Expression of the DNA-repair protein XRCC1 and DNA-repair enzyme Ku70 mRNA in the rat cerebral cortex following global cerebral ischemia/reperfusion Is this correlated with neuroprotective effects of theanine? *

Rongliang Xue, Wenchao Chen, Ning Wang, Shasha Li

Department of Anesthesiology, Second Affiliated Hospital, Medical College of Xi’an Jiaotong University, Xi’an  710004, Shaanxi Province, China

Rongliang Xue, Professor, Chief physician, Department of Anesthesiology, Second Affiliated Hospital, Medical College of Xi’an Jiaotong University, Xi’an  710004, Shaanxi Province, China

Corresponding author: Rongliang Xue, Professor, Chief physician, Department of Anesthesiology, Second Affiliated Hospital, Medical College of Xi’an Jiaotong University, Xi’an  710004, Shaanxi Province, China    
E-mail: xuerl@China.com

Supported by: the National Natural Science Foundation of China, No. 30571790*

Abstract
BACKGROUND: Numerous studies have shown that apoptosis, caused by cerebral ische-mia/reperfusion injury, is a dynamic evolving process that occurs in a time-dependent fashion. Non-apoptotic cells can result in apoptosis, with a prolonged reperfusion period and an accumulation of DNA damage. Recent studies have indicated that theanine has a protective effect on the brain. Nonetheless, there is no research relating to whether theanine is neuroprotective following brain injury.
OBJECTIVE: To investigate the effects of theanine on apoptosis in the cerebral cortex and mRNA expression of the DNA repair protein, XRCC1, and DNA repair enzyme, Ku70, at various time points following global cerebral ischemia/reperfusion in rats.
DESIGN, TIME AND SETTING: A randomized, controlled animal study was performed at the Pharmacological Laboratory of Animal Experimental Center of Xi’an Jiaotong University, China, from April to October 2007.
MATERIALS: Theanine (Wuhan Remote Technology Co., Ltd., China), XRCC1, Ku70 and GAPDH primers (Shanghai Sangon Biological Engineering Technology & Services Co., Ltd., China) were used in this study.
METHODS: A total of 108 healthy, male Sprague Dawley rats were randomly divided into sham operation, ischemia/reperfusion and theanine groups. Each group was further divided into 6 sub-groups, with six rats in each, according to reperfusion time (2, 6, 12, 24, 48 and 72 hours). Rat models of global cerebral ischemia/reperfusion were established by the four-vessel occlusion method. Theanine (1 000 mg/kg) was injected into the caudal vein of rats in the theanine group immediately after surgery. Saline (4 mL/kg) was used in the sham operation and ische-mia/reperfusion groups. The same dose was administered every 24 hours.
MAIN OUTCOME MEASURES: Flow cytometry was employed to determine apoptosis of cerebral cortical neurons. Real-time reverse transcription-polymerase chain reaction was applied to quantify the expression of XRCC1 and Ku70 mRNA in the cerebral cortex.
RESULTS: The apoptotic rate in the ischemia/reperfusion group was significantly greater compared with the sham operation group at various time points (P < 0.05). The apoptotic rate in the theanine group was significantly diminished compared with the ischemia/reperfusion group, but significantly increased compared with the sham operation group at 6, 12, 24, 48 and 72 hours (P < 0.05). Ex-pression of XRCC1 and Ku70 mRNA was significantly decreased in the ischemia/reperfusion group compared with the sham operation group at 2, 6, 12, 24, 48 and 72 hours (P < 0.05). The relative expression of XRCC1 mRNA in the theanine group was significantly increased compared with the ischemia/reperfusion group, but significantly reduced compared with the sham operation group at 6, 12, 24, 48 and 72 hours (P < 0.05). The relative expression of Ku70 mRNA was significantly lower in the theanine group compared with the sham operation group at 2, 6 and 12 hours (P < 0.05) and significantly greater than the ischemia/reperfusion group at 6, 12, 24, 48 and 72 hours (P < 0.05).
CONCLUSION: The apoptotic rate in rats following global cerebral ischemia/reperfusion injury reaches a peak at 24 hours after reperfusion; however, the levels of DNA repair protein began to decrease after only 2 hours of reperfusion. Theanine antagonizes the decrease in XRCC1 and Ku70 mRNA expression in the rat cerebral cortex during global cerebral ischemia/reperfusion injury.
Key Words: brain; ischemia/reperfusion injury; XRCC1; Ku70; theanine

INTRODUCTION
   
The DNA repair protein XRCC1 potentially plays multiple roles in the discrimination and repair of single strand DNA breaks[1], with each role being extremely important in efficiently eliminating the breakage. Based on research over the last decade, the most prominent characteristic of XRCC1 is its ability to interact with other DNA repair proteins[2]. Although it has no enzyme activity, it is capable of interacting with other enzymes involved in each stage of the DNA repair pathway.
Ku70, a widely expressed nucleoprotein in cells, interacts with a variety of double-strand DNA fragments. It is only activated following binding with Ku80 to form a stable complex[3]. In non-homologous end joining, the association of Ku and the DNA-dependent protein kinase catalytic subunit forms DNA-dependent protein kinase[4]. Ku connects to either end of fragmented DNA to correctly locate and activate the DNA-dependent protein kinase catalytic subunit. Furthermore, Ku has weak helicase activity[5], which continues to unwind fragmented DNA. Subsequently, nuclease or polymerase enzymes trim the ends and Lig4/XRCC1 connects the terminal ends[6].
Theanine is an amide compound, with the chemical name N-acetyl-γ-L glutamine. It occurs abundantly in the tea tree plant. It is a non-protein free amino acid. Theanine can protect against cerebral infarction[7], inhibit glutamic acid excitotoxicity[8-9], decrease blood pressure, protect blood vessels, improve microcirculation and reduce neuronal apoptosis[10-12].
Many harmful factors of cerebral ischemia/reperfusion injury can aggravate DNA brittleness and DNA injury in neurons, directly causing neuronal death through necrosis or programmed cell death, eventually resulting in apoptosis. Accordingly, we aimed to determine the effects of theanine on DNA repair and apoptosis to determine its clinical value in the protection and treatment of cerebral ischemia.
In this study, we sought to analyze the effects of theanine on the relationship of the time-dependent expression of the DNA repair protein, XRCC1, and the DNA repair enzyme, Ku70, in the cerebral cortical apoptosis in the rat model of global cerebral ischemia/reperfusion injury. We also investigated the molecular biological mechanisms behind theanine neuroprotection.

MATERIALS AND METHODS

Design
A randomized controlled animal study.  
Time and setting
This experiment was performed at the Pharmacological Laboratory of Animal Experimental Center of Xi’an Jiaotong University, China, from April to October 2007.   
Materials
A total of 108 healthy, clean, male Sprague Dawley rats, aged 55–65 days, weighing (300 ± 20) g, were obtained from the Animal Experimental Center of Xi’an Jiaotong University (license No. SCXK (Shaan) 2007-001). All rats were bred under strict biological experimental conditions for 1 week. Preoperative examinations were performed to ensure they had no abnormalities. Rats were fasted overnight before surgery, with free access to water. All rats were randomized into sham operation, ischemia/reperfusion and theanine groups. Each group was divided into six subgroups (n = 6) according to reperfusion time (2, 6, 12, 24, 48 and 72 hours). Protocols were performed in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of the People’s Republic of China[13].
Main reagents and equipment used in this study are as follows: 

Methods
Establishment of rat models of global cerebral ischemia/reperfusion injury
Rat models of global cerebral ischemia/reperfusion injury were established by the four-vessel occlusion method[14]. Briefly, all rats were intraperitoneally administered with 10% chloral hydrate 30 mg/kg, fixed in the supine position, and a median incision was made at the first cervical vertebrae under the occipital bone. The muscle was separated, layer by layer, to expose the transverse foramen in the transverse process of the first cervical vertebrae, and a hot probe was placed into the transverse foramen to cauterize the bilateral vertebral arteries and permanently occlude them. Postoperatively, each rat was individually housed at 20–25 °C and continuously irradiated for 24 hours using a 60 W incandescent lamp. At day 2 following surgery, rats were anesthetized in the same way as before and fixed in the supine position. A median incision was made at the neck to separate the bilateral common carotid arteries which were subsequently occluded by bulldog clamps. Within 1 minute, the rats developed mydriasis, pale eyeballs, lip cyanosis and tachypnea. After 6 minutes, the bulldog clamps were loosened to restore cerebral blood flow. Any rat that did not develop the above-described symptoms or developed hemiplegia was eliminated. Rats in the sham operation group had their bilateral carotid and vertebral arteries exposed without cauterization or occlusion. Rats from each group were sacrificed at 2, 6, 12, 24, 48 and 72 hours following surgery.
Administration
The sham operation group was administered with saline (4 mL/kg); the ischemia/reperfusion group was administered saline (4 mL/kg); and the theanine group was administered 25% theanine solution (4 mL/kg) (i.e.,  1 000 mg/kg).This was done by injection through the caudal vein and repeated every 24 hours.
Detection of apoptosis utilizing flow cytometry
A total of six rats from each group were intraperitoneally anesthetized and sacrificed to obtain the brain. The pia mater and arachnoid mater were isolated and placed in a petri dish supplemented with 0–4 °C PBS on ice. The cerebral cortex (0.5 cm × 0.5 cm) between the optic chiasma and mamillary body was isolated and stored on ice in a centrifuge tube containing 0–4 °C PBS. Single cell suspensions were made within 30 minutes and the cell density was adjusted to 1×106/mL. A total of 100 μL of cell suspension was placed in a 5 mL test tube and mixed with 5 μL Annexin V/FITC with 10 μL propidium iodide (20 μL/mL). The mixture was incubated in the dark at room temperature for 15 minutes. 400 μL PBS was added to the Eppendorf reaction tube and analyzed by flow cytometry.
Time-course expression of XRCC1 and Ku70 by real-time PCR  
Samples were prepared under the same conditions as the samples collected for flow cytometry. They were placed in freezing tubes and preserved in a liquid nitrogen container at –196 °C. A total of 30–50 mg cerebral cortex of rats from each group was obtained for total RNA extraction using Trizol reagent. Reverse transcription was performed using Two-Step RT-PCR kit on PE9600 PCR apparatus to obtain template cDNA. In accordance with the gene sequence of XRCC1 (16758175), Ku70 (144445949), GAPDH (110347607) from GenBank (http://www.ncbi.nlm.nih.gov/Genbank/index.html), Beacon designer4.0 was used to design primers for real-time PCR as follows: 

A 25 μL reaction system was created for real-time PCR[14]. Using SYBR Green PCR MIX fluorescer, the reaction system was created prior to PCR was conducted on an iQ5 Realtime Fluorescence Quantitative PCR Detection System. The amplification conditions are as follows:   95 °C for 10 seconds, followed by 40 cycles of 95 °C for 5 seconds, 60 °C for 15 seconds and 72 °C for 10 seconds.
The phase of fluorescence intensity tested by PCR Detection System was established in the annealing stage of amplification. A temperature of 60 °C for 15 seconds was adopted as annealing phase. After 40 cycles of amplification, the system analyzed the fluorescence (DRn) of each reaction tube per circle and a kinetic curve of amplification for each reaction tube was graphed. Based on the kinetic curve, we defined the cycle threshold (Ct) value of the amplification cycle number for each sample tube when fluorescence intensity reached a threshold value. The Ct value was inversely proportional to the initial copy number of the template DNA. The initial data (Ct value) was converted into the relative expression value of each gene (2-ΔΔCt).
Main outcome measures
The following parameters were measured: apoptosis of cerebral cortical neurons and the expression of XRCC1 and Ku70 mRNA in the cerebral cortex.
Statistical analysis
Measurement data were expressed as Mean ± SD. All data were analyzed using SPSS 13.0 software packages (SPSS, Chicago, IL, USA). The difference among groups was compared using one-way analysis of variance and LSD-t test. A value of P < 0.05 was considered statistically significant.

RESULTS

Quantitative analysis of experimental animals
The success rate of model establishment and death rates were approximately 70% and 25%, respectively. Dead rats and unsuccessful models were replaced by new animals. A total of 108 Sprague Dawley rats were included in the final analysis.
Analysis of apoptosis
Results of flow cytometry indicated that the apoptotic rate in the sham operation group was very low. This was significantly increased in the ischemia/reperfusion group at 6 hours after reperfusion, reaching a peak at 24 hours. This increase was significantly different between the groups at various time points (P < 0.05). The apoptotic rate in the theanine group was significantly diminished compared with the ischemia/reperfusion group, but significantly increased compared with the sham operation group at 6, 12, 24, 48 and 72 hours (P < 0.05) (Table 1).  

Time-course expression of XRCC1 mRNA
Real-time PCR results demonstrated that the relative expression of XRCC1 mRNA was very high in the sham operation group. The relative expression of XRCC1 mRNA was significantly decreased in the ischemia/reperfusion group after 6 hours of reperfusion, and remained low up to 72 hours. The relative expression of XRCC1 mRNA in the ischemia/reperfusion group was significantly decreased compared with the sham operation group at various time points (P < 0.05). The relative expression of XRCC1 mRNA in the theanine group was significantly increased compared with the ischemia/reperfusion group, but significantly diminished compared with the sham operation group at 6, 12, 24, 48 and 72 hours (P < 0.05) (Table 2, Figures 1, 2).
Time-course expression of Ku70 mRNA
Real-time PCR demonstrated that the relative expres-sion of Ku70 mRNA was very high in the sham opera-tion group. The relative expression of Ku70 mRNA in the ischemia/reperfusion group started to decrease at 2 hours after reperfusion, reaching a minimum at 6 hours, and gradually increased up to 72 hours. The relative expression of Ku70 mRNA in the ischemia/reperfusion group was significantly decreased compared with the sham operation group at various time points (P < 0.05). The relative expression of Ku70 mRNA was signifi-cantly decreased in the theanine group compared with the sham operation group at 2, 6 and 12 hours (P < 0.05). The relative expression of Ku70 mRNA was sig-nificantly higher in the theanine group compared with the ischemia/ reperfusion group at 6, 12, 24, 48 and 72 hours (P < 0.05) (Table 3, Figures 3, 4).

DISCUSSION

Results from this study indicate that XRCC1 is normally expressed in the cerebral cortex of normal adult Sprague Dawley rats. XRCC1 expression in the cerebral cortex began to decrease 2 hours after global cerebral ischemia/reperfusion, and remained low up to 72 hours. Combined with results from flow cytometry, we determined that XRCC1 expression and the apoptotic rate show a negative correlation. Furthermore, the decrease in XRCC1 expression was prior to the occurrence of apoptosis. This is identical to the results of a previous study[14] that tested XRCC1 expression in the rat hippocampus after global cerebral ischemia/reperfusion using immunohistochemical method. At 6 hours after cerebral ischemia/reperfusion and intravenous injection of theanine into the caudal vein, XRCC1 expression in the ischemic cortex was greater in the theanine group compared with the ischemia/reperfusion group and this expression remained significantly different up to 72 hours. Results from this study reveal that Ku70 expression in the cerebral cortex of rats is decreased at 2 hours after ischemia/reperfusion, reaching a minimum at 6 hours. Moreover, neuronal apoptosis is increased at 2 hours after ischemia/reperfusion, reaching a peak at 24 hours, and showing subsequent gradual reduction. This suggests that the decrease in Ku70 protein expression in the injured region, occurring in the early stage of ischemia/reperfusion, persistently exists, which is in agreement with results from a previous study[15]. At 6 hours after cerebral ischemia/reperfusion and intravenous injection of theanine via the caudal vein, Ku70 expression in the ischemic cortex was significantly greater in the theanine group compared with the ischemia/reperfusion group, and remained up to 72 hours after reperfusion. This suggests that the ability to repair DNA double-strand break was higher in the theanine group compared with the ischemia/reperfusion group.
Recently, many researchers have investigated the cerebral protective activity of theanine[16-18]. In 1995, Nozawa et al[19] discovered that theanine could inhibit glutamic acid-induced death of rat neurons cultured in vitro. Kakuda et al[20] showed that theanine pretreatment produces noticeable protective effects on delayed neuronal death in the hippocampal CA1 region of gerbils with temporal telencephalic ischemia. Wang et al[21] confirmed that pre- and post-treatment with theanine significantly increases the expression of neuron specific enolase in the brain of acute cerebral ischemia rabbits. Furthermore, theanine can traverse the blood-brain barrier and protect the ischemic brain by inhibiting the release of the excitatory amino acid[22-23]. Through the use of global cerebral ischemia models, we have found that the apoptotic rate in the cerebral cortex is significantly lower in the theanine group compared with the ischemia/reperfusion group at 6 hours after ischemia/reperfusion and remains decreased up to 72 hours. The difference is not significant 2 hours after ischemia/reperfusion; which may correlate with the pharmacokinetics of theanine.
In this experiment, the expression of XRCC1 and Ku70 began to decline 2 hours following global cerebral ischemia/reperfusion, with a subsequent gradual increase in the apoptotic rate, indicating a loss of DNA-repair. Although the apoptotic rate reached the maximum 24 hours after ischemia/reperfusion, the DNA repair protein began to decrease during the early stages of reperfusion (2 hours). Thus, if we can inhibit or delay the decrease in XRCC1 and Ku70, it may be possible to reduce the extent of neuronal damage.
Our results confirm that during global cerebral ischemia/reperfusion, theanine can upregulate the expression of XRCC1 and Ku70 in the cerebral cortex, thereby reducing neuronal apoptosis. Overall, theanine inhibited the decrease in the DNA repair protein XRCC1 and Ku70, strengthened the ability to repair DNA single-/double-strand breaks and inhibited apoptosis.

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 (Edited by Liu HJ/Qiu Y/Song LP)