植、伤寒沙门菌对肠道的感染等。另有报道指出用链霉素水溶液(150、300、450 mg/L,自由饮用)处理C57BL/6小鼠2 d,并没有显著改变肠道总细菌的丰度,而是导致了细菌群落结构的变化[13]。鉴于硫酸链霉素的特性以及肠道菌群与PD发生的密切关系,我们考虑是否可以应用硫酸链霉素调节PD小鼠的肠道菌群,从而缓解PD的症状,因此进行了以下实验:采用1-甲基-4-苯基-1, 2, 3, 6-四氢吡啶(1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine,MPTP)和丙磺舒(probenecid,P)联合用药作用于C57BL/6成年小鼠,建立慢性PD模型[14],同时自由饮用硫酸链霉素水溶液(500 μg/mL),分析硫酸链霉素对肠道菌群的影响以及对PD小鼠症状的改善。

1 材料和方法 1.1 实验动物与主要试剂

SPF级雄性C57BL/6小鼠,40只,9周龄,体重(22–25) g,购自北京维通利华实验动物技术有限公司【SCXK(京)2016-0006】。饲养条件:室温23±2 ℃,湿度55%±5%,12 h明暗交替,自由饮水和摄食。1-甲基-4-苯基-1, 2, 3, 6-四氢吡啶(MPTP,Sigma公司);丙磺舒、硫酸链霉素(大连美伦生物技术有限公司);酪氨酸羟化酶抗体(Abcam公司);兔SP试剂盒(北京中杉金桥生物技术有限公司,SP-9001);革兰阴性菌脂多糖检测试剂盒(北京金山川科技发展有限公司);反转录、荧光定量试剂盒等分子生物学相关试剂均购自宝生物工程(大连)有限公司;其余试剂为分析纯品。

1.2 小鼠疾病模型构建及给药

40只9周龄C57BL/6雄性小鼠适应性饲养1周后,随机分为四组(表 1):正常对照组(CK)、帕金森病模型组(PD)、硫酸链霉素对照组(STR)和硫酸链霉素干预帕金森病模型组(PD_STR)。慢性PD模型构建与给药(图 1)如下:PD组腹腔注射丙磺舒(12.5 mg/mL,20 mL/kg小鼠体重),30 min后皮下注射MPTP (2.5 mg/mL,10 mL/kg小鼠体重),自由饮用无菌纯净水;CK组腹腔注射丙磺舒,30 min后皮下注射与MPTP等量的生理盐水,自由饮用无菌纯净水;STR组腹腔注射丙磺舒,30 min后皮下注射与MPTP等量的生理盐水,自由饮用硫酸链霉素水溶液(500 μg/mL);PD_STR组腹腔注射丙磺舒,30 min后皮下注射MPTP,自由饮用硫酸链霉素水溶液(500 μg/mL)。构造PD模型期间丙磺舒和MPTP每间隔3.5 d注射1次,共10次,造模结束后,STR和PD_STR组继续自由饮用硫酸链霉素水溶液3周。

表 1. 小鼠分组及给药 Table 1. Mice grouping and processing
Groups CK STR PD PD_STR
p.o. Sterile water STR (500 μg/mL) Sterile water STR (500 μg/mL)
i.p. Probenecid Probenecid Probenecid Probenecid
s.c. NS NS MPTP MPTP
CK: control group; STR: STR treated group; PD: MPTP/p induced Parkinson’s disease model mice; PD_STR: STR treated PD model mice. p.o.: oral, i.p.: intraperitoneal injection; s.c.: subcutaneous injection, n=10.

图 1 动物模型构建 Figure 1 Animal model construction. CK: control group; STR: STR treated group; PD: MPTP/p induced Parkinson's disease model mice; PD_STR: STR treated PD model mice; n=10.

1.3 行为学测定

在实验第5周末(第36–37天)和8周末(第57–58天),通过转棒和爬杆实验进行小鼠神经行为学测试(图 1-B)。转棒实验参照Petroske等[15]的方法,略作修改:共7个递增的旋转速度(8、10、12、16、20、23、25 r/min),每个速度的测量时间为150 s,每2个转速之间间隔至少10 min,以减轻小鼠所受的压力与疲劳。以转速为横坐标,以动物在转棒上停留的时间为纵坐标作图,计算每只动物累积的行为学活性得分,并转化成曲线下面积(area under curve,AUC)进行统计学分析。爬杆实验参照Heng等[16]的方法,适应训练后,测试小鼠自杆顶爬至杆底部后肢着地的总时间(T-total),测试3次,每次间隔至少10 min,如果小鼠出现中途停顿或反向攀爬,则重新测试。

1.4 样品的收集

粪便样品:于实验第56天把每只小鼠单独放于干净的无菌鼠笼内,让其自由活动,排出粪便后,立即收集到无菌的2.0 mL EP管中,–80 ℃保存。

血液、脑组织和结肠上皮细胞:处理小鼠前一天晚上(第58天)禁食不禁水12 h,每组随机取6只小鼠于异氟烷深度麻醉前20 min灌胃2.5%的伊文思蓝溶液(溶解于1.5%的羧甲基纤维素钠) 0.3 mL,深度麻醉后迅速打开胸腔,快速心脏灌注生理盐水,待右心耳流出的液体清亮为止;每组其余4只小鼠均心脏取血,用于检测血清内毒素(lipopolysaccharide,LPS)。所有小鼠后续操作均为:快速剥离出完整的小鼠脑组织及结肠组织,脑组织于4%的多聚甲醛溶液中固定,结肠组织测量长度后提取肠上皮细胞于–80 ℃备用[17];最后剥离出胃幽门至盲肠上端的小肠组织。

1.5 免疫组织检测小鼠脑组织酪氨酸羟化酶(tyrosine hydroxylase, TH)的表达

1.5.1 制备石蜡切片: 取出固定好的脑组织进行常规处理后包埋于石蜡中,连续冠状切片(厚4 μm),保存4 ℃备用。每个组织每个部位各选取5张切片进行TH免疫组织化学染色。

1.5.2 免疫组织化学染色: 脑切片置于0.01 mol/L枸橼酸钠溶液(pH=6.0)微波修复抗原,PBS漂洗3 min×3次;3%过氧化氢室温孵育10 min阻断内源性过氧化物酶,PBS漂洗3 min×3次;正常山羊血清室温封闭30 min,甩去多余的血清,滴加TH一抗(1:800) 4 ℃过夜;PBS漂洗3 min×3次,滴加生物素标记的二抗,37 ℃孵育30 min,PBS漂洗3 min×3次。滴加辣根酶标记链霉卵白素工作液,37 ℃孵育30 min,PBS漂洗3 min×3次;DAB显色,封片,拍照。

1.6 结肠长度与小肠传导率的测定

将肠组织移至干净的白纸上,调整为一条直线,切勿拉伸,避免不必要的误差,测量结肠长度;同法测量胃幽门至伊文思蓝远端的距离和小肠全长,两者的比值为伊文思蓝的小肠推进百分率,即小肠传导率[18]

1.7 结肠上皮细胞炎性细胞因子丰度的RT-PCR检测

利用TRNzol-A+试剂提取结肠上皮细胞的总mRNA,反转录为cDNA。使用Step One Plus System (ABI)进行GAPDH、TNF-α、IL-6、IL-1β的实时定量PCR (qPCR),按照如下条件上机:95 ℃ 30 s;95 ℃ 5 s,60 ℃ 30 s,40个循环。最后使用ΔΔCt方法计算表达水平的倍数变化[19]

1.8 内毒素的检测

取小鼠血清样品0.1 mL直接加入反应主剂(C因子、B因子、凝固酶原及凝固蛋白原)中,溶解后使用微量加样器转移至10 mm×75 mm标注玻璃反应管中(勿产生气泡),插入到MB 80真菌/细菌动态检测系统中进行反应,反应结束后系统自动计算出血清中LPS的含量[20]

1.9 肠道菌群结构分析

从实验第8周(第56天)收集的粪便样品中每组随机挑取6个,共计24个进行16S rRNA基因的高通量测序,采用细菌16S rRNA基因V3-V4区引物[338F(forward primer):ACTCCTACGGGA GGCAGCAG;806R(reverse primer):GGACTACH VGGGTWTCTAAT]对粪便DNA进行PCR扩增。对PCR产物进行回收、纯化和定量。根据Illumina MiSeq平台(Illumina,San Diego,USA)标准操作规程将纯化后的PCR产物构建PE2*300的文库。利用该平台进行测序,原始数据使用Trimmomatic软件质控,FLASH软件进行拼接;使用UPARES软件(version 7.1 http://drive5.com/uparse/),根据97%的相似度对序列进行OTU聚类;使用UCHIME软件剔除嵌合体。利用RDP classifier (http://rdp.cme.msu.edu/)对每条序列进行物种分类注释,比对Silva数据库(http://www.arbsilva.de/),置信水平设置为70%,从而对每个OTU进行物种分类,得到不同分类水平(门、纲、目、科和属)上的细菌分类信息。然后根据每个OTU中序列数目,得到每个样本OTU丰度表进行后续分析(上海美吉生物医药科技有限公司)。原始数据已上传至美国国立生物技术信息中心(NCBI)SRA(The Sequence Read Archive)数据库,登录号为SPR159120。

1.10 统计学方法

使用SPSS 19.0软件进行数据分析,通过单因素ANOVA进行统计学分析。数据表示为mean± SD,P<0.05表示差别有统计学意义。采用Graphad Prism 5.01软件作图。

2 结果和分析 2.1 硫酸链霉素和MPTP/p对小鼠体重的影响

实验过程中每周末对小鼠进行称重,小鼠体重变化随时间呈逐渐增长趋势,且组间差异均不显著,说明造模药物和硫酸链霉素的使用对小鼠体重无影响。结果见图 2

图 2 不同组小鼠体重随时间的变化趋势 Figure 2 The trend of body weight change in different groups of mice over time. CK: control group; STR: STR treated group; PD: MPTP/p induced Parkinson's disease model mice; PD_STR: STR treated PD model mice; n=10.

2.2 硫酸链霉素对PD小鼠相关症状的改善

2.2.1 硫酸链霉素可改善PD小鼠运动功能障碍: 转棒实验结果如表 2所示,实验第5周末PD组小鼠累积运动活性得分较CK组低(P<0.05),PD_STR组较PD组无明显改善;实验第8周末PD组小鼠累积运动活性得分仍较CK组低(P<0.01),但PD_STR组较PD组显著升高(P<0.05)。爬杆测试结果显示在实验第5周末和第8周末,PD组小鼠完成爬杆实验所需时间较CK组长(P<0.01),而PD_STR组较PD组所需时间显著缩短(P<0.05)。因此硫酸链霉素可改善PD小鼠的运动功能障碍。

表 2. 各组小鼠转棒测试AUC和爬杆总时间(x±SD,n=10) Table 2. The AUC of rotating rod test and the total time of pole test (x±SD, n=10)
Groups AUC T-total/s
5th week 8th week 5th week 8th week
CK 1607.40±190.81a 1503.94±182.08a 6.4±0.82c 6.55±0.79c
STR 1486.20±175.37a 1408.20±221.49ab 6.95±1.09c 6.96±0.73bc
PD 1025.00±101.71b 909.18±85.14c 9.65±1.11a 10.30±1.31a
PD_STR 1099.20±124.09b 1171.18±125.32b 8.07±0.78b 8.27±0.55b
Significant differences (P<0.05) between treatments are indicated by the letters a, b, or c. CK: control group; STR: STR treated group; PD: MPTP/p induced Parkinson’s disease model mice; PD_STR: STR treated PD model mice; n=10.

2.2.2 硫酸链霉素可减少PD小鼠黑质纹状体中多巴胺能神经元及其纤维的丢失: TH是合成DA过程中的限速酶,TH+细胞可代表DA能神经元。PD组小鼠黑质部位DA神经元的数量和纹状体DA能神经元纤维的光密度较CK组极显著减少(P<0.01),而硫酸链霉素的使用均可改善PD小鼠黑质纹状体系统中DA能神经元和神经纤维的减少(P<0.05)(图 3)。

图 3 黑质纹状体系统中TH的免疫组织化学染色 Figure 3 Immunohistochemical staining of TH in the nigrostriatal system. A: representative IHC staining for TH neurons in the SNpc; B: representative IHC staining for TH fibers in the striatum; C: quantitative analysis of the number of TH-positive cells in the SNpc; D: quantitative analysis of the optical density of TH-positive fibers in the Striatum. Significant differences (P < 0.05) between treatments are indicated by the letters a, b, or c. CK: control group; STR: STR treated group; PD: MPTP/p induced Parkinson's disease model mice; PD_STR: STR treated PD model mice; n=10.

2.2.3 硫酸链霉素对PD小鼠胃肠道功能的影响: 测量小鼠结肠长度和小肠转运速率评估各组小鼠的胃肠道功能。结果如图 4-A所示,PD组和PD_STR组小鼠结肠长度较CK组显著变短(P<0.05)。PD组小鼠小肠传导速率与CK组相比极显著变慢(P<0.01),但PD_STR组小鼠与CK组差异不显著(图 4-B),因此硫酸链霉素可在一定程度上改善PD小鼠的胃肠道功能。

图 4 各组小鼠结肠长度和小鼠小肠传导率 Figure 4 Colon length and intestinal transit rate in each group of mice. A: the colon length of each group mice; B: the intestinal transit rate of each group mice. Significant differences (P < 0.05) between treatments are indicated by the letters a, b, or c. CK: control group; STR: STR treated group; PD: MPTP/p induced Parkinson's disease model mice; PD_STR: STR treated PD model mice; n=10.

2.2.4 硫酸链霉素可改善PD小鼠的肠道炎症: PD组TNF-α、IL-6、IL-1β mRNA表达增高(PD vs. CK,P<0.01),STR作用于慢性PD小鼠可显著降低炎症细胞因子mRNA表达水平(PD_STR vs. PD,P<0.05)(图 5)。

图 5 结肠上皮细胞炎性细胞因子mRNA相对丰度检测结果 Figure 5 The relative abundance of inflammatory cytokines mRNA in the colonic epithelial cells. A: TNF-α; B: IL-6; C: IL-1β. Significant differences (P < 0.05) between treatments are indicated by the letters a, b, or c. CK: control group; STR: STR treated group; PD: MPTP/p induced Parkinson's disease model mice; PD_STR: STR treated PD model mice; n=4.

2.2.5 硫酸链霉素可降低PD小鼠血清内毒素的含量: PD组小鼠血清LPS含量极显著高于其他各组小鼠(P<0.001),而PD_STR组与CK组差异不显著(图 6),说明硫酸链霉素可显著降低PD组小鼠血清内毒素的含量。

图 6 血清内毒素检测结果 Figure 6 Serum LPS content. ***: P < 0.001. CK: control group; STR: STR treated group; PD: MPTP/p induced Parkinson's disease model mice; PD_STR: STR treated PD model mice; n=4.

2.3 硫酸链霉素对PD小鼠肠道菌群的影响

2.3.1 Alpha (α)多样性分析: 按照最小样本序列进行抽平分析,每个样品均取25419条序列,每个序列平均长度为437 bp,97%相似度水平的OTU有568个。基于OTU水平的α多样性分析结果如表 3所示:Coverage指数在各样本中最低为99.75%,表明本次测序结果可代表样品中微生物的真实情况;Shannon指数表明,PD组小鼠肠道菌群较CK组更具多样性;Shannon和Chao指数表明STR的应用降低了小鼠肠道菌群的多样性。

表 3. 高通量测序微生物多样性分析 Table 3. High-throughput sequencing microbial diversity analysis
Groups Coverage Shannon Chao
CK 0.9975±0.0002a 4.1199±0.1988b 437.7808±46.5193a
STR 0.9988±0.0001a 3.4893±0.2654c 245.0009±29.4946b
PD 0.9979±0.0003a 4.5182±0.1697a 444.2088±22.4568a
PD_STR 0.9988±0.0004a 3.6478±0.1563c 255.6389±31.8720b
Significant differences (P<0.05) between treatments are indicated by the letters a, b, or c. CK: control group; STR: STR treated group; PD: MPTP/p induced Parkinson’s disease model mice; PD_STR: STR treated PD model mice; n=6.

2.3.2 Beta (β)多样性分析: 基于bray_curtis的3D-PCA分析显示PC1、PC2和PC3的贡献值分别为46.61%、13.44%、9.59%,表明各组样品重复性较好(图 7-A);基于OTU水平的相似性分析(analysis of similarities, ANOSIM)结果R值为0.6837,P=0.001,表明组间细菌群落结构具有显著性差异(图 7-B)。

图 7 基于OTU水平的β多样性分析 Figure 7 Beta diversity analysis based on OTU level. A: 3D-PCA analysis; B: ANOSIM analysis (R=0.6837, P=0.001). CK: control group; STR: STR treated group; PD: MPTP/p induced Parkinson's disease model mice; PD_STR: STR treated PD model mice; n=6.

2.3.3 物种组成分析: 进一步分析各组小鼠肠道菌群组成,发现与CK组相比,PD组Bacteroidetes丰度显著降低(P<0.05),而STR处理后,PD_STR组Bacteroidetes丰度上升;CK、STR、PD与PD_STR组F/B比值分别为0.8857±0.1037、0.6911±0.1391、1.3037±0.0815与0.8241±0.2029,表明PD组在门水平上就出现了肠道菌群紊乱,硫酸链霉素可以改善PD小鼠的菌群紊乱(图 8-A8-B)。在科的水平上,与CK组相比,PD组Ruminococcaceae丰度(17.35%±1.40% vs. 12.53%± 1.29%,P<0.01)极显著升高,Deferribacteraceae (1.00%±0.26% vs. 0.27%±0.09%,P<0.05)丰度显著升高,Prevotellaceae (6.28%±0.56% vs. 12.89%± 1.72%,P<0.01)丰度极显著降低;硫酸链霉素处理可逆转PD小鼠肠道中这些菌群丰度的改变,RuminococcaceaePrevotellaceae的丰度在PD_STR组中与CK相比均无显著性差异(图 8-C)。在属的分类水平上,相对丰度超过1%的菌属有21个。与CK组相比,PD组小鼠肠道菌群中Prevotellaceae_UCG-001 (3.97%±0.39% vs. 9.61%±1.30%,P<0.01)丰度极显著降低,Oscillibacter (1.13%±0.21% vs. 0.51%±0.07%,P<0.01)丰度极显著升高;硫酸链霉素处理后(PD_STR),与PD组相比Prevotellaceae_UCG-001 (9.82%±1.74% vs. 3.97%±0.39%,P<0.05)丰度显著升高,Ruminococcaceae_UCG-014 (0.55%±0.26% vs. 10.11%±1.39%,P<0.01)丰度极显著降低、norank_f__Lachnospiraceae (1.50%±0.24% vs. 3.56%±0.60%,P<0.05)丰度显著降低(图 8-D)。

图 8 不同分类学水平的细菌群落组成分析 Figure 8 Analysis of bacterial flora composition at different taxonomic levels. A: relative abundance of phyla > 1% in different groups; B: the ratio of Firmicutes/Bacteroidetes; C: relative abundance of family > 1% in different groups; D: relative abundance of genus > 1% in different groups. The abscissa is the group name, the ordinate is the proportion of the species in the group of samples, the columns of different colors represent different species, and the length of the column represents the proportion of the species (A and C). CK: control group; STR: STR treated group; PD: MPTP/p induced Parkinson's disease model mice; PD_STR: STR treated PD model mice; n=6.

2.3.4 物种差异分析: LEfSe分析采用线性判别分析(linear discriminant analysis,LDA)估计每个物种丰度对差异效果影响的大小。利用该分析(LDA=3.0)筛选出各组特征性菌科,结果如图 9-A所示,CK组的特征菌科为EubacteriaceaeErysipelotrichaceaeRhodospirillaceae;PD组的特征菌科为RuminococcaceaeLactobacillaceaeEnterobacteriaceaeRikenellaceaeVerrucomicrobiaceae等;PD_STR组的特征菌科为Christensenellaceae;STR组的特征菌科为AerococcaceaeAerococcaceae。属水平的LEfSe分析(LDA=3.5)显示CK组的特征菌属为AlloprevotellaEubacterium_ruminantium_group、Lachnospiraceae_UCG_008、Ruminococcus_2;PD组的特征菌属为Ruminococcaceae_UCG_014、LactobacillusAlistipesAkkermansiaLachnospiraceae_UCG_001;PD_STR组的特征菌属为Prevotellaceae_UCG_001、OscillibacterRuminiclostridium_6、Ruminiclostridium_5;STR组的特征菌属为norank_f_Bacteroidales_S24_7_ group、LachnoclostriudiumRuminiclostridiumButyricicoccusMarvinbryantia (图 9-B)。

图 9 科和属水平的差异物种分析 Figure 9 The analysis of differential species at the level of family and genus. The LDA score obtained by LDA analysis (linear regression analysis), the larger the LDA score, the greater the influence of bacteria abundance on the difference effect. CK: control group; STR: STR treated group; PD: MPTP/p induced Parkinson's disease model mice; PD_STR: STR treated PD model mice; n=6.

3 讨论

帕金森症的发病率呈逐年递增的趋势,目前临床上只能缓解症状,达不到治愈的目的。揭示PD的发病机制,寻找治愈PD的方法一直是医学研究的方向。随着肠道微生态研究的深入,发现肠道微生物群紊乱不仅影响运动症状,还影响PD动物的脑功能[12, 21]。慢性MPTP/p小鼠模型常用于研究DA能神经元变性的机制[15]。在本实验中,构建了MPTP/p慢性小鼠模型,通过使用硫酸链霉素干预肠道菌群评估肠道细菌在慢性PD发生发展中的作用。实验结果显示慢性PD小鼠与正常小鼠相比无论是爬杆还是转棒测试都表现出较低的运动活性;且在脑组织免疫组化结果显示黑质DA能神经元丢失过多,纹状体DA能神经元纤维明显减少。这符合之前的研究结果[22-23],然而硫酸链霉素应用改善了慢性PD小鼠的运动损伤,同时也缓解了脑组织黑质部DA能神经元的丧失和纹状体中DA能神经元纤维的减少,说明硫酸链霉素对慢性PD小鼠具有一定的保护作用。

除了目前已经明确的运动障碍外,PD常出现各种非运动症状(non-motor symptoms,NMS)特征,如胃肠道功能障碍、认知障碍、睡眠障碍等。研究表明98.6%的PD患者至少存在一种NMS[24],胃肠道功能障碍最常见,50%–90%的PD患者有便秘症状[25-27]。PD的便秘在很大程度上是由于食物或代谢物在肠道转运时间延长,据报道PD患者较正常对照小肠转运时间延长[28]。本研究中,慢性PD小鼠表征小肠转运时间的小肠传导率显著低于正常小鼠;有趣的是,PD_STR组小鼠小肠传导率与正常小鼠和PD小鼠均无显著性差异,表明应用硫酸链霉素后慢性PD小鼠的肠道功能障碍得到了一定的改善。

最近的一项研究指出肠道炎症是PD发病机制的沉默驱动因素,慢性促炎免疫活性越来越多被认为是神经退行性疾病的基本要素,并且PD患者肠道中的炎症似乎与PD发病机理特别相关[29]。PD患者肠道存在低度炎性反应已得到证实,其特征为促炎细胞因子表达增高,肠神经中神经胶质细胞失调[30]。在本研究中,我们通过小鼠结肠上皮促炎细胞因子(TNF-α、IL-6和IL-1β) mRNA丰度检测验证了小鼠结肠炎症的存在。硫酸链霉素药动学特性为口服吸收较差,但在本研究中其应用在一定程度上抑制了炎症的进展,这可能与其干扰肠道菌群密切相关[31]

鉴于PD患者出现的肠道微生态失调[32-33],我们对小鼠的粪便样品进行了细菌16S rRNA的高通量测序分析,结果亦表明:与正常对照小鼠相比,慢性PD小鼠肠道细菌群落结构发生了显著改变。Sun等的研究表明PD小鼠与正常小鼠相比肠道菌群中Firmicutes丰度显著降低,Proteobacteria丰度显著升高[21];而Unger等的研究证实PD患者与健康对照者相比粪便微生物中Bacteroidetes丰度显著下降[34]。本研究结果表明慢性PD小鼠肠道细菌Firmicutes/Bacteroidetes比值较正常小鼠显著升高,而硫酸链霉素的干预可逆转F/B比值使其接近正常小鼠,说明硫酸链霉素具有调节FirmicutesBacteroidetes丰度来重新平衡肠道菌群的能力。

Prevotellaceae可参与肠道黏膜层中黏蛋白的合成,并通过可溶性纤维的发酵产生具有营养神经细胞和抗炎作用的短链脂肪酸[35],而Prevotellaceae的减少可导致肠黏液减少和肠道通透性增加,增加局部和全身对细菌抗原和LPS的易感性,从而诱导大量的α-突触核蛋白的过表达和错误折叠[35-36]。本研究中PD小鼠与健康小鼠相比,Prevotellaceae丰度显著降低,硫酸链霉素应用后Prevotellaceae丰度则显著增加,且PD相关症状明显改善,Prevotellaceae可能是PD发生发展的关键功能菌群。

最近研究表明在PD患者的肠道中AlistipesLactobacilusAkkermansia丰度较正常对照者显著升高[5, 32, 37-38]。而本实验中属水平的LEfSe分析结果亦显示LactobacillusAlistipesAkkermansia是PD组小鼠肠道中的特征性菌群,同时也发现PD小鼠肠道中参与黏蛋白降解的Akkermansia菌丰度较其他三组小鼠有增加趋势,这些菌是否参与了PD的发生发展以及硫酸链霉素是否通过这些菌对肠道功能进行了调节亦有待深入探究。

由肠道微生物的变化引起的炎症将导致血脑屏障完整性的变化,血液中的LPS更易进入脑内[39],而LPS已被证明可引起黑质多巴胺能神经元的炎症[40]。Elena等将LPS注射到两种最常见的LRRK2基因突变的小鼠体内时,导致了小鼠强烈的免疫反应,炎症细胞因子丰度升高,且这些升高的细胞因子通过血脑屏障进入大脑,导致小胶质细胞活化并破坏运动所涉及的大脑区域,表明革兰阴性菌感染对于帕金森的发病可能有重要促进作用[41]。本研究结果显示慢性PD小鼠血清LPS含量明显高于正常小鼠水平,硫酸链霉素干预后血清LPS含量显著降低;硫酸链霉素的使用可能抑制了部分革兰阴性菌的生长,从根源上减少了LPS的产生,从而对PD的发生发展具有抑制作用。

综上所述,慢性PD小鼠存在肠道菌群失调,口服硫酸链霉素可改善慢性PD小鼠的肠道炎症反应,延缓脑内DA能神经元及其纤维的损伤,进而改善慢性小鼠的运动功能障碍,这可能与其对肠道菌群的调节作用有关。后续工作将聚焦于与PD相关的功能菌群,深入揭示硫酸链霉素改善PD症状的具体机制。

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