基于妊娠期应用中药安全性评价体系探讨黄芩的胚胎毒性

Study on embryo toxicity of scutellaria using safety evaluation system of traditional Chinese medicine in pregnancy

关键词:  妊娠期用药  中药  胚胎毒性  黄芩

Key words:  using of drugs in pregnancy  Chinese medicine  embryo toxicity  scutellaria

摘要:

[目的] 利用妊娠期应用中药安全性评价体系,应用胚胎干细胞实验(EST)方法,通过对中药-含药血清-胚胎蓄积组分的综合评价,揭示黄芩的胚胎毒性,验证妊娠期应用中药安全性评价体系的客观性.[方法]分别将胚胎干细胞D3系(ES-D3)和胚胎成纤维细胞(Balb/c 3T3)在不同浓度黄芩培养液和20%黄芩含药血清培养液中培养,噻唑蓝(MTT)法检测细胞活性,绘制细胞生长曲线,分别计算50%的ES细胞和3T3细胞增殖受抑制的黄芩浓度,即IC₅₀D3和IC₅₀3T3.利用悬滴-悬浮-贴壁方法,体外培养ES细胞向心肌细胞分化,实时定量PCR(Q-PCR)方法检测肌球蛋白重链基因(β-MHC)的表达量,计算50%的ES细胞分化受抑制的黄芩浓度,即ID₅₀D3.利用生物统计公式,预测黄芩及其含药血清的胚胎毒性.[结果]黄芩的IC₅₀D3、IC₅₀3T3、ID₅₀D3分别为25.58、42.20、0.28 μg/mL,预测其具有强胚胎毒性.低、高剂量黄芩含药血清对3T3细胞和ES细胞增殖均呈现抑制作用,ES细胞分化为心肌细胞β-MHC表达量为空白血清表达量的32.5%.[结论]黄芩存在胚胎毒性,应用中药-含药血清的综合评价方法,能够客观准确评价中药的胚胎毒性.

Abstract:

[Objective] To assess the embryotoxicity of scutellaria and to confirm the objectivity of evaluation system of traditional Chinese medicine in pregnancy, taking evaluation system of traditional Chinese medicine in pregnancy, using embryonic stem cell test (EST) in vitro and through the comprehensive evaluation of traditional Chinese medicine, drug-containing serum and embryo volume fractions.[Methods] D3 cell line of EST(ES-D3) and embryo fibroblast cell (Balb/c 3T3) were cultured respectively with different concentration of scutellaria and 20% scutellaria containing serum. The cell morphology was observed with phase contrast microscope and the absorbance of the resulted coloured solution was measured at 570 nm in a microplate reader. According to the concentration-effect curve, 50% inhibition of growth was obtained and the viability of ES-D3 cells and 3T3 cells was demonstrated (IC₅₀D3 and IC₅₀3T3). Using hanging-drop-suspension-attachment method ES cells were cultured in different concentration of scutellaria for proliferation to myocardial cell. Then the expression of myosin heavy chains genes (β-MHC) was detected and the concentration of scutellaria, which could inhibit the ES proliferation for 50% was calculated ID₅₀D3). Using a biologic statistic formula the embryo toxicity of scutellaria and it-containing serum was predicted. [Results] The half-maximal proliferation-inhibition concentration (IC₅₀) of scutellaria on ES cell line was as follows: IC₅₀D3, IC₅₀3T3 and ID₅₀3D3 were 25.58, 42.20 and 0.28 μg/mL respectively. It was predicted that they had heavy embryo toxicity. All low and high doses of scutellaria-containing serum presented inhibitory effect for proliferation on 3T3 and ES cell. The expression of differentiation of ES cells into β-MHC of myocardial cells was 32.5% of blank serum. [Conclusion] Scutellaria has embryo toxicity. The comprehensive evaluation system using traditional Chinese medicine-containing serum can objectively and accurately evaluate the embryotoxicity of traditional Chinese medicine.