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Neuroprotection against ischemic brain injury by SP600125 via suppressing the extrinsic and intrinsic pathways of apoptosis
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Neuroprotection against ischemic brain injury by SP600125 via suppressing the extrinsic and intrinsic pathways of apoptosis Qiu-Hua Guana,b , Dong-Sheng Pei b , Xiao-Mei Liua , Xiao-Tian Wanga , Tian-Le Xub,c , Guang-Yi Zhanga,b,⁎ a
Research Center for Biochemistry and Molecular Biology, Xuzhou Medical College, Xuzhou 221002, PR China Department of Neurobiology and Biophysics, School of Life Science, University of Science and Technology of China, Hefei 230027, PR China c Institute of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, PR China b
A R T I C LE I N FO
AB S T R A C T
Article history:
Our previous studies and the others have strongly suggested that JNK signaling pathway
Accepted 22 March 2006
plays a critical role in ischemic brain injury. Here, we reported that SP600125, a potent, cell-
Available online 3 May 2006
permeable, selective, and reversible inhibitor of c-Jun N-terminal kinase (JNK), potently decrease neuronal apoptosis induced by global ischemia/reperfusion in the vulnerable
Keywords:
hippocampal CA1 subregion. As a result, SP600125 diminished the increased phosphorylation
c-Jun N-terminal protein kinase
of c-Jun and the increased expression of FasL induced by ischemia/reperfusion in the vulnerable
(JNK)
hippocampal CA1 subregion. At the same time, through inhibiting phosphorylation of Bcl-2 and
Cerebral ischemia/reperfusion
the release of Bax from Bcl-2/Bax dimers, SP600125 attenuated Bax translocation to mitochondria
Extrinsic and intrinsic pathways
and the release of cytochrome c induced by ischemia/reperfusion (I/R). Furthermore, the
of apoptosis
activation of caspase-3 induced by ischemia/reperfusion was also significantly suppressed by
SP600125
preinfusion of SP600125. Importantly, the same neuropotective effect was showed by administration of SP600125 both before and after ischemia. Thus, our findings imply that SP600125 can inhibit the activation of JNK signaling pathway and induce neuroprotection against ischemia/reperfusion in rat hippocampal CA1 region via suppressing the extrinsic and intrinsic pathways of apoptosis. Taken together, these results indicate that targeting the JNK pathway provides a promising therapeutic approach for ischemic brain injury. © 2006 Elsevier B.V. All rights reserved.
1.
Introduction
Neuronal apoptosis has been implicated in cerebral ischemia. However, the apoptotic process is regulated by many intracellular signaling pathways, including the JNK signaling pathway (Chang and Karin, 2001; Davis, 2000; Lin, 2003; Shaulian and Karin, 2002). The c-Jun N-terminal protein kinase (JNK) signaling pathway is implicated in neuronal
apoptosis triggered by focal or global ischemia (Irving and Bamford, 2002). Furthermore, the study by Kuan et al. (2003), based on gene-knockout approaches, convincingly demonstrated the critical role of JNK3, the neural-specific JNK, in ischemia-induced apoptosis in the brain. However, the downstream mechanism that accounts for the proapoptotic actions of JNK during cerebral ischemia/reperfusion remains to be investigated in detail. Understanding the
⁎ Corresponding author. Research Center for Biochemistry and Molecular Biology, Xuzhou Medical College, 84 West Huai-hai Road, Xuzhou, Jiangsu, 221002, PR China. Fax: +86 516 574 8486. E-mail address: [email protected] (G.-Y. Zhang). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.03.086
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molecular mechanisms by which JNK regulates apoptosis should provide insights into treatment of cerebral ischemia. In mammals, signaling cascades culminating in apoptotic cell death can be divided into two broad categories: the “intrinsic” (i.e., mitochondria) and the “extrinsic” (i.e., death receptor) pathways (Ashkenazi and Dixit, 1998; Cohen, 1997; Green and Reed, 1998; Thornberry and Lazebnik, 1998). In the intrinsic pathway, various proapoptotic signals induce the release of mitochondrial proteins, such as cytochrome c, through some proapoptotic Bcl-2 family proteins which may form a pore and/or open permeability transition pore. Bax is a potent regulator of mitochondria-dependent apoptosis. The translocation of Bax to mitochondria causes the loss of mitochondrial membrane potential and the full opening of permeability transition pore (PTP) and results in the release of cytochrome c. Once released, cytochrome c interacts with Apaf-1 and deoxyadenosine triphosphate forming the apoptosome and leading to activation of cytochrome-c-dependent caspase cascade, resulting in apoptosis. In contrast, so-called “extrinsic” pathway signals, such as those mediated by death receptors of the TNF receptor superfamily, activate the caspase cascade more directly. For example, interaction of Fas with its ligand (FasL) triggers formation of a deathinducing signaling complex (DISC), which in turn recruits and activates caspase-8. Caspase-8 then activates other procaspases, culminating in cleavage of cellular substrates, and apoptosis. The recent development of chemical inhibitors of the JNK pathway has greatly accelerated our understanding of the downstream signaling pathway of JNK in ischemic neurodegeneration. Two effective inhibitors, CEP-1347 (previously called KT7515) and SP600125, have been recently reported. The actions of CEP-1347 (previously called KT7515) have been attributed to the inhibition of the JNK upstream activators, the mixed lineage kinases (Saporito et al., 2002). SP600125 has been reported to function as a reversible ATP competitive inhibitor of JNK MAPKs (Bennett et al., 2001; Han et al., 2001), which shows 300-fold selectivity of inhibition of JNK over the extracellular signal-regulated kinases (ERKs) and p38 MAPKs, the closest kinase relatives of JNK. In the present study, we showed that SP600125, a potent, cell-permeable, selective, and reversible inhibitor of c-Jun N-terminal kinase (JNK), could potently decrease neuronal apoptosis induced by global ischemia/reperfusion in the vulnerable hippocampal CA1 subregion via inhibiting the extrinsic and intrinsic pathways of apoptosis.
2.
Results
2.1. The effect of SP600125 on the increased expression of FasL induced by global ischemia/reperfusion in hippocampal CA1 To elucidate the involvement of Fas-receptor-mediated pathway in the apoptotic program during global ischemia/reperfusion injury, the expression of FasL and Fas was analyzed by Western blotting. The expression of FasL was significantly increased from 3 h to 1 day reperfusion, but the expression of Fas was not significantly increased at various time points after
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15 min of ischemia (Fig. 1A). In the present study, we examined the effect of SP600125 on the expression of FasL and Fas. As shown in Fig. 1B, results of Western blotting revealed that the increased expression of FasL at 6 h reperfusion was significantly suppressed by preinfusion of SP600125. The same dose of vehicle 1% DMSO did not affect the increase on the expression of FasL. The protein level of Fas was not affected by SP600125 and vehicle 1% DMSO.
Fig. 1 – Effects of pretreatment with SP600125 on the increased expression of FasL and Fas. (A) Time courses of the expression of FasL and Fas in hippocampal CA1 derived from sham-treated rats or rats at various time points after 15 min of ischemia. (B) Effects of pretreatment with SP600125 on the increases of expression of FasL and Fas induced by 6 h of reperfusion following transient brain ischemia (I/R6h) in hippocampal CA1. Western blot probed with antibodies to FasL and Fas. Bands corresponding to FasL and Fas were scanned, and the intensities were represented as folds vs. sham control. Data are the mean ± SD and were expressed as folds vs. respective sham. aP < 0.05 vs. sham; bP < 0.05 vs. 6 h after 15 min of ischemia groups (n = 6 rats).
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2.2. The effect of SP600125 on the phosphorylation of c-Jun induced by global ischemia/reperfusion in hippocampal CA1 A JNK-dependent element in the Fas ligand promoter that binds c-Jun and ATF2 has been identified (Faris et al., 1998). Our previous studies showed that activated JNK induced neuronal death via phosphorylating c-Jun and promoting its transcription activity (Guan et al., 2005). In the present study, we examined the effect of SP600125 on phosphorylation of cJun by immunohistochemical method (Fig. 2). In the sham group (Figs. 2A, B), week c-Jun immunoreactivity was detected in the nucleus of hippocampal CA1. Six hours reperfusion after ischemia (Figs. 2C, D), p-c-Jun immunoreactivity significantly increased compared with the sham group. There was no inhibitory effect of intracerebral ventricular preinfusion of 1% DMSO on p-c-Jun immunoreactivity 6 h reperfusion after
ischemia (Figs. 2E, F). Administration of SP600125 20 min before cerebral ischemia (Figs. 2G, H) and 1 h after cerebral ischemia (Figs. 2I, J) significantly inhibited p-c-Jun immunoreactivity 6 h reperfusion after ischemia.
2.3. The effect of SP600125 on the release Bax from Bcl-2/Bax dimers induced by global ischemia/reperfusion in hippocampal CA1 Our previous studies showed that activated JNK induced neuronal death via phosphorylating Bcl-2 and inhibiting its prosuvival activity (Guan et al., 2005). In this study, we found that phosphorylated Bcl-2 was not shown to interact with Bax during ischemia and reperfusion in hippocampal CA1 (data not shown). Furthermore, our results showed that ischemia/ reperfusion decreased interaction of Bcl-2 with Bax and SP600125 inhibited the decreased interaction of Bcl-2 with
Fig. 2 – Immunohistochemical staining of phosphorylation of c-Jun in coronal sections of hippocampus and the inhibitory effect of SP600125 on phosphorylation of c-Jun. Example of immunohistochemical staining sections of the hippocampi of sham-operated rats (A, B) and rats subjected to 15 min of ischemia followed by 6 h of reperfusion (C, D) and rats subjected to 15 min of ischemia followed by 6 h of reperfusion with administration of DMSO (E, F), 30 μg/10 μl of SP600125 20 min before ischemia (G, H) and 60 min after ischemia (I, J). Data were obtained from six independent animals in each experimental group, and the results of a typical experiment are presented. Boxed areas in left column are shown at higher magnification in right column. A, C, E, G, I: ×40; B, D, F, H, J: ×400. Scale bar in I = 200 μm; scale bar in J = 10 μm.
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Bax (Fig. 3). Therefore, we speculate that ischemia/reperfusion could promote the release of Bax from Bcl-2/Bax dimers via JNK phosphorylating Bcl-2.
2.4. The effect of SP600125 on translocation of Bax to mitochondria and the release of cytochrome c from mitochondria induced by global ischemia/reperfusion in hippocampal CA1 To elucidate the involvement of “intrinsic”-mediated apoptotic pathway during global ischemia/reperfusion injury and the action of JNK activity on Bax translocation and the release of cytochrome c, expression of Bax and cytochrome c in mitochondria and cytosol was examined by Western blotting. We first determine whether Bax translocates from cytosol to mitochondria after ischemia. Using Western blotting analysis and subcellular fraction, we found that the expression of Bax was significantly increased from 3 h to 1 day after ischemia in the mitochondria, but the expression of Bax was not significantly decreased at various time points after 15 min of ischemia in cytosol (Fig. 4A). We supposed that the overwhelming majority of Bax were located in cytosol, thus partial translocation of Bax did not obviously affect the total protein level of Bax in cytosol. Moreover, we examined whether the inhibition of JNK by SP600125 contributes to attenuating Bax
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translocation. The inhibitory effect of SP600125 on Bax translocation in the mitochondrial fraction reached a statistical difference 6 h after reperfusion compared with that of vehicle 1% DMSO (Fig. 4B). Cytochrome c immunoreactivity was evident as a single band of molecular mass of 15 kDa in the cytosolic fraction of the hippocampal CA1 6 h to 3 days after global ischemia. However, it was barely detected in the sham CA1 subregion. A significant amount of mitochondrial cytochrome c was detected in the controls and decreased after ischemia, corresponding to a marked increase in the cytosolic fraction (Fig. 4C). Moreover, we examined whether the activation of JNK signaling pathway contributes to the release of cytochrome c. The inhibitory effect of SP600125 on the release of cytochrome c in the cytosol fraction reached a statistical difference 6 h after reperfusion compared with the vehicle 1% DMSO (Fig. 4D). To elucidate whether other mitochondrial proteins were released from mitochondria, we determined the cytochrome c oxidase level in the cytosolic and mitochondrial fraction using cytochrome c oxidase subunit IV antibody. The cytochrome c oxidase subunit IV was examined only in the mitochondrial fraction but not in the cytosolic fraction in sham, ischemia/reperfusion and application of drug groups (Figs. 4A, B, C and D). These results suggested that cytochrome c oxidase was not coreleased with cytochrome c from mitochondria.
2.5. The effect of SP600125 on the activation of caspase-3 induced by global ischemia/reperfusion in hippocampal CA1 Caspase-3 is one of the key executioners in extrinsic and intrinsic apoptotic pathways. It is a cytosolic protein as an inactive 32 kDa proenzyme and is activated by proteolytic cleavage into the 20 kDa (p20) and 11 kDa (p11) active subunits. In this study, we examined the activation of caspase-3 during ischemia/reperfusion and the action of inhibition of JNK activity by SP600125 on activation of caspase-3. Immunohistochemical observation was carried out using anti-cleaved caspase-3 antibody. As shown in Fig. 5, weak cleaved caspase-3 immunoreactivity was detected in the cytosol of hippocampal CA1 in the sham group (Figs. 5A, B), cleaved caspase-3 immunoreactivity significantly increased 6 h reperfusion after ischemia (Figs. 5C, D) compared with the sham group. There was no inhibitory effect of intracerebral ventricular preinfusion of 1% DMSO on cleaved caspase-3 immunoreactivity 6 h reperfusion after ischemia (Figs. 5E, F). Cleaved caspase-3 immunoreactivity was significantly inhibited at 6 h reperfusion by administration of SP600125 20 min before cerebral ischemia (Figs. 5G, H) and 1 h after cerebral ischemia (Figs. 5I, J).
2.6. Neuroprotective role of SP600125 against cerebral ischemia Fig. 3 – Effects of SP600125 on altered interactions Bcl-2 with Bax induced by ischemia/reperfusion in hippocampal CA1. (A) Co-immunoprecipitation analysis of interactions between Bcl-2 and Bax. (B) Bands corresponding to Bax and Bcl-2 were scanned, and the intensities were represented as folds vs. sham control. Data were expressed as mean ± SD (n = 6). a,c P < 0.05 vs. respective sham; b,dP < 0.05 vs. respective vehicle treatment group.
To investigate whether pretreatment of SP600125 would have neuroprotection against ischemia-induced apoptotic cell death, adult Sprague–Dawley rats were subjected to 15 min ischemia followed by 5 days reperfusion. Rats were pretreated with SP600125 or 1% DMSO by cerebral ventricular injection 20 min before ischemia and 1 h after ischemia. After 3 days reperfusion, rats were perfusion-fixed with
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Fig. 4 – Effects of pretreatment with SP600125 on the translocation of Bax and the release of cytochrome c. (A) Time courses of the expression of Bax in cytosol and mitochondria in hippocampal CA1 derived from sham-treated rats or rats at various time points after 15 min of ischemia. (B) Effects of pretreatment with SP600125 on the expression of Bax induced by 6 h of reperfusion following transient brain ischemia (I/R6h) in cytosol and mitochondria in hippocampal CA1. (C) Western blot analysis of cytochrome c and cytochrome oxidase 4 in rat hippocampal CA1 subregion. Cytochrome c immunoreactivity is evident as a single band of molecular mass (15 kDa) in the cytosolic and mitochondrial fraction 6 h to 3 days after ischemia, but not in the sham CA1 tissue. (D) Effects of pretreatment with SP600125 on the expression of cytochrome c induced by 6 h of reperfusion following transient brain ischemia (I/R6h) in cytosol and mitochondria in hippocampal CA1. Cytochrome oxidase 4 (COX4) was strongly expressed in the mitochondrial fraction and did not decrease after ischemia, but virtually no immunoreactivity was seen in the cytosolic fraction in both the sham and ischemic CA1 subregions. Bands corresponding to Bax were scanned, and the intensities were represented as folds vs. sham control. Data are the mean ± SD and were expressed as folds vs. respective sham. aP < 0.05 vs. sham; bP < 0.05 vs. 6 h after 15 min of ischemia group (n = 6 rats).
paraformaldehyde and TUNEL staining was used to examine the apoptosis of CA1 pyramidal cells in hippocampus (Fig. 6). A significant number of positive cells were observed 3 days after ischemia (Figs. 6Ac, d). Some of them showed characteristic appearances, such as shrunken, condensed nuclei and apoptotic bodies. However, others showed lightly stained, large and swollen nuclei. We excluded the cells with the latter features from the TUNEL positive cells because they might have contained necrotic cells. As the results shown, administration of SP600125 20 min before cerebral ischemia significantly decreased TUNEL-positive cells (Figs. 6Ag, h). Administration of SP600125 1 h after cerebral ischemia also significantly decreased TUNEL-positive cells (Figs. 6Ai, j). At the same time, as the control, 1% DMSO did not show any protection (Figs. 6Ae, f).
3.
Discussion
Our early study and the others strongly suggest that activation of JNK signaling may play a critical role in brain ischemia injury (Gao et al., 2005; Guan et al., 2005; Irving and Bamford, 2002; Kuan et al., 2003). Therefore, JNK is an important therapeutic target for prevention of neuronal death induced by brain ischemia. In the present study, results from histological assessment showed that transient global cerebral ischemia lead to cell apoptosis of hippocampal CA1 pyramidal neurons at 3 days after ischemia and application of SP600125 could prevent hippocampal CA1 neurons from apoptosis after cerebral ischemia/reperfusion. Moreover, the protective effect of SP600125 shown here is likely clinically
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Fig. 5 – Immunohistochemical staining of expression of cleaved caspase-3 in coronal sections of hippocampus and the inhibitory effect of SP600125 on expression of cleaved caspase-3. Example of immunohistochemical staining sections of the hippocampi of sham-operated rats (A, B) and rats subjected to 15 min of ischemia followed by 6 h of reperfusion (C, D), and rats subjected to 15 min of ischemia followed by 6 h of reperfusion with administration of DMSO (E, F), 30 μg/10 μl of SP600125 20 min before ischemia (G, H) and 60 min after ischemia (I, J). Data were obtained from six independent animals in each experimental group, and the results of a typical experiment are presented. Boxed areas in the left column are shown at higher magnification in right column. A, C, E, G, I: ×40; B, D, F, H, J: ×400. Scale bar in I = 200 μm; scale bar in J = 10 μm.
relevant as significant neuroprotection was achieved when SP600125 was administered either before or after the onset of ischemia. Our previous studies indicated that SP600125, a reversible ATP-competitive JNK inhibitor, rescued transient brain ischemia/reperfusion-induced neuronal death in rat hippocampal CA1 subregion via suppressing the activation of nuclear substrate (c-Jun) and the phosphorylation of non-nuclear substrate (Bcl-2) induced by ischemic insult (Guan et al., 2005). Activated JNK phosphorylates the transcription factor cJun and leads to increase AP-1 transcription activity to modulate transcription of a number of genes, which lead to apoptosis. For example, a JNK-dependent element in the Fas ligand promoter that binds c-Jun and ATF2 has been identified (Faris et al., 1998). Results from our current studies showed
that pretreatment of SP600125 could diminish the increased expression of FasL and interaction of FasL with Fas induced by ischemia and reperfusion. Previous studies indicated that neuronal protection was conferred by a c-Jun mutant lacking JNK phosphoacceptor sites, which inhibited FasL induction by withdrawal of survival factors in PC12 cells (Le-Niculescu et al., 1999). Furthermore, Martin-Villalba et al. (1999) showed that FasL mRNA and protein were induced after cerebral ischemia and reduced by the treatment of FK506, which prevented the phosphorylation of c-Jun. In addition, Padosch et al. (2003) reported that FasL was induced in the thalamus but not in the hippocampus after global cerebral ischemia. However, Gao et al. studies in a murine model of focal ischemia and reperfusion showed that SP600125 attenuated ischemia-induced expression of Fas, but not the expression
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of FasL (Gao et al., 2005). Taken together, these data suggested that, through c-Jun/AP-1-mediated transcriptional regulation, activation of JNK could enhance the expression of FasL or Fas, which can ultimately contribute to Fas-mediated apoptosis.
Now, accumulating evidences suggest that mitochondriamediated apoptotic pathway is one of the downstream mechanisms by which JNK promotes neuronal cell death (Carboni et al., 2005; Kuan et al., 2003; Okuno et al., 2004; Putcha et al., 2003). Consistent with these studies, our
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results also showed that SP600125 inhibited the translocation of Bax to mitochondria and the release of cytochrome c induced by global ischemia and reperfusion. Recent studies have suggested that activated JNK may directly interact with mitochondria and results in the release of cytochrome c and Smac from mitochondria (Schroeter et al., 2003). The underlying mechanism for this direct effect of JNK is unclear, but it may involve JNK-mediated phosphorylation of certain Bcl-2 family members. There is a lack of evidence that activated JNK may directly phosphorylate Bax. Therefore, JNK may phosphorylate other Bcl-2 family members leading to the translocation of Bax to mitochondria. For example, it has been reported that JNK phosphorylation of Bim-related members of the Bcl-2 family induced Baxdependent apoptosis (Lei and Davis, 2003; Okuno et al., 2004). Other results also revealed that JNK activation caused serine phosphorylation of 14-3-3, a cytoplasmic sequestration protein of Bax, leading to Bax disassociation from 14-3-3 and subsequent translocation to mitochondria (Gao et al., 2005). Moreover, recent results suggest that activated JNK phosphorylated Bcl-2 and were associated with the phosphorylated form of Bcl-2 in the control of apoptosis following paclitaxel treatment (Brichese et al., 2004). Our previous studies have also indicated that JNK promoted transient brain ischemia/reperfusion-induced neuronal death via phosphorylation Bcl-2 and decreasing the anti-apoptotic function of Bcl-2 (Kuan et al., 2003). Phosphorylation inactivates Bcl-2, thus promoting apoptosis, possibly by freeing Bax from Bcl-2/Bax dimers (Haldar et al., 1996). The Bcl-2/Bax heterodimer is the active component for death protection, but in response to apoptotic stimulation, Bax is freed from Bcl-2/Bax dimers and forms ion channels and pores in mitochondrial membranes by large homoligomers and results in the release of cytochrome c. Current study found that phosphorylated Bcl-2 did not interact with Bax, resulting in the decrease of interaction of Bcl-2 with Bax during ischemia and reperfusion in hippocampal CA1. Our results also showed that SP600125 inhibited the phosphorylation of Bcl-2 and increased the interaction of Bcl-2 with Bax. Therefore, JNK could trigger the release of Bax from Bcl2/Bax dimers and subsequent translocation of Bax to mitochondria via phosphorylation of Bcl-2 during brain ischemia and reperfusion. Taken together, these data suggest that, through phosphorylating Bcl-2 leading to the release of Bax from Bcl-2/Bax dimers, activation of JNK may increase the translocation of Bax to mitochondria and the subsequent release of cytochrome c, which can ultimately contribute to mitochondria-mediated apoptosis. Apoptosis may be executed via two predominant effector pathways, which eventually converge at the level of caspase-3
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activation (Hengartner, 2000). Recent data highlight the pivotal role of caspase-3 in the execution of ischemia-induced apoptosis (Zheng et al., 2003). Increased caspase-3-like protease activation in the hippocampal CA1 region that may be responsible for the delayed neuronal cell death after cerebral ischemia has been recently reported (Kuan et al., 2003). Caspase-3 inhibitors can prevent delayed neuronal death in the hippocampus after transient cerebral ischemia (Chang and Karin, 2001). Thus, caspase-3 is a key step in the execution process of apoptosis, and its inhibition can block apoptotic cell death. Our studies prove that activation of JNK may enhance the activation of caspase-3, which might take effect at the final step of the apoptotic cascade. In summary, the present study confirms that application of SP600125 effectively protects transient brain ischemia/ reperfusion-induced neuronal apoptosis in rat hippocampal CA1 region via suppressing the “intrinsic” (i.e., mitochondria) and the “extrinsic” (i.e., death receptor) apoptotic pathways. The study also identifies the release of Bax from Bcl-2/Bax dimers via phosphorylation of Bcl-2 as a novel mechanism through which JNK may activate the mitochondrial apoptosis signaling pathway in ischemic neurons. Taken together, these results indicate that targeting the JNK pathway provides a promising therapeutic approach for ischemic brain injury.
4.
Experimental procedures
4.1.
Animal surgical procedures
Adult male Sprague–Dawley rats (Shanghai Experimental Animal Center, Chinese Academy of Science) weighing 250– 300 g were given free access to food and water before surgery. Transient brain ischemia (15 min) was induced by the fourvessel occlusion method (4-VO) as described previously (Pulsinelli and Brierley, 1979). Briefly, under anesthesia with chloral hydrate (300–350 mg/kg, i.p.), vertebral arteries were electrocauterized and common carotid arteries were exposed. Rats were allowed to recover for 24 h and fasted overnight. Ischemia was induced by occluding the common arteries with aneurysm clips. Animals meeting the criteria of a completely flat bitemporal electroencephalograph, maintenance of dilated pupils and the absence of a corneal reflex during ischemia were selected for the present experiments. Carotid artery blood flow was restored by releasing the clips. During ischemia and reperfusion, rectal temperature was maintained at about 37 °C. The sham operation was performed using the same surgical exposure procedures except for occlusion of the carotid artery.
Fig. 6 – Representative hippocampal photomicrographs of TUNEL staining counterstained with methyl green and quantitative analyses of TUNEL-positive cells. (A) Rats were subjected to sham-operated (a, b) and 15 min of ischemia followed by 3 days of reperfusion (c, d) and rats subjected to 15 min of ischemia followed by 3 day of reperfusion with administration of DMSO (E, F), 30 μg/10 μl of SP600125 20 min before ischemia (g, h) and 60 min after ischemia (i, j). Some cells had shrunken, darkly stained nuclei and apoptotic bodies (arrowheads). Others had lightly stained, large and swollen nuclei. The cells with the latter features were not counted as TUNEL-positive cells. Data were obtained from six independent animals, and the results of a typical experiment are presented. Boxed areas in left column are shown at higher magnification in right column. A, C, E, G, I: ×40; B, D, F, H, J: ×400. Scale bar in I = 200 μm; scale bar in J = 10 μm. (B) Quantitative analyses of TUNEL-positive cells. All values shown are mean ± SD (n = 6). aP < 0.05 vs. sham; bP < 0.05 vs. 3 days after 15 min of ischemia group.
44 4.2.
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Tissue preparation
Rats were decapitated at specified time points after reperfusion under anesthesia, and the hippocampi were separated into CA1 and CA3/DG from hippocampal fissure and CA1 and CA3/DG were rapidly frozen in liquid nitrogen. Frozen tissue samples were homogenized in 1:10 (w/v) ice-cold homogenization buffer C containing 50 mM MOPS [3-(N-morpholino) ropanesulfonic acid, pH 7.4], 100 mM KCl, 320 mM sucrose, 0.5 mM MgCl, 0.2 mM dithiothreitol, phosphatase and protease inhibitors [20 mM β-glycerophosphate, 20 mM sodium pyrophosphate, 50 mM NaF, and 1 mM each of EGTA, EDTA, sodium orthovanadate, pnitrodomains phenyl phosphate (PNPP), phenylmethylsulfonyl fluoride (PMSF) and benzamidine, and 5 μg/ml each of aprotinin, leupeptin and pepstatin A]. The homogenates were centrifuged at 800 × g for 10 min at 4 °C. Supernatants were collected, and protein concentrations were determined by the method of Lowry et al. (1951). When necessary, cytosol fractions and nuclear fractions were extracted with some modifications of previously described procedure. Briefly, hippocampal CA1 tissue samples were homogenized in 1:10 (w/v) ice-cold homogenization buffer A containing 10 mM HEPES, pH 7.9, 0.5 mM MgCl2, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 50 mM NaF, 5 mM dithiothreitol (DTT), 10 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1% NP40 and enzyme inhibitors [1 mM benzamidine, pnitrodomains phenyl phosphate (PNPP), phenylmethylsulfonyl fluoride (PMSF) and 5 μg/ml each of aprotinin, leupeptin and pepstatin A] and then were centrifuged at 1000 × g for 10 min at 4 °C. Supernatants as cytosolic part were collected, and protein concentrations were determined by the method of Lowry et al. The nuclear pellets were extracted with homogenization buffer B containing 20 mM HEPES, pH 7.9, 20% glycerol, 420 mM NaCl, 0.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT and enzyme inhibitors for 30 min at 4 °C with constant agitation. After centrifugation at 12,000 × g for 15 min at 4 °C, supernatants as nuclear parts were collected and protein concentrations were determined. Samples were stored at −80 °C and were thawed only once. When necessary, the hippocampal CA1 was immediately isolated to prepare mitochondrial fractions. All procedures were conducted in a cold room. Nonfrozen brain tissue was used to prepare mitochondrial fractions because freezing tissue causes release of cytochrome c from mitochondria. The hippocampal CA1 tissues were homogenized in 1:10 (w/v) cold homogenization buffer C. The homogenates were centrifuged at 1000 × g for 10 min at 4 °C. The pellets were discarded, and supernatants were centrifuged at 17,000 × g for 20 min at 4 °C to get the cytosolic fraction in the supernatants and the crude mitochondrial fraction in the pellets. The protein concentrations were determined by the method of Lowry et al.
4.3.
Administration of drugs
When necessary, SP600125 (30 μg/10 μl) or vehicle (DMSO) was administered to rats by intracerebral ventricular infusion 20 min before ischemia or 1 h after ischemia. For administration of drugs, rats were positioned in a stereotaxic apparatus (Hamilton Company, USA). Drug infusion (10 μl) was performed using a microinjector through a preimplanted cannula
in the left cerebral ventricle (from the bregma: anteroposterior, −0.8 mm; lateral, 1.5 mm; depth, 3.5 mm).
4.4.
Immunoprecipitation and immunoblot
Tissue homogenates (400 μg of protein) were diluted four-fold with 50 mM HEPES buffer (pH 7.4), containing 10% glycerol, 150 mM NaCl, 1% Triton X-100, 0.5% NP-40, and 1 mM each of EDTA, EGTA, PMSF and Na3VO4. Samples were preincubated for 1 h with 20 μl protein A sepharose CL-4B (Amersham, Uppsala, Sweden) at 4 °C and then centrifuged to remove proteins adhered nonspecifically to protein A. The supernatants were incubated with 1–2 μg primary antibodies for 4 h or overnight at 4 °C. Protein A was added to the tube for another 2-h incubation. Samples were centrifuged at 10,000 × g for 2 min at 4 °C, and the pellets were washed with immunoprecipitation buffer for three times. Bound proteins were eluted by boiling at 100 °C for 5 min in SDS-PAGE loading buffer and then isolated by centrifuge. The supernatants were used for immunoblot analysis. Proteins were separated on polyacrylamide gels and then electrotransferred onto a nitrocellulose membrane (Amersham, Buckinghamshire, UK). After being blocked for 3 h in Tris-buffered saline with 0.1% Tween20 (TBST) and 3% bovine serum albumin (BSA), membranes were incubated overnight at 4 °C with primary antibodies in TBST containing 3% BSA. Membranes were then washed and incubated with alkalinephosphatase-conjugated secondary antibodies in TBST for 2 h and developed using NBT/BCIP color substrate (Promega, Madison, USA). The density of the bands on the membrane was scanned and analyzed with an image analyzer (LabWorks Software, UVP Upland, CA, USA).
4.5.
Histological assessment and immunohistochemistry
Rats were perfusion-fixed with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4) under anesthesia after 5 days of ischemia/reperfusion. Brains were removed quickly and further fixed with the same fixation solution at 4 °C overnight. Post-fixed brains were embedded by paraffin followed by preparation of coronal sections 5 μm thick using a microtome. The paraffin-embedded brain sections were deparaffinized with xylene and rehydrated by ethanol at graded concentrations of 100:70% (v/v) followed by washing with water. The sections were stained with 0.1% (w/v) cresyl violet and were examined with light microscopy, and the number of surviving hippocampal CA1 pyramidal cells per 1 mm length was counted as the neuronal density. Immunoreactivity was determined by the avidin–biotin– peroxidase method. Briefly, sections were deparaffinized with xylene and rehydrated by ethanol at graded concentrations and distilled water. High-temperature antigen retrieval was performed in 1 mM citrate buffer. To block endogenous peroxidase activity, sections were incubated for 30 min in 1% H2O2. After being blocked with 5% (v/v) normal goat serum in PBS for 1 h at 37 °C, sections were incubated with rabbit polyclonal antibodies against FasL (1:100) or cleaved caspase-3 protein (1:100) or mouse monoclonal antibody against p-c-Jun (1:50) at 4 °C for 2 days. These sections were then incubated with biotinylated goat-anti-rabbit/mouse secondary antibody
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overnight and subsequently with avidin-conjugated horseradish peroxidase for 1 h at 37 °C. Finally, sections were incubated with peroxidase substrate diaminobenzidine (DAB) until desired stain intensity develops. TUNEL staining was performed using an ApopTag® Peroxidase In Situ Apoptosis Detection Kit according to the manufacturer's protocol with minor modifications. The paraffin-embedded coronal sections were deparaffinized and rehydrated and then treated with protease K at 20 μg/ml for 15 min at room temperature. Sections were incubated with reaction buffer containing TdT enzyme and at 37 °C for 1 h. After washing with stop/wash buffer, sections were treated with anti-digoxigenin conjugate for 30 min at room temperature and subsequently developed color in peroxidase substrate. The nuclei were lightly counterstained with 0.5% methyl green.
4.6.
Antibodies and reagents
Mouse monoclonal anti-p-c-Jun (sc-822), rabbit polyclonal anti-FasL (sc-6237), rabbit polyclonal anti-Bcl-2 (sc-492) and rabbit polyclonal anti-Fas (sc-1023) were purchased from Santa Cruz Biotechnology. Rabbit polyclonal anti-Bax (#2772), rabbit polyclonal anti-cytochrome c (#4272) and rabbit polyclonal anti-cleaved caspase-3 (#9661) were obtained from Cell Signal Biotechnology. The secondary antibodies used in our experiment were goat anti-mouse IgG, goat antirabbit IgG and donkey anti-goat IgG. They were from Sigma. ApopTag® Peroxidase In Situ Apoptosis Detection Kit (S7100) was purchased from Chemicon.
4.7.
Statistical evaluation
Values were expressed as mean ± SD and obtained from six independent rats in every group. Statistical analysis of the results was carried out by Student's t test or one-way analysis of the variance (ANOVA) followed by the Duncan's new multiple range method or Newman–Keuls test. P values <0.05 were considered significant.
Acknowledgment This work was supported by a grant from the Key Project of the National Natural Science Foundation of China (No. 30330190).
REFERENCES
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Carboni, S., Antonsson, B., Gaillard, P., Gotteland, J.P., Gillon, J.Y., Vitte, P.A., 2005. Control of death receptor and mitochondrial-dependent apoptosis by c-Jun N-terminal kinase in hippocampal CA1 neurons following global transient ischaemia. J. Neurochem. 92, 1054–1060. Chang, L., Karin, M., 2001. Mammalian MAP kinase signalling cascade. Nature 410, 37–40. Cohen, G.M., 1997. Caspases: the executioners of apoptosis. Biochem. J. 326, 1–16. Davis, R.J., 2000. Signal transduction by the JNK group of MAP kinases. Cell 103, 239–252. Faris, M., Latinis, K.M., Kempiak, S.J., Koretzky, G.A., Nel, A., 1998. Stress-induced Fas ligand expression in T cells is mediated through a MEK kinase 1-regulated response element in the Fas ligand promoter. Mol. Cell. Biol. 18, 5414–5424. Gao, Y.Q., Signore, A.P., Yin, W., Cao, G.D., Yin, X.M., Sun, F.Y., Luo, Y.M., Graham, S.H., Chen, J., 2005. Neuroprotection against focal ischemic brain injury by inhibition of c-Jun N-terminal kinase and attenuation of the mitochondrial apoptosis-signaling pathway. J. Cereb. Blood Flow Metab. 25, 694–712. Green, D.R., Reed, J.C., 1998. Mitochondria and apoptosis. Science 281, 1309–1312. Guan, Q.H., Pei, D.S., Zhang, Q.G., Hao, Z.B., Xu, T.L., Zhang, G.Y., 2005. The neuroprotective action of SP600125, a new inhibitor of JNK, on transient brain ischemia/ reperfusion-induced neuronal death in rat hippocampal CA1 via nuclear and non-nuclear pathways. Brain Res. 1035, 51–59. Haldar, S., Chintapalli, J., Croce, C.M., 1996. Taxol induces bcl-2 phosphorylation and death of prostate cancer cells. Cancer Res. 56, 1253–1255. Han, Z., Boyle, D.L., Chang, L., Bennett, B., Karin, M., Yang, L., Manning, A.M., Firestein, G.S., 2001. c-Jun N-terminal kinase is required for metalloproteinase expression and joint destruction in inflammatory arthritis. J. Clin. Invest. 108, 73–81. Hengartner, M.O., 2000. The biochemistry of apoptosis. Nature (London) 407, 770–775. Irving, E.A., Bamford, M., 2002. Role of mitogen and stress activated kinases in ischemic injury. J. Cereb. Blood Flow Metab. 22, 631–647. Kuan, C.Y., Whitmarsh, A.J., Yang, D.D., Liao, G., Schloemer, A.J., Dong, C., Bao, J., Banasiak, K.J., Haddad, G.G., Flavell, R.A., Davis, R.J., Rakic, P., 2003. A critical role of neural-specific JNK3 for ischemic apoptosis. Proc. Natl. Acad. Sci. U. S. A. 100, 15184–15189. Lei, K., Davis, R.J., 2003. JNK phosphorylation of Bim-related members of the Bcl2 family induces Bax-dependent apoptosis. Proc. Natl. Acad. Sci. U. S. A. 100, 2432–2437. Le-Niculescu, H., Bonfoco, E., Kasuya, Y., Claret, F.X., Green, D.R., Karin, M., 1999. Withdrawal of survival factors results in activation of the JNK pathway in neuronal cells leading to Fas ligand induction and cell death. Mol. Cell. Biol. 19, 751–763. Lin, A., 2003. Activation of the JNK signaling pathway: breaking the break on apoptosis. BioEssays 25, 1–8. Lowry, O.H., Rosebrough, H.J., Farr, A.L., 1951. Protein measurement with Folin-Phenol reagent. J. Biol. Chem. 193, 265–275. Martin-Villalba, A., Herr, I., Jeremias, I., Hahne, M., Brandt, R., Vogel, J., Schenkel, J., Herdegen, T., Debatin, K.M., 1999. CD95 ligand (Fas-L/APO-1L) and tumor necrosis factor-related apoptosis-inducing ligand mediate ischemia-induced apoptosis in neurons. J. Neurosci. 19, 3809–3817. Okuno, S., Saito, A., Hayashi, T., Chan, P.H., 2004. The c-Jun N-terminal protein kinase signaling pathway mediates Bax activation and subsequent neuronal apoptosis through
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interaction with Bim after transient focal cerebral ischemia. J. Neurosci. 24, 7879–7887. Padosch, S.A., Popp, E., Vogel, P., Bottiger, B.W., 2003. Altered protein expression levels of Fas/CD95 and Fas ligand in differentially vulnerable brain areas in rats after global cerebral ischemia. Neurosci. Lett. 338, 247–251. Pulsinelli, W.A., Brierley, J.B., 1979. A new model of bilateral hemispheric ischemia in the unanesthetized rat. Stroke 10, 267–272. Putcha, G., Le, S., Frank, S., Besirli, C., Clark, K., Chu, B., Alix, S., Youle, R., LaMarche, A., Maroney, A., 2003. JNK-mediated BIM phosphorylation potentiates BAX-dependent apoptosis. Neuron 38, 899–914. Saporito, M.S., Hudkins, R.L., Maroney, A.C., 2002. Discovery of CEP-1347/KT-7515, an inhibitor of the JNK/SAPK pathway for
the treatment of neurodegenerative diseases. Prog. Med. Chem. 40, 23–62. Schroeter, H., Boyd, C.S., Ahmed, R., Spencer, J.P., Duncan, R.F., Rice-Evans, C., Cadenas, E., 2003. c-Jun N-terminal kinase (JNK)-mediated modulation of brain mitochondria function: new target proteins for JNK signaling in mitochondrion-dependent apoptosis. Biochem. J. 372, 359–369. Shaulian, E., Karin, M., 2002. AP-1 as a regulator of cell life and death. Nat. Cell Biol. 4, E131–E136. Thornberry, N.A., Lazebnik, Y., 1998. Caspases: enemies within. Science 281, 1312–1316. Zheng, Z., Zhao, H., Steinberg, G.K., Yenari, M.A., 2003. Cellular and molecular events underlying ischemia-induced neuronal apoptosis. Drug News Perspect. 16, 497–503.
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Neuroprotection against ischemic brain injury by SP600125 via suppressing the extrinsic and intrinsic pathways of apoptosis Qiu-Hua Guana,b , Dong-Sheng Pei b , Xiao-Mei Liua , Xiao-Tian Wanga , Tian-Le Xub,c , Guang-Yi Zhanga,b,⁎ a
Research Center for Biochemistry and Molecular Biology, Xuzhou Medical College, Xuzhou 221002, PR China Department of Neurobiology and Biophysics, School of Life Science, University of Science and Technology of China, Hefei 230027, PR China c Institute of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, PR China b
A R T I C LE I N FO
AB S T R A C T
Article history:
Our previous studies and the others have strongly suggested that JNK signaling pathway
Accepted 22 March 2006
plays a critical role in ischemic brain injury. Here, we reported that SP600125, a potent, cell-
Available online 3 May 2006
permeable, selective, and reversible inhibitor of c-Jun N-terminal kinase (JNK), potently decrease neuronal apoptosis induced by global ischemia/reperfusion in the vulnerable
Keywords:
hippocampal CA1 subregion. As a result, SP600125 diminished the increased phosphorylation
c-Jun N-terminal protein kinase
of c-Jun and the increased expression of FasL induced by ischemia/reperfusion in the vulnerable
(JNK)
hippocampal CA1 subregion. At the same time, through inhibiting phosphorylation of Bcl-2 and
Cerebral ischemia/reperfusion
the release of Bax from Bcl-2/Bax dimers, SP600125 attenuated Bax translocation to mitochondria
Extrinsic and intrinsic pathways
and the release of cytochrome c induced by ischemia/reperfusion (I/R). Furthermore, the
of apoptosis
activation of caspase-3 induced by ischemia/reperfusion was also significantly suppressed by
SP600125
preinfusion of SP600125. Importantly, the same neuropotective effect was showed by administration of SP600125 both before and after ischemia. Thus, our findings imply that SP600125 can inhibit the activation of JNK signaling pathway and induce neuroprotection against ischemia/reperfusion in rat hippocampal CA1 region via suppressing the extrinsic and intrinsic pathways of apoptosis. Taken together, these results indicate that targeting the JNK pathway provides a promising therapeutic approach for ischemic brain injury. © 2006 Elsevier B.V. All rights reserved.
1.
Introduction
Neuronal apoptosis has been implicated in cerebral ischemia. However, the apoptotic process is regulated by many intracellular signaling pathways, including the JNK signaling pathway (Chang and Karin, 2001; Davis, 2000; Lin, 2003; Shaulian and Karin, 2002). The c-Jun N-terminal protein kinase (JNK) signaling pathway is implicated in neuronal
apoptosis triggered by focal or global ischemia (Irving and Bamford, 2002). Furthermore, the study by Kuan et al. (2003), based on gene-knockout approaches, convincingly demonstrated the critical role of JNK3, the neural-specific JNK, in ischemia-induced apoptosis in the brain. However, the downstream mechanism that accounts for the proapoptotic actions of JNK during cerebral ischemia/reperfusion remains to be investigated in detail. Understanding the
⁎ Corresponding author. Research Center for Biochemistry and Molecular Biology, Xuzhou Medical College, 84 West Huai-hai Road, Xuzhou, Jiangsu, 221002, PR China. Fax: +86 516 574 8486. E-mail address: [email protected] (G.-Y. Zhang). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.03.086
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molecular mechanisms by which JNK regulates apoptosis should provide insights into treatment of cerebral ischemia. In mammals, signaling cascades culminating in apoptotic cell death can be divided into two broad categories: the “intrinsic” (i.e., mitochondria) and the “extrinsic” (i.e., death receptor) pathways (Ashkenazi and Dixit, 1998; Cohen, 1997; Green and Reed, 1998; Thornberry and Lazebnik, 1998). In the intrinsic pathway, various proapoptotic signals induce the release of mitochondrial proteins, such as cytochrome c, through some proapoptotic Bcl-2 family proteins which may form a pore and/or open permeability transition pore. Bax is a potent regulator of mitochondria-dependent apoptosis. The translocation of Bax to mitochondria causes the loss of mitochondrial membrane potential and the full opening of permeability transition pore (PTP) and results in the release of cytochrome c. Once released, cytochrome c interacts with Apaf-1 and deoxyadenosine triphosphate forming the apoptosome and leading to activation of cytochrome-c-dependent caspase cascade, resulting in apoptosis. In contrast, so-called “extrinsic” pathway signals, such as those mediated by death receptors of the TNF receptor superfamily, activate the caspase cascade more directly. For example, interaction of Fas with its ligand (FasL) triggers formation of a deathinducing signaling complex (DISC), which in turn recruits and activates caspase-8. Caspase-8 then activates other procaspases, culminating in cleavage of cellular substrates, and apoptosis. The recent development of chemical inhibitors of the JNK pathway has greatly accelerated our understanding of the downstream signaling pathway of JNK in ischemic neurodegeneration. Two effective inhibitors, CEP-1347 (previously called KT7515) and SP600125, have been recently reported. The actions of CEP-1347 (previously called KT7515) have been attributed to the inhibition of the JNK upstream activators, the mixed lineage kinases (Saporito et al., 2002). SP600125 has been reported to function as a reversible ATP competitive inhibitor of JNK MAPKs (Bennett et al., 2001; Han et al., 2001), which shows 300-fold selectivity of inhibition of JNK over the extracellular signal-regulated kinases (ERKs) and p38 MAPKs, the closest kinase relatives of JNK. In the present study, we showed that SP600125, a potent, cell-permeable, selective, and reversible inhibitor of c-Jun N-terminal kinase (JNK), could potently decrease neuronal apoptosis induced by global ischemia/reperfusion in the vulnerable hippocampal CA1 subregion via inhibiting the extrinsic and intrinsic pathways of apoptosis.
2.
Results
2.1. The effect of SP600125 on the increased expression of FasL induced by global ischemia/reperfusion in hippocampal CA1 To elucidate the involvement of Fas-receptor-mediated pathway in the apoptotic program during global ischemia/reperfusion injury, the expression of FasL and Fas was analyzed by Western blotting. The expression of FasL was significantly increased from 3 h to 1 day reperfusion, but the expression of Fas was not significantly increased at various time points after
37
15 min of ischemia (Fig. 1A). In the present study, we examined the effect of SP600125 on the expression of FasL and Fas. As shown in Fig. 1B, results of Western blotting revealed that the increased expression of FasL at 6 h reperfusion was significantly suppressed by preinfusion of SP600125. The same dose of vehicle 1% DMSO did not affect the increase on the expression of FasL. The protein level of Fas was not affected by SP600125 and vehicle 1% DMSO.
Fig. 1 – Effects of pretreatment with SP600125 on the increased expression of FasL and Fas. (A) Time courses of the expression of FasL and Fas in hippocampal CA1 derived from sham-treated rats or rats at various time points after 15 min of ischemia. (B) Effects of pretreatment with SP600125 on the increases of expression of FasL and Fas induced by 6 h of reperfusion following transient brain ischemia (I/R6h) in hippocampal CA1. Western blot probed with antibodies to FasL and Fas. Bands corresponding to FasL and Fas were scanned, and the intensities were represented as folds vs. sham control. Data are the mean ± SD and were expressed as folds vs. respective sham. aP < 0.05 vs. sham; bP < 0.05 vs. 6 h after 15 min of ischemia groups (n = 6 rats).
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2.2. The effect of SP600125 on the phosphorylation of c-Jun induced by global ischemia/reperfusion in hippocampal CA1 A JNK-dependent element in the Fas ligand promoter that binds c-Jun and ATF2 has been identified (Faris et al., 1998). Our previous studies showed that activated JNK induced neuronal death via phosphorylating c-Jun and promoting its transcription activity (Guan et al., 2005). In the present study, we examined the effect of SP600125 on phosphorylation of cJun by immunohistochemical method (Fig. 2). In the sham group (Figs. 2A, B), week c-Jun immunoreactivity was detected in the nucleus of hippocampal CA1. Six hours reperfusion after ischemia (Figs. 2C, D), p-c-Jun immunoreactivity significantly increased compared with the sham group. There was no inhibitory effect of intracerebral ventricular preinfusion of 1% DMSO on p-c-Jun immunoreactivity 6 h reperfusion after
ischemia (Figs. 2E, F). Administration of SP600125 20 min before cerebral ischemia (Figs. 2G, H) and 1 h after cerebral ischemia (Figs. 2I, J) significantly inhibited p-c-Jun immunoreactivity 6 h reperfusion after ischemia.
2.3. The effect of SP600125 on the release Bax from Bcl-2/Bax dimers induced by global ischemia/reperfusion in hippocampal CA1 Our previous studies showed that activated JNK induced neuronal death via phosphorylating Bcl-2 and inhibiting its prosuvival activity (Guan et al., 2005). In this study, we found that phosphorylated Bcl-2 was not shown to interact with Bax during ischemia and reperfusion in hippocampal CA1 (data not shown). Furthermore, our results showed that ischemia/ reperfusion decreased interaction of Bcl-2 with Bax and SP600125 inhibited the decreased interaction of Bcl-2 with
Fig. 2 – Immunohistochemical staining of phosphorylation of c-Jun in coronal sections of hippocampus and the inhibitory effect of SP600125 on phosphorylation of c-Jun. Example of immunohistochemical staining sections of the hippocampi of sham-operated rats (A, B) and rats subjected to 15 min of ischemia followed by 6 h of reperfusion (C, D) and rats subjected to 15 min of ischemia followed by 6 h of reperfusion with administration of DMSO (E, F), 30 μg/10 μl of SP600125 20 min before ischemia (G, H) and 60 min after ischemia (I, J). Data were obtained from six independent animals in each experimental group, and the results of a typical experiment are presented. Boxed areas in left column are shown at higher magnification in right column. A, C, E, G, I: ×40; B, D, F, H, J: ×400. Scale bar in I = 200 μm; scale bar in J = 10 μm.
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Bax (Fig. 3). Therefore, we speculate that ischemia/reperfusion could promote the release of Bax from Bcl-2/Bax dimers via JNK phosphorylating Bcl-2.
2.4. The effect of SP600125 on translocation of Bax to mitochondria and the release of cytochrome c from mitochondria induced by global ischemia/reperfusion in hippocampal CA1 To elucidate the involvement of “intrinsic”-mediated apoptotic pathway during global ischemia/reperfusion injury and the action of JNK activity on Bax translocation and the release of cytochrome c, expression of Bax and cytochrome c in mitochondria and cytosol was examined by Western blotting. We first determine whether Bax translocates from cytosol to mitochondria after ischemia. Using Western blotting analysis and subcellular fraction, we found that the expression of Bax was significantly increased from 3 h to 1 day after ischemia in the mitochondria, but the expression of Bax was not significantly decreased at various time points after 15 min of ischemia in cytosol (Fig. 4A). We supposed that the overwhelming majority of Bax were located in cytosol, thus partial translocation of Bax did not obviously affect the total protein level of Bax in cytosol. Moreover, we examined whether the inhibition of JNK by SP600125 contributes to attenuating Bax
39
translocation. The inhibitory effect of SP600125 on Bax translocation in the mitochondrial fraction reached a statistical difference 6 h after reperfusion compared with that of vehicle 1% DMSO (Fig. 4B). Cytochrome c immunoreactivity was evident as a single band of molecular mass of 15 kDa in the cytosolic fraction of the hippocampal CA1 6 h to 3 days after global ischemia. However, it was barely detected in the sham CA1 subregion. A significant amount of mitochondrial cytochrome c was detected in the controls and decreased after ischemia, corresponding to a marked increase in the cytosolic fraction (Fig. 4C). Moreover, we examined whether the activation of JNK signaling pathway contributes to the release of cytochrome c. The inhibitory effect of SP600125 on the release of cytochrome c in the cytosol fraction reached a statistical difference 6 h after reperfusion compared with the vehicle 1% DMSO (Fig. 4D). To elucidate whether other mitochondrial proteins were released from mitochondria, we determined the cytochrome c oxidase level in the cytosolic and mitochondrial fraction using cytochrome c oxidase subunit IV antibody. The cytochrome c oxidase subunit IV was examined only in the mitochondrial fraction but not in the cytosolic fraction in sham, ischemia/reperfusion and application of drug groups (Figs. 4A, B, C and D). These results suggested that cytochrome c oxidase was not coreleased with cytochrome c from mitochondria.
2.5. The effect of SP600125 on the activation of caspase-3 induced by global ischemia/reperfusion in hippocampal CA1 Caspase-3 is one of the key executioners in extrinsic and intrinsic apoptotic pathways. It is a cytosolic protein as an inactive 32 kDa proenzyme and is activated by proteolytic cleavage into the 20 kDa (p20) and 11 kDa (p11) active subunits. In this study, we examined the activation of caspase-3 during ischemia/reperfusion and the action of inhibition of JNK activity by SP600125 on activation of caspase-3. Immunohistochemical observation was carried out using anti-cleaved caspase-3 antibody. As shown in Fig. 5, weak cleaved caspase-3 immunoreactivity was detected in the cytosol of hippocampal CA1 in the sham group (Figs. 5A, B), cleaved caspase-3 immunoreactivity significantly increased 6 h reperfusion after ischemia (Figs. 5C, D) compared with the sham group. There was no inhibitory effect of intracerebral ventricular preinfusion of 1% DMSO on cleaved caspase-3 immunoreactivity 6 h reperfusion after ischemia (Figs. 5E, F). Cleaved caspase-3 immunoreactivity was significantly inhibited at 6 h reperfusion by administration of SP600125 20 min before cerebral ischemia (Figs. 5G, H) and 1 h after cerebral ischemia (Figs. 5I, J).
2.6. Neuroprotective role of SP600125 against cerebral ischemia Fig. 3 – Effects of SP600125 on altered interactions Bcl-2 with Bax induced by ischemia/reperfusion in hippocampal CA1. (A) Co-immunoprecipitation analysis of interactions between Bcl-2 and Bax. (B) Bands corresponding to Bax and Bcl-2 were scanned, and the intensities were represented as folds vs. sham control. Data were expressed as mean ± SD (n = 6). a,c P < 0.05 vs. respective sham; b,dP < 0.05 vs. respective vehicle treatment group.
To investigate whether pretreatment of SP600125 would have neuroprotection against ischemia-induced apoptotic cell death, adult Sprague–Dawley rats were subjected to 15 min ischemia followed by 5 days reperfusion. Rats were pretreated with SP600125 or 1% DMSO by cerebral ventricular injection 20 min before ischemia and 1 h after ischemia. After 3 days reperfusion, rats were perfusion-fixed with
40
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Fig. 4 – Effects of pretreatment with SP600125 on the translocation of Bax and the release of cytochrome c. (A) Time courses of the expression of Bax in cytosol and mitochondria in hippocampal CA1 derived from sham-treated rats or rats at various time points after 15 min of ischemia. (B) Effects of pretreatment with SP600125 on the expression of Bax induced by 6 h of reperfusion following transient brain ischemia (I/R6h) in cytosol and mitochondria in hippocampal CA1. (C) Western blot analysis of cytochrome c and cytochrome oxidase 4 in rat hippocampal CA1 subregion. Cytochrome c immunoreactivity is evident as a single band of molecular mass (15 kDa) in the cytosolic and mitochondrial fraction 6 h to 3 days after ischemia, but not in the sham CA1 tissue. (D) Effects of pretreatment with SP600125 on the expression of cytochrome c induced by 6 h of reperfusion following transient brain ischemia (I/R6h) in cytosol and mitochondria in hippocampal CA1. Cytochrome oxidase 4 (COX4) was strongly expressed in the mitochondrial fraction and did not decrease after ischemia, but virtually no immunoreactivity was seen in the cytosolic fraction in both the sham and ischemic CA1 subregions. Bands corresponding to Bax were scanned, and the intensities were represented as folds vs. sham control. Data are the mean ± SD and were expressed as folds vs. respective sham. aP < 0.05 vs. sham; bP < 0.05 vs. 6 h after 15 min of ischemia group (n = 6 rats).
paraformaldehyde and TUNEL staining was used to examine the apoptosis of CA1 pyramidal cells in hippocampus (Fig. 6). A significant number of positive cells were observed 3 days after ischemia (Figs. 6Ac, d). Some of them showed characteristic appearances, such as shrunken, condensed nuclei and apoptotic bodies. However, others showed lightly stained, large and swollen nuclei. We excluded the cells with the latter features from the TUNEL positive cells because they might have contained necrotic cells. As the results shown, administration of SP600125 20 min before cerebral ischemia significantly decreased TUNEL-positive cells (Figs. 6Ag, h). Administration of SP600125 1 h after cerebral ischemia also significantly decreased TUNEL-positive cells (Figs. 6Ai, j). At the same time, as the control, 1% DMSO did not show any protection (Figs. 6Ae, f).
3.
Discussion
Our early study and the others strongly suggest that activation of JNK signaling may play a critical role in brain ischemia injury (Gao et al., 2005; Guan et al., 2005; Irving and Bamford, 2002; Kuan et al., 2003). Therefore, JNK is an important therapeutic target for prevention of neuronal death induced by brain ischemia. In the present study, results from histological assessment showed that transient global cerebral ischemia lead to cell apoptosis of hippocampal CA1 pyramidal neurons at 3 days after ischemia and application of SP600125 could prevent hippocampal CA1 neurons from apoptosis after cerebral ischemia/reperfusion. Moreover, the protective effect of SP600125 shown here is likely clinically
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41
Fig. 5 – Immunohistochemical staining of expression of cleaved caspase-3 in coronal sections of hippocampus and the inhibitory effect of SP600125 on expression of cleaved caspase-3. Example of immunohistochemical staining sections of the hippocampi of sham-operated rats (A, B) and rats subjected to 15 min of ischemia followed by 6 h of reperfusion (C, D), and rats subjected to 15 min of ischemia followed by 6 h of reperfusion with administration of DMSO (E, F), 30 μg/10 μl of SP600125 20 min before ischemia (G, H) and 60 min after ischemia (I, J). Data were obtained from six independent animals in each experimental group, and the results of a typical experiment are presented. Boxed areas in the left column are shown at higher magnification in right column. A, C, E, G, I: ×40; B, D, F, H, J: ×400. Scale bar in I = 200 μm; scale bar in J = 10 μm.
relevant as significant neuroprotection was achieved when SP600125 was administered either before or after the onset of ischemia. Our previous studies indicated that SP600125, a reversible ATP-competitive JNK inhibitor, rescued transient brain ischemia/reperfusion-induced neuronal death in rat hippocampal CA1 subregion via suppressing the activation of nuclear substrate (c-Jun) and the phosphorylation of non-nuclear substrate (Bcl-2) induced by ischemic insult (Guan et al., 2005). Activated JNK phosphorylates the transcription factor cJun and leads to increase AP-1 transcription activity to modulate transcription of a number of genes, which lead to apoptosis. For example, a JNK-dependent element in the Fas ligand promoter that binds c-Jun and ATF2 has been identified (Faris et al., 1998). Results from our current studies showed
that pretreatment of SP600125 could diminish the increased expression of FasL and interaction of FasL with Fas induced by ischemia and reperfusion. Previous studies indicated that neuronal protection was conferred by a c-Jun mutant lacking JNK phosphoacceptor sites, which inhibited FasL induction by withdrawal of survival factors in PC12 cells (Le-Niculescu et al., 1999). Furthermore, Martin-Villalba et al. (1999) showed that FasL mRNA and protein were induced after cerebral ischemia and reduced by the treatment of FK506, which prevented the phosphorylation of c-Jun. In addition, Padosch et al. (2003) reported that FasL was induced in the thalamus but not in the hippocampus after global cerebral ischemia. However, Gao et al. studies in a murine model of focal ischemia and reperfusion showed that SP600125 attenuated ischemia-induced expression of Fas, but not the expression
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of FasL (Gao et al., 2005). Taken together, these data suggested that, through c-Jun/AP-1-mediated transcriptional regulation, activation of JNK could enhance the expression of FasL or Fas, which can ultimately contribute to Fas-mediated apoptosis.
Now, accumulating evidences suggest that mitochondriamediated apoptotic pathway is one of the downstream mechanisms by which JNK promotes neuronal cell death (Carboni et al., 2005; Kuan et al., 2003; Okuno et al., 2004; Putcha et al., 2003). Consistent with these studies, our
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results also showed that SP600125 inhibited the translocation of Bax to mitochondria and the release of cytochrome c induced by global ischemia and reperfusion. Recent studies have suggested that activated JNK may directly interact with mitochondria and results in the release of cytochrome c and Smac from mitochondria (Schroeter et al., 2003). The underlying mechanism for this direct effect of JNK is unclear, but it may involve JNK-mediated phosphorylation of certain Bcl-2 family members. There is a lack of evidence that activated JNK may directly phosphorylate Bax. Therefore, JNK may phosphorylate other Bcl-2 family members leading to the translocation of Bax to mitochondria. For example, it has been reported that JNK phosphorylation of Bim-related members of the Bcl-2 family induced Baxdependent apoptosis (Lei and Davis, 2003; Okuno et al., 2004). Other results also revealed that JNK activation caused serine phosphorylation of 14-3-3, a cytoplasmic sequestration protein of Bax, leading to Bax disassociation from 14-3-3 and subsequent translocation to mitochondria (Gao et al., 2005). Moreover, recent results suggest that activated JNK phosphorylated Bcl-2 and were associated with the phosphorylated form of Bcl-2 in the control of apoptosis following paclitaxel treatment (Brichese et al., 2004). Our previous studies have also indicated that JNK promoted transient brain ischemia/reperfusion-induced neuronal death via phosphorylation Bcl-2 and decreasing the anti-apoptotic function of Bcl-2 (Kuan et al., 2003). Phosphorylation inactivates Bcl-2, thus promoting apoptosis, possibly by freeing Bax from Bcl-2/Bax dimers (Haldar et al., 1996). The Bcl-2/Bax heterodimer is the active component for death protection, but in response to apoptotic stimulation, Bax is freed from Bcl-2/Bax dimers and forms ion channels and pores in mitochondrial membranes by large homoligomers and results in the release of cytochrome c. Current study found that phosphorylated Bcl-2 did not interact with Bax, resulting in the decrease of interaction of Bcl-2 with Bax during ischemia and reperfusion in hippocampal CA1. Our results also showed that SP600125 inhibited the phosphorylation of Bcl-2 and increased the interaction of Bcl-2 with Bax. Therefore, JNK could trigger the release of Bax from Bcl2/Bax dimers and subsequent translocation of Bax to mitochondria via phosphorylation of Bcl-2 during brain ischemia and reperfusion. Taken together, these data suggest that, through phosphorylating Bcl-2 leading to the release of Bax from Bcl-2/Bax dimers, activation of JNK may increase the translocation of Bax to mitochondria and the subsequent release of cytochrome c, which can ultimately contribute to mitochondria-mediated apoptosis. Apoptosis may be executed via two predominant effector pathways, which eventually converge at the level of caspase-3
43
activation (Hengartner, 2000). Recent data highlight the pivotal role of caspase-3 in the execution of ischemia-induced apoptosis (Zheng et al., 2003). Increased caspase-3-like protease activation in the hippocampal CA1 region that may be responsible for the delayed neuronal cell death after cerebral ischemia has been recently reported (Kuan et al., 2003). Caspase-3 inhibitors can prevent delayed neuronal death in the hippocampus after transient cerebral ischemia (Chang and Karin, 2001). Thus, caspase-3 is a key step in the execution process of apoptosis, and its inhibition can block apoptotic cell death. Our studies prove that activation of JNK may enhance the activation of caspase-3, which might take effect at the final step of the apoptotic cascade. In summary, the present study confirms that application of SP600125 effectively protects transient brain ischemia/ reperfusion-induced neuronal apoptosis in rat hippocampal CA1 region via suppressing the “intrinsic” (i.e., mitochondria) and the “extrinsic” (i.e., death receptor) apoptotic pathways. The study also identifies the release of Bax from Bcl-2/Bax dimers via phosphorylation of Bcl-2 as a novel mechanism through which JNK may activate the mitochondrial apoptosis signaling pathway in ischemic neurons. Taken together, these results indicate that targeting the JNK pathway provides a promising therapeutic approach for ischemic brain injury.
4.
Experimental procedures
4.1.
Animal surgical procedures
Adult male Sprague–Dawley rats (Shanghai Experimental Animal Center, Chinese Academy of Science) weighing 250– 300 g were given free access to food and water before surgery. Transient brain ischemia (15 min) was induced by the fourvessel occlusion method (4-VO) as described previously (Pulsinelli and Brierley, 1979). Briefly, under anesthesia with chloral hydrate (300–350 mg/kg, i.p.), vertebral arteries were electrocauterized and common carotid arteries were exposed. Rats were allowed to recover for 24 h and fasted overnight. Ischemia was induced by occluding the common arteries with aneurysm clips. Animals meeting the criteria of a completely flat bitemporal electroencephalograph, maintenance of dilated pupils and the absence of a corneal reflex during ischemia were selected for the present experiments. Carotid artery blood flow was restored by releasing the clips. During ischemia and reperfusion, rectal temperature was maintained at about 37 °C. The sham operation was performed using the same surgical exposure procedures except for occlusion of the carotid artery.
Fig. 6 – Representative hippocampal photomicrographs of TUNEL staining counterstained with methyl green and quantitative analyses of TUNEL-positive cells. (A) Rats were subjected to sham-operated (a, b) and 15 min of ischemia followed by 3 days of reperfusion (c, d) and rats subjected to 15 min of ischemia followed by 3 day of reperfusion with administration of DMSO (E, F), 30 μg/10 μl of SP600125 20 min before ischemia (g, h) and 60 min after ischemia (i, j). Some cells had shrunken, darkly stained nuclei and apoptotic bodies (arrowheads). Others had lightly stained, large and swollen nuclei. The cells with the latter features were not counted as TUNEL-positive cells. Data were obtained from six independent animals, and the results of a typical experiment are presented. Boxed areas in left column are shown at higher magnification in right column. A, C, E, G, I: ×40; B, D, F, H, J: ×400. Scale bar in I = 200 μm; scale bar in J = 10 μm. (B) Quantitative analyses of TUNEL-positive cells. All values shown are mean ± SD (n = 6). aP < 0.05 vs. sham; bP < 0.05 vs. 3 days after 15 min of ischemia group.
44 4.2.
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Tissue preparation
Rats were decapitated at specified time points after reperfusion under anesthesia, and the hippocampi were separated into CA1 and CA3/DG from hippocampal fissure and CA1 and CA3/DG were rapidly frozen in liquid nitrogen. Frozen tissue samples were homogenized in 1:10 (w/v) ice-cold homogenization buffer C containing 50 mM MOPS [3-(N-morpholino) ropanesulfonic acid, pH 7.4], 100 mM KCl, 320 mM sucrose, 0.5 mM MgCl, 0.2 mM dithiothreitol, phosphatase and protease inhibitors [20 mM β-glycerophosphate, 20 mM sodium pyrophosphate, 50 mM NaF, and 1 mM each of EGTA, EDTA, sodium orthovanadate, pnitrodomains phenyl phosphate (PNPP), phenylmethylsulfonyl fluoride (PMSF) and benzamidine, and 5 μg/ml each of aprotinin, leupeptin and pepstatin A]. The homogenates were centrifuged at 800 × g for 10 min at 4 °C. Supernatants were collected, and protein concentrations were determined by the method of Lowry et al. (1951). When necessary, cytosol fractions and nuclear fractions were extracted with some modifications of previously described procedure. Briefly, hippocampal CA1 tissue samples were homogenized in 1:10 (w/v) ice-cold homogenization buffer A containing 10 mM HEPES, pH 7.9, 0.5 mM MgCl2, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 50 mM NaF, 5 mM dithiothreitol (DTT), 10 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1% NP40 and enzyme inhibitors [1 mM benzamidine, pnitrodomains phenyl phosphate (PNPP), phenylmethylsulfonyl fluoride (PMSF) and 5 μg/ml each of aprotinin, leupeptin and pepstatin A] and then were centrifuged at 1000 × g for 10 min at 4 °C. Supernatants as cytosolic part were collected, and protein concentrations were determined by the method of Lowry et al. The nuclear pellets were extracted with homogenization buffer B containing 20 mM HEPES, pH 7.9, 20% glycerol, 420 mM NaCl, 0.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT and enzyme inhibitors for 30 min at 4 °C with constant agitation. After centrifugation at 12,000 × g for 15 min at 4 °C, supernatants as nuclear parts were collected and protein concentrations were determined. Samples were stored at −80 °C and were thawed only once. When necessary, the hippocampal CA1 was immediately isolated to prepare mitochondrial fractions. All procedures were conducted in a cold room. Nonfrozen brain tissue was used to prepare mitochondrial fractions because freezing tissue causes release of cytochrome c from mitochondria. The hippocampal CA1 tissues were homogenized in 1:10 (w/v) cold homogenization buffer C. The homogenates were centrifuged at 1000 × g for 10 min at 4 °C. The pellets were discarded, and supernatants were centrifuged at 17,000 × g for 20 min at 4 °C to get the cytosolic fraction in the supernatants and the crude mitochondrial fraction in the pellets. The protein concentrations were determined by the method of Lowry et al.
4.3.
Administration of drugs
When necessary, SP600125 (30 μg/10 μl) or vehicle (DMSO) was administered to rats by intracerebral ventricular infusion 20 min before ischemia or 1 h after ischemia. For administration of drugs, rats were positioned in a stereotaxic apparatus (Hamilton Company, USA). Drug infusion (10 μl) was performed using a microinjector through a preimplanted cannula
in the left cerebral ventricle (from the bregma: anteroposterior, −0.8 mm; lateral, 1.5 mm; depth, 3.5 mm).
4.4.
Immunoprecipitation and immunoblot
Tissue homogenates (400 μg of protein) were diluted four-fold with 50 mM HEPES buffer (pH 7.4), containing 10% glycerol, 150 mM NaCl, 1% Triton X-100, 0.5% NP-40, and 1 mM each of EDTA, EGTA, PMSF and Na3VO4. Samples were preincubated for 1 h with 20 μl protein A sepharose CL-4B (Amersham, Uppsala, Sweden) at 4 °C and then centrifuged to remove proteins adhered nonspecifically to protein A. The supernatants were incubated with 1–2 μg primary antibodies for 4 h or overnight at 4 °C. Protein A was added to the tube for another 2-h incubation. Samples were centrifuged at 10,000 × g for 2 min at 4 °C, and the pellets were washed with immunoprecipitation buffer for three times. Bound proteins were eluted by boiling at 100 °C for 5 min in SDS-PAGE loading buffer and then isolated by centrifuge. The supernatants were used for immunoblot analysis. Proteins were separated on polyacrylamide gels and then electrotransferred onto a nitrocellulose membrane (Amersham, Buckinghamshire, UK). After being blocked for 3 h in Tris-buffered saline with 0.1% Tween20 (TBST) and 3% bovine serum albumin (BSA), membranes were incubated overnight at 4 °C with primary antibodies in TBST containing 3% BSA. Membranes were then washed and incubated with alkalinephosphatase-conjugated secondary antibodies in TBST for 2 h and developed using NBT/BCIP color substrate (Promega, Madison, USA). The density of the bands on the membrane was scanned and analyzed with an image analyzer (LabWorks Software, UVP Upland, CA, USA).
4.5.
Histological assessment and immunohistochemistry
Rats were perfusion-fixed with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4) under anesthesia after 5 days of ischemia/reperfusion. Brains were removed quickly and further fixed with the same fixation solution at 4 °C overnight. Post-fixed brains were embedded by paraffin followed by preparation of coronal sections 5 μm thick using a microtome. The paraffin-embedded brain sections were deparaffinized with xylene and rehydrated by ethanol at graded concentrations of 100:70% (v/v) followed by washing with water. The sections were stained with 0.1% (w/v) cresyl violet and were examined with light microscopy, and the number of surviving hippocampal CA1 pyramidal cells per 1 mm length was counted as the neuronal density. Immunoreactivity was determined by the avidin–biotin– peroxidase method. Briefly, sections were deparaffinized with xylene and rehydrated by ethanol at graded concentrations and distilled water. High-temperature antigen retrieval was performed in 1 mM citrate buffer. To block endogenous peroxidase activity, sections were incubated for 30 min in 1% H2O2. After being blocked with 5% (v/v) normal goat serum in PBS for 1 h at 37 °C, sections were incubated with rabbit polyclonal antibodies against FasL (1:100) or cleaved caspase-3 protein (1:100) or mouse monoclonal antibody against p-c-Jun (1:50) at 4 °C for 2 days. These sections were then incubated with biotinylated goat-anti-rabbit/mouse secondary antibody
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overnight and subsequently with avidin-conjugated horseradish peroxidase for 1 h at 37 °C. Finally, sections were incubated with peroxidase substrate diaminobenzidine (DAB) until desired stain intensity develops. TUNEL staining was performed using an ApopTag® Peroxidase In Situ Apoptosis Detection Kit according to the manufacturer's protocol with minor modifications. The paraffin-embedded coronal sections were deparaffinized and rehydrated and then treated with protease K at 20 μg/ml for 15 min at room temperature. Sections were incubated with reaction buffer containing TdT enzyme and at 37 °C for 1 h. After washing with stop/wash buffer, sections were treated with anti-digoxigenin conjugate for 30 min at room temperature and subsequently developed color in peroxidase substrate. The nuclei were lightly counterstained with 0.5% methyl green.
4.6.
Antibodies and reagents
Mouse monoclonal anti-p-c-Jun (sc-822), rabbit polyclonal anti-FasL (sc-6237), rabbit polyclonal anti-Bcl-2 (sc-492) and rabbit polyclonal anti-Fas (sc-1023) were purchased from Santa Cruz Biotechnology. Rabbit polyclonal anti-Bax (#2772), rabbit polyclonal anti-cytochrome c (#4272) and rabbit polyclonal anti-cleaved caspase-3 (#9661) were obtained from Cell Signal Biotechnology. The secondary antibodies used in our experiment were goat anti-mouse IgG, goat antirabbit IgG and donkey anti-goat IgG. They were from Sigma. ApopTag® Peroxidase In Situ Apoptosis Detection Kit (S7100) was purchased from Chemicon.
4.7.
Statistical evaluation
Values were expressed as mean ± SD and obtained from six independent rats in every group. Statistical analysis of the results was carried out by Student's t test or one-way analysis of the variance (ANOVA) followed by the Duncan's new multiple range method or Newman–Keuls test. P values <0.05 were considered significant.
Acknowledgment This work was supported by a grant from the Key Project of the National Natural Science Foundation of China (No. 30330190).
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