Neural-tube defects (NTDs) are one type of the most serious birth defects. Studies have shown that inositol deficiency is closely related to the occurrence of NTDs. Bone morphogenetic protein (BMP)-mediated Smad signaling pathways have been implicated in neurogenesis and neural-tube closure. However, the role of the BMP/Smad pathway in inositol-deficiency-induced NTDs remains unclear. Inositol-deficiency models in C57 mice and mouse neural stem cells (mNSCs) were induced with Li₂CO₃ treatment or inositol withdrawal. The role of the BMP/Smad pathway in the regulation of cell proliferation and the development of NTDs was determined utilizing qRT-PCR, HE staining, Western blot, immunostaining, MTT assay, EdU staining, and flow cytometry. The intraperitoneal injection of Li₂CO₃ at Embryonic Day 7.5 induced the occurrence of NTDs. The mRNA levels of Bmp2, Bmp4, Smad1, Smad5, Smad8 and Runx2, the phosphorylation of Smad1/5/8, and the nuclear translocation of Runx2 were significantly increased in NTD embryonic brain tissues and mNSCs exposed to Li₂CO₃ or an inositol-free medium, which were suppressed by BMP receptor selective inhibitor LDN-193189. The Li₂CO₃-induced phosphorylation of Smad1/5/8 was inhibited by inositol supplementation. Cell proliferation was significantly promoted by Li₂CO₃ exposure or the absence of inositol in mNSCs, which was reversed by LDN-193189. These results suggest that the activation of the BMP/Smad signaling pathway might play an important role in the development of NTDs induced by maternal Li₂CO₃ exposure via inositol deficiency.

Lithium carbonate (Li₂CO₃) is a first-line pharmacotherapy for bipolar disorder (BPD) in female patients of childbearing age, although it is associated with embryonic teratogenicity. Lithium exposure in batteries and pharmaceuticals has been a toxicity factor for women’s reproductive health in energy, environment, and fitness fields. Neural tube defects (NTDs) are the severest reproductive outcome of maternal Li₂CO₃ exposure. Currently, there is a lack of effective prevention for Li₂CO₃ neurotoxicity. Our previous study revealed that lithium-induced murine NTDs correlated with enhanced proliferation and impaired inositol biosynthesis. In this study, 400 mg/kg inositol was used to treat the lithium-induced murine NTDs after calculating the toxicity using ADMETlab 3.0 and Deep-PK. We found that inositol supplementation ameliorated fetal neural phenotypes, crown-rump length, and body weight and rescued aberrant cell proliferation and dysregulated the GSK3β/β-catenin/CyclinD1 pathway in both animal and cellular models. The affinity of GSK3β-inositol interaction was analyzed and validated using molecular dynamics and surface plasmon resonance. GSK3β-edited cell models further revealed that inositol altered GSK3β post-transcriptional status and phosphorylation, consequently modulating the GSK3β/β-catenin/CyclinD1 signaling cascade. Overall, our study clarified that inositol alleviates lithium-induced NTDs via the GSK3β/β-catenin/CyclinD1 pathway, revealed the direct molecular interaction between inositol and GSK3β, and highlighted the potential of inositol as a therapeutic prevention against lithium-induced embryonic neurotoxicity for childbearing female BPD patients.