槲皮素拮抗大气细颗粒物PM_(2.5)对大鼠胚胎毒性的体外研究

Antagonistic effect of quercetin on PM_(2.5) toxicity in the rat's embryonic development in vitro

目的:探讨槲皮素对大气细颗粒物(fine particulate matter,PM_(2.5))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PM_(2.5)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PM_(2.5)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 PM_(2.5)染毒,然后用不同剂量(0.1、0.5、1.0、5.0μmol/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:PM_(2.5)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PM_(2.5)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PM_(2.5)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。1.0μmol/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1.0μmol/L剂量组。结论:适量补充槲皮素对PM_(2.5)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。

Objective: To explore the antagonistic effect of quercetin on fine particulate matter( PM_(2.5))-induced embryonic developmental toxicity in vitro. Methods: PM_(2.5)was collected on glass fiber filters by PM_(2.5)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 PM_(2.5)suspension was filtered into a 10 cm glass dish through 8 layers of sterile carbasus and stored at-80 ℃ until freeze drying. Frozen PM_(2.5)suspension was dried by vacuum freeze-drying. In vitro postimplantation 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 PM_(2.5),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 cytometry using Mito SOXTMRed staining. Results: An obvious antagonistic effect was achieved through quercetin 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 PM_(2.5)group. Although quercetin at the doses of 0. 1 μmol/L,0. 5 μmol/L,5. 0 μmol/L also exhibited protective effects against PM_(2.5)-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 PM_(2.5)in the uterus of the rats. Quercetin provides an effective strategy for the prevention of PM_(2.5)-induced embryonic developmental toxicity. Clearance of mitochondrial ROS may be one of its mechanisms.