帕宁Ⅰ号方调节肠道菌群改善PD小鼠行为学与神经递质的作用机制  

Mechanism of Pa′ning Formula I in Improving Behavior and Neurotransmitters in PD Mice by Regulating Gut Microbiota

作　　者：陈静文 周胜强 陈彦君 王瑞珍 万钰玲 辛凤 曾雯 刘芳[2] CHEN Jingwen;ZHOU Shengqiang;CHEN Yanjun;WANG Ruizhen;WAN Yuling;XIN Feng;ZENG Wen;LIU Fang(Hunan University of Chinese Medicine,Changsha Hunan China 410208;The Affiliated Hospital of Hunan Academy of Traditional Chinese Medicine,Changsha Hunan China 410006)

机构地区：[1]湖南中医药大学,湖南长沙410208 [2]湖南省中医药研究院附属医院,湖南长沙410006

出　　处：《中医学报》2024年第7期1357-1367,共11页Acta Chinese Medicine

基　　金：湖南省临床医疗技术创新引导项目(2021SK51010);湖南省中医药科研计划项目(2021083,C2022011);湖南省卫生健康委员会科研计划项目(D202319019427);中国中医科学院学部委员学术传承与传播专项项目(CI2022E014XB);长沙市自然科学基金项目(kq2208144)。

摘　　要：目的:基于肠道菌群探索帕宁Ⅰ号方改善帕金森病(Parkinson′s disease,PD)小鼠行为学和神经递质水平的作用机制。方法:从60只小鼠中随机选取10只作为正常组,其余小鼠腹腔注射MPTP(30 mg·kg^(-1))建立PD模型。将造模成功的50只小鼠随机分为模型组、美多芭组、帕宁Ⅰ号方低剂量组、中剂量组及高剂量组,每组10只。第6次注射MPTP后开始灌胃,连续14 d,每3天进行1次爬杆实验、旷场实验和步态分析实验。采用UPLC-HRMS法鉴定帕宁Ⅰ号方的活性成分,ELISA法检测纹状体多巴胺(dopamine,DA)含量,免疫组化法检测纹状体酪氨酸羟化酶(tyrosine hydroxylase,TH)表达水平,16S rRNA高通量测序技术检测肠道菌群结构。结果:与正常组比较,模型组小鼠的末次爬杆时间和步宽增加,运动路程和步长减少(P<0.0001);与模型组比较,帕宁Ⅰ号方低剂量组、中剂量组、高剂量组及美多芭组小鼠的末次爬杆时间和步宽减少,运动路程和步长增加(P<0.0001)。帕宁Ⅰ号方的化学成分主要包括苯丙氨酸、3-吲哚乳酸、牡荆素等。与正常组比较,模型组小鼠纹状体DA含量下降(P<0.0001);与模型组比较,帕宁Ⅰ号方中剂量组、高剂量组及美多芭组小鼠的纹状体DA含量升高(P<0.0001)。免疫组化结果显示,与正常组比较,模型组小鼠纹状体TH表达水平降低(P<0.0001);与模型组比较,帕宁Ⅰ号方高剂量组和美多芭组小鼠纹状体TH表达水平升高(P<0.01)。与正常组比较,模型组小鼠Turicibacter、Odoribacter的相对丰度增加,Lactobacillus、Bifidobacterium、Prevotella的相对丰度减少。与模型组比较,帕宁Ⅰ号方高剂量组和美多芭组小鼠Prevotellaceae_Prevotella、Lactobacillus、Bifidobacterium、Prevotella的相对丰度增加。Staphylococcus、Turicibacter、Rikenella相对丰度与爬杆时间呈正相关(P<0.05);Prevotella_Prevotella相对丰度与步长呈正相关(P<0.05);Prevotella相对丰度与爬杆时间呈负相Objective:To explore the mechanism of Pa′ningⅠFormula in improving the behavior and neurotransmitter levels of Parkinson′s disease(PD)mice based on gut microbiota.Methods:Ten mice were randomly selected as the normal group,and the remaining mice were intraperitoneally injected with MPTP(30 mg·kg^(-1))to establish a PD model.Fifty mice with successful modeling were randomly divided into model group,Madopar group,low-dose Pa′ning FormulaⅠgroup,medium-dose Pa′ning FormulaⅠgroup and high-dose Pa′ning FormulaⅠgroup,with 10 mice in each group.After the sixth injection of MPTP,the rats were given intragastric administration for 14 consecutive days,and the climbing pole test,open field test and gait analysis test were performed every 3 days.The active ingredients of Pa′ning FormulaⅠwere identified by UPLC-HRMS,the content of dopamine(DA)in striatum was detected by ELISA,and the expression of tyrosine hydroxylase(TH)in striatum was detected by immunohistochemistry.In addition,16S rRNA high-throughput sequencing technology was used to detect the structure of gut microbiota.Results:Compared with that of the normal group,the last pole climbing time and step width of the model group were increased,while the distance and step length were decreased significantly(P<0.0001).Compared with that of the model group,the last pole climbing time and step width were significantly decreased,while the distance and step length were increased in the low-dose,medium-dose,and high-dose Pa′ning FormulaⅠgroups and Madopar group significantly(P<0.0001).The chemical components of Pa′ning FormulaⅠmainly include phenylalanine,3-indole lactic acid,vitexin,etc.Compared with that of the normal group,the content of DA in striatum of the model group was decreased significantly(P<0.0001).Compared with that of the model group,the content of DA was increased significantly in striatum of the Pa′ning FormulaⅠmedium-dose group,high dose group and Madopar group(P<0.0001).It is showed in the immunohistochemistry results that th

关 键 词：帕宁Ⅰ号方 帕金森病 肠道菌群 多巴胺 酪氨酸羟化酶 小鼠 

分 类 号：R285.5[医药卫生—中药学]