Razi Journal of Medical Sciences

fa تاثیر تغییرات ساب کلینیکال هورمون‌های تیروئیدی مادری بر بیان ژن در مغز در حال تکوین رت The Effect of Subclinical Alterations in Maternal Thyroid Hormone Levels on Gene Expression in the Developing Rat Brain ژنتیک Genetic پژوهشي Research <div style="text-align: justify;">زمینه و هدف: هورمون‌های تیروئیدی نقش مهمی در تکوین سیستم عصبی مرکزی ایفا می‌کنند. این هورمون‌ها از طریق اتصال به گیرنده‌های هسته‌ای تیروئید (Thyroid Receptors; TRs) بیان ژن‌های هدف را در سیستم عصبی در حال تکوین تنظیم می‌کنند. همچنین تغییرات ناشی از هورمون‌های تیروئیدی در میزان مصرف غذا و آب توسط رت مادر می‌تواند از طریق سازوکارهای اپی‌ژنتیکی بر بیان برخی فاکتورهای نوروتروفیک اثر گذاشته و رشد سیستم عصبی در حال تکوین را تحت تأثیر قرار دهد. روش کار: در این مطالعه، کم‌کاری تیروئیدی تحت‌بالینی با استفاده از متی‌مازول (Methimazole; MMI) در غلظت‌های صفر قسمت در میلیون (ppm) به‌عنوان گروه کنترل و 50، 75 و 100 ppm به‌عنوان گروه‌های هیپوتیروئید القا شد. متی‌مازول از روز سوم آبستنی تا روز بیستم پس از تولد از طریق آب آشامیدنی در اختیار رت‌های مادر قرار گرفت. در یک گروه دیگر، همراه با 50 ppm متی‌مازول، لووتیروکسین (Levothyroxine; L-T4) با دوز 200 میکروگرم در لیتر از طریق گاواژ تجویز شد. تغییرات سرمی هورمون‌های تیروئیدی مادر، تغییرات وزن بدن رت‌های مادر در دوران آبستنی و وزن نوزادان در روز تولد اندازه‌گیری شد. همچنین با استفاده از تکنیک واکنش زنجیره‌ای پلیمراز کمی در زمان واقعی (Quantitative Real-Time Polymerase Chain Reaction; qRT-PCR)، بیان نسبی ژن‌های فاکتور نوروتروفیک مشتق از مغز (Brain-Derived Neurotrophic Factor; BDNF)، نوروتروفین-3 (Neurotrophin-3; NT-3)، فاکتور رشد عصبی (Nerve Growth Factor; NGF) و ژن لنفوم سلول B نوع 2 (B-cell Lymphoma-2; Bcl-2) در مغز در حال تکوین رت‌های نوزاد بررسی شد. یافته‌ها: نتایج نشان داد که در شرایط هیپوتیروئیدی، افزایش وزن رت‌های مادر آبستن کاهش یافت. همچنین وزن نوزادان در زمان تولد و روند افزایش وزن آن‌ها در دوره نوزادی نسبت به گروه کنترل کاهش پیدا کرد. علاوه بر این، بیان نسبی ژن‌های BDNF، 3NT و 2-Bcl در روز بیستم پس از تولد افزایش یافت، در حالی که بیان نسبی ژن NGF در مقایسه با گروه کنترل کاهش نشان داد. نتیجه‌گیری: یافته‌های این مطالعه نشان داد که بیان ژن‌های دخیل در تکوین سیستم عصبی مرکزی به شدت به تغییرات سطح هورمون‌های تیروئیدی مادری وابسته است. این تغییرات می‌تواند با تأثیر بر تنظیم فاکتورهای نوروتروفیک و مسیرهای بقا و مرگ سلولی، موجب اختلال در ریخت‌زایی و عملکرد طبیعی سیستم عصبی مرکزی در حال تکوین شود.</div> <div style="text-align: justify;"> Background & Aims: Thyroid hormones are essential regulators of central nervous system (CNS) development, maturation, and functional organization. During critical developmental periods, they influence neuronal proliferation, migration, differentiation, synaptogenesis, and synaptic plasticity (1). Maternal hypothyroxinemia or thyroxine (T4) deficiency during pregnancy may cause irreversible structural and functional abnormalities in the fetal brain, leading to long-term neurodevelopmental impairment (2–4). Even mild disturbances in maternal thyroid hormone levels can produce persistent morphological, synaptic, and behavioral alterations (5). Maternal thyroid hormones affect fetal brain development through direct and indirect mechanisms. After entering the brain, T4 is converted to the biologically active hormone triiodothyronine (T3) by deiodinase enzymes, mainly in glial cells. T3 then binds to nuclear thyroid hormone receptors and regulates genes involved in CNS maturation and neuronal differentiation (6–8). Although many thyroid hormone-responsive genes have been identified, several downstream targets involved in neurodevelopment and neuroprotection remain poorly characterized (9–11). Neurotrophic factors such as brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and nerve growth factor (NGF) regulate neuronal survival, differentiation, axonal growth, and synaptic plasticity. The anti-apoptotic protein Bcl-2 also supports neuronal survival during development. This study investigated the effects of methimazole-induced maternal thyroid dysfunction on the expression of BDNF, NT-3, NGF, and Bcl-2 in the developing rat brain using quantitative real-time PCR (qRT-PCR). BDNF protein levels were also measured to determine whether maternal thyroid disruption induces compensatory or maladaptive responses in offspring brain tissue. Methods: Sixty pregnant Wistar rats (200–250 g) were obtained on gestational day 3 (GD3) and maintained under controlled conditions with free access to food and water. Maternal hypothyroidism was induced by administering methimazole (MMI) in drinking water at 0 ppm (control), 50 ppm, 75 ppm, or 100 ppm from GD3 to postnatal day 20 (PN20). An additional group received 50 ppm MMI plus levothyroxine (T4; 200 µg/L) by daily oral gavage. Animals were housed individually. Maternal body weight was recorded throughout pregnancy, and offspring body weight was measured from birth to PN20. On PN20, maternal serum concentrations of TSH, total T3, total T4, and free T4 were measured by ELISA. Brain tissues were collected from randomly selected offspring, and the cerebral cortex, hippocampus, cerebellum, brainstem, and olfactory bulb were dissected. Total RNA was extracted, and cDNA was synthesized from 5 µg RNA. Relative expression of BDNF, NT-3, NGF, and Bcl-2 was measured using SYBR Green-based qRT-PCR, with HPRT as the housekeeping gene. Reactions were performed in triplicate, and expression was calculated using the 2−ΔΔCt method. BDNF protein concentrations were determined by rat-specific ELISA in brain homogenates. Data were analyzed using one-way ANOVA followed by Tukey’s post hoc test or generalized linear model analysis. Results are presented as mean ± SEM, with p ≤ 0.05 considered statistically significant. Results: MMI produced a significant dose-dependent disruption of maternal thyroid function. Serum T3, T4, and free T4 levels decreased significantly, whereas TSH increased markedly in all MMI-treated groups compared with controls (p < 0.0001), confirming hypothyroidism. Maternal weight gain was significantly reduced in all treated groups (F = 85.891, p < 0.0001), particularly after GD15. A significant dose-by-gestational-age interaction was observed (F = 24.06, p < 0.001; R² = 0.618), indicating that the effects of MMI varied over time. Levothyroxine partially restored maternal weight gain. Offspring body weight was also significantly reduced after MMI exposure (F = 146.230, p < 0.0001). Growth impairment became evident from postnatal day 3 and persisted through PN20, with a significant dose-by-age interaction (F = 70.072, p < 0.01). Birth weight in the 50 ppm group did not differ significantly from controls, whereas the 75 and 100 ppm groups showed significant intrauterine growth restriction-like outcomes. At the molecular level, BDNF mRNA expression was not significantly altered (p = 0.078). In contrast, NGF expression was significantly downregulated in all experimental groups (p < 0.0001). NT-3 and Bcl-2 showed dose-dependent compensatory increases, and Bcl-2 was significantly upregulated in the 50 ppm group (p < 0.0001). Despite the absence of a significant change in BDNF transcript levels, BDNF protein was significantly reduced in the 100 ppm group (p = 0.0386), suggesting post-transcriptional regulation. Multivariate analysis confirmed significant overall group effects on neurotrophic and apoptotic gene expression (p < 0.001), particularly in the NGF and Bcl-2 pathways. Conclusion: Methimazole-induced maternal hypothyroidism disrupts maternal thyroid hormone homeostasis, impairs maternal weight gain, restricts offspring growth, and alters neurodevelopmental molecular pathways in the developing brain. The dose-dependent reduction in thyroid hormones and elevation of TSH confirmed successful induction of hypothyroidism, while partial improvement following levothyroxine treatment suggests that hormone replacement may reduce, but not completely prevent, developmental consequences. The consistent reduction in NGF expression indicates that this neurotrophic pathway is highly sensitive to thyroid hormone availability. In contrast, increased NT-3 and Bcl-2 expression at lower MMI doses may represent compensatory neuroprotective responses that support neuronal survival under moderate hormonal stress. The reduction in BDNF protein without a corresponding significant change in BDNF mRNA suggests regulation at the translational or protein-degradation level and demonstrates that transcriptional findings alone may not accurately reflect functional changes. Overall, maternal thyroid dysfunction produces dose-dependent growth and molecular abnormalities in offspring. Developing brain tissue appears capable of activating limited compensatory mechanisms, but these responses become insufficient under severe thyroid hormone disruption. These findings highlight the importance of maintaining adequate maternal thyroid function during pregnancy and early postnatal development and identify NGF, Bcl-2, NT-3, and BDNF as differentially responsive components of thyroid hormone-dependent neurodevelopment. </div> متی‌مازول, بیان ژن, BDNF, NGF, NT-3, Bcl-2, سیستم عصبی مرکزی, کم‌کاری تیروئیدی تحت‌بالینی, qRT-PCR Methimazole, Gene expression, BDNF, NGF, NT-3, Bcl-2, Central nervous system, Subclinical hypothyroidism, qRT-PCR 1 18 http://rjms.iums.ac.ir/browse.php?a_code=A-10-5201-1&slc_lang=fa&sid=1 Husein Nemati حسین نعمتی 3900319475328460091270 3900319475328460091270 No PhD Student, Department of Biology, SR.C., Islamic Azad University, Tehran, Iran دانشجوی دکتری، گروه زیست‌شناسی، واحد علوم و تحقیقات، دانشگاه آزاد اسلامی، تهران، ایران Kazem Parivar کاظم پریور kazemparivar1941@gmail.com 3900319475328460091266 3900319475328460091266 Yes Professor, Department of Biology, SR.C., Islamic Azad University, Tehran, Iran استاد، گروه زیست‌شناسی، واحد علوم و تحقیقات، دانشگاه آزاد اسلامی، تهران، ایران Delaram Doroud دلارام درود 3900319475328460091267 3900319475328460091267 No Associate Professor, Immunotherapy & LeishmaniaVaccine Research Department, Pasteur Institute of Iran, Tehran, Iran دانشیار، بخش ایمنوتراپی و تحقیقات واکسن لیشمانیا، انستیتو پاستو ایران، تهران، ایران Nasin Hayati Rudbari نسیم حیاتی رودباری 3900319475328460091268 3900319475328460091268 No Associate Professor, Department of Biology, SR.C., Islamic Azad University, Tehran, Iran دانشیار، گروه زیست‌شناسی، واحد علوم و تحقیقات، دانشگاه آزاد اسلامی، تهران، ایران Mohamad Saeed Nabiuni محمد نبیونی 3900319475328460091269 3900319475328460091269 No Professor, Department of Cell and Molecular Biology, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran استاد، گروه آموزشی سلولی-مولکولی، دانشکده علوم زیستی، دانشگاه خوارزمی، تهران، ایران