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Recombinant Osteopontin Improves Neurological Functional Recovery and Protects Against Apoptosis via PI3K/Akt/GSK-3β Pathway Following Intracerebral Hemorrhage

Recombinant Osteopontin Improves Neurological Functional Recovery and Protects Against Apoptosis... ANIMAL STUDY e-ISSN 1643-3750 © Med Sci Monit, 2018; 24: 1588-1596 DOI: 10.12659/MSM.905700 Received: 2017.06.09 Recombinant Osteopontin Improves Neurological Accepted: 2017.09.04 Published: 2018.03.18 Functional Recovery and Protects Against Apoptosis via PI3K/Akt/GSK-3b Pathway Following Intracerebral Hemorrhage Authors’ Contrib ution: AE 1 Wenqian Zhang 1 Department of Surgery, Hebei Medical University, Shijiazhuang, Hebei, P.R. China Study Design A 2 Department of Neurosurgery, Tangshan Gongren Hospital, Tangshan, Hebei, BF 2 Ying Cui Data Collection B P.R. China AF 3,4 Junling Gao Statistical Analysis C 3 School of Basic Medical Science, North China University of Science and Data Interpr etation D CD 3,4 Ran Li Technology, Tangshan, Hebei, P.R. China Manuscript Preparation E 4 Hebei Key Laboratory for Chronic Diseases, Tangshan Key Laboratory for BF 3,4 Xiaohua Jiang Literature Search F Preclinical and Basic Research on Chronic Diseases, Tangshan, Hebei, P.R. China D 3,4 Yanxia Tian Funds Collection G BC 2 Kaijie Wang AE 1,2 Jianzhong Cui Corresponding Author: Jianzhong Cui, e-mail: [email protected] Source of support: This study was supported by a grant from the Natural Science Foundation of Hebei, China (grant no. H2012401071 and H2014105079) Background: This study aimed to investigate the potential neuroprotective effect of recombinant osteopontin (r-OPN) on apop - totic changes via modulating phosphoinositide-3-kinase/Akt/glycogen synthase kinase 3 beta (PI3K/Akt/GSK-3b) signaling in a rat model of intracerebral hemorrhage (ICH). Material/Methods: We subjected 10–12-week-old Sprague-Dawley male rats (n=120) to injection of autologous blood into the right basal ganglia to induce ICH or sham surgery. ICH animals received vehicle administration, r-OPN (4 μL/pup), or r-OPN combined with phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin (86 ng/pup) at 30 min af- ter injury. Neurological scores and rotarod latencies were evaluated on days 1–5 post-ICH. Brain water content was evaluated on days 1–3 post-ICH. The number of apoptotic cells changes were evaluated by terminal de- oxynucleotidyl transferase-mediated 2-deoxyuridine 5-triphosphate-biotin nick-end labeling (TUNEL) and he- matoxylin staining. Apoptosis-related proteins Bcl-2, Bax, and cleaved caspase-3 (CC3), and the phosphoryla- tion levels of Akt and GSK-3b were assayed by Western blot. Results: Neurological deficits, rotarod latencies, and brain water content following ICH were reduced in the r-OPN group compared to the vehicle group. r-OPN also attenuated cell death in ICH. Furthermore, treatment with r-OPN sig- nificantly increased p-Akt expression and decreased p-GSK-3 b. These effects were associated with a decrease in the Bax/Bcl-2 ratio and the suppression of CC3 at 24 h after ICH. Importantly, all the beneficial effects of r-OPN in ICH were abrogated by the PI3K inhibitor wortmannin. Conclusions: r-OPN may provide a wide range of neuroprotection by suppressing apoptosis through the PI3K/Akt/GSK-3b signaling pathway after ICH. MeSH Keywords: Apoptosis • Cerebral Hemorrhage • Osteopontin • Phosphatidylinositol 3-Kinases Full-text PDF: https://www.medscimonit.com/abstract/index/idArt/905700 3313 — 5 46 Indexed in: [Current Contents/Clinical Medicine] [SCI Expanded] [ISI Alerting System] This work is licensed under Creative Common Attribution- [ISI Journals Master List] [Index Medicus/MEDLINE] [EMBASE/Excerpta Medica] NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) [Chemical Abstracts/CAS] Zhang W. et al.: Recombinant osteopontin improves neurological functional recovery… ANIMAL STUDY © Med Sci Monit, 2018; 24: 1588-1596 Background to improve neurological outcomes and behavioral outcomes by inhibiting apoptosis in a rat model of ICH. The PI3K/Akt/ Intracerebral hemorrhage (ICH) accounted for 9% to 27% of all GSK-3b pathway was hypothesized and tested as the possi- strokes worldwide in the last decade, with poor functional out- ble mechanism of r-OPN effects. come and high early case fatality [1–3]. Despite this devastat- ing personal and societal impact, ICH remains an understudied disease with little improvement in patient outcomes over the Material and Methods last 20 years [4]. Studies have demonstrated that ICH-induced neurological damage can be divided into primary and second- Animals and ICH Model ary brain injury. Primary injury evoked by the physical destruc- tion due to rapid hematoma formation in the brain parenchy- Adult male Sprague-Dawley rats (11–13 weeks, 280–320 g) ma leads to brain tissue compression or disruption, as well were used in the study. The procedures for this study were as elevated intracranial pressure. Subsequent development of approved by the Animal Care and Use Committee of Hebei various parallel pathological processes such as neuroinflam - Medical University and were carried out in accordance with mation, neuronal apoptosis, and excitotoxicity contribute to the Guide for the Chinese Council on Animal Protection. All secondary injury [5,6]. Apoptosis is a prominent form of cell rats were housed under conditions of controlled temperature death associated with ICH in the perihematomal region [7,8]. and humidity. Rats had free access to food and water before ICH has long been reported to cause neuronal apoptosis lead- and after surgery or sham treatment. Our injury model was ing to declines in neurological functions [9]. Considering intact adapted from a previously described model of ICH in rats [23]. neurons as the basis of normal neurological functions, inhibi- The rats were anesthetized with 0.4% pentobarbital sodium tion of neuronal apoptosis and alleviating neurological deficits intraperitoneally. Through the tail vein, we removed 50 ml of are helpful to the patient’s long-term prognosis. autologous whole blood. Using stereotaxic guidance, we ad- ministered autologous whole blood into the right caudate pu- Osteopontin (OPN) is a secreted extracellular matrix (ECM) gly- tamen. The stereotactic coordinates were: 0.2 mm anterior, 3.0 coprotein that is involved in both physiological and pathological mm lateral to bregma, and 5.5 mm ventral. The sham group processes in a wide range of tissues [10]. There is compelling only had needle insertion. After slowly withdrawing the injec- evidence that endogenous OPN is the most highly upregulat- tion needle 10 min after injection, sealed with zinc phosphate ed factor after central nervous system disease [11,12]. It can cement, the incision was closed and disinfected with local io- regulate inflammation, tissue remodeling, and cell survival in dine. The animals were allowed to recover spontaneous ven- the field of stroke injury [10,13,14]. For example, recombinant tilation. In the course of the establishment of the experimen- osteopontin (r-OPN) reduced cortical neuron death via the ac- tal group in the ICH model, a total of 10 rats died. tivation the phosphatidylinositol 3-kinase (PI3K) -Akt signal- ing pathway kinase and increased expression of pro-survival Experimental groups and treatment genes in ischemic stroke [13]. Moreover, r-OPN provided neu- roprotection by anti-apoptotic action in subarachnoid hemor- We randomly assigned 150 adult rats to 5 groups (n=30): rhage and exerted an anti-inflammatory effect by downregulat - the sham group, the vehicle (ICH+vehicle) group, the r-OPN ing inducible NO synthase (iNOS) in ICH [14,15]. Nevertheless, (ICH+r-OPN) group, r-OPN+Wort (ICH+r-OPN+ wortmannin) until now, no published study investigated the effect of r-OPN group, and the Wort (ICH+Wortmannin) group. Mouse r-OPN administration on the apoptotic changes associated with re- (0.1 μg in 1 μL of sterile PBS; EMD chemicals, La Jolla, CA) or covery of neurological function during ICH. vehicle (1 μL; sterile PBS) were intracerebroventricularly inject - ed at 1 h after ICH. The PI3K inhibitor wortmannin (15 μg/kg; Glycogen synthase kinase 3b (GSK-3b) is a serine/threonine Sigma-Aldrich, St Louis, MO) was administered before the in- kinase known to be important in cell proliferation and apop- duction of ICH. In the sham, the needle was inserted, with no tosis signaling pathways [16,17]. The stimulated GSK-3b has administration of drug. been found to exacerbate brain injury in experimental ischemic stroke, traumatic brain injury, and ICH [18–20]. The pro-apop- Brain water content totic role of GSK-3b has previously been shown to be negatively regulated through the phosphoinositol-3-kinase (PI3-kinase)- Rats were anesthetized and euthanized on days 1–3 after ICH Akt survival pathway [21,22]. Further studies of r-OPN and in- to coincide with the point of maximum apoptotic changes. The terconnected anti-apoptotic mechanisms will help to develop procedure was carried out as previously described [24]. Brains new therapeutic targets to reduce cell death and to explore were sectioned in the mid-sagittal plane, and hemispheres were neuroprotective properties after ICH. In this study, we inves- weighed immediately (‘‘wet’’ weight). Then, the hemispheres tigated the role of r-OPN in amelioration of brain edema and were dried at 80°C for 24 h, and ‘‘dry’’ weight was measured. Indexed in: [Current Contents/Clinical Medicine] [SCI Expanded] [ISI Alerting System] This work is licensed under Creative Common Attribution- [ISI Journals Master List] [Index Medicus/MEDLINE] [EMBASE/Excerpta Medica] NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) [Chemical Abstracts/CAS] Zhang W. et al.: Recombinant osteopontin improves neurological functional recovery… ANIMAL STUDY © Med Sci Monit, 2018; 24: 1588-1596 We calculated water content as a percentage of wet weight hydrate, rats were sacrificed at different time points after sur - ([wet weight–dry weight]/[wet weight]×100). gery. Brain tissue, except for the caudate putamen tissues sur- rounding the hematoma, was instantly frozen at –80°C until Neurological score use. To detect protein expression, an appropriate amount of frozen brain tissue samples was minced with eye scissors in Modified neurological severity score (mNSS) is a composite ice. Then, the tissue was homogenized in cold lysis buer ff and of motor tests, sensory tests, beam balance tests, and reflex - then clarified by centrifuging (12000×g for 20 min at 4°C) be - es absent [25]. In our study, it was used to grade various as- fore taking the supernatant. Protein concentration was de- pects of neurobehavioral function on days 1–5 after ICH. Lower termined using the Bradford method. The samples were sub- scores indicate better function (normal score=0), and higher jected to SDS-polyacrylamide gel electrophoresis to resolve scores indicate more severe injury (maximal deficit score=18). and were transferred onto a polyvinylidene fluoride (PVDF) filter membrane by a transfer apparatus at 350 mA for 2 h. Rotarod test Following blocked with 5% fat-free dry milk, the membrane was then incubated overnight with primary antibody at 4°C, An accelerating automated rotarod was used to measure the including Actin (anti-goat, 1: 2000, Santa Cruz Biotechnology), effects of therapeutic intervention on vestibulomotor func - p-GSK-3b (anti-rabbit, 1: 1000, Abcam), GSK-3b (anti-rabbit, tion [26]. The speed was slowly increased from 4 to 40 rpm 1: 1000, Abcam), p-Akt (anti-mouse, 1: 1000, Abcam), Akt (anti- within 5 min. When the rats were dropped twice from the mouse, 1: 1000, Abcam), cleaved caspase-3 (CC3, anti-mouse, ladder of the device, the experiment ended. On 3 days before 1: 1000; Cell Signaling), caspase-3 (anti-mouse, 1: 1000; Cell the ICH injury, the rats were pretrained, and the average time Signaling), Bcl-2 (anti-rabbit, 1: 1000, Cell Signaling Technology), elapsed from the rotation of the cylinder was recorded as the and Bax (anti-mouse, anti-1000, Cell Signaling Technology). baseline latency. To assess early motor outcome, mice under- Nitrocellulose membranes were then labeled with secondary went rotarod testing on days 1–5 after injury. Average laten- antibodies for 1 h at room temperature. cy to fall from the rod was recorded. Statistical analyses Apoptosis analysis by terminal deoxynucleotidyl transferase-mediated (dUTP) nick-end labeling (TUNEL) All data are presented as the mean ±SD and were analyzed staining using SPSS 17.0. The data were analyzed by one-way ANOVA, followed by Tukey’s post hoc analysis. Differences were con - A modified method for analysis of apoptosis in paraffin-em - sidered statistically significant for values of P<0.05 bedded sections using TUNEL staining has been described pre- viously [28]. Briefly, the rats that were randomly selected for anesthetization with chloral hydrate were perfused transcar- Results dially with ice-cold saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer, to pre-fix the brain tissue. Brain tis - r-OPN alleviated brain edema and attenuated neurofunction- sue specimen from the cerebral hemorrhage area were excised al deficits and fixed in 4% paraformaldehyde for 48 h, embedded in par - affin, and then cut into serial sections (5-µm). After deparaf - The brain water content was analyzed in the injured hemi- finization and rehydration, the paraffin sections were rinsed sphere on days 1–3 after ICH induction. The brain water con- in 0.1 M phosphate-buffered saline (PBS) twice and incubat - tent was significantly increased in the vehicle group compared ed with Proteinase K working solution (10 µg/ml in 10 mM with the sham group (P<0.05, Figure 1). The rats treated with Tris/HCl, pH 7.5–8.0) for 15 min at 37°C. After being washed r-OPN showed reduced brain edema content compared with in PBS again, the sections were treated with green fluoresce - the vehicle group (P<0.05, Figure 1). However, the rats re- in-labeled dUTP solution that contains 10% TdT. Finally, the ceiving r-OPN combined with PI3K inhibitor wortmannin (r- paraffin sections were counterstained with DAPI. In apoptotic OPN+Wort) did not have reduced brain edema compared to cells, TUNEL-positive cells that exhibit green fluorescent gran - the r-OPN group (P<0.05, Figure 1). Wortmannin did not in- ules were determined using a microscope and were quantified crease brain edema when administered alone compared with by randomly selecting 5 microscopic fields to count. the vehicle group (P>0.05, Figure 1). There was no significant difference in brain water content between the vehicle group Western blot analysis and r-OPN+Wort group (P>0.05, Figure 1). The mNSS scores and rotarod testing provide a comprehensive evaluation of neu- Western blot analysis was carried out as described previous- rological functions in rats. There was a significant increase in ly [25]. Briefly, after administration of an overdose of chloral mNSS score of the vehicle group compared to the sham group Indexed in: [Current Contents/Clinical Medicine] [SCI Expanded] [ISI Alerting System] This work is licensed under Creative Common Attribution- [ISI Journals Master List] [Index Medicus/MEDLINE] [EMBASE/Excerpta Medica] NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) [Chemical Abstracts/CAS] Zhang W. et al.: Recombinant osteopontin improves neurological functional recovery… ANIMAL STUDY © Med Sci Monit, 2018; 24: 1588-1596 significantly longer than that of vehicle-treated mice ( P<0.05, Sham 85 Figure 2B), whereas r-OPN+Wort group animals lagged be- Vehicle r-OPN & * hind the r-OPN group (P<0.05, Figure 2B). Wortmannin did r-OPN+Wort * not induce a significant decrease of rotarod latencies when Wort 80 * & * administered alone compared with the vehicle group (P>0.05, # * Figure 2B). There was no significant difference in rotarod la - tencies between the vehicle group and r-OPN+Wort group (P>0.05, Figure 2B). r-OPN administration attenuated the number of apoptotic cells at 24 h after ICH To examine the role of r-OPN in apoptosis, TUNEL/DAPI stain- 1 23 ing revealed a high density of positively stained cells with- in the hemorrhagic lesion itself as well as in the surrounding Figure 1. Brain water content was reduced in r-OPN mice at periphery. TUNEL staining was localized with the nuclei mark- days 1–3 after ICH. Data in bar graphs are expressed as er DAPI (Figure 3A). Quantitative analysis showed region- mean ±SEM. (* P<0.05 compared with sham, P<0.05 al TUNEL-positive cell differences between the groups. Few compared with vehicle, P<0.05 compared with r-OPN, TUNEL-positive apoptotic cells were found in the sham group. n=6–8/group). In vehicle groups, the apoptotic index in the perihematomal area was found to be significantly increased compared with over 5 days after ICH (P<0.05, Figure 2A). r-OPN significantly those in the sham group (P<0.05, Figure 3B). r-OPN significant - decreased mNSS scores compared to the vehicle group (P<0.05, ly decreased TUNEL-positive cells compared with the vehicle Figure 2A). When r-OPN was combined with the PI3K inhibitor group (P<0.05, Figure 3B). Administration of r-OPN combined wortmannin (r-OPN+Wort), mNSS scores were higher than in with wortmannin treatment increased the number of apop- the r-OPN group (P<0.05, Figure 2A). Wortmannin did not in- totic cells, which abolished the anti-apoptotic effect of r-OPN crease mNSS when administered alone compared with the ve- alone (P<0.05, Figure 3B). There was no significant difference hicle group (P>0.05, Figure 2A). There was no significant dif - in the number of apoptotic cells between the vehicle group ference in mNSS between the vehicle group and r-OPN+Wort and r-OPN+Wort group (P>0.05, Figure 3B). group (P>0.05, Figure 2A). ICH induced a significant decrease in rotarod latencies compared to the sham group (P<0.05, Figure 2B). The rotarod latencies of r-OPN-treated mice were A B 18 Sham 350 Vehicle r-OPN r-OPN+Wort Wort & 150 6 & pre 1 2 345 pre 1 2 345 Days post ICH or Sham Days post ICH or Sham Figure 2. Neurological function showed short-term improvement in r-OPN mice over 5 days following ICH, based on neurobehavioral function and rotarod motor test. The r-OPN treatment significantly decreased the mNSS scores compared with vehicle group at 1–5 days after injury (A). The vestibulomotor performance by rotarod testing was also significantly improved in r-OPN group at 1–5 days after injury (B). Data are presented as the mean ±SEM (* P<0.05 compared with sham, P<0.05 compared with vehicle, P<0.05 compared with r-OPN, n=6–8/group). Indexed in: [Current Contents/Clinical Medicine] [SCI Expanded] [ISI Alerting System] This work is licensed under Creative Common Attribution- [ISI Journals Master List] [Index Medicus/MEDLINE] [EMBASE/Excerpta Medica] NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) [Chemical Abstracts/CAS] mNSS Brain water content (%) Rotarod latency (Second) Zhang W. et al.: Recombinant osteopontin improves neurological functional recovery… ANIMAL STUDY © Med Sci Monit, 2018; 24: 1588-1596 A B TUNELDAPI Merged Sham Vehicle 40 # r-OPN Sham Vehicle r-OPN r-OPN+Wort r-OPN+Wort Figure 3. Co-staining of TUNEL (green) and DAPI (blue) in the perihematomal cerebral cortex at 24 h after ICH. TUNEL-positive cells were barely detected in the sham group but were widely distributed in the perihematomal area after ICH. r-OPN decreased the amounts of TUNEL-positive cells, which was blocked by wortmannin. Scale bar 50 μm ( A). ICH increased the number of TUNEL-positive cells compared with sham group. r-OPN remarkably attenuated the number of TUNEL-positive cells, whereas wortmannin combined with r-OPN reversed the decreasing trend compared with only r-OPN treatment (B). Data # & are presented as the mean ±SEM (* P<0.05 compared with sham, P<0.05 compared with vehicle, P<0.05 compared with r-OPN, n=4–5/group). r-OPN downregulated the ratio of Bax/Bcl-2 and decreased r-OPN activated the PI3K-Akt signaling pathway and the CC3 (cleaved caspase-3) at 24 h after ICH decreased protein expressions of p-GSK-3b at 24 h after ICH Bcl-2 is an anti-apoptotic factor and is important for cell surviv- al, while Bax promotes apoptosis. A low ratio of Bax and Bcl- Akt is a prosurvival kinase and is activated by phosphorylation 2 favors cell survival. Western blot analysis revealed a remark- at Ser473 via the PI3K pathway, which is thought to be one of ably increased ratio of Bax/Bcl-2 in the vehicle group compared the molecules that promote cell survival and prevent apoptosis. with the sham group (P<0.05, Figure 4A). r-OPN treatment sig- The pro-apoptotic role of GSK-3b is an important downstream nificantly down-regulated the ratio of Bax/Bcl-2 compared with target of the Akt signaling pathway. Phosphorylation of GSK-3b the vehicle group (P<0.05, Figure 4A). r-OPN restored Bax and on the inactivating residue serine-9 by Akt results in GSK-3b Bcl-2 expression levels after ICH, and this protection was hin- inactivation. Western blot analysis showed decreased p-Akt dered by wortmannin (r-OPN+Wort) administration (P<0.05, expression after ICH compared with sham (P<0.05, Figure 5A). Figure 4A). There was no significant difference in the ratio of r-OPN administration dramatically increased p-Akt expression Bax/Bcl-2 between the vehicle group and r-OPN+Wort group compared with the vehicle group (P<0.05, Figure 5A), and this (P>0.05, Figure 4A). CC3 is an important marker of apoptosis. expression was reversed by PI3K inhibitor wortmannin (r- Western blot analysis revealed a remarkably increase of CC3 OPN+Wort) administration (P<0.05, Figure 5A). There was no in the vehicle group compared with the sham group (P<0.05, statistical difference in p-Akt between the vehicle group and Figure 4B). r-OPN treatment significantly decreased the level r-OPN+Wort group (P>0.05, Figure 5A). The expression of total of CC3 compared with the vehicle group (P<0.05, Figure 4B), Akt was constant in control and experimental groups (P>0.05, whereas treatment with wortmannin combined with r-OPN clear- Figure 5A). Western blot analysis showed ICH dramatically in- ly increased CC-3 expression compared with r-OPN at 24 h af- creased the levels of p-GSK-3b compared with sham (P<0.05, ter ICH (P<0.05, Figure 4B). There was no significant difference Figure 5B). Treatment with r-OPN significantly decreased the in CC3 between the vehicle group and the r-OPN+Wort group levels of p-GSK-3b compared with the vehicle group (P<0.05, (P>0.05, Figure 4B). The expression of total caspase-3 was con- Figure 5B), whereas, treatment with r-OPN plus wortmannin stant in the control and experimental groups (P>0.05, Figure 4B). obviously increased GSK-3b levels compared with r-OPN alone Indexed in: [Current Contents/Clinical Medicine] [SCI Expanded] [ISI Alerting System] This work is licensed under Creative Common Attribution- [ISI Journals Master List] [Index Medicus/MEDLINE] [EMBASE/Excerpta Medica] NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) [Chemical Abstracts/CAS] TUNEL positive cell/mm Zhang W. et al.: Recombinant osteopontin improves neurological functional recovery… ANIMAL STUDY © Med Sci Monit, 2018; 24: 1588-1596 A B Sham Vehicle r-OPN r-OPN+Wort Sham Vehicle r-OPN r-OPN+Wort Cleaved Bax Caspase-3 Bcl-2 β-actin β-actin Sham Vehicle r-OPN r-OPN+Wort Caspase-3 β-actin 3.0 1.2 Cleaved caspase-3 Caspase-3 2.5 1.0 2.0 0.8 # 1.5 0.6 1.0 0.4 0.5 0.2 0.0 0.0 Sham Vehicle r-OPN r-OPN+Wort Sham Vehicle r-OPN r-OPN+Wort Figure 4. r-OPN affects Bcl-2 family and cleaved caspase-3 protein expression 24 h after ICH or sham. Representative Western blots and densitometric quantification of Bax/Bcl-2 (A). Representative Western blots and densitometric quantification and cleaved caspase-3/b-actin and total caspase-3/b-actin at 24 h after ICH (B). Data are presented as the mean ±SEM (* P<0.05 # & compared with sham, P<0.05 compared with vehicle, P<0.05 compared with r-OPN, n=6–8/group). (P<0.05, Figure 5B). There was no significant difference in p- cells [31]. There is compelling evidence that OPN can, in a va- GSK-3b between the vehicle group and r-OPN+Wort group riety of situations, help cells survive an otherwise lethal in- (P>0.05, Figure 5B). The expression of total GSK-3b were con- sult [12]. Endogenous OPN induction has consistently been stant in control and experimental groups (P>0.05, Figure 5B). found to have protective effects on ischemic injuries involving the brain and other organs [32–34]. A previous study revealed that r-OPN alleviates brain damage in a rat model of ICH by in- Discussion hibiting the expression of inflammation-related iNOS, thereby suppressing matrix metalloproteinase (MMP)-9 activation [10]. In the present study, we found that r-OPN improves function- Moreover, r-OPN markedly reduced the infarct size via anti- al outcomes, alleviates brain edema, and reduces cell apop- apoptotic actions in a transient focal cerebral ischemia mouse tosis by regulating apoptosis-related PI3K/Akt/GSK-3b sig- model [14]. Experimentally applied ICH induced neurological naling pathways in a preclinical rat model of ICH. ICH induced deficits and brain edema accompanied by neuronal apoptosis initial hematoma expansion and resultant mass effect injury, in rats [7]. However, the effect of r-OPN on apoptosis associ - secondary to the hematoma resulting in ischemia, edema, in- ated with neurological recovery in ICH remains poorly eluci- tense inflammation, and ultimately cell death and neurologi - dated. In the current study, r-OPN alleviated apoptosis within cal deficits [5,29]. Apoptosis is a prominent form of cell death the hemorrhagic lesion itself as well as in the surrounding pe- in the perihematomal region after ICH [8]. As apoptosis is a riphery at 24 h after ICH. r-OPN also significantly attenuated reversible process, research has been focused on the apoptot- ICH-induced brain edema on days 1–3 after ICH, and improved ic pathways involved in neuronal death after ICH to identify short-term neurological function outcome of mNSS scores and important therapeutic targets [30]. OPN is a phosphorylated rotarod test on days 1–5 after ICH induction. Previous stud- glycoprotein, which is a soluble cytokine capable of stimulat- ies demonstrated that OPN contains a conserved arginine-gly- ing signal transduction pathways in many different types of cine-aspartic acid (RGD) amino acid sequence initiating cell Indexed in: [Current Contents/Clinical Medicine] [SCI Expanded] [ISI Alerting System] This work is licensed under Creative Common Attribution- [ISI Journals Master List] [Index Medicus/MEDLINE] [EMBASE/Excerpta Medica] NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) [Chemical Abstracts/CAS] Bax/Ccl-2 ratio (protein) Relative cleaved caspase-3 and caspase-3 level (protein) Zhang W. et al.: Recombinant osteopontin improves neurological functional recovery… ANIMAL STUDY © Med Sci Monit, 2018; 24: 1588-1596 A B Sham Vehicle r-OPN r-OPN+Wort Sham Vehicle r-OPN r-OPN+Wort p-Akt p-GSK-3β β-actin β-actin Sham Vehicle r-OPN r-OPN+Wort Sham Vehicle r-OPN r-OPN+Wort Akt GSK-3β β-actin β-actin 1.2 1.4 p-Akt p-GSK-β Akt GSK-3β 1.2 1.0 1.0 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 0.0 0.0 Sham Vehicle r-OPN r-OPN+Wort Sham Vehicle r-OPN r-OPN+Wort Figure 5. r-OPN affects p-Akt and p-GSK-3 b protein expression 24 h after ICH or sham. Representative Western blots and densitometric quantification of p-Akt/ b-actin and total Akt/b-actin (A). Representative Western blots and densitometric quantification of p-GSK-3 b/b-actin and total GSK-3b/b-actin (B). Data are presented as the mean ±SEM (* P<0.05 compared # & with sham, P<0.05 compared with vehicle, P<0.05 compared with r-OPN, n=6–8/group). signaling events upon integrin binding, and it promotes sur- CC3 is an important marker of apoptosis [40]. In the present vival through the PI3K/Akt signaling pathway [35]. In addition, study, ICH induced an imbalance of Bax/Bcl-2 and increased suppression of the apoptosis has been suggested to reduce the CC3 expression. r-OPN downregulated the ratio of Bax/Bcl- brain edema and improve functional outcomes in experimen- 2 expression more toward the levels found in the sham group tally induced ICH [36]. Therefore, we explored whether the and also decreased the expression of CC3. We used TUNEL to neuroprotective mechanism of r-OPN was mediated by sup- detect apoptotic cell death and observed a significantly re - pressing the cell apoptosis-related PI3K/Akt/GSK-3b signaling duced density of TUNEL-positive cells in the r-OPN treatment pathway. Animals receiving r-OPN treatment showed neuro- group. r-OPN inhibited the apoptosis during the process of protective effects compared with vehicle group. When we ad - ICH, whereas r-OPN plus the wortmannin treatment upregu- ministered PI3K inhibitor wortmannin and r-OPN, neurological lated the ratio of Bax/Bcl-2, increased the expression of CC3, impairment and brain edema were not alleviated compared and the number of TUNEL-positive cells increased significant - with the r-OPN group. Our findings further confirm previous ly. Therefore, we explored whether the neuroprotection and findings that wortmannin alone did not worsen the behavior - the cell apoptosis suppression mechanism of r-OPN were me- al outcome and brain edema compared with the vehicle group diated via the PI3K/Akt signal pathway. after ICH [36]. These results indicate that r-OPN exerts neu- roprotective effects through the PI3K/Akt signaling pathway. Akt, a serine/threonine kinase, is a primary mediator of the downstream effects of phosphatidylinositol-3 kinase (PI3K), co - At the molecular level, the Bcl-2 family proteins play impor- ordinating a variety of intracellular signals and controlling cell tant roles in intrinsic apoptotic pathway [37,38]. The family responses to extrinsic stimuli and regulating cell proliferation is divided into anti-apoptotic proteins such as Bcl-2 and pro- and survival [42]. The pro-apoptotic role of GSK-3b is an im- apoptotic proteins. Bax induces apoptosis, and Bax/Bcl-2 ra- portant downstream target of the Akt signaling pathway [43]. tio is often used to represent the extent of apoptosis [39,40]. GSK-3b has been shown to participate in apoptosis in several Indexed in: [Current Contents/Clinical Medicine] [SCI Expanded] [ISI Alerting System] This work is licensed under Creative Common Attribution- [ISI Journals Master List] [Index Medicus/MEDLINE] [EMBASE/Excerpta Medica] NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) [Chemical Abstracts/CAS] Relative p-Akt and Akt level (protein) Relative p-GSK-3β and GSK-3β level (protein) Zhang W. et al.: Recombinant osteopontin improves neurological functional recovery… ANIMAL STUDY © Med Sci Monit, 2018; 24: 1588-1596 cell types and is known to be an upstream regulator of pro- chemotactic for the endothelial cells, and help promote new grammed cell death, which is particularly abundant in the blood vessel formation in vitro [10]. Thrombin is produced in CNS [44,45]. Dysregulation of GSK-3b activity is believed to the brain immediately after ICH [46]. Whether r-OPN follow- play a key role in the pathogenesis of central nervous system ing thrombin cleavage has positive effects on neuroprotection chronic and acute disorders such as ischemic stroke, and trau- after ICH in vivo should be further explored. matic brain injury [18,20]. Akt activation promotes cell survival by phosphorylation and subsequent inactivation of GSK-3b [21]. In the present study, r-OPN treatment significantly increased Conclusions the expression of activated Akt (p-Akt, Ser473) at 24 h after ICH, which thereby successively reduced the expression of ac- In summary, our study demonstrated that r-OPN protected tivated GSK-3b (p-GSK-3b, Ser9). r-OPN treatment reduced cell against ICH injury and improved neurological function by re- apoptosis and expression of apoptosis-related proteins. When ducing apoptosis in vivo. Moreover, r-OPN exerted its anti-apop- we used PI3K inhibitor wortmannin combined with r-OPN, the totic effect through the PI3K/Akt/GSK-3 b signaling pathway. In levels of activated Akt protein and activated GSK-3b protein conclusion, the elucidation of the neuroprotective action of r- were similar to levels in the vehicle group. The wortmannin plus OPN in the current study adds to a growing literature suggest- r-OPN treatment did not reduce the incidence of cell apoptosis ing the potential beneficial role for this factor in ICH. after ICH in rats. Our results support r-OPN-mediated neuro- protection in a rat model of ICH, primarily via activation of the Conflicts of interest PI3K/Akt, and inhibited GSK-3b. However, certain physiologi- cal processes involving OPN partly depend on its cleavage by None. thrombin [10]. Thrombin-cleaved OPN fragments are strongly References: 1. Steiner T, Alshahi SR, Beer R et al: European Stroke Organisation (ESO) 15. 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Stroke, 2009; 40(3 Suppl. 1): S88–89 Indexed in: [Current Contents/Clinical Medicine] [SCI Expanded] [ISI Alerting System] This work is licensed under Creative Common Attribution- [ISI Journals Master List] [Index Medicus/MEDLINE] [EMBASE/Excerpta Medica] NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) [Chemical Abstracts/CAS] http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Medical Science Monitor : International Medical Journal of Experimental and Clinical Research Pubmed Central

Recombinant Osteopontin Improves Neurological Functional Recovery and Protects Against Apoptosis via PI3K/Akt/GSK-3β Pathway Following Intracerebral Hemorrhage

Zhang, Wenqian; Cui, Ying; Gao, Junling; Li, Ran; Jiang, Xiaohua; Tian, Yanxia; Wang, Kaijie; Cui, Jianzhong
Medical Science Monitor : International Medical Journal of Experimental and Clinical Research , Volume 24 – Mar 18, 2018
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10.12659/MSM.905700
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Abstract

ANIMAL STUDY e-ISSN 1643-3750 © Med Sci Monit, 2018; 24: 1588-1596 DOI: 10.12659/MSM.905700 Received: 2017.06.09 Recombinant Osteopontin Improves Neurological Accepted: 2017.09.04 Published: 2018.03.18 Functional Recovery and Protects Against Apoptosis via PI3K/Akt/GSK-3b Pathway Following Intracerebral Hemorrhage Authors’ Contrib ution: AE 1 Wenqian Zhang 1 Department of Surgery, Hebei Medical University, Shijiazhuang, Hebei, P.R. China Study Design A 2 Department of Neurosurgery, Tangshan Gongren Hospital, Tangshan, Hebei, BF 2 Ying Cui Data Collection B P.R. China AF 3,4 Junling Gao Statistical Analysis C 3 School of Basic Medical Science, North China University of Science and Data Interpr etation D CD 3,4 Ran Li Technology, Tangshan, Hebei, P.R. China Manuscript Preparation E 4 Hebei Key Laboratory for Chronic Diseases, Tangshan Key Laboratory for BF 3,4 Xiaohua Jiang Literature Search F Preclinical and Basic Research on Chronic Diseases, Tangshan, Hebei, P.R. China D 3,4 Yanxia Tian Funds Collection G BC 2 Kaijie Wang AE 1,2 Jianzhong Cui Corresponding Author: Jianzhong Cui, e-mail: [email protected] Source of support: This study was supported by a grant from the Natural Science Foundation of Hebei, China (grant no. H2012401071 and H2014105079) Background: This study aimed to investigate the potential neuroprotective effect of recombinant osteopontin (r-OPN) on apop - totic changes via modulating phosphoinositide-3-kinase/Akt/glycogen synthase kinase 3 beta (PI3K/Akt/GSK-3b) signaling in a rat model of intracerebral hemorrhage (ICH). Material/Methods: We subjected 10–12-week-old Sprague-Dawley male rats (n=120) to injection of autologous blood into the right basal ganglia to induce ICH or sham surgery. ICH animals received vehicle administration, r-OPN (4 μL/pup), or r-OPN combined with phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin (86 ng/pup) at 30 min af- ter injury. Neurological scores and rotarod latencies were evaluated on days 1–5 post-ICH. Brain water content was evaluated on days 1–3 post-ICH. The number of apoptotic cells changes were evaluated by terminal de- oxynucleotidyl transferase-mediated 2-deoxyuridine 5-triphosphate-biotin nick-end labeling (TUNEL) and he- matoxylin staining. Apoptosis-related proteins Bcl-2, Bax, and cleaved caspase-3 (CC3), and the phosphoryla- tion levels of Akt and GSK-3b were assayed by Western blot. Results: Neurological deficits, rotarod latencies, and brain water content following ICH were reduced in the r-OPN group compared to the vehicle group. r-OPN also attenuated cell death in ICH. Furthermore, treatment with r-OPN sig- nificantly increased p-Akt expression and decreased p-GSK-3 b. These effects were associated with a decrease in the Bax/Bcl-2 ratio and the suppression of CC3 at 24 h after ICH. Importantly, all the beneficial effects of r-OPN in ICH were abrogated by the PI3K inhibitor wortmannin. Conclusions: r-OPN may provide a wide range of neuroprotection by suppressing apoptosis through the PI3K/Akt/GSK-3b signaling pathway after ICH. MeSH Keywords: Apoptosis • Cerebral Hemorrhage • Osteopontin • Phosphatidylinositol 3-Kinases Full-text PDF: https://www.medscimonit.com/abstract/index/idArt/905700 3313 — 5 46 Indexed in: [Current Contents/Clinical Medicine] [SCI Expanded] [ISI Alerting System] This work is licensed under Creative Common Attribution- [ISI Journals Master List] [Index Medicus/MEDLINE] [EMBASE/Excerpta Medica] NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) [Chemical Abstracts/CAS] Zhang W. et al.: Recombinant osteopontin improves neurological functional recovery… ANIMAL STUDY © Med Sci Monit, 2018; 24: 1588-1596 Background to improve neurological outcomes and behavioral outcomes by inhibiting apoptosis in a rat model of ICH. The PI3K/Akt/ Intracerebral hemorrhage (ICH) accounted for 9% to 27% of all GSK-3b pathway was hypothesized and tested as the possi- strokes worldwide in the last decade, with poor functional out- ble mechanism of r-OPN effects. come and high early case fatality [1–3]. Despite this devastat- ing personal and societal impact, ICH remains an understudied disease with little improvement in patient outcomes over the Material and Methods last 20 years [4]. Studies have demonstrated that ICH-induced neurological damage can be divided into primary and second- Animals and ICH Model ary brain injury. Primary injury evoked by the physical destruc- tion due to rapid hematoma formation in the brain parenchy- Adult male Sprague-Dawley rats (11–13 weeks, 280–320 g) ma leads to brain tissue compression or disruption, as well were used in the study. The procedures for this study were as elevated intracranial pressure. Subsequent development of approved by the Animal Care and Use Committee of Hebei various parallel pathological processes such as neuroinflam - Medical University and were carried out in accordance with mation, neuronal apoptosis, and excitotoxicity contribute to the Guide for the Chinese Council on Animal Protection. All secondary injury [5,6]. Apoptosis is a prominent form of cell rats were housed under conditions of controlled temperature death associated with ICH in the perihematomal region [7,8]. and humidity. Rats had free access to food and water before ICH has long been reported to cause neuronal apoptosis lead- and after surgery or sham treatment. Our injury model was ing to declines in neurological functions [9]. Considering intact adapted from a previously described model of ICH in rats [23]. neurons as the basis of normal neurological functions, inhibi- The rats were anesthetized with 0.4% pentobarbital sodium tion of neuronal apoptosis and alleviating neurological deficits intraperitoneally. Through the tail vein, we removed 50 ml of are helpful to the patient’s long-term prognosis. autologous whole blood. Using stereotaxic guidance, we ad- ministered autologous whole blood into the right caudate pu- Osteopontin (OPN) is a secreted extracellular matrix (ECM) gly- tamen. The stereotactic coordinates were: 0.2 mm anterior, 3.0 coprotein that is involved in both physiological and pathological mm lateral to bregma, and 5.5 mm ventral. The sham group processes in a wide range of tissues [10]. There is compelling only had needle insertion. After slowly withdrawing the injec- evidence that endogenous OPN is the most highly upregulat- tion needle 10 min after injection, sealed with zinc phosphate ed factor after central nervous system disease [11,12]. It can cement, the incision was closed and disinfected with local io- regulate inflammation, tissue remodeling, and cell survival in dine. The animals were allowed to recover spontaneous ven- the field of stroke injury [10,13,14]. For example, recombinant tilation. In the course of the establishment of the experimen- osteopontin (r-OPN) reduced cortical neuron death via the ac- tal group in the ICH model, a total of 10 rats died. tivation the phosphatidylinositol 3-kinase (PI3K) -Akt signal- ing pathway kinase and increased expression of pro-survival Experimental groups and treatment genes in ischemic stroke [13]. Moreover, r-OPN provided neu- roprotection by anti-apoptotic action in subarachnoid hemor- We randomly assigned 150 adult rats to 5 groups (n=30): rhage and exerted an anti-inflammatory effect by downregulat - the sham group, the vehicle (ICH+vehicle) group, the r-OPN ing inducible NO synthase (iNOS) in ICH [14,15]. Nevertheless, (ICH+r-OPN) group, r-OPN+Wort (ICH+r-OPN+ wortmannin) until now, no published study investigated the effect of r-OPN group, and the Wort (ICH+Wortmannin) group. Mouse r-OPN administration on the apoptotic changes associated with re- (0.1 μg in 1 μL of sterile PBS; EMD chemicals, La Jolla, CA) or covery of neurological function during ICH. vehicle (1 μL; sterile PBS) were intracerebroventricularly inject - ed at 1 h after ICH. The PI3K inhibitor wortmannin (15 μg/kg; Glycogen synthase kinase 3b (GSK-3b) is a serine/threonine Sigma-Aldrich, St Louis, MO) was administered before the in- kinase known to be important in cell proliferation and apop- duction of ICH. In the sham, the needle was inserted, with no tosis signaling pathways [16,17]. The stimulated GSK-3b has administration of drug. been found to exacerbate brain injury in experimental ischemic stroke, traumatic brain injury, and ICH [18–20]. The pro-apop- Brain water content totic role of GSK-3b has previously been shown to be negatively regulated through the phosphoinositol-3-kinase (PI3-kinase)- Rats were anesthetized and euthanized on days 1–3 after ICH Akt survival pathway [21,22]. Further studies of r-OPN and in- to coincide with the point of maximum apoptotic changes. The terconnected anti-apoptotic mechanisms will help to develop procedure was carried out as previously described [24]. Brains new therapeutic targets to reduce cell death and to explore were sectioned in the mid-sagittal plane, and hemispheres were neuroprotective properties after ICH. In this study, we inves- weighed immediately (‘‘wet’’ weight). Then, the hemispheres tigated the role of r-OPN in amelioration of brain edema and were dried at 80°C for 24 h, and ‘‘dry’’ weight was measured. Indexed in: [Current Contents/Clinical Medicine] [SCI Expanded] [ISI Alerting System] This work is licensed under Creative Common Attribution- [ISI Journals Master List] [Index Medicus/MEDLINE] [EMBASE/Excerpta Medica] NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) [Chemical Abstracts/CAS] Zhang W. et al.: Recombinant osteopontin improves neurological functional recovery… ANIMAL STUDY © Med Sci Monit, 2018; 24: 1588-1596 We calculated water content as a percentage of wet weight hydrate, rats were sacrificed at different time points after sur - ([wet weight–dry weight]/[wet weight]×100). gery. Brain tissue, except for the caudate putamen tissues sur- rounding the hematoma, was instantly frozen at –80°C until Neurological score use. To detect protein expression, an appropriate amount of frozen brain tissue samples was minced with eye scissors in Modified neurological severity score (mNSS) is a composite ice. Then, the tissue was homogenized in cold lysis buer ff and of motor tests, sensory tests, beam balance tests, and reflex - then clarified by centrifuging (12000×g for 20 min at 4°C) be - es absent [25]. In our study, it was used to grade various as- fore taking the supernatant. Protein concentration was de- pects of neurobehavioral function on days 1–5 after ICH. Lower termined using the Bradford method. The samples were sub- scores indicate better function (normal score=0), and higher jected to SDS-polyacrylamide gel electrophoresis to resolve scores indicate more severe injury (maximal deficit score=18). and were transferred onto a polyvinylidene fluoride (PVDF) filter membrane by a transfer apparatus at 350 mA for 2 h. Rotarod test Following blocked with 5% fat-free dry milk, the membrane was then incubated overnight with primary antibody at 4°C, An accelerating automated rotarod was used to measure the including Actin (anti-goat, 1: 2000, Santa Cruz Biotechnology), effects of therapeutic intervention on vestibulomotor func - p-GSK-3b (anti-rabbit, 1: 1000, Abcam), GSK-3b (anti-rabbit, tion [26]. The speed was slowly increased from 4 to 40 rpm 1: 1000, Abcam), p-Akt (anti-mouse, 1: 1000, Abcam), Akt (anti- within 5 min. When the rats were dropped twice from the mouse, 1: 1000, Abcam), cleaved caspase-3 (CC3, anti-mouse, ladder of the device, the experiment ended. On 3 days before 1: 1000; Cell Signaling), caspase-3 (anti-mouse, 1: 1000; Cell the ICH injury, the rats were pretrained, and the average time Signaling), Bcl-2 (anti-rabbit, 1: 1000, Cell Signaling Technology), elapsed from the rotation of the cylinder was recorded as the and Bax (anti-mouse, anti-1000, Cell Signaling Technology). baseline latency. To assess early motor outcome, mice under- Nitrocellulose membranes were then labeled with secondary went rotarod testing on days 1–5 after injury. Average laten- antibodies for 1 h at room temperature. cy to fall from the rod was recorded. Statistical analyses Apoptosis analysis by terminal deoxynucleotidyl transferase-mediated (dUTP) nick-end labeling (TUNEL) All data are presented as the mean ±SD and were analyzed staining using SPSS 17.0. The data were analyzed by one-way ANOVA, followed by Tukey’s post hoc analysis. Differences were con - A modified method for analysis of apoptosis in paraffin-em - sidered statistically significant for values of P<0.05 bedded sections using TUNEL staining has been described pre- viously [28]. Briefly, the rats that were randomly selected for anesthetization with chloral hydrate were perfused transcar- Results dially with ice-cold saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer, to pre-fix the brain tissue. Brain tis - r-OPN alleviated brain edema and attenuated neurofunction- sue specimen from the cerebral hemorrhage area were excised al deficits and fixed in 4% paraformaldehyde for 48 h, embedded in par - affin, and then cut into serial sections (5-µm). After deparaf - The brain water content was analyzed in the injured hemi- finization and rehydration, the paraffin sections were rinsed sphere on days 1–3 after ICH induction. The brain water con- in 0.1 M phosphate-buffered saline (PBS) twice and incubat - tent was significantly increased in the vehicle group compared ed with Proteinase K working solution (10 µg/ml in 10 mM with the sham group (P<0.05, Figure 1). The rats treated with Tris/HCl, pH 7.5–8.0) for 15 min at 37°C. After being washed r-OPN showed reduced brain edema content compared with in PBS again, the sections were treated with green fluoresce - the vehicle group (P<0.05, Figure 1). However, the rats re- in-labeled dUTP solution that contains 10% TdT. Finally, the ceiving r-OPN combined with PI3K inhibitor wortmannin (r- paraffin sections were counterstained with DAPI. In apoptotic OPN+Wort) did not have reduced brain edema compared to cells, TUNEL-positive cells that exhibit green fluorescent gran - the r-OPN group (P<0.05, Figure 1). Wortmannin did not in- ules were determined using a microscope and were quantified crease brain edema when administered alone compared with by randomly selecting 5 microscopic fields to count. the vehicle group (P>0.05, Figure 1). There was no significant difference in brain water content between the vehicle group Western blot analysis and r-OPN+Wort group (P>0.05, Figure 1). The mNSS scores and rotarod testing provide a comprehensive evaluation of neu- Western blot analysis was carried out as described previous- rological functions in rats. There was a significant increase in ly [25]. Briefly, after administration of an overdose of chloral mNSS score of the vehicle group compared to the sham group Indexed in: [Current Contents/Clinical Medicine] [SCI Expanded] [ISI Alerting System] This work is licensed under Creative Common Attribution- [ISI Journals Master List] [Index Medicus/MEDLINE] [EMBASE/Excerpta Medica] NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) [Chemical Abstracts/CAS] Zhang W. et al.: Recombinant osteopontin improves neurological functional recovery… ANIMAL STUDY © Med Sci Monit, 2018; 24: 1588-1596 significantly longer than that of vehicle-treated mice ( P<0.05, Sham 85 Figure 2B), whereas r-OPN+Wort group animals lagged be- Vehicle r-OPN & * hind the r-OPN group (P<0.05, Figure 2B). Wortmannin did r-OPN+Wort * not induce a significant decrease of rotarod latencies when Wort 80 * & * administered alone compared with the vehicle group (P>0.05, # * Figure 2B). There was no significant difference in rotarod la - tencies between the vehicle group and r-OPN+Wort group (P>0.05, Figure 2B). r-OPN administration attenuated the number of apoptotic cells at 24 h after ICH To examine the role of r-OPN in apoptosis, TUNEL/DAPI stain- 1 23 ing revealed a high density of positively stained cells with- in the hemorrhagic lesion itself as well as in the surrounding Figure 1. Brain water content was reduced in r-OPN mice at periphery. TUNEL staining was localized with the nuclei mark- days 1–3 after ICH. Data in bar graphs are expressed as er DAPI (Figure 3A). Quantitative analysis showed region- mean ±SEM. (* P<0.05 compared with sham, P<0.05 al TUNEL-positive cell differences between the groups. Few compared with vehicle, P<0.05 compared with r-OPN, TUNEL-positive apoptotic cells were found in the sham group. n=6–8/group). In vehicle groups, the apoptotic index in the perihematomal area was found to be significantly increased compared with over 5 days after ICH (P<0.05, Figure 2A). r-OPN significantly those in the sham group (P<0.05, Figure 3B). r-OPN significant - decreased mNSS scores compared to the vehicle group (P<0.05, ly decreased TUNEL-positive cells compared with the vehicle Figure 2A). When r-OPN was combined with the PI3K inhibitor group (P<0.05, Figure 3B). Administration of r-OPN combined wortmannin (r-OPN+Wort), mNSS scores were higher than in with wortmannin treatment increased the number of apop- the r-OPN group (P<0.05, Figure 2A). Wortmannin did not in- totic cells, which abolished the anti-apoptotic effect of r-OPN crease mNSS when administered alone compared with the ve- alone (P<0.05, Figure 3B). There was no significant difference hicle group (P>0.05, Figure 2A). There was no significant dif - in the number of apoptotic cells between the vehicle group ference in mNSS between the vehicle group and r-OPN+Wort and r-OPN+Wort group (P>0.05, Figure 3B). group (P>0.05, Figure 2A). ICH induced a significant decrease in rotarod latencies compared to the sham group (P<0.05, Figure 2B). The rotarod latencies of r-OPN-treated mice were A B 18 Sham 350 Vehicle r-OPN r-OPN+Wort Wort & 150 6 & pre 1 2 345 pre 1 2 345 Days post ICH or Sham Days post ICH or Sham Figure 2. Neurological function showed short-term improvement in r-OPN mice over 5 days following ICH, based on neurobehavioral function and rotarod motor test. The r-OPN treatment significantly decreased the mNSS scores compared with vehicle group at 1–5 days after injury (A). The vestibulomotor performance by rotarod testing was also significantly improved in r-OPN group at 1–5 days after injury (B). Data are presented as the mean ±SEM (* P<0.05 compared with sham, P<0.05 compared with vehicle, P<0.05 compared with r-OPN, n=6–8/group). Indexed in: [Current Contents/Clinical Medicine] [SCI Expanded] [ISI Alerting System] This work is licensed under Creative Common Attribution- [ISI Journals Master List] [Index Medicus/MEDLINE] [EMBASE/Excerpta Medica] NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) [Chemical Abstracts/CAS] mNSS Brain water content (%) Rotarod latency (Second) Zhang W. et al.: Recombinant osteopontin improves neurological functional recovery… ANIMAL STUDY © Med Sci Monit, 2018; 24: 1588-1596 A B TUNELDAPI Merged Sham Vehicle 40 # r-OPN Sham Vehicle r-OPN r-OPN+Wort r-OPN+Wort Figure 3. Co-staining of TUNEL (green) and DAPI (blue) in the perihematomal cerebral cortex at 24 h after ICH. TUNEL-positive cells were barely detected in the sham group but were widely distributed in the perihematomal area after ICH. r-OPN decreased the amounts of TUNEL-positive cells, which was blocked by wortmannin. Scale bar 50 μm ( A). ICH increased the number of TUNEL-positive cells compared with sham group. r-OPN remarkably attenuated the number of TUNEL-positive cells, whereas wortmannin combined with r-OPN reversed the decreasing trend compared with only r-OPN treatment (B). Data # & are presented as the mean ±SEM (* P<0.05 compared with sham, P<0.05 compared with vehicle, P<0.05 compared with r-OPN, n=4–5/group). r-OPN downregulated the ratio of Bax/Bcl-2 and decreased r-OPN activated the PI3K-Akt signaling pathway and the CC3 (cleaved caspase-3) at 24 h after ICH decreased protein expressions of p-GSK-3b at 24 h after ICH Bcl-2 is an anti-apoptotic factor and is important for cell surviv- al, while Bax promotes apoptosis. A low ratio of Bax and Bcl- Akt is a prosurvival kinase and is activated by phosphorylation 2 favors cell survival. Western blot analysis revealed a remark- at Ser473 via the PI3K pathway, which is thought to be one of ably increased ratio of Bax/Bcl-2 in the vehicle group compared the molecules that promote cell survival and prevent apoptosis. with the sham group (P<0.05, Figure 4A). r-OPN treatment sig- The pro-apoptotic role of GSK-3b is an important downstream nificantly down-regulated the ratio of Bax/Bcl-2 compared with target of the Akt signaling pathway. Phosphorylation of GSK-3b the vehicle group (P<0.05, Figure 4A). r-OPN restored Bax and on the inactivating residue serine-9 by Akt results in GSK-3b Bcl-2 expression levels after ICH, and this protection was hin- inactivation. Western blot analysis showed decreased p-Akt dered by wortmannin (r-OPN+Wort) administration (P<0.05, expression after ICH compared with sham (P<0.05, Figure 5A). Figure 4A). There was no significant difference in the ratio of r-OPN administration dramatically increased p-Akt expression Bax/Bcl-2 between the vehicle group and r-OPN+Wort group compared with the vehicle group (P<0.05, Figure 5A), and this (P>0.05, Figure 4A). CC3 is an important marker of apoptosis. expression was reversed by PI3K inhibitor wortmannin (r- Western blot analysis revealed a remarkably increase of CC3 OPN+Wort) administration (P<0.05, Figure 5A). There was no in the vehicle group compared with the sham group (P<0.05, statistical difference in p-Akt between the vehicle group and Figure 4B). r-OPN treatment significantly decreased the level r-OPN+Wort group (P>0.05, Figure 5A). The expression of total of CC3 compared with the vehicle group (P<0.05, Figure 4B), Akt was constant in control and experimental groups (P>0.05, whereas treatment with wortmannin combined with r-OPN clear- Figure 5A). Western blot analysis showed ICH dramatically in- ly increased CC-3 expression compared with r-OPN at 24 h af- creased the levels of p-GSK-3b compared with sham (P<0.05, ter ICH (P<0.05, Figure 4B). There was no significant difference Figure 5B). Treatment with r-OPN significantly decreased the in CC3 between the vehicle group and the r-OPN+Wort group levels of p-GSK-3b compared with the vehicle group (P<0.05, (P>0.05, Figure 4B). The expression of total caspase-3 was con- Figure 5B), whereas, treatment with r-OPN plus wortmannin stant in the control and experimental groups (P>0.05, Figure 4B). obviously increased GSK-3b levels compared with r-OPN alone Indexed in: [Current Contents/Clinical Medicine] [SCI Expanded] [ISI Alerting System] This work is licensed under Creative Common Attribution- [ISI Journals Master List] [Index Medicus/MEDLINE] [EMBASE/Excerpta Medica] NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) [Chemical Abstracts/CAS] TUNEL positive cell/mm Zhang W. et al.: Recombinant osteopontin improves neurological functional recovery… ANIMAL STUDY © Med Sci Monit, 2018; 24: 1588-1596 A B Sham Vehicle r-OPN r-OPN+Wort Sham Vehicle r-OPN r-OPN+Wort Cleaved Bax Caspase-3 Bcl-2 β-actin β-actin Sham Vehicle r-OPN r-OPN+Wort Caspase-3 β-actin 3.0 1.2 Cleaved caspase-3 Caspase-3 2.5 1.0 2.0 0.8 # 1.5 0.6 1.0 0.4 0.5 0.2 0.0 0.0 Sham Vehicle r-OPN r-OPN+Wort Sham Vehicle r-OPN r-OPN+Wort Figure 4. r-OPN affects Bcl-2 family and cleaved caspase-3 protein expression 24 h after ICH or sham. Representative Western blots and densitometric quantification of Bax/Bcl-2 (A). Representative Western blots and densitometric quantification and cleaved caspase-3/b-actin and total caspase-3/b-actin at 24 h after ICH (B). Data are presented as the mean ±SEM (* P<0.05 # & compared with sham, P<0.05 compared with vehicle, P<0.05 compared with r-OPN, n=6–8/group). (P<0.05, Figure 5B). There was no significant difference in p- cells [31]. There is compelling evidence that OPN can, in a va- GSK-3b between the vehicle group and r-OPN+Wort group riety of situations, help cells survive an otherwise lethal in- (P>0.05, Figure 5B). The expression of total GSK-3b were con- sult [12]. Endogenous OPN induction has consistently been stant in control and experimental groups (P>0.05, Figure 5B). found to have protective effects on ischemic injuries involving the brain and other organs [32–34]. A previous study revealed that r-OPN alleviates brain damage in a rat model of ICH by in- Discussion hibiting the expression of inflammation-related iNOS, thereby suppressing matrix metalloproteinase (MMP)-9 activation [10]. In the present study, we found that r-OPN improves function- Moreover, r-OPN markedly reduced the infarct size via anti- al outcomes, alleviates brain edema, and reduces cell apop- apoptotic actions in a transient focal cerebral ischemia mouse tosis by regulating apoptosis-related PI3K/Akt/GSK-3b sig- model [14]. Experimentally applied ICH induced neurological naling pathways in a preclinical rat model of ICH. ICH induced deficits and brain edema accompanied by neuronal apoptosis initial hematoma expansion and resultant mass effect injury, in rats [7]. However, the effect of r-OPN on apoptosis associ - secondary to the hematoma resulting in ischemia, edema, in- ated with neurological recovery in ICH remains poorly eluci- tense inflammation, and ultimately cell death and neurologi - dated. In the current study, r-OPN alleviated apoptosis within cal deficits [5,29]. Apoptosis is a prominent form of cell death the hemorrhagic lesion itself as well as in the surrounding pe- in the perihematomal region after ICH [8]. As apoptosis is a riphery at 24 h after ICH. r-OPN also significantly attenuated reversible process, research has been focused on the apoptot- ICH-induced brain edema on days 1–3 after ICH, and improved ic pathways involved in neuronal death after ICH to identify short-term neurological function outcome of mNSS scores and important therapeutic targets [30]. OPN is a phosphorylated rotarod test on days 1–5 after ICH induction. Previous stud- glycoprotein, which is a soluble cytokine capable of stimulat- ies demonstrated that OPN contains a conserved arginine-gly- ing signal transduction pathways in many different types of cine-aspartic acid (RGD) amino acid sequence initiating cell Indexed in: [Current Contents/Clinical Medicine] [SCI Expanded] [ISI Alerting System] This work is licensed under Creative Common Attribution- [ISI Journals Master List] [Index Medicus/MEDLINE] [EMBASE/Excerpta Medica] NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) [Chemical Abstracts/CAS] Bax/Ccl-2 ratio (protein) Relative cleaved caspase-3 and caspase-3 level (protein) Zhang W. et al.: Recombinant osteopontin improves neurological functional recovery… ANIMAL STUDY © Med Sci Monit, 2018; 24: 1588-1596 A B Sham Vehicle r-OPN r-OPN+Wort Sham Vehicle r-OPN r-OPN+Wort p-Akt p-GSK-3β β-actin β-actin Sham Vehicle r-OPN r-OPN+Wort Sham Vehicle r-OPN r-OPN+Wort Akt GSK-3β β-actin β-actin 1.2 1.4 p-Akt p-GSK-β Akt GSK-3β 1.2 1.0 1.0 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 0.0 0.0 Sham Vehicle r-OPN r-OPN+Wort Sham Vehicle r-OPN r-OPN+Wort Figure 5. r-OPN affects p-Akt and p-GSK-3 b protein expression 24 h after ICH or sham. Representative Western blots and densitometric quantification of p-Akt/ b-actin and total Akt/b-actin (A). Representative Western blots and densitometric quantification of p-GSK-3 b/b-actin and total GSK-3b/b-actin (B). Data are presented as the mean ±SEM (* P<0.05 compared # & with sham, P<0.05 compared with vehicle, P<0.05 compared with r-OPN, n=6–8/group). signaling events upon integrin binding, and it promotes sur- CC3 is an important marker of apoptosis [40]. In the present vival through the PI3K/Akt signaling pathway [35]. In addition, study, ICH induced an imbalance of Bax/Bcl-2 and increased suppression of the apoptosis has been suggested to reduce the CC3 expression. r-OPN downregulated the ratio of Bax/Bcl- brain edema and improve functional outcomes in experimen- 2 expression more toward the levels found in the sham group tally induced ICH [36]. Therefore, we explored whether the and also decreased the expression of CC3. We used TUNEL to neuroprotective mechanism of r-OPN was mediated by sup- detect apoptotic cell death and observed a significantly re - pressing the cell apoptosis-related PI3K/Akt/GSK-3b signaling duced density of TUNEL-positive cells in the r-OPN treatment pathway. Animals receiving r-OPN treatment showed neuro- group. r-OPN inhibited the apoptosis during the process of protective effects compared with vehicle group. When we ad - ICH, whereas r-OPN plus the wortmannin treatment upregu- ministered PI3K inhibitor wortmannin and r-OPN, neurological lated the ratio of Bax/Bcl-2, increased the expression of CC3, impairment and brain edema were not alleviated compared and the number of TUNEL-positive cells increased significant - with the r-OPN group. Our findings further confirm previous ly. Therefore, we explored whether the neuroprotection and findings that wortmannin alone did not worsen the behavior - the cell apoptosis suppression mechanism of r-OPN were me- al outcome and brain edema compared with the vehicle group diated via the PI3K/Akt signal pathway. after ICH [36]. These results indicate that r-OPN exerts neu- roprotective effects through the PI3K/Akt signaling pathway. Akt, a serine/threonine kinase, is a primary mediator of the downstream effects of phosphatidylinositol-3 kinase (PI3K), co - At the molecular level, the Bcl-2 family proteins play impor- ordinating a variety of intracellular signals and controlling cell tant roles in intrinsic apoptotic pathway [37,38]. The family responses to extrinsic stimuli and regulating cell proliferation is divided into anti-apoptotic proteins such as Bcl-2 and pro- and survival [42]. The pro-apoptotic role of GSK-3b is an im- apoptotic proteins. Bax induces apoptosis, and Bax/Bcl-2 ra- portant downstream target of the Akt signaling pathway [43]. tio is often used to represent the extent of apoptosis [39,40]. GSK-3b has been shown to participate in apoptosis in several Indexed in: [Current Contents/Clinical Medicine] [SCI Expanded] [ISI Alerting System] This work is licensed under Creative Common Attribution- [ISI Journals Master List] [Index Medicus/MEDLINE] [EMBASE/Excerpta Medica] NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) [Chemical Abstracts/CAS] Relative p-Akt and Akt level (protein) Relative p-GSK-3β and GSK-3β level (protein) Zhang W. et al.: Recombinant osteopontin improves neurological functional recovery… ANIMAL STUDY © Med Sci Monit, 2018; 24: 1588-1596 cell types and is known to be an upstream regulator of pro- chemotactic for the endothelial cells, and help promote new grammed cell death, which is particularly abundant in the blood vessel formation in vitro [10]. Thrombin is produced in CNS [44,45]. Dysregulation of GSK-3b activity is believed to the brain immediately after ICH [46]. Whether r-OPN follow- play a key role in the pathogenesis of central nervous system ing thrombin cleavage has positive effects on neuroprotection chronic and acute disorders such as ischemic stroke, and trau- after ICH in vivo should be further explored. matic brain injury [18,20]. Akt activation promotes cell survival by phosphorylation and subsequent inactivation of GSK-3b [21]. In the present study, r-OPN treatment significantly increased Conclusions the expression of activated Akt (p-Akt, Ser473) at 24 h after ICH, which thereby successively reduced the expression of ac- In summary, our study demonstrated that r-OPN protected tivated GSK-3b (p-GSK-3b, Ser9). r-OPN treatment reduced cell against ICH injury and improved neurological function by re- apoptosis and expression of apoptosis-related proteins. When ducing apoptosis in vivo. Moreover, r-OPN exerted its anti-apop- we used PI3K inhibitor wortmannin combined with r-OPN, the totic effect through the PI3K/Akt/GSK-3 b signaling pathway. In levels of activated Akt protein and activated GSK-3b protein conclusion, the elucidation of the neuroprotective action of r- were similar to levels in the vehicle group. The wortmannin plus OPN in the current study adds to a growing literature suggest- r-OPN treatment did not reduce the incidence of cell apoptosis ing the potential beneficial role for this factor in ICH. after ICH in rats. Our results support r-OPN-mediated neuro- protection in a rat model of ICH, primarily via activation of the Conflicts of interest PI3K/Akt, and inhibited GSK-3b. However, certain physiologi- cal processes involving OPN partly depend on its cleavage by None. thrombin [10]. Thrombin-cleaved OPN fragments are strongly References: 1. Steiner T, Alshahi SR, Beer R et al: European Stroke Organisation (ESO) 15. 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Stroke, 2009; 40(3 Suppl. 1): S88–89 Indexed in: [Current Contents/Clinical Medicine] [SCI Expanded] [ISI Alerting System] This work is licensed under Creative Common Attribution- [ISI Journals Master List] [Index Medicus/MEDLINE] [EMBASE/Excerpta Medica] NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) [Chemical Abstracts/CAS]

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Medical Science Monitor : International Medical Journal of Experimental and Clinical Research – Pubmed Central

Published: Mar 18, 2018

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