Expression of CD137 in the cerebral artery after experimental subarachnoid hemorrhage in rats: A pilot study

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Expression of CD137 in the cerebral artery after experimental subarachnoid hemorrhage in rats: A pilot study Jian Zhang, Gang Chen, Dai Zhou, Zhong Wang⁎ Department of Neurosurgery, The First Affiliated Hospital of Soochow University, 188 Shizi Street, Suzhou 215006, Jiangsu Province, PR China

A R T I C LE I N FO

AB S T R A C T

Article history:

Inflammation and immunity play a crucial role in the pathogenesis of cerebral vasospasm after

Accepted 15 February 2011

subarachnoid hemorrhage (SAH). CD137 is recognized as an independent costimulatory

Available online 23 February 2011

molecule of T cells and activator of monocytes. A growing body of evidence indicates that CD137 is vital for inflammation and immunity. Therefore, this study aimed to investigate the

Keywords:

expression of CD137 in the basilar artery in a rat SAH model and to clarify the potential role of

CD137

CD137 in cerebral vasospasm. A total of 107 rats were randomly divided into four groups:

Inflammation

control group; day 3, day 5, and day 7 groups. Day 3, day 5, and day 7 groups were all SAH groups.

Vasospasm

The animals in SAH groups were subjected to injection of autologous blood into cisterna magna

Subarachnoid hemorrhage

twice on day 0 and day 2 and were sacrificed on days 3, 5, and 7, respectively. Cross-sectional

Rat

area of basilar artery was measured and the CD137 expression was assessed by quantitative real-time PCR, Western blot and immunohistochemistry. The cross-sectional area of basilar artery was found to be 57,944± 5581 μm2 in control group, 26,100 ± 2639 μm2 in day 3, 19,723 ± 2412 μm2 in day 5, and 28,800 ± 2980 μm2 in day 7 group, respectively. The basilar artery exhibited vasospasm after SAH and became more severe on day 5. The elevated mRNA and protein of CD137 were detected after SAH and peaked on day 5. CD137 is increasingly expressed in a parallel time course to the development of cerebral vasospasm in a rat experimental model of SAH. These findings indicate the possible role of CD137 in the pathogenesis of cerebral vasospasm after SAH. © 2011 Elsevier B.V. All rights reserved.

1.

Introduction

Cerebral vasospasm is a common and potentially incapacitating complication of subarachnoid hemorrhage (SAH). It has been demonstrated to be a significant predictor of adverse outcome and the leading potentially treatable cause of death and disability in patients who experience aneurysmal SAH (Kassell et al., 1985, 1990). Despite its clinical significance and the extensive research efforts placed into elucidating its pathogenesis and therapy, vasospasm remains as an incompletely

understood and important clinical problem. However, increasing evidence indicates that inflammation and immunity accompanying SAH may contribute to the development and maintenance of cerebral vasospasm (Dumont et al., 2003; Chaichana et al., 2009). Some pathological changes associated with inflammation and immunity, such as the leukocyte recruitment, infiltration and activation, have been detected in the arterial wall in many studies of cerebral vasospasm (Findlay et al., 1989; Peterson et al., 1990a, 1990b; Handa et al., 1991). Inflammation and, more

⁎ Corresponding author. Fax: +86 512 65462663. E-mail address: [email protected] (Z. Wang). 0006-8993/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2011.02.049

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specifically, leukocyte–endothelial cell interactions appear to play a critical role in vasospasm (Gallia and Tamargo, 2006). Recent studies aimed at dissecting the cellular and molecular basis of the inflammatory response accompanying SAH and cerebral vasospasm have provided a promising groundwork for future studies. CD137, a member of the tumor necrosis factor receptor superfamily, is recognized as an independent costimulatory molecule of T cells and activator of monocytes (Langstein et al., 1998; Vinay and Kwon, 1999). It was isolated from activated T lymphocytes in mice and humans (Kwon and Weissman, 1989; Schwarz et al., 1993). CD137 is now recognized as being expressed far more broadly than first recognized (Wang et al., 2009). A previous study indicated that CD137 is expressed on blood vessel walls at sites of inflammation (Drenkard et al., 2007). Accumulating evidences have shown an important role for CD137 signaling in autoimmune disease (Sun et al., 2002a, 2002b; Foell et al., 2004; Vinay et al., 2004; Polte et al., 2006). As

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yet, however, there was no study to explore the expression and role of CD137 in the cerebral vasospasm after SAH. Therefore, the purpose of this study was to investigate the time course of CD137 expression in the arterial wall after SAH and to clarify the possible role for CD137 during cerebral vasospasm.

2.

Results

2.1.

General observation and mortality

There were no significant differences among the groups in terms of body weight, arterial pH, PO2, PCO2, or mean arterial blood pressure (P > 0.05, data not shown). Mortality rate of rat in control group was 0 (0/24), it was 11.11% (3/27 rats) in day 3 group, 14.29% (4/28) in day 5 group, and 14.29% (4/28) in day 7 group.

Fig. 1 – Representative cross-sections of basilar arteries of the different groups (scale bar, 100 μm). A indicates the control group; B, day 3 group; C, day 5 group; D, day 7 group. Severe vasospasm and thicken media were observed in the day 5 (C) group, and moderate in the day 3 (B) and day 7 (D) groups. Bottom: bar diagram representing the average cross-sectional area of the basilar arteries from the different groups. There is a significant difference in the basilar artery cross-sectional area between the SAH groups and control group (E). Bars represent the mean ± SD (n = 6, each group). *P < 0.05 vs. control group.

202 2.2.

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Histopathological study

No vasospasm was observed in the control group. Severe morphological vasospasm was observed in the basilar artery in the day 5 group, characterized by a corrugated internal elastic lamina, a thicken vessel wall, and contracted smooth muscle cell. Moderate vasospasm could be observed in the basilar arteries in both the day 3 and 7 group (Fig. 1).

2.3.

The basilar artery cross-sectional area

As shown in Fig. 1E, morphometric analysis of the vessels revealed that the mean cross-sectional area of the basilar artery in the day 5 group (19,723 ± 2,412 μm2, mean ± SD) showed extremely significant (P < 0.05) reduction compared to the control group (57,944 ± 5581 μm2). A moderate reduction was detected in the day 3 group (26,100 ± 2639 μm2) as compared to the normal control value (P < 0.05). And so it is between the day 7 group (28,800 ± 2980 μm2) and the control group (P < 0.05).

2.4.

Immunohistochemistry for CD137 expression

Immunohistochemistry study, which was performed to ascertain the immunoreactivity of CD137, revealed that, in the control group, no or weak expression of CD137 was observed in the basilar artery. Increased CD137-positive cells in the SAH groups could be found in the basilar arteries (Fig. 2). Semiquantitative analysis showed the low CD137 immunoreactivity in the control group with the average score of 0.50. While in the day 3, day 5 and day 7 groups, it was 2.67, 3.50 and 1.83, respectively (Fig. 2E). Compared to the control group, the level of CD137 immunoreactivity was significantly up-regulated in the endothelial cells and smooth muscle cells in all SAH groups (P < 0.05).

2.5.

Expression mRNA levels of CD137 after SAH

Expression patterns of CD137 mRNA as determined by realtime PCR were summarized in Fig. 3. Very low level of CD137

Fig. 2 – Immunohistochemical staining for CD137 on cross sections of basilar arteries (scale bar, 25 μm). Few CD137 positive cells were observed in the control group (A). Increased CD137 positive cells in the SAH groups were present mainly in the endothelial cells and smooth muscle cells on days 3, 5 and 7 (B–D, respectively). Bottom: bar diagram representing the semi-quantitative analysis CD137 expression of the basilar arteries from all groups. Significant differences were found between the SAH groups and control group (E). Bars represent the mean ± SD (n = 6, each group). *P < 0.05 vs. control group.

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mRNA was found in control artery, and significantly increased mRNA expression of CD137 in the artery was detected in the SAH groups as compared with that of the control group (P < 0.05).

2.6.

Western blot analysis for CD137 expression

As shown in Fig. 4, CD137 protein expressed at a low level in the control group. While in the SAH groups, the level of CD137 was significantly increased. There was a statistically significant difference between control group and each SAH group (P < 0.05).

3.

Discussion

Delayed or chronic vasospasm is the leading cause of morbidity and mortality after aneurysmal SAH (Solenski et al., 1995; Suarez et al., 2006). Although cerebral vasospasm after SAH has been the subject of substantial research interest, the underlying pathogenic mechanisms remain obscure. In the present study we have demonstrated, for the first time, that CD137 was activated in the basilar arterial wall during cerebral vasospasm after experimental SAH in rats. The enhanced expression of CD137 could be detected on day 3, peaked on day 5, and recovered on day 7. This will lead to the hypothesis that upregulation of CD137 in the cerebral arterial wall may be important in the development of cerebral vasospasm after SAH. A growing body of evidence supports the critical role of inflammation in the development and maintenance of cerebral vasospasm. Earlier studies suggest that various constituents of the inflammatory response, including adhesion molecules, cytokines, leukocytes, immunoglobulins, and complement, may be critical in the pathogenesis of cerebral vasospasm (Dumont et al., 2003; Chaichana et al., 2009). Several drugs aimed at limiting the inflammatory response associated with cerebral vasospasm after SAH have been used with varying levels of success, including nonsteroidal antiinflammatory agents, cyclosporine A, FK-506, methylprednisolone and other anti-inflammatory drugs (Chyatte, 1989; Peterson et al., 1990a, 1990b; Nagata et al., 1993; Nishizawa et al., 1993; Thai et al., 1999). Furthermore, some other therapies

Fig. 3 – The mRNA expression of CD137 in the basilar arteries in all groups. SAH could induce a marked increase of CD137 mRNA expression in the rat basilar arteries. Bars represent the mean ± SD (n = 6, each group). *P < 0.05 vs. control group.

Fig. 4 – (A) Representative autoradiogram of CD137 expression in basilar arteries. We observed CD137 at 32 kDa and a loading control GAPDH at 37 kDa. It shows that the expression of CD137 protein increased in the SAH groups and peaked approximately on day 5. (B) Quantitative analysis of the Western blot results for CD137. It shows that CD137 levels in SAH groups are significantly higher than that in control group. Bars represent the mean ± SD (n = 6, each group). *P < 0.05 vs. control group.

targeting the inflammatory cascade also showed some efficacy in the animal models (Bavbek et al., 1998; Ono et al., 1998; Sasaki et al., 2004; Lin et al., 2005; Yatsushige et al., 2005). However, the outcomes of these studies have been equivocal (Nishizawa et al., 1993; Manno et al., 1997; Hop et al., 2000). Currently, standard practice for SAH does not include anti-inflammatory therapy. Partial efficacy of anti-inflammatory therapies thus far may be explained, at least in part, by the fact that the precise mechanism of inflammatory response accompanying SAH has not been entirely elucidated (Crowley et al., 2008). Further understanding of this inflammation-based disease is required to formulate effective treatment strategies for preventing or reversing the development of this devastating condition. As is known to all, the tumor necrosis factor receptor (TNFR)/TNF superfamily represents a key group of receptor/ ligands for controlling life and death in the immune system (Locksley et al., 2001). Among TNFR superfamily members, CD137 (also known as 4-1BB and ILA) is recognized as an independent costimulatory molecule of T cells (Pollok et al., 1993; Vinay and Kwon, 1999). However, some studies have indicated that CD137 is now recognized as being expressed far more broadly than first recognized, including expression on regulatory T cells, follicular dendritic cells, dendritic cells, differentiating myeloid-lineage cells, monocytes, immunoglobulin E-stimulated mast cells, eosinophils, neutrophils, activated natural killer T cells, and activated NK cells (Langstein et al., 1998; Heinisch et al., 2001; Gavin et al., 2002; Pauly et al., 2002; Wilcox et al., 2002a, 2002b; Vinay et al., 2004; Lee et al., 2005; Nishimoto et al., 2005; Lee et al., 2008). Interestingly, recent studies have identified expression of CD137 on nonhematopoietic cells under disease conditions. These include endothelial cells, smooth muscle cells, and cardiac myocytes (Seko et al., 2001; Drenkard et al., 2007; Olofsson et al., 2008; Saiki et al., 2008). CD137 ligand (CD137L; also known as 4-1BBL), a member of TNF superfamily, is

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constitutively expressed on professional antigen-presenting cells (dendritic cells, monocyte/macrophages, and B cells) and upregulated by their respective activation stimuli (Kwon et al., 2000, 2002; Croft, 2009). Like CD137, CD137L can be expressed in cardiovascular components such as endothelial cells, smooth muscle cells, and cardiomyocytes (Langstein et al., 1998; Seko et al., 2001; Drenkard et al., 2007; Saiki et al., 2008). Such expression pattern implies the possibility that CD137/ CD137L interaction may be involved in multiple steps in varied innate and immune responses. It is generally accepted that blood vessel walls are sites of essential biological processes, such as blood coagulation and leukocyte extravasation. Endothelial cells, located between circulating blood and vessel wall, play an essential role in the vascular inflammation and immunity (Luscher and Barton, 1997; Biedermann, 2001). What is more, a few studies demonstrated that vascular smooth muscle displays elements of an innate immune response when stimulated both in vitro and in vivo (Sasaki et al., 2004; Jimenez et al., 2005). It is conceivable that vascular smooth muscle is an important site within the vessel for pathogen activation. As mentioned above, CD137 is known as a costimulator of T cells, where it activates nuclear factor-κB and promotes proliferation and cytokine production (Watts, 2005). Furthermore, binding of CD137L to CD137 can activate B cells and monocytes through bidirectional signaling (Langstein et al., 1998, 1999, 2000; Pauly et al., 2002). Therefore, it is tempting to presume that CD137 expression could activate adherent immune cells, which are recruited to the blood vessel walls through an activated endothelium, and increase their capacity for diapedesis and cytokine production, ultimately adding to the inflammatory milieu in the vascular wall which could promote local inflammation. In fact, recent studies have indicated that the inflammation and immunity could be triggered and amplified by up-regulation of the CD137 in the endothelial cells and vascular smooth muscle cells, and reverse signaling through CD137L enhances monocyte migratory activity (Drenkard et al., 2007; Olofsson et al., 2008). In our present study we showed that the CD137 expression in the endothelial cells and the medial layer of the smooth muscle cells of basilar artery after SAH was significantly elevated. Thus, we could speculate that the up-regulated expression of CD137 in the cerebral artery might contribute to the development of inflammation in the cerebral arteries after SAH and the signal pathway involved might play a putative role in the pathogenesis of cerebral vasospasm. Considering that CD137/CD137L interactions are important in the induction of inflammation, blocking of CD137/CD137L should inhibit the progression of inflammatory diseases. Recently, blockade of CD137/CD137L interaction with monoclonal antibody has been administrated to inhibit the immune or chronic inflammatory response in vivo. A study by Sun et al. demonstrated that treatment with an agonistic monoclonal antibody to CD137 blocks lymphadenopathy and spontaneous autoimmune diseases in Fas-deficient MRL/lpr mice, ultimately leading to their prolonged survival (Sun et al., 2002a, 2002b). Using a murine asthma model, Polte et al. demonstrated for the first time that the capacity of anti-CD137 monoclonal antibody to ameliorate allergic asthma (Polte et al., 2006). Saiki et al. blocked the CD137 pathway with CD137Ig every 7 days for

8 weeks significantly attenuates graft arterial disease in cardiac allografts (Saiki et al., 2008). Furthermore, a previous study used a murine model of herpetic stromal keratitis indicated that blocking CD137/ CD137L interactions by introducing monoclonal antibody against CD137L prevents herpetic stromal keratitis (Seo et al., 2003). Indeed, there are many studies, aiming at blocking of CD137/CD137L interaction, had achieved inspiring outcome in suppression the progression of inflammatory diseases (Foell et al., 2003; Wang et al., 2003; Foell et al., 2004; Vinay et al., 2004; Haga et al., 2009). However, it is not well known whether the blocking effects are due to the absence of signaling through CD137 or CD137L. It still needs further studies. In summary, our observations suggest that enhanced expression of CD137 in the cerebral arteries in the experimental SAH model may contribute to the progression of cerebral vasospasm, while the exact role of CD137 in the cerebral vasospasm calls for further study. It may be possible that blocking of CD137/ CD137L interaction may attenuate the development of cerebral vasospasm.

4.

Experimental procedures

4.1.

Animals and experiment design

All experiments were performed according to the protocol evaluated and approved by the Animal Care and Use Committees of Soochow University. Male Sprague–Dawley rats (300 to 350 g) were assigned randomly to 4 groups: intracisternal saline injection was performed in the animals of control group (n = 24). The animals in day 3, day 5, and day 7 groups were subjected to experimental SAH twice on day 0 and 2 and were killed on days 3, 5, and 7, respectively (n = 24 for each group). The animals in control group were sacrificed one day after all the procedures. Six rats in each group were sacrificed with the fixation–perfusion method. The basilar arteries were taken for hematoxylin and eosin (H&E) and immunohistochemical staining. Another six rats were used for RNA isolation and the other twelve rats for protein isolation.

4.2.

Rat double-hemorrhage model

The double-hemorrhage model was induced by a posterior craniocervical approach (Delgado et al., 1985). Procedures were performed as previously described (Meguro et al., 2001). Briefly, the animal was anesthetized by intraperitoneal injection of pentobarbital (50 mg/kg) on day 0 and was allowed to breathe spontaneously and body temperature was approximately maintained at 37 °C using a heading pad. A small suboccipital incision was made using microsurgical dissection, exposing the arch of the atlas, the occipital bone, and the atlantooccipital membrane. The cisterna magna was tapped using a 27-gauge needle, and 0.3 ml of cerebral spinal fluid was gently aspirated. Freshly drawn autologous nonheparinized blood (0.3 ml) from the tail artery was then injected aseptically into the cisterna magna over a period of 2 min. Immediately after the injection of blood, the hole was sealed with glue to prevent fistula and then the incision was sutured. To permit blood distribution around the basal arteries, the rats were then placed in a head-down prone position at a 30-degree angle for

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30 min. Forty-eight hours after the first injection (day 2), a second one was performed in the same manner. To the animals in the control group the same technique was applied, but with injection of sterile saline instead of blood. Twenty milliliters of 0.9% NaCl was injected subcutaneously right after the operation to prevent dehydration. After operation procedures, the rats were then returned to their cages and the room temperature kept at 23± 1 °C. All animals were housed in a light–dark cycle environment with free access to food and water.

4.3.

Perfusion–fixation

On days 3, 5, and 7, the rats (n = 6) scheduled for death were reanesthetized deeply with pentobarbital (100 mg/kg, IP). By means of transthoracic cannulation of the left ventricle, they were perfused with 300 ml of 0.1 mol/L phosphate-buffered saline solution, pH 7.4, and then reperfused with 300 ml of 4% paraformaldehyde in 0.1 mol/L phosphate-buffered saline under a pressure of 100 cm H2O. After perfusion–fixation, the whole brain with the basilar artery was immediately removed and immersed in the same fixative solution for 24 h. Animals in the control group were killed using the same procedure.

4.4.

Measurement of basilar artery cross-sectional area

The entire length of basilar artery was divided into 3 parts: proximal, middle, and distal thirds. At each midpoint of these parts, about 3 mm artery tissue with brain stems was dissected out and embedded in paraffin. All 5-μm-thick sections were cut and mounted on glass slides for measurement of blood vessel cross-sectional area and immunohistochemical examination. To assess the degree of chronic vasospasm, 5-μm-thick sections were deparaffinized, hydrated, washed and stained with H&E. In the SAH animals, the basilar artery usually appeared in an oval or other irregular shape. To correct for vessel deformation and off-transverse sectioning, cross-sectional area of each basilar artery was determined by measuring the circumference of the vessel lumen and calculating the area as a generalized circle based on the measured circumference (area= p2/4π, where p = perimeter). The cross-sectional areas were determined by using the National Institutes of Health image analyzer ImageJ (version 1.32).

4.5.

Immunohistochemical examination

To assess the localization of CD137 expression, immunohistochemistry for CD137 was performed. Briefly, sections were deparaffinized and rehydrated in graded concentrations of ethanol to distilled water. Sections were placed in 10 mmol/l citrate buffer (pH 6.0), and heated in microwave oven at 95 °C for 30 min. Primary antibody (anti-CD137, diluted 1:200,

Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) was incubated for 1 h at room temperature, followed by a 15-min wash in PBS. Then, the sections were incubated with horseradish peroxidase (HRP)-conjugated IgG (diluted 1:500, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) for 60 min at room temperature. DAB–H2O2 solution was used to visualize CD137. Finally, sections were counterstained with hematoxylin, and observed under a light microscope. For the negative control, the same procedure was performed with the exception that the primary antibody was omitted. The intensity of CD137 labeling (5 grades) on each layer of the vessel wall was graded semiquantitatively as follows: “0” indicates that there were no detectable positive cells; “1” indicates very low density of positive cells; “2” indicates a moderate density of positive cells; “3” indicates the higher, but not maximal density of positive cells; and “4” indicates the highest density of positive cells.

4.6.

Harvest of cerebral arteries

The rats for RNA and protein isolation from the various groups were anesthetized and then decapitated. The brains were quickly removed and chilled in ice-cold bicarbonate buffer solution. With the aid of a microscope, the basilar arteries and circle of Willis arteries were carefully dissected free from each brain, cleared of connective tissue, and immediately snap frozen in liquid nitrogen until use.

4.7.

RNA isolation and quantitative real-time PCR

Total RNAs were isolated from sample cerebral arteries from each group (n = 6) with the use of TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) as per manufacturer's instructions. The concentration and quality of the RNA in each sample were determined by gel visualization and spectrophotometric analysis (OD260/280). The quantity of RNA was measured using the OD260. The isolated RNA was stored at −80 °C until analyzed. RNA was reversely transcribed to cDNA using M-MLV Reverse Transcriptase (Promega, Madison, WI, USA) and oligo (dT)15 primers. The primers were synthesized by Sangon Biotech (Shanghai, China) and the sequences used were from a database at NCBI for rat CD137 and β-actin as shown in Table 1 Quantitative real-time PCR analysis was performed by using the ABI PRISM 7300 real-time PCR system (Applied Biosystems, Foster City ,CA, USA), applying real-time SYBR Green PCR technology. The reaction mixtures contained 2 μl cDNA, 0.4 μl ROX Reference Dye and 10 μl SYBR Green I (Takara Bio Inc, Shiga, Japan), 1 μl of each forward and reverse primer (1 μM) and nuclease-free water to a final volume of 20 μl. After denaturation at 95 °C for 30 s, 40 PCR cycles were performed, each consisting of a denaturation step (95 °C, 5 s)

Table 1 – PCR primer sequences. Target gene CD 137 β-actin

Sense primer (5′ to 3′)

Antisense primer (5′ to 3′)

5′-AAGCCrrGCTCCTCTACCCA-3′ 5′-CGHCCCAAGCCACAGTH-3′ 5′-CAGGTCATCA CTATCGGCAA T- 3′ 5′-GAGGTCITIACGGATGTCAAC -3′

Annealing temperature Number of Size (bp) (°C) cycles 62 62

40 40

147 144

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and an annealing step (62 °C, 34 s). Total RNA concentrations from each sample were normalized by the quantity of β-actin mRNA, and the expression levels of target genes were evaluated by the ratio of the number of target mRNA to β-actin mRNA. All samples were analyzed in triplicate.

4.8.

Western blot analysis

Cerebrovascular protein lysates from the different groups were compared. Cerebral arteries from 2 animals were pooled for each group of experiment. The frozen arteries from each group were homogenized at 4 °C with ultrasonic waves in lysis buffer containing 8 M urea, 4% CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-1 propanesulfonate), 40 mM Tris base and 65 mM DTT (dithiothreitol). Lysates were centrifuged at 25,000g for 60 min at 4 °C. The protein concentration of the supernatants was determined by the Bradford method using the Nanjing Jiancheng (NJJC) protein assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Equal amounts (30 μg per lane) of protein were loaded in each lane of SDS-PAGE, electrophoresed, and transferred to a nitrocellulose membrane. The membrane was blocked with 3% nonfat dry milk for 1 h at room temperature and subsequently incubated with primary antibody against rat CD137 (diluted 1:200, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) overnight at 4 °C followed by 3 × 5 min wash with Tween-TBS (TBST). After incubation with the primary antibody, the nitrocellulose membranes were washed with TBST and incubated with the appropriate horseradish peroxidase-labeled secondary antibodies for 1 h at room temperature followed by 5 × 5 min wash with TBST. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (diluted in1:6000, Sigma-Aldrich, Inc., St. Louis, MO, USA) was used as a loading control. An enhanced chemiluminescence system (Amersham, Arlington Heights, IL, USA) was used to visualize the protein bands and were exposed to X-ray film. The optical density of the resulting bands was determined by standard scanning densitometry, with normalization of densitometry measures to GAPDH.

4.9.

Statistical analysis

All data were presented as mean ± SD. SAS 8.0 was used for statistical analysis of the data. The measurements were subjected to one-way ANOVA, followed by the Dunnett test. A value of P < 0.05 was considered statistically significant.

Acknowledgments This work was supported by the 135 grant from Health Department of Jiangsu Province, China.

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