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Keywords:

  • Akt;
  • cerebral ischemia;
  • heme oxygenase 1;
  • nuclear factor-E2-related factor 2;
  • oxidative stress;
  • SHPS-1

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

J. Neurochem. (2012) 122, 834–843.

Abstract

Src homology 2 domain–containing protein tyrosine phosphatase substrate–1 (SHPS-1), also known as Signal-regulatory protein alpha (SIRPα) or SIRPA is a transmembrane protein that is predominantly expressed in neurons, dendritic cells, and macrophages. This study was conducted to investigate the role of SHPS-1 in the oxidative stress and brain damage induced by acute focal cerebral ischemia. Wild-type (WT) and SHPS-1 mutant (MT) mice were subjected to middle cerebral artery occlusion (60 min) followed by reperfusion. SHPS-1 MT mice had significantly reduced infarct volumes and improved neurological function after brain ischemia. In addition, neural injury and oxidative stress were inhibited in SHPS-1 MT mice. The mRNA and protein levels of the antioxidant genes nuclear factor-E2-related factor 2 (Nrf2) and heme oxygenase 1 were up-regulated in SHPS-1 MT mice. The SHPS-1 mutation suppressed the phosphorylation of SHP-1 and SHP-2 and increased the phosphorylation of Akt and GSK3β. These results provide the first demonstration that SHPS-1 plays an important role in the oxidative stress and brain injury induced by acute cerebral ischemia. The activation of Akt signaling and the up-regulation of Nrf2 and heme oxygenase 1 likely account for the protective effects that were observed in the SHPS-1 MT mice.

Abbreviations used
HO-1

heme oxygenase 1

Nrf2

nuclear factor-E2-related factor 2

ROS

reactive oxygen species

SHPS-1 MT

SHPS-1 mutant

tMCAO

transient middle cerebral artery occlusion

Stroke is the second leading cause of mortality and the most common cause of long-term disability worldwide (Mathers et al. 2009). Over 15 million people suffer from stroke each year, and approximately 80–85% of these cases are ischemic stroke. Accumulating evidence from the past two decades has suggested that the oxidative stress associated with the excessive production of reactive oxygen species (ROS) has a profound effect on ischemic stroke pathogenesis (El Kossi and Zakhary 2000; Kelly et al. 2008; Lei et al. 2011). ROS have direct cellular effects, such as lipid peroxidation, protein denaturation, and DNA and RNA damage, which result in tissue destruction and cell death. ROS also act in several signal transduction pathways, including intrinsic and extrinsic caspase activation and nuclear factor kappa B (NF-κB) activation, which may lead to excessive cell apoptosis and inflammatory gene expression (Chan 2001; Allen and Bayraktutan 2009). In recent years, continuous efforts have been made to modulate oxidative stress after ischemic stroke. Although some free radical scavenging agents and radical trapping agents have shown therapeutic potential in animal models, they have failed in clinical trials. In the present study, we found that the deletion of the signal regulatory protein (SIRP) family member SHPS-1 inhibits oxidative damage and mitigates brain injury after ischemic stroke.

SHPS-1, which is also known as SIRP α or SIRPA, is a transmembrane protein that consists of three domains: an immunoglobulin (Ig)-like extracellular domain, a transmembrane domain, and an intracellular domain (Yamao et al. 1997; Oshima et al. 2002). The intracellular domain of SHPS-1 contains two immunoreceptor tyrosine-based inhibition motifs (ITIMs) with four tyrosine residues, which can be phosphorylated by growth factors and integrins that mediate cell adhesion to extracellular matrix proteins (Galbaugh et al. 2010; Kapoor and O’Rourke 2010; Shen et al. 2010). The intracellular tyrosine-phosphorylated sites of SHPS-1 then bind to and activate two Src homology-2 (SH2) domain-containing protein tyrosine phosphatases, SHP-1 and SHP-2. The activated SHP-1 and SHP-2 act on multiple signaling molecules and modulate downstream signal transduction via dephosphorylation.

The MAPK, JAK/STAT, and PI3K-Akt pathways have been reported to be modulated by SHP-1 or SHP-2 under diverse biological states. Akt, an important member of Arg-directed kinases, plays a central role in mediating critical cellular responses including cell survival, metabolism, angiogenesis, and transcriptional regulation. GSK3β is a direct substrate of Akt, which can be directly phosphorylated by activated Akt at Ser 9. Previous studies have demonstrated that Akt activation plays an important role in the expression and activation of nuclear factor-E2-related factor 2 (Nrf2) (Martin et al. 2004; Bak et al. 2012). Nrf2 is an important transcription factor that induces the expression of a number of genes including those that encode for several antioxidant enzymes, and it plays a physiological role in the regulation of oxidative stress.

SHPS-1 is predominantly expressed in neurons, dendritic cells, and in macrophages, and previous studies have shown that SHPS-1 is involved in a variety of physiological processes, including the regulation of immune cells, the self-recognition of red blood cells, macrophage multinucleation, vascular smooth muscle proliferation, neuronal development, and survival (Ikeda et al. 2006; Mitsuhashi et al. 2008; Sobota et al. 2008). To date, studies have not examined the role of SHPS-1 in ischemic stroke or the impact of SHPS-1 on oxidative stress after cerebral ischemia. Identifying the functional role and the mechanisms of SHPS-1 in the stroke pathological process may provide potential treatment targets for ischemic stroke.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Animals

All the animal procedures were approved by the Wuhan University Animal Ethics Committee. The generation of SHPS-1 mutant (MT) mice was described previously (Inagaki et al. 2000; Ohnishi et al. 2010). The SHPS-1 MT mice lack most of the cytoplasmic region instead of the wild-type protein. The SHPS-1 MT mice were backcrossed to the C57BL/6J background for > 10 generations. The present study used male WT and SHPS-1 MT mice on a C57BL/6J background that were between 10 and 12 weeks of age.

Mouse transient focal cerebral ischemia model

The procedure for transient middle cerebral artery occlusion (tMCAO) has been previously described (Connolly et al. 1997). Briefly, the mice were anesthetized with 2.5–3% isoflurane in O2. The rectal temperature was maintained at 37 ± 0.5°C with a heating pad. A probe was fixed to the skull (2 mm posterior and 5 mm lateral to the bregma) and connected to a laser Doppler flow meter (Periflux System 5010; Perimed, Sweden) to continuously monitor cerebral blood flow (CBF). For tMCAO, a 6-0 silicon-coated monofilament surgical suture (Doccol, Redland, CA, USA) was inserted into the left external carotid artery, advanced into the internal carotid artery, and wedged into the cerebral arterial circle to obstruct the origin of the MCA (middle cerebral artery). An interruption of the cerebral blood flow in the MCA territory was confirmed by documenting a > 80% decline in the relative cerebral blood flow. The filament was left in place for 60 min and then withdrawn. A return to > 70% of basal cerebral blood flow within 10 min of suture withdrawal confirmed the reperfusion of the MCA territory.

Indian ink staining

The Indian ink staining was utilized to show gross cerebral vasculatures of WT mice and SHPS-1 MT mice (Fujii et al. 1997). Animals were perfused under sodium pentobarbital (Sigma, St. Louis, MO, USA) anesthesia (50 mg/kg IP) with 10 mL of physiological saline followed by 2 mL of preheated Indian ink staining solution via the left cardiac ventricle until the tissues (e.g., tongue, lips, and gums) turned black. The Indian ink staining solution contained 10% (W/V) gelatin (Amresco, Solon, OH, USAyy), 50% (V/V) Indian ink (Solarbio, Beijing, China). After decapitation, the brains were carefully removed into 10% buffered formalin for 24 h before examination. The vessels of the circle of Willis and their branches were photographed using Nikon D700 digital camera.

Neurological deficit scores

Three days after tMCAO, neurological deficits were assessed using a 9-point scale (Xia et al. 2006). A lack of neurological deficit was scored as 0, and left forelimb flexion when suspended by the tail or failure to fully extend the right forepaw was scored as 1. Left shoulder adduction when suspended by the tail was scored as 2, and reduced resistance to a lateral push toward the left side was scored as 3. Spontaneous movement in all directions with circling to the left that was only exhibited when the animal was pulled by the tail was scored as 4, spontaneously circling or walking solely to the left was scored as 5 and only walking when stimulated was scored as 6. A lack of response to stimulation was scored as 7, and stroke-related death was scored as 8.

Measurement of infarct volume

Infarct volume and swelling were measured at 24 h after tMCAO by 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) staining. After being anesthetized by sodium pentobarbital (50 mg/kg IP), the mice were killed by cervical dislocation. Brains were cut into 1-mm-thick coronal sections using a mouse brain matrix and stained with 2% TTC (Sigma, St. Louis, MO) in phosphate buffer (pH 7.4) for 15 min at 37°C. After staining, the sections were transferred to a 10% formalin solution and fixed overnight. Fixed sections were photographed, and the volume of the infarct area was quantified using Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA). To correct for the effect of edema, the area of infarction was measured by subtracting the area of the non-lesioned hemisphere from the area of the lesioned hemisphere. The volume of infarction was calculated by integrating the lesioned areas from the seven measured levels of the brain.

Immunofluorescence staining

Mice were anesthetized with sodium pentobarbital and perfused through the left ventricle with 0.1 mol/L sodium phosphate buffer under 100 mmHg of pressure for 5 min. This was followed by perfusion with a fixative solution that contained 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4) for 15 min. The brains were carefully removed, post-fixed for 6–8 h in the same fixative solution at 25°C, and immersed overnight in a 0.1 mol/L phosphate buffer that contained 30% sucrose at 4°C. The brains were embedded in OCT, and serial frontal sections were cut with a cryostat microtome. For immunofluorescence staining, the sections were washed in PBS containing 10% goat serum. The sections were incubated with anti-4-Hydroxynonenal (4HNE, ab48506, Abcam, Cambridge, MA) or anti-8-Hydroxyguanosine (8OHdG, sc-66036, Santa cruz, CA), anti-Nrf2 (ab31163, Abcam, Cambridge, MA) primary antibodies overnight at 4°C. For NeuN (MAB377, Millipore, Billerica, MA) immunofluorescence staining, the sections were washed in PBS containing 10% goat serum and 0.1% Triton X-100. After washing, the sections were incubated in the anti-NeuN antibody for 2 h prior to incubation in secondary antibody for 1 h at 37°C. After the sections were washed in PBS, they were incubated with secondary antibody for 1 h. Finally, the nuclei were labeled with DAPI. Visualization was performed under a fluorescence microscope OLYMPUS DX51 (Olympus, Japan) with DP2-BSW Ver. 2.2 software, and the image analysis was performed with Image-Pro Plus 6.0 software.

TUNEL staining

Six hours after the onset of ischemia, the brains were collected and sliced as described above. After the NeuN immunofluorescence staining was completed, TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) staining was performed using the In Situ Cell Death Detection Kit (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer’s protocol. We also labeled the nuclei with DAPI, and DNA fragmentation was quantified under high-power magnification (200X). An investigator who was blinded to the study condition calculated the percentage of DAPI-positive cells that were also TUNEL-positive.

Tissue preparation

For quantitative real-time PCR (qRT-PCR) and western blotting analysis, mice were anesthetized with sodium pentobarbital (50 mg/kg IP) and perfused through the left ventricle with cold sodium phosphate. After perfusion, the brains were quickly removed. To collect tissue in an unbiased manner that globally reflected the infarct, the olfactory bulbs and the front and back 1 mm of brain tissue were excised from each animal. The remaining left hemisphere was collected (including the infarct area and the peri-infarct area). The brain tissues were immediately frozen in liquid nitrogen and transferred to a −80°C freezer for storage.

Quantitative real-time PCR

Total RNA was prepared from snap-frozen tissue specimens using TRIzol reagent (Invitrogen, Carlsbad, CA) and was reverse transcribed into cDNA using 2 μg of RNA from each sample and the Transcriptor First Strand cDNA Synthesis Kit (Roche, Indianapolis, IN). To examine the relative mRNA expression of Nrf2 and heme oxygenase 1 (HO-1), the specific mRNA expression levels were normalized to GAPDH. Quantitative RT-PCR analysis was performed using the LightCycler 480 SYBR Green 1 Master Mix (Roche, Indianapolis, IN) and the LightCycler 480 QPCR System (Roche, Indianapolis, IN). The following sequence-specific primers were used:

Nrf2 forward: 5′-ATGATGGACTTGGAGTTGCC-3′;

Nrf2 reverse: 5′-TCCTGTTCCTTCTGGAGTTG-3′;

HO-1 forward: 5′-AGGAGATAGAGCGCAACAAGCAGA-3′;

HO-1 reverse: 5′-CCAGTGAGGCCCATACCAGAAG-3′.

Western blotting

Western blotting was conducted to determine the activation state of programmed cell death using cleaved caspase 8 (Asp387), caspase 8, cleaved caspase 3 (Asp175), and caspase 3 (all these antibodies were from Cell Signaling Technology; Beverly, MA, USA). We also examined the expression of Nrf2 (Bioworld Technology, Minneapolis, MN, USA) and HO-1 (Cell Signaling Technology, Beverly, MA, USA). In addition, we examined the phosphorylation of SHP-1 (Bioworld Technology, Minneapolis, MN), SHP-2 (Bioworld Technology, Minneapolis, MN), Akt (Cell Signaling Technology, Beverly, MA) and GSK3β (Cell Signaling Technology, Beverly, MA). For the western blot analysis, 50 μg of protein extract was separated on an 8–12% SDS-PAGE gel and subsequently transferred to a PVDF membrane (Millipore, Bedford, MA). Membrane blocking (5% skimmed milk powder), washes (PBS) and secondary antibody (goat anti-rabbit IRDye 800CW or goat anti-mouse IRDye 800CW, LI-COR Biosciences, Lincoln, NE) incubations were all performed at 25°C for 1 h, whereas the primary antibodies were allowed to incubate overnight at 4°C. The protein signals were detected using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE). All the specific protein expression levels were normalized to GAPDH.

Statistical analysis

Data are expressed as the means ± SEM. Differences among the groups were determined by two-way anova followed by a post hoc Tukey test. Comparisons between the two groups were performed by the unpaired Student’s t-test. A p-value of < 0.05 was accepted as the level of statistical significance.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

SHPS-1 deficiency decreased the infarct volume at 24 h after tMCAO

Gross cerebral vasculatures showed by Indian ink staining. There was no macroscopic difference in the vessels of the circle of Willis and their branches between WT mice and SHPS-1 MT mice (Fig. 1a). According to the laser Doppler flowmetry monitoring, the CBF was similar between the WT and the SHPS-1 MT mice during both ischemia and reperfusion (Fig. 1b). The infarct volume was determined using TTC staining. The infarct volume was 30.77 ± 4.12 mm3 in the SHPS-1 MT mice, which was 30% smaller than the volume in the WT mice (47.18 ± 3.10 mm3) (Fig. 1c). The edema volume percentage was 7.03 ± 1.43% in the SHPS-1 MT mice, and was 7.93 ± 2.09% in the WT mice, there was no statistical significance between the two groups.

image

Figure 1.  The SHPS-1 deficiency reduced the infarct volume in a mouse transient middle cerebral artery occlusion model. (a) Cerebral vascular anatomy. Representative images showing gross cerebral vasculatures stained by India ink (= 3 for WT and MT mice). (b) There was no significant difference in the CBF of the SHPS-1 mutant mice and the WT mice during the ischemia and reperfusion process. (c) The SHPS-1 deficiency significantly reduced the infarct volume (*< 0.05 vs. WT, = 7 for each group) and slightly reduced edema formation.

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Neurological function was improved in SHPS-1 mutant mice

On the basis of previous study (Xia et al. 2006), the neurological deficits were assessed 24 h after tMCAO using a 9-point scale. The neurological deficit score was 4.00 ± 0.31 in the SHPS-1 MT mice compared with 5.29 ± 0.18 in the WT mice (Fig. 2), which suggested that neurological function improved in the SHPS-1 MT mice following transient cerebral ischemia.

image

Figure 2.  Neurological function was improved in the SHPS-1 mutant mice. Neurological deficits were assessed 24 hours after transient middle cerebral artery occlusion using a 9-point scale. The SHPS-1 deficiency significantly improved neurological function following transient middle cerebral artery occlusion (*< 0.05 vs. WT, = 7 for each group).

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Neuronal injury was inhibited in the SHPS-1 mutant mice

Neuronal injury was detected with TUNEL and NeuN staining 6 h after tMCAO. There was an abundance of TUNEL-positive cells in the infarct and peri-infarct area. In addition, NeuN immunofluorescence staining revealed that most of the TUNEL-positive cells also expressed NeuN, which indicated that the injured cells were mainly neurons. We calculated the number of TUNEL-positive nuclei and total nuclei (DAPI labeled), and the TUNEL-positive rate was calculated with the following formula: TUNEL-positive rate (%)=TUNEL-positive nuclei/total nuclei. The TUNEL-positive rate in the peri-infarct area in the SHPS-1 MT mice was 36.73 ± 0.04%, which was much lower than that of the WT mice (63.58 ± 0.41%) (Fig. 3a). Western blot analysis revealed that the protein levels of cleaved caspase 3 and cleaved caspase 8 were much lower in the SHPS-1 MT mice compared with the WT mice (Fig. 3b).

image

Figure 3.  Neuronal injury was inhibited in the SHPS-1 mutant mice. (a) TUNEL and NeuN double staining shows that, most TUNEL-positive nuclei can overlap with NeuN positive nuclei, indicates that the majority of TUNEL-positive cells were neurons. The TUNEL-positive nuclei numbers and total nuclei numbers were counted, TUNEL-positive rate (%)=TUNEL-positive nuclei number/total nuclei number. SHPS-1 mutation significantly reduced the TUNEL-positive rate in the peri-infarct area (*< 0.05 vs. WT, n = 4 for each group). (b) western blot analysis shows the protein levels of cleaved caspase 3 (c-caspase 3), caspase 3, cleaved caspase 8 (c-caspase 8) and caspase 8. All the caspase proteins were normalized with GAPDH. The protein levels of c-caspase 3 and c-caspase 8 were reduced in the SHPS-1 mutant mice (*< 0.05 vs. WT, #< 0.05 vs. sham, n = 6 for each group).

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Oxidative injury was attenuated in the SHPS-1 mutant mice

8OHdG is a biomarker of oxidative DNA damage, and 4 HNE is a stable product of lipid peroxidation and a key mediator of oxidative stress-induced cell death (Imai et al. 2001; Kawai et al. 2011). Immunofluorescence staining for 8OHdG and 4HNE was used to estimate the oxidative injury. The 8OHdG- or 4HNE-positive cells were counted under a 200X field. The numbers of both the 8OHdG-positive cells and the 4HNE-positive cells were reduced by approximately 50% in the SHPS-1 MT mice compared with the WT mice (Fig. 4a).

image

Figure 4.  The oxidative injury was attenuated in the SHPS-1 MT (mutant)mice. (a) Immunofluorescence staining indicates that the numbers of both 8OHdG-positive cells (red) and 4HNE-positive cells (red) in the peri-infarct area were significantly reduced in the SHPS-1 MT mice, blue is nucleus stained by DAPI. (*< 0.05 vs. WT, n = 4 for each group). (b) Quantitative real-time PCR and western blot analysis indicate that the mRNA and protein levels of the anti-oxidative genes Nrf2 and heme oxygenase 1 were up-regulated in the SHPS-1 MT mice (*< 0.05 vs. WT, #< 0.05 vs. sham, = 6 for each group). (c) Immunofluorescence staining reveals that the Nrf2 positive cell number and light intensity of Nrf2 are increased in the peri-infarct area of SHPS-1 MT mice, in addition, the expression of Nrf2 protein is more concentrated in the nucleus in SHPS-1 MT mice.

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The expression levels of the antioxidant genes Nrf2 and HO-1 were elevated in SHPS-1 mutant mice

Nrf2 is an important transcription factor that regulates the expression of a large number of antioxidant genes (Wang et al. 2011). HO-1 is a target gene of Nrf2 and plays a protective role in cerebral ischemic injury via an anti-oxidative mechanism (Kweon et al. 2006). Quantitative real-time PCR and western blot analysis revealed that the mRNA and protein levels of Nrf2 and HO-1 were elevated in the brains of the ischemic SHPS-1 MT mice compared with the WT mice (Fig. 4b). Immunofluorescence staining showed that the Nrf2 positive cell numbers and the fluorescence intensity of Nrf2 were increased in SHPS-1 MT mice, indicating that the expression of Nrf2 was up-regulated in the ischemic brain of SHPS-1 MT mice. Immunofluorescence staining also showed that, the expression of Nrf2 was more concentrated in the nucleus in SHPS-1 MT mice, implying that there was more Nrf2 transferred into the nucleus in SHPS-1 MT mice (Fig. 4c).

The deficiency in SHPS-1 led to increased Akt activation

Phosphorylated SHP-1, SHP-2, Akt, and GSK3β were detected using western blot analysis. The phosphorylation of SHP-1 and SHP-2 was inhibited in the SHPS-1 MT mice after cerebral ischemia, whereas the phosphorylation of Akt and GSK3β was markedly elevated. The total protein levels of SHP-1, SHP-2, Akt, and GSK3β were not changed in the SHPS-1 MT mice compared with the WT mice (Fig. 5).

image

Figure 5.  The SHPS-1 deficiency led to increased Akt activation. Western blot analysis indicates that the phosphorylation of SHP-1 and SHP-2 (p-SHP-1, p-SHP-2) was inhibited in the ischemic brain of SHPS-1 MT (mutant) mice, whereas the phosphorylation of Akt and GSK3β (p-Akt, p-GSK3β) was elevated in the SHPS-1 MT mice compared with the WT mice (*< 0.05 vs. WT, # < 0.05 vs. sham, = 6 for each group). The total protein levels of SHP-1, SHP-2, Akt and GSK3β were not changed in the SHPS-1 MT mice.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This study suggests that SHPS-1 plays an important role in the pathological process of focal cerebral ischemia. In the SHPS-1 MT mice, the infarct volume was decreased, neurological function was improved, neuronal apoptosis was reduced, the activities of caspase 3 and caspase 8 were inhibited, oxidative injury was attenuated and the expression levels of the antioxidant genes Nrf2 and HO-1 were up-regulated. The phosphorylation of SHP-1 and SHP-2 was also inhibited, and the activity of Akt was enhanced in the SHPS-1 MT mice.

SHPS-1 is a ubiquitously expressed receptor-type transmembrane glycoprotein that is abundantly expressed in the brain (Yamao et al. 1997; Oshima et al. 2002). Previous studies (Stofega et al. 2000; Ikeda et al. 2006) have demonstrated that SHPS-1 plays a key role in the negative regulation of tyrosine kinase-coupled cellular responses that are induced by cell adhesion, growth factors and/or insulin. SHPS-1 has been shown to be involved in various biological functions, such as cell migration, phagocytosis, and mast and dendritic cell activation. In the brain, SHPS-1 has been identified as a neural adhesion molecule that participates in brain-derived neurotrophic factor (BDNF)-mediated neuronal survival via Akt activation (Araki et al. 2000). In addition, transfection with wild-type and mutant SHPS-1 both have been shown to enhance Akt activation in neurons (Araki et al. 2000). CD47 is a ligand of SHPS-1, Koshimizu et al. and Xing et al. suggested that the activation of CD47 by its activating peptide induces oxidative injury and cytotoxicity in cultured neurons (Koshimizu et al. 2002; Xing et al. 2009); however, previous studies have not shown that SHPS-1 is involved in oxidative stress.

Oxidative stress has been identified as an important factor in the pathological process of stroke because it has direct effects on cellular injury and activates downstream signaling pathways, which may aggravate the post-ischemic injury (El Kossi and Zakhary 2000; Kelly et al. 2008; Lei et al. 2011). In the present study, the biomarkers of oxidative damage (i.e., 8OHdG and 4HNE) were markedly decreased in the brains of the ischemic SHPS-1 MT mice, which suggested that oxidative damage is mitigated by the SHPS-1 mutation. The severity of oxidative stress depends on the balance between antioxidants and pro-oxidants. We estimated the expression of the antioxidant genes Nrf2 and HO-1. Nrf2 is an important transcription factor that regulates the expression of a large number of antioxidant genes (Venugopal and Jaiswal 1996; Solis et al. 2002; Kweon et al. 2006), and HO-1 is a target gene of Nrf2 that plays a protective role in cerebral ischemic injury via an anti-oxidative mechanism (Kweon et al. 2006; Aztatzi-Santillán et al. 2010; Kim et al. 2010). The mRNA and protein levels of Nrf2 and HO-1 were markedly up-regulated in the brains of the ischemic SHPS-1 MT mice, which indicated that the SHPS-1 mutation mitigated oxidative stress, likely through the up-regulation of an anti-oxidative mechanism.

The number of TUNEL-positive neurons decreased, and the protein levels of cleaved caspase 3 and caspase 8 were down-regulated in the SHPS-1 knockout mice in this study. TUNEL detects DNA fragmentation which appeared in programmed cell death. Caspases, or cysteine-aspartic proteases or cysteine-dependent aspartate-directed proteases are a family of cysteine proteases that play essential roles in programmed cell death, necrosis, and inflammation. Caspase 8 is involved in the programmed cell death induced by Fas and various apoptotic stimuli. Activated caspase 8 cleaves and activates downstream effector caspases, such as caspase 1, caspase 3, caspase 6, and caspase 7. Caspase 3 ultimately elicits the morphological hallmarks of apoptosis, including DNA fragmentation and cell shrinkage. Several studies have demonstrated that oxidative stress-induced neural injury is mediated by caspase 8 and caspase 3 (Russell et al. 2002; Wang et al. 2002). In the present study, the inhibition of neural injury appeared to be attributed to the mitigation of oxidative stress in the SHPS-1 MT mice.

We observed that the phosphorylation of SHP-1 and SHP-2 was inhibited after cerebral ischemia in the SHPS-1 MT mice. SHP-1 and SHP-2 are SH2 domain-containing non-transmembrane protein tyrosine phosphatases. When SHPS-1 is activated by growth factors, cell adhesion or other stimuli, the phosphorylated cytoplasmic region binds to and activates SHP-1 and SHP-2. SHP-1 and SHP-2 have been implicated in several signaling pathways, such as the MAPK, JAK/STAT, and PI3K-Akt pathways (Dubois et al. 2006; Pandey et al. 2009; Won et al. 2011). In addition, SHP-1 and SHP-2 have been shown to interact with a variety of signaling intermediates, and they dephosphorylate associated signaling molecules, which negatively regulate the local signal pathways. In the present study, higher levels of Akt phosphorylation were detected in the SHPS-1 MT mice, which was likely because of decreased activity of SHP-1 and SHP-2 (Zhang et al. 2002; Lodeiro et al. 2011).

In previous studies, it has been demonstrated that the PI3K/Akt signaling pathway is involved in the expression and activation of Nrf2 and that the induction of Nrf2 protein expression contributes to the transcriptional activity of Nrf2 (Martin et al. 2004; Bak et al. 2012). Not only does it affect the accumulation of Nrf2, but the PI3K/Akt signaling pathway also contributes to the activation of Nrf2 by promoting transfer of cytoplasmic Nrf2 to the nucleus (Wang et al. 2008). GSK-3β is a downstream signaling molecule of the PI3K/Akt pathway whose activity can be inhibited by Akt-mediated phosphorylation at Ser9. A previous study demonstrated that GSK-3β down-regulated Nrf2 activity; thus, phosphorylation of GSK-3β can increase the activity of Nrf2 (Rojo et al. 2008). In the present study, the mRNA level and protein level of Nrf2 were up-regulated in SHPS-1 MT mice, the expression of Nrf2 protein was more concentrated in the nucleus in SHPS-1 MT mice, the mRNA, and protein level of Nrf2 targeted gene HO-1 were also up-regulated in SHPS-1 MT mice. The increasing expression and activity of Nrf2 is probably attributed to the phosphorylation of Akt/GSK-3β.

In conclusion, this study suggested that the SHPS-1 deficiency protects the brain from acute ischemic injury. Oxidative stress was mitigated and neural injury was inhibited in SHPS-1 MT mice. In addition, we demonstrated that Akt was activated in the brains of ischemic SHPS-1 MT mice. The activated Akt signaling may account for the up-regulation of Nrf2 and HO-1 and may eventually lead to the decline of oxidative stress. These findings may provide a new therapeutic target for the treatment of acute ischemic stroke.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The SHPS-1 mutant mice were provided by the RIKEN BRC through the National Bio-Resource Project of The MEXT Japan. This work was supported in part by National Natural Science Foundation of China (NO. 81100230), National Science and Technology Support Project (NO. 2011BAI15B02).

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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