南京农业大学学报  2018, Vol. 41 Issue (6): 1100-1106   PDF    
http://dx.doi.org/10.7685/jnau.201711039
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文章信息

杨春, 李惠芳, 杨树, 方茜, 马文强, 赵茹茜
YANG Chun, LI Huifang, YANG Shu, FANG Xi, MA Wenqiang, ZHAO Ruqian
母鼠妊娠期和哺乳期日粮添加甜菜碱对子代雌性大鼠机体铁代谢的影响及其机制
Effects of maternal betaine supplementation during gestagation and lactation on iron metablism in female offsprings of rats and its mechanism
南京农业大学学报, 2018, 41(6): 1100-1106
Journal of Nanjing Agricultural University, 2018, 41(6): 1100-1106.
http://dx.doi.org/10.7685/jnau.201711039

文章历史

收稿日期: 2017-11-28
母鼠妊娠期和哺乳期日粮添加甜菜碱对子代雌性大鼠机体铁代谢的影响及其机制
杨春 , 李惠芳 , 杨树 , 方茜 , 马文强 , 赵茹茜     
南京农业大学农业农村部动物生理生化重点实验室/动物医学院, 江苏 南京 210095
摘要[目的]本文旨在研究母鼠日粮饲喂甜菜碱对子代雌性大鼠机体铁代谢的影响及其机制。[方法]随机选取56只10周龄Sprague Dawley雌性大鼠,与同种雄鼠自然交配,配种成功后,孕鼠随机分为对照组和甜菜碱组。对照组孕鼠在怀孕第1~21天饲喂基础日粮,甜菜碱组孕鼠同期饲喂在基础日粮上添加质量分数为1%甜菜碱的混合日粮。出生雌性大鼠于(3周龄)断奶后饲喂基础日粮至16周龄。记录13~16周龄子代雌性大鼠生产性能,测定16周龄子代雌性大鼠血液及组织铁代谢相关指标并分析其肝脏铁代谢相关基因及蛋白的表达。[结果]与对照相比,母体添加甜菜碱对子代雌性大鼠生长性能无明显影响,但显著或极显著降低16周龄子代雌性大鼠的血红蛋白含量(P < 0.05)、血清铁含量、转铁蛋白结合铁含量和总铁结合力(P < 0.01),促进肝脏铁摄取相关蛋白二价金属离子转运体(DMT1)及转铁蛋白受体1(TFR1)蛋白表达(P < 0.05)。[结论]母体饲喂甜菜碱降低了子代雌性大鼠血清铁水平,但对肝脏铁含量没有显著影响。肝脏铁的稳态与甜菜碱诱导的肝脏铁摄取通路相关蛋白(TFR1和DMT1)的高表达密切相关。
关键词甜菜碱   大鼠   铁代谢   
Effects of maternal betaine supplementation during gestagation and lactation on iron metablism in female offsprings of rats and its mechanism
YANG Chun, LI Huifang, YANG Shu, FANG Xi, MA Wenqiang , ZHAO Ruqian    
Key Laboratory of Animal Physiology and Biochemistry, Ministry of Agriculture and Rural Affairs, Nanjing Agricultural University, Nanjing 210095, China
Abstract: [Objectives] This paper aimed to study the effects of maternal dietary betaine supplementation during gestagation and lactation on the iron metabolism in female offspring of rats. [Methods] Fifty-six 10 week-old Sprague Dawley female rats were randomly selected, mated and divided into two groups:control group (Con) and betaine group (Bet). Female rats were fed with basal diet and trail diet (basal diet plus 1% betaine)during pregnancy and lactation, respectively. The female offsprings of rats were selected and provided with basal diet from weaning up to 16-weeks. The growth performance was recorded during the experimental periods. The blood iron parameters, tissue iron content and hepatic iron metabolism related genes and protein expression were measured at the end of the experiment. [Results] Maternal betaine supplementation had no significant effect on growth performance, greatly decreased hemoglobin concentration, serum iron, transferrin-bound iron and total iron binding capacity (P < 0.01), but significantly enhanced hepatic iron-uptake-related proteins DMT1 (divalent metal transperfer 1) and TFR1 (transferrin receptor protein 1)expression (P < 0.05). [Conclusions] Maternal betaine exposure during pregnancy and lactation decreases serum iron content, but does not affect hepatic iron content in female offsprings of rats. Maintenance of hepatic iron homeostasis is strong associated with high expression of DMT1 and TFR1 induced by betaine in female offsprings of rats.
Keywords: betaine    rats    iron metabolism   

铁是动物机体必需的微量元素, 是血红蛋白、肌红蛋白和机体多种代谢酶重要组分, 参与机体细胞生长、增殖和分裂等过程[1-3]。动物机体铁代谢稳态对于机体十分重要, 畜禽铁代谢紊乱影响机体生长发育, 严重时会影响畜禽生产性能。肝脏是机体铁稳态调控的主要器官, 具有贮存铁和调节机体铁稳态的作用[4]。参与铁代谢的铁蛋白(ferritin)和转铁蛋白(transferrin)均由肝脏合成, 并通过铁调素(hepcidin)-膜铁转运蛋白(ferroportin, FPN)途径调节机体铁稳态。hepcidin与FPN在细胞表面结合形成hepeidin-FPN复合物, 引发FPN内化作用而进入细胞, 并在溶酶体发生降解, 从而抑制FPN表达调控肝脏铁释放[4]。甜菜碱(betaine)化学结构为三甲胺乙内酯, 它广泛存在于动植物中。甜菜碱混合日粮能够显著改善动物饲料的适口性, 对动物有促进脂肪代谢、抗炎、调节机体渗透压作用, 可以缓解应激, 保护动物肠道, 提高畜禽酮体品质[5-10]

有研究表明, 母鼠从妊娠到哺乳期间补充胆碱能够改善子鼠缺铁现象及缺铁引起的疾病[11-13]。甜菜碱具备胆碱的部分功能[14], 通过向机体提供甲基, 增强子代功能基因启动子区和组蛋白的甲基化水平从而调控子代基因表达[15-16], 孕期母体中循环的甜菜碱可以经胎盘转运到胎儿体内[17-18]。孕期母猪饲喂甜菜碱显著提高了仔猪肝脏3-羟基-3-甲基戊二酸单酰CoA还原酶(3-hydroxy-3-methylglutaryl coenzyme A reductase, HMGCR)基因启动子区CpG岛和组蛋白H3赖氨酸甲基化水平, 抑制胆固醇代谢关键基因HMGCR基因转录, 从而调控仔猪胆固醇代谢[19]。母猪饲喂甜菜碱日粮也可提高仔猪磷酸烯醇式丙酮酸羧激酶1(phosphoenolpyruvate carboxykinase, PEPCK1)启动子区CpG岛和组蛋白甲基化程度, 抑制肝脏糖异生关键基因PEPCK1 的表达, 影响新生仔猪肝脏糖异生[20]

甜菜碱作为营养添加剂被广泛应用在畜牧生产中, 甜菜碱对糖、脂、固醇代谢的调控研究已有报道, 但是鲜少有关于铁代谢的研究。研究表明, 甜菜碱可作为甲基供体, 而动物机体肝脏铁调控相关基因启动子区也存在甲基化修饰位点[21], 探讨母体饲喂甜菜碱与子代铁代谢关系能够为铁的畜禽生产应用提供参考依据。铁稳态对于畜禽生长发育及生产性能至关重要, 因此本试验通过在母鼠妊娠及哺乳期间饲喂甜菜碱, 研究母体日粮添加甜菜碱对子代雌性大鼠铁代谢的影响并探讨甜菜碱对铁代谢的作用机制。

1 材料与方法 1.1 试验仪器与试剂 1.1.1 试验仪器

电消解仪(DigiBlock ED54, LabTech), 原子吸收分光光度计(ICE3500, Thermo Fisher, Waltham), 全自动血球分析仪(BC-2800, 迈瑞医疗, 深圳), 全自动生化分析仪(7020, Hitachi High-Tech Cropration), Real-time PCR系统(Mx3000, Stratagene, 美国), 凝胶成像系统(VersaDoc 4000MP, BIO-RAD, 美国)。

1.1.2 试验试剂

甜菜碱(B2629, Sigma), 血清铁检测试剂盒(6063-2012)、不饱和铁结合力检测试剂盒(6062-2012, Shino-test corporation), Trizol试剂(15596026, Thermo Fisher), D6110A反转录(RT)试剂盒、实时荧光定量PCR(qPCR)试剂盒(TaKaRa公司)。

1.2 试验方法

选取56只10周龄Sprague Dawley雌性大鼠(购于江苏大学实验动物研究中心), 与同种雄性大鼠自然交配, 见阴道栓为配种成功。配种成功后将怀孕雌性大鼠随机分为对照组和甜菜碱组。对照组孕鼠从怀孕到哺乳期间饲喂基础日粮, 试验组雌性大鼠同期饲喂在基础日粮基础上添加质量分数为1%甜菜碱的日粮。记录子代雌性大鼠初生体质量。子代大鼠出生后由各自母鼠哺乳, 断奶后均饲喂基础日粮。13周龄时每组随机选取14只子代雌性大鼠, 饲养至16周龄。期间自由饮水和采食。饲养试验结束后将16周龄子代雌性大鼠禁食12 h, 称体质量, 麻醉, 迅速采集血液及组织样品(肝脏、脾脏及心脏)。制备血清样品, 并贮存于-20 ℃待用。记录肝脏、心脏、脾脏各组织器官质量, 并将采集肝脏组织样品于-80 ℃贮存, 备用。

1.3 测定指标 1.3.1 生长性能测定

在子代雌性大鼠出生时记录初生体质量、13周龄体质量、16周龄体质量及13~16周龄平均日采食量。

1.3.2 血液铁相关指标及组织铁含量测定

饲料、血清及组织(肝脏、脾脏及心脏)样品经电热消解仪消解后, 采用原子吸收分光光度计测定饲料、血清及组织中铁含量。应用全自动血球分析仪检测全血的血红蛋白(hemoglobin, Hb)含量; 应用全自动生化分析仪检测血清转铁蛋白结合铁(transferrin-bound iron, Tf-Fe)和不饱和铁结合力(unsaturated iron binding capacity, UIBC), 计算总铁结合力(total iron binding capacity, TIBC)和转铁蛋白饱和度(transferrin saturation, TS)。根据平均采食量及饲料铁含量计算子代雌性大鼠的日平均铁摄入量。

1.3.3 RT-qPCR检测肝脏铁代谢相关基因的表达

用Trizol试剂提取子代大鼠肝脏总RNA, 反转录后应用qPCR试剂盒检测肝脏铁代谢相关基因mRNA的表达水平。反应条件:95 ℃, 10 min; 95 ℃, 30 s; 62 ℃, 20 s; 72 ℃, 20 s, 共40个循环。检测与肝脏铁转运、摄取、贮存、释放及铁调控相关的基因,包括转铁蛋白(transferrin, Tf)、转铁蛋白受体1(transferrin receptor 1, Tfr1 )、转铁蛋白受体2(transferrin receptor 2, Tfr2 )、二价金属离子转运体(divalent metal transporter 1, Dmt1 )、锌铁转运体14(ZRT/IRT-like protein 14, Zip14 )、铁蛋白重链(ferritin heavy chain, Fth)、铁蛋白轻链(ferritin light chain, Ftl)、膜铁转运蛋白(ferroportin, Fpn)及铁调素(hepcidin)基因, 内参基因为酸性核糖体磷酸化蛋白P0(acidic ribosomal phosphoprotein P0, Arbp)。引物由上海捷瑞生物工程有限公司合成, 引物序列见表 1

表 1 荧光定量PCR(qPCR)的引物[22] Table 1 Primers used for real-time fluorescence quantitive PCR
基因
Gene
基因序列号
GenBank accession No.
引物对序列(5′→3′)
Primer pairs sequence
片段长度/bp
Fragment size
退火温度/℃
Annealing temperature
Tf NM_001013110.1 ATCAGACTCCAGCATCAAC/CCAATACACAGGTCACAGA 198 62
Tfr1 NM_022712.1 CACTTACGGTCAGCACTT/CACAACTCACTGGACTTAGA 114 62
Tfr2 NM_001105916.1 GTTGGTGGTTGGTGAAGA/ACATAGTGCGTGTCAGTC 240 62
Dmt1 NM_013173.2 TCACTTGGTCCTCGTTCT/TCACTAACAGCCTCCTTATAG 139 62
Zip14 NM_001107275.1 CCACCATTCTCCAGCAGT/GACAAGGCGATGAAGTAAGT 149 62
Fth NM_012848.2 GTCACTACTGGAACTTCACA/TTCAGGTAATGCGTCTCAAT 216 62
Ftl NM_022500.4 GCAGAAGCCATCTCAAGA/TTCCAAGAAGTCACAGAGG 197 62
Fpn NM_133315.2 AGGAAGGATGCTGTGGAT/R:TGTCAAGAGGAGGCTGTT 115 62
Hepcidin NM_053469.1 GAAGGCAAGATGGCACTA/TTACAGCAGAAGAGGCATAT 221 62
Arbp NM_022402.2 TAGAGGGTGTCCGCAATGTG/CAGTGGGAAGGTGTAGTCAGTC 217 62
注: Tf:转铁蛋白基因Transferrin gene; Tfr1 :转铁蛋白受体1基因Transferrin receptor 1 gene; Tfr2 :转铁蛋白受体2基因Transferrin receptor 2 gene; Dmt1 :二价金属离子转运体基因Divalent metal transporter 1 gene; Zip14 :锌铁转运体14基因ZRT/IRT-like protein 14 gene; Fth:铁蛋白重链基因Ferritin heavy chain gene; Ftl:铁蛋白轻链基因Ferritin light chain gene; Fpn:膜铁转运蛋白基因Ferroportin gene; Hepcidin:铁调素基因Hepcidin gene; Arbp:酸性核糖体磷酸化蛋白基因P0 Acidic ribosomal phosphoprotein P0 gene.下同。The same as follows.
1.3.4 Western blot检测肝脏铁代谢相关蛋白表达

利用蛋白裂解液RIPA提取子代雌性大鼠肝脏蛋白, BCA法进行蛋白定量, 95 ℃、10 min变性, 于-80 ℃贮存备用。以Tubulin-α为内参, 蛋白样品经SDS-PAGE电泳、硝酸纤维素膜转膜、封闭(脱脂奶粉40 g·L-1, 2 h), 加一抗4 ℃孵育过夜, 用TBST洗膜3次, 每次10 min, 用辣根过氧化物酶(HRP)标记的二抗室温孵育2 h, 用TBST再次洗膜后进行化学发光, 应用凝胶成像系统测定蛋白条带灰度值。肝脏铁代谢相关蛋白表达以目的蛋白条带与Tubulin-α条带灰度值的比值表示[23]。使用相关抗体详细信息如下:TFR1(10084-2-AP, Proteintech, 英国), TF(HPA001527)、TFR2(SAB2700078, Sigma, 美国), DMT1(ab55735)、ZIP14(ab123988)、FTL(ab69090)、Hepcidin(ab105098)(Abcam, 英国), FPN(sc-49668, Santa Cruz, 美国), FTH(BS6175)、Tubulin-α(BS1966)、HRP标记的羊抗鼠的二抗(BS10043)、羊抗鼠的二抗(BS12478)、兔抗羊的二抗(BS30503)(Bioworld, 美国)。

1.4 数据统计

试验数据采用SPSS 19.0软件进行单因素方差分析(one-way ANOVA, LSD), 结果以平均值±标准误(x±SE)表示。

2 结果与分析 2.1 母体添加甜菜碱对子代雌性大鼠生长性能的影响

表 2可知:母体添加甜菜碱对子代雌性大鼠初生体质量、13周龄体质量和16周体质量无显著影响(P>0.05)。

表 2 母体添加甜菜碱对子代雌性大鼠生长性能的影响 Table 2 Effects of maternal betaine supplementation on growth performance in female offsprings of rats
指标
Items
对照组
Control group
甜菜碱组
Betaine group
初生体质量/g Birth body weight 6.58±0.05(n=202) 6.46±0.04(n=215)
13周体质量/g 13-week body weight 338.42±5.60(n=14) 335.36±4.92(n=14)
16周体质量/g 16-week body weight 351.07±4.66(n=14) 344.79±5.80(n=14)
2.2 母体添加甜菜碱对子代雌性大鼠日平均采食量及铁摄入量的影响

表 3可知:母体添加甜菜碱对子代雌性大鼠13周至16周日平均采食量和日平均铁摄入量均无显著影响(P>0.05)。

表 3 母体添加甜菜碱对子代雌性大鼠日平均采食量和铁摄入量的影响(n=14) Table 3 Effects of maternal betaine supplementation on average daily food intake and average daily iron intake in female offsprings of rats
指标
Items
对照组
Control group
甜菜碱组
Betaine group
日平均采食量/(g·d-1)Average daily food intake 20.46±0.43 19.49±0.65
日平均铁摄入量/(mg·d-1)Average daily iron intake 7.92±0.17 7.55±0.25
2.3 母体添加甜菜碱对子代雌性大鼠血液及组织铁指标的影响

表 4可知:与对照组相比, 母体添加甜菜碱显著和极显著降低了子代雌性大鼠血红蛋白含量(P < 0.05)、血清铁含量、转铁蛋白结合铁含量和总铁结合力(P < 0.01), 对血清不饱和铁结合力、转铁蛋白饱和度和组织铁含量没有显著影响(P>0.05)。

表 4 母体添加甜菜碱对子代雌性大鼠血液及组织铁指标的影响(n=9) Table 4 Effects of maternal betaine supplementation on blood and tissue iron parameters in female offsprings of rats
指标
Items
对照组
Control group
甜菜碱组
Betaine group
血液Blood
  血红蛋白含量/(g·L -1) Hb content 139.60±1.28 134.44±0.93 *
  血清铁含量/(μg·mL -1) Serum iron content 9.23±0.44 6.14±0.10 **
  转铁蛋白结合铁含量/(μmol·L -1) TF-Fe content 45.29±2.12 29.93±1.76 **
  不饱和铁结合力/(μmol·L -1) UIBC 39.16±2.66 32.19±2.17
  总铁结合力/(μmol·L -1) TIBC 86.73±2.36 62.12±1.75 **
  转铁蛋白饱和度/% TS 54.38±2.45 48.33±2.83
组织Tissue
  肝脏铁含量/(μg·g -1) Iron content of liver 198.93±9.25 210.60±9.96
  脾脏铁含量/(μg·g -1) Iron content of spleen 20.28±0.78 18.83±0.32
  心脏铁含量/(μg·g -1) Iron content of heart 20.93±1.77 18.74±0.44
Note: Hb:Hemoglobin; TF-Fe:Transferrin-bound iron; UIBC:Unsaturated iron bonding capacity; TIBC:Total iron binding capacity; TS:Transferrin saturation. * P < 0.05; * * P < 0.01. The same as follows.
2.4 母体添加甜菜碱对子代雌性大鼠肝脏铁代谢相关基因的影响

图 1可知:与对照组相比, 母体添加甜菜碱组子代雌性大鼠肝脏Dmt1Zip14基因相对表达量显著增加(P < 0.05), 而其他基因表达没有显著变化(P>0.05)。

图 1 母体添加甜菜碱对子代雌性大鼠肝脏铁代谢相关基因表达的影响 Figure 1 Effect of maternal betaine supplementation on iron metabolism related genes expression of liver in female offsprings of rats
2.5 母体添加甜菜碱对子代雌性大鼠肝脏铁代谢相关蛋白的影响

图 2可知:与对照组相比, 母体添加甜菜碱后子代雌性大鼠肝脏铁摄取相关蛋白DMT1和铁转运相关受体(TFR1)蛋白含量显著上升(P < 0.05), 而其他蛋白含量均无显著变化(P>0.05)。

图 2 母体添加甜菜碱对子代雌性大鼠肝脏铁代谢相关蛋白表达的影响 Figure 2 Effect of maternal betaine supplementation on iron metabolism related proteins expression of liver in female offsprings of rats
3 讨论

甜菜碱在畜牧生产中的应用十分广泛, 日粮添加质量分数为0.05%~0.25%的甜菜碱不影响生长猪增重[24-26]; 肉鸡日粮添加质量分数为0.1%甜菜碱不影响肉鸡1~7周龄生长性能[27], 日粮中添加质量分数为0.2%的甜菜碱可显著提高42日龄肉鸡出栏体质量[28]。卢霞等[29]报道, 用15~60 mg·kg-1甜菜碱水溶液灌胃大鼠, 对2~8周龄大鼠体质量未产生显著影响。有关甜菜碱对子代的影响研究结果表明, 母猪饲料中添加质量分数为0.3%的甜菜碱对窝产仔猪数、窝重和新生仔猪体质量均无显著影响[20], 而皋黄鸡种蛋内注射甜菜碱(每枚2.5 mg)显著增加了孵育后4~8周龄肉鸡体质量[30]。本试验在母鼠孕期及哺乳期饲喂质量分数为1%的甜菜碱日粮[31-32], 为避免性别差异, 从对照组及试验组中随机选取子代雌性大鼠进行试验, 结果表明, 母鼠孕期及哺乳期饲喂甜菜碱日粮对子代雌性大鼠初生体质量及16周体质量没有影响, 推测甜菜碱对动物生长性能的影响可能与动物品种及甜菜碱添加剂量有关。

机体全身性铁稳态依赖于机体对铁的吸收、转运、贮存、释放及调节作用。肝脏是铁贮存及调控器官, 其通过分泌hepcidin抑制FPN和DMT1, 精密调控动物机体十二指肠铁吸收、机体各组织铁摄取和释放, 进而调控机体全身性铁稳态。肝脏经由2条途径摄取体内的非血红素铁, 即转铁蛋白结合铁途径和非转铁蛋白结合铁途径。TFR1/2介导细胞转铁蛋白结合铁摄取, DMT1和ZIP14介导非转铁蛋白结合铁摄取。肝脏摄取的铁进入肝细胞内铁池, 再与胞浆内铁蛋白结合贮存在铁蛋白轻链(FTL)中, FPN是目前唯一已知存于在细胞表面的铁转出相关蛋白, 它将胞浆铁池中的铁运入循环血液[33]。机体内的铁大部分用来合成血红蛋白并最终形成红细胞, 血红素是血红蛋白的重要成分, 肝脏是血红素合成的主要器官。已有报道表明, 机体缺铁会引起肝细胞表面TFR1合成增加并通过肝脏调控作用维持铁稳态[34]。本试验结果显示, 母体从怀孕到哺乳期饲喂1%甜菜碱日粮能显著降低子代雌性大鼠血清总铁、转铁蛋白结合铁水平以及血红蛋白浓度, 脾脏组织铁含量也呈降低趋势, 提示母鼠孕期及哺乳期饲喂1%甜菜碱可能造成子代缺铁, 但肝组织铁含量无显著变化。从蛋白及基因表达水平检测结果发现, 子代雌性大鼠肝细胞中的铁贮存相关蛋白(FTL、FTH)和肝细胞表面铁释放相关蛋白(FPN)均无明显变化, 而子鼠肝细胞铁摄取相关蛋白(TFR1和DMT1)表达量显著增加。

母体从怀孕到哺乳期间饲喂1%甜菜碱日粮可能造成子代雌性大鼠铁缺乏, 诱导肝脏通过提高TFR1和DMT1表达量增加肝脏铁摄取能力, 维持细胞铁稳态, 同时TFR1可能作为信号传递分子参与机体全身性铁稳态中。日粮中甜菜碱含量较高时甜菜碱主要聚集在肝脏, 反之主要聚集在小肠[10], 母体添加高剂量甜菜碱可能影响子代肝脏基因甲基化修饰, 调节子代肝脏铁调控通路, 而低剂量甜菜碱日粮与前者可能存在不同作用机制, 关于母体饲喂不同剂量甜菜碱对子代机体铁代谢的影响及其作用机制有待进一步研究。

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