左归丸抵抗高糖抑制植入前胚胎发育的研究

Research on the inhibition of high glucose to preimplantation embryo development and the resistant effect of Zuogui Pills

目的:观察滋阴补肾方左归丸对受高糖抑制的小鼠植入前胚胎的影响。方法:体外培养小鼠胚胎,调葡萄糖浓度至40mmol/L,设正常组、模型组和给药组。观察2-细胞期和囊胚期的胚胎,收集囊胚期的胚胎,DAPI染色做囊胚细胞计数,用2-细胞率、囊胚率和囊胚细胞数评估胚胎发育质量。收集囊胚时期各组的胚胎培养液,做GC-MS代谢检测和数据分析。结果:形态学结果显示,与正常组比较,模型组囊胚率、囊胚细胞数均降低(P<0.05);与模型组比较,给药组囊胚率升高,囊胚细胞数降低(P<0.01)。GC-MS代谢组学结果显示,左归丸含药血清干预后,胚胎培养液中的β-羟基丁酸、尿素、丙氨酸、甘氨酸、缬氨酸、亮氨酸、花生四烯酸、丝氨酸、亚油酸、苏糖酸增加,羟胺、山梨糖、丙酮酸、核糖酸、苯乙醛、羟基天冬氨酸、乙酰丝氨酸减少。结论:高糖抑制胚胎发育,左归丸含药血清促进胚胎发育。滋阴补肾左归丸调节妊娠糖尿病母体宫内高糖环境,抵抗高糖抑制,提高植入前胚胎质量的关键途径是亚油酸代谢通路,甘氨酸、丝氨酸、苏氨酸代谢通路,缬氨酸、亮氨酸、异亮氨酸合成代谢通路。

Objective： To observe the inhibition of high glucose to preimplantation embryo development in mice and the resistant effect of Zuogui Pills. Methods： Mouse embryos were cultured in vitro and the glucose concentration was increased to 40 mmol/L. Embryos were divided into normal group, model group and treatment group. The 2-cell embryos and blastocysts were observed. The blastocyst-derived embryos were collected. The blastocysts were counted by DAPI staining. The embryonic developmental quality was evaluated by the rate of 2-cell, blastocyst and blastocyst. Embryonic fluid in each group was collected during the blastocyst stage and GC-MS metabolic analysis and data analysis were performed. Results： Morphological results showed that the blastocyst rate and the number of blastocyst cells in the model group decreased compared with that of the normal group（P〈0.05）. Compared with the model group, the blastocyst rate of the treatment group increased（P〈0.05）, however the blastocyst cell number showed a significant decrease（P〈0.01）. The results of GC-MS metabolomics showed that interfered by Zuogui Pill serum, β-hydroxybutyric acid, urea, alanine, glycine, valine, leucine, arachidonic acid, serine, linoleic acid and threonic acid were increased, while hydroxylamine, sorbose, pyruvic acid, riboic acid, phenylacetaldehyde, hydroxyaspartate and acetylserine were decreased. Conclusion： High glucose could inhibit embryonic development and Zuogui Pills containing serum could promote embryonic development. Zuogui Pills which were nourishing yin for tonifying kidney regulated the intrauterine high glucose environment in gestational diabetes mellitus, played a role in the resistance to high glucose, and the key ways to improve the quality of preimplantation embryos were linoleic acid metabolic pathway, glycine, serine and threonine metabolic pathway and valine, leucine and isoleucine anabolic pathways.