Table_4_Bhlhe40/Sirt1 Axis-Regulated Mitophagy Is Implicated in All-Trans Retinoic Acid-Induced Spina Bifida Aperta.XLS

Neural tube defects (NTDs) are the most severe congenital malformations that result from failure of neural tube closure during early embryonic development, and the underlying molecular mechanisms remain elusive. Mitophagy is the best-known way of mitochondrial quality control. However, the role and regulation of mitophagy in NTDs have not yet been elucidated. In this study, we used an all-trans retinoic acid (ATRA)-induced rat model to investigate mitophagy and its underlying mechanism in spina bifida aperta (SBA). The results of western blot, immunofluorescence and RT-qPCR analyses indicated that mitophagy was impaired and Sirt1 was downregulated in SBA. Administration of resveratrol-a strong specific Sirt1 activator-activated Sirt1, thus attenuating autophagy suppression and ameliorating SBA. RNA-sequencing and bioinformatics analysis results indicated that transcriptional regulation played an important role in NTDs. A luciferase reporter assay was performed to demonstrate that the transcription factor Bhlhe40 directly bound to and negatively regulated Sirt1 expression. Further, we discovered that the Bhlhe40/Sirt1 axis regulated mitophagy in neural stem cells. Collectively, our results for the first time demonstrate that Bhlhe40/Sirt1 axis regulated mitophagy is implicated in ATRA-induced SBA. Our findings provide new insights into pathogenesis of NTDs and a basis for potential therapeutic targets for NTDs.

神经管缺陷（NTDs）是胚胎早期发育过程中神经管闭合失败所致的最严重先天性畸形，其潜在分子机制尚不明确。线粒体自噬是线粒体质量控制的已知最佳途径。然而，线粒体自噬在NTDs中的作用及其调控机制尚未阐明。在本研究中，我们利用全反式视黄酸（ATRA）诱导的大鼠模型，探讨了线粒体自噬及其在开放性脊柱裂（SBA）中的潜在机制。Western blot、免疫荧光和RT-qPCR分析的结果表明，SBA中自噬受损且Sirt1表达下调。给予白藜芦醇——一种强效的Sirt1激活剂——能够激活Sirt1，从而减轻自噬抑制并改善SBA。RNA测序和生物信息学分析结果表明，转录调控在NTDs中起着重要作用。通过荧光素酶报告基因检测证实，转录因子Bhlhe40直接结合并负向调控Sirt1的表达。进一步研究揭示，Bhlhe40/Sirt1轴调控神经干细胞的线粒体自噬。综上所述，我们的研究结果首次揭示了Bhlhe40/Sirt1轴调控的线粒体自噬在ATRA诱导的SBA中的潜在作用。我们的发现为NTDs的发病机制提供了新的见解，并为潜在的治疗靶点奠定了基础。