[Mechanistic investigation of fibroblast growth factor 1-mediated regulation of cell proliferation and apoptosis in retinoic acid-induced neural tube defects in mice].

Objective: To investigate the molecular mechanism by which fibroblast growth factor 1 (FGF1) participates in neural tube defects (NTD) through regulating cell proliferation in a retinoic acid (RA)-induced mouse model, and to elucidate the role of FGF1 during neural tube closure in mice. Methods: An NTD mouse model was established using RA induction, with mice divided into a treatment group and a control group (n=9 each). Pregnant mice in the treatment group received 28 mg/kg RA via gavage at embryonic day 7.5 (E7.5), while the control group received sesame oil. Embryonic brain vesicle tissues were collected at E8.5, E9.5, and E10.5 for RNA and protein extraction to assess gene expression. Embryonic brain sections from E9.5 and E10.5 were prepared for pathological examination and gene expression analysis. At the mouse embryonic brain tissue level, quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to validate FGF1 mRNA expression, and immunohistochemistry (IHC) was performed to detect FGF1 protein expression. An FGF1 knockdown plasmid was constructed, and cell proliferation was assessed in vitro. Western blot was used to measure the expression of the proliferation marker proliferating cell nuclear antigen (PCNA), 5-Ethynyl-2'-deoxyuridine (EdU) staining was employed to evaluate cell proliferation, and flow cytometry was conducted to analyze apoptosis. Results: The RA treatment group of mice showed delayed embryonic development, abnormal brain structure, and unclosed neural tubes, inducing the formation of NTD mouse models. At the animal level, both the mRNA and protein expressions of FGF1 were abnormally highly expressed in the brain tissues of NTD mouse embryos at embryonic day 9.5 (E9.5) and E10.5 (P<0.001). At the cellular level, FGF1 was significantly highly expressed in HT-22 cells treated with RA (P<0.001), while its expression was significantly downregulated after FGF1 knockdown (P<0.001); the protein expression of PCNA (a cellular proliferation marker) in RA-treated cells was significantly increased (P<0.01), and this expression was significantly decreased after FGF1 gene knockdown in the RA-treated group (P<0.01). EdU staining experiments also showed that excessive cell proliferation occurred after RA treatment (P<0.001), whereas cell proliferation was reduced in RA-treated cells with FGF1 knockdown (P<0.001); flow cytometry analysis indicated that the proportion of apoptotic cells increased significantly after RA intervention (P<0.001), and this proportion continued to increase after FGF1 knockdown (P<0.05). Conclusions: FGF1 was significantly upregulated in the RA-induced NTD model. Overexpression of FGF1 inhibited cell proliferation and induced apoptosis. Knockdown of FGF1 restored the reduced cell proliferation caused by RA intervention and further increased RA-induced apoptosis. It provides a new perspective on the mechanism of NTD and may serve as a potential biomarker for clinical diagnosis.