莪术胚胎期暴露对仔鼠神经行为的影响及氧化损伤机制

Effects of embryonic exposure to Rhizoma Curcumae on neurobehavior in offspring mice and oxidative damage mechanisms

目的观察正常和血瘀证孕小鼠子代莪术神经发育毒性的差异,为中药辨证减毒提供实验依据。方法采用冰水浸入法制备寒凝血瘀小鼠模型。C57BL/6野生小鼠和Nrf2基因敲除(Nrf2-KO)小鼠分为对照组和莪术暴露组,孕小鼠从妊娠第5天至18天灌胃给予莪术水煎液。检测子代小鼠前肢悬挂和空中翻正达标日龄,分光光度法检测子代小鼠脑组织MDA含量,实时定量PCR检测子代小鼠脑组织Nox4 mRNA表达,蛋白质印迹法检测Nox4蛋白表达。结果与正常对照组比较,正常小鼠灌胃莪术导致子代前臂悬挂和空中翻正达标日龄显著延长和脑组织MDA显著增加(P<0.05),而血瘀证小鼠未见显著差异(P>0.05)。与对照组比较,莪术增加了正常和血瘀证小鼠子代脑组织Nox4 mRNA和蛋白表达(P<0.05),而对ERK1/2磷酸化水平无显著影响(P>0.05)。Nrf2基因敲除增加了血瘀证孕小鼠子代前臂悬挂和空中翻正的达标日龄和脑组织MDA含量(P<0.05)。结论莪术对正常小鼠子代的神经毒性效较血瘀证小鼠子代明显,莪术神经发育毒性的"有故无殒"现象可能与Nrf2核转位有关。

Objective This study aims at evaluating the differences of developmental neurotoxicity between normal and blood-stasis pregnant mice treated with Rhizoma Curcumae, and provide data for syndrome differentiation-based attenuating toxicity of Chinese medicinal herb. Methods Blood stasis mice model was induced by being immersed in ice water. C57 BL/6 wild-type(WT) mice and Nrf2 knock out(KO) mice were randomized into control groups and Rhizoma Curcumae exposure groups. The pregnant mice were administered with Rhizoma Curcumae during pregnant day 5 to day 18. The neurodevelopment reflex was examined by the positive occurring time of turned over in air appeared and the forelimb hanging tests. Measurement of MDA in brain of the offspring mice were performed by Colorimetric Assays. Nox4 mRNA and protein expression in brain of the offspring mice were examined by Real-Time RT-PCR and Western blotting respectively. Results Compared with the normal control group, C57 BL/6 mice treated with Rhizoma Curcumae(10.0 g/kg) extend the positive occurring time of turned over in air appeared and the forelimb hanging tests of offspring and increased MDA content in brain. We could not find any significant change in that of blood-stasis pregnant mice offspring compared with the normal control group. Compared with the control group, mRNA and protein expression of Nox4, but not ERK1/2 phosphorylation, up-regulated significantly in brain of the offspring of mice treated with Rhizoma Curcumae(10.0 g/kg). Knock out Nrf2 using CRISPR/Cas9 extended the positive occurring time of turned over in air appeared and the forelimb hanging tests and increased MDA content of blood-stasis pregnant mice offspring. Conclusion Developmental neurotoxicity of the normal pregnant mice exposure with Rhizoma Curcumae was stronger than that of the blood-stasis pregnant mice. The nuclear translocation of Nrf2 activation play important roles in "YOU-GU-WU-YUN" phenomenon of Rhizoma Curcumae.