Ning Wang, Dongpi Wang, Zhong Lü, Xuan Chen, Long Lin, Zhiyong Hu

1Children's Hospital of Zhejiang University School of Medicine, Hangzhou 310003, Zhejiang Province, China

2Huqingyu Hall Traditional Chinese Medicine Museum, Hangzhou 310000, Zhejiang Province, China

3Red Cross Hospital, Hangzhou 310000, Zhejiang Province,China

lNTRODUCTlON

Xuefuzhuyu decoction (XFZYD), invented by Qingren Wang from the Qing dynasty,China, may improve microcirculation hemodynamics and inhibit cytochrome C release from neuronal mitochondria in rats with cerebral hemorrhage, thus preventing cellular apoptosis[1]. Astragalus, a traditional Chinese herb, has been shown to attenuate free radical damage[2-3]and significantly increase cerebral blood flow in animals following brain injury[4]. Through lipid peroxidation, astragalus may improve membrane micro-viscosity, reduce microvascular resistance, and promote microcirculation, thereby improving hemodynamics in brain tissues[5]. Nerve growth factor (NGF) maintains the survival and function of nerve cells and promotes the repair of damaged nerve fibers; therefore,NGF is considered to be a hallmark of the regeneration and repair of damaged nerves.

The present study sought to elucidate the following issues: (1) the efficacy of combined therapy of XFZYD and astragalus for hypoxic ischemic encephalopathy;(2) whether combined therapy is superior to treatment with XFZYD alone; (3) the dose-dependence of astragalus in combined therapy; and (4) how combined therapy related to the expression of endogenous NGF.

RESULTS

Quantitative analysis of treated animals

A total of 128 Sprague-Dawley rats were included in this experiment. Four animals showed no neurological deficit after induction of hypoxic-ischemia, fifteen rats died after anesthesia, and twenty-nine rats died after induction of hypoxic-ischemia. The remaining 80 rats were involved in the analysis. These successful models were randomly divided into five groups: normal control, model and treatment 1, 2, 3 groups.

After hypoxic-ischemic brain injury models were established, the model group and treatment 1, 2, 3 groups were respectively treated with normal saline, XFZYD, XFZYD plus 10 g astragalus, and XFZYD plus 40 g astragalus.

XFZYD plus astragalus treatment reduced damage in the hypoxic-ischemic brain

Hematoxylin-eosin staining showed that normal control rats exhibited intact morphology of nerve cells, with even and dense intercellular substance. At day 1 after hypoxic-ischemic brain injury, cortical neurons in the parietal, frontal and partial temporal areas tended to be ischemic, with the following characteristics: apparent hyperplasia of microglia; neuronal condensation; nuclear condensation and homogenization; deepened staining;cytoplasm vacuolization; loose mesenchyme; polygonal or fan-shape neurons enclosed by bubbled nerve fibers with normal brain cells visible; and vascular ischemia-like changes including luminal narrowing and congestion, as well as enlarged stroma (Figure 1).

Figure 1 Morphological changes in the hippocampus on day 6 after XFZYD plus astragalus treatment in rats with hypoxic-ischemic brain injury (hematoxylin-eosin staining).

In the hippocampal dentate gyrus, cells were deeply stained and unevenly distributed with patches containing no cells, cellular disruption or drop-out was visible, the cellular layer was reduced in size, cell bodies were swollen, and nuclear lysis and fragmentation were seen.

On day 6, these symptoms of cerebral hypoxia-ischemia were slightly improved in the model group. The above pathological changes improved significantly after treatment with XFZYD and astragalus, and the morphology and structure of neurons appeared to be restored (Figure 1, supplementary Figure 1 online).

XFZYD plus astragalus treatment increased the expression of NGF mRNA in the hypoxic-ischemic brain

Reverse transcription-PCR showed that the NGF mRNA expression level in rats with hypoxic brain injury was significantly higher than that in the normal control group(P ＜ 0.05), and that the expression levels were greatly up-regulated after XFZYD plus 10 g astragalus treatment compared with the model group (P ＜ 0.05). However,there was no significant difference in the expression levels among treatment 1, 2, 3 groups (Figure 2, Table 1).

Figure 2 Nerve growth factor (505 bp) and β-actin(375 bp) gene fragments amplified in brain tissue of rats with hypoxic-ischemic brain injury after XFZYD plus astragalus treatment, as detected by gel electrophoresis.Lanes 1, 6: blank controls; lanes 2-5: β-actin mRNA normal control group, XFZYD group, XFZYD plus 10 g astragalus group, XFZYD plus 40 g astragalus group on day 6; lanes 7-10: nerve growth factor mRNA normal control group, XFZYD group, XFZYD plus 10 g astragalus group, XFZYD plus 40 g astragalus group on day 6.Marker: 100 bp marker. XFZYD: Xuefuzhuyu decoction.

Table 1 Nerve growth factor mRNA expression in brain tissues of rats with hypoxic-ischemic brain injury after Xuefuzhuyu decoction plus astragalus treatment, as detected by TaqMan reverse transcription-PCR (2-△△CT)

XFZYD plus astragalus treatment improved neurological function in rats with hypoxic-ischemic brain injury

After modeling, rats exhibited varying degrees of neurological deficits. Neurological function was significantly improved after treatments compared with the model group on day 6 (P ＜ 0.05), but no significant difference was found among the three treatment groups(Table 2).

Table 2 Zea Longa scores for assessment of neurological deficits in hypoxic-ischemic brain injury rats after Xuefuzhuyu decoction plus astragalus treatment (n)

DlSCUSSlON

Permanent bilateral common carotid artery ligation can cause acute cerebral ischemia, then the basilar artery loop can regulate blood flow, form a collateral circulation, improve and compensate for the cerebral ischemia-induced injury. The whole procedure results in a chronic cerebral hypoperfusion and subsequent hypoxic-ischemic injury[7]. After acute ischemia, pathological and morphological changes in brain tissue are a hallmark of brain damage, and are also the most reliable indicator for assessing pharmacodynamics. The experimental results revealed no significant differences in pathological changes on day 1 after brain injury among groups except for the normal control group. This finding confirms the success of model establishment.

The postoperative neurological deficits and pathological damage of brain tissue were improved to varying degrees on day 6. With the postoperative recovery time prolonged, the surviving rats exhibited a stronger capacity for self-recovery from brain injury. XFZYD plus 10 g astragalus treatment has been shown to significantly improve neurological deficits, although there were significant differences in the degrees of improvement of neurological deficits and pathological damage between the two XFZYD plus astragalus treatment groups and the model group on day 1. This finding suggested that XFZYD plus astragalus plays an apparent protective role in hypoxic-ischemic brain injury, and the effect is superior to that of XFZYD alone.

Xu et al[8]found that NGF expression reached a peak on day 1 after cortex ischemia and that NGF expression was still detected in the puerarin treatment group on day 14 in rat models with middle cerebral artery occlusion. The present experiment obtained similar results. A prospective clinical study of Chiaretti et al[9]showed a high level of NGF expression following early traumatic brain injury, and the severity of injury was positively correlated with the prognosis of neuronal repair. A high level of NGF, which is an endogenous self-protection reflex involving a series of biochemical and molecular cascades after traumatic brain injury, indicates a good prognosis. The present study suggested that brain tissue could promote NGF synthesis within a short term after hypoxic-ischemic injury, and that XFZYD combined with different doses of astragalus could enhance NGF mRNA transcription in brain tissue after hypoxic-ischemic brain injury, and maintain it for a long time, thereby contributing to the recovery of hypoxic-ischemic neurons and an improvement of prognosis.

In summary, the combination of XFZYD plus different dosages astragalus plays a neuroprotective role by increasing NGF mRNA expression in the brain tissues of Sprague-Dawley rats after hypoxic-ischemic brain injury,and the optimal dosage of astragalus is 10 g. However,further studies are required to conclusively determine how long the increased expression of NGF mRNA can be maintained.

MATERlALS AND METHODS

Design

A randomized controlled animal experiment.

Time and setting

The experiment was performed from December 2007 to April 2008 at the Center Laboratory and Graduate Laboratory, Children’s Hospital of Zhejiang University School of Medicine, China.

Materials

Animals

Eighty healthy Sprague-Dawley rats, aged 5 weeks,weighing 100 g, half males and half females, of clean grade, were provided by the Animal Center, Zhejiang Academy of Medical Sciences, China, under the license No. SCXK(Zhejiang)2003-0001.

XFZYD

XFZYD was prepared by the Department of Pharmacy,Traditional Chinese Medicine Hospital of Zhejiang Province, China, consisting of Angelica 9 g, rehmannia dried rhizome 9 g, peach seed 12 g, safflower 9 g, fructus aurantii 6 g, red peony 6 g, Bupleurum 3 g, licorice 3 g,Campanulaceae 6 g, Szechwan Lovage Rhizome 5 g,and twotoothed Achyranthes root 9 g. The first formula was decocted for 30-40 minutes, and the second one was decocted for 40-50 minutes; a mixture of these two decoctions (40 mL) was the resultant liquid used in experiments. There was 2 g of crude drug in each 1 mL of liquid.

Astragalus

XFZYD (10 and 40 g) was added to astragalus (Department of Pharmacy, Traditional Chinese Medicine Hospital of Zhejiang Province, China).

Methods

Establishment of hypoxic-ischemic encephalopathy models

Sprague-Dawley rats were anesthetized with intraperitoneal injections of ketamine. The bilateral common carotid artery was separated and ligated using a No. 3 silk (1-0) suture. The normal control group only underwent bilateral common carotid artery isolation,without suture. At 6 hours after the operation,neurological functions were assessed based on Zea Longa scores[6]. Model rats scoring 1-4 were included in the experiment.

XFZYD combined with different doses of astragalus or alone

The model group and treatment 1, 2, and 3 groups were respectively treated with normal saline, XFZYD, XFZYD plus 10 g astragalus, and XFZYD plus 40 g astragalus at 1 mL/100 g per day. The normal control group received normal saline at 1 mL/100 g per day.

Neurobehaviors of rats with hypoxic-ischemic brain injury as detected by Zea Longa scores

The neurological functions of 8 rats in each group were evaluated on the basis of Zea Longa scores on days 1 and 6[6].

Hematoxylin-eosin staining of brain tissues of hypoxic-ischemic brain injury

Rats were anesthetized by intraperitoneal injection of ketamine and the abdominal venous blood was sampled. Immediately after sampling, rats were decapitated and brains were removed. Coronal brain tissues[10]were cut into two pieces on ice. One piece was fixed with 10% neutral formalin and paraffin embedding, for observation of pathological changes under light microscope by hematoxylin-eosin staining;the other piece of fresh brain tissue was utilized for reverse transcription-PCR.

NGF expression in brain tissues of rats with hypoxic-ischemic brain injury as detected by reverse transcription-PCR

Primers were synthesized by Shanghai Invitrogen Biotechnology Co., Ltd., China and used according to instructions of the SYBR PrimeScript reverse transcription-PCR kit (TaKaRa Company). NGF and internal reference β-actin primers were designed based on the Genbank sequence[11]. The two primers were designed to anneal to bases 328-347 and 813-832; the expected amplification product was 505 bp. Sense sequence: 5'-GAT CGG CGT ACA GGC AGA AC-3';antisense sequence: 5'-GGC TCG GCA CTT GGT CTC AA-3'. β-actin primers were used as a control. Sense sequence: 5'-GCC ATG TAC GTA GCC ATC CA-3'; antisense sequence: 5'-GAA CCG CTC ATT GCC GAT AG-3'. The expected PCR product length was 375 bp.NGF and β-actin genes were amplified by 3-step PCR,as follows[12]. Step 1: 95°C denaturation for 5 minutes,one cycle. Step 2: 30 cycles of 95°C denaturation for 40 seconds, 57°C annealing for 40 seconds, and 72°C extension for 45 seconds. Step 3: 72°C extension for 10 minutes, one cycle. After PCR, real-time PCR amplification curves and melting curves were confirmed to calculate and compare the Ct values of target genes with β-actin as an internal reference. PCR amplification products were randomly selected for 2% agarose (containing ethidium bromide 0.5 mg/mL) gel electrophoresis,to determine whether the amplified bands were the target gene band.

Statistical analysis

Data were analyzed with SPSS 10.0 statistical software(SPSS, Chicago, IL, USA) for statistical analysis. Measurement data are expressed as mean ± SD. Comparisons between the groups was performed using one-way analysis of variance, and paired samples were compared using the paired Student’s t-test. Count data were compared between groups using the Kruskal-Wallis rank sum test; paired samples were compared using the Mann-Whitney test. A level of P ＜ 0.05 was considered statistically significant.

Author contributions:Ning Wang was responsible for the experimental implementation, data collection, data acquisition and integration, manuscript drafting, study proposal and design,and obtaining funding. Dongpi Wang was responsible for the experimental implementation, data collection, manuscript drafting. Zhong Lü validated the manuscript, provided support technology and instructed the experimenters. Xuan Chen was the article writer and technical instructor. Long Lin was responsible for the pathological data collection, data analysis, statistical analysis, and providing technical support. Zhiyong Hu provided instructions regarding the experimental methods and techniques, and supervised preparation of the manuscript.

Conflicts of interest:None declared.

Funding:This study was financially sponsored by a grant from the Bureau of Traditional Chinese Medicine, Zhejiang Province,China (491040-W50434).

Ethical approval:This study was approved by the Animal Ethics Committee, Children’s Hospital of Zhejiang University School of Medicine, China.

Supplementary information:Supplementary data associated with this article can be found, in the online version, by visiting www.nrronline.org, and entering Vol. 6, No. 21, 2011 after selecting the “NRR Current Issue” button on the page.

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