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Introduction

Subarachnoid hemorrhage (SAH) is a common cerebrovascular disease, accounting for ~5% of stroke cases, and there are 30,000 new cases annually in the US (1). Since the American Heart Association (AHA) issued treatment guidelines for SAH in 2004 (2), progress has been made in the diagnostic methods, endovascular therapy, surgery and perioperative management of SAH; however, the prognosis of SAH remains unsatisfactory, with a mortality and disability rate of up to 45% (3,4).

A number of studies have shown that early brain injury (EBI) is a major factor in the poor prognosis of the patients with SAH, suggesting that the timely and effective intervention and treatment of EBI may improve the clinical prognosis of patients with SAH (5,6). Following SAH, EBI pathogenesis is complex, while a large number of studies indicate that oxidative stress plays an important role in the development of EBI (7–9).

Akt, also known as protein kinase B (PKB), is a serine/threonine protein kinase that is involved in cell survival and apoptosis (10). Studies have shown that apoptosis-like changes occur in the basilar artery endothelial cells, vascular wall cells proliferate widely, and the expression levels of Akt and caspase-3 in basilar arterial wall tissues are modulated at different time points following SAH, indicating that the Akt pathway is involved in the apoptosis of basilar arterial endothelial cells subsequent to SAH (11,12).

The nuclear transcription factor nuclear factor (NF)-κВ and cyclooxygenase are involved in the inflammatory response of the central nervous system, which also plays an important role in the pathogenesis of cerebral vasospasm after SAH (13,14). A previous study has demonstrated the ability of baincalein to alleviate EBI through involvement in a Toll-like receptor 4/NF-κB-mediated inflammatory pathway in rats following SAH (15). Furthermore, ceftriaxone has been found to alleviate EBI in a rat model of SAH via the phosphatidylinositide 3-kinase (PI3K)/Akt/NF-κB signaling pathway (16).

The phenolic compound 3,4-dihydroxyphenylethanol (DOPET), also known as hydroxytyrosol, a natural compound that can be extracted from olives, has numerous biological and pharmacological activities; studies conducted in recent years have confirmed that hydroxytyrosol has strong antioxidant activity, promotes the reduction of blood pressure, prevents cardiovascular disease and has anticancer activity; thus, hydroxytyrosol is a natural antioxidant considered worthy of research and vigorous development (17,18). However, the neuroprotective effects of DOPET on SAH-induced EBI have not been investigated. In the present study, it was hypothesized that DOPET might attenuate EBI and improve neurological outcomes through the suppression of oxidative stress, Akt and NF-κВ following SAH in rats.

Materials and methods

Chemicals and reagent

DOPET (purity 98%) was acquired from Sigma-Aldrich (St. Louis, MO, USA) and its molecular structure is shown in Fig. 1. Malondialdehyde (MDA), superoxide dismutase (SOD), acetyltransferase (CAT) glutathione peroxidase (GSH-PX), bicinchoninic acid (BCA) and caspase-3 kits were purchased from Beyotime Institute of Biotechnology (Nanjing, China).

Figure 1. Chemical structure of 3,4-dihydroxyphenylethanol.

Animals

Adult male Sprague-Dawley rats (250–300 g) were purchased from the Animal Center of Taian Central Hospital (Taian, China). The rats were acclimated in a humidified room with a 12-h light/dark cycle at ~25°C, and free access to food and water prior to the experiment. All experimental procedures were approved by the Animal Care and Use Committee of Taian Central Hospital and performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (19).

A total of 40 rats were randomly divided into four equal groups: Sham group (n=10), SAH group (n=10), SAH + vehicle group (n=10) and SAH + DOPET group (n=10). In the sham group, normal rats received sodium pentobarbital [0.1 ml/100 g, intraperitoneally (i.p.); Sigma-Aldrich]; in the SAH group, SAH model rats did not receive any treatment; in the SAH + vehicle group, SAH model rats received sodium pentobarbital (0.1 ml/100 g, i.p.) 24 h after modeling; in the SAH + DOPET group, SAH model rats received 100 mg/kg DOPET 24 h after modeling once a day for 6 weeks. The dose of DOPET was chosen according to a previous study (20). All parameters were investigated at 24 h after SAH.

Rat SAH model and mortality

The rat SAH model was created by an endovascular perforation method as described previously (21). Briefly, rats were anesthetized with an injection of sodium pentobarbital (50 mg/kg, i.p.). Firstly, the left common, external and internal carotid arteries were revealed. Then, a sharpened 4-0 monofilament nylon suture was inserted into the left internal carotid artery through the external carotid artery. The artery was perforated at the bifurcation of the anterior and middle cerebral artery to create the SAH model. Sham-operated rats underwent a similar procedure, without perforation. Mortality rate was calculated 24 h after SAH. After 6 weeks of DOPET treatment, surviving rats were anesthetized using sodium pentobarbital [0.1 ml/100 g, intraperitoneally (i.p.)] and sacrificed via decollation.

Assessment of neurological status by testing blood-brain barrier (BBB) permeability

Evans blue (EB) dye (2%; ~4 ml/kg; Shanghai Hengyuan Biological Technology Co., Ltd., Shanghai, China) was injected over 2 min into the right femoral vein and allowed to circulate for 1 h. Rats were re-anesthetized and perfused with saline to remove intravascular EB dye. Then, the proportion of brain tissue that was stained with EB was observed with a Biodropsis BD-2000 spectrophotometer (BD Biosciences, Franklin Lakes, NJ, USA) at 620 nm.

Evaluation of brain water content

Brain samples were weighed, dried in an oven (80°C) for 72 h and then weighed again. The brain water content (%) was calculated as follows: [(wet weight - dry weight)/wet weight] × 100 (22).

Assessment of oxidative stress

Left basal cortical samples were obtained and incubated with 100 µl tissue lysis buffer (Shanghai Sangon Biological Engineering Co., Ltd., Shanghai, China) for 30 min on ice. Homogenates were centrifuged at 12,000 × g for 10 min at 4°C. Supernatants were collected to assess the content of malondialdehyde (MDA), and the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-PX). These indices were measured using commercial kits, according to the manufacturer's protocols.

Western blot analysis of Akt and NF-κB

Left basal cortical samples were obtained and incubated with 100 µl tissue lysis buffer for 30 min on ice. Homogenates were centrifuged at 12,000 × g for 10 min at 4°C. Supernatants were collected and the protein concentration was determined using a BCA kit. Equal quantities of protein were loaded on 12% sodium dodecyl sulfate-polyacrylamide gels, subjected to electrophoresis, and then transferred to polyvinylidene fluoride membranes (0.22 µm; Millipore, Temecula, CA, USA). After blocking non-specific binding with phosphate-buffered saline containing 5% non-fat milk for 2 h, the membranes were incubated with anti-p-Akt (1:1,000; sc-135650), anti-NF-κB p65 (1:1,000; sc-372) and anti-β-actin (1:3,000; sc-130656; all Santa Cruz Biotechnology, Inc., Dallas, TX, USA) rabbit polyclonal antibodies overnight at 4°C. After incubation, the membrane was detected by incubating with horseradish peroxidase-conjugated anti-rabbit IgG (1:10,000; 7074; Cell Signaling Technology, Inc., Danvers, MA, USA) for 2 h. The band intensity was determined using an Image Quant LAS4000 mini gel image analysis system (GE Healthcare, Arlington Heights, IL, USA).

Assessment of caspase-3 activities

Left basal cortical samples were obtained from the rats and incubated with 100 µl tissue lysis buffer for 30 min on ice. Homogenates were centrifuged at 12,000 × g for 10 min at 4°C. Supernatants were collected and the protein concentration was performed using a BCA kit. Reaction buffer (Ac-DEVD-pNA for caspase-3) was added to equal quantities of protein and incubated at 37°C for 2 h in the dark. Caspase-3 activities were determined by measuring the absorbance at 405 nm.

Statistical analysis

Values were expressed as the means ± standard deviations. Data were analyzed using analysis of variance, followed by Tukey-Kramer multiple comparisons test. P<0.05 was considered to indicate a statistically significant difference.

Results

Mortality and effects of DOPET on BBB permeability

The mortality rate after surgery was 0/35 (0%) in the sham group, 8/35 (22.86%) in the SAH group, 7/35% (20.00%) in the SAH + vehicle group and 7/35% (20.00%) in the SAH + DOPET group. No significant inter-group difference was identified between the SAH, SAH + vehicle and SAH + DOPET groups (P>0.05; Fig. 2A). SAH insult significantly induced BBB permeability in comparison with that in the sham group. Notably, the BBB permeability of the SAH group was very similar to that of the SAH + vehicle group (P>0.05; Fig. 2B). Following DOPET administration, BBB permeability was significantly reduced as compared with that of the SAH + vehicle group (Fig. 2B).

Figure 2. Mortality and effects of DOPET on BBB permeability. Evaluation of (A) mortality rate and (B) BBB permeability. **P<0.01 vs. the sham group; #P<0.01 vs. the SAH group. BBB, blood-brain barrier; SAH, subarachnoid hemorrhage; DOPET, 3,4-dihydroxyphenylethanol (100 mg/kg).

Effects of DOPET on brain water content

The effect of DOPET on brain water content was determined by measuring the change in brain weight before and after drying. As shown in Fig. 3, SAH increased brain water content in comparison with that in the sham group. However, no significant inter-group difference was found between the SAH group and the SAH + vehicle group with regard to brain water content (P>0.05; Fig. 3). Furthermore, DOPET administration effectively reduced brain water content as compared with that of the SAH + vehicle group (Fig. 3).

Figure 3. Effects of DOPET on brain water content. **P<0.01 vs. the sham group; #P<0.01 vs. the SAH group. SAH, subarachnoid hemorrhage; DOPET, 3,4-dihydroxyphenylethanol (100 mg/kg).

Effects of DOPET on oxidative stress

To further elucidate the anti-oxidative effects of DOPET, the activities of SOD, CAT and GSH-PX were measured. The MDA contents of cortical samples from the SAH model rats were increased in comparison with those of the sham group (Fig. 4A). No significant difference was identified between the SAH group and the SAH + vehicle group (P>0.05; Fig. 4A). However, DOPET markedly decreased MDA levels in comparison with those in the SAH + vehicle group (P<0.01; Fig. 4A). Following SAH modeling, the activities of SOD, CAT and GSH-PX were restrained as compared with those of the sham group (Fig. 4B–D). Notably, the activities of SOD, CAT and GSH-PX in the SAH group were very similar to those in the SAH + vehicle group (P>0.05; Fig. 4B–D). Furthermore, pretreatment with DOPET augmented the activities of SOD, CAT and GSH-PX in comparison with those of the SAH + vehicle group (Fig. 4B–D).

Figure 4. Effects of DOPET on oxidative stress. Anti-oxidative effects of DOPET on (A) the concentration of malondialdehyde (MDA) and the activities of (B) superoxide dismutase (SOD), (C) catalase (CAT) and (D) glutathione peroxidase (GSH-PX) in SAH model rats. **P<0.01 vs. the sham group; #P<0.01 vs. the SAH group. SAH, subarachnoid hemorrhage group; DOPET, 3,4-dihydroxyphenylethanol (100 mg/kg).

Effects of DOPET on Akt expression

Akt signaling pathways are associated with apoptosis in the rat SAH model (23). In comparison with the sham group, Akt protein expression levels in the SAH group were increased (Fig. 5). No significant difference in Akt expression was observed between the SAH group and the SAH + vehicle group (P>0.05; Fig. 5). Administration of DOPET notably boosted Akt protein expression in comparison with that of the SAH + vehicle group (Fig. 5). These results indicate that DOPET treatment induced Akt protein expression following experimental SAH.

Figure 5. Effects of DOPET on Akt expression. (A) Representative western blot of Akt protein expression and (B) densitometric analysis of Akt protein expression levels. **P<0.01 vs. the sham group; #P<0.01 vs. the SAH group. SAH, subarachnoid hemorrhage group; DOPET, 3,4-dihydroxyphenylethanol (100 mg/kg).

Effects of DOPET on NF-κB expression

To elucidate the anti-inflammatory effects of DOPET, NF-κB protein expression was evaluated using western blot analysis. As shown in Fig. 6, NF-κB protein expression in the cerebral cortex following SAH was increased as compared with that of the sham group (Fig. 6). However, no significant difference between the SAH group and SAH + vehicle group was identified (P>0.05; Fig. 6). By contrast, the administration of DOPET partially abrogated NF-κB protein expression in comparison with that of the SAH + vehicle group (Fig. 6).

Figure 6. Effects of DOPET on NF-κB expression. (A) Representative western blot of NF-κB protein expression and (B) densitometric analysis of NF-κB protein expression levels. **P<0.01 vs. the sham group; #P<0.01 vs. the SAH group. SAH, subarachnoid hemorrhage; DOPET, 3,4-dihydroxyphenylethanol (100 mg/kg); NF-κB, nuclear factor-κB.

Effects of DOPET on caspase-3 activity

Since caspase-3 has a crucial role in apoptosis (24), whether the administration of DOPET regulates cell apoptosis in the rat SAH model was examined. As shown in Fig. 7, SAH insult induced caspase-3 activity in comparison with that of the sham group. No significant difference in caspase-3 activity was observed between the SAH group and the SAH + vehicle group (P>0.05; Fig. 7). Furthermore, pretreatment with DOPET markedly decreased caspase-3 activity as compared with that of the SAH + vehicle group (Fig. 7).

Figure 7. Effects of DOPET on caspase-3 activity. **P<0.01 compared with sham group; #P<0.01 compared with SAH group. Sham, sham group; SAH, subarachnoid hemorrhage group; SAH+vehicle, SAH+vehicle group; SAH+DOPET, SAH+3,4-Dihydroxyphenylethanol (DOPET, 100 mg/kg)-treated.

Discussion

SAH is a commonly occurring cerebrovascular disease with rapid onset and development, and the prognosis is usually poor with high morbidity and mortality, making it a serious threat to the quality of life and safety of patients; therefore, timely and effective treatment measures for patients with SAH are vital (25). In the present study, DOPET administration significantly reduced BBB permeability and the brain water content of SAH model rats. Schaffer et al (26) reported the neuroprotective effects of a DOPET-containing extract on brain cells in vitro and ex vivo. These results demonstrated that DOPET was able to attenuate EBI and improve the neurological outcomes of rats with SAH. Cabrerizo et al (27) showed that the neuroprotective effects of DOPET occurred via the inhibition of oxidative stress and inflammatory mediators in rat brain slices subjected to hypoxia reoxygenation.

EBI is the main cause of high mortality and morbidity of patients with SAH, and oxidative stress has been demonstrated to play a key role in the development of EBI (5,28,29). The balance of the oxidation and antioxidant systems in the body is broken following SAH; oxidative damage exceeds the capacity of the body's antioxidant defense system, and a large number of oxygen free radicals induce lipid peroxidation, protein denaturation and DNA damage, causing damage to the BBB and pathological changes such as cell death in the brain (28). In the present study, DOPET markedly attenuated the MDA contents and enhanced the activities of SOD, CAT and GSH-PX in the brains of SAH model rats. Zheng et al (30) suggested that DOPET administration improved neurogenesis and cognitive function via the reduction of oxidative stress in prenatally stressed offspring. Granados-Principal et al (31) demonstrated that DOPET ameliorated the oxidative stress associated with doxorubicin-induced cardiotoxicity in rats with breast cancer.

The activation of second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP3) by protein kinase B occurs in the plasma membrane; PIP3 together with the intracellular signals of Akt and phosphoinositide dependent kinase-1 (PDK1) prompts the PDK1-activated phosphorylation of Akt on Ser308, thereby activating Akt protein (23). The phosphorylation of Akt activates or inhibits its downstream proteins, such as caspase-9, NF-κВ, glycogen synthase kinase-3, forkhead in rhabdomyosarcoma, p21Cipl and p27Kip1, so as to regulate cell proliferation, differentiation and apoptosis (32). In the current study, the administration of DOPET resulted in a significant increase in Akt protein expression levels in the brains of SAH model rats. This is consistent with a previous study, which showed that the potential protective effect provided by DOPET against oxidative kidney cell injury may be attributed to its interactions with these important intracellular signaling pathways (33). DOPET has also been shown to induce antioxidant enzymes via extracellular regulated PI3K/Akt pathways in HepG2 cells (34).

The nuclear transcription factor NF-κВ has been shown to be an important inflammatory mediator in brain tissue during the pathophysiological occurrence of cerebral vasospasm (35). NF-κВ is mainly expressed in vascular endothelial cells and cell nuclei of the outer membrane, and its expression level is positively correlated with the degree of vascular spasm (36). Cell adhesion molecules play an important role in the process by which leukocytes cross the blood vessel wall to reach adventitia-surrounding areas, and NF-κВ may be involved in the process of cerebral vasospasm initiation by regulating the expression of cell adhesion molecules (37). In the present study, the administration of DOPET restrained NF-κB protein expression following SAH. This is consistent with a study conducted by Zhang et al (38), which suggested that treatment with DOPET significantly suppressed NF-κB expression in THP-1 cells. These results support the implication of the Akt/NF-κB pathway in the neuroprotective effect of DOPET in the EBI after SAH. In addition to a reduction in neural apoptosis, caspase-3 activity was also ameliorated following DOPET administration in the present study. The results were consistent with previous findings; for example, a study conducted by Anter et al (39) reported that hydroxytyrosol restrained caspase-3-dependent proapoptotic effects in a human tumoral cell line, namely HL60 cells.

In summary, to the best of our knowledge, this is the first study to investigate the effects of DOPET treatment on SAH. Notably, the current study demonstrated that DOPET reduced BBB permeability and brain water content, inhibited oxidative stress, activated Akt expression, and suppressed NF-κB expression and neuronal apoptosis after SAH. The optimum time window of treatment, exact neuroprotective effect and mechanisms of DOPET on SAH treatment require elucidation in further studies.

References