Chinese Medical E-ournals Database

Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition) ›› 2017, Vol. 13 ›› Issue (03): 281 -286. doi: 10.3877/cma.j.issn.1673-5250.2017.03.007

Special Issue:

Original Article

Regulation mechanism of AMP-activated protein kinase signaling pathway in hypoxic-ischemic neuronal apoptosis in neonate rats

Deyuan Li1, Yang Wang1, Lili Luo1, Lina Qiao1,()   

  1. 1. Department of Pediatrics, Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, West China Second University Hospital, Chengdu 610041, Sichuan Province, China
  • Received:2017-02-01 Revised:2017-04-08 Published:2017-06-01
  • Corresponding author: Lina Qiao
  • About author:
    Corresponding author: Qiao Lina, Email:
Objective

To study expression levels of AMP-activated protein kinase (AMPK), FOXO3a transcription factor and apoptotic protein cleaved caspase-3 (CC3) in the neonate rat brains with hypoxic-ischemic brain damage (HIBD), and to explore the mechanisms of neuroprotective effects of AMPK/FOXO3a signaling pathway inhibition on hypoxic-ischemic neuronal apoptosis in neonate rats.

Methods

Sixty-four postnatal day 7 SD rats were divided into 4 groups by random number table method: hypoxia-ischemia group (HI group), the sham controls group (sham group), AMPK specific inhibiter Compound C-treated group (treated group), and dimethyl sulfoxide (DMSO) vehicle group (control group), respectively. And 16 SD rats in each group. Modeling methods were as followed. For HI group, the right common carotid artery of SD rats was exposed and ligated by ether anesthesia, and the SD rates were exposed to hypoxia in a chamber filled with 8% oxygen (balanced with 92% nitrogen) for 2.5 h. For sham group, the right common carotid artery of SD rats was surgically exposed without ligation and did not receive hypoxia treatment. For treated group, Compound C was intracerebroventricularly injected into the injured cerebral hemisphere of SD rats 30 min prior to treatment by 8% oxygen (balanced with 92% nitrogen) for 2.5 h. For control group, the SD rates received intracerebroventricular injections of DMSO with the equal amount of compound C in treated group, and 30 min later, the SD rats received hypoxic-ischemic treatment as treated group. SD rats′ cerebral cortexes of 4 groups were collected at 24 h after modeling success. Western blotting method was used to detect the protein expression of AMPK, p-AMPKα, total FOXO3a, nuclear and cytoplasmic FOXO3a and CC3 in cerebral cortexes of SD rats modeling in each group. In situ nick-end labeling (TUNEL) staining of was used to detect the apoptotic cells.

Results

①There were no significant differences between HI group and sham group, control group and sham group in expression levels of AMPK protein and total FOXO3a protein in rat′s cerebral cortexes (P>0.05). However, expression levels of p-AMPKα protein and nuclear protein of FOXO3a in HI group and control group all were much higher than those in sham group, and the cytoplasmic protein of FOXO3a were evidently lower than that in sham group, which up-regulated expression level of CC3 protein after hypoxic-ischemic in HI group and control group compared with sham group, and all the differences were statistically significant (P<0.01). ②Expression levels of p-AMPKα protein and nuclear protein of FOXO3a in treated group were obviously lower than those in HI group and control group, and expression level of cytoplasmic protein was much higher than those in HI group and control group, and meanwhile leading to the decrease of CC3 protein after hypoxic-ischemic in treated group compared with HI group and control group, and all the differences were statistically significant (P<0.01). ③Apoptosis levels of neuronal cell (expression levels of TUNEL positive cells) in HI group and control group both were higher than that in sham group, and both the differences were statistically significant (P<0.01). TUNEL positive cells were obviously reduced in treated group compared with those in HI group and control group, and all the differences were statistically significant (P<0.01).

Conclusions

AMPK activity increased in the neonate rat brain with HIBD. AMPK activity inhibition can inhibit FOXO3a translocation from cytoplasm to nucleus, down-regulate the level of CC3 protein, leading to the reduction of neuronal apoptosis.

图1 Western印迹法检测4组造模成功24 h后SD大鼠(n=16)大脑皮质AMPK蛋白及p-AMPKα蛋白表达水平结果(图1A:电泳图;图1B:统计学柱状图)
图2 Western印迹法检测4组造模成功24 h后SD大鼠(n=16)大脑皮质细胞核FOXO3a蛋白、细胞质FOXO3a蛋白、总FOXO3a蛋白及CC3表达水平结果(图2A:电泳图;图2B:统计学柱状图)
图3 4组造模成功24 h后SD大鼠(n=16)大脑皮质神经元凋亡细胞表达情况(图3A~3D 分别为:sham组、HI组、对照组、干预组SD大鼠大脑皮质神经元细胞DAPI染色结果;图3E~3H 分别为:sham组、HI组、对照组、干预组SD大鼠大脑皮质凋亡神经元细胞TUNEL染色结果)(高倍镜)
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