【作者】 曾惠；

【导师】 舒为群；

【作者基本信息】 第三军医大学 ， 劳动卫生与环境卫生学， 2007， 博士

【摘要】 随着人们生活水平的提高,消耗高能和精制食物逐渐增加,饮食中矿物质含量不断下降:而饮用软水在全世界也越来越多。长期饮用矿物质较低的软水对健康有无负面影响,各家观点不一,争论热烈。其中主要存在两种观点,一方认为:人体矿物质仅5%来源于饮水,绝大部分可以从食物补充。饮水的意义仅在补充水分。另一方则认为:水中矿物元素生物利用率较食物高,在食物矿物质元素摄入不足的情况下,饮水中矿物质元素的贡献是非常有意义的,尤其是在生长期、孕期和哺乳期等特殊时期;而饮用软水能加速机体固有矿物质的排出,增加机体对环境毒物的吸收,所以长期饮用纯净水可能会诱发疾病。但是,上述观点都缺乏系统、科学的研究依据。因此,系统研究长期饮用纯净水后大鼠的生物学效应,对指导科学饮水、维护人们的生命和健康具有重要意义。我室前期研究显示,饲料中矿物质元素足够的情况下,长期饮用纯净水对大鼠一般生长发育情况未见明显影响;但能够诱导大鼠血脂、肝重、肝体指数、MDA、ET-1含量及AI值显著升高。另据报道,饮用低钙镁的软水,通常与低出生体重相关。提示我们低矿物质饮水可能影响繁殖功能和氧化应激。另外,我室前期研究发现长期饮用纯净水后,大鼠血清中Ca、Mg、K、Na、Fe、Mn、P、Zn、Cu、Sr、Cr、Mo、F、Li、Ni、Al等16种宏量及微量元素中,雌鼠血清中镁含量显著降低,而其它元素（如钙元素）含量未见明显改变。目前国内外相关研究多为普通或标准饲料条件下的动物研究,尚未见关于饲料缺镁条件下饮用纯净水相关生物学效应的研究和报道。因此,我们选择几乎不含矿物质的纯净水作为软水的代表,并主要以饮水中镁元素为观察对象,建立了饲料缺镁和标准饲料两个动物模型,分别研究和探讨了饮用纯净水后大鼠重要脏器镁含量、繁殖功能、免疫功能、肝脏蛋白表达及其仔鼠生长发育、免疫功能等几种重要生物学效应及其机制,以期为了解长期饮用软水对人体的健康效应及饮水中矿物质元素（镁）的依赖程度提供理论依据。方法:1、饲料缺镁（参考AIN-93G配方并对镁含量进行控制）动物模型:正常饲料按照美国国家卫生研究院及国立营养研究所饲料配方（AIN-93G）,矿物质混合物仅调整镁含量为需要量（0.4g/kg）。低镁饲料按照AIN-93G配方,矿物质混合物仅调整镁含量为需要量的50%（0.2g/kg）。正常水:经陶瓷芯过滤的自来水。镁12.7mg/L,TDS 175mg/L,电导率350μS/cm,COD低于检测限。纯净水:符合饮用瓶装纯净水国家标准（GB17324-98）。镁0.015mg/L,TDS1.16mg/L,电导率2.28μS/cm,COD低于检测限。断乳SD大鼠104只（雌68只,雄36只）,随机分为四组（每组雌17只,雄9只）,分别为:Ⅰ:食正常饲料饮正常水:文中简称正常饲料正常水Ⅱ:食正常饲料饮纯净水:文中简称正常饲料纯净水Ⅲ:食缺镁饲料饮正常水:文中简称缺镁饲料正常水Ⅳ:食缺镁饲料食纯净水:文中简称缺镁饲料纯净水饲喂至性成熟（5周）、交配（雌雄比2:1交配2周）,怀孕（3周）,哺乳直至仔鼠断乳（3周）,共13周。实验期间,①观察长期饮用纯净水对大鼠一般情况、繁殖功能（雄鼠生育率和雌鼠受孕率、妊娠分娩时间、产仔数、活仔数、死仔数、仔鼠平均窝重、外观畸形率、性别比率、出生存活率、哺育4天成活率）。②记录仔鼠耳廓分离、出毛、门齿萌出、张耳日龄,作为生理发育指标;③记录仔鼠平面翻正反射、负趋地性反射、悬崖回避反射达标日龄和空中翻正反射级别作为神经行为发育指标;④观察仔鼠前肢握力、行走能力作为运动能力指标;利用Morris水迷宫实验观察仔鼠学习记忆能力。⑤记录大鼠脏器系数（雄鼠、母鼠、仔鼠）;用生化法测定血清蛋白含量（雄鼠、母鼠、仔鼠）;用MTT法测定ConA和LPS刺激后仔鼠（21日龄）脾淋巴细胞增殖活力,用分光光度计法测定尾静脉注射印度墨汁后的仔鼠（21日龄）腹腔巨噬细胞吞噬功能,观察腹腔注射绿脓杆菌后仔鼠（21日龄）存活率。⑥大鼠镁含量测定:血清镁用甲基麝香草酚蓝法测定;已除去脂肪结缔组织的大鼠心脏、肝、脾、左腿胫骨,用微波消解仪消解后用原子吸收分光光度计测定。⑦采用SPSS13.0统计软件对实验数据进行统计分析。所有计数资料用χ²检验,所有计量资料除特殊注明外均以均数±标准差（M±SD）表示,用2×2析因设计方差分析。2、标准饲料（GB14924-2001）动物模型:断乳SD雄鼠20只随机分为两组:Ⅰ标准饲料饮正常水（水质同上）Ⅱ标准饲料饮纯净水（水质同上）饲喂28周后活杀取材。每周称量大鼠体重一次。利用2D电泳筛选出肝脏蛋白差异表达点,再用质谱分析法（MALDI-TOF）对差异蛋白点进行鉴定、通过MSCOT搜索差异蛋白点的相关信息。结果与讨论一、饲料缺镁动物模型（一）成功建立了饲料缺镁动物模型:成功地诱导出动物的镁缺乏:与正常饲料比较,饲料缺镁（正常水和纯净水）组亲代大鼠喂养至2周时出现由于缺镁引起的皮肤炎症;正常饲料（正常水和纯净水）组大鼠未出现。同时观察到,与正常饲料比较,缺镁饲料（正常水和纯净水）能够显著性降低亲代雄鼠肝镁含量、雌鼠血镁含量和骨镁含量、仔鼠肝镁含量。（二）饮用纯净水对大鼠及仔鼠重要脏器镁元素含量的影响:1、雄鼠:与正常饲料相比,饲料缺镁能够显著性降低雄鼠的肝镁含量（p＜0.05）。无论饲料缺镁与否,与饮用正常水相比较,饮用纯净水具有降低雄鼠的血镁、增高肝镁和脾镁含量的趋势（p＞0.05）。提示在雄鼠体内很可能发生了镁元素的重分布。即饮用纯净水使雄鼠机体摄入的镁量相对减少,镁可以从较丰富的器官（如肝等）迁移到它脏器中,以维持机体正常生理需要。由于镁元素在雄鼠机体内的重分布机制,使饮水中较小浓度镁对大鼠及其仔鼠重要脏器镁元素含量的影响难以充分显示出。2、雌鼠:与正常饲料相比,饲料缺镁能够显著性降低雌鼠的血镁和骨镁浓度（P＜0.05）。无论饲料缺镁与否,与饮用正常水相比较,饮用纯净水具有降低雌鼠的血镁和骨镁的趋势（p＞0.05）。由此可见,饲料缺镁和饮用纯净水可能降低雌鼠血镁和骨镁元素含量。提示,亲代雌鼠在经历妊娠、哺乳期后,机体很可能因镁元素消耗量大、摄入不足而改变骨质成分,即:亲代雌鼠由于饲料缺镁和/或饮用纯净水,机体镁元素摄入量相对减少,又由于妊娠、哺乳等对镁需求量较大,导致血镁浓度降低,不断动员骨镁释放入血,满足其它重要脏器生理需要,致使血镁和骨镁维持较低水平。3、仔鼠:与正常饲料相比,亲代大鼠饲料缺镁能够显著性降低仔鼠肝镁含量（p＜0.05）;无论饲料缺镁与否,与饮用正常水相比较,亲代大鼠饮用纯净水对仔鼠脏器镁元素含量未见显著性影响,但具有降低仔鼠骨镁含量的趋势。说明亲代母鼠由于饲料缺镁摄入镁元素量减少,在母鼠体内经镁动员不足以维持乳汁中镁元素的正常含量时,仔鼠从母鼠乳汁中摄取镁元素的量减少,导致仔鼠出现肝镁含量显著下降。这可能存在两种机制:①由于仔鼠从母鼠乳汁中摄入的镁元素量少,直接致使肝脏镁元素含量减少;②仔鼠体内镁的重分布,如肝镁和骨镁动员,来满足其它脏器需求。（三）饮用纯净水对大鼠繁殖功能的影响实验中4组大鼠繁殖功能（雄鼠生育率和雌鼠受孕率、妊娠分娩时间、产仔数、活仔数、死仔数、仔鼠平均窝重、外观畸形率、性别比率、出生存活率、哺育4天成活率）未见显著性差异。说明在饲料缺镁和饮用纯净水条件下,亲代大鼠能够提供足够的镁元素来维持正常的繁殖功能。其原因可能是:体内存在镁动员机制,在大鼠机体镁摄入水平降低或不足时,能够动员体镁优先供给大鼠生殖系统来保证正常的繁殖功能。（四）饮用纯净水对仔鼠生长发育的影响1、与正常饲料相比,亲代饲料缺镁对仔鼠出生体重未见显著性影响,但随着时间的延长,亲代母鼠饲料缺镁可显著性延缓仔鼠体重增长。究其原因可能也如前面我们所分析,仔鼠不直接饮用水,其营养来源主要是母乳;当母鼠镁缺乏或不足时,能够进行机体镁动员,优先满足繁殖功能,保证了仔鼠出生至哺乳前期的正常生长发育（出生体重正常）。随着哺乳时间延长至哺乳中后期,亲代母鼠因饲料缺镁而持续的低镁摄入,经体镁动员仍无法满足仔鼠生长发育的需要,从而显著性延缓仔鼠的生长发育（体重增长）。2、无论饲料缺镁与否,与饮用正常水相比较,亲代母鼠饮用纯净水对仔鼠生长发育未见显著性影响,但具有延缓仔鼠生长发育的趋势。提示,亲代母鼠饮用纯净水也可能延缓仔鼠生长发育;可能是因为饲料因素对亲代大鼠缺镁的贡献远大于饮水因素,所以与饮用正常水相比,亲代母鼠饮用纯净水对仔鼠生长发育的影响较轻,未能观察到显著性差异,只观察到了延缓仔鼠生长发育的趋势。3、哺乳前期未观察到亲代雌鼠饮用纯净水和饲料缺镁对仔鼠表面翻正和负趋地性等神经行为发育指标的显著性影响,提示哺乳前期母鼠乳汁中镁含量能够维持仔鼠正常的神经行为发育;但到哺乳中后期,与正常饲料相比,饲料缺镁（纯净水和正常水）可显著性降低仔鼠断崖回避和空中翻正测试成绩。说明随着哺乳时间的延长,母鼠持续低水平的镁摄入,乳汁中镁含量已降低,不足以维持仔鼠正常神经行为发育的需要。与饮用正常水相比,未观察到亲代大鼠饮用纯净水对此现象有加剧或减轻作用。4、与正常饲料相比,亲代母鼠饲料缺镁可显著降低仔鼠的运动能力测试成绩;无论饲料缺镁与否,与饮用正常水相比,亲代大鼠饮用纯净水对仔鼠运动能力未见显著影响,但仍能观察到降低仔鼠运动能力（行走能力测试成绩）的趋势。提示,亲代大鼠饲料缺镁和饮用纯净水均可能会降低仔鼠的运动能力。5、亲代母鼠饮用纯净水和饲料缺镁对仔鼠与空间记忆相关的神经组织（如海马等）发育指标未见显著性影响,对记忆能力、感觉、知觉、视觉发育指标也未见显著性影响。（五）饮用纯净水对大鼠免疫功能的影响观察了亲代大鼠皮肤炎症、免疫器官脏器系数、血清蛋白和仔鼠免疫器官脏器系数、血清蛋白、腹腔巨噬细胞吞噬功能、脾淋巴细胞增殖功能、白细胞分类计数、对绿脓杆菌攻击后的宿主抵抗力等指标后发现:与正常饲料相比,缺镁饲料的雄性和雌鼠皮肤、双耳出现红斑、充血和水肿;雌鼠脾脏脏器系数显著性增大（P＜0.05）,血清球蛋白含量显著性降低（p＜0.01）;但饮用纯净水对此现象未见加剧作用。饮用纯净水和饲料缺镁对仔鼠免疫功能均未见显著性影响,但可观察到饮用纯净水具有降低仔鼠腹腔巨噬细胞吞噬功能的趋势（p＞0.05）。说明,亲代大鼠（尤其是母鼠）因镁摄入量不足而引起免疫力明显低下,但只具有降低仔鼠免疫功能的趋势（p＞0.05）。推测其原因是:当母鼠镁缺乏或不足时,能够进行机体镁动员,优先满足繁殖功能（妊娠、哺乳等）,维持乳汁中镁元素相对正常水平,因而对仔鼠的免疫功能影响较小,可能对巨噬细胞吞噬能力具有远期效应。（六）饮用纯净水对大鼠营养状况（白蛋白含量）的影响与正常饲料相比,饲料缺镁对亲代雄鼠血清白蛋白未见显著性影响;与饮用正常水相比较,饮用纯净水对亲代雄鼠血清白蛋白含量未见显著性影响;但饮用纯净水和饲料缺镁（第四组与前三组相比较）在降低雄鼠血清总蛋白和白蛋白含量中具有交互作用（p＜0.05）。说明在饲料缺镁条件下,饮用纯净水能影响雄鼠血清白蛋白的代谢（p＜0.05）。与正常饲料相比,饲料缺镁能显著性降低亲代母鼠血清白蛋白含量;与饮用正常水相比较,饮用纯净水对雌鼠血清白蛋白含量未见显著影响;两者间也未见交互效应。提示由于母鼠营养需求较高,饲料缺镁引起的机体镁缺乏程度较高,其贡献远大于饮用纯净水的效应,故不能观察到饮用纯净水对雌鼠血清蛋白含量的显著性影响。亲代母鼠饲料缺镁和饮用纯净水对仔鼠血清白蛋白含量未见显著性影响。提示母鼠能通过机体镁元素（营养素）的动员来满足仔鼠营养（白蛋白含量）需要。二、标准饲料动物模型饮用纯净水对雄鼠肝脏蛋白质组的影响及其机制:应用2D电泳技术分析鉴定出饮用纯净水和正常水大鼠肝脏组织间的差异表达蛋白点304个。对其中15个差异较大的点进行MSADI-TOF-MS鉴定,得到14个蛋白质,其中评分大于49分的蛋白是:精氨酸酶、ATP合酶β亚单位、MMSDH（甲基丙二酸半醛脱氢酶）、bFGF（成纤维细胞生长因子）、regucalcin（钙调素）和CAT（过氧化氢酶）。1、精氨酸酶是肝脏鸟氨酸循环中的一种分解酶,可将精氨酸分解成尿素和鸟氨酸。内源性精氨酸在NOS催化下通过脱氨基作用生成一氧化氮（NO）或相似的含氮化合物,而NO可被超氧阴离子破坏。提示,可能由于饮用纯净水诱导大鼠肝脏向氧化应激发展,不断消耗NO,机体NO水平降低;机体为维持正常代谢,反馈性抑制精氨酸酶表达,减少精氨酸分解为尿素,相对增加精氨酸脱氨基作用,促进NO的生成。此推论与本室前期研究发现饮用纯净水大鼠血清NO、尿酸、尿素氮、肌酐含量显著性下降相一致。2、ATP合酶β亚单位是ATP合酶组成部分,ATP合酶的三个催化区均位于3个β亚基上。在纯净水组肝脏中有表达而正常水中无表达,提示饮用纯净水能够增加ATP合酶表达水平。其可能机制为:由于饮用纯净水促进大鼠向氧化应激发展,线粒体脂质过氧化增强,产生MDA等脂质过氧化物及裂解物,抗自由基损伤作用减弱,导致线粒体膜流动性下降,从而引起定位在线粒体膜上的ATP合酶活性降低。ATP合酶活性降低,反馈性增加ATP合酶表达水平。这也与前述“MMSDH表达减少,可抑制MDA分解,导致MDA聚集”相一致。3、MMSDH是CoA依赖的一个线粒体酶,是醛脱氢酶超家族成员,在MDA代谢通路中具有重要作用。此酶在正常饮水组表达,纯净水组不表达,提示大鼠肝脏中由MMSDH催化的丙二酸单酰CoA半醛向丙二酸单酰CoA的转化受阻,丙二酸半醛向丙二酸单酰CoA半醛的转化受阻,丙二酸半醛聚集,从而可能抑制MDA向丙二酸半醛的转化,导致MDA聚集。此机制与我室前期发现饮用纯净水大鼠血清MDA含量显著增加相一致。据报道,用化学剂诱导的Arabidopsis细胞氧化应激中,观察到甲基丙二酸半醛脱氢酶显著降低。因此,很有必要对这一机制进一步研究。4、碱性成纤维细胞生长因子（bFGF）是一类通过与细胞膜特异性受体结合发挥作用,调节细胞生长的肽类。在炎性疾病的发生中,bFGF表达上调,主要促进NO的生成,通过细胞因子网络释放PEG₂,诱发自由基的产生。本实验中,饮用纯净水组大鼠肝脏中bFGF有表达而正常水组无表达,说明饮用纯净水可能诱导了大鼠肝脏炎性反应。这与我室前期研究发现长期饮用纯净水组大鼠肝细胞灶性坏死及炎细胞浸润相一致。5、调钙素（regucalcin）是一高度保守的钙结合蛋白。在肝组织中能刺激Ca²⁺向胞外转运,促进肝细胞线粒体和微粒体对Ca²⁺的吸收,维持肝细胞质内Ca²⁺的动态平衡。饮用纯净水组大鼠肝脏中Regucalcin高表达,而正常水组低表达。提示纯净水组大鼠肝细胞内可能存在Ca²⁺水平稳态失调。其可能原因是:①饮用纯净水可能相对减少了钙的吸收;②可能由于饮用纯净水促使大鼠向氧化应激发展,自由基生成增加,破坏线粒体膜和细胞膜等膜性结构,使定位细胞膜上的Ca²⁺主动转运功能受损,致使细胞内钙稳态失调。6、过氧化氢酶（CAT）主要的生理功能就是参与活性氧代谢过程,在消除O₂代谢副产物H₂O₂方面起着重要作用。饮用纯净水组肝脏中CAT无表达,提示此酶的抑制可能与饮用纯净水导致的氧化应激的发生有关。其具体机制有待于进一步研究。结论:饲料缺镁和饮用纯净水均可能诱导大鼠机体镁元素重分布,使重要脏器镁含量发生不同程度的改变:与正常饲料相比,饲料缺镁可引起亲代大鼠重要脏器镁含量（雄鼠的肝镁、雌鼠的血镁和骨镁）显著降低;与饮用正常水相比,饮用纯净水具有降低雄鼠血镁、雌鼠血镁和骨镁,增高雄鼠肝镁、脾镁含量的趋势。所以,经历妊娠、哺乳等特殊生理时期的雌鼠饮用纯净水（尤其是在饲料缺镁条件下）可能会因骨镁过度动员而影响骨质成份。由于母鼠能够通过机体镁动员优先满足繁殖和哺乳,所以未见饲料缺镁和饮用纯净水对大鼠繁殖功能的显著性影响。妊娠和哺乳前期,亲代母鼠能够通过机体镁动员来满足仔鼠生长发育需求,所以仔鼠出生至哺乳前期的生长发育未见显著性影响。由于饲料缺镁条件下母鼠持续的镁摄入不足,随着哺乳时间延长至哺乳中后期,亲代母鼠已不能通过持续的体镁动员来满足仔鼠生长发育所需,因而可显著性迟滞仔鼠体重增长和神经发育;与饮用正常水相比,饮用纯净水也具有迟滞仔鼠体重增长和神经行为发育的趋势。镁在大鼠免疫功能中具有重要作用,饲料缺镁（纯净水和正常水）能够显著性降低亲代大鼠免疫功能,但亲代母鼠可通过机体镁动员优先满足繁殖和哺乳需要,从而缓解对仔鼠免疫功能的影响;饮用纯净水对亲代大鼠及其仔鼠免疫功能未见显著性影响,但具有降低仔鼠免疫功能（巨噬细胞吞噬能力）的趋势,其具体机制尚需进一步研究。围产期母鼠对镁元素需求量较大,因此与正常饲料相比,饲料缺镁（正常水和纯净水）可显著性降低雌鼠血清蛋白含量;在饲料正常镁含量条件下饮用纯净水对雌性和雄鼠血清白蛋白含量未见显著性影响,但在饲料缺镁条件下饮用纯净水可显著性降低雄鼠血清总蛋白和白蛋白含量;由于大鼠体内存在营养素（镁）动员优先保证繁殖和哺乳的机制,亲代母鼠饲料缺镁和饮用纯净水对仔鼠血清蛋白含量均未见显著性影响。白蛋白是评价蛋白质合成功能的常用指标之一,几乎全部在肝脏中合成。所以饮用纯净水可能会影响亲代雄鼠肝脏蛋白质合成。此外,在标准饲料条件下,饮用纯净水可能会使肝脏出现氧化应激、钙稳态失调和炎性反应,具体表现为肝脏表达MMSDH、CAT消失,可使肝脏MDA聚积,发生脂质过氧化（氧化应激）,从而可能破坏膜的流动性,使定位于线粒体膜上的ATP合酶功能降低,反馈性促使ATP合酶（β亚基）合成增加;而氧化应激又可使NO不断消耗,NO含量下降又可反馈性抑制精氨酸酶表达,降低精氨酸酶催化精氨酸水解为尿素的水平,从而增加精氨酸脱氨基生成NO的水平:同时,脂质过氧化引起的膜性结构受损,能够使定位于细胞膜上的钙离子主动转运功能受抑,从而可能会产生钙稳态失调,促使regucalin表达增加;此外,脂质过氧化与炎症密不可分,bFGF表达出现说明了饮用纯净水可能使大鼠肝脏发生了炎性反应。综上所述,无论饲料缺镁与否,饮用纯净水对大鼠重要脏器镁含量、繁殖功能、免疫功能及其仔鼠重要脏器镁含量、学习记忆和营养状态未见显著性影响,但具有影响机体正常镁分布、延迟子代生长发育、降低子代免疫功能的趋势;在饲料缺镁条件下,能够显著性降低大鼠血清总蛋白和白蛋白含量,导致大鼠营养不良的发生。在标准饲料条件下,饮用纯净水可能会使大鼠肝脏与出现氧化应激、钙稳态失调和炎性反应相关的蛋白表达发生改变。结合我室前期相关研究发现（饮用纯净水可致血脂升高、氧化应激等效应）和本实验研究结果,我们认为纯净水不宜作为敏感人群长期专一的饮用水,尤其是孕妇、婴幼儿、老人和体弱多病者等。更多还原

【Abstract】 With the improvement of people’s living standard,consuming high-energy and refined food is common in the world.Then the mineral matter in the food kept on decreasing. However,drinking soft water is prevailed in the world.And the hard effect due to low calcium or magnesium of soft water is still debated.Some one supports the hypothesis that the purpose of drinking water is supplement water,because the mineral mainly came from diet.The other supports hypothesis that the mineral matter in water is more bioavailability to a higher content,soft water can accelerate the mineral water elimination from body and increase dietary intake of toxic metals,people may be sickness due to drinking purified water long period.Then,study the biological effect of soft water on rat is useful to guide drinking waterThe research in our department reported that purified water can not affect on the growth and development of rat when the mineral mater sufficient in food,but it can induce blood rat,liver weight,liver to body weight,MDA,ET-1 increased significantly.Another study indicated a significant trend toward a decreased risk of having a child of very low birth weight(VLBW) with increasing magnesium levels in drinking water.It suggested that soft water might exert on the reproduce function and induce oxidative stress.Furthermore, our department reported that the serum magnesium of female rat was decreased significantly but other serum elements were not,such as Ca、Mg、K、Na、Fe、Mn、P、Zn、Cu、Sr、Cr、Mo、F、Li、Ni.In order to gain a better understanding of the effect of the soft drinking water(especially the magnesium in water),we study the effect of the purified water on rat with dietary magnesium deficient,magnesium sufficient and magnesium plentiful.Methods:1.The model of the magnesium content controlled strict in animal feeds(according to AIN-93G):Control diet:all the micronutrients are sufficient for good health,according to AIN-93M-GX Mineral Mix(DYETS #210050) except Mg2+content is 0.4g/kg,according to Estimated Average Requirement(EAR,from Nutrition Rehabilitation Centre,NRC) for rats.Magnesium deficient-diet:Mg2+ content is approx.0.2g/kg,a half of the Estimated Average Requirement(EAR,from NRC) for rats.Control drinking water:total dissolved solids(TDS) 175mg/L,conductivity 350μS/cm,chemical oxygen demand(COD) under detection.Purified drinking water.TDS 1.16mg/L,conductivity 2.28μS/cm,COD under detection.Weaning SD rat(68 female,36male) were assigned into 4 group by the randomization method,group 1 fed with control diet and control water,group 2 fed with control diet and purified water,group 3 fed with magnesium deficient diet and control water,group 4 fed with magnesium deficient diet and purified water throughout the entire study respectively.The animals were weighed individually on experimental day 0,7,14,21,28, and 35 during the preconception period.At 9 weeks of age(the rats were sexual mature), two females were paired with one male(2:1)from the same treatment group for a period of 14 days until mating was confirmed by observation of a copulatory plug or the presence of sperm in a vaginal rinse.The day that mating was confirmed was recorded as gestation day (GD) 0.The mated females were weighed on days 0,6,12,18 of gestation and 0,4,7,14 and 21 of lactation.All offspring were examined for behavior development during the lactation period.The body weight,reproduction(fertility rate of male,conception rate, delivery time,number of litters,number of pups,number of live pups,umber of dead pups, number of external abnormalities,average litter size,total sex ratio),offspring’s physical development parameters(pinna detachment,incisor eruption,development of fur,eye opening,auditive channel opening),behavioral parameters(surface righting ability,negative geotaxis,air righting reflex,walking test and forepaw grip strength),spatial learning were observed in the prenatal toxicity test.The serum protein,serum magnesium concentration was assayed with biochemical method.The magnesium content in organ was detected with atomic absorption spectrophotometer.The spleen cell proliferation was determined with MTT.The function of peritoneal macrophage was determined by Indian ink.And the host’s resistance was determined by bacterium attack.Statistical analyses were performed using SPSS 12.0 Statistical Software.The data for quantitative,continuous variables(e.g.,body weights) collected from all rats were analyzed by a factorial model to examine the main effects of diet(control or Mg deficient diet) and water(control or purified water) and their interactions.For the hidden platform test,a repeated measure ANOVA was used to determine the significance of the difference between the groups.The frequency data were analyzed by nonparametric x2-test.Unless otherwise noted,data presented were for the mean values±standard error of the mean.In all the experiments,P≤0.05 was the criterion for statistical significance.2.The model of the common animal feeds(according to GB14924-2001):Weaning SD rat(20 male) were assigned into 2 groups by the randomization method,group 1 fed with control water,group 2 fed with purified water 28 weeks.The Rat liver proteins were detected with 2D electrophoresis and MALDI-TOF-MS.The main results and conclusions are summarized as follows:Part one:The model of the magnesium content controlled strict in animal feeds (according to AIN-93G):The effect of the purified water was decrypted as follows:1.The model of magnesium deficiency was established successfully.The magnesium concentration of male’s liver,female’s serum and bone and offspring’s liver decreased significantly in the rat fed with magnesium deficient diet but not in the control diet.2.The effect of purified water on the magnesium content(1) Male rat:Magnesium deficiency in animal feeds decreased the liver magnesium content significantly.There was a decreased trend in the serum magnesium content, increased trend in the liver and spleen magnesium content in the rat drinking purified water, although this did not achieve statistical significance.It suggested that the magnesium redistribute,and it is hard to observe the effect ion of magnesium in water.(2) Female rat:Magnesium deficiency in animal feeds decreased the serum and bone magnesium content significantly.There was a decreased trend in the serum and bone magnesium content in the rat drinking purified water,although this did not achieve statistical significance.It suggested that the magnesium redistribute,and study the biological effect on bone of female is interesting.(3) Offspring:Magnesium deficiency in maternal animal feeds decreased the pups’ liver magnesium content significantly.There were no significantly differences in the magnesium content of the pups which maternal drinking purified water whatever fed with control diet or magnesium deficient diet.It suggested that the maternal can not afford the quantity of the magnesium for pups’ development when maternal magnesium deficiency.3.The effect of purified water on the reproduction functionNo differences were noted for number of litters,pups,live pups,dead pups,average litter size,average litter weight,total sex ratio,and average sex ratio in the groups.It suggested that the maternal can afford the quantity of the magnesium to maintain the reproduction function.4.The effect of purified water on the F1-pupsMagnesium deficiency in maternal animal feeds decreased the offspring’s body weight. The behavior deviations in F1-pups,defined by decreases in the performance of cliff avoidance reflex(day),air righting(degree),walking tests(score),grip strength(s),learning and memory show a decreased trend in the groups,although there were no significantly differences.It seems that maternal fed with magnesium deficiency diet can retard the offspring’s behavior development;drinking purified might aggravate the retardation.5.The effect of purified water on the immune functionMagnesium deficiency in animal feeds decreased serum protein significantly in F0-female,but it had no effect on the serum protein in F0-male and pups.Magnesium deficiency and drinking purified water in maternal animal feeds had no effect on the spleen cell proliferation,the function of peritoneal macrophage and the host’s resistance.But we observed maternal drinking purified water can decrease the function of peritoneal macrophage although this did not achieve statistical significance.It suggested studying the immune function of purified water is useful.6.The effect of purified water on the albuminMagnesium deficiency in animal feeds decreased albumin significantly in F0-female, but it had no effect on the albumin in F0-male alone.Magnesium deficiency diet with purified water decreased albumin significantly in F0-male(there were interaction between the magnesium deficiency diets with purified water).Part two:The model of the magnesium content plentiful in animal feeds:The effect of the purified water was decrypted as follows: 1.It had no effect on the body weight,organ-to-body weight.2.We identify 304 proteins with different expressing level between group fed with control water and group fed with purified water by 2D electrophoresis.We chosen 15 proteins to identify by MALDI-TOF-MS,and got 14 differential proteins,including MMSDH,Arginase -1,ATP synthase beta subunit,CAT,bFGF,regucalcin.(1) Arginase -1 participates in the urea cycle.It catalyzes arginine to ornithin and urea. NOS can catalyze arginine to NO.And NO decreased in the rat which drinking purified water.So,Arginase -1 express lower in the group 2 might inhibit the urea cycle,increase the NO.(2) ATP synthase beta subunit is an ingredient of ATP synthase.ATP synthase beta subunit express in group 2 but not in group 1.It might be the mitochondrial membrane damaged by oxidative stress in rat liver induced by purified water,and then the ATP synthase activity decreased feedback to increase the ATP synthase beta subunit expression.(3) MMSDH is a Coenzyme A dependent mitochondrial enzyme,a member of aldehyde dehydrogenase.It is important in the MDA metabolism.It expressed in the group 1but not in group 2,indicated that MDA would aggregation in liver.It is similar to our previous study.(4) BFGF is a peptide to regulate the cell growth.It increased in the inflammatory disease.It express in group 2 but not in group 1,indicated there were inflammatory reaction in the rat liver which drinking purified water.Our previous study also reported hepatic cell was focal necrosis and leukocyte infiltration.(5) Regucalcin is an calcium binding protein.In liver,it maintains calcium homeostasis in hepatocyte intracytoplasm.It express higher in group 2 indicated the absorption of calcium decreased or the calcium homeostasis disturbance due to the membrane damaged by oxidative stress.(6) CAT participate in the ROS metabolic process。It expressed in the group 1 but not in group 2,indicated that relation to the oxidative stress in liver induced by purified water.Conclusion:1.Magnesium deficiency in diets and drinking purified water might induce the magnesium redistribute in rat body,and the magnesium level of the organ changed in different degree.The maternal serum magnesium and bone magnesium decreased significantly should be pay more attention,it suggested the bone constituent of females especially which experience pregnancy and breast feed are easy to change due to drinking purified water.2.F0-female mobilize the organism magnesium to maintain the reproduction and nurse offspring,so there were no significant differences in the rat whatever fed with magnesium deficient diet or control diet and purified water or control water.But at later period of the lactation day,maternal fed with magnesium deficient diet cannot afford magnesium to the need of pups,so the pups weight gain and development delayed.Maternal drinking purified water whatever fed with control diet or magnesium deficient diet might lag the pups weight gain and development.3.Magneium exerts an important role in the immune function.Magnesium deficient diet can decreased the F0-rat(especially the female rat) immune function significantly. Although maternal fed with magnesium deficient diet,it can mobilize the magnesium to afford the pups,the pups immune function did not decrease.But maternal drinking purified water might decrease the pups’ immune function,and the mechanism was not unknown.4.The albumin decreased significantly in F0-male but not in F0-female which fed with magnesium deficient diet,the reason might be the need of the magnesium in female more than the male during gestation and lactation period.Because the magnesium level in water was lower,so we did not observed the albumin decreased in the female rat which drinking purified water whatever having control diet or magnesium deficiency diet.But the albumin decreased significantly in F0-male rat drinking purified water and consuming magnesium deficient diet.5.It might be oxidative stress,calcium homeostasis disturbance and inflammatory reaction in liver of the rat drinking purified water under common diet.Because we observed the MMSDH、CAT expression disappeared in the rat liver which drinking purified water, which might induce MDA accumulate,lipid peroxidation(oxidative stress),then damage the cell membrane.The mitochondrial membrane damaged might affect the ATP synthase activity decreased,then feedback to increase the ATP synthase beta subunit expression.And the membrane damaged might induce calcium homeostasis disturbance,so the regucalcin express higher in the rat liver which drinking purified water.NO decreased in the rat which drinking purified water,then Arginase -1 express lowers in the rat liver which drinking purified water inorder to inhibit the urea cycle,increase the NO.It is reported that oxidative stress are relate to inflammatory reaction,we also observed the bFGF express in the rat liver which drinking purified water but not in the rat which drinking control water.In a word,drinking purified water should be carefully considered,especially for susceptible population.更多还原