不同水平锌缺乏对大鼠胚胎心脏发育的影响及其可能机制的探讨
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摘要
目的
锌是人体必需微量元素之一,孕期母体锌内稳态对正常妊娠及胎儿的生长发育极为重要,锌缺乏可导致机体功能障碍、行为异常及胎儿出生缺陷等。先天性心脏病是新生儿先天缺陷中最常见的疾病之一,本研究通过构建不同水平的缺锌孕鼠模型,探讨缺锌对大鼠胚胎心脏的发育毒性及其作用阈剂量和剂量-效应关系,同时探讨其作用的可能机制,旨在为临床锌的正确补充和监测及心脏发育畸形的预防提供实验依据。
方法
采用健康清洁级SD雌性大鼠50只,随机分成5组:1极低锌组、2低锌组、3中等低锌组、4边缘低锌组、5常锌组,饲以含锌量不同的饲料。锌耗竭性饲养25天后交配,至孕第19天时剖腹取鼠胚心,同时留取孕鼠静脉血及胎盘标本。病理切片观察胎鼠心脏畸形情况;原子吸收法测定孕鼠血锌值;碱性磷酸酶试剂盒检测孕鼠血清AKP活性;RT—PCR法检测胎盘中MT1及ZNT1 mRNA的相对表达量。
结果
1.各低锌组孕鼠血锌值和血清AKP活性与常锌组比较均有显著性差异(均P<0.05),随饲料含锌量的增加而增加,且血清AKP活性增加更为明显。
2.各组鼠胚均发现吸收胎、畸胎及死胎,中等以上低锌组与常锌组比较发生率明显增加(均P<0.01)。
3.中等以上低锌组与常锌组比较,胚心畸形率均明显增高(均P<0.05);各低锌组比较,也有显著性差异(均P<0.05)。
4.中等以上低锌组胎盘中MT1及ZNT1 mRNA的相对表达丰度较常锌组均明显降低(均P<0.05)。
5.心脏发育异常鼠胚胎盘中MT1及ZNT1 mRNA的相对表达丰度较正常胚胎明显降低(均P<0.01)。
结论
1.缺锌配方饲料饲养后大鼠的一般表现及血锌值和血清AKP值显著降低,说明缺锌大鼠模型成功。
2.同等低锌情况下,血清AKP活性较血锌值变化明显,表明血清AKP活性与血锌值相结合能够较准确地反映机体内的锌水平。
3.中等以上低锌组胚鼠不良胚胎及心脏畸形发生增加,且随低锌程度加重而增加。提示孕鼠低锌对胚胎有致畸作用,且毒性作用存在较明显的剂量-效应关系,对中等以上缺锌的防治应该引起重视。
4.中等以上低锌组胚鼠胎盘中MT1 mRNA表达减少,提示低锌可能通过降低机体锌储备及抗氧化能力,影响胚胎心脏的正常发育。
5.中等以上低锌组胚鼠胎盘中ZNT1 mRNA表达减少,表明母鼠低锌可使胎盘的锌转运能力降低,减少鼠胚血锌供应,加重高氧化应激状态,增加胚鼠心脏畸形率。
6.心脏畸形胚鼠较正常胚鼠胎盘中MT1及ZNT1 mRNA表达减少,提示母鼠低锌只有影响到胎盘中的MT1及ZNT1等锌相关蛋白的表达时才有可能导致胚鼠心脏畸形发生。
Objective
Zinc is one of the essential trace elements, relating to many fuctions of the cells. Zinc deficiency can lead to organic dysfunction, dystrophy and fetal dysplasia. Gravidas require more zinc. Homeostasis of zinc is very important to normal pregnancy and fetal development during pregnancy. Congenital heart disease(CHD) is one of the most common diseases of human birth defects, which is seriously endangering infant,s health. This research established rat models of zinc deficiency through feeding them in diet with different content of zinc. It aims to study the effect of zinc deficiency on the development of heart in rat fetus and the relation between dose and effect; and the possible mechanism was also investigated. The object is to provide an experimental data for possible zinc supplement and prevention of CHD in clinic.
Methods
50 SD-healthy adult female rats were randomly divided into 5 groups, zinc acutely deficient group, zinc deficient group, zinc moderately deficient group, zinc marginally deficient group, normal zinc group, feeding in diet with different content of zinc. They were fed 25 days for depleting the zinc pool in the body, and mated with male rats. They were underwent feeding in diet with different content of zinc during pregnancy. All the pregnant rats were killed on the 19th day of pregnancy, the embryonic heart were sliced, and the heart defects observed. Blood level of zinc was determined by atomic absorption spectrophotometer and the serum AKP activity was determined by AKP kit. The expression of MT1 and ZNT1 mRNA in placenta was examined by RT-PCR.
Results
1. As compared with the normal zinc group, the serum AKP activity and blood zinc content in all the zinc deficient groups were significantly lowered (all P<0.05). Under the same conditions the changes of serum AKP activity were more obvious.
2. In every group, it was found different extents of absorptive embryos, abnormal embryos and stillbirths. As compared with the normal zinc group, the incidences were significantly higher in the zinc acutely deficient group, zinc deficient group, and zinc moderately deficient group (all P<0.05).
3. Embryo heart malformations were mainly hypoevolutial, atrial absence, ventricular septal defect, ventricular defect, and so on. As compared with the normal zinc group, embryo heart malformations were significantly higher in the zinc acutely deficient group, zinc deficient group, and zinc moderately deficient group (all P<0.01).
4. The MT1 and ZNT1 mRNA levels in placenta were both significantly decreased in the zinc acutely deficient group, zinc deficient group and zinc moderately deficient group vs. normal zinc group. (all P<0.05).
5. The MT1 and ZNT1 mRNA levels in placenta were both significantly decreased in the cardiac anomaly groups vs. the normal group (P<0.01).
Conclusions
1. The serum AKP activity and blood zinc content of rats reduced after feeding the rats with zinc-deficient diet of Flanagan formula. It indicated that the model is successful.
2. Serum AKP activity is a sensitive indicator of zinc in body; combination with the blood level of zinc can be more accurately reflecting the level of zinc.
3. Over moderately deficient zinc in the maternal body can increase heart malformations in embryos. The toxicity of zinc deficiency in embryos has a significant dose-effect relation. We must have a regard for preventing and curing over moderately deficiency of zinc.
4. Rats with over moderate zinc deficiency show increased heart malformations in embryos. The mechanism may include down-regulating the expression of MT1 mRNA in placenta and reducing the anti-oxidation in the body.
5. As an output of zinc in cells, ZNT1 can influence the utilization of zinc in the body. Zinc deficiency in the body will reduce the expression of ZNT1, cause a reduced blood zinc content and decreased expression of MTs. All the above results can exacerbate the state of high oxidative stress in the body and increase heart malformations in embryos.
6. It can't cause heart malformations in embryos unless zinc deficiency has influenced the expression of zinc-relating proteinums, such as MTs and ZNTs.
锌是人体必需微量元素之一,孕期母体锌内稳态对正常妊娠及胎儿的生长发育极为重要,锌缺乏可导致机体功能障碍、行为异常及胎儿出生缺陷等。先天性心脏病是新生儿先天缺陷中最常见的疾病之一,本研究通过构建不同水平的缺锌孕鼠模型,探讨缺锌对大鼠胚胎心脏的发育毒性及其作用阈剂量和剂量-效应关系,同时探讨其作用的可能机制,旨在为临床锌的正确补充和监测及心脏发育畸形的预防提供实验依据。
方法
采用健康清洁级SD雌性大鼠50只,随机分成5组:1极低锌组、2低锌组、3中等低锌组、4边缘低锌组、5常锌组,饲以含锌量不同的饲料。锌耗竭性饲养25天后交配,至孕第19天时剖腹取鼠胚心,同时留取孕鼠静脉血及胎盘标本。病理切片观察胎鼠心脏畸形情况;原子吸收法测定孕鼠血锌值;碱性磷酸酶试剂盒检测孕鼠血清AKP活性;RT—PCR法检测胎盘中MT1及ZNT1 mRNA的相对表达量。
结果
1.各低锌组孕鼠血锌值和血清AKP活性与常锌组比较均有显著性差异(均P<0.05),随饲料含锌量的增加而增加,且血清AKP活性增加更为明显。
2.各组鼠胚均发现吸收胎、畸胎及死胎,中等以上低锌组与常锌组比较发生率明显增加(均P<0.01)。
3.中等以上低锌组与常锌组比较,胚心畸形率均明显增高(均P<0.05);各低锌组比较,也有显著性差异(均P<0.05)。
4.中等以上低锌组胎盘中MT1及ZNT1 mRNA的相对表达丰度较常锌组均明显降低(均P<0.05)。
5.心脏发育异常鼠胚胎盘中MT1及ZNT1 mRNA的相对表达丰度较正常胚胎明显降低(均P<0.01)。
结论
1.缺锌配方饲料饲养后大鼠的一般表现及血锌值和血清AKP值显著降低,说明缺锌大鼠模型成功。
2.同等低锌情况下,血清AKP活性较血锌值变化明显,表明血清AKP活性与血锌值相结合能够较准确地反映机体内的锌水平。
3.中等以上低锌组胚鼠不良胚胎及心脏畸形发生增加,且随低锌程度加重而增加。提示孕鼠低锌对胚胎有致畸作用,且毒性作用存在较明显的剂量-效应关系,对中等以上缺锌的防治应该引起重视。
4.中等以上低锌组胚鼠胎盘中MT1 mRNA表达减少,提示低锌可能通过降低机体锌储备及抗氧化能力,影响胚胎心脏的正常发育。
5.中等以上低锌组胚鼠胎盘中ZNT1 mRNA表达减少,表明母鼠低锌可使胎盘的锌转运能力降低,减少鼠胚血锌供应,加重高氧化应激状态,增加胚鼠心脏畸形率。
6.心脏畸形胚鼠较正常胚鼠胎盘中MT1及ZNT1 mRNA表达减少,提示母鼠低锌只有影响到胎盘中的MT1及ZNT1等锌相关蛋白的表达时才有可能导致胚鼠心脏畸形发生。
Objective
Zinc is one of the essential trace elements, relating to many fuctions of the cells. Zinc deficiency can lead to organic dysfunction, dystrophy and fetal dysplasia. Gravidas require more zinc. Homeostasis of zinc is very important to normal pregnancy and fetal development during pregnancy. Congenital heart disease(CHD) is one of the most common diseases of human birth defects, which is seriously endangering infant,s health. This research established rat models of zinc deficiency through feeding them in diet with different content of zinc. It aims to study the effect of zinc deficiency on the development of heart in rat fetus and the relation between dose and effect; and the possible mechanism was also investigated. The object is to provide an experimental data for possible zinc supplement and prevention of CHD in clinic.
Methods
50 SD-healthy adult female rats were randomly divided into 5 groups, zinc acutely deficient group, zinc deficient group, zinc moderately deficient group, zinc marginally deficient group, normal zinc group, feeding in diet with different content of zinc. They were fed 25 days for depleting the zinc pool in the body, and mated with male rats. They were underwent feeding in diet with different content of zinc during pregnancy. All the pregnant rats were killed on the 19th day of pregnancy, the embryonic heart were sliced, and the heart defects observed. Blood level of zinc was determined by atomic absorption spectrophotometer and the serum AKP activity was determined by AKP kit. The expression of MT1 and ZNT1 mRNA in placenta was examined by RT-PCR.
Results
1. As compared with the normal zinc group, the serum AKP activity and blood zinc content in all the zinc deficient groups were significantly lowered (all P<0.05). Under the same conditions the changes of serum AKP activity were more obvious.
2. In every group, it was found different extents of absorptive embryos, abnormal embryos and stillbirths. As compared with the normal zinc group, the incidences were significantly higher in the zinc acutely deficient group, zinc deficient group, and zinc moderately deficient group (all P<0.05).
3. Embryo heart malformations were mainly hypoevolutial, atrial absence, ventricular septal defect, ventricular defect, and so on. As compared with the normal zinc group, embryo heart malformations were significantly higher in the zinc acutely deficient group, zinc deficient group, and zinc moderately deficient group (all P<0.01).
4. The MT1 and ZNT1 mRNA levels in placenta were both significantly decreased in the zinc acutely deficient group, zinc deficient group and zinc moderately deficient group vs. normal zinc group. (all P<0.05).
5. The MT1 and ZNT1 mRNA levels in placenta were both significantly decreased in the cardiac anomaly groups vs. the normal group (P<0.01).
Conclusions
1. The serum AKP activity and blood zinc content of rats reduced after feeding the rats with zinc-deficient diet of Flanagan formula. It indicated that the model is successful.
2. Serum AKP activity is a sensitive indicator of zinc in body; combination with the blood level of zinc can be more accurately reflecting the level of zinc.
3. Over moderately deficient zinc in the maternal body can increase heart malformations in embryos. The toxicity of zinc deficiency in embryos has a significant dose-effect relation. We must have a regard for preventing and curing over moderately deficiency of zinc.
4. Rats with over moderate zinc deficiency show increased heart malformations in embryos. The mechanism may include down-regulating the expression of MT1 mRNA in placenta and reducing the anti-oxidation in the body.
5. As an output of zinc in cells, ZNT1 can influence the utilization of zinc in the body. Zinc deficiency in the body will reduce the expression of ZNT1, cause a reduced blood zinc content and decreased expression of MTs. All the above results can exacerbate the state of high oxidative stress in the body and increase heart malformations in embryos.
6. It can't cause heart malformations in embryos unless zinc deficiency has influenced the expression of zinc-relating proteinums, such as MTs and ZNTs.
引文
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[3] Konig K, Will J C, Berger F, et al.Familial congenital heart disease, progressive atrioventricular block and the cardiac homeobox transcription factor gene NKX2.5: Identification of a novel mutation [J].Clin Res Cardiol, 2006; 95:499-503.
[4] Janet C King, Determinants of maternal zinc status during pregnancy [J]. American Journal of Clinical Nutrition, 2000; 71(5):1334S-1343S.
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[1] K. Konig, J.C. Will, F. Berger, et al. Familial congenital heart disease, progressive atrioventricular block and the cardiac homeobox transcription factor gene NKX2.5: identification of a novel mutation [J]. Clin Res Cardiol, 2006; 95:499–503.
[2] Pamela J. Fraker,.Roles for Cell Death in Zinc Deficiency [J]. J.Nutr, 2005; 135:359-362.
[3] Paola Llinas, Michel Masella, Torgny Stigbrand et al. Structural studies of human alkaline phosphatase in complex with strontium: Implication for its secondary effect in bones[J].Protein Science, 2006; 15:1691-1700.
[4] Palmiter, R. D. & Huang, L. Efflux and compartmentalization of zinc by members of the SLC30 family of solute carriers. Pflugers Arch. Eur[J]. J. Physiol, 2004; 447:744-751.
[5] Eide, D. J. The SLC39 family of metal ion transporters. Pflugers Arch. Eur[J]. J. Physiol, 2004; 447:796-800.
[6] Karl B.Andree, Jihye Kim, Catherine P.Kirschke et al. Investigation of Lymphocyte Gene Expression for Use as Biomarkers for Zinc Status in Humans[J],J.Nutr, 2004; 134:1716-1723.
[7] Carl L. Keen, Lynn A. Hanna, Louise Lanoue, et al. Developmental Consequences of Trace Mineral Deficiencies in Rodents: Acute and Long-Term Effects[J] . J. Nutr. 2003;133:1477S-1480S.
[8] Duffy J.Y., Overmann G.J., Keen C.L., et al.Cardiac Abnormalities Induced by Zinc Deficiency Are Associated With Alterations in the Expression of Genes Regulated by the Zinc-Finger Transcription Factor GATA-4[J],Birth Defects Research (Part B). 2004; 71:102–109.
[9] James Y., James Kang, Metallothionein Redox Cycle and Function[J]. Experimental Biology and Medicine.2006; 231:1459-1467.
[10] Xiaoping Yang, Thomas A. Doser, Cindy X. Fang,et al. Metallothionein prolongs survival and antagonizes senescence-associated cardiomyocyte diastolic dysfunction: role of oxidative stress [J]. FASEB J, 2006; 20: 1024 - 1026.
[11] Jianxun Wang, MS; Ye Song, MD, PhD; Laila Elsherif, PhD; et al.Cardiac Metallothionein Induction Plays the Major Role in the Prevention of Diabetic Cardiomyopathy by Zinc Supplementation[J]. Circulation, 2006; 113:544-554.
[12] Kathy J. Jenkins, Adolfo Correa, Jeffrey A. Feinstein, et al,.Noninherited Risk Factors and Congenital Cardiovascular Defects [J]. Circulation, 2007; 115: 2995 -3014.
[13] Carl L. Keen, Michael S. Clegg, Lynn A. Hanna, et al.The Plausibility of Micronutrient Deficiencies Being a Significant Contributing Factor to the Occurrence of Pregnancy Complications[J]. J Nutr, 2003; 133:1597S-1605S.
[2]杨安峰、王平等,大鼠的解剖和组织[M],科学出版社,1985年6月第一版,统一书号:13031-2886:108-109。
[3] Konig K, Will J C, Berger F, et al.Familial congenital heart disease, progressive atrioventricular block and the cardiac homeobox transcription factor gene NKX2.5: Identification of a novel mutation [J].Clin Res Cardiol, 2006; 95:499-503.
[4] Janet C King, Determinants of maternal zinc status during pregnancy [J]. American Journal of Clinical Nutrition, 2000; 71(5):1334S-1343S.
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[1] K. Konig, J.C. Will, F. Berger, et al. Familial congenital heart disease, progressive atrioventricular block and the cardiac homeobox transcription factor gene NKX2.5: identification of a novel mutation [J]. Clin Res Cardiol, 2006; 95:499–503.
[2] Pamela J. Fraker,.Roles for Cell Death in Zinc Deficiency [J]. J.Nutr, 2005; 135:359-362.
[3] Paola Llinas, Michel Masella, Torgny Stigbrand et al. Structural studies of human alkaline phosphatase in complex with strontium: Implication for its secondary effect in bones[J].Protein Science, 2006; 15:1691-1700.
[4] Palmiter, R. D. & Huang, L. Efflux and compartmentalization of zinc by members of the SLC30 family of solute carriers. Pflugers Arch. Eur[J]. J. Physiol, 2004; 447:744-751.
[5] Eide, D. J. The SLC39 family of metal ion transporters. Pflugers Arch. Eur[J]. J. Physiol, 2004; 447:796-800.
[6] Karl B.Andree, Jihye Kim, Catherine P.Kirschke et al. Investigation of Lymphocyte Gene Expression for Use as Biomarkers for Zinc Status in Humans[J],J.Nutr, 2004; 134:1716-1723.
[7] Carl L. Keen, Lynn A. Hanna, Louise Lanoue, et al. Developmental Consequences of Trace Mineral Deficiencies in Rodents: Acute and Long-Term Effects[J] . J. Nutr. 2003;133:1477S-1480S.
[8] Duffy J.Y., Overmann G.J., Keen C.L., et al.Cardiac Abnormalities Induced by Zinc Deficiency Are Associated With Alterations in the Expression of Genes Regulated by the Zinc-Finger Transcription Factor GATA-4[J],Birth Defects Research (Part B). 2004; 71:102–109.
[9] James Y., James Kang, Metallothionein Redox Cycle and Function[J]. Experimental Biology and Medicine.2006; 231:1459-1467.
[10] Xiaoping Yang, Thomas A. Doser, Cindy X. Fang,et al. Metallothionein prolongs survival and antagonizes senescence-associated cardiomyocyte diastolic dysfunction: role of oxidative stress [J]. FASEB J, 2006; 20: 1024 - 1026.
[11] Jianxun Wang, MS; Ye Song, MD, PhD; Laila Elsherif, PhD; et al.Cardiac Metallothionein Induction Plays the Major Role in the Prevention of Diabetic Cardiomyopathy by Zinc Supplementation[J]. Circulation, 2006; 113:544-554.
[12] Kathy J. Jenkins, Adolfo Correa, Jeffrey A. Feinstein, et al,.Noninherited Risk Factors and Congenital Cardiovascular Defects [J]. Circulation, 2007; 115: 2995 -3014.
[13] Carl L. Keen, Michael S. Clegg, Lynn A. Hanna, et al.The Plausibility of Micronutrient Deficiencies Being a Significant Contributing Factor to the Occurrence of Pregnancy Complications[J]. J Nutr, 2003; 133:1597S-1605S.