The Expression Of Nitric Oxide Synthase In Cerebral Cortex Of Congential Hypothyroidism Mice Model

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 ModelHealth 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-PCRThe 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.ImmunohistochemistryTissue 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 NOSThe 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.