先天性甲状腺功能减低仔鼠大脑皮层一氧化氮合酶表达的变化
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摘要
目的：
先天性甲状腺功能减低症(先天性甲低)是由于甲状腺激素(TH)合成不足所造成的一种儿科常见的内分泌代谢性疾病,是造成儿童智力障碍的重要原因之
所以一直是儿童内分泌医务人员关注的重点。随着分子生物学的发展,对TH生理作用有了更深入了解,TH在细胞核内与其受体(TR)结合,作用于靶基因启动子的甲状腺激素反应元件(TREs),在转录水平调节靶基因的表达,然而甲低具体通过影响哪些基因导致儿童智力障碍目前尚不十分清楚。信号蛋白和相关的功能蛋白是细胞各种信息传导及各种功能代谢活动的执行者,是神经细胞生长、分化等活动的基础,也是神经感觉、运动、学习和记忆等功能活动形成机制的重要环节,深入研究TH对脑组织信号系统相关蛋白的基因学调节,能更全面揭示甲状腺调控脑发育机制和先天性甲低的发病机制。
神经系统经典的细胞信号传递主要指突触部位的神经递质传递,以往研究表明TH参与多种神经递质的合成、降解,以及其受体表达和功能。近来,研究表明神经系统还存在一些非经典的信号传导系统,如一氧化氮(NO),它也介导了神经系统一些重要的功能。
NO是一种细胞信息分子,具有亲脂性,微溶于水的,既可以透过细胞的水相,也可以透过脂相。NO的半衰期很短,可能仅几秒钟。一氧化氮合酶(nitric oxide synthase, NOS)是NO合成中唯一的、关键的限速酶,它主要有三种表型,神经型(nNOS)、诱导型(iNOS)和内皮型(eNOS),另外还有线粒体型(mtNOS)。nNOS和eNOS分别存在于神经元和血管内皮细胞内,其持续的产生低浓度的NO,主要参与脏器血流的调节和神经的信息传递等。iNOS几乎存在于所有组织中,在病理状况下由多种细胞因子或炎症刺激靶细胞大量表达,产生过量NO,主要参与炎症和免疫反应。中枢神经系统,NO作为一种新的细胞间信息交换的载体,兼有第二信使和神经传递的功能,它可从突触后迅速扩散到突触前末梢,发挥各种生理功能,因此脑神经元产生的NO往往是中枢神经系统的一个反馈信息分子,现已证明NO对多种神经功能活动均具有重要的影响。
由于NO的化学性质极不稳定,体内NO生成的主要限速因素是NOS,故常需依赖于对NOS的研究来了解NO的分布和量。Ueta等研究发现甲低可导致成年大鼠下丘脑视上核和室旁核NOS基因的表达明显下降,但未见先天性甲低对大脑皮层不同亚型NOS表达影响的报道。
在此,我们通过建立先天性甲状腺功能减低症的动物模型,检测先天性甲低仔鼠大脑皮层nNOS、iNOS和eNOS的表达,了解先天性甲低对仔鼠大脑皮层nNOS、iNOS知eNOS表达的影响。
方法：
1.模型建立和分组
清洁级健康成年C57BL/6J合笼交配后自行产子,按处理条件不同分甲低组和对照组。
甲低组：合笼第10天开始给予母鼠含0.03%甲巯咪唑的饮用水,一直到仔鼠出生7天后。
对照组：一直用清洁饮用水喂养,其它实验控制因素同甲低组。
在仔鼠出生1、7、14和21天麻醉后处死,生理盐水灌注,留取大脑皮层标本。
2. Real time RT-PCR检测
采用Real time RT-PCR检测nNOS、iNOS和eNOS的mRNA水平。采用AxyPrep总RNA小量制备试剂盒提取脑组织总RNA,采用自行设计引物和Takara荧光PCR试剂盒进行PCR扩增,其中nNOS采用两步法,eNOS和iNOS采用三步法。目的基因mRNA水平采用ΔCt表示,并应用ΔCt进行组间比较,ACt=Ct(目的基因)-Ct (GAPDH),ΔCt越小表明mRNA水平越高。
3.免疫组化
新鲜脑组织予10%中性福尔马林固定和石蜡包埋,采用"EnvisionTM二步法”进行免疫组化检测。
结果：
先天性甲低对NOS表达的影响
甲低组nNOSmRNA较对照组下降,在1和7天差异在边界水平,14天差异具有显著性意义。免疫组化显示大脑皮层的nNOS表达以神经元胞体阳性为主,神经纤维基本阴性。与对照组比较,甲低组大脑皮层nNOS免疫阳性较低。
甲低组iNOSmRNA明显升高,在1和7天差异具有显著性意义。免疫组化显示大脑皮层的iNOS表达,以神经元胞体尤为丰富,神经纤维基本阴性。与对照组比较,甲低组大脑皮层iNOS免疫阳性明显高。
免疫组化显示大脑皮层和脑膜微小血管上皮的eNOS丰富,而神经细胞和神经纤维均阴性。对照组与甲低组间eNOSmRNA的差异均无显著性意义。
讨论：
先天性甲低是儿科常见的内分泌代谢性疾病之一,可导致脑机能发生不可逆损害。信息传递是神经系统学习、记忆和思维等高级活动的基础,神经科学家已对神经元化学突触间的信息处理和传递功能进行了比较广泛而深入的研究,然而神经系统还有不少非经典的信息传递途径,如NO信号系统,研究表明它也介导了神经系统一些重要的功能。那么,先天性甲低是否会通过影响大脑NO信号系统表达导致脑功能机能障碍呢?目前尚无相关报道。
NO作为一种新的信号传递物质,其与甲状腺激素关系的研究成为一个新的热点。由于NO的化学性质极不稳定,半衰期短,体内NOS是NO合成中关键的限速酶,故常依赖于对NOS的研究来了解NO的分布和量,我们对其3种主要亚型表达情况进行了研究。
本研究证实nNOS在中枢神经系统表达,主要在神经元的胞浆。研究还发现先天性甲低新生小鼠大脑皮层nNOS的mRNA表达下降,在停用MMI水后7天时最为明显,说明TH可通过调节大脑皮质NO含量及NO/CG信号转导通路的活性来发挥对中枢神经系统的作用,甲低可通过减少nNOS表达降低神经元NO合成,影响中枢神经系统发育和神经系统的机能。本研究表明,生理情况下仔鼠中枢神经系统可有微量的iNOS表达,甲低时iNOS表达增加,主要分布部分神经元和胶质细胞,提示其NO的主要作用应不在于生理调节,而很可能是参与一种损伤机制。NO除作为信使外,也是种活泼的自由基,过量NO与超氧化物结合可生成具更高毒性的过氧化物硝酸根阴离子,这些分子可能通过自分泌或旁分泌的方式在继发损害的过程中起作用,能造成脂质膜损伤、抑制细胞能量代谢和DNA的合成修复,并引发细胞凋亡和坏死。因此,iNOS产生过量的NO则可能也是甲低时脑内神经元凋亡增加的原因之一。虽然临床上已有许多有关甲状腺功能疾病与血压异常有关的报道,我们也证实eNOS在大脑皮层和脑膜微小血管内皮有表达,但未发现先天性甲低对大脑皮层eNOSmRNA表达的影响,提示甲低血压等异常并不通过颅内血管内皮细胞分泌NO来调节。
先天性甲低是如何导致NOS表达变化的呢?目前尚不明了。TH主要通过与TR结合调节基因转录而发挥作用,TR属于配体依赖性转录因子,它通过与特定的TREs结合而发挥作用。我们分析小鼠NOS启动子序列,发现他们的启动子均有TREs或TREs结构类似的DNA序列,提示TH可能直接参与调节这些基因的表达。然而,本研究发现两组间仅nNOS和iNOS表达有差异,而eNOS表达没有变化,且表达有差异的时间点也不一致。这些差异提示TH对基因调控可能具有时空的差异,即TH可能仅在特定时间调控特定组织或细胞的特定基因转录。
TREs按其对基因转录影响可分为正性和负性TREs。正性TERs在没有结合配体的TR对靶基因的基础转录有抑制作用,激活性配体(如T3)与TR结合后促进靶基因的转录,负性TER则发挥相反的作用。本研究发现先天性甲低小鼠大脑皮层nNOS下降。我们推测nNOS基因启动子区域存在的TRE是正性的。然而iNOS表达的改变与nNOS相反。我们推测这可能与：nNOS下降导致神经细胞元间信号交流能力下降有关,还可能还与甲低导致细胞因子和凋亡增加有关。
总之,本研究发现先天性甲低仔鼠大脑皮层nNOS表达下降和iNOS表达上升,提示先天性甲低影响神经细胞NO系统,NO系统异常可能是先天性甲低导致脑功能障碍的机制之一。另外,TH对基因调控可能具有时空性。
Background:
Congenital hypothyroidism (CH), is a common pediatric endocrine disease.It due to insufficient secretion of thyroid hormone (TH). It is also a main cause of childhood mental retarded and draws more and more attention from endocrine pediatrician. With the development of molecular biology, physiology of TH has been better understood. TH binds its receptor (TH receptor, TR) in the nucleus, which plays a role on regulation the target gene transcriptional level by recognition the thyroid hormone response elements (TREs) in the promoters. However, the candidate genes which be regulated by TH and cause mental retardation in children with CH are still unclear. The signaling proteins and related proteins have effect on the information transfer and the various metabolic activity, and then on nerve cell growth and differentiation. They also take part in the mechanisms of sensory, motor, learning, memory and other activities. Hence, study the effect of TH on the signaling proteins and its related proteins may help us to better understand the effect of TH on brain development and the mechanism of CH.
The classic information transfer in nervous system is the neurotransmitter transmittion throught the synapses, or the chemical synapses. Previous studies showed that TH had effect on the synthesis and degradation of neurotransmitters, and on the expression and function of their receptors. Recently, studies found several several non-classic information transfer pathway in the nervous system, such as nitric oxide (NO) signal transduction systems, which play important role on some nervous activities.
NO is lipophilic and slightly soluble in water. It can pass through both the aqueous phase and lipid-phase. NO has a very short half-life with a few seconds. NO synthase (NOS) are the only critical rate-limiting enzymes on NO synthesis and mainly include 3 isoforms, neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS). The nNOS and eNOS exist in the neurons and vascular endothelial cells respectively, produce low concentrations of NO and involved in blood flow regulation and information transmission. The iNOS exists in almost all organizations. It is induced by inflammatory cytokines and produces large NO in pathological conditions, and mainly involved in inflammation and immune response. In central nervous system, NO acts as a new carrier of intercellular information exchange with both second messenger and neurotransmitter function. It can quickly spread from postsynaptic endings to presynaptic endings, acting as feedback information. It is involved in a wide range of important neurological activity. As NO is extremely unstable and NOS is the only rate-limiting factors of NO synthesis in vivo, NOS was usually used to represent the distribution and quantity of NO in research. Ueta et al. found that NOS expression were reduced on supraoptic nucleus and paraventricular nucleus in adult rat with hypothyroidism. However, no study about the 3 different types of NOS on cerebral cortex with CH was reported.
Here, we measure the nNOS, iNOS and eNOS levels on cerebral cortex in mice model of congenital hypothyroidism to investigate the role of TH on brain development and the mechanism of CH.
Objectives:
To investigate the effect of TH on NOS and the mechanism of CH by measuring the iNOS, nNOS and eNOS expression on cerebral cortex with CH.
Materials and Methods:
Animal Model
Health adult C57BL/6J mice mated together. They were divided into CH group and control group according to the different intervention. The control group is definited as feeding of clean drinking water. The CH group is definted as feeding 0.03% Thiamazole contained water from the 10th day after mate to 7th day after the offsprings'birth. The offsprings were sacrificed on day 1,7,14 and 21. Saline perfusion was performed and the cerebral cortex was collected.
Real time RT-PCR
The level of NOS mRNA was measured by real time RT-PCR. Total RNA was extracted using AxyPrep Mutilsoure Total RNA Miniprep Kit. Self-designed primers and SYBR PrimeScriptTM PCR Kit were used real time PCR. Two-step method was used for nNOS while three-step method was used for eNOS and iNOS measurement. ACt, calculated as Ct of target gene minus Ct ofβ-actin, was presented for target gene mRNA levels.
Immunohistochemistry
Tissue was fixed in 10% neutral formalin and embeded in paraffin. "EnvisionTM two-step" method was used for immunohistochemistry.
Results:
The effect of CH on the expression of NOS
The levels of nNOS mRNA in CH group were lower than these in the controls with significant difference on day 14 and marginal difference on day 1 and 7. Immunohistochemistry showed expression of nNOS were positive in the neurons in cerebral cortex.
In CH group, significantly higher iNOS mRNA was noted on day 1 and 7. Immunohistochemistry showed that iNOS was positive in cerebral cortex, especially in neurons body.
Immunohistochemistry showed that eNOS was positive in the small vessels and capillary in the cerebral cortex while the nerve cells and fibers were negative. The levels of eNOS mRNA has no difference between two groups.
Discussion:
CH is a common pediatric endocrine diseases and cause irreversible brain damage. Information transmission in the nervous system is the basis of other advanced activity. Many neuroscientists have focused the research on the synapses, which has been believed to have important role in the information processing and transmission. However, there are several non-classical pathways for information transmission in the nervous system (e.g. NO signal systems), which participate some important activities in the nervous system. Whether CH associated mental retard is related with disorder of NO signaling system is still unclear.
NO is a new signaling molecular. As its instability and short half-life, NOS, the only key rate-limiting enzyme in vivo, was usually used to represent the distribution and quantity of NO in research.
In this study, nNOS was confirmed to express in the central nervous system, especially in neuronal cytoplasm. We also found that nNOS mRNA was decreased in cerebral cortex in CH, which suggests that TH regulates the activity of NO/cGMP signal transduction pathway in the cerebral cortex. NO synthesis is reduced in CH by reducing the nNOS expression in neurons, and then affects the development and function of the nervous system. Our study showed traces iNOS expression in physiological circumstances and much higher level in CH mice, which suggested that the primary role of NO should not be physiological regulation, but likely to be involved in a damage mechanism. Except for the signal transmission, NO is also a free radical species, excessive NO combines to the superoxide and generates nitrate anion with higher peroxide toxicity. These molecules can induce the secondary damage through autocrine or paracrine manner, including membrane lipid damage, inhibition of energy metabolism and DNA repair, and trigger apoptosis and necrosis. Therefore, iNOS upregulation and excessive NO generation may be also a mechanism of neuronal apoptosis in CH.
There were some reports about the relationship between thyroid dysfunction and blood pressure abnormalities. In this study, we confirmed that eNOS was located in the vascular endothelial in the cerebral cortex. However, the levels of eNOS mRNA were not significantly different between two groups, suggesting CH do not affect the eNOS in intracranial vascular endothelial cells.
How CH affects the expression of NOS is still unclear. TH regulates gene transcription mainly through binding to TR, a ligand-dependent transcription factor, which recognized TREs in the promoter of target gene. We analyzed of mouse promoter sequence of mice NOS and found TREs structure or similar DNA sequence in their promoters. It suggests that TH can directly regulate the transcription of these genes. However, our study found that only nNOS and iNOS were different while the eNOS expression were similar between the two groups. This difference suggests the regulation of TH on gene transcription may be spatial and temporal control.
The TREs were divided into positive and negative TREs according to their function. The positive TREs inhibit gene transcription without TR ligand binding. TR combinated with activated-ligand (e.g. T3) facilitates target gene transcription. The negative TER exerts an opposite role. We found that the nNOS was downregulated in CH, which suggests TREs in this gene is positive one and lower TH inhibits nNOS gene transcription. It is interesting that the iNOS was upregulated in CH. Moreover, the upregulated iNOS might be also associated with increased cytokines produce and apoptosis in CH.
In summary, our study showed that the expressions of nNOS was downregulated and iNOS was upregulated in the cerebral cortex in CH, suggesting that CH affect NO system. The abnormalities of NO system might be part of the mechanisms of mental retarded in CH. In addition, the regulation of TH on gene transcription has a spatial and temporal control.
先天性甲状腺功能减低症(先天性甲低)是由于甲状腺激素(TH)合成不足所造成的一种儿科常见的内分泌代谢性疾病,是造成儿童智力障碍的重要原因之
所以一直是儿童内分泌医务人员关注的重点。随着分子生物学的发展,对TH生理作用有了更深入了解,TH在细胞核内与其受体(TR)结合,作用于靶基因启动子的甲状腺激素反应元件(TREs),在转录水平调节靶基因的表达,然而甲低具体通过影响哪些基因导致儿童智力障碍目前尚不十分清楚。信号蛋白和相关的功能蛋白是细胞各种信息传导及各种功能代谢活动的执行者,是神经细胞生长、分化等活动的基础,也是神经感觉、运动、学习和记忆等功能活动形成机制的重要环节,深入研究TH对脑组织信号系统相关蛋白的基因学调节,能更全面揭示甲状腺调控脑发育机制和先天性甲低的发病机制。
神经系统经典的细胞信号传递主要指突触部位的神经递质传递,以往研究表明TH参与多种神经递质的合成、降解,以及其受体表达和功能。近来,研究表明神经系统还存在一些非经典的信号传导系统,如一氧化氮(NO),它也介导了神经系统一些重要的功能。
NO是一种细胞信息分子,具有亲脂性,微溶于水的,既可以透过细胞的水相,也可以透过脂相。NO的半衰期很短,可能仅几秒钟。一氧化氮合酶(nitric oxide synthase, NOS)是NO合成中唯一的、关键的限速酶,它主要有三种表型,神经型(nNOS)、诱导型(iNOS)和内皮型(eNOS),另外还有线粒体型(mtNOS)。nNOS和eNOS分别存在于神经元和血管内皮细胞内,其持续的产生低浓度的NO,主要参与脏器血流的调节和神经的信息传递等。iNOS几乎存在于所有组织中,在病理状况下由多种细胞因子或炎症刺激靶细胞大量表达,产生过量NO,主要参与炎症和免疫反应。中枢神经系统,NO作为一种新的细胞间信息交换的载体,兼有第二信使和神经传递的功能,它可从突触后迅速扩散到突触前末梢,发挥各种生理功能,因此脑神经元产生的NO往往是中枢神经系统的一个反馈信息分子,现已证明NO对多种神经功能活动均具有重要的影响。
由于NO的化学性质极不稳定,体内NO生成的主要限速因素是NOS,故常需依赖于对NOS的研究来了解NO的分布和量。Ueta等研究发现甲低可导致成年大鼠下丘脑视上核和室旁核NOS基因的表达明显下降,但未见先天性甲低对大脑皮层不同亚型NOS表达影响的报道。
在此,我们通过建立先天性甲状腺功能减低症的动物模型,检测先天性甲低仔鼠大脑皮层nNOS、iNOS和eNOS的表达,了解先天性甲低对仔鼠大脑皮层nNOS、iNOS知eNOS表达的影响。
方法：
1.模型建立和分组
清洁级健康成年C57BL/6J合笼交配后自行产子,按处理条件不同分甲低组和对照组。
甲低组：合笼第10天开始给予母鼠含0.03%甲巯咪唑的饮用水,一直到仔鼠出生7天后。
对照组：一直用清洁饮用水喂养,其它实验控制因素同甲低组。
在仔鼠出生1、7、14和21天麻醉后处死,生理盐水灌注,留取大脑皮层标本。
2. Real time RT-PCR检测
采用Real time RT-PCR检测nNOS、iNOS和eNOS的mRNA水平。采用AxyPrep总RNA小量制备试剂盒提取脑组织总RNA,采用自行设计引物和Takara荧光PCR试剂盒进行PCR扩增,其中nNOS采用两步法,eNOS和iNOS采用三步法。目的基因mRNA水平采用ΔCt表示,并应用ΔCt进行组间比较,ACt=Ct(目的基因)-Ct (GAPDH),ΔCt越小表明mRNA水平越高。
3.免疫组化
新鲜脑组织予10%中性福尔马林固定和石蜡包埋,采用"EnvisionTM二步法”进行免疫组化检测。
结果：
先天性甲低对NOS表达的影响
甲低组nNOSmRNA较对照组下降,在1和7天差异在边界水平,14天差异具有显著性意义。免疫组化显示大脑皮层的nNOS表达以神经元胞体阳性为主,神经纤维基本阴性。与对照组比较,甲低组大脑皮层nNOS免疫阳性较低。
甲低组iNOSmRNA明显升高,在1和7天差异具有显著性意义。免疫组化显示大脑皮层的iNOS表达,以神经元胞体尤为丰富,神经纤维基本阴性。与对照组比较,甲低组大脑皮层iNOS免疫阳性明显高。
免疫组化显示大脑皮层和脑膜微小血管上皮的eNOS丰富,而神经细胞和神经纤维均阴性。对照组与甲低组间eNOSmRNA的差异均无显著性意义。
讨论：
先天性甲低是儿科常见的内分泌代谢性疾病之一,可导致脑机能发生不可逆损害。信息传递是神经系统学习、记忆和思维等高级活动的基础,神经科学家已对神经元化学突触间的信息处理和传递功能进行了比较广泛而深入的研究,然而神经系统还有不少非经典的信息传递途径,如NO信号系统,研究表明它也介导了神经系统一些重要的功能。那么,先天性甲低是否会通过影响大脑NO信号系统表达导致脑功能机能障碍呢?目前尚无相关报道。
NO作为一种新的信号传递物质,其与甲状腺激素关系的研究成为一个新的热点。由于NO的化学性质极不稳定,半衰期短,体内NOS是NO合成中关键的限速酶,故常依赖于对NOS的研究来了解NO的分布和量,我们对其3种主要亚型表达情况进行了研究。
本研究证实nNOS在中枢神经系统表达,主要在神经元的胞浆。研究还发现先天性甲低新生小鼠大脑皮层nNOS的mRNA表达下降,在停用MMI水后7天时最为明显,说明TH可通过调节大脑皮质NO含量及NO/CG信号转导通路的活性来发挥对中枢神经系统的作用,甲低可通过减少nNOS表达降低神经元NO合成,影响中枢神经系统发育和神经系统的机能。本研究表明,生理情况下仔鼠中枢神经系统可有微量的iNOS表达,甲低时iNOS表达增加,主要分布部分神经元和胶质细胞,提示其NO的主要作用应不在于生理调节,而很可能是参与一种损伤机制。NO除作为信使外,也是种活泼的自由基,过量NO与超氧化物结合可生成具更高毒性的过氧化物硝酸根阴离子,这些分子可能通过自分泌或旁分泌的方式在继发损害的过程中起作用,能造成脂质膜损伤、抑制细胞能量代谢和DNA的合成修复,并引发细胞凋亡和坏死。因此,iNOS产生过量的NO则可能也是甲低时脑内神经元凋亡增加的原因之一。虽然临床上已有许多有关甲状腺功能疾病与血压异常有关的报道,我们也证实eNOS在大脑皮层和脑膜微小血管内皮有表达,但未发现先天性甲低对大脑皮层eNOSmRNA表达的影响,提示甲低血压等异常并不通过颅内血管内皮细胞分泌NO来调节。
先天性甲低是如何导致NOS表达变化的呢?目前尚不明了。TH主要通过与TR结合调节基因转录而发挥作用,TR属于配体依赖性转录因子,它通过与特定的TREs结合而发挥作用。我们分析小鼠NOS启动子序列,发现他们的启动子均有TREs或TREs结构类似的DNA序列,提示TH可能直接参与调节这些基因的表达。然而,本研究发现两组间仅nNOS和iNOS表达有差异,而eNOS表达没有变化,且表达有差异的时间点也不一致。这些差异提示TH对基因调控可能具有时空的差异,即TH可能仅在特定时间调控特定组织或细胞的特定基因转录。
TREs按其对基因转录影响可分为正性和负性TREs。正性TERs在没有结合配体的TR对靶基因的基础转录有抑制作用,激活性配体(如T3)与TR结合后促进靶基因的转录,负性TER则发挥相反的作用。本研究发现先天性甲低小鼠大脑皮层nNOS下降。我们推测nNOS基因启动子区域存在的TRE是正性的。然而iNOS表达的改变与nNOS相反。我们推测这可能与：nNOS下降导致神经细胞元间信号交流能力下降有关,还可能还与甲低导致细胞因子和凋亡增加有关。
总之,本研究发现先天性甲低仔鼠大脑皮层nNOS表达下降和iNOS表达上升,提示先天性甲低影响神经细胞NO系统,NO系统异常可能是先天性甲低导致脑功能障碍的机制之一。另外,TH对基因调控可能具有时空性。
Background:
Congenital hypothyroidism (CH), is a common pediatric endocrine disease.It due to insufficient secretion of thyroid hormone (TH). It is also a main cause of childhood mental retarded and draws more and more attention from endocrine pediatrician. With the development of molecular biology, physiology of TH has been better understood. TH binds its receptor (TH receptor, TR) in the nucleus, which plays a role on regulation the target gene transcriptional level by recognition the thyroid hormone response elements (TREs) in the promoters. However, the candidate genes which be regulated by TH and cause mental retardation in children with CH are still unclear. The signaling proteins and related proteins have effect on the information transfer and the various metabolic activity, and then on nerve cell growth and differentiation. They also take part in the mechanisms of sensory, motor, learning, memory and other activities. Hence, study the effect of TH on the signaling proteins and its related proteins may help us to better understand the effect of TH on brain development and the mechanism of CH.
The classic information transfer in nervous system is the neurotransmitter transmittion throught the synapses, or the chemical synapses. Previous studies showed that TH had effect on the synthesis and degradation of neurotransmitters, and on the expression and function of their receptors. Recently, studies found several several non-classic information transfer pathway in the nervous system, such as nitric oxide (NO) signal transduction systems, which play important role on some nervous activities.
NO is lipophilic and slightly soluble in water. It can pass through both the aqueous phase and lipid-phase. NO has a very short half-life with a few seconds. NO synthase (NOS) are the only critical rate-limiting enzymes on NO synthesis and mainly include 3 isoforms, neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS). The nNOS and eNOS exist in the neurons and vascular endothelial cells respectively, produce low concentrations of NO and involved in blood flow regulation and information transmission. The iNOS exists in almost all organizations. It is induced by inflammatory cytokines and produces large NO in pathological conditions, and mainly involved in inflammation and immune response. In central nervous system, NO acts as a new carrier of intercellular information exchange with both second messenger and neurotransmitter function. It can quickly spread from postsynaptic endings to presynaptic endings, acting as feedback information. It is involved in a wide range of important neurological activity. As NO is extremely unstable and NOS is the only rate-limiting factors of NO synthesis in vivo, NOS was usually used to represent the distribution and quantity of NO in research. Ueta et al. found that NOS expression were reduced on supraoptic nucleus and paraventricular nucleus in adult rat with hypothyroidism. However, no study about the 3 different types of NOS on cerebral cortex with CH was reported.
Here, we measure the nNOS, iNOS and eNOS levels on cerebral cortex in mice model of congenital hypothyroidism to investigate the role of TH on brain development and the mechanism of CH.
Objectives:
To investigate the effect of TH on NOS and the mechanism of CH by measuring the iNOS, nNOS and eNOS expression on cerebral cortex with CH.
Materials and Methods:
Animal Model
Health adult C57BL/6J mice mated together. They were divided into CH group and control group according to the different intervention. The control group is definited as feeding of clean drinking water. The CH group is definted as feeding 0.03% Thiamazole contained water from the 10th day after mate to 7th day after the offsprings'birth. The offsprings were sacrificed on day 1,7,14 and 21. Saline perfusion was performed and the cerebral cortex was collected.
Real time RT-PCR
The level of NOS mRNA was measured by real time RT-PCR. Total RNA was extracted using AxyPrep Mutilsoure Total RNA Miniprep Kit. Self-designed primers and SYBR PrimeScriptTM PCR Kit were used real time PCR. Two-step method was used for nNOS while three-step method was used for eNOS and iNOS measurement. ACt, calculated as Ct of target gene minus Ct ofβ-actin, was presented for target gene mRNA levels.
Immunohistochemistry
Tissue was fixed in 10% neutral formalin and embeded in paraffin. "EnvisionTM two-step" method was used for immunohistochemistry.
Results:
The effect of CH on the expression of NOS
The levels of nNOS mRNA in CH group were lower than these in the controls with significant difference on day 14 and marginal difference on day 1 and 7. Immunohistochemistry showed expression of nNOS were positive in the neurons in cerebral cortex.
In CH group, significantly higher iNOS mRNA was noted on day 1 and 7. Immunohistochemistry showed that iNOS was positive in cerebral cortex, especially in neurons body.
Immunohistochemistry showed that eNOS was positive in the small vessels and capillary in the cerebral cortex while the nerve cells and fibers were negative. The levels of eNOS mRNA has no difference between two groups.
Discussion:
CH is a common pediatric endocrine diseases and cause irreversible brain damage. Information transmission in the nervous system is the basis of other advanced activity. Many neuroscientists have focused the research on the synapses, which has been believed to have important role in the information processing and transmission. However, there are several non-classical pathways for information transmission in the nervous system (e.g. NO signal systems), which participate some important activities in the nervous system. Whether CH associated mental retard is related with disorder of NO signaling system is still unclear.
NO is a new signaling molecular. As its instability and short half-life, NOS, the only key rate-limiting enzyme in vivo, was usually used to represent the distribution and quantity of NO in research.
In this study, nNOS was confirmed to express in the central nervous system, especially in neuronal cytoplasm. We also found that nNOS mRNA was decreased in cerebral cortex in CH, which suggests that TH regulates the activity of NO/cGMP signal transduction pathway in the cerebral cortex. NO synthesis is reduced in CH by reducing the nNOS expression in neurons, and then affects the development and function of the nervous system. Our study showed traces iNOS expression in physiological circumstances and much higher level in CH mice, which suggested that the primary role of NO should not be physiological regulation, but likely to be involved in a damage mechanism. Except for the signal transmission, NO is also a free radical species, excessive NO combines to the superoxide and generates nitrate anion with higher peroxide toxicity. These molecules can induce the secondary damage through autocrine or paracrine manner, including membrane lipid damage, inhibition of energy metabolism and DNA repair, and trigger apoptosis and necrosis. Therefore, iNOS upregulation and excessive NO generation may be also a mechanism of neuronal apoptosis in CH.
There were some reports about the relationship between thyroid dysfunction and blood pressure abnormalities. In this study, we confirmed that eNOS was located in the vascular endothelial in the cerebral cortex. However, the levels of eNOS mRNA were not significantly different between two groups, suggesting CH do not affect the eNOS in intracranial vascular endothelial cells.
How CH affects the expression of NOS is still unclear. TH regulates gene transcription mainly through binding to TR, a ligand-dependent transcription factor, which recognized TREs in the promoter of target gene. We analyzed of mouse promoter sequence of mice NOS and found TREs structure or similar DNA sequence in their promoters. It suggests that TH can directly regulate the transcription of these genes. However, our study found that only nNOS and iNOS were different while the eNOS expression were similar between the two groups. This difference suggests the regulation of TH on gene transcription may be spatial and temporal control.
The TREs were divided into positive and negative TREs according to their function. The positive TREs inhibit gene transcription without TR ligand binding. TR combinated with activated-ligand (e.g. T3) facilitates target gene transcription. The negative TER exerts an opposite role. We found that the nNOS was downregulated in CH, which suggests TREs in this gene is positive one and lower TH inhibits nNOS gene transcription. It is interesting that the iNOS was upregulated in CH. Moreover, the upregulated iNOS might be also associated with increased cytokines produce and apoptosis in CH.
In summary, our study showed that the expressions of nNOS was downregulated and iNOS was upregulated in the cerebral cortex in CH, suggesting that CH affect NO system. The abnormalities of NO system might be part of the mechanisms of mental retarded in CH. In addition, the regulation of TH on gene transcription has a spatial and temporal control.
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