阻燃剂磷酸三丁酯对斑马鱼早期发育的影响

李瑶, 朱晶颖, 李尧, 陈丽梅, 朱鹏飞, 丁新良, 周伟杰

李瑶, 朱晶颖, 李尧, 陈丽梅, 朱鹏飞, 丁新良, 周伟杰. 阻燃剂磷酸三丁酯对斑马鱼早期发育的影响[J]. 环境与职业医学, 2024, 41(12): 1376-1383. DOI: 10.11836/JEOM24391
引用本文: 李瑶, 朱晶颖, 李尧, 陈丽梅, 朱鹏飞, 丁新良, 周伟杰. 阻燃剂磷酸三丁酯对斑马鱼早期发育的影响[J]. 环境与职业医学, 2024, 41(12): 1376-1383. DOI: 10.11836/JEOM24391
LI Yao, ZHU Jingying, LI Yao, CHEN Limei, ZHU Pengfei, DING Xinliang, ZHOU Weijie. Effects of flame retardant tributyl phosphate on early development of zebrafish[J]. Journal of Environmental and Occupational Medicine, 2024, 41(12): 1376-1383. DOI: 10.11836/JEOM24391
Citation: LI Yao, ZHU Jingying, LI Yao, CHEN Limei, ZHU Pengfei, DING Xinliang, ZHOU Weijie. Effects of flame retardant tributyl phosphate on early development of zebrafish[J]. Journal of Environmental and Occupational Medicine, 2024, 41(12): 1376-1383. DOI: 10.11836/JEOM24391

阻燃剂磷酸三丁酯对斑马鱼早期发育的影响

基金项目: 南京医科大学无锡医学中心2023年“揭榜挂帅”项目(WMCJ202302);无锡市水质健康研究队列建设项目(WMCC202318)
详细信息
    作者简介:

    李瑶(2000—),女,硕士生;E-mail:1468784489@qq.com

    通讯作者:

    周伟杰,E-mail:wxcdczwj@163.com

  • 中图分类号: R114

Effects of flame retardant tributyl phosphate on early development of zebrafish

Funds: This study was funded.
More Information
  • 摘要:
    背景

    磷酸三丁酯(TBP)作为一种有机磷酸酯阻燃剂被广泛应用,然而TBP在低浓度暴露下对水生生物毒性研究有限。

    目的

    以斑马鱼作为模式动物,探讨阻燃剂TBP对斑马鱼早期发育的影响。

    方法

    将受精后2 h(2 hpf)的斑马鱼胚胎随机分为4组,分别为0.01%二甲基亚砜(DMSO)对照组和TBP染毒组(0.02、0.2、2 μg·L−1)。染毒时间为2 hpf至120 hpf,分别观察斑马鱼胚胎72 hpf孵化率、畸形率、心率和体长,24~29 hpf卷尾频率、96 hpf运动能力和120 hpf存活率。染毒结束后利用酶联免疫法检测幼鱼全身三碘甲状腺原氨酸(T3)及甲状腺素(T4)含量,采用实时荧光定量聚合酶链式反应(q-PCR)法检测下丘脑-垂体-甲状腺轴(HPT)和神经发育相关基因的表达水平。

    结果

    TBP染毒组斑马鱼胚胎均出现心率下降(P<0.001),0.02、2 μg·L−1染毒组的存活率下降(P<0.05),2 μg·L−1染毒组畸形率上升(P<0.05),主要表现为心包水肿。各组斑马鱼胚胎卷尾频率在25 hpf达到最高,各染毒组的卷尾频率低于对照组(P<0.001)。运动行为实验中,暗周期0.02、0.2 μg·L−1染毒组斑马鱼游泳速度下降(P<0.05),在光周期0.2、2 μg·L−1染毒组斑马鱼游泳速度明显下降(P<0.05)。与对照组相比,0.2 μg·L−1染毒组幼鱼T3水平上升(P<0.05)。q-PCR结果表明,染毒组HPT轴相关基因甲状腺激素受体(trα、$tr\beta $)、甲状腺球蛋白(tg)、钠碘共转运体(nis)表达水平下调,甲状腺转运蛋白(ttr)在0.02 μg·L−1染毒组上调,碘甲腺原氨酸脱碘酶2(dio2)在0.02 μg·L−1染毒组下调(P<0.05);染毒组神经发育相关基因乙酰胆碱酯酶(ache)下调,髓鞘碱性蛋白(mbp)和ELAV样神经元特异性RNA结合蛋白3(elavl3)在0.02 μg·L−1染毒组也下调(P<0.05)。

    结论

    TBP暴露可导致斑马鱼早期发育异常,表现为孵化期和早幼期的发育毒性、甲状腺内分泌破坏和神经毒性。

     

    Abstract:
    Background

    Tributyl phosphate (TBP) is widely used as an organophosphate flame retardant. However, there are limited studies on the toxicity of TBP to aquatic organisms at low levels of exposure.

    Objective

    To investigate the effects of TBP on early development of zebrafish (Danio rerio).

    Methods

    Zebrafish embryos were randomly divided into four groups at 2 h post-fertilisation (2 hpf), namely, the 0.01% dimethyl sulfoxide (DMSO) control group and TBP exposure groups (0.02, 0.2 and 2 μg·L−1). The exposure time was from 2 hpf to 120 hpf and the hatching rate, malformation rate, heart rate and body length of zebrafish embryos at 72 hpf, the frequency of tail curling at 24-29 hpf, the locomotor ability at 96 hpf and the survival rate at 120 hpf were evaluated, respectively. The whole-body triiodothyronine (T3) and tetraiodothyronine (T4) levels of juvenile fish were measured by enzyme immunoassay at the end of the infection, and the expression levels of hypothalamic-pituitary-thyroid axis (HPT) and neurodevelopmental-related genes were detected by quantitative real-time PCR (q-PCR).

    Results

    The heart rates of zebrafish embryos were significantly decreased in all TBP-treated groups (P<0.001), the survival rates of the 0.02 and 2 μg·L−1 TBP groups were significantly decreased (P<0.05), and the malformation rate of the 2 μg·L−1 treated group was significantly increased (P<0.05), which was mainly manifested by pericardial oedema. The frequency of tail curling of zebrafish embryos in all groups reached the highest at 25 hpf, which was significantly lower (P<0.001) in all exposure groups than in the control group (P<0.001). In the locomotor behaviour experiments, the swimming speed of zebrafish larvae in the dark cycle was significantly decreased in the 0.02 and 0.2 μg·L−1 TBP groups (P<0.05), and similar results were found for the light cycle in the 0.2 and 2 μg·L−1 TBP groups (P<0.05). Compared with the control group, the T3 level of zebrafish juveniles in the 0.2 μg·L−1 TBP group increased significantly (P<0.05). The q-PCR results showed that the expression levels of HTP axis-related genes [thyroid hormone receptors (trα, $tr\beta $), thyroglobulin (tg), and sodium/iodide co-transporter (nis)] were significantly down-regulated in the exposure groups, the expression level of transthyretin (ttr) was significantly up-regulated in the 0.02 μg·L−1 TBP group, and the iodothyronine deiodinase 2 (dio2) expression level was significantly down-regulated in the 0.02 μg·L−1 TBP group (P<0.05); the neurodevelopment-related gene acetylcholinesterase (ache) was significantly down-regulated in the exposure groups, and the expression levels of myelin basic protein (mbp) and Elav like neuron-specific RNA binding protein 3 (elavl3) were significantly down-regulated in the 0.02 μg·L−1 TBP group (P<0.05).

    Conclusion

    TBP exposure can lead to early developmental abnormalities in zebrafish, manifested as developmental toxicity, thyroid endocrine disruption and neurotoxicity during hatching and early juvenile stages.

     

  • 帕金森病(Parkinson's disease)是世界上患病人数增长最快的神经系统疾病,从1990年到2015年,全球帕金森病患者人数增加了118%,达到620万人[1]。在工业化和老龄化的影响下,预计到2040年,患病人数可高达1200多万[2]。数据显示,我国1990年至2016年间调整年龄后的帕金森病患病率增长了一倍,是全世界增长幅度最大的国家[3]。帕金森病与多巴胺缺乏以及运动和非运动障碍相关。许多环境和遗传因素影响帕金森病的风险,这些因素集中于特定的途径,例如线粒体功能障碍、氧化应激、蛋白质聚集、自噬受损和神经炎症[4]。帕金森病前驱期(prodromal Parkinson's disease, pPD)是指存在帕金森病神经退行性变早期症状或体征的阶段,pPD的非运动症状包括嗅觉减退、视觉异常、抑郁和焦虑、便秘、白天过度嗜睡以及快速眼动睡眠行为障碍[5]。胃肠道微生物群作为肠道微生物群、神经发育和神经疾病之间的联系,在帕金森病的发展中发挥着重要作用,肠道中的初始发病机制可能会导致pPD的发展[6]。膳食因素对肠道微生态有着极其重要的影响,可能影响帕金森病的发病风险。

    大型前瞻性队列研究以及流行病学研究的荟萃分析证据表明,长期食用红肉,尤其是加工肉与总死亡率[7]、心血管疾病[7-8]、胰腺癌[9]和2型糖尿病[10]的风险增加相关。2型糖尿病是帕金森病的危险标志之一[11]。加工肉制品因其高钠含量、胆固醇和游离脂肪酸而影响健康,在烹饪过程中,脂质可以与蛋白质反应,形成高级脂质氧化终产物,而神经炎症和氧化应激影响着帕金森病的发生发展[12]。红肉及加工肉制品可能增加帕金森病风险,但目前缺乏大型人群研究证据证明红肉及加工肉制品摄入量与帕金森病以及pPD的相关性。

    本研究通过分析“神经系统疾病专病社区队列研究”基线和随访的调查数据,探索红肉及加工肉制品摄入与pPD的关系,以进一步揭示膳食与帕金森病之间的关系。

    本研究基于中国疾病预防控制中心营养与健康所的“神经系统疾病专病社区队列研究”项目,该项目于2018年采取多阶段整群随机抽样的方法在河北、浙江、湖南和陕西省4个省份抽取调查样本开展基线调查。每个省份抽取城市和县各2个调查点,每个城市调查点选取1个城市居委会和1个郊区村居委会,每个县城调查点选取1个县城居委会和1个农村,利用调查问卷、体格检查和生物样本采集等方式收集研究对象的相关信息[13-14]。项目已通过中国疾病预防控制中心营养与健康所的伦理审查(No.2017-020),调查开始前所有调查对象均由本人签署知情同意书。

    项目在2020年对13443名参加基线调查的对象进行随访,其中603名调查对象失访,12840位研究对象完成随访。选择参与基线及随访调查并且具有完整的人口学信息、膳食调查信息以及帕金森病相关风险因素信息的10348名55岁及以上成年人作为研究对象,剔除基线调查中总能量摄入异常(总能量摄入<2 090或>20 900 kJ·d−1)者183名,剔除长期饮用酒精、滥用药物以及确诊精神疾病并服用相关药物者162人,共纳入研究对象10003名。

    该研究利用食物频率调查表收集研究对象过去12个月所食用的食物种类、食用频次以及摄入量等信息,了解研究对象长期的食物消费习惯。本研究选取肉类中“瘦猪肉、肥猪肉、牛肉/羔羊肉/羊肉/其他非加工肉类”这三类红肉以及“加工肉制品”的食用频率和平均每次食用量,计算2018年基线红肉及加工肉制品每日摄入量。本研究根据基线红肉及加工肉制品摄入量的四分位数分组。利用《中国食物成分表》(第6版)[15]将食物以及食用油和调味品的消费量转换成能量摄入量(kJ·d−1)。

    利用生活方式调查问卷以及健康风险因素筛查调查问卷,以经培训的专业调查人员面对面询问调查对象的方式收集用于评估pPD的相关信息。本研究根据国际运动障碍协会(The International Parkinson and Movement Disorder Society, MDS)2019年发布的更新版MDS pPD诊断标准[16]选择风险标志(性别为男性、普通杀虫剂暴露、职业性溶剂暴露、无咖啡因摄入史、无吸烟史、2型糖尿病、低身体活动以及男性低血尿酸水平)与前驱期标志(嗅觉减退、便秘、过度日间嗜睡、症状性低血压、男性勃起功能障碍、泌尿功能障碍和抑郁)。其中:男性低血尿酸水平指尿酸浓度<297 μmol·L−1;2型糖尿病诊断为空腹血糖浓度≥7.0 mmol·L−1,或自报糖尿病确诊以及正在接受治疗。利用每周各项身体活动(休闲性体育活动、交通性/职业性/家务性身体活动)相应的代谢当量(metabolic equivalent, MET)与每周参与各项身体活动的时间(h·周−1)计算而得身体活动水平(MET·h·周−1[17-18],低身体活动水平为身体活动量<1.0 MET·h·周−1

    利用各个研究对象不考虑任何风险因素时的(即“先验”)疾病概率,然后将研究对象各个风险/前驱标志阳性或阴性对应的似然比值相乘得到总似然比值,当标志的结果数据缺失时则将该项似然比值记为1,将总似然比值与先验概率相结合计算得出后验疾病概率[11](本研究将后验概率作为连续性变量并定义为pPD风险水平)。

    根据人口学信息以及经济因素将本研究涉及的相关基线协变量进行分组,其中年龄分为55~64岁、65~74岁、75岁及以上三组,教育水平分为文盲、小学及以下、初中及以上三组,收入水平根据家庭人均月收入分为<1000元、1000~3999元、≥4000元三组。根据调查对象的户口所在地分为城市和农村。利用体格检查测量的身高和体重计算体质量指数(body mass index, BMI)。根据研究对象是否有工作、是否饮酒、是否有非甾体抗炎药使用史分别分为两组。膳食能量、新鲜蔬菜、水果、乳制品以及其他肉类摄入量以连续型变量进行调整。

    定性和定量变量分别用个数(构成比)和中位数(M)及第25、75百分位数(P25P75)表示。分别利用卡方检验和Kruskal-Wallis秩和检验对不同红肉及加工肉制品摄入水平的基线人口学特征等相关协变量分布差异进行单因素分析。由于pPD后验概率呈偏态分布,对后验概率行自然对数转换后做进一步分析。利用多重线性回归分析研究对象基线时红肉及加工肉摄入量与随访时pPD危险水平的关联。根据风险/前驱期标志特征数将其分为三组(≤2个、3~5个和≥6个),利用多项logit回归分析红肉及加工肉制品摄入量与风险/前驱期标志特征数的关系。检验水准α=0.05。使用SAS 9.4软件进行数据分析。

    研究纳入男性4305人,女性5698人。其中52.58%为农村人口,17.62%的研究对象有工作,61.34%的研究对象家庭人均月收入为1000~3999元。调查对象平均每日摄入红肉及加工肉制品28.57 g,根据红肉及加工肉制品摄入量四分位数分组的基线特征见表1。红肉及加工肉制品摄入量最高四分位(Q4)组的研究对象平均每日膳食能量、新鲜蔬菜、新鲜水果以及其他肉类摄入量较高。仅非甾体抗炎药使用史在不同红肉及加工肉制品摄入水平组差异无统计学意义。

    表  1  不同红肉及加工肉制品摄入水平研究对象的基线特征分布(n=10003)
    Table  1.  Baseline characteristics of subjects by intake levels of red meat and processed meat (n=10003)
    特征(Characteristic)合计(Total)Q1
    (n=2517)
    Q2
    (n=2617)
    Q3
    (n=2370)
    Q4
    (n=2499)
    P
    红肉及加工肉制品摄入量(Red meat and
    processed meat intake)/(g·d−1),MP25P75
    28.57(11.43,60.48) 5.00(1.64,8.00) 19.81(14.52,24.76) 42.86(34.32,50.68) 105.53(80.99,152.74) <0.001
    性别(Gender),n(%) 0.002
     男(Male) 4305(43.04) 1057(41.99) 1082(41.35) 1010(42.62) 1156(46.26)
     女(Female) 5698(56.96) 1460(58.01) 1535(58.65) 1360(57.38) 1343(53.74)
    年龄/岁(Age/years),n(%) <0.001
     55~64 4169(41.68) 961(38.18) 1035(39.55) 1036(43.72) 1137(45.50)
     65~74 4114(41.13) 1051(41.76) 1103(42.15) 949(40.04) 1011(40.45)
     ≥75 1720(17.19) 505(20.06) 479(18.30) 385(16.24) 351(14.05)
    教育水平(Education level),n(%) <0.001
     文盲(Illiteracy) 1644(16.43) 482(19.15) 442(16.89) 354(14.94) 366(14.64)
     小学以及下(Primary school and below) 4268(42.67) 1205(47.87) 1097(41.92) 931(39.28) 1035(41.42)
     初中及以上(Middle school and above) 4091(40.90) 830(32.98) 1078(41.19) 1085(45.78) 1098(43.94)
    家庭人均月收入/元(Monthly household income per capita/yuan),n(%) <0.001
     <1000 2352(23.51) 943(37.47) 608(23.23) 426(17.97) 375(15.00)
     1000~3999 6136(61.34) 1336(53.08) 1688(64.50) 1517(64.01) 1595(63.83)
     ≥4000 1515(15.15) 238(9.46) 321(12.27) 427(18.02) 529(21.17)
    城乡(Residential area),n(%) <0.001
     农村(Rural) 5260(52.58) 1455(57.81) 1336(51.05) 1134(47.85) 1335(53.42)
     城市(Urban) 4743(47.42) 1062(42.19) 1281(48.95) 1236(52.15) 1164(46.58)
    工作(Active employment),n(%) <0.001
     无(No) 8240(82.38) 2088(82.96) 2251(86.01) 1988(83.88) 1913(76.55)
     有(Yes) 1763(17.62) 429(17.04) 366(13.99) 382(16.12) 586(23.45)
    饮酒(Drinking),n(%) <0.001
     无(No) 8362(83.59) 2228(88.52) 2254(86.13) 1900(80.17) 1980(79.23)
     有(Yes) 1641(16.41) 289(11.48) 363(13.87) 470(19.83) 519(20.77)
    BMI/(kg·m−2),M(P25P75) 24.06(21.78,26.37) 24.08(21.63,26.44) 24.02(21.79,26.37) 24.22(21.89,26.60) 23.92(21.78,26.09) 0.034
    非甾体抗炎药使用史(Use of non-steroidal anti-inflammatory drugs),n(%) 0.252
     无(No) 9512(95.09) 2390(94.95) 2473(94.50) 2258(95.27) 2391(95.68)
     有(Yes) 491(4.91) 127(5.05) 144(5.50) 112(4.73) 108(4.32)
    膳食能量摄入(Total energy intake)/(kJ·d−1),
    M(P25P75)
    6263.19
    (4839.94,8094.61)
    5081.42
    (4170.18,6456.09)
    5580.76
    (4452.95,6923.71)
    6472.56
    (5209.91,7902.50)
    8313.98
    (6823.68,10526.33)
    <0.001
    新鲜蔬菜摄入(Fresh vegetables intake)/(g·d−1),
    M(P25P75)
    170.00(85.07,294.52) 86.9(38.57,165.71) 133.84(75.24,235.19) 187.14(114.29,294.83) 289.14(200.19,404.95) <0.001
    新鲜水果摄入(Fresh fruits intake)/(g·d−1),
    M(P25P75)
    40.76(16.14,85.71) 19.05(6.67,45.71) 34.29(14.29,71.43) 50.74(23.63,100.00) 61.33(33.33,114.29) <0.001
    乳制品摄入(Dairy products intake)/(g·d−1),
    M(P25P75)
    0.00(0.00,65.45) 0.00(0.00,42.86) 0.00(0.00,77.53) 0.00(0.00,84.85) 0.00(0.00,42.86) <0.001
    其他肉类摄入(Other meat intake)/(g·d−1),
    M(P25P75)
    16.31(5.00,42.86) 3.53(0.68,9.01) 14.00(5.64,28.57) 23.12(10.18,48.22) 48.57(19.14,82.86) <0.001
    后验概率(Post-test probability)/%,M(P25P75) 0.74(0.39,1.57) 0.78(0.42,1.75) 0.74(0.40,1.57) 0.73(0.37,1.57) 0.64(0.36,1.32) <0.001
    风险/前驱标志特征数(Number of risk/
    prodromal markers),M(P25P75)
    3(2,4) 3(3,5) 3(2,4) 3(2,4) 3(2,4) 0.057
    [注] 本研究中红肉及加工肉制品摄入数据有较多相同的数值,尤其是在四等分点处;实际的划分点是25.16%、51.32%、75.02%、100%。[Note] In this study, there were many similar values in the intake data of red meat and processed meat products, especially at the fourth grade point. The actual quartile sites were 25.16%, 51.32%, 75.02%, and 100%.
    下载: 导出CSV 
    | 显示表格

    在2020年随访时,研究对象中仅26.20%有不超过2个风险/前驱标志特征,总体pPD后验概率水平的MP25P75)为0.74%(0.42%,1.49%)。红肉及加工肉制品摄入量Q4组的研究对象中77.58%有3个及以上的风险/前驱标志特征,有6个及以上的风险/前驱标志特征的占7.6%。见表2

    表  2  不同红肉及加工肉制品摄入水平研究对象随访帕金森病前驱期情况分布
    Table  2.  Distribution of follow-up prodromal Parkinson's disease by intake levels of red meat and processed meat among subjects
    变量(Variable)合计(Total)Q1
    (n=2517)
    Q2
    (n=2617)
    Q3
    (n=2370)
    Q4
    (n=2499)
    P
    先验概率(Prior probability)/% 0.026
    M(P25P75) 2.00
    (1.25,2.50)
    2.00
    (1.25,2.50)
    2.00
    (1.25,2.50)
    2.00
    (1.25,2.50)
    2.00
    (1.25,2.50)
    Mean(SD) 2.10
    (1.00)
    2.08
    (1.00)
    2.07
    (1.01)
    2.10
    (0.99)
    2.14
    (1.01)
    风险/前驱标志特征
    数(Number of risk/
    prodromal markers),
    M(P25P75)
    3
    (2,4)
    3
    (2,4)
    3
    (2,4)
    3
    (2,4)
    3
    (3,4)
    0.040
    风险/前驱标志特征数分布(Distribution of the number of risk/prodromal
    markers),n(%)
    <0.001
     ≤2 2621
    (26.20)
    680
    (27.02)
    707
    (27.02)
    674
    (28.44)
    560
    (22.41)
     3~5 6680
    (66.78)
    1661
    (65.99)
    1741
    (66.53)
    1529
    (64.51)
    1749
    (69.99)
     ≥6 702
    (7.02)
    176
    (6.99)
    169
    (6.46)
    167
    (7.05)
    190
    (7.60)
    后验概率(Post-test
    probability)/%,
    M(P25P75)
    0.74
    (0.42,1.49)
    0.74
    (0.41,1.38)
    0.74
    (0.40,1.38)
    0.74
    (0.42,1.41)
    0.78
    (0.42,1.61)
    0.001
    下载: 导出CSV 
    | 显示表格

    多重线性回归分析结果显示,研究对象红肉及加工肉制品摄入量与pPD风险水平关系存在统计学意义相关性(P<0.05)(表3)。模型2在调整人口学特征的模型1的基础上进一步调整饮酒状况、BMI、膳食能量摄入量、新鲜蔬菜摄入量、新鲜水果摄入量、乳制品摄入量以及其他肉类摄入量,结果显示红肉及加工肉制品摄入量越大,则研究对象随访期患pPD风险水平越高(b=0.021,P=0.048)。调整所有混杂因素后,结果依然具有统计学相关性(b=0.021,P=0.042)。

    表  3  红肉及加工肉制品摄入与研究对象随访帕金森病前驱期风险水平关联的多重线性回归分析
    Table  3.  Multiple linear regression analysis on the relationship between red meat and processed meat intake and risk levels of prodromal Parkinson’s disease at follow-up visit among subjects
    模型(Model)后验概率(Post-test probability)
    bP
    模型1(Model 1)0.0270.003
    模型2(Model 2)0.0210.048
    模型3(Model 3)0.0210.042
    [注] 使用取自然对数的后验概率进行线性回归分析。模型1调整了教育程度、收入水平、城乡和工作状况;模型2进一步调整了饮酒、BMI、膳食能量摄入、新鲜蔬菜摄入量、新鲜水果摄入量、乳制品和其他肉类摄入量;模型3进一步调整了非甾体抗炎药的使用和取自然对数的基线后验概率。[Note] Results from linear regression analyses using log-transformed data for post-test probability of prodromal Parkinson's disease. Model 1 adjusts education, income level, residential area, and active employment. Model 2 adjusts drinking, BMI, total energy intake, fresh vegetables intake, fresh fruits intake, dairy products intake, and other meat intake on the basis of Model 1. Model 3 additionally adjusts use of non-steroidal anti-inflammatory drugs and the log-transformed post-test probability at baseline on the basis of Model 2.
    下载: 导出CSV 
    | 显示表格

    根据风险/前驱标志特征个数将其分为三组(≤2、3~5、≥6),利用多项logit回归分析不同红肉及加工肉制品摄入量与风险/前驱标志特征数的关系(表4)。结果显示,以研究对象出现3~5个标志特征者与≤2个标志者相比,所有模型趋势检验均有统计学意义;以红肉及加工肉制品摄入量Q1组为参照,在所有模型中Q4组研究对象的风险更高(模型1:OR=1.274,95%CI:1.115~1.456;模型2:OR=1.185,95%CI:1.016~1.382;模型3:OR=1.185,95%CI:1.015~1.382)。而研究对象出现不少于6个标志特征者与≤2个者相比,Q4组的风险仅在模型1中高于Q1组(OR=1.310,95%CI:1.032~1.664);经协变量调整后,均无统计学意义。

    表  4  红肉及加工肉制品摄入与研究对象随访帕金森病前驱期标志特征数的关联
    Table  4.  Associations between red meat and processed meat intake and number of markers of prodromal Parkinson's disease at follow-up visit among subjects
    模型(Model)风险/前驱期标志特征数(Number of risk/prodromal markers)
    3~5 vs. ≤2≥6 vs. ≤2
    模型1(Model 1)
     Q11.0001.000
     Q21.010(0.890,1.146)0.916(0.722,1.162)
     Q30.930(0.817,1.059)0.947(0.744,1.205)
     Q4 1.274(1.115,1.456)* 1.310(1.032,1.664) *
     P-trend<0.001 0.004
    模型2(Model 2)
     Q11.0001.000
     Q20.998(0.879,1.133)0.894(0.704,1.136)
     Q30.909(0.795,1.039)0.900(0.702,1.154)
     Q4 1.185(1.016,1.382) *1.133(0.862,1.490)
     P-trend0.0140.170
    模型3(Model 3)
     Q11.0001.000
     Q20.998(0.879,1.133)0.895(0.704,1.137)
     Q30.909(0.795,1.039)0.903(0.704,1.157)
     Q4 1.185(1.015,1.382) *1.137(0.864,1.495)
     P-trend0.0140.163
    [注] 模型1调整了教育程度、收入水平、城乡和工作状况;模型2进一步调整了饮酒、BMI、膳食能量摄入、新鲜蔬菜摄入量、新鲜水果摄入量、乳制品和其他肉类摄入量;模型3进一步调整了非甾体抗炎药的使用和基线标志特征数。*:P<0.05。[Note] Model 1 adjusts education, income level, residential area, and active employment. Model 2 adjusts drinking, BMI, total energy intake, fresh vegetables intake, fresh fruits intake, dairy products intake, and other meat intake on the basis of Model 1. Model 3 additionally adjusts use of non-steroidal anti-inflammatory drugs and number of prodromal Parkinson' disease markers at baseline on the basis of Model 2. *: P<0.05.
    下载: 导出CSV 
    | 显示表格

    本研究对“神经系统疾病专病社区队列研究”项目中10003名基线人群进行了为期2年的完整随访,分析了我国四省55岁及以上成年人基线红肉及加工肉制品摄入与随访pPD风险水平以及pPD标志特征数的关系。研究发现2018年我国四省55岁及以上成年人平均每日红肉及加工肉制品摄入量为28.57 g,与我国相关研究相比摄入量偏低[19]。2020年pPD后验概率为0.74%,风险/前驱期标志中位数为3个。结果表明,基线红肉及加工肉制品摄入量Q4组的研究对象后验概率中位数最高,并且风险/前驱标志特征数不低于6个的人群占比较多。此外,基线红肉及加工肉制品摄入量与pPD风险水平相关。基线红肉及加工肉制品摄入水平Q4组的人群随访期出现风险/前驱标志的可能较大。

    Zapala等[20]对年龄51~82岁的59名帕金森病患者和108名无神经变性的健康对照者进行了比较,结果显示帕金森病患者比健康对照组更常食用红肉(P<0.015),同时研究也发现肠道菌群与食物消费的关系,例如组织蛋白酶普氏菌的高丰度与红肉消费的低频率有关。红肉等肉制品是蛋白质、氨基酸、维生素(烟酸、维生素B6和维生素B12)以及铁和锌等矿物质的主要来源[21]。动物研究发现在A53T突变体α-突触核蛋白小鼠中,高膳食铁补充可诱导严重的疾病表型,在突变体α-突触核蛋白和衰老的影响下导致脑内多巴胺能神经元的脆弱性[22]。美国一项以平均年龄63岁的1053名自我报告的特发性帕金森病患者为调查对象的研究显示红肉消费与帕金森病进展相关,尤其是牛肉,但是其他高脂肪肉类与帕金森病进展无关,可能由于红肉富含膳食铁[23]。然而中国的一项病例-对照研究发现膳食铁摄入量相差不大,饮用水和空气中金属污染导致的铁暴露更为突出[24]。红肉占肉类摄入总量的比例、其他肉类如富含不饱和脂肪酸的鱼类的摄入量、富含植物化学物的蔬果摄入量以及各个膳食因素间的相互作用都可能影响pPD的患病风险。红肉及加工肉制品对帕金森病及其前驱期影响的潜在机制或相关的剂量-反应关系仍需要进行大型前瞻性研究以探索与证明。

    本研究存在着一定的局限性。首先红肉及加工肉制品摄入评估是基于半定量食物频率调查表,虽然能反映研究对象的通常摄入状况,但对过去一年的膳食摄入会存在回忆偏倚;本研究中红肉只包括猪肉、牛肉/羔羊肉/羊肉/其他非加工肉类,没有包含猪、牛和羊内脏,可能会低估红肉的摄入量;将其他肉类摄入量纳入模型进行回归分析可在一定程度上调整红肉摄入评估的偏差。本研究的基线特征显示,红肉及加工肉制品摄入量高的研究对象其膳食能量、蔬菜水果以及其他肉类的每日平均摄入量也较高,而本研究中受限于资料的完整性,这些膳食因素间的相互作用以及其对pPD发生产生的影响未能全面分析。并且,本研究仅选取了MDS pPD诊断标准中部分风险/前驱标志进行分析,可能的快速眼动睡眠行为障碍、多基因风险评分等重要标志未纳入。

    目前,帕金森病仍以其特有的运动症状来定义,还没有生物标志能够对帕金森病确诊前的任何概念阶段进行高灵敏度和特异性的可靠诊断,从大脑外开始的潜在神经变性的发展过程与发展速度仍然未知,因此了解pPD能够帮助我们深入了解帕金森病的所有异质性。本研究显示成年人红肉及加工肉制品摄入量与pPD风险水平相关,较高的红肉及加工肉制品摄入量可能是存在风险/前驱标志的潜在影响因素。

  • 图  1   TBP暴露对斑马鱼胚胎发育的影响($\bar x \pm s$,n=3)

    A~D:72 hpf;E:120 hpf。与对照组比较,*:P<0.05,**:P<0.001。

    Figure  1.   The effects of TBP exposure on zebrafish embryo development ($\bar x \pm s$, n=3)

    A-D: 72 hpf; E:120 hpf. Compared with the control group, *: P<0.05; **: P<0.001.

    图  2   TBP暴露对斑马鱼心脏形态的影响($\bar \chi \pm s $,n=3)

    暴露时间:72 hpf;红色箭头:心包水肿。

    Figure  2.   The effects of TBP exposure on zebrafish heart morphology ($\bar \chi \pm s $, n=3)

    Exposure time: 72 hpf. Red arrow: Pericardial edema.

    图  3   TBP暴露对斑马鱼运动行为的影响($\bar x \pm s$,n=3)

    A:24~29 hpf;B:96 hpf。与对照组比较,*:P<0.05,**:P<0.001。

    Figure  3.   The effects of TBP exposure on zebrafish locomotor behaviour ($\bar x \pm s$, n=3)

    A: 24-29 hpf; B:96 hpf. Compared with the control group, *: P<0.05; **: P<0.001.

    图  4   TBP暴露对斑马鱼甲状腺激素含量的影响($\bar x \pm s$,n=3)

    A~B:T3、T4。*:与对照组比较,P<0.05。

    Figure  4.   The effects of TBP exposure on zebrafish thyroid hormone content ($\bar x \pm s$, n=3)

    A-B: T3 , T4. *: Compared with the control group, P<0.05.

    图  5   TBP暴露对斑马鱼HPT轴及神经发育相关基因表达的影响($\bar x \pm s$,n=3)

    A~B:HPT轴相关基因、神经发育相关基因。*:与对照组比较,P<0.05。

    Figure  5.   The effects of TBP exposure on the expression of genes related to zebrafish HPT axis and neurodevelopment ($\bar x \pm s$, n=3)

    A-B: HPT axis-related genes and neurodevelopment-related genes. *: Compared with the control group, P<0.05.

    表  1   斑马鱼荧光定量PCR引物序列

    Table  1   The sequences of zebrafish qPCR primers

    目的基因
    (Target gene)

    引物序列
    (Primer sequence)
    β肌动蛋白($\beta $-actin) F: 5′-CAGTGCCCATCTACGAGGGTTAT-3′
    R: 5′-CGGCTGTGGTGGTGAAGGAGT-3′
    甲状腺激素受体α(Thyroid hormone receptor α, trα) F: 5′-CGAGAAGTGTCAGGAGAT-3′
    R: 5′-GTTCGTCACCTTCATCAG-3′
    甲状腺激素受体β(Thyroid hormone receptor β, tr$\beta $) F: 5′-ACTTGGACGATTCAGAGG-3′
    R: 5′-CCTTGTGCTTACGGTAGT-3′
    甲状腺球蛋白(Thyroglobulin, tg) F: 5′-GTGAAGAGGATGGTGAGT-3′
    R: 5′-GATGGCTGGTTGAATGAC-3′
    钠碘共转运体(Sodium-iodide transporter, nis) F: 5′-GGTGGCATGAAGGCTGTAAT-3′
    R: 5′-GATACGGCATCCATTGTTGG-3′
    甲状腺转运蛋白(Transthyretin, ttr) F: 5′-CTCCTGGTGTGTATCGGGTG-3′
    R: 5′-AGGATGTCAGTCATGTGCCTT-3′
    尿苷二磷酸葡萄糖醛酸转移酶(Udp-glucuronosyltransferase family 1 member A1, ugt1ab) F: 5′-CCACCAAGTCTTTCCGTGTT-3′
    R: 5′-GCAGTCCTTCACAGGCTTTC-3′
    碘甲状腺原氨酸脱碘酶1(Iodothyronine deiodinase 1, dio1) F: 5′-CTGGACCGACAGAAGACGAG-3′
    R: 5′-TGCGACATTGCTGAAGTCCT-3′
    碘甲状腺原氨酸脱碘酶2(Iodothyronine deiodinase 2, dio2) F: 5′-CTCGGACACTTGGCTTGACT-3′
    R: 5′-TTGGATCAGGACGGAGAGGT-3′
    乙酰胆碱酯酶(Acetylcholinesterase, ache) F: 5′-CCCTCCAGTGGGTACAAGAA-3′
    R: 5′-GGGCCTCATCAAAGGTAACA-3′
    髓鞘碱性蛋白(Myelin basic protein, mbp) F: 5′-AATCAGCAGGTTCTTCGGAGGAGA-3′
    R: 5′-AAGAAATGCACGACAGGGTTGACG-3′
    突触素Ⅱa(Synapsin Ⅱa, syn2a) F: 5′-GTGACCATGCCAGCATTTC-3′
    R: 5′-TGGTTCTCCACTTTCACCTT-3′
    生长相关蛋白43(Growth associated protein 43, gap43) F: 5′-TGCTGCATCAGAAGAACTAA-3′
    R: 5′-CCTCCGGTTTGATTCCATC-3′
    ELAV样神经元特异性RNA结合蛋白3
    (ELAV like neuron-specific RNA binding protein 3, elavl3)
    F: 5′-AGACAAGATCACAGGCCAGAGCTT-3′
    R: 5′-TGGTCTGCAGTTTGAGACCGTTGA-3′
    胶质纤维酸性蛋白(Glial fibrillary acidic protein, gfap) F: 5′-GGATGCAGCCAATCGTAAT-3′
    R: 5′-TTCCAGGTCACAGGTCAG-3′
    音猬因子a(Sonic hedgehog signaling molecule a, shha) F: 5′-GCAAGATAACGCGCAATTCGGAGA-3′
    R: 5′-TGCATCTCTGTGTCATGAGCCTGT-3′
    下载: 导出CSV
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  • 收稿日期:  2024-08-01
  • 录用日期:  2024-10-21
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