Pregnant Sprague-Dawley rats were randomly divided into three groups. On the 10th day of gestation, pregnant rats were intragastrically injected with all-trans retinoic acid (ATRA) (60 mg/kg) to induce MMC fetal rats. The same amount of olive oil was put into the control group to create normal fetal rats. The rats in the rescue group were given folic acid (40 mg/kg) by gavage 0.5 and 12 hr after ATRA therapy. Bladders were obtained via cesarean section on embryonic day E20.5 and examined for MMC. The histology of the fetuses was examined using hematoxylin and eosin staining, and immunohistochemistry (IHC) was utilized to determine the expression of α-smooth muscle actin (α-SMA) and neuron-specific nuclear-binding protein (NeuN). Furthermore, the levels of neuromuscular development-related and apoptotic proteins were determined by western blotting.
The incidence of MMC in the model group was 60.6% (20/33) while it was much lower in the rescue group (21.4%). In comparison to the model group, the weight and crown-rump length of the fetal rats in the rescue group were significantly improved. IHC revealed that there was no significant difference in the expression of α-SMA and NeuN between the control and ATRA groups, while the expression levels decreased significantly in the MMC group. Western blot analysis showed that there was no significant difference between the model and ATRA groups, but the expression of the α-SMA protein and the β3-tubulin was much lower in the MMC group than in the control group. After the administration of folic acid, the α-SMA and β3-tubulin proteins considerably increased in the folic acid-rescued MMC group and folic acid-rescued ATRA group. Meanwhile, in the control group, the expression of cleaved caspase-3 in the bladder tissue was significantly higher, and the expression of poly (ADP-ribose) polymerase (PARP) protein was significantly lower compared to the control group. Folic acid therapy reduced cleaved caspase-3 expression while increasing PARP expression in comparison to the MMC group.
NB in MMC fetal rats is associated with the reduction of bladder nerve and smooth muscle-related protein synthesis. However, folic acid therapy can help improve these functional deficiencies. Folic acid also exhibits strong anti-apoptotic properties against NB in MMC fetal rats.
方法:妊娠SD大鼠随机分为3组。在妊娠的第10天,孕鼠胃内注射全反式维甲酸(ATRA)(60mg/kg)诱导MMC胎鼠。将等量的橄榄油放入对照组以造就正常胎鼠。拯救组大鼠在ATRA治疗后0.5和12小时通过管饲法给予叶酸(40mg/kg)。在胚胎日E20.5通过剖宫产获得膀胱,并检查MMC。使用苏木精和伊红染色检查胎儿的组织学,免疫组织化学(IHC)用于确定α-平滑肌肌动蛋白(α-SMA)和神经元特异性核结合蛋白(NeuN)的表达。此外,通过蛋白质印迹法测定神经肌肉发育相关蛋白和凋亡蛋白的水平.
结果:模型组MMC的发生率为60.6%(20/33),而抢救组则低得多(21.4%)。与模型组相比,抢救组胎鼠体重和冠-臀长均有明显改善。IHC显示,对照组和ATRA组之间α-SMA和NeuN的表达没有显着差异,而MMC组的表达水平显著下降。Westernblot分析显示模型组与ATRA组之间无显著差异,但是MMC组的α-SMA蛋白和β3-微管蛋白的表达远低于对照组。服用叶酸后,在叶酸拯救的MMC组和叶酸拯救的ATRA组中,α-SMA和β3-微管蛋白显著增加.同时,在对照组中,caspase-3在膀胱组织中的表达明显增高,与对照组相比,聚(ADP-核糖)聚合酶(PARP)蛋白的表达显着降低。与MMC组相比,叶酸治疗减少切割的caspase-3表达,同时增加PARP表达。
结论:MMC胎鼠的NB与膀胱神经和平滑肌相关蛋白合成的减少有关。然而,叶酸治疗可以帮助改善这些功能缺陷。叶酸在MMC胎鼠中还表现出针对NB的强的抗凋亡特性。