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β-catenin signaling pathway in hippocampal neurons
Biochemical and Biophysical Research Communications xxx (2017) 1e9
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Mild hypothermia protects against oxygen glucose deprivation/ reoxygenation-induced apoptosis via the Wnt/b-catenin signaling pathway in hippocampal neurons Tianen Zhou a, 1, Yanran Liang b, 1, Longyuan Jiang a, Tao Yu a, Chaotao Zeng a, Enxiang Tao b, * a b
Department of Emergency, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China Department of Neurology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 March 2017 Accepted 27 March 2017 Available online xxx
Mild hypothermia is thought to be one of the most effective therapies for cerebral ischemia/reperfusion injuries. Our previous research revealed that mild hypothermia inhibits the activation of caspase-3 and protects against oxygen glucose deprivation/reoxygenation (OGD/R)-induced injury in hippocampal neurons. However, the mechanisms behind the activation of caspase-3 remain unclear. The aims of this study were to determine whether the protective effects of mild hypothermia were exerted through the Wnt/b-catenin signaling pathway. We found that, under OGD/R conditions, the pathway was down regulated, but mild hypothermia induced the reactivation of the Wnt/b-catenin signaling pathway, which had been suppressed by OGD/R injury. Mild hypothermia also caused the down regulation of the expression of apoptosis promoting proteins (Bax cleaved caspase-3), up-regulated the expression of apoptosis inhibiting proteins (Bcl-2), and ameliorated OGD/R injury-induced apoptosis. The protective effects of mild hypothermia were blocked by DKK1 (an antagonist of the canonical Wnt signaling pathway). Taken together, these results indicate that the Wnt/b-catenin signaling pathway mediates the protective effects of mild hypothermia against OGD/R-induced apoptosis. Our study provides evidence that mild hypothermia reactivates the Wnt/b-catenin signaling pathway, which is suppressed by OGD/R injury, in hippocampal neurons and protects neurons from OGD/R-induced apoptosis via the reactivation of the Wnt/b-catenin signaling pathway, ultimately suggesting that mild hypothermia could have therapeutic effects on OGD/R-induced apoptosis. © 2017 Elsevier Inc. All rights reserved.
Keywords: Mild hypothermia OGD/R Apoptosis Wnt/b-catenin
1. Introduction Mild hypothermia has been proven to be one of the most effective therapies for hypoxic-ischemic encephalopathy (HIE) without severe side effects [1,2]. The protective mechanism of mild hypothermia on HIE is complex, although many studies have found that it improves neurological function by reducing neuronal apoptosis [3,4]. Our previous research found that mild hypothermia could inhibit the activation of caspase-3 (an effector molecule implementing apoptosis) that was induced by oxygen glucose
* Corresponding author. Department of Neurology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, 107# Yanjiangxi Road Yuexiu District, Guangzhou 510120, China. E-mail address: [email protected] (E. Tao). 1 These authors contributed equally to this article.
deprivation/reoxygenation (OGD/R) in rat hippocampal neurons [5]. However, the mechanisms for the activation of caspase-3 remain unclear. Recent studies have shown that the Wnt/b-catenin signaling pathway is likely involved in ischemia/reperfusion(I/R) injury pathogenesis. Several studies have reported that, under ischemichypoxic conditions, the Wnt/b-catenin pathway is downregulated and cytoplasmic becatenin is degraded, which reduces the transcription of numerous target genes, including proapoptotic and inflammatory factors [6e8]. Moreover, the association between the canonical Wnt pathway and apoptosis-dependent cellular death has also been widely reported. Previous studies have indicated that the activation of the Wnt/b-catenin pathway protects Mang cell lines against I/R injury by attenuating apoptosis [9,10]. A few studies have also indicated that mild hypothermia can regulate the Wnt/b-catenin signaling pathway under I/R conditions.
http://dx.doi.org/10.1016/j.bbrc.2017.03.153 0006-291X/© 2017 Elsevier Inc. All rights reserved.
Please cite this article in press as: T. Zhou, et al., Mild hypothermia protects against oxygen glucose deprivation/reoxygenation-induced apoptosis via the Wnt/b-catenin signaling pathway in hippocampal neurons, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.03.153
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Hanfeng Zhang reported that hypothermia may block molecular signaling downstream of GSK 3b and stop the degradation of bcatenin [11]. However, whether mild hypothermia can protect neurons from apoptosis via the Wnt/b-catenin signaling pathway has remained poorly understood and has not been fully elucidated. The mechanisms behind the effects of mild hypothermia on the Wnt/b-catenin signaling pathway also need further analysis. In this study, we used the OGD/R model of rat hippocampal neurons to simulate I/R injury and HIE. The aim of our study was to test whether mild hypothermia could protect neurons from OGD/Rreduced apoptosis via the Wnt/b-catenin signaling pathway. 2. Materials and methods 2.1. Cell culture Hippocampal neurons in primary culture were used for our study. Suckling rats born within 1d to 3d prior to the experiments were selected (provided by the Experimental Animal Center of Sun Yat-sen University). After disinfecting, the cerebrum tissues of rats were quickly removed and hippocampal tissues were separated and placed in a centrifuge tube containing Dulbecco's Modified Eagle's Medium (DMEM)/F12 medium (Gibco Company, U.S.) and were gently scattered. Then, the hippocampal neurons were inoculated into a 6 cm culture dish with DMEM/F12 containing 10% fetal bovine serum (Hyclone Company, USA) at a density of 1 106 cells per dish, and then incubated in incubator at 37 C. After 24 h, the medium was completely replaced with a medium containing 2% B27 (Gibco Company, USA). On the third day, cytosine arabinoside at a final concentration of 5 mmol/L was added for 24 h, in order to inhibit glial cells. On the 8th day, immunochemical fluorescence identification of neuronal microtubule-associated protein 2 (MAP2) was employed. After the immunochemical fluorescence identification of neuronal MAP-2, we confirmed that hippocampal neurons accounted for over 90% of all of the cultured cells. The results of the identification of neuronal MAP-2 were shown in our previous article [5]. 2.2. Oxygen glucose deprivation and reoxygenation (OGD/R) Cell cultures were subjected to OGD injury using a protocol described previously [5]. In brief, the OGD cell culture medium was replaced with a glucose-free balanced salt solution (BSS) containing 130 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 26 mM NaHCO3, 0.8 mM MgCl2, and 1.18 mM NaH2PO4. It was then transferred to an anaerobic chamber (PLAS & LABS, MI, USA), equilibrated for 10 min with a continuous flux of gas (95% N2/5% CO2), and humidified at 37 C for 6 h. To terminate OGD assays, cultures were carefully washed with DMEM containing 10% fetal bovine serum and OGD cells were reoxygenated in an incubator (74% N2/21% O2/5% CO2) at 37 C or 32 C for 12 h or 24 h. Control cells were incubated in DMEM containing 10% fetal bovine serum in a normoxic incubator for the same period of time. 2.3. Grouping and drug treatment The experimental cells were randomly divided into five groups: the Control, OGD/R, OGD/R þ mild hypothermia (OGD/R þ MH), OGD/Rþ mild hypothermia þ Dickkopf-1 (OGD/R þ MH þ DKK1), and OGD/R þ Dickkopf-1 (OGD/R þ DKK1). Next, 100 ng/ml Dkk1, an antagonist of the canonical Wnt signaling pathway (Sigma, St Louis, MO, USA), was immediately added after OGD for 24 h. The duration of OGD was 6 h. The temperature for the induction of mild hypothermic was 32 C and the duration was either 12 h or 24 h after OGD. All the neurons were detected 24 h after reoxygenation.
2.4. Western blot analysis Cell lysates were diluted in 5 SDS buffer and denatured at 100 C for 3 min. Proteins were electrophoresed on a 12% SDSepolyacrylamide gel and transferred onto polyvinylidene difluoride membranes using a Bio-Rad transblot apparatus. After incubation at room temperature for 1 h in blocking solution (5% nonfat dry milk), membranes were incubated overnight at 4 C with primary antibodies, including Wnt1 (Santa Cruz Biotechnology, California, USA), b-catenin (ABclonal Biotech, Hubei, China), GSK-3b (ABclonal Biotech, Hubei, China), cleaved caspase-3 (Abcam, Cambridge, MA, USA), Bcl-2 (Cell Signaling Technology, USA), Bax (Cell Signaling Technology, USA), or b-actin (Sigma, St Louis, MO, USA) antibodies, followed by goat anti-rabbit or antimouse IgG (Beyotime Biotech, Jiangsu, China). Immunoreactive proteins were detected by enhanced chemiluminescence. All data were normalized to the levels of b-actin that were used as the loading control, and the amount of immunoreactivity was expressed relative to the corresponding control. 2.5. Immunofluorescence Neurons were post-fixed in 4% paraformaldehyde for 20 min and incubated in 0.3% Triton X-100 for 20 min. They were then washed 3 times in PBS, blocked with 5% BSA solution for 1 h, and incubated with a primary antibody overnight at 4 C. The primary antibodies were anti-MAP2 (APPLYGEN, Biotech, Beijing, China), anti-b-catenin (ABclonal Biotech, Hubei, China), and anti-GSK-3b (ABclonal Biotech, Hubei, China). Afterward, they were incubated for 1 h with FITC-conjugated secondary antibodies (Beyotime Biotech, Jiangsu, China). The nuclei were stained with DAPI (Sigma, St Louis, MO, USA) and the slides were left to dry overnight at room temperature. Immunostaining was examined using a laser confocal microscope (Leica, Heidelberg, Germany) and then images were analyzed by Image-Pro Plus 6.0 software package (Bethesda, MD,USA). 2.6. Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay TUNEL staining was performed using an in situ apoptosis detection kit (YEASEN, Shanghai, China) according to the manufacturer's protocols. Neurons were seeded into 6-well plates and fixed with 4% paraformaldehyde for 20 min, before being incubated in 0.3% Triton X-100 for 10 min. Afterward, the neurons were stained as per the TUNEL method. Images were acquired with a fluorescence microscope (LEICA DM4000B, LEICA, Germany). Cells in five randomly selected high-power fields(400 ) were evaluated and the TUNEL-positive cells were counted and analyzed by ImagePro Plus 6.0 software package (Bethesda, MD,USA). 2.7. Measurement of apoptotic rate In order to assess the apoptotic rate of neurons, a flow cytometer was employed. After reoxygenation for 24 h, the various groups of cells were collected and resuspended with phosphate buffer solution. Annexin VeFITC (Austria Bender Medsystems Company) and PI (America Sigma Company) were added and mixed uniformly. Next, the apoptotic rate was detected with the flow cytometer (America Becton Dickinson Company). 2.8. Measurement of lactic acid dehydrogenase (LDH) release rate After OGD, cytoplasmic LDH was partially released into the culture liquid. A higher LDH release rate is related to more serious
Please cite this article in press as: T. Zhou, et al., Mild hypothermia protects against oxygen glucose deprivation/reoxygenation-induced apoptosis via the Wnt/b-catenin signaling pathway in hippocampal neurons, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.03.153
T. Zhou et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e9
cell injury. After OGD and reoxygenation for 24 h, the medium was collected and LDH activity was determined using a commercially available assay kit (Beyotime Biotech, Jiangsu, China), according to the manufacturer's protocol [12,13]. First, 50 mL of the culture liquid was removed for the detection of extracellular LDH activity. The cell lysate with Triton-100 was then added into the culture dish and the supernatant liquid was collected to detect the total cell LDH activity. LDH release rate (%) ¼ extracellular LDH activity/total LDH activity. 2.9. Measurement of cell viability In order to assess the viability of neurons, a quantitative colorimetric MTT assay was employed. Briefly, cells were seeded into 96well plates (1 104 cells per well). Then, 24 h after reoxygenation, 20 mL of the MTT solution (5 mg/mL, Amresco Company, USA) was added into each well. The neurons were incubated at 37 C for 4 h in a humidified atmosphere and, finally, the absorbances of each well at 490 nm were measured using an ELISA 96-well plate reader (BioRad Laboratories, CA, USA). Results are expressed as the percentage of viable cells detected following OGD compared to the normoxic control plates. 2.10. Statistical analysis The measurement data were expressed as the mean ± standard deviation (X±s). Parametric unpaired two-tailed Student's t-tests (to compare two groups) or 1-way ANOVA (for more than two groups) were used for data with a normal distribution. The variance homogeneity test was then conducted. All calculations were performed using SPSS 13.0 statistical software. Differences with P < 0.05 were considered statistically significant. 3. Results 3.1. Mild hypothermia induced the reactivation of the Wnt/bcatenin signaling pathway, which had been suppressed by OGD/R injury in hippocampal neurons The expression level of Wnt/b-catenin in the OGD/R group was significantly lower than in the control. Mild hypothermia (continuing for 24 h after OGD) significantly promoted the Wnt/b-
3
catenin expression levels, compared to the OGD/R neurons not treated with mild hypothermia (Fig. 1A and B). These results indicate that OGD/R suppresses the activity of Wnt/becatenin, which can be recovered by mild hypothermia. 3.2. Dkk1 blocks the effects of mild hypothermia on the Wnt/bcatenin signaling pathway in hippocampal neurons following OGD/ R injury Our results showed that the protein levels of Wnt-1 and b -catenin were significantly decreased and that GSK-3b was significantly increased in OGD/R-treated cells. By contrast, when the OGD/R neurons were treated with mild hypothermia for 24 h, the protein levels of Wnt-1 and b-catenin increased significantly and GSK-3b decreased significantly. Furthermore, when the OGD/R neurons were synchronously treated with mild hypothermia and DKK1, the protein levels of Wnt-1 and b-catenin were significantly lower and GSK-3b was significantly higher than those treated with only mild hypothermia (Fig. 2A and B). The immunofluorescence results were consistent with the protein levels (Fig. 2C and D). These indicate that the effects of mild hypothermia are mediated through the reactivation of the Wnt/b-catenin signaling pathway. 3.3. Wnt/b-catenin mediates the expression levels of apoptosisrelated proteins and the protective effects of mild hypothermia against OGD/R-induced apoptosis Our results showed that the expression levels of apoptosisrelated proteins (Bax, cleaved caspase-3, and cleaved PARP) had increased significantly and that Bcl-2 had decreased significantly in OGD/R-treated cells. By contrast, when the OGD/R neurons were treated with mild hypothermia for 24 h, the protein levels of Bax, cleaved caspase-3, and cleaved PARP decreased significantly and Bcl-2 increased significantly. Furthermore, when the OGD/R neurons were synchronously treated with mild hypothermia and DKK1, the protein levels of all three increased significantly and Bcl-2 was decreased significantly compared to neurons treated with only mild hypothermia (Fig. 3A, B, C, D). Our results also showed that there were significantly more apoptotic neurons in OGD/R-treated cells than when OGD/R neurons were treated with mild hypothermia for 24 h. Furthermore, when OGD/R neurons were synchronously treated with mild hypothermia and DKK1, there were
Fig. 1. Effects of mild hypothermia on Wnt/b-catenin signaling following OGD/R in rat hippocampal neurons. (A) Representative Western blot images showing the expression of Wnt-1, becatenin, and GSK-3b in different groups. (B) A bar graph showing the quantification of Wnt-1/b-actin, becatenin/b-actin, and GSK-3b/b-actin in different groups. The data represent the mean ± SD (n ¼ 3). *P < 0.05 compared to the control group; △P < 0.05 compared to the OGD/R group.
Please cite this article in press as: T. Zhou, et al., Mild hypothermia protects against oxygen glucose deprivation/reoxygenation-induced apoptosis via the Wnt/b-catenin signaling pathway in hippocampal neurons, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.03.153
Fig. 2. Dkk1 blocks the effects of mild hypothermia via the Wnt/b-catenin signaling pathway in hippocampal neurons following OGD/R injury. (A) Representative images of Western blots showing the expression of Wnt-1, becatenin, and GSK-3b in different groups. (B) A bar graph showing the quantification of Wnt-1/b-actin, becatenin/b-actin, and GSK-3b/b-actin in different groups. (C) Dual immunofluorescence staining for b -catenin (red) and DAPI (blue) or GSK-3b (red) and DAPI (blue) in hippocampal neurons. a. Control; b.OGD/R; c.OGD/R þ MH(24 h); d. OGD/R þ MH(24 h)þDkk1; e.OGD/R þ Dkk1. (D) A bar graph showing the relative immunofluorescence intensity of becatenin and GSK-3b in different groups. The data represent the mean ± SD (n ¼ 3). *P < 0.05 compared to the control group; △P < 0.05 compared to the OGD/R group; #P < 0.05 compared to the OGD/ R þ MH group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: T. Zhou, et al., Mild hypothermia protects against oxygen glucose deprivation/reoxygenation-induced apoptosis via the Wnt/b-catenin signaling pathway in hippocampal neurons, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.03.153
Fig. 3. Wnt/b-catenin mediates the expression levels of apoptosis-related proteins and the protective effects of mild hypothermia against OGD/R-induced apoptosis. (A) Representative Western blot images showing the expression of Bax and Bcl-2 in different groups. (B) A bar graph showing the quantification of Bax/b-actin and Bcl-2/b-actin in different groups. (C) Representative Western blot images showing the expression of cleaved caspase-3 and PARP in different groups. (D) A bar graph shows the quantification of cleaved caspase-3/b-actin and PARP/b-actin in different groups. (E) Scatter diagram of PI/Annexin V gating from different groups. a. Control; b.OGD/R; c.OGD/R þ MH(24 h); d.OGD/ R þ MH(24 h)þDkk1; e. OGD/R þ Dkk1.(F) A bar graph shows the quantification of apoptotic cells. The apoptosis rate ¼ [Annexin V(þ)PI(-) cells þ Annexin V(þ) PI(þ) cells]/total cells 100%.(G) Representative TUNEL images showing the apoptotic hippocampal nuclei in the different groups. a. Control;b.OGD/R;c.OGD/R þ MH(24h); d. OGD/R þ MH(24h)þ Dkk1;e.OGD/R þ Dkk1.(H) A bar graph showing the percentage of TUNEL-positive cells in different groups. The data represent the mean ± SD (n ¼ 3). *P < 0.05 compared to the control group; △P < 0.05 compared to the OGD/R group; #P < 0.05 compared to the OGD/R þ MH group.
Please cite this article in press as: T. Zhou, et al., Mild hypothermia protects against oxygen glucose deprivation/reoxygenation-induced apoptosis via the Wnt/b-catenin signaling pathway in hippocampal neurons, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.03.153
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Fig. 3. (continued).
significantly more apoptotic neurons than in those treated only with mild hypothermia (Fig. 3E and F). The TUNEL results were consistent with the Scatter diagram of PI/Annexin V (Fig. 3G and H). These results indicate that Wnt/b-catenin mediates the expression levels of apoptosis-related proteins and the protective effects of mild hypothermia against OGD/R-induced apoptosis.
3.4. Dkk1 blocks the protective effects of mild hypothermia against OGD/R-induced injury When observed with electron microscopy, the organelles in the OGD/R group were damaged or degraded, the mitochondrias were clearly swollen, and many cavities were observed. When the OGD/R neurons were treated with mild hypothermia for 24 h, there were fewer injured organelles. When the OGD/R neurons were synchronously treated with mild hypothermia and DKK1, the organelle injury was deteriorated again. (Fig. 4A). Our results also show that the LDH release rate increased significantly and that neuron viability decreased significantly in OGD/R-treated cells. By contrast, when the OGD/R neurons were treated with mild hypothermia for 24 h, the LDH release rate decreased significantly and neuron viability increased significantly. Furthermore, when the OGD/R neurons were synchronously treated with mild hypothermia and DKK1, the LDH release rate increased significantly and neuron viability decreased significantly compared to neurons treated with only mild hypothermia (Fig. 4B and C). These results indicate that Wnt/b-catenin mediates the protective effects of mild hypothermia against OGD/R-induced injury in hippocampal neurons.
4. Discussion Since the 1980's, researchers have been progressively elucidating the protective effects of mild hypothermia on experimental cerebral ischemia and craniocerebral injury. Currently, mild hypothermia is still a key treatment for cerebral ischemia/reperfusion injuries, as it has long-term effects on multiple cell signaling pathways, including necrotic and apoptotic pathways [14e17]. Nevertheless, the mechanistic details involved behind these effects remain unclear. Here we provide further evidence that mild hypothermia protects against oxygen glucose deprivation/ reoxygenation-induced apoptosis via the Wnt/b-catenin signaling pathway in hippocampal neurons. In order to investigate the effect of mild hypothermia on cerebral ischemia/reperfusion injury, we successfully cultivated hippocampal neurons. The mature neuronal cells cultivated in vitro keep all the physiological characteristics of neurons in vivo, and they have therefore been widely cultivated to replace in vivo neuron experiments [18,19]. Experimental in vitro models are often used to study molecular biology mechanisms and the biochemical and morphological changes related to processes of degeneration, because they allow for easier and more precise control of the extracellular environment compared to in vivo models [20]. For the past few years, the OGD model of neurons has been generally accepted as a suitable model to imitate cerebral ischemic-hypoxic injuries and has been widely used in studies of hypoxic-ischemic encephalopathy. Several studies have reported that, under ischemic-hypoxic conditions, the Wnt/b-catenin pathway is down-regulated and
Please cite this article in press as: T. Zhou, et al., Mild hypothermia protects against oxygen glucose deprivation/reoxygenation-induced apoptosis via the Wnt/b-catenin signaling pathway in hippocampal neurons, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.03.153
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Fig. 4. Effects of mild hypothermia and DKK1 on hippocampal neurons following OGD/R injury. (A) Injury of organelles observed with electron microscopy. a.Control; b.OGD/R; c. OGD/R þ MH(24 h); d.OGD/R þ MH(24 h)þDkk1; e.OGD/R þ Dkk1. In the control group, organelles were undamaged and regularly arranged. In the OGD/R group, the organelles were damaged or degraded. In the OGD/R þ MH(24 h) group, the severity of organelle injury was reduced. In the OGD/R þ MH(24 h)þDKK1 group, the organelle injury was deteriorated again.(Black arrows show swollen mitochondria). (B) A bar graph shows the quantification of cell viability. The cell viability was evaluated by the MTT value of different groups divided by the MTT value of the Control 100%. (C) A bar graph shows the quantification of LDH release. The LDH release rate (%) was evaluated by the extracellular LDH activity divided by the total LDH activity 100%. The data represent the mean ± SD (n ¼ 3). *P < 0.05 compared to the control group; △P < 0.05compared to the OGD/R group; # P < 0.05 compared to the OGD/R þ MH group.
cytoplasmic becatenin is degraded, which reduces c-myc and cyclin D1 gene transcription, among others [21e23]. These findings are consistent with our observations, which show that the protein expression levels of Wnt/b-catenin in the OGD/R group decreased significantly, while the protein expression levels of GSK3b increased significantly. b-catenin can be phosphorylated by GSK 3b and is then degraded by ubiquitin-proteasome system (UPS). In detail, it may be that OGD/R suppresses the expression of Wnt molecules and increases the expression of GSK3b, which both contribute to the degradation of b-catenin. Our results also show that mild hypothermia (24 h) significantly promoted the expression level of Wnt/b-catenin in hippocampal neurons after OGD/R injury. Meanwhile, the effects of mild hypothermia can be blocked by DKK1 (an antagonist of the canonical Wnt signaling pathway). These results indicate that mild hypothermia prevents the OGD/Rinduced suppression of Wnt/b-catenin in hippocampal neurons. In other words, mild hypothermia reactivates the Wnt/b-catenin signaling pathway, which was suppressed by OGD/R injury. These
findings are partially consistent with those of Hanfeng Zhang [11], who reported that hypothermia blocks molecular signaling downstream from GSK 3b and stops the degradation of b-catenin. Suppression of the Wnt/n-catenin signaling pathway has been shown to correlate with apoptosis in Mang cell lines [24,25]. The association between the canonical Wnt pathway and apoptosisdependent cellular death has also been widely reported. Zheng R [26] found that curcumin inhibits gastric carcinoma cell growth and induces apoptosis by suppressing the Wnt/b-catenin signaling pathway. Zhang RX [27] found that ursolic acid inhibits proliferation and induces apoptosis by inactivating the Wnt/b-catenin signaling pathway. b-catenin is the downstream effector of the Wnt signaling pathway, which regulates cell proliferation, differentiation, and apoptosis. b-catenin silencing increases the protein expression of phosphorylated-p53, the pro-apoptotic protein Bax and active caspase-3, while decreasing the expression of the antiapoptotic protein, Bcl-2. Caspases, as members of the cysteine protease family, play a critical role in apoptosis [28]. Cleaved
Please cite this article in press as: T. Zhou, et al., Mild hypothermia protects against oxygen glucose deprivation/reoxygenation-induced apoptosis via the Wnt/b-catenin signaling pathway in hippocampal neurons, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.03.153
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caspase-3 is an effector molecule that implements apoptosis. It can induce the cleavage of Poly(ADP-ribose) polymerase (PARP). PARP is a family of proteins involved in a number of cellular processes, mainly involving DNA repair and programmed cell death. Cleavage of PARP occurs at aspartic acid 214 and glycine 215, separating PARP into two 24kDA and 89kDA segments. The smaller moiety includes the zinc finger motif requisite in DNA binding, which is the initiating signal for apoptosis. The Wnt/b-catenin pathway regulates the expression of a number of genes involved in apoptosis. Therefore, we further investigated the effect of mild hypothermia on the Wnt/b-catenin pathway and apoptosis in hippocampal neurons following OGD/R injury. Our results show that mild hypothermia causes the downregulation of the expression of apoptosis promoting proteins (Bax, cleaved caspase-3) and the up-regulation of the expression of apoptosis inhibiting proteins (Bcl-2), thereby decreasing OGD/Rinduced injury. Meanwhile, the effects of mild hypothermia can be blocked by DKK1. We also found that mild hypothermia increased neuron viability and decreased the release of LDH in hippocampal neurons following OGD/R injury. At the same time, the protective effects of mild hypothermia were also blocked by DKK1. This indicates that the Wnt/b-catenin signaling pathway mediates the protective effects of mild hypothermia against OGD/ R-induced apoptosis and injury. In other words, these results imply that mild hypothermia protects neurons from OGD/Rinduced apoptosis and injury via the reactivation of the Wnt/bcatenin signaling pathway. Moreover, our study provides further evidence that the Wnt/b-catenin signaling pathway mediates the expression levels of apoptosis-related proteins. We found that suppression of the Wnt/b-catenin signaling pathway was correlated with increased apoptosis in hippocampal neurons after OGD/ R injury. In conclusion, we have demonstrated here that mild hypothermia induces the reactivation of the Wnt/b-catenin signaling pathway, which is suppressed by OGD/R injury, in hippocampal neurons and protects neurons from OGD/R-induced apoptosis via the reactivation of the Wnt/b-catenin signaling pathway. While further studies are necessary in order to elucidate the detailed sequence of reactions based on the upregulation of Wnt/b-catenin, the present findings further the general understanding of the mechanisms underlying the protective effect of mild hypothermia on OGD/R injury via the Wnt/b-catenin signaling pathway. Conflict of interest The authors declare no potential conflicts of interest. We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work. There is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in the manuscript entitled“Mild hypothermia protects against oxygen glucose deprivation/reoxygenation-induced apoptosis via the Wnt/ b-catenin signaling pathway in hippocampal neurons”. Acknowledgements This work was supported by the Medical Research Fund of Guangdong Province (Grant No. A2015392), the National Natural Science Foundation of China (Grant No. 81503052), the Public Welfare Research and Capacity Building Project of Guangdong Province (Grant No. 2014A020212164), the Basic and Applied Basic Research Project of Guangdong Province (Grant No. 2016A030313322), and the Guangzhou Science and Technology Research Project (Grant No. 201607010219).
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T. Zhou et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e9 focal cerebral ischemia induced by electroacupuncture pretreatment, Neurochem. Int. 97 (2016) 124e132. [25] Yuzhen Xu, Qian Wang, Dongsheng Li, Zhenghua Wu, Dawei Li, Kaili Lu, Yuwu Zhao, Yongning Sun, Protective effect of lithium chloride against hypoglycemia-induced apoptosis in neuronal pc12 cell, Neuroscience 330 (2016) 100e108. [26] R. Zheng, Q. Deng, Y. Liu, P. Zhao, Curcumin inhibits gastric carcinoma cell growth and induces apoptosis by suppressing the wnt/b-catenin signaling
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Mild hypothermia protects against oxygen glucose deprivation/ reoxygenation-induced apoptosis via the Wnt/b-catenin signaling pathway in hippocampal neurons Tianen Zhou a, 1, Yanran Liang b, 1, Longyuan Jiang a, Tao Yu a, Chaotao Zeng a, Enxiang Tao b, * a b
Department of Emergency, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China Department of Neurology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 March 2017 Accepted 27 March 2017 Available online xxx
Mild hypothermia is thought to be one of the most effective therapies for cerebral ischemia/reperfusion injuries. Our previous research revealed that mild hypothermia inhibits the activation of caspase-3 and protects against oxygen glucose deprivation/reoxygenation (OGD/R)-induced injury in hippocampal neurons. However, the mechanisms behind the activation of caspase-3 remain unclear. The aims of this study were to determine whether the protective effects of mild hypothermia were exerted through the Wnt/b-catenin signaling pathway. We found that, under OGD/R conditions, the pathway was down regulated, but mild hypothermia induced the reactivation of the Wnt/b-catenin signaling pathway, which had been suppressed by OGD/R injury. Mild hypothermia also caused the down regulation of the expression of apoptosis promoting proteins (Bax cleaved caspase-3), up-regulated the expression of apoptosis inhibiting proteins (Bcl-2), and ameliorated OGD/R injury-induced apoptosis. The protective effects of mild hypothermia were blocked by DKK1 (an antagonist of the canonical Wnt signaling pathway). Taken together, these results indicate that the Wnt/b-catenin signaling pathway mediates the protective effects of mild hypothermia against OGD/R-induced apoptosis. Our study provides evidence that mild hypothermia reactivates the Wnt/b-catenin signaling pathway, which is suppressed by OGD/R injury, in hippocampal neurons and protects neurons from OGD/R-induced apoptosis via the reactivation of the Wnt/b-catenin signaling pathway, ultimately suggesting that mild hypothermia could have therapeutic effects on OGD/R-induced apoptosis. © 2017 Elsevier Inc. All rights reserved.
Keywords: Mild hypothermia OGD/R Apoptosis Wnt/b-catenin
1. Introduction Mild hypothermia has been proven to be one of the most effective therapies for hypoxic-ischemic encephalopathy (HIE) without severe side effects [1,2]. The protective mechanism of mild hypothermia on HIE is complex, although many studies have found that it improves neurological function by reducing neuronal apoptosis [3,4]. Our previous research found that mild hypothermia could inhibit the activation of caspase-3 (an effector molecule implementing apoptosis) that was induced by oxygen glucose
* Corresponding author. Department of Neurology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, 107# Yanjiangxi Road Yuexiu District, Guangzhou 510120, China. E-mail address: [email protected] (E. Tao). 1 These authors contributed equally to this article.
deprivation/reoxygenation (OGD/R) in rat hippocampal neurons [5]. However, the mechanisms for the activation of caspase-3 remain unclear. Recent studies have shown that the Wnt/b-catenin signaling pathway is likely involved in ischemia/reperfusion(I/R) injury pathogenesis. Several studies have reported that, under ischemichypoxic conditions, the Wnt/b-catenin pathway is downregulated and cytoplasmic becatenin is degraded, which reduces the transcription of numerous target genes, including proapoptotic and inflammatory factors [6e8]. Moreover, the association between the canonical Wnt pathway and apoptosis-dependent cellular death has also been widely reported. Previous studies have indicated that the activation of the Wnt/b-catenin pathway protects Mang cell lines against I/R injury by attenuating apoptosis [9,10]. A few studies have also indicated that mild hypothermia can regulate the Wnt/b-catenin signaling pathway under I/R conditions.
http://dx.doi.org/10.1016/j.bbrc.2017.03.153 0006-291X/© 2017 Elsevier Inc. All rights reserved.
Please cite this article in press as: T. Zhou, et al., Mild hypothermia protects against oxygen glucose deprivation/reoxygenation-induced apoptosis via the Wnt/b-catenin signaling pathway in hippocampal neurons, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.03.153
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Hanfeng Zhang reported that hypothermia may block molecular signaling downstream of GSK 3b and stop the degradation of bcatenin [11]. However, whether mild hypothermia can protect neurons from apoptosis via the Wnt/b-catenin signaling pathway has remained poorly understood and has not been fully elucidated. The mechanisms behind the effects of mild hypothermia on the Wnt/b-catenin signaling pathway also need further analysis. In this study, we used the OGD/R model of rat hippocampal neurons to simulate I/R injury and HIE. The aim of our study was to test whether mild hypothermia could protect neurons from OGD/Rreduced apoptosis via the Wnt/b-catenin signaling pathway. 2. Materials and methods 2.1. Cell culture Hippocampal neurons in primary culture were used for our study. Suckling rats born within 1d to 3d prior to the experiments were selected (provided by the Experimental Animal Center of Sun Yat-sen University). After disinfecting, the cerebrum tissues of rats were quickly removed and hippocampal tissues were separated and placed in a centrifuge tube containing Dulbecco's Modified Eagle's Medium (DMEM)/F12 medium (Gibco Company, U.S.) and were gently scattered. Then, the hippocampal neurons were inoculated into a 6 cm culture dish with DMEM/F12 containing 10% fetal bovine serum (Hyclone Company, USA) at a density of 1 106 cells per dish, and then incubated in incubator at 37 C. After 24 h, the medium was completely replaced with a medium containing 2% B27 (Gibco Company, USA). On the third day, cytosine arabinoside at a final concentration of 5 mmol/L was added for 24 h, in order to inhibit glial cells. On the 8th day, immunochemical fluorescence identification of neuronal microtubule-associated protein 2 (MAP2) was employed. After the immunochemical fluorescence identification of neuronal MAP-2, we confirmed that hippocampal neurons accounted for over 90% of all of the cultured cells. The results of the identification of neuronal MAP-2 were shown in our previous article [5]. 2.2. Oxygen glucose deprivation and reoxygenation (OGD/R) Cell cultures were subjected to OGD injury using a protocol described previously [5]. In brief, the OGD cell culture medium was replaced with a glucose-free balanced salt solution (BSS) containing 130 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 26 mM NaHCO3, 0.8 mM MgCl2, and 1.18 mM NaH2PO4. It was then transferred to an anaerobic chamber (PLAS & LABS, MI, USA), equilibrated for 10 min with a continuous flux of gas (95% N2/5% CO2), and humidified at 37 C for 6 h. To terminate OGD assays, cultures were carefully washed with DMEM containing 10% fetal bovine serum and OGD cells were reoxygenated in an incubator (74% N2/21% O2/5% CO2) at 37 C or 32 C for 12 h or 24 h. Control cells were incubated in DMEM containing 10% fetal bovine serum in a normoxic incubator for the same period of time. 2.3. Grouping and drug treatment The experimental cells were randomly divided into five groups: the Control, OGD/R, OGD/R þ mild hypothermia (OGD/R þ MH), OGD/Rþ mild hypothermia þ Dickkopf-1 (OGD/R þ MH þ DKK1), and OGD/R þ Dickkopf-1 (OGD/R þ DKK1). Next, 100 ng/ml Dkk1, an antagonist of the canonical Wnt signaling pathway (Sigma, St Louis, MO, USA), was immediately added after OGD for 24 h. The duration of OGD was 6 h. The temperature for the induction of mild hypothermic was 32 C and the duration was either 12 h or 24 h after OGD. All the neurons were detected 24 h after reoxygenation.
2.4. Western blot analysis Cell lysates were diluted in 5 SDS buffer and denatured at 100 C for 3 min. Proteins were electrophoresed on a 12% SDSepolyacrylamide gel and transferred onto polyvinylidene difluoride membranes using a Bio-Rad transblot apparatus. After incubation at room temperature for 1 h in blocking solution (5% nonfat dry milk), membranes were incubated overnight at 4 C with primary antibodies, including Wnt1 (Santa Cruz Biotechnology, California, USA), b-catenin (ABclonal Biotech, Hubei, China), GSK-3b (ABclonal Biotech, Hubei, China), cleaved caspase-3 (Abcam, Cambridge, MA, USA), Bcl-2 (Cell Signaling Technology, USA), Bax (Cell Signaling Technology, USA), or b-actin (Sigma, St Louis, MO, USA) antibodies, followed by goat anti-rabbit or antimouse IgG (Beyotime Biotech, Jiangsu, China). Immunoreactive proteins were detected by enhanced chemiluminescence. All data were normalized to the levels of b-actin that were used as the loading control, and the amount of immunoreactivity was expressed relative to the corresponding control. 2.5. Immunofluorescence Neurons were post-fixed in 4% paraformaldehyde for 20 min and incubated in 0.3% Triton X-100 for 20 min. They were then washed 3 times in PBS, blocked with 5% BSA solution for 1 h, and incubated with a primary antibody overnight at 4 C. The primary antibodies were anti-MAP2 (APPLYGEN, Biotech, Beijing, China), anti-b-catenin (ABclonal Biotech, Hubei, China), and anti-GSK-3b (ABclonal Biotech, Hubei, China). Afterward, they were incubated for 1 h with FITC-conjugated secondary antibodies (Beyotime Biotech, Jiangsu, China). The nuclei were stained with DAPI (Sigma, St Louis, MO, USA) and the slides were left to dry overnight at room temperature. Immunostaining was examined using a laser confocal microscope (Leica, Heidelberg, Germany) and then images were analyzed by Image-Pro Plus 6.0 software package (Bethesda, MD,USA). 2.6. Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay TUNEL staining was performed using an in situ apoptosis detection kit (YEASEN, Shanghai, China) according to the manufacturer's protocols. Neurons were seeded into 6-well plates and fixed with 4% paraformaldehyde for 20 min, before being incubated in 0.3% Triton X-100 for 10 min. Afterward, the neurons were stained as per the TUNEL method. Images were acquired with a fluorescence microscope (LEICA DM4000B, LEICA, Germany). Cells in five randomly selected high-power fields(400 ) were evaluated and the TUNEL-positive cells were counted and analyzed by ImagePro Plus 6.0 software package (Bethesda, MD,USA). 2.7. Measurement of apoptotic rate In order to assess the apoptotic rate of neurons, a flow cytometer was employed. After reoxygenation for 24 h, the various groups of cells were collected and resuspended with phosphate buffer solution. Annexin VeFITC (Austria Bender Medsystems Company) and PI (America Sigma Company) were added and mixed uniformly. Next, the apoptotic rate was detected with the flow cytometer (America Becton Dickinson Company). 2.8. Measurement of lactic acid dehydrogenase (LDH) release rate After OGD, cytoplasmic LDH was partially released into the culture liquid. A higher LDH release rate is related to more serious
Please cite this article in press as: T. Zhou, et al., Mild hypothermia protects against oxygen glucose deprivation/reoxygenation-induced apoptosis via the Wnt/b-catenin signaling pathway in hippocampal neurons, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.03.153
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cell injury. After OGD and reoxygenation for 24 h, the medium was collected and LDH activity was determined using a commercially available assay kit (Beyotime Biotech, Jiangsu, China), according to the manufacturer's protocol [12,13]. First, 50 mL of the culture liquid was removed for the detection of extracellular LDH activity. The cell lysate with Triton-100 was then added into the culture dish and the supernatant liquid was collected to detect the total cell LDH activity. LDH release rate (%) ¼ extracellular LDH activity/total LDH activity. 2.9. Measurement of cell viability In order to assess the viability of neurons, a quantitative colorimetric MTT assay was employed. Briefly, cells were seeded into 96well plates (1 104 cells per well). Then, 24 h after reoxygenation, 20 mL of the MTT solution (5 mg/mL, Amresco Company, USA) was added into each well. The neurons were incubated at 37 C for 4 h in a humidified atmosphere and, finally, the absorbances of each well at 490 nm were measured using an ELISA 96-well plate reader (BioRad Laboratories, CA, USA). Results are expressed as the percentage of viable cells detected following OGD compared to the normoxic control plates. 2.10. Statistical analysis The measurement data were expressed as the mean ± standard deviation (X±s). Parametric unpaired two-tailed Student's t-tests (to compare two groups) or 1-way ANOVA (for more than two groups) were used for data with a normal distribution. The variance homogeneity test was then conducted. All calculations were performed using SPSS 13.0 statistical software. Differences with P < 0.05 were considered statistically significant. 3. Results 3.1. Mild hypothermia induced the reactivation of the Wnt/bcatenin signaling pathway, which had been suppressed by OGD/R injury in hippocampal neurons The expression level of Wnt/b-catenin in the OGD/R group was significantly lower than in the control. Mild hypothermia (continuing for 24 h after OGD) significantly promoted the Wnt/b-
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catenin expression levels, compared to the OGD/R neurons not treated with mild hypothermia (Fig. 1A and B). These results indicate that OGD/R suppresses the activity of Wnt/becatenin, which can be recovered by mild hypothermia. 3.2. Dkk1 blocks the effects of mild hypothermia on the Wnt/bcatenin signaling pathway in hippocampal neurons following OGD/ R injury Our results showed that the protein levels of Wnt-1 and b -catenin were significantly decreased and that GSK-3b was significantly increased in OGD/R-treated cells. By contrast, when the OGD/R neurons were treated with mild hypothermia for 24 h, the protein levels of Wnt-1 and b-catenin increased significantly and GSK-3b decreased significantly. Furthermore, when the OGD/R neurons were synchronously treated with mild hypothermia and DKK1, the protein levels of Wnt-1 and b-catenin were significantly lower and GSK-3b was significantly higher than those treated with only mild hypothermia (Fig. 2A and B). The immunofluorescence results were consistent with the protein levels (Fig. 2C and D). These indicate that the effects of mild hypothermia are mediated through the reactivation of the Wnt/b-catenin signaling pathway. 3.3. Wnt/b-catenin mediates the expression levels of apoptosisrelated proteins and the protective effects of mild hypothermia against OGD/R-induced apoptosis Our results showed that the expression levels of apoptosisrelated proteins (Bax, cleaved caspase-3, and cleaved PARP) had increased significantly and that Bcl-2 had decreased significantly in OGD/R-treated cells. By contrast, when the OGD/R neurons were treated with mild hypothermia for 24 h, the protein levels of Bax, cleaved caspase-3, and cleaved PARP decreased significantly and Bcl-2 increased significantly. Furthermore, when the OGD/R neurons were synchronously treated with mild hypothermia and DKK1, the protein levels of all three increased significantly and Bcl-2 was decreased significantly compared to neurons treated with only mild hypothermia (Fig. 3A, B, C, D). Our results also showed that there were significantly more apoptotic neurons in OGD/R-treated cells than when OGD/R neurons were treated with mild hypothermia for 24 h. Furthermore, when OGD/R neurons were synchronously treated with mild hypothermia and DKK1, there were
Fig. 1. Effects of mild hypothermia on Wnt/b-catenin signaling following OGD/R in rat hippocampal neurons. (A) Representative Western blot images showing the expression of Wnt-1, becatenin, and GSK-3b in different groups. (B) A bar graph showing the quantification of Wnt-1/b-actin, becatenin/b-actin, and GSK-3b/b-actin in different groups. The data represent the mean ± SD (n ¼ 3). *P < 0.05 compared to the control group; △P < 0.05 compared to the OGD/R group.
Please cite this article in press as: T. Zhou, et al., Mild hypothermia protects against oxygen glucose deprivation/reoxygenation-induced apoptosis via the Wnt/b-catenin signaling pathway in hippocampal neurons, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.03.153
Fig. 2. Dkk1 blocks the effects of mild hypothermia via the Wnt/b-catenin signaling pathway in hippocampal neurons following OGD/R injury. (A) Representative images of Western blots showing the expression of Wnt-1, becatenin, and GSK-3b in different groups. (B) A bar graph showing the quantification of Wnt-1/b-actin, becatenin/b-actin, and GSK-3b/b-actin in different groups. (C) Dual immunofluorescence staining for b -catenin (red) and DAPI (blue) or GSK-3b (red) and DAPI (blue) in hippocampal neurons. a. Control; b.OGD/R; c.OGD/R þ MH(24 h); d. OGD/R þ MH(24 h)þDkk1; e.OGD/R þ Dkk1. (D) A bar graph showing the relative immunofluorescence intensity of becatenin and GSK-3b in different groups. The data represent the mean ± SD (n ¼ 3). *P < 0.05 compared to the control group; △P < 0.05 compared to the OGD/R group; #P < 0.05 compared to the OGD/ R þ MH group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: T. Zhou, et al., Mild hypothermia protects against oxygen glucose deprivation/reoxygenation-induced apoptosis via the Wnt/b-catenin signaling pathway in hippocampal neurons, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.03.153
Fig. 3. Wnt/b-catenin mediates the expression levels of apoptosis-related proteins and the protective effects of mild hypothermia against OGD/R-induced apoptosis. (A) Representative Western blot images showing the expression of Bax and Bcl-2 in different groups. (B) A bar graph showing the quantification of Bax/b-actin and Bcl-2/b-actin in different groups. (C) Representative Western blot images showing the expression of cleaved caspase-3 and PARP in different groups. (D) A bar graph shows the quantification of cleaved caspase-3/b-actin and PARP/b-actin in different groups. (E) Scatter diagram of PI/Annexin V gating from different groups. a. Control; b.OGD/R; c.OGD/R þ MH(24 h); d.OGD/ R þ MH(24 h)þDkk1; e. OGD/R þ Dkk1.(F) A bar graph shows the quantification of apoptotic cells. The apoptosis rate ¼ [Annexin V(þ)PI(-) cells þ Annexin V(þ) PI(þ) cells]/total cells 100%.(G) Representative TUNEL images showing the apoptotic hippocampal nuclei in the different groups. a. Control;b.OGD/R;c.OGD/R þ MH(24h); d. OGD/R þ MH(24h)þ Dkk1;e.OGD/R þ Dkk1.(H) A bar graph showing the percentage of TUNEL-positive cells in different groups. The data represent the mean ± SD (n ¼ 3). *P < 0.05 compared to the control group; △P < 0.05 compared to the OGD/R group; #P < 0.05 compared to the OGD/R þ MH group.
Please cite this article in press as: T. Zhou, et al., Mild hypothermia protects against oxygen glucose deprivation/reoxygenation-induced apoptosis via the Wnt/b-catenin signaling pathway in hippocampal neurons, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.03.153
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Fig. 3. (continued).
significantly more apoptotic neurons than in those treated only with mild hypothermia (Fig. 3E and F). The TUNEL results were consistent with the Scatter diagram of PI/Annexin V (Fig. 3G and H). These results indicate that Wnt/b-catenin mediates the expression levels of apoptosis-related proteins and the protective effects of mild hypothermia against OGD/R-induced apoptosis.
3.4. Dkk1 blocks the protective effects of mild hypothermia against OGD/R-induced injury When observed with electron microscopy, the organelles in the OGD/R group were damaged or degraded, the mitochondrias were clearly swollen, and many cavities were observed. When the OGD/R neurons were treated with mild hypothermia for 24 h, there were fewer injured organelles. When the OGD/R neurons were synchronously treated with mild hypothermia and DKK1, the organelle injury was deteriorated again. (Fig. 4A). Our results also show that the LDH release rate increased significantly and that neuron viability decreased significantly in OGD/R-treated cells. By contrast, when the OGD/R neurons were treated with mild hypothermia for 24 h, the LDH release rate decreased significantly and neuron viability increased significantly. Furthermore, when the OGD/R neurons were synchronously treated with mild hypothermia and DKK1, the LDH release rate increased significantly and neuron viability decreased significantly compared to neurons treated with only mild hypothermia (Fig. 4B and C). These results indicate that Wnt/b-catenin mediates the protective effects of mild hypothermia against OGD/R-induced injury in hippocampal neurons.
4. Discussion Since the 1980's, researchers have been progressively elucidating the protective effects of mild hypothermia on experimental cerebral ischemia and craniocerebral injury. Currently, mild hypothermia is still a key treatment for cerebral ischemia/reperfusion injuries, as it has long-term effects on multiple cell signaling pathways, including necrotic and apoptotic pathways [14e17]. Nevertheless, the mechanistic details involved behind these effects remain unclear. Here we provide further evidence that mild hypothermia protects against oxygen glucose deprivation/ reoxygenation-induced apoptosis via the Wnt/b-catenin signaling pathway in hippocampal neurons. In order to investigate the effect of mild hypothermia on cerebral ischemia/reperfusion injury, we successfully cultivated hippocampal neurons. The mature neuronal cells cultivated in vitro keep all the physiological characteristics of neurons in vivo, and they have therefore been widely cultivated to replace in vivo neuron experiments [18,19]. Experimental in vitro models are often used to study molecular biology mechanisms and the biochemical and morphological changes related to processes of degeneration, because they allow for easier and more precise control of the extracellular environment compared to in vivo models [20]. For the past few years, the OGD model of neurons has been generally accepted as a suitable model to imitate cerebral ischemic-hypoxic injuries and has been widely used in studies of hypoxic-ischemic encephalopathy. Several studies have reported that, under ischemic-hypoxic conditions, the Wnt/b-catenin pathway is down-regulated and
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Fig. 4. Effects of mild hypothermia and DKK1 on hippocampal neurons following OGD/R injury. (A) Injury of organelles observed with electron microscopy. a.Control; b.OGD/R; c. OGD/R þ MH(24 h); d.OGD/R þ MH(24 h)þDkk1; e.OGD/R þ Dkk1. In the control group, organelles were undamaged and regularly arranged. In the OGD/R group, the organelles were damaged or degraded. In the OGD/R þ MH(24 h) group, the severity of organelle injury was reduced. In the OGD/R þ MH(24 h)þDKK1 group, the organelle injury was deteriorated again.(Black arrows show swollen mitochondria). (B) A bar graph shows the quantification of cell viability. The cell viability was evaluated by the MTT value of different groups divided by the MTT value of the Control 100%. (C) A bar graph shows the quantification of LDH release. The LDH release rate (%) was evaluated by the extracellular LDH activity divided by the total LDH activity 100%. The data represent the mean ± SD (n ¼ 3). *P < 0.05 compared to the control group; △P < 0.05compared to the OGD/R group; # P < 0.05 compared to the OGD/R þ MH group.
cytoplasmic becatenin is degraded, which reduces c-myc and cyclin D1 gene transcription, among others [21e23]. These findings are consistent with our observations, which show that the protein expression levels of Wnt/b-catenin in the OGD/R group decreased significantly, while the protein expression levels of GSK3b increased significantly. b-catenin can be phosphorylated by GSK 3b and is then degraded by ubiquitin-proteasome system (UPS). In detail, it may be that OGD/R suppresses the expression of Wnt molecules and increases the expression of GSK3b, which both contribute to the degradation of b-catenin. Our results also show that mild hypothermia (24 h) significantly promoted the expression level of Wnt/b-catenin in hippocampal neurons after OGD/R injury. Meanwhile, the effects of mild hypothermia can be blocked by DKK1 (an antagonist of the canonical Wnt signaling pathway). These results indicate that mild hypothermia prevents the OGD/Rinduced suppression of Wnt/b-catenin in hippocampal neurons. In other words, mild hypothermia reactivates the Wnt/b-catenin signaling pathway, which was suppressed by OGD/R injury. These
findings are partially consistent with those of Hanfeng Zhang [11], who reported that hypothermia blocks molecular signaling downstream from GSK 3b and stops the degradation of b-catenin. Suppression of the Wnt/n-catenin signaling pathway has been shown to correlate with apoptosis in Mang cell lines [24,25]. The association between the canonical Wnt pathway and apoptosisdependent cellular death has also been widely reported. Zheng R [26] found that curcumin inhibits gastric carcinoma cell growth and induces apoptosis by suppressing the Wnt/b-catenin signaling pathway. Zhang RX [27] found that ursolic acid inhibits proliferation and induces apoptosis by inactivating the Wnt/b-catenin signaling pathway. b-catenin is the downstream effector of the Wnt signaling pathway, which regulates cell proliferation, differentiation, and apoptosis. b-catenin silencing increases the protein expression of phosphorylated-p53, the pro-apoptotic protein Bax and active caspase-3, while decreasing the expression of the antiapoptotic protein, Bcl-2. Caspases, as members of the cysteine protease family, play a critical role in apoptosis [28]. Cleaved
Please cite this article in press as: T. Zhou, et al., Mild hypothermia protects against oxygen glucose deprivation/reoxygenation-induced apoptosis via the Wnt/b-catenin signaling pathway in hippocampal neurons, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.03.153
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T. Zhou et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e9
caspase-3 is an effector molecule that implements apoptosis. It can induce the cleavage of Poly(ADP-ribose) polymerase (PARP). PARP is a family of proteins involved in a number of cellular processes, mainly involving DNA repair and programmed cell death. Cleavage of PARP occurs at aspartic acid 214 and glycine 215, separating PARP into two 24kDA and 89kDA segments. The smaller moiety includes the zinc finger motif requisite in DNA binding, which is the initiating signal for apoptosis. The Wnt/b-catenin pathway regulates the expression of a number of genes involved in apoptosis. Therefore, we further investigated the effect of mild hypothermia on the Wnt/b-catenin pathway and apoptosis in hippocampal neurons following OGD/R injury. Our results show that mild hypothermia causes the downregulation of the expression of apoptosis promoting proteins (Bax, cleaved caspase-3) and the up-regulation of the expression of apoptosis inhibiting proteins (Bcl-2), thereby decreasing OGD/Rinduced injury. Meanwhile, the effects of mild hypothermia can be blocked by DKK1. We also found that mild hypothermia increased neuron viability and decreased the release of LDH in hippocampal neurons following OGD/R injury. At the same time, the protective effects of mild hypothermia were also blocked by DKK1. This indicates that the Wnt/b-catenin signaling pathway mediates the protective effects of mild hypothermia against OGD/ R-induced apoptosis and injury. In other words, these results imply that mild hypothermia protects neurons from OGD/Rinduced apoptosis and injury via the reactivation of the Wnt/bcatenin signaling pathway. Moreover, our study provides further evidence that the Wnt/b-catenin signaling pathway mediates the expression levels of apoptosis-related proteins. We found that suppression of the Wnt/b-catenin signaling pathway was correlated with increased apoptosis in hippocampal neurons after OGD/ R injury. In conclusion, we have demonstrated here that mild hypothermia induces the reactivation of the Wnt/b-catenin signaling pathway, which is suppressed by OGD/R injury, in hippocampal neurons and protects neurons from OGD/R-induced apoptosis via the reactivation of the Wnt/b-catenin signaling pathway. While further studies are necessary in order to elucidate the detailed sequence of reactions based on the upregulation of Wnt/b-catenin, the present findings further the general understanding of the mechanisms underlying the protective effect of mild hypothermia on OGD/R injury via the Wnt/b-catenin signaling pathway. Conflict of interest The authors declare no potential conflicts of interest. We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work. There is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in the manuscript entitled“Mild hypothermia protects against oxygen glucose deprivation/reoxygenation-induced apoptosis via the Wnt/ b-catenin signaling pathway in hippocampal neurons”. Acknowledgements This work was supported by the Medical Research Fund of Guangdong Province (Grant No. A2015392), the National Natural Science Foundation of China (Grant No. 81503052), the Public Welfare Research and Capacity Building Project of Guangdong Province (Grant No. 2014A020212164), the Basic and Applied Basic Research Project of Guangdong Province (Grant No. 2016A030313322), and the Guangzhou Science and Technology Research Project (Grant No. 201607010219).
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Please cite this article in press as: T. Zhou, et al., Mild hypothermia protects against oxygen glucose deprivation/reoxygenation-induced apoptosis via the Wnt/b-catenin signaling pathway in hippocampal neurons, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.03.153