妊娠期莪术暴露对子代神经发育的影响及凋亡机制

Effect of maternal exposure to Curcumae Rhizoma during pregnancy on neurodevelopment and apoptosis mechanism in offspring

莪术是妊娠禁中药,寒凝血瘀证是莪术的对应证候。目前,莪术的发育毒性是否与其对证使用有关尚不清楚。为了验证中医"有故无殒,亦无殒"理论,该研究采用冰水浸入法制备野生小鼠和Nrf2敲除小鼠寒凝血瘀小鼠模型。于小鼠妊娠第5～18天连续14 d灌胃给予莪术水煎液,通过检测子代小鼠神经行为学和小鼠脑组织凋亡相关分子,比较正常和血瘀证孕小鼠子代莪术神经发育毒性的差异。结果显示,与正常对照组比较,正常小鼠灌胃莪术导致子代平面翻正达标日龄显著延长和脑组织caspase-3和caspase-9活性显著增加(P<0.05),而血瘀证小鼠无显著差异。与对照组比较,莪术对正常和血瘀证小鼠子代脑组织Bcl-2表达和p38 MAPK磷酸化水平无显著影响。Nrf2基因敲除增加了血瘀证孕鼠子代平面翻正达标日龄和脑组织caspase-3和caspase-9活性(P<0.05)。总之,莪术对正常小鼠的毒性较血瘀证小鼠子代明显,Nrf2分子参与莪术神经发育毒性的有故无殒现象,但上游信号作用机制值得进一步研究。

Curcumae Rhizoma is a Chinese medicinal herb that is contraindicated during pregnancy. Cold-congelation and blood-stasis are corresponding syndromes to Curcumae Rhizoma. Whether syndrome-based treatment is associated with developmental neurotoxicity of Curcumae Rhizoma remains to be unclear. To verify the theory of traditional Chinese medicine of 'syndrome-based treatment during pregnancy', the present study induced the mice blood stasis model by immersing mice in ice water. Pregnant C57 BL/6 wild type(WT) mice and pregnant Nrf2 knock out(KO) mice were randomly divided into control groups and Rhizoma Curcumae exposure groups. The mice were exposed to Rhizoma Curcumae during day 5 to day 18 after pregnancy. The neurodevelopment was examined to evaluate the differences of developmental neurotoxicity between normal and blood-stasis pregnant mice exposed to Rhizoma Curcumae. caspase-3 and caspase-9 activity in brain of the offspring were measured by colorimetric assays. Bcl-2 mRNA and protein expression in brain of the offspring were examined by Real-time RT-PCR and Western blot, respectively. According to the findings, C57 BL/6 mice exposed to Rhizoma Curcumae(10.0 g·kg-1) had a longer positive occurring time of the surface righting reflex test of offspring and higher caspase-3 and caspase-9 activities in brain of offspring, compared with the normal control group, but with no significant change in those of blood-stasis pregnant mice offspring. However, mice exposed to Rhizoma Curcumae(10.0 g·kg-1) showed no change in Bcl-2 gene expression and p38 MAPK phosphorylation in brain of the offspring. Nrf2 gene knockout using CRISPR/Cas9 resulted in a longer positive occurring time of the surface righting reflex test of offspring and higher caspase-3 and caspase-9 activities in brain of offspring. In conclusion, developmental neurotoxicity of the blood-stasis pregnant mice exposed to Rhizoma Curcumae was weaker than that of the normal pregnant mice. Nrf2 activation involved in the phenomenon of Rhizoma Curcumae of 'syndrome-based treatment during pregnancy', but the upstream signal pathway mechanism value shall be further investigated.