Journal of Aquaculture Development

fa ارزیابی سمیت عصاره گیاه زنجبیل (Zingiber officinale) در تکامل سیستم عصبی جنین گورخر ماهی (Danio rerio) بر ژن‌های NeuroD ،Ngn1 و Shh با استفاده از روش Real-time PCR Evaluation of the toxicity of Zingiber officinale plant extract on the development of the nervous system of zebrafish embryos on NeuroD, Ngn1, and Shh genes using Real-time PCR method تخصصي Special پژوهشي Research <div style="text-align: justify;">زنجبیل (Zingiber officinale) گیاهی از تیره زنجبیل است که به دلیل خواص آنتی‌‌اکسیدانی و ضد التهابی به عنوان یک گیاه دارویی به طور گسترده مورد استفاده قرار می‌گیرد. این مطالعه به منظور ارزیابی اثرات عصاره زنجبیل بر بیان ژن‌های نوروژنیک NeuroD، Ngn1 و Shh در جنین‌های گورخر ماهی (Danio rerio) انجام شد.  عصاره زنجبیل با استفاده از روش خیساندن در اتانول تهیه شد.  جنین‌‌های گورخر ماهی به مدت 24، 48 و 72 ساعت پس از لقاح در معرض مجموعه‌ای از غلظت‌ها (76/0 تا 28/2 میکروگرم بر میلی‌ لیتر) قرارگرفتند. جنین‌ها از نظر ریخت شناسی، مرگ و میر و ویژگی ‌های بافت‌ شناسی ارزیابی شدند. از PCR در زمان واقعی برای اندازه‌گیری رونویسی ژن استفاده شد. نتایج نشان داد غلظت‌های کم تا متوسط هیچ سمیت رشدی قابل توجهی ایجاد نکردند. تجزیه و تحلیل بافت‌شناسی شواهدی از ساختارهای سالم مغز، مانند تکتوم بینایی و مخچه ارائه داد. قرار گرفتن در معرض غلظت ‌های بالاتر LC50 ≈ 1.6 (میکروگرم در میلی‌لیتر) باعث کاهش قابل توجهی در بیان NeuroD، Ngn1 و Shh  ضریب تعیین (p<0.05) شد و همچنین به عنوان یک اثر مهاری "وابسته به دوز" با اهمیت فراوان در رشد عصبی نشان داده شد. بطورکلی می‌توان گفت عصاره زنجبیل در دوزهای درمانی بی‌خطر به نظر می‌رسد، اما در دوزهای بالاتر، عصاره زنجبیل باعث تغییرات عصبی-رشدی شد. با توجه به عوارض جانبی نهفته، حفظ دوزهای مناسب عصاره‌های گیاهی برای اهداف دارویی در دوره رشد اولیه بسیار مهم است.</div> <div style="text-align: justify;">Introduction: Medicinal plants have long been an integral part of human therapies and, even today, they remain an essential source of modern pharmaceutical agents. A considerable number of contemporary drugs are either derived from natural products or designed to mimic their structures )Mustafa et al., 2017).  Although modern synthetic chemistry has enabled the development of novel and exclusive formulations that could not have been achieved solely through natural compounds, it still provides only limited access to the vast chemical and structural diversity found in plant-derived metabolites (Bode and Dong, 2011). Ginger (Zingiber officinale) a plant from the ginger family, is widely used as a medicinal herb due to its antioxidant and anti-inflammatory properties. Ginger is one of the most widely used medicinal plants worldwide. Its rhizome is commonly employed both as a culinary spice and as a principal herbal medicine, due to its abundant bioactive compounds—particularly pungent phenols such as [6]-gingerol and [6]-shogaol—and its numerous therapeutic properties. Notably, ginger exhibits anti-inflammatory, antioxidant, analgesic, and other pharmacological activities (Bischoff-Kont and Fürst, 2021, Zhang et al., 2021). It has been used to treat a variety of conditions, ranging from digestive disorders to arthritis. In particular, ginger is well recognized for alleviating nausea and vomiting, making it popular among pregnant women and chemotherapy patients. These medicinal properties contribute to ginger’s reputation as a natural remedy and help explain the growing scientific interest in its potential health benefits. Nevertheless, these bioactivities also raise important questions regarding its mechanisms of action and the boundaries of safe consumption (Belgacem et al., 2016).   This study aimed to evaluate the effects of ginger extract on the expression of neurogenic genes—NeuroD, Ngn1, and Shh—in zebrafish (Danio rerio) embryos. Materials and Methods: A sample of ginger was purchased from Barij Essence Company (Tehran, Iran). After authentication and registration in the herbarium of the Faculty of Pharmacy, Islamic Azad University, Tehran (voucher code: 621-PMP/A), the plant material was prepared for extraction (Ferri-Lagneau et al., 2012). Adult zebrafish were obtained from the Faculty of Veterinary Medicine, University of Tehran, and maintained under standard laboratory conditions at 28.5 °C with a 14 h light/10 h dark photoperiod. Natural spawning was allowed, and embryos were cultured in embryo (Modarresi Chahardehi et al., 2020). For histological analysis, embryos treated with different concentrations were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned at 5 µm, and stained with H&E. Sections were examined under a light microscope (Fan et al., 2010). Brains of embryos treated with LC50 and a lower concentration were dissected, and RNA was extracted. cDNA synthesis was performed using the Pars Tous cDNA synthesis kit (Mashhad, Iran). cDNA quality was verified by conventional PCR amplification of the GAPDH reference gene, and products were visualized on agarose gel. Expression levels of NeuroD, Ngn1, and Shh were analyzed by quantitative Real-Time PCR using SYBR Green chemistry on a Rotor-Gene Q system in a final reaction volume of 20 µL (Fan et al., 2010). Primers were designed using Primer3Plus software. Statistical analyses were performed using GraphPad Prism software (version 7.0). One-way ANOVA, Dunnett’s test, and independent t-tests were applied, and p < 0.05 was considered statistically significant (Fan et al., 2010). Results: The results showed that ginger extract induced a significant, dose- and time-dependent increase in zebrafish embryo mortality (p<0.05). Notably, substantial mortality was observed even at concentrations below the LC50 (1.6 µg/mL), with the highest mortality occurring at the LC50 itself (p<0.01). These findings indicate cumulative and delayed effects of the extract on embryonic development. Also, the results indicated ginger extract exhibited altered expression of key neurodevelopmental genes, particularly the transcription factors NeuroD and Neurogenin1 (Ngn1), which regulate neuronal differentiation, as well as the morphogen Sonic hedgehog (Shh), which is essential for neural patterning. These gene expression changes occurred in embryos that were morphologically normal, indicating that only subtle modifications in neurogenesis, rather than overt teratogenesis, had taken place. While no gross malformations were observed at moderate concentrations of ginger extract, the altered expression of NeuroD/Ngn1 and Shh cannot be conclusively interpreted from zebrafish embryonic morphology alone. Nonetheless, the findings suggest the possibility of slight delays or shifts in neural specification. Exposure to higher concentrations around the LC50 (~1.6 µg/mL) caused a significant decrease in NeuroD, Ngn1, and Shh expression (p<0.05), demonstrating a dose-dependent inhibitory effect on neurodevelopment. Histological analysis of zebrafish embryonic brains revealed that the telencephalon, cerebellum, eyes, and gills in the control group appeared normal and free of malformations, indicating healthy development and providing a baseline for comparison with treated groups. The telencephalon in control embryos exhibited regularly arranged neurons and normal morphology, with no signs of tissue damage or degeneration, reflecting intact neural tissue suitable for comparison with embryos treated with ginger extract. Exposure of embryonic zebrafish brains to the LC50 dose of ginger extract induced histopathological alterations in the cerebellum, eyes, and gills. These changes included reduced cell density, disorganized layering, and disrupted tissue architecture. Severe neural damage in the telencephalon, including neuronal cell death and extensive vacuolization, was observed at the LC50 dose, indicating cellular destruction and high-dose neurotoxicity, which negatively affected neuronal differentiation and underscore the need for caution when using high doses. In contrast, treatment with a sub-LC50 concentration of ginger extract did not adversely affect critical structures such as the telencephalon, cerebellum, eyes, or gills. Tissue integrity was preserved, with no signs of extensive cell death or severe vacuolization, suggesting relative safety at lower doses. Mild tissue death and vacuolization in the telencephalon were noted at sub-LC50 doses; these changes appeared subtle and reversible, indicating limited cellular effects without widespread tissue damage below the toxicity threshold. Conclusion: Ginger extract appears safe at therapeutic doses but induces neurodevelopmental alterations at higher concentrations. Considering potential side effects, maintaining appropriate dosing of herbal extracts during early developmental stages is critical for medicinal purposes. The present study demonstrated that acute exposure to concentrations ranging from low to high induced changes in gene expression, yet no embryonic malformations or losses were observed. Furthermore, there were no indications of severe developmental damage at these exposure levels, consistent with previous studies. This study provides new evidence that ginger extract can modulate neurodevelopmental gene networks in vivo and calls for a more balanced perspective. The traditional and medical applications of ginger are relatively well supported, and its impact on developmental biology deserves further investigation. Such systematic inquiry will help ensure that recommendations for ginger use, particularly during pregnancy and in relation to the brain and nervous system, are informed by both mechanistic data and clinical experience.</div> زنجبیل, جنین ماهی گور خری, NeuroD, Ngn1, Shh Zingiber officinale, Zebrafish embryo, NeuroD, Ngn1, Shh 35 54 http://aqudev.lahijan.iau.ir/browse.php?a_code=A-10-501-6&slc_lang=fa&sid=1 Farnaz Vosough1 فرناز وثوق1 100319475328460012210 100319475328460012210 No Tahereh Naji1 طاهره ناجی1 dr_naji@iau.ir 100319475328460012211 100319475328460012211 Yes Saeed Mohammadi Motamed سعید محمدی معتمد 100319475328460012212 100319475328460012212 No گروه فارماکوگنوزی، واحد علوم پزشکی تهران، دانشگاه آزاد اسلامی، تهران، ایران Nikoo Nasoohi1 نیکو نصوحی1 100319475328460012213 100319475328460012213 No