Research on the Effect of Electroacupuncture on MDM2 Expression in a Hypoxic-Ischemic Encephalopathy Rat Model*

Aim: To explore the effect of electroacupuncture (EA) on the expression of mdm2 following hypoxic-ischemic encephalopathy (HIE). Method: The hypoxic-ischemic models were established by colligating bilateral common carotid artery in new-bore SD rats aged 7 days. All rats were randomly divided into sham-surgery, model, sham-EA, EA, antagonist and EA plus antagonist group which subdivided 1d, 3d, 7d, and 21d time-phases. Ultra-structure of neurons was observed using TEM, real-time PCR was used to determine the level of mdm2 mRNA, and the expression of mdm2 was tested with Western Blot. Results: TEM shown that At 21d, the damnification of neurons had evidently recovery with electroacupuncture treatment. The down-regulation of mdm2 was observed in electroacupuncture group, which was significantly lower than that in model groups (P<0.05). Conclusion: Electroacupuncture could obviously lighten the damage of ultrastructure of neurons to protect the neurons from HIE by inhibiting the expression of mdm2.


INTRODUCTION
Hypoxic-ischemic encephalopathy is the brain injury in the neonate caused by perinatal asphyxia, can result in varying degrees of residual symptoms, even acute neonatal death and chronic nervous system damage [1]. HIE can lead to DNA damage and mutation of neurons, and the gene expression of mdm2 in neurons has been reported [2]. As an effective treatment on the nervous system dysfunction left by HIE, acupuncture can promote the proliferation, differentiation of neural stem cells, axonal sprouting, growth and extension, so as to achieve the effect of building neural networks [3]. In this study, the authors aimed at exploring the effect and mechanism of electroacupuncture on hypoxic-ischemic encephalopathy by observing the effect of electroacupuncture on mdm2 expression in neurons of HIE rats, and provide theoratical and experimental basis for clinical treatment.

Experimental animals
Total of 250 Sprague-Dawley rats aged 7d of either sex, weighing 14.0 to 18.0g, were provided by Experimental Animal Center of Qingdao Drug Inspection Institute (SCXK (LU) 20140001). All rats were housed at the ambient temperature of 20-25°C, and fed with their mothers.

Animal grouping
Each brood was randomly divided into two groups,

Establishment of models
Rats was anesthetized with ether and fixed later.

Statistical Analysis
SPSS 17.0 software was applied for the statistical analysis. The data was expressed as mean ± standard error ( s x  ). Factorial analysis was applied in the multi-group comparison after the homogeneity test of variances; and t-test were applied for the two-group comparison. Values were considered to be significant when P was less than 0.05.

Results
Ultrastructure of neurons (Fig.1) Sham-surgery group: The structure of neurons in sham-surgery group was regular with distinct outline and cytoplasm, and the chromatin was uniform ( Fig.   1 A).
Model group: At 1st day after hypoxic-ischemic injury, the neurons have integrated membrane, unevenly distributed chromatin and mild swelling mitochondrion (Fig.1 B1). At 3rd day, the damage of neurons increased with blurred membrane, dissolved nuclear membrane, agglutinated chromatin, and a large number of vacuoles. Apoptotic neurons can be observed (Fig.1 B2). For the self-healing of neurons, at 7th day after hypoxic-ischemic injury, the membrane of neurons was intact with a little loose nucleoplasm and uniform chromatin (Fig.1 B3). The morphology of neurons at 21st day after hypoxic-ischemic injury was similar to that of 7th day (Fig.1 B4).

Sham-EA group:
The pathological change of neurons at each period of sham-EA group was similar to that of model group (Fig.1 C1-4).
EA group: At 1st day after hypoxic-ischemic injury, the ultrastructure of neurons was similar to that of model group, and the cell body was slightly swelling with blurred nuclear membrane, indistinct nucleolus and micro-bubbles around nucleus can be observed ( Fig.1 D1). Slightly swelling cell body, vague nuclear membrane and nucleolus and perinuclear micro-vacuoles could also be observed in neurons at the subgroup of 3rd day, but the degree of neurons damage was lighter than that of model group (Fig.1   D2). At 7th day, the swelling of cell body was lightened, and the nuclear membrane and nucleolus became clear (Fig.1 D3). At 21st day of EA group, the ultrastructure of neurons was similar to that of sham-surgery group. The membrane of neurons was intact with a little loose nucleoplasm and generally uniform chromatin. And a few of slightly swelling mitochondria and dilated rough endoplasmic reticulum can be observed (Fig.1 D4).

Antagonist plus EA group: Compared with neurons
at each period of EA group, signs of necrosis and degeneration were more serious than those in antagonist plus EA group (Fig.1 E1-4).

Antagonist group:
The damage of neurons at each period was more serious than that of model group.
The cytomembrane and nuclear membrane were indistinct, karyopyknosis, chromatin condensation, a large number of vacuoles in cytoplasm, dilated rough endoplasmic reticulum and degranulation were more obvious ( Fig.1 F1-4).
The results of TEM showed that: at 3rd day after hypoxic-ischemic injury, the damage of neurons was obvious, and the damage became alleviated with the

Discussion
As a conservative proto-oncogene [6], murine double minute 2 (mdm2) gene can inhibit the transcriptional activity of p53 [7]and inhibit the DNA repair of damaged cells [8]by promoting the degradation of p53.
Mdm2 is widely researched in the field of cancer with few studies reported in hypoxic-ischemic areas.
The overexpression of mdm2 for some incentives could led to abnormal hyperplasia and canceration of cells [9,10]. As a negative regulator of p53 factor, mdm2 and p53 constitute a degradation-trans activation circulation path [11]. can lead to the disintegration of axonal growth cones and the elimination of the amino acid sequence of p53 in the nucleus, furtherly suggest that p53 can promote the growth of axons [12].
In this experiment, the expression of mdm2 in the neurons of model rats began to be down-regulated from 1st day to 21th day after cerebral ischemia-hypoxia, but the downward trend slowed down gradually, suggesting that the body launch the mechanism of self-protection and self-healing after cerebral ischemia-hypoxia, but the protection mechanism decreases with the extension of time. In