槲皮素拮抗大气细颗粒物PM2.5对大鼠胚胎毒性的体外研究

目的：探讨槲皮素对大气细颗粒物（fine particulate matter,PM2.5）发育毒性的拮抗作用及可能机制。方法：于2015年12月至2016年3月以大气采样器将北京市某地区采暖期PM2.5收集在石英滤膜上,超声震动后将洗脱液过滤,冷冻干燥后得到PM2.5样品。采用体外全胚胎培养模型,将胎龄9.5 d的SD大鼠胚胎以50 mg/L PM2.5染毒,然后用不同剂量（0.1、0.5、1.0、5.0 μmol/L）的槲皮素干预,培养48 h后对大鼠胚胎生长发育和组织器官形态分化进行评分。染毒结束后,剥离卵黄囊,制备胚胎单细胞悬液,通过流式细胞仪检测细胞线粒体内活性氧（reactive oxygen species,ROS）的生成情况。结果：PM2.5染毒后,大鼠体外胚胎的卵黄囊直径、头长、颅臀长和体节数明显减少,各组织器官形态学评分明显降低,细胞线粒体内ROS水平显著升高。槲皮素干预后,对PM2.5致胚胎生长发育阻滞有明显改善作用,可使胚胎卵黄囊直径、颅臀长、头长增大,体节数增多,卵黄囊血管分化情况得到改善,并对PM2.5所致的尿囊、体屈、心脏、后脑、中脑、前脑、听觉、视觉、嗅觉、腮弓、上颌突、前肢芽及后肢芽分化抑制均有不同程度的改善,细胞内线粒体ROS含量也显著降低。1.0 μmol/L槲皮素干预效果最佳,其他3个剂量组的槲皮素干预也有一定的保护作用,但效果均不及1.0 μmol/L剂量组。结论：适量补充槲皮素对PM2.5致胚胎发育毒性有拮抗作用,清除线粒体内ROS可能是其机制之一。

Antagonistic effect of quercetin on PM2.5 toxicity in the rat's embryonic development in vitro

Objective: To explore the antagonistic effect of quercetin on fine particulate matter (PM2.5)-induced embryonic developmental toxicity in vitro.Methods: PM2.5 was collected on glass fiber filters by PM2.5 samplers during the heating period of Dec.2015 to Mar.2016 in an area of Haidian District, Beijing City.The sampled filters were cut into 1 cm×3 cm pieces followed by sonication.The PM2.5 suspension was filtered into a 10 cm glass dish through 8 layers of sterile carbasus and stored at-80 ℃ until freeze drying.Frozen PM2.5 suspension was dried by vacuum freeze-drying.In vitro post-implantation whole embryo culture was used in this study.Pregnant rats with 9.5 gestation days (GD) were killed by cervical dislocation and the uteri were removed into sterile Hank's solution.The embryos with intact yolk sacs and ecto placental cones were induced by PM2.5, and then subjected to intervention of quercetin at the doses of 0.1 μmol/L, 0.5 μmol/L, 1.0 μmol/L and 5.0 μmol/L, respectively.At the end of the 48 h culture period, the cultures were terminated, and all embryos were removed from the culture bottles and placed in prewarmed Hank's solution for evaluation.Morphological evaluation of the embryos was conducted under a stereomicroscope using the morphologic scoring system by Brown and Fabro.The mitochondrial reactive oxygen species (ROS) level was detected by FACSCalibur flow cyto-metry using MitoSOXTM Red staining.Results: An obvious antagonistic effect was achieved through querce-tin at the dose of 1.0 μmol/L, which could result in an increase of visceral yolk sac (VYS) diameter, crown-rump length and head length, somite number, and the differentiation of visceral yolk sac vascular vessels.The scores of allantois, flexion, heart, hind brain, midbrain, forebrain, auditory system, visual system, olfactory system, branchialarch, maxillary process, forelimb bud and hindlimb bud also revealed a significant increase and the relative mitochondrial ROS level of embryonic cells was significantly decreased when compared with PM2.5 group.Although quercetin at the doses of 0.1 μmol/L, 0.5 μmol/L, 5.0 μmol/L also exhibited protective effects against PM2.5-induced embryonic developmental toxicity, the protective effect was weaker when compared with the dose of 1.0 μmol/L.Conclusion: Quercetin at proper dose may be of great benefit for the development of embryos exposed to PM2.5 in the uterus of the rats.Quercetin provides an effective strategy for the prevention of PM2.5-induced embryonic developmental toxicity.Clearance of mitochondrial ROS may be one of its mechanisms.