TLR4-mediated MyD88-dependent signaling pathway is activated by cerebral ischemia–reperfusion in cortex in mice

Available online at

www.sciencedirect.com Biomedicine & Pharmacotherapy 63 (2009) 442e450

Original article

TLR4-mediated MyD88-dependent signaling pathway is activated by cerebral ischemiaereperfusion in cortex in mice Yin Gao a,b, Xiubin Fang a,*, Yue Tong a, Yuli Liu a, Baohui Zhang a a

Department of Neurobiology, Basic Medical College, China Medical University, Shenyang 110001, China b Department of anatomy, Qiqihar Medical University, Qiqihar 161042, China Received 2 June 2008; accepted 12 June 2008 Available online 11 July 2008

Abstract To study whether the signaling pathway is activated in the inflammatory reaction of cerebral ischemiaereperfusion and its mechanism. The mice were randomly divided into sham group, ischemiaereperfusion group and TLR4-blocked group with different time points of reperfusion 12 h, 24 h, 48 h and 72 h group. We observed the different expression of TLR4 mRNA and MyD88 mRNA, activation of NF-kB and the TNFa and IL-1b protein levels in each group at different time point after ischemiaereperfusion. Mice cerebral ischemia was induced by occlusion of common carotid arteries (CCA) bilaterally. TLR4 signaling pathway could be inhibited by specific anti-TLR4 binding protein to prevent TLR4 from interacting with its receptors. We determined the result of TLR4 antibodies-blocking and mice cerebral ischemiaereperfusion injuries by Western blot, and evaluated neuronal damage in cortex. We also determined the expression of TLR4 mRNA and MyD88 mRNA by in situ hybridization (ISH), the activation of NF-kB by EMSA, and the expression of TNF-a protein by Western blot. Anti-TLR4 binding TLR4 receptors before reperfusion was effective; There was distinct difference among each group respecting neuronal damage; The expression of TLR4 mRNA and MyD88 mRNA, the activation of NF-kB, and the expression of TNF-a protein showed clear difference as well. LR4-mediated MyD88dependent signaling pathway activated by ischemiaereperfusion may be involved in the mechanism of ischemiaereperfusion through upregulation of NF-kB and TNF-a . Ó 2008 Published by Elsevier Masson SAS. Keywords: TLR4; Ischemia; Reperfusion; Antibodies-blocking

1. Introduction The membrane-bound toll-like receptor 4 (TLR4)-mediated MyD88-dependent signaling pathway play an essential role in the detection and elimination of invading microbes [1]. TLR4 is a transmembrane receptor with a cytoplasmic Toll/interleukin-1 receptor (TIR) homology domain [2]. The TIR domain can interact with the adapter protein myeloid differentiation factor88 (MyD88) [3]. Upon stimulation, TLR4 triggers the signaling pathway, which culminates in the activation of the transcription factor NF-kB (nuclear factor-kB), and results in expression of genes involved in innate and inflammatory responses including the production of pro-inflammatory * Corresponding author. Tel./fax: þ86 24 23252582. E-mail address: [email protected] (X. Fang). 0753-3322/$ - see front matter Ó 2008 Published by Elsevier Masson SAS. doi:10.1016/j.biopha.2008.06.028

cytokines such as TNF-a [4,5]. Studies have shown that innate immune and inflammatory responses play a critical role in cerebral ischemiaereperfusion injury [6]. Recent evidence suggests that TLR4-mediated NF-kB signaling contributes to myocardial ischemiaereperfusion injury [7,8]. Considering the role of TLR4-mediated MyD88-dependent signaling pathway, we infer that TLR4 signaling pathway may be involved in the damage of cerebral ischemiaereperfusion. Little is known about this aspect. To investigate whether TLR4 signaling pathway is activated by ischemiaereperfusion, we examined the expression of TLR4 mRNA and MyD88 mRNA, the activation of NF-kB and the expression of TNF-a and IL-1b protein levels in the cortex in the present study. We used the TLR4blocked model in mice. TLR4 signaling pathway can be inhibited using specific TLR4 antibodies (anti-TLR4) binding protein to prevent TLR4 from interacting with its receptors

Y. Gao et al. / Biomedicine & Pharmacotherapy 63 (2009) 442e450

[9]. The results of blocked TLR4 was evaluated by Western blot analysis for TLR4 protein levels. 2. Materials and methods 2.1. Chemicals and reagents The following primary antibodies were used: rabbit polyclonal anti-TNF-a and monoclonal anti-TLR4 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). EMSA kit and horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG were also obtained from Santa Cruz Biotechnology. ISH kit was purchased from Nippon Gene Biotechnology (Nippon Gene, Toyama, Japan). All other reagents were purchased from Sigma unless mentioned. 2.2. Experimental animals

443

Each group mice (n ¼ 6) were sacrificed by pentobarbital sodium overdose at 12 h, 24 h, 48 h and 72 h, respectively. The brains were removed and stored at 80 C for isolation of cellular proteins. Nuclear extracts of cortex tissues were used for analysis of NF-kB activation by electrophoretic mobility shift assay (EMSA). Cellular proteins of the right cortex tissues were used for analysis of TNF-a protein levels and the result of the TLR4-blocked by Western blot. At 12 h, 24 h, 48 h and 72 h reperfusion, each group mice (n ¼ 6) were anesthetized with intraperitoneal of 2% pentobarbital sodium and transcardially perfused with saline followed by of 4% paraformaldehyde in PBS (0.1 mol/L, pH 7.4) and post-fixed [7]. The brains were removed, embedded in paraffin and cut into sections (7 mm). The right cortex formation slides were detected in situ hybridization for analysis of TLR4 mRNA and MyD88 mRNA levels and evaluation of neuronal damage in the right cortex by hematoxylineosin (HeE) staining.

Healthy Kunming mice (8 weeks old, weight 18e22 g) were supplied by the Experiment Animal Center of China Medical University. All mice which expressed normal functional TLR4 were bred in our laboratory, and kept in-house under pathogen-free condition. The animal procedures were approved by the Animal Care and Use Committee of the China Medical University (SYSC 2003-009). The mice in the present study were randomly divided into three groups: sham (S group, n ¼ 72), ischemiaereperfusion (I group, n ¼ 72) and TLR4blocked (T group, n ¼ 72) with different time points of reperfusion 12 h, 24 h, 48 h and 72 h group (n ¼ 18/group).

According to the stereotaxic mouse brain atlas, we took out the right cortex tissues. The 0e4 neuropathological score (NPS) was used as previously described [11]: score 0, no neurologic impairment; score 1, scattered injured neurons in cortex; score 2, moderate damage in cortex (less than half of cortex cells affected); score 3, severe damage to cortex (more than half of the cortex cells affected); and score 4, extensive cell damage in cortex. The sections were stained with hematoxylineosin (HeE), and detected by light microscope.

2.3. Animal surgical procedures

2.5. TLR4 antibodies-blocking

Animal surgical procedures were performed according to the strict sterility standard. Animals were anesthetized with intraperitoneal of 2% pentobarbital sodium (40 mg/kg) (Forene, Abbott GmbH, Wiesbaden, Germany). The cerebral ischemiaereperfusion was induced with previously described method [10]. Briefly, cerebral ischemia was induced by occlusion of common carotid arteries (CCA) bilaterally. The CCA were gently isolated and clamped with microsurgical clamps. Cerebral ischemia was maintained for 12 min and reperfusion started when the clamps were removed. We kept the temperature at about 25  C during and after surgery, exposed animals in incandescent lamp to keep their rectal temperature at about 37  C until its pallanesthesia (Mini Vent 845, Hugo Sachs Elektronik, March-Hugstetten, Germany). The ischemic condition and the sufficiency of reperfusion were confirmed by cerebral blood flow detected by the Laser Doppler Perfusion Monitor (BS EN 60825-1, Devon, England). Two minutes before reperfusion, the anti-TLR4 (10 mg/ml) was injected into the right CCA using a microsyringe in 2 min for blocking TLR4 receptors while 0.9% NaCl was injected into the right CCA of I group animals with the same dose and method. The mice were placed in the cage at 25 C for the following 3 h and then returned to the animal care room. Sham animals received the same surgical procedures except for occluding common carotid arteries bilaterally and injecting the medicine.

TLR4 antibodies-blocking was performed as described previously [10]. Two minutes before reperfusion, the anti-TLR4 (10 mg/ml) was injected into the right CCA of T group animals using a microsyringe (B/BRAUN8714843, Germany) in 2 min for blocking TLR4 receptors. The result of blocking was detected by Western blot.

2.4. Evaluation of neuronal damage in the cortex

2.6. In situ hybridization (ISH) The dioxigenin (DIG)-labeled mRNA probe for TLR4 and MyD88 were obtained from the PubMed and purchased from Haoyang Bio. (Tianjing, China). The sequences of TLR4 mRNA: 50 -TCAGAATAAGAACAGCAACCACTAAAGC-30 . The sequences of MyD88 mRNA: 50 -GCTTCTCGGACTCCT GGTTCTGCTGCTTA-30 , 50 -CCTGCCTCTACCTCCCAAAT GCTGAAA-30 . ISH was performed as described previously [12]. Paraffin-embedded specimens were sliced at a 7 mm thickness. These sections were mounted on poly-L-lysine-treated (Vector Lab, USA) slides, deparaffinized and rehydrated. The sections were blocked by being immersed in H2O2 for 20 min at room temperature. They were further digested with P/E (proteinase K 1 mg/ml, pepsin 20 mg/ml, EDTA0.1 mg/ml, pH 6.4) for 30 min at room temperature, and then were incubated in prehybridization solution for 1 h at 37  C in a humidified chamber. Slides were washed thrice in 0.2  SSC (50 mM NaCl,

444

Y. Gao et al. / Biomedicine & Pharmacotherapy 63 (2009) 442e450

3 mM Na citrate), 5 min each. For hybridization, each slide was incubated in hybridization solution (8 mg/ml) for 4 h at 37  C in a chamber humidified, and then sections were washed thrice in 0.2  SSC, 2  SSC, and 0.1 mol TBS (NaCl 8.5 mg/ml, Tris 1.2 mg/ml, pH 7.5), 5 min each, respectively. Each slide was carried out by diluted solution containing the anti-DIG (DB7405), and then was subjected to the solution containing the high sensitive peroxidase-chain-avidin for 45 min at 37  C in a chamber humidified, respectively. The mean optical density (MOD) of positive cells was evaluated by Motic Images Advanced 3.2.

optical density (MOD) of ISH section were evaluated by means of the Motic Images Advanced 3. 2 (BX51 þ PM10SP e 35, OLYMPUS, Japan). Values were expressed as means  standard deviation (X  S.D.). All data were analyzed with SPSS13.0 software. For tests of significance between groups, one-way analysis of variance (ANOVA) were performed. Significance was regarded as p < 0.05.

2.7. Electrophoretic mobility shift assay (EMSA)

To evaluate the result of TLR4 antibodies-blocking, Western blot analysis was carried out to study the TLR4 protein levels in each group. Fig. 1A shows that the protein levels of TLR4 in I group was obviously higher than those in T group and in sham group. The IDV of TLR4 in I group was higher than that in T group at 12 h, 24 h, 48 h and 72 h, respectively, (p < 0.05) (Fig. 1B). In the present study, the results demonstrated that the protein levels of TLR4 in I group was expressed excessively and anti-TLR4 binding TLR4 receptors before reperfusion was effective.

Preparation of nuclear extracts and gel mobility shift assays were performed according to methods described previously [13]. The sequence of the NF-kB-binding oligonucleotide used as a Biotin-probe was 50 -TCGACAGAGGGGACTTTCCGAGAGGC-30 . Equal amounts sample of nuclear extracts (7 mg) from right cortex in each group at each time point was incubated with binding reaction [binding buffer 1.5 ml, poly (dI:dC) 0.5 ml, dH2O, biotin-probe 0.5 ml] for 20 min at room temperature, then were electrophoresed on 6.5% polyacrylamide gel (40% Acrylamide/Bis 3.3 ml, 50% glycerol 1.0 ml, dH2O, TEMED 20 ml) on ice at 180 V for 60 min. Competition was performed by adding 3.3 ml unlabeled DNA along with the biotin-probe. The DNA-protein complexes and unbound probe were separated electrophoretically on 6.5% polyacrylamide gel in 0.5  TBE buffer. Block the membrane with 15 ml blocking buffer for 30 min. Chemiluminescence was developed by the system of Chemiluminence-imager (Chemi Imager 5500, USA). The membrane was exposed to X-ray film at room temperature for 3e5 min. NF-kB activity was examined by the optical density (OD) of the band.

3. Results 3.1. Western blot for TLR4 antibodies-blocking

A 12h

24h

48h

72h

2.8. Western blot for TNF-a

2.9. Statistical analysis The optical density (OD) values of EMSA band, the integrated density value (IDV) of Western blot band and the mean

B

0.7 I group T group

0.6

S group

IDV of TLR4 protein

For total cellular protein, cells were lysed in buffer containing 25 mM Hepes, pH 7.5, 0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 20 mM b-glycerophosphate, 0.5 mM DTT, 1 mM sodium orthovanadate, 0.1 mM okadaic acid, and 1 mM phenylmethylsulfonyl fluoride. Protein concentrations of the cell lysates were determined by Lowry method [14] with bovine serum albumin as standard, and the supernatants were boiled in SDS sample buffer for 5 min. Equal amounts of lysate protein were run on 12% SDS-PAGE and electrophoretically transferred to PVDF membrane (AmershamePharmacia Biotech). After blocking, the blots were incubated with specific primary antibody overnight at 4  C and further incubated for 1 h with HRP-conjugated secondary antibody. Bound antibodies were detected by ECL kit with a Lumino Image Analyzer (TAITEC).

0.5

0.4

0.3

0.2

0.1

0 12h

24h

48h

72h

Fig. 1. Expression of TLR4 protein of each group in mice right cortex at ischemiaereperfusion 12 h, 24 h, 48 h and 72 h, respectively by Western blot: (A) the IDV of TLR4 in each group with different reperfusion time point; (B) data were expressed as mean  S.D. from five independent animals (n ¼ 6). I group vs S group p < 0.05; I group vs T group p < 0.05.

Y. Gao et al. / Biomedicine & Pharmacotherapy 63 (2009) 442e450

445

Fig. 2. The sections of 4% paraformaldehyde-fixed, paraffin-embedded right cortex were stained with hematoxylineosin (HeE) to reveal cortex region (200): (A), representative histology section of 48 h after ischemiaereperfusion in T group and normal neurons in S group were shown, while the above changes were obviously palliated in T group (n ¼ 6).

3.2. Hematoxylineosin (HeE) for evaluation of neuronal damage in the right cortex We observed the tissue sections of right cortex and assessed cerebral histological changes of mice caused by ischemiaereperfusion under light microscope. Neuronal damage was evaluated qualitatively by a pathologist blinded to the treatment groups. In S group, neurons were arranged orderly and intact in shape, but the neurons and glial cells were swollen, shrunken cell bodies, triangulated, pyknotic, the cellular inter-space widened and arranged asymmetrically in I group, while obviously palliated in T group ( p < 0.05). The evident changes in I group were found at 24 h after ischemiaereperfusion, reaching the maximum at 48 h, then decreasing gradually at 72 h (Fig. 2, Table 1).

decreasing gradually at 72 h (Fig. 3A). The expression of MyD88 mRNA shows the similar result. (Fig. 4A). The mean optical density values (MOD) of positive cells for TLR4 mRNA in T group was lower than those in I group ( p < 0.05) (Fig. 3B). The MOD of positive cells for MyD88 mRNA in T group was lower than those in I group ( p < 0.05) (Fig. 4B). Both MOD of TLR4 mRNA and MyD88 mRNA in I group were higher than those in S group at each time point after reperfusion ( p < 0.05. Fig. 3B, Fig. 4B). The results suggested that TLR4 in right cortex was activated by ischemiaereperfusion and blocked by antiTLR4. The down expression of MyD88 mRNA by anti-TLR4 suggests that MyD88 might be involved in the TLR4-mediated signal pathway activated by ischemiaereperfusion. 3.4. Activation of NF-kB

3.3. Expression of TLR4 mRNA and MyD88 mRNA by in situ hybridization (ISH) To investigate whether TLR4 and MyD88 were activated by ischemiaereperfusion, we examined the expression of TLR4 mRNA and MyD88 mRNA in each group at each time point after ischemiaereperfusion. Expression of TLR4 mRNA and MyD88 mRNA were determined by in situ hybridization (ISH) method. There was apparent expression of TLR4 mRNA and MyD88 mRNA in right cortex in I group and T group. The positive products for TLR4 mRNA were found at 12 h, reaching the peak at 48 h after reperfusion, then Table 1 Neuropathologic lesion score following cerebral ischemiaereperfusion in each group by (HeE) method Reperfusion time (h)

I

T

S

12 24 48 72

0.7  0.05* 1.4  0.06* 2.6  0.21* 1.9  0.03*

0.2  0.02*# 0.6  0.06*# 1.2  0.07*# 0.9  0.07*#

0.1  0.03 0.1  0.04 0.2  0.01 0.2  0.02

The table shows the neuropathological score data (mean  S.D.) from cerebral ischemiaereperfusion induced by occlusion of common carotid arteries (CCA) bilaterally. The score given was assessed by using a 0e4 grading scale: score 0, no neurologic impairment; score 1, scattered injured neurons in cortex; score 2, moderate damage in cortex (less than half of cortex cells affected); score 3, severe damage to cortex cells (more than half of cortex cells affected); score 4, extensive cell damage in cortex. Compared with S group. * p < 0.05; Compared with I group, # p < 0.05.

When the electrophoretic gel mobility shift assay (EMSA) was used to examine the activation of NF-kB in right cortex in each group at each time point after ischemiaereperfusion, no protein product of NF-kB was observed in S group. However, a significant activation of NF-kB in I group was detected. In I group, the band of NF-kB became detectable at 12 h, and reached the maximal level at 48 h after reperfusion, then decreased gradually at 72 h. Nevertheless, activation of NF-kB in T group at each time point after ischemiaereperfusion was lower than those in I group ( p < 0.05. Fig. 5A,B). In the TLR4-mediated MyD88-dependent signaling pathway, NFkB has been reported to be activated by MyD88 in earlier period [15,16]. Comparison of these results to those in Fig. 5 showed that the activation of NF-kB in I group induced by MyD88 was first detected at 12 h and maximal at 48 h, indicating clearly the nuclear translocation of NF-kB. The present results suggest that ischemiaereperfusion activated the expression of NF-kB, NF-kB might be involved in the TLR4mediated signal pathway activated by ischemiaereperfusion. 3.5. Expression of TNF-a protein levels Nuclear translocation of NF-kB has been reported to induce the expression of inflammatory factors [17,18]. After the nuclear translocation of NF-kB has been detected, to investigate whether inflammatory factors were induced, we then determined the TNF-a protein levels by Western blot in right cortex

Y. Gao et al. / Biomedicine & Pharmacotherapy 63 (2009) 442e450

446

A

I group

T group

S group

12h

24h

48h

72h

B

0.5

MOD of TLR4 mRNA

0.45 0.4 0.35 0.3

I group

0.25

T group

0.2

S group

0.15 0.1 0.05 0 12h

24h

48h

72h

Fig. 3. Expression of TLR4 mRNA by in situ hybridization (ISH) in right cortex in each group (400). The positive products for TLR4 mRNA showed brown, and mainly expressed in cellular membrane. TLR4 mRNA positive products in right cortex in different groups after ischemiaereperfusion at 12 h, 24 h, 48 h and 72 h, respectively, (A), the MOD of positive cells for TLR4 mRNA in each group, (B), data were expressed as mean  S.D. from six independent animals (n ¼ 6). I group vs S group p < 0.05; I group vs T group p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

in each group at each time point. The results from Western blotting analysis of TNF-a showed that they were found at 12 h, reaching the peak at 48 h after reperfusion, then decreasing gradually in I group (Fig. 6A),which was coincident with

the expression of NF-kB, TLR4 mRNA, MyD88 mRNA and NF-kB. Furthermore, the TNF-a protein levels in T group and S group at each time point after ischemiaereperfusion were lower than those in I group ( p < 0.05. Fig. 6B).

Y. Gao et al. / Biomedicine & Pharmacotherapy 63 (2009) 442e450

A

I group

T group

447

S group

12h

24h

48h

72h

B MOD of MyD88 mRNA

0.14 0.12 0.1 I group

0.08

T group 0.06

S group

0.04 0.02 0

12h

24h

48h

72h

Fig. 4. Expression of MyD88 mRNA by in situ hybridization (ISH) in right cortex in each group (400). The positive products for MyD88 mRNA showed brown, and mainly expressed in cytoplasm. MyD88 mRNA positive products in right cortex in different groups after ischemiaereperfusion at 12 h,24 h,48 h and 72 h, respectively, (A), the MOD of positive cells for MyD88 mRNA in each group; (B), data were expressed as mean  S.D. from six independent animals (n ¼ 6). I group vs S group p < 0.05; I group vs T group p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

4. Discussion and conclusion Cerebral ischemiaereperfusion injury is a complex pathophysiological process including a cascade reaction such as releasing of inflammatory cytokines [19,20]. TLR4 is type I

transmembrane protein that contains a large, leucine-rich repeat in an extracellular region and a Toll/IL-1 receptor homology (TIR) domain in a cytoplasmic region [21,22]. TLR4 is a key component of the innate immune system, functioning as pattern recognition receptor that recognises the

72hS

72hT

72hI

48hT

48hS

48hI

24hT

24hS

24hI

negative control

positive control

12hT

12hI

A

12hS

Y. Gao et al. / Biomedicine & Pharmacotherapy 63 (2009) 442e450

448

NF-κB

NS

Free probe

B

7

OD of EMSA

6 5 I group

4

T group 3

S group

2 1 0

12h

24h

48h

72h

Fig. 5. Identification of the activated NF-kB in the DNA-protein complex by EMSA. Equal amounts sample of nuclear extracts (7 mg) from right cortex in each group at each time point was incubated with Binding Reaction, The DNA-protein complexes and unbound probe (Free probe) were separated electrophoretically on 6.5% polyacrylamide gel. NS indicated the nonspecific DNA-binding band: (A), the OD of EMSA in each group; (B), data were expressed as mean  S.D. from six independent animals (n ¼ 6). I group vs S group p < 0.05; I group vs T group p < 0.05.

lipopolysaccharide (LPS), one of the most immunostimulatory glycolipids constituting the outer membrane of the Gram-negative bacteria [23]. After being stimulated by some microbial agents, TLR4-mediated MyD88-dependent signaling pathway triggers a cascade of cellular signals which leads to inflammatory cytokines expression such as TNF-a [24,25]. There is increasing evidence supporting the notion that a link existed between innate immune/inflammatory responses and ischemia-induced neuronal damage [20]. However, whether TLR4-mediated MyD88-dependent signaling pathway was activated in cortex after ischemiaereperfusion has not been reported. In present study, TLR4 signaling pathway can be inhibited by specific TLR4 antibodies (anti-TLR4) binding protein to prevent TLR4 from interacting with its receptors. The molecular weight of TLR4 is 89 kD, which could through the BBB (bloodebrain barrier) after 2 min ischemia [26]. Mice cerebral ischemia was induced by occlusion of common carotid arteries (CCA) bilaterally. The mice in T group and I group were induced through 12 min of ischemia and sacrificed at different reperfusion time points. Reports have indicated that the effect of inhibiting TLR4 induced by specific antibodies-binding (antibodies-blocking) and transgenic method was affinis [10]. To evaluate the result of TLR4 antibodiesblocking in our study, TLR4 protein levels in each group were detected by Western blot. Results showed that the

protein levels of TLR4 in I group was obviously higher than those in T group and in sham group.(Fig. 1A). The IDV of TLR4 in I group was higher than that in T group at 12 h, 24 h, 48 h and 72 h, respectively (P < 0.05) (Fig. 1B). The results demonstrated that anti-TLR4 binding TLR4 receptors, before reperfusion, significantly prevented TLR4 receptors from receiving the reperfusion induced stimulation in present studies. Moreover, to determine the result of mice cerebral ischemiaereperfusion injuries by occlusion of CCA bilaterally, the tissue sections of right cortex were observed and the neuropathological scores were analyzed in each group. Obvious neuronal damage was observed in cortex in I group at each reperfusion time points compared with those in S group. The evident changes of the sections of right cortex in I group were found at 24 h after ischemiaereperfusion, reaching the maximum at 48 h, then decreasing gradually at 72 h (Fig. 2, Table 1). Above data demonstrated that the method of TLR4 antibodies-blocking and mice cerebral ischemia injuries induced by occlusion of CCA bilaterally were effective in the present study. Some researchers have verified that the signal sent out by the cells in injured and stressed condition, as endogenic ligands, can activate TLR4 [15,27,28]. Studies show TLR4 participates in cerebral ischemiaereperfusion injury [29]. However, it is still unclear about its mechanism. Our data in expression of TLR4 mRNA and MyD88 mRNA indicated

Y. Gao et al. / Biomedicine & Pharmacotherapy 63 (2009) 442e450

A

I group

T group

S group

12h

24h

48h

72h

B

0.9

IDV of TNF-α protein

0.8 I group

0.7

T group

0.6

S group

0.5 0.4 0.3 0.2 0.1 0

12h

24h

48h

72h

Fig. 6. Expression of TNF-a protein in each group in mice right cortex by Western blot at ischemiaereperfusion 12 h, 24 h, 48 h and 72 h, respectively, (A) the IDV of TNF-a in each group with different reperfusion time point; (B) data were expressed as mean  S.D. from six independent animals (n ¼ 6). I group vs S group p < 0.05; I group vs T group p < 0.05.

that TLR4 was activated by cerebral ischemiaereperfusion and downregulation of MyD88 by blocking TLR4 (Fig. 4). Studies have found that TLR4-mediated MyD88-dependent signaling pathway was essential for NF-kB activation [30]. MyD88 recruitment to the TIR domain of TLR4 results in activation of NF-kB which produces pro-inflammatory cytokines, such as tumor necrosis factor a (TNF-a). TNF-a, a representative inflammatory cytokine, has been reported to play an important role in the cerebral ischemiaereperfusion injury [31]. In the present study, we found that the activation of NF-kB in I group induced by TLR4eMyD88 was first detected at 12 h and maximal at 48 h, indicate clearly the nuclear translocation of NF-kB. However, TLR4 receptors blocked by antiTLR4 resulted in downregulation of NF-kB, TNF-a which suggest that TLR4-mediated MyD88-dependent signaling pathway activated by ischemiaereperfusion may be involved in the mechanism of ischemiaereperfusion through upregulation of NF-kB and TNF-a. Our study suggests that TLR4blocked method may be applicable for the treatment of cerebral ischemiaereperfusion injury.

449

References [1] Akira S. Toll-like receptors: lessons from knockout mice. J Biochem Soc Trans 2000;28:551e6. [2] Medzhitov R, Preston-Hurlburt P, Janeway Jr CA. A human homologue of the Drosophila toll protein signals activation of adaptive immunity. Nature 1997;388:394e7. [3] Kawai T, Takeuchi O, Fujita T, Inoue J, Muhlradt PF, Sato S. Lipopolysaccharide stimulates the MyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expression of a subset of lipopolysaccharide-inducible genes. J Immunol 2001;167:5887e94. [4] Janeway Jr , Medzhitov R. Innate immune recognition. Annu Rev Immunol 2002;20:197e216. [5] Hallenbeck JM. The many faces of TNF in stroke. Nat Med 2002;8: 1363e8. [6] Lambertsen KL, Gregersen R, Meldgaard M, Clausen BH, Heibol EK, Ladeby R, et al. A role for interferon-gamma in focal cerebral ischemia in mice. J Neuropathol Exp Neurol 2004;63:942e55. [7] Hua F, Ma J, Li Y, Ha T, Xia Y, Kelley J, et al. The development of a novel mouse model of transient global cerebral ischemia. Neurosci Lett 2006;400:69e74. [8] Hua F, Ma J, Ha TZ, Xia Y, Kelley J, Williams DL, et al. Activation of Toll-like receptor 4 signaling contributes to hippocampal neuronal death following global cerebral ischemia/reperfusion. J Neuroimmunol 2007; 190:101e11. [9] Wu YQ, Fang ZP, Guo WZ, Xi J. Toll like receptor 4 mediates lipopolysaccharide-induced cell activation in human endothelial Cells. Prog Biochem Biophys 2002;29:407e10. [10] Li CF, Ha TZ, Liu LP, Browder W, Kao RL. Adenosine prevents activation of transcription factor and enhances activator protein-1 binding activity in ischemic rat heart. Surgery 2000;127:161e9. [11] Lee SR, Tsuji K, Lo EH. Role of matrix metalloproteinases in delayed euronal damage after transient global cerebral ischemia. J Neurosci 2004;24:671e8. [12] Koga K, Osuga Y, Yano T, Ikezuki Y, Yoshino O, Hirota Y, et al. Evidence for the presence of angiogenin in human testis. J Androl 2004; 25:369e74. [13] Ha T, Li YH, Hua F, Ma J, Gao X, Kelley J, et al. Reduced cardiac hypertrophy in toll-like receptor 4-deficient mice following pressure overload. Cardiovas Res 2005;68:224e34. [14] Gu ZL, Jiang Q, Zhang GY. Diphosphorylation of extracellular signalregulated kinases and c-Jun N-terminal protein kinases in brain ischemic tolerance in rat. Brain Res 2000;860:157e60. [15] Kariko K, Ni H, Capodici J, Lamphier M, Weissman D. mRNA is an endogenous ligand for toll-like receptor 3. J Biol Chem 2000;279:12542e50. [16] Yamamoto M, Takeda K, Akira S. TIR domain-containing adaptors define the specificity of TLR signaling. Mol Immunol 2004;40:861e8. [17] Ninomiya Tsuji J, Kishimoto K, Hiyama A, Lnoue J, Cao Z, Matsumoto K. The kinase TAK1 can activate the NIK-1 kappaB as well as the MAP kinase cascade in the IL-1 signalling pathway. Nature 1999;398:252e6. [18] Suzuki N, Suzuki S, Duncan GS, Millar DG, Wada T, Mirtsos C, et al. Severe impairment of interleukin-1 and toll-like receptor signalling in mice lacking IRAK-4. Nature 2002;416:750e6. [19] Chan PH. Reactive oxygen radicals in signaling and damage in the ischemic brain. J Cereb Blood Flow Metab 2001;21:2e14. [20] Chen WY, Raung SL, Kuo JS, Chen CJ, et al. Association of immune responses and ischemic brain infarction in rat. Neuroreport 2001;12:1943e7. [21] Beutler B, Jiang Z, Georgel P, Crozat K, Croker B, Rutschmann S, et al. Genetic analysis of host resistance: toll-like receptor signaling and immunity at large. Annu Rev Immunol 2006;24:353e89. [22] Kaisho T, Akira S. Toll-like receptor function and signaling. J Allergy Clin Immunol 2006;117:979e87. [23] Sarah LD, Luke AJ, O’Neill L. Toll-like receptors: from the discovery of NFkB to new insights into transcriptional regulations in innate immunity. Biochem Pharmacol 2006;72:1102e13. [24] Monick MM, Yarovinsky TO, Powers LS, Butler NS, Carter AB, Gudmundsson G, et al. Respiratory syncytial virus up-regulates TLR4

450

Y. Gao et al. / Biomedicine & Pharmacotherapy 63 (2009) 442e450

and sensitizes airway epithelial cells to endotoxin. J Biol Chem 2003; 278:53035e44. [25] Fearon D, Locksley R. The instructive role of innate immunity in the acquired immune response. Science 1996;272:50e4. [26] Zhang NZ, Zhang HJ, Wang M. A study on the relationship between expression of matrix metalloproteinase-9 in rat brain and permeability of blood brain barrier in pathogenesis of epilepsy. Chinese J Clin Neuroscience 2008;1:10e4. [27] Akira S, Takeda K, Kaisho T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immunol 2001;8:675e80.

[28] Beg Amer A. Endogenous ligands of toll-like receptors: implications for regulating inflammatory and immune responses. Trends Immunol 2002;23:509e12. [29] Cao CX, Yang QW, Lv FL, Cui J, Fu HB, Wang JZ. Reduced cerebral ischemiaereperfusion injury in toll-like receptor 4 deficient mice. Biochem Biophys Res Commun 2007;353:509e14. [30] Seki E, Tsutsui H, Iimuro Y, Naka T, Son G, Akira S, et al. Contribution of toll-like receptor/myeloid differentiation factor 88 signaling to murine liver regeneration. Hepatology 2005;41:443e50. [31] Baeuerle PA, Henkel T. Function and activation of NF kappa B in the immune system. Annu Rev Immunol 1994;12:141e79.