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Background: Intracerebral hemorrhage (ICH) remains with high morbidity and mortality due to lack effective treatment. Urocortin, as a novel anti-inflammatory neuropeptide, has exhibited antiinflammatory and neuroprotective effects on ICH. However, the mechanisms underlying urocortin involved in the protective effects
are still not clear. Therefore, in this study, ICH was induced by an infusion of autologous blood into the unilateral striatum of anesthetized rats. At 1 hour after the induction of ICH, UCN was infused into the lateral ventricle on the ipsilateral side. We found that the UCN, administered in the ipsilateral lateral ventricle, was able to penetrate into the injured striatum. Posttreatment with UCN could reduce the injury area and brain edema and improve neurological deficits. Moreover, our in-vitro experiment indicated UCN could inhibit the apoptosis of neuron cell line N2a and SH-SY5Y by up-regulating VEGF expression via CRFR2. Thus, our data imply that UCN may be a therapeutic agent in the protection against ICH.
Key words: UCN; Intracerebral hemorrhage; CRFR2; VEGF
Intracerebral hemorrhage (ICH), which results from the spontaneous rupture of intracranial vessels or hemorrhage transformation of acute ischemic stroke, occurs frequently as a major complication of thrombolytic therapy for acute ischemic stroke. ICH accounts for approximately 15% of stroke incidents in Western populations and an even higher proportion, up to 20-30%, in Asian populations . After the occurrence of ICH, various changes, including brain edema formation, blood clot formation, and release of toxic blood components and inflammatory cytokines, worsen neurological deficits [2, 3]. Edema leads to an expansion of brain volume that causes a negative result on ICH outcomes. In the clinical setting in select patients, medical management for those who have suffered an ICH is only limited to supportive care or hematoma evacuation. No effective drugs can increase survival after ICH.
Corticotropin-releasing factor (CRF) family, is composed of CRF, urocortin1 (UCN1), UCN2 and UCN3, which are identified as about 40-amino acid neuropeptides and bind to their two known receptors, CRF receptor 1 (CRFR1) and CRFR2 [4-6]. These receptors are G-protein coupled, and their activation can mediate responses via protein kinase signaling pathways [7, 8]. CRF has a 10-fold higher affinity for CRFR1 than for CRFR2.UCN1 has equal affinity for both receptors, and UCN2 & UCN3 are specific for CRFR2 . Both CRFRs are distributed in central nervous system (CNS) and periphery nervous system (PNS) [10-12], but the two subtypes display quite different pharmacological profiles . It has become apparent that CRF pathway and its components are involved in a wide array of physiological and potentially pathophysiological processes, and the CRF pathway presents, therefore, a very promising clinical therapeutic target.
Previous studies indicated that UCN alleviated inflammation and neurotocity by microglial activation, suggesting its anti-inflammatory and neuroprotective effects in brain injuries, but these actions are absent in CRFR2-deficient mice [13, 14]. However, both CRF and UCN also activate CRFR1 in the pituitary and thus stimulate the hypothalamic-pituitary-adrenal (HPA) axis, complicating the potential use of these peptides for treatment of ICH. UCN was reported to have protective effects against ICH in vivo, but its mechanism is largely unknown.
In this study, we investigate the effects of UCN on ICH in vivo, and demonstrate the roles of UCN on neuron cells in vitro, and its possible mechanisms will also be demonstrated.
Materials and Methods
Cells and Animals
The neurological cell lines N2a and SH-SY5Y were obtained from American Type Culture Collection. Cells were grown in RPMI- 1640,with 10% fetal bovine serum and 1% penicillin- streptomycin, and incubated at 37â„ƒ in a humidified atmosphere of 95% air and 5% CO2. Adult male Sprague-Dawley rats, weighing from 200 to 250 g, were purchased from Soochow University Laboratory Animal Center (Suzhou, China), and enrolled in the study. This project was approved by the Committee of Experimental Animals of Soochow University and the procedures were followed according to the routine animal care guidelines. All experimental procedures complied with the National Institute of Health (NIH) Guidelines for the care and use of laboratory animals.
The rats were anesthetized intraperitoneally with chloral hydrate (0.4 g/kg). 100 µl of autologous blood (taken from the tail vein, infused within 60 seconds) were infused via a 30-gauge needle into the striatum (0.0 mm posterior, 3.0 mm right, 5.0 mm anterior to the bregma on the surface of the dura mater) . The needle remained in place for an additional 10 minutes to prevent backflow. The craniotomies were sealed with bone wax. The rats were allowed to recover in individual cages in the animal center and had free access to food and water under a light/dark cycle of 12/12 hours. The rats were randomly divided into the following five groups: Group 1, sham ICH control group (n = 8). The rats were infused with 1 L saline (0.1 L/minute for 10 minutes) into the striatum. Group 2, ICH saline group, blood infusion model (n = 8). One hour after induction of ICH by autologous blood (100 µL) infused into the striatum, the rats received an infusion of saline (0.5 L/minute for 10 minutes) into the lateral ventricle, ipsilateral to the hemorrhagic striatum. Group 3, ICH UCN group, blood infusion model (n = 8). One hour after the induction of ICH by autologous blood infusion in the same manner as Group 2, UCN (5 µg in 5 L saline) was infused (0.5 L/minute for 10 minutes) into the lateral ventricle.
Evaluation of Neurological Function
An mNSS for experimental rats was evaluated by an investigator blinded to the treatment scheme before ICH (Day 0), and on Days 1, 3, and 7 after treatment. Neurological function was graded on a scale of 0–18 (normal score, 0; maximal deficit score, 18). The mNSS is a composite test of motor, sensory, and balance functions . The rats with an abnormal mNSS (score > 0) before ICH were excluded from the experiment.
Cell Viability Assay
For analysis of cell viability, cells were seeded in 96-well plates with 6 × 103 cells/well. After incubation with UCN for 24 hr, cell viability was determined by Cell Count Kit- 8 (CCK-8) assay (Donjindo Laboratories, Kumamoto, Japan), which was performed as described by Morita and coworkers . Absorbance readings at a wavelength of 450 nm and reference wavelength of 650 nm were taken on a spectrophotometer.
Enzyme-linked immunosorbent assay (ELISA)
Rat VEGF level was analyzed by ELISA using the Quantitative VEGF ELISA kit (BioSource, Camarillo, California, USA). According to the manufacturer’s protocol, the assay was designed to measure natural VEGF levels in tissue supernate. The brain tissue was lysated by the buffer according to the manufacturer’s protocol, the supernatants were collected to measure VEGF expression level. For cells, equal numbers of N2a and SH-SY5Y cells were seeded in 24-well plates (1× 105 cells/well) and incubated in medium containing 2% FBS at 37â-¦C for 24 hr with the indicated concentration of each peptide or reagent. The medium was then aspirated, and VEGF levels in the supernatants were measured. All analyses were carried out in triplicate. The concentration of VEGF in unknown samples was determined by comparing the optical density of the samples to the standard curve and reported in pg/mL.
Brain tissue or cell samples were lysated by RIPA buffer, protein concentration was determined using a Bradford assay. Total 50 μg proteins was separated on 10% SDS gels and transferred to PVDF membranes (Millipore, USA). Following transferring, the membranes were washed three times in TBST (0.1% Tween-20) and then blocked for 1 h at room temperature with 5% milk in TBST. The membranes were blotted with a 1:1000 dilution of anti-p-JNK1/2, anti-t-JNK1/2, anti-p-p38, anti-t-p38, anti-p-ERK1/2, anti-t-ERK1/2, anti-p-Akt or anti-t-Akt (1:1,000 dilution; CST, USA). Membranes were subsequently washed four times in TBST and then subjected to the appropriate HRP-conjugated secondary antibody for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence regent (ECL kit, Amersham, Piscataway, NJ, USA) and densitometry analysis was performed using Bio-Rad Quantity-one software.
All data were reported as mean ± S.E.M and statistical analysis was performed using SPSS 13.0. One-way or two-way ANOVA with a Bonferroni post-hoc test was used to determine if any significant differences existed between groups for individual parameters. The level of significance was defined as p < 0.05.
The effects of UCN on the neurological phenotype of rat ICH models
Figure 1 shows a comparison of mNSS among administrations of saline and UCN (5 mg) in autologous blood infusion-induced ICH rats. The UCN administrations produced a dose-dependent reduction in the mNSS (an improvement from neurological deficits), most effectively at 5 mg (p < 0.001 vs ICH + saline group). These data demonstrated that post treatment with UCN significantly reduced the ICH-induced neurological deficits over 7 days. The most effective dosage of UCN (5 mg) was adopted for further experiments.
UCN diminishes the injured area of brain
We examined brain water contents of whole brain and cerebellum in the sham ICH control, ICH + saline, and ICH + UCN groups (Fig. 2). Whole brain water content in the sham ICH rats (n = 5) was 75.25 ± 4.67%. In the ICH + saline group, the water content of the whole brain was increased to 82.75 ± 5.71% (p < 0.05). In the ICH + UCN group, however, the brain water content was reduced to 76.5 ± 3.69% (p < 0.05). The brain water content of the cerebellum was essentially not affected among these 3 groups (6 rats in each group). These findings indicated that ICH produced cerebral edema on the whole brain, and that unilateral UCN posttreatment significantly reduced the cerebral edema (Fig. 2).
UCN promotes the survival of neurological cells
Next, we explore the effect of UCN on the survival of neurological cell lines N2a and SY5Y. After incubation with different concentration of UCN, it is found that the survival rates of the cells increased concentration-dependently; furthermore, these effects can be abolished by co-incubate with CRFR2 antagonist anti-Svg-30, but CRFR1 antagonist NBI-27914 had no effects (Fig. 3). These data demonstrated UCN can promote the survival of neurological cells via CRFR2.
UCN up-regulates VEGF expression in ICH models
Brain tissue VEGF levels were compared in baseline 30 min following ischemia, 2 hr or 4 hr following reperfusion of all groups. The levels of VEGF at 2 hr or 4 hr after ICH in UCN groups were significantly increased compared with saline groups (p < 0.01) (Fig. 4A). Furthermore, in the UCN + anti-Svg-30 group, the levels of VEGF had no significant differences compared with saline groups after 4 hr reperfusion (p < 0.05), but the expression of VEGF also increased when pretreatment with NBI-27914 (Fig. 4B), suggesting UCN’s effect on VEGF expression was mediated by CRFR2, not CRFR1.
UCN up-regulates VEGF expression by ERK1/2 and p38 via CRFR2
In order to further investigate the effects of UCN on the VEGF expression of neurological cells, we used N2a and SH-SY5Y cell lines to treat with UCN. Consistent with our in-vivo data, the results showed UCN significantly increased the expression of VEGF, but these effect could be abolished by CRFR2 antagonist anti-Svg-30, but not NBI-27914, indicating UCN could upregulate the VEGF expression via CRFR2 (Fig. 5A).
Next we tried to explore the possible pathways how UCN make effects to reduce the VEGF expression. We detected some important signaling pathways of UCN-CRFR2, and found that the expressions of phosphorylated JNK1/2 and p38 were increased after UCN’s treatment on the neurological cells. These effects could be abolished when pretreatment with CRFR2 antagonist anti-Svg-30 (Fig. 5B), but the expressions of ERK1/2 and Akt remained unchanged (data not shown). These results indicate UCN may upregulate VEGF by activating JNK1/2 and p38 phosphorylation via CRFR2. In order to confirm this hypothesis, we used JNK1/2 inhibitor TAT-JBD peptide or p38 inhibitor SB-203580 to eliminate the functions of JNK1/2 or p38, and it was found that the level of VEGF expression was similar with control (without UCN treatment) when pretreated with TAT-JBD peptide or SB-203580 (Fig. 5C) These data show UCN’s effects may be mediated by JNK1/2 and p38 pathways.
This study demonstrates that posttreatment of UCN through the lateral cerebral ventricle can significantly reduce the neurological functional deficits, brain edema and injury size caused by ICH in the rats. Moreover, our data also uncover that UCN’s benefit effects against ICH may be mediated by JNK1/2 and p38 phosphorylation via down-regulating VEGF expression of neurological cells. Therefore, these findings indicate that UCN may have a potential therapeutic application in ICH injury and possibly other tissue injuries of different causes.
The effect of UCN for the treatment of tissue injuries is rarely investigated. Urocortin can protect injuries of cardiac myocytes in vitro and heart in vivo [18-20] and restore nigrostriatal function after endotoxin-induced neuroinflammation in vivo . We demonstrate that posttreatment with UCN at doses of 0.05, 0.5, and 5 mg can protect against ICH-induced injury dose-dependently. The mechanisms underlining the effects of UCN in alleviating the aforementioned tissue injuries is probably due to the down-regulation of VEGF. Previous in vitro study also demonstrated that femtomolar concentrations of UCN could inhibit TNF-¡ production in cultured microglia treated with endotoxin . Furthermore, Wang et al. found that UCN could inhibit the tumor growth by inhibiting the secretion of VEGF [23, 24]. These studies indicate that the effect of UCN in reducing brain edema in the in vivo ICH injuries may be due to its VEGF regulatory effects.
Brain edema can be substantial and deleterious, as ICH-induced brain edema is associated with significant midline shift and increased intracranial pressure, which can result in brain herniation [25, 26]. Different approaches have been taken experimentally to reduce the edema after ICH, but these approaches are limited for clinical use. To these concerns, peak edema occurs 3 days after ICH , while our study shows that posttreatment with UCN reduces the brain edema (water content) on Day 3 after ICH. These data suggest that UCN has the potential for further clinical use.
Only femtomolar concentrations of UCN were needed to inhibit TNF-¡ production in cultured microglia of 0.05, 0.5, and 5 mg per rat, about 0.01, 0.1, and 1 nmol per rabbit via the lateral cerebral ventricle. The present investigation did not intend to determine the concentrations of UCN in the lateral cerebral ventricle or in the blood. Nevertheless, whether UCN can pass across the CSF-brain barrier should be addressed. Although the permeability of UCN, a 4.7-kD peptide, across the BBB is less than that for other peptides and larger proteins, physiologically the barrier across CSF and brain is relatively less than that across the BBB. In our experiment, intracerebroventricularly administered UCN effectively reduces the ICH-induced brain injuries while similarly administered fluorescence-labeled UCN is identified in the cell nucleus of the striatum, indicating that UCN can readily penetrate across the barrier between CSF and brain. Whether UCN can readily penetrate across the barrier between CSF and brain as well as the BBB is further evident.
Though the two corticotropin-releasing factor receptors (CRFR1 and CRFR2) are isolated in cerebral microvessels, and mediate UCN transport, here, we indicated that CRFR2 take the major roles of UCN benefit effects on ICH, because CRFR2 antagonist anti-Svg-30 abolished these effects, but not CRFR1 antagonist NBI-27914. Previously, the experiment indicated UCN inhibited tumoral angiogenesis in vitro and in vivo in hepatocellar carcinoma via CRFR2  and showed loss of CRFR2 led to pathophysiological processes due to alteration of anti-angiogenic signaling pathways . Moreover, it was investigated that activation of CRFR2 by UCN2 strikingly inhibited the growth and vascularization of mice in vivo . Furthermore, UCN2 directly inhibited the proliferation of mice LLCC in vitro . These observations imply CRFR2 activation takes important roles in many pathophysiological procedures, including inflammation. In the present study, it was shown that UCN protected the brain against ICH, while CRFR2 selective antagonist anti-Svg-30 abolished these effects. This investigation indicated UCN improves neurological phenotype and decrease the brain edema post-ICH via CRFR2.
MAPK signaling family, as inflammatory signal transduction pathways in ICH , was determined in our study. We found no effects on phosphorylation of ERK1/2 by UCN, which were consistent with previous reports [30-32]. Interestingly, previous literatures reported CRFR2 induced the activation of p38 signaling to make its diversely biological effects , and UCN2 activated p38 phosphorylation to increase COX-2 and ICAM-1 expression of vascular smooth muscle cells . Our data also showed that the activation of CRFR2 by UCN obviously promoted the phosphorylation of p38 in neurological cells. JNK1/2 is commonly activated in inflammatory tissues, and recognized as one of central signaling molecules in regulating brain stroke [35, 36]. In this study, phosphorylation of JNK1/2 was most potently blocked by the CRFR2 agonist.
Taken together, our study showed CRFR2 expression in human brain, and the activation of CRFR2 by UCN protect the brain against ICH. Furthermore, the activation of CRFR2 could up-regulate the secretion of VEGF via CRFR2 in neurological cells. Therefore, CRFR2 may be a potential target in ICH, moreover, its ligand UCN might become new endocrine biological therapeutic approach to ICH.