Xanthotoxol Exerts Neuroprotective Effects Via Suppression of the Inflammatory Response in a Rat Model of Focal Cerebral Ischemia
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- He, W., Chen, W., Zhou, Y. et al. Cell Mol Neurobiol (2013) 33: 715. doi:10.1007/s10571-013-9939-2
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We previously found that xanthotoxol, one of the major active ingredients in Cnidium monnieri (L.) Cusson, exerts protective effects in a rat model of focal cerebral ischemia/reperfusion injury by alleviating brain edema, inhibiting the neutrophil infiltration, and decreasing the expression of intercellular adhesion molecule-1 (ICAM-1) and E-selectin. The present study was designed to further determine the possible mechanisms of action of neuroprotective properties of xanthotoxol after cerebral ischemia. Transient focal cerebral ischemia/reperfusion model in male Sprague–Dawley rats was induced by 2-h middle cerebral artery occlusion followed by 24-h reperfusion. Xanthotoxol (5 and 10 mg/kg) or vehicle were administered intraperitoneally at 1 and 12 h after the onset of ischemia. At 24 h after reperfusion, we assessed the effect of xanthotoxol on the blood–brain barrier (BBB) permeability, the production of pro-inflammatory mediators such as interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-8, nitric oxide (NO), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and the p65 subunit of the transcription factor, nuclear factor-κB (NF-κB) in the cortex after ischemic insult. The results showed that xanthotoxol treatment significantly attenuated BBB disruption, reduced the IL-1β, TNF-α, IL-8 and NO level, and attenuated the iNOS activity compared with vehicle-treated animals. Further, xanthotoxol treatment also significantly prevented the ischemia/reperfusion-induced increase in the protein expression of iNOS, COX-2, and the nuclear NF-κB p65. These results, taken together with those of our previous study, suggest that the neuroprotection may be attributed to the ability of xanthotoxol to attenuate the expression of pro-inflammatory mediators and thereby inhibit the inflammatory response after cerebral ischemia.
KeywordsXanthotoxolCerebral ischemia/reperfusionInflammatory response
Cerebral inflammation has been suggested to play an important role in the pathogenesis and progression of ischemic stroke (Amantea et al. 2009; Jin et al. 2010; Kleinig and Vink 2009). There is increasing evidence that cerebral ischemia leads to significantly elevated levels of pro-inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6, and the chemokines, IL-8, macrophage inflammatory protein-1α (MIP-1α), and monocyte chemotactic protein-1 (MCP-1). These mediators can enhance the expression of the intercellular adhesion molecule-1 (ICAM-1), P-selectin, and E-selectin on cerebral endothelial cells and leukocytes, and facilitate the adhesion and transendothelial migration of leukocytes (del Zoppo et al. 2000; Huang et al. 2006; Rodríguez-Yáñez and Castillo 2008; Wang et al. 2007). In addition, it has been demonstrated that the inducible effector enzymes such as inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), and the inducible transcription factors such as nuclear factor-κB (NF-κB) are markedly up-regulated and activated in ischemic brain regions (Collino et al. 2006; Wang et al. 2007). Then, these pro-inflammatory mediators and transcriptional factors induce multiple inflammatory cascades and contribute to the progression of brain damage following ischemic insult. Therefore, anti-inflammation treatment has been proposed to reduce ischemic damage and improve outcomes after an ischemic insult.
Xanthotoxol is a coumarin compound which is extracted from a Chinese herb Cnidium monnieri (L.) Cusson (Xiang and Fu 1984). Research studies have demonstrated that xanthotoxol possesses anti-inflammatory (Lian et al. 1998), antioxidant (Ng et al. 2000), sedative (Sethi et al. 1992), antiarrhythmic (Lian et al. 1996), Calcium antagonistic (Liu et al. 2005; Zeng et al. 2003), and analgesic (Shangguan et al. 1997) properties. We previously found that xanthotoxol exerts protective effects in a rat model of focal cerebral ischemia/reperfusion injury by reducing lesion volume, decreasing brain edema, improving neurological deficit score, inhibiting the neutrophil infiltration, and suppressing the expressions of intercellular adhesion molecule-1 (ICAM-1) and E-selectin (He et al. 2009). However, the possible mechanisms of action of xanthotoxol after cerebral ischemia/reperfusion injury have not been fully elucidated. In the present study, we aim to further investigate the mechanisms of action of neuroprotective properties of xanthotoxol. Therefore, we assessed the effects of xanthotoxol on the blood–brain barrier (BBB) permeability, the production of the pro-inflammatory mediators, such as IL-1β, TNF-α, IL-8, NO, iNOS, and COX-2, as well as the nuclear NF-κB p65 subunit expression following focal cerebral ischemia–reperfusion injury.
Materials and Methods
Chemicals and Animals
Xanthotoxol (purity ≥98 %) was a compound purchased from Shanghai Shunbo Biotech CO., LTD., (Shanghai, China). The powder was dissolved in a small volume dimethylsulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA), and its concentration was diluted with warm normal saline and was adjusted to the required working concentration just before use. The final concentration of DMSO was 25 %.
Adult male Sprague–Dawley rats weighing 210–260 g (n = 100) were obtained from the Experimental Animal Center, Gannan Medical College. The animals were maintained on a 12-h light/dark cycle and had free access to food and water and adapted to these conditions for at least 7 days before experiments. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1985) and were approved by the Experimental Animal Ethics Committee of Gannan Medical College (No. 2009006). According to these guidelines, efforts were made to minimize animal suffering and to reduce the number of subjects used.
Model of Cerebral Ischemia
Rats were anesthetized with an intraperitoneal injection of 10 % chloral hydrate (350 mg/kg). Transient focal cerebral ischemia was induced as described by Longa et al. (1989). In brief, the right carotid rejoin was exposed through a ventral midline cervical incision. The right common carotid artery, external carotid artery (ECA), and internal carotid artery (ICA) were isolated. The pterygopalatine branch of ICA was ligated to prevent incorrect insertion of the occluder filament. A 5-cm length of fish nylon thread (Φ 0.23 mm), tip of which had been rounded by heating near a flame, was introduced from the ECA into the ICA until mild resistance was felt (18–19 mm). In this way, the thread occluded the origin of the middle cerebral artery (MCA). At 2 h after MCA occlusion, the thread was removed to allow reperfusion of the ischemic area. The sham-operated animals were subjected to the same surgical procedures, and the MCA was not occluded. The rectal temperature was maintained at 37–38 °C with a heat lamp and heating pad during the operation. The room temperature was controlled in the range of 25–27 °C throughout the experimental procedure.
Rats were randomly divided into four groups: sham-operated group, ischemia/reperfusion (I/R) model group, I/R+ xanthotoxol (5 and 10 mg/kg) groups. Drugs were administered i.p. at 1 and 12 h after the onset of ischemia. Sham-operated and I–R model groups received vehicle (25 % DMSO in normal saline) 10 mL/kg according to the same protocol.
Brain Edema Measurement
The rats were anesthetized with 10 % chloral hydrate (350 mg/kg, ip) and brains were removed at 24 h after reperfusion (n = 5 for each experimental group). The brain samples were weighed to obtain the wet weight and were then dried at 105 °C for 24 h before measuring dry weight. Brain water content (%) was calculated by the wet and dry method as follows: [(wet weight−dry weight)/wet weight] ×100.
Brain Infarct Size Measurement
The brains were stained with 2,3,5-triphenyltetrazolium chloride (TTC, Sigma-Aldrich, St. Louis, MO, USA) to determine the brain infarct size. At 24 h after reperfusion, rats (n = 5 for each experimental group) were decapitated under deep anesthesia with 10 % chloral hydrate (350 mg/kg, ip), and brains were removed and sectioned coronally at a thickness of 2 mm and incubated in 2 % TTC at 37 °C for 30 min. Brain slices were then stored in 4 % paraformaldehyde overnight before analysis. The data were expressed as the percentage of the infarction size/the ipsilateral hemisphere size.
BBB Permeability Measurement
BBB permeability was evaluated by Evans blue extravasation. Rats (n = 5 for each experimental group) were injected with Evans blue (Sigma-Aldrich, St. Louis, MO, USA; 2 % in saline, 3 ml/kg) through the tail vein at 18 h after reperfusion. Six hours later, rats were anesthetized with 10 % chloral hydrate (350 mg/kg, ip) and transcardially perfused with saline to remove the intravascular dye. Brains were quickly removed and separated the ipsilateral hemispheres, and then weighed and homogenized in N, N-dimethylformamide (2.0 ml, Sigma-Aldrich, St. Louis, MO, USA), incubated at 37 °C for 72 h and then centrifuged at 15,000×rpm for 15 min. The supernatant was measured for absorbance at 620 nm by spectrophotometry. The extravasation was expressed as μg of Evans blue per g of wet tissue weight.
Preparation of Brain Samples for ELISA and Biochemical Analysis
ELISA Analysis of IL-1β, TNF-α, and IL-8 Levels
The levels of TNF-α, IL-1β, and IL-8 using commercially available rat enzyme-linked immunosorbent assay (ELISA) kits (TNF-α, IL-1β from R&D systems; IL-8 from BlueGene) according to the manufacturer’s instructions. All data are expressed as pg of cytokine per mg protein.
NO Levels and iNOS Activity Assay
NO levels and iNOS activity were evaluated using the assay kits from Jiancheng-Bioeng Ins. (China) according to the manufacturer′s instruction. Protein concentration was determined by the Coomassie blue protein-binding assay using bovine serum albumin as a standard.
Western Blot Analysis
To investigate the effects of xanthotoxol on the expressions of iNOS, COX-2, and NF-κB p65 protein in the ischemic core area of the cortex, rats (n = 4 per group) were decapitated at 24 h after reperfusion. Isolated brain tissues were homogenized in an ice-cold Tris buffer (50 mmol/l Tris, pH 7.4, 150 mmol/l NaCl, 0.5 % Triton X-100, 1 mmol/l edetic acid, 1 mol/l phenylmethylsulfonyl fluoride (PMSF), and 5 mg/l aprotinin), centrifuged at 12,000×rpm at 4 °C for 30 min. The supernatant was used for western blot analyses of iNOS and COX-2 expressions. In the case of the NF-κB subunit p65, analyses were carried out on nuclear extracts. In brief, total proteins from the cerebral cortical tissue were resuspended in buffer solution (10 mmol/l HEPES, pH7.5, 10 mmol/l KCl, 2 mmol/l MgCl2, 1 mmol/l vanadate, 1 mmol/l PMSF, 0.5 mmol/l DTT, 1 μg/ml aprotinin, and 10 μg/ml leupeptin) and centrifuged at 14,000×rpm for 10 min. The pelleted nuclei were incubated with extraction buffer (20 mmol/l HEPES, pH 7.9, 0.2 mmol/l EDTA, 1.5 mmol/l MgCl2, 300 mmol/l NaCl, 1 mmol/l EGTA, 1 mmol/l DTT, 0.5 mmol/l PMSF, 20 % glycerol, 5 μg/ml aprotinin, and 2.5 μg/ml leupeptin) on ice for 30 min followed by centrifugation at 15,000×rpm for 20 min at 4 °C, and the supernatant was used as nuclear extract.
Protein samples (15 μg) were separated on 8 % sodium dodecyl sulfate-polyacylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. The membrane was then blocked for unspecific binding for 2 h at 37 °C with phosphate-buffered saline (PBS) containing 0.1 % Tween-20 (PBST) and 5 % skimmed milk, and thereafter incubated overnight at 4 °C with primary antibodies: rabbit anti-iNOS, rabbit anti-COX-2, and rabbit anti- NF-κB p65 (1:500 dilution, Santa Cruz, USA) or mouse GAPDH antibody (1:8,000 dilution). After washing with PBST, the membranes were incubated with the secondary antibody (Goat Anti Rabbit IgG/HRP (1:10,000)) at room temperature for 1 h. The protein bands were visualized with ECL western blot detection reagents (Amersham, USA). The protein expression was normalized to GAPDH expression, and the normalized values are then expressed as the normalized folds with respect to sham-operated group.
Data are expressed as mean ± SD. Comparisons of multiple groups were performed using one-way analysis of variance (ANOVA) followed by the student–Newman–Keuls test. Statistical significant was determined when P < 0.05.
Effects of Xanthotoxol on the Brain Edema and Infarct Size Following I/R Injury
Effects of Xanthotoxol on BBB Permeability
Effects of Xanthotoxol on the Levels of Cytokines Following I/R Injury
Effects of Xanthotoxol on the iNOS Activity and NO Content in the Cortex Following I/R Injury
Effects of Xanthotoxol on the Expression of iNOS and COX-2 Protein in the Cortex Following I/R Injury
Effects of Xanthotoxol on the NF-κB p65 Levels in the Nucleus of Cortex Following I/R Injury
The results of this study show that xanthotoxol treatment is able to significantly attenuate the BBB disruption; decrease the levels of pro-inflammatory cytokines IL-1β, TNF-α, and chemokine IL-8; reduce the NO levels and iNOS activity; inhibit the protein expression of iNOS and COX-2 up-regulation; and suppress the nuclear translocation of NF-κB p65 after focal cerebral ischemia–reperfusion injury.
It is well known that within hours after the ischemic insult, the levels of pro-inflammatory mediators, such as IL-1β, TNF-α, and IL-8 markedly increased in the ipsilateral hemisphere of the brain. Subsequently, these mediators enhance the expressions of the ICAM-1 and E-selectin on endothelial cells and leukocytes, and facilitate the adhesion and transendothelial migration of leukocytes. IL-8 can also activate neutrophil infiltration. By these mechanisms, acute cerebral ischemia triggers the inflammatory cascade which causes dysfunction of BBB, brain edema, and further neuronal injury. Accordingly, inhibiting the expressions of IL-1β, TNF-α, and IL-8 appears to reduce ischemic brain damage (Khan et al. 2004; Villa et al. 2007). Consistent with these previous findings, we found here that cerebral ischemia markedly increased the production of these pro-inflammatory mediators which were effectively suppressed by treatment with xanthotoxol. Therefore, we presume that the suppression of pro-inflammatory mediators may explain the inhibition in ischemia-induced cell adhesion molecules, up-regulation, neutrophil infiltration, and edema formation previously observed following xanthotoxol treatment.
NO synthesized by iNOS modulates inflammatory responses and is involved in the regulation of immune reactions. Overproduction of NO by iNOS has been implicated in the pathological processes of cerebral inflammation and tissue damage caused by ischemic stroke (Toda et al. 2009). Inhibition of iNOS expression has been shown to protect the brain damage by reducing levels of NO and subsequently decreasing the generation of ONOO− after cerebral ischemia–reperfusion injury (Danielisova et al. 2011; Khan et al. 2004). Our present study shows that xanthotoxol significantly attenuates ischemia-induced NO levels, increase in the lesioned hemisphere. Furthermore, compared with sham-operated group controls, both iNOS activity and protein expression were increased after cerebral ischemia insult. However, treatment with xanthotoxol resulted in a significant inhibition of both the activity and protein expression of iNOS.
COX-2 is the key enzyme involved in arachidonic acid metabolism and plays an important role in inflammatory reaction (Vane et al. 1998). It has been demonstrated that cerebral ischemia can lead to an increase in the expression and activity of the COX-2, an enzyme reaction products of which contribute to the evolution of ischemic brain injury (Nogawa et al. 1997). In addition, the experimental evidences suggest that NO produced by iNOS activates COX-2 and increases the cytotoxic effects of this enzyme after focal cerebral ischemia (Nogawa et al. 1998; Salvemini et al. 1993). It has also been reported that COX-2 selective inhibitor protects against ischemic brain damage, especially reduces BBB disruption, leukocyte infiltration, and brain edema following transient focal cerebral ischemia (Candelario-Jalil et al. 2007). In the present study, xanthotoxol significantly reduced COX-2 up-regulations induced by cerebral ischemia. These results suggest that neuroprotective effects of xanthotoxol after cerebral ischemia might be attributable to interrupting inflammatory response by the inhibition of COX-2 expression.
The transcription factor NF-κB is a key regulator of the expression of downstream target genes that are involved in cell survival and inflammation (Tas et al. 2009). It has been demonstrated that neuronal activation of NF-κB during cerebral ischemia contributes to the ischemic tissue damage (Zhang et al. 2005). The ischemia-induced NF-κB activation induces the expression of many pro-inflammatory mediators, such as cytokines (e.g., IL-1β and TNF-α), chemokines IL-8, adhesion molecules (e.g., ICAM, VCAM, and E-selectin), and inducible effector enzymes (e.g., iNOS and COX-2). The present study shows that the protein level of NF-κB p65 in the nucleus at 24 h after reperfusion in the ipsilateral hemisphere cortex was increased significantly after I/R injury compared with sham-operated group. However, treatment with xanthotoxol significantly prevented this increase in NF-κB p65 level induced by I/R injury. On the basis of these data, we suggest that xanthotoxol can suppress the expression of the NF-κB p65 subunit in the nucleus of the ipsilateral hemisphere cortex, thereby leading to a reduction in the production of pro-inflammatory mediators and decrease in the inflammation after cerebral ischemic insult.
In conclusion, the present study, together with our previous results (He et al. 2009), demonstrates that xanthotoxol exerts neuroprotective effects on the cerebral ischemia–reperfusion injury, and this effect is related to the anti-inflammatory action by suppression of the production of pro-inflammatory mediators following cerebral ischemia.
This study was supported by Grants (to Wei He) from the National Natural Science Foundation of China (NSFC, 81060269) and from the Science and Technology Foundation of Educational Department of Jiangxi Province of China (GJJ08391).