叶酸通过抑制氧化应激和Wnt/β-catenin信号通路预防神经管畸形的机制研究

Mechanism of folic acid in preventing neural tube defects by inhibiting oxidative stress and Wnt/β-catenin signaling pathway

目的 探索叶酸预防神经管畸形的机制及其与Wnt/β-catenin信号通路和氧化应激之间的相关性。方法 将C57BL/6小鼠30只按照雌雄=2∶1合笼过夜,次日清晨检出阴栓的雌鼠视为怀孕0.5 d。孕鼠随机分成对照组(Control)、模型组(Model)、叶酸低剂量组(FA-L)和叶酸高剂量组(FA-H),每组5只。FA-L组(2.0μg/mL)、FA-H组(10.0μg/mL)、NTDs模型组和正常组(生理盐水)按25μL/(g·d)体积灌胃。孕7.5 d时一次性腹腔注射12.5 mg/kg的5-氟尿嘧啶建立NTDs模型(对照组注射等体积生理盐水)。孕12.5 d孕鼠眼眶后静脉丛采血,断颈处死,体视显微镜下观察胎鼠畸形及神经管发育情况。检测胎鼠脑组织中GSH含量,SOD和GSH-Px活力,Wnt1、Frizzled-4、Gsk-3β、β-catenin mRNA相对表达量及孕鼠血清Hcy含量。结果 与对照组比较,5-FU诱导的NTDs模型组胎鼠脑组织GSH含量、SOD和GSH-Px活力及Gsk-3β mRNA相对表达量均显著降低(P<0.05),同时Wnt1、Frizzled-4、β-catenin mRNA相对表达量及孕鼠血清Hcy含量显著增高(P<0.05),而在5-FU诱导同时增补不同剂量叶酸的FA-L组和FA-H组中,上述指标变化得到不同程度缓解。结论 增补叶酸可能通过抑制氧化应激和降低Hcy水平调控Wnt/β-catenin信号通路,对5-FU诱导的胚胎NTDs有预防作用。

Objective To investigate the mechanism of folic acid in preventing and controlling neural tube defects(NTDs)by inhibiting oxidative stress and Wnt/β-catenin signaling pathway.Methods Thirty C57BL/6 mice were caged ac-cording to the ratio of female to male=2∶1 at 6:00 p.m,and the female mice with vaginal plugs were regarded as pregnant 0.5 days(E0.5)in the next morning.Pregnant mice were randomly divided into the Control group,Model group,Low-dose folic acid group(FA-L)and High-dose folic acid group(FA-H)with 5 mice in each group.The pregnant mice in the FA-L and FA-H group were given folic acid solution(2.0μg/mL)and(10.0μg/mL)by gavage daily,while the pregnant mice in the Control and Model group were given physiological saline by the same way with the volume of 25μL/(g·d).5-FU(12.5 mg/kg)was injected intraperitoneally into pregnant mice in the NTDs models on gestational day 7.5.The Control group was intrap-eritoneally injected with equal volume of normal saline.All pregnant mice were euthanized by cervical dislocation and care-fully observed by stereomicroscope on gestational day 12.5.The content of GSH,the activity of SOD and GSH-Px,the relative expression of Wnt1,Frizzled-4,Gsk-3β,β-catenin mRNA in embryonic brain tissue,and the serum Hcy level in pregnant mice were detected.Results Compared with the Control group,the content of GSH,the activity of SOD and GSH-Px,the relative expression of Gsk-3βin the NTDs models were significantly decreased(P<0.05),while the relative expression of Wnt1,Friz-zled-4,β-catenin mRNA and the serum Hcy level in pregnant mice were significantly increased(P<0.05).However,the above variables induced by 5-FU,in the FA-L and FA-H groups,were been mitigated to varying degrees.Conclusion Folic acid supplementation may regulate the Wnt/β-catenin signaling pathway by inhibiting oxidative stress and reducing Hcy levels,and has a preventive effect on 5-FU-induced NTDs in embryos.