Protective effect of Angelica sinensis on cerebral neurons from rat embryos under hypoxia

摘要

BACKGROUND: The enhanced expression of c-Fos protein in nerve cells after hypoxia is the marker for converting extracellular hypoxia information to intracellular changes at hypoxia, and it is suspected that the increase of c-Fos protein can lead to the synthesis and excretion of related neurotrophic factor and nerve growth factor. However, it is still unclear what functional changes of nerve cells are induced by the increase of c-Fos protein at hypoxia, and whether it is good for the survival of damaged neurons.OBJECTIVE: To observe the expression of c-Fos in the cerebral neurons from embryos of rats with hypoxia in uterus, and investigate the pathway for the protective effect of Angelica sinensis injection on the cerebral neurons from rat embryos under hypoxia.DESIGN: A completely randomized controlled study.SETTING: Department of Histology and Embryology, Luzhou Medical College.MATERIALS: Twelve female Wistar rats in oestrum and 1 male adult Wistar rat with body mass of 220 to 250 g were selected. Rabbit-anti-rat neuro-specific enolase (NSE) and rabbit-anti-rat c-Fos were purchased from Wuhan Boster Biological Technology Co., Ltd.; Double-staining kit was bought from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. Angelica sinensis injection was produced by the Department of Pharmacy, the Second Affiliated Hospital of Hubei Medical University.METHODS: The experiments were completed in the experimental animal center and the Department of Histology and Embryology of Luzhou Medical College from December 2004 to December 2005. ① Twelve adult female Wistar rats in oestrum and 1 male Wistar rat were housed in one rearing cage. The appearance of vaginal embolus at 8:00 in the next morning was recorded as 0 day of pregnancy and the rats were recorded for 15 days, and they were divided randomly into three groups, control group (n =4), hypoxia group (n =4)and Angelica group (n =4). The pregnant rats in the hypoxia group were firstly injected with saline (8 mL/kg),then put into 2 L wide-mouthed bottle containing 100 g sodalime, and then the lid of the bottle was closed tightly to induce hypotonic hypoxia for 1 hour followed by 1-hour re-oxygenation. The pregnant rats were killed under anesthesia, and then fetuses were taken out by rapid cesarean. Part of the brain tissues were exposed and then fixed in formaldehyde (40 g/L). The pregnant rats in the Angelica group were treated the same as those in the hypoxia group except that saline was replaced by 250 g/L Angelica sinensis injection which was injected via caudal vein (8 mL/kg). The rats in the control group were injected with saline (8 mL/kg) slowly via caudal vein, but not put into the wide-mouthed bottle for hypoxia, and then the brain tissues were removed and fixed as those in the hypoxia group after 1 hour. ② Twenty embryos from rats were chosen randomly in each group and then routinely embedded in paraffin. Paraffin sections of 4 μm thick were prepared through the anterior fontanelle of head of the fetal rats. The sections were immunohistologically stained with c-Fos/NSE. ③ The one-way analysis of variance (ANOVA) was used to compare the differences of measurement data among the groups, and the q test was applied in the two-two comparison.MAIN OUTCOME MEASURES: The numbers of c-Fos and c-Fos/NSE positive neurons in cerebrum from rat embryos were observed.RESULTS: ① Numbers of NSE positive neurons in cerebrum of rat embryos in the control group, hypoxia group and Angelica group were (84.3 ± 9.0), (90.2 ± 12.5) and (86.7 ± 9.7) cells/high power field (P ＞ 0.05).② The number of c-Fos/NSE positive neurons was more in the hypoxia group than in the control group and Angelica group [(38.4±5.28), (11.35±2.67), (20.65±4.07) cells/high power field, q =29.17, 19.14, P ＜0.05].CONCLUSION: Hypoxia can stimulate the expression of c-Fos in cerebral neurons from rat embryos.Angelica sinensis injection could reducing the damage of hypoxia to neurons and play a neuroprotective role by decreasing the expression of c-Fos protein in hypoxic neurons.