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Resveratrol relieves Angiostrongylus cantonensis – Induced meningoencephalitis by activating sirtuin-1
Acta Tropica 173 (2017) 76–84
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Resveratrol relieves Angiostrongylus cantonensis – Induced meningoencephalitis by activating sirtuin-1 An-Chih Chena,1, Ling-Yuh Shyub,1, Yue-Loong Hsinc, Ke-Min Chenb, Shih-Chan Laib, a b c
MARK ⁎
Department of Neurology, Chung-Shan Medical University Hospital, Taichung 402, Taiwan Department of Parasitology, Chung Shan Medical University, Taichung 402, Taiwan Neurology Department, Chung Shan Medical University, Taichung, Taiwan
A R T I C L E I N F O
A B S T R A C T
Keywords: Angiostrongylus cantonensis Meningoencephalitis Resveratrol Sirtuin-1
Resveratrol, a natural herbal compound found in high levels in grapes and red wine, is frequently used as activator of sirtuin-1. This study investigated the potential function of sirtuin-1 in regulating angiostrongyliasis meningoencephalitis in resveratrol-treated mice. Mice were subjected to meningoencephalitis to study the protective effect of resveratrol against meningoencephalitis and investigate the effects of sirtuin-1 activation on brain. Results demonstrated that sirtuin-1 level decreased in mice with meningoencephalitis and significantly increased in resveratrol-treated mice. Moreover, resveratrol treatment significantly reduced eosinophil counts, p65, Interferon-γ, interleukin (IL)-5, IL-33, and tumor necrosis factor-α levels, matrix metalloproteinase-9 activity, claudin-5 degradation, and blood–brain barrier permeability. By contrast, the anti-inflammatory factor IL10 was significantly increased in resveratrol-treated mice. Resveratrol treatment was partially beneficial in controlling the pathological processes of angiostrongyliasis meningoencephalitis. The results demonstrate the neuroprotective and anti-inflammatory effects of resveratrol against Angiostrongylus cantonensis-induced eosinophilic meningoencephalitis in mice. Treatment with sirtuin-1 agonist was given within a therapeutic window after A. cantonensis infection.
1. Introduction Angiostrongylus cantonensis (rat lungworm) is a parasitic nematode that causes severe dysfunction of the central nervous system (CNS) and induces eosinophilic meningitis or meningoencephalitis (Mackerras and Sandars, 1954; Hsu et al., 1990). The levels of pro-inflammatory biomarkers, such as interleukin (IL)-1β, IL-5, tumor necrosis factor (TNF)α, and matrix metalloproteinase (MMP)-9, are significantly increased in A. cantonensis-infected mice (Chen and Lai, 2007). Blood–CSF barrier dysfunction is associated with degradation of tight junction proteins and extracellular matrix as a result of MMP-9 activation in angiostrongyliasis meningoencephalitis (Chiu and Lai, 2013; Shyu et al., 2015). When infected with A. cantonensis, mice manifest marked systemic and CSF eosinophilia, reaching a peak level at around day 20. Eosinophil is cytotoxic in A. cantonensis infection, and a prominent infiltration of eosinophils occurs around the worm surface (Sugaya and Yoshimura, 1988; Tseng et al., 2004). Moreover, MMP-9 leads to blood–brain barrier (BBB) leakage in mice with A. cantonensis-induced eosinophilic meningoencephalitis (Chiu and Lai, 2014). Protein acetylation is a post-translational modification and an ⁎
1
important transcriptional regulatory mechanism. This process is controlled by histone acetyltransferases and histone deacetylases (Glozak et al., 2005). Sirtuin-1 is a member of sirtuins, which constitute the class III histone deacetylases (Milne and Denu, 2008). The neuroprotective role of sirtuin-1 has been demonstrated in models of various neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease (Jeong et al., 2011; Donmez et al., 2012). Resveratrol (3,5,4ʹ-trihydroxy-trans-stilbene), a natural agonist of sirtuin-1, is a naturally occurring phytoalexin that is present in skin of red grapes and a component of red wine (Nakata et al., 2012). Resveratrol acts as an activator of sirtuin-1 and demonstrates neuroprotective effects in various models (Bradamante et al., 2004; Meng et al., 2015). Accumulated evidence has documented that resveratrol exerts anti-neuroinflammation action by downregulating various pro-inflammatory biomarkers, such as IL-1β and TNF-α in neurodegenerative diseases (Foti Cuzzola et al., 2011). Moreover, resveratrol treatment is partially beneficial in controlling pathologic processes in Chagas disease (Wan et al., 2016). Therefore, we hypothesized that resveratrol can attenuate inflammation by upregulating sirtuin-1 in mice with eosinophilic meningoencephalitis.
Corresponding author at: Department of Parasitology, Chung Shan Medical University, 110, Section 1, Chien-Kuo North Road, Taichung 402, Taiwan. E-mail address: [email protected] (S.-C. Lai). An-Chih Chen and Ling-Yuh Shyu contributed equally to this work and both are first author.
http://dx.doi.org/10.1016/j.actatropica.2017.05.023 Received 17 January 2017; Received in revised form 17 May 2017; Accepted 19 May 2017 Available online 22 May 2017 0001-706X/ © 2017 Elsevier B.V. All rights reserved.
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Fig. 1. Effects of resveratrol on sirtuin-1 levels. (a) Time-course studies on sirtuin-1 levels in mouse brain. β-actin was used as loading control. (b) Quantification and normalization to β-actin showed that sirtuin-1 level was significantly reduced (*P < 0.05) on days 15, 20, and 25 after Angiostrongylus cantonensis infection. (c) Influence of resveratrol treatment on sirtuin-1 after A. cantonensis infection. (d) Quantification and normalization to βactin showed that sirtuin-1 level significantly increased (#P < 0.05) in A. cantonensis-infected mice (Infected) compared with that in uninfected controls. Mice treated with resveratrol (Res) alone, albendazole (ABZ) alone, or resveratrol-albendazole (Res + ABZ) significantly increased (*P < 0.05) the sirtuin-1 level compared with that in untreated A. cantonensis-infected mice. Bars represent the mean ± S.D. of three independent experiments performed in duplicate.
Fig. 2. Effects of resveratrol treatment on p65 levels in mouse brain. Protein levels of p-p65 (a) and p65 (c). β-actin was used as loading control. Quantification and normalization to β-actin showed that p-p65 (b) and p65 (d) were significantly increased (#P < 0.05) in Angiostrongylus cantonensisinfected mice (Infected) compared with that in uninfected controls. Treatment with resveratrol (Res) alone, albendazole (ABZ) alone, or resveratrol-albendazole (Res + ABZ) significantly reduced the levels of p-p65 and p65 (*P < 0.05) compared with those in untreated A. cantonensis-infected mice. Bars represent the mean ± S.D. of three independent experiments performed in duplicate.
2. Materials and methods
The present study investigated the deacetylation mechanisms during A. cantonensis infection. We aimed to determine whether treatment with sirtuin-1 agonist will be beneficial in improving the inflammatory response in angiostrongyliasis meningoencephalitis, providing clinical evaluation of the therapeutic strategy against A. cantonensis-induced eosinophilic meningoencephalitis.
2.1. Experimental animals Male BALB/c mice (specific pathogen-free grade and 5-week old) were purchased from the National Laboratory Animal Center, Taipei, Taiwan. The mice were maintained in an alternating 12h–12 h light77
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Fig. 3. Effects of resveratrol on the levels of IFN-γ, IL-5, IL-10, IL-33, and TNF-α. Concentrations of (a) IFN-γ, (b) IL-5, (c) IL-10, (d) IL-33, and (e) TNF-α were significantly increased (#P < 0.05) in Angiostrongylus cantonensis-infected mice (Infected) compared with those in uninfected controls. Concentrations of IFN-γ, IL-5, IL-33, and TNF-α in mice treated with resveratrol (Res) alone, albendazole (ABZ) alone, or resveratrol-albendazole (Res + ABZ) were significantly lower (*P < 0.05) than those in untreated A. cantonensis-infected mice. By contrast, IL-10 was significantly increased in mice treated with resveratrol alone (Res), albendazole alone (ABZ), or resveratrol-albendazole (Res + ABZ) compared with that in meningoencephalitis mice. Bars represent the mean ± S.D. of three independent experiments performed in duplicate.
dark cycle and provided with Purina Laboratory Chow and water ad libitum. The mice were kept in our laboratory for more than one week prior to experimental infection. All procedures involving animal use and care were approved by the Institutional Animal Care and Use Committee of Chung-Shan Medical University, Taiwan and performed in accordance with the institutional guidelines for animal experiments.
purchased from Sigma (St. Louis, MO, USA). Goat anti-mouse albumin polyclonal antibody was purchased from Bethyl Laboratories (Montgomer, USA). Horseradish peroxidase (HRP)-conjugated antirabbit IgG, HRP-conjugated anti-goat IgG, and HRP-conjugated antimouse IgG were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).
2.2. Antibodies
2.3. Larval preparation
The anti-mouse monoclonal antibody p-p65 and p65 generated in rabbit was purchased from Cell Signaling Technology (Beverly, MA). Goat anti-mouse claudin-5 polyclonal antibody and rabbit anti-mouse sirtuin-1 polyclonal antibody were purchased from Santa Cruz Biotechnology (CA, USA). Anti-mouse β-actin monoclonal antibody was
Infective larvae (L3) of A. cantonensis were originally isolated from wild giant African snails (Achatina fulica). The snails were bred for several months and then infected with A. cantonensis L1 by cycling through rats (definitive host) in Wufeng Experimental Farm (Taichung, Taiwan). Infective larvae (L3) were recovered by using the method 78
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Fig. 4. Effects of resveratrol on activity of matrix metalloproteinase (MMP)-9. MMP-9 bands (94 kDa) were detected in mouse (a) brains and (b) CSF. Quantitative analysis showed that MMP-9 levels were significantly increased (#P < 0.05) in Angiostrongylus cantonensis-infected mice (Infected) compared with those in uninfected controls. Mice treated with resveratrol (Res) alone, albendazole (ABZ) alone, or resveratrol-albendazole (Res + ABZ) showed significantly lower (*P < 0.05) MMP-9 than the untreated A. cantonensis-infected mice. Bars represent the mean ± S.D. of three independent experiments performed in duplicate.
2.5. Treatment of animals
developed by Parsons and Grieve (1990) with modification. The snail shells were crushed and then the tissues were homogenized with pepsin-HCl solution (pH of 1–2, 500 IU pepsin/g tissue) and digested through agitation at 37 °C in a water bath for 2 h. The sediments were clarified through series of washing with double-distilled water to clearly see the larvae, which were counted under a microscope. The morphological criteria of A. cantonensis L3 were confirmed according to the method described by Hou et al. (2004).
A total of 72 mice were randomly divided into six groups (12 mice/ group). Food and water were withheld for 12 h before infection. The uninfected control mice were orally administered with distilled water only on day 10 PI. All other groups were infected with 30 A. cantonensis larvae, including the untreated infected control mice, which were orally treated with distilled water only on day 10 PI. The experimental mice were separately treated with DMSO alone (10 mg/kg/day), resveratrol alone (10 mg/kg/day) albendazole alone (10 mg/kg/day), and albendazole-resveratrol (10 mg each/kg/day) for 7 consecutive days starting on day 10 PI. All groups were sacrificed on day 22 PI; the brains and CSF of the mice were collected for biochemical analysis.
2.4. Animal infection A total of 30 male BALB/c mice were randomly allocated into six groups (control, D5, D10, D15, D20, and D25), each group contains five mice. Food and water were withheld for 12 h before infection. The mice in five experimental groups (D5, D10, D15, D20, and D25) were infected with 30 A. cantonensis larvae via oral inoculation and then sacrificed on days 5, 10, 15, 20, or 25 post-inoculation (PI). Control mice received water only and sacrificed on day 25 PI. Brains of the mice were quickly removed and frozen at −80 °C.
2.6. CSF collection The mice were anesthetized intraperitoneally with urethane (1.25 g/kg). Each mouse was placed in a stationary instrument where the body was laid 135° relative to the head. The skin on the neck was shaved and swabbed three times with 70% ethanol. The subcutaneous tissues and muscles were separated. A capillary tube was inserted 79
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Fig. 7. Effects of resveratrol on eosinophil counts. Eosinophil counts significantly increased (#P < 0.05) in Angiostrongylus cantonensis-infected mice (Infected) compared with those in uninfected controls. Mice treated with resveratrol (Res) alone, albendazole (ABZ) alone, or resveratrol-albendazole (Res + ABZ) showed significantly lower (*P < 0.05) eosinophil than the untreated A. cantonensis-infected mice. Bars represent the mean ± S.D. of three independent experiments performed in duplicate.
Fig. 5. Effects of resveratrol on BBB permeability. Mouse BBB permeability was detected by performing extravasation of Evans blue during Angiostrongylus cantonensis infection. Evans blue dye units were significantly increased (#P < 0.05) in A. cantonensis-infected mice (Infected) compared with those in uninfected controls. Mice treated with resveratrol (Res) alone, albendazole (ABZ) alone, or resveratrol-albendazole (Res + ABZ) showed significantly lower (*P < 0.05) Evans blue dye units than the untreated A. cantonensisinfected mice. Bars represent the mean ± S.D. of three independent experiments performed in duplicate.
through the dura mater into cisterna magna and then CSF was poured into the capillary tube. The CSF was subsequently injected into a 0.5 mL Eppendorf tube and centrifuged at 3000g for 5 min at 4 °C. The supernatants were collected in a new 0.5 mL Eppendorf tube and then frozen at −80 °C.
2.7. Western blot analysis The mouse brains were homogenized with RIPA lysis buffer (150 mM sodium chloride, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS, and 50 mM Tris, pH 7.5) that contains a protease inhibitor cocktail (Sigma, MO, USA). The homogenates were centrifuged at 12,000g for 10 min at 4 °C. Protein concentration was determined by using protein assay kits (Bio-Rad, USA). The homogenates were mixed with an equal volume of sample buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 5% 2-mercaptoethanol, and 0.05% bromophenol blue) and boiled at 95 °C for 5 min. The mixtures were electrophoretically separated on SDS-polyacrylamide gels (PAGE) and then transferred onto polyvinyl difluoride membranes. The membranes were blocked with 5% non-fat milk in PBS (containing 0.1% Tween 20 and PBST) for 1 h at room temperature and then incubated with primary antibodies at 37 °C for 1 h. Subsequently, the membranes were washed three times with PBST followed by incubation with HRP-conjugated secondary antibody (1:10,000 dilution) at 37 °C for 1 h. Reactive protein bands were visualized by performing enhanced chemiluminescence (Amersham, Little Chalfont, Bucks, UK). Bands were quantitatively analyzed using a computer-assisted imaging densitometer system (UN-SCAN-ITTM gel Version 5.1, Silk Scientific, USA). Densitometric scanning result was presented as the ratio of the band density of target protein to that of the loading control (β-actin or albumin).
Fig. 6. Effects of resveratrol on claudin-5 levels in CSF. (a) Protein bands of claudin-5 obtained from mice CSF. Albumin was used as loading control. (b) Claudin-5 level was significantly increased (#P < 0.05) in Angiostrongylus cantonensis-infected mice (Infected) compared with that in uninfected controls. Mice treated with resveratrol (Res) alone, albendazole (ABZ) alone, or resveratrol-albendazole (Res + ABZ) showed a significantly lower (*P < 0.05) claudin-5 level than the untreated A. cantonensis-infected mice. Bars represent the mean ± S.D. of three independent experiments performed in duplicate.
2.8. Cytokine assay Production of cytokines (IFN-γ, IL-5, IL-10, IL-33, and TNF-α) in CSF was assayed using commercial enzyme-linked immunosorbent assay (ELISA) kits (Biosource, Camarillo, CA, USA) according to the manufacturer’s instructions. CSF samples for cytokine assays were pooled given that the amounts of CSF harvested from each mouse were frequently insufficient for individual assays.
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Fig. 8. Light microscopic observations. (a) Brain of uninfected mice; no Angiostrongylus cantonensis nor hemorrhage was observed in the brain. (b) Brain of untreated infected mice; young adult worms (arrowheads) and hemorrhage were evident. (c) Brain of DMSO-treated mice; young adult worms (arrowheads) and hemorrhage can be observed. (d) Brain of resveratrol-treated mice; the brain shows a significantly reduced amount of hemorrhage. Arrowheads indicate young adult worms. (e) Brain of albendazole-treated mice; no young adult worms were found in the brain and the amount of hemorrhage was significantly reduced. (f) Brain of mice treated with the combination of resveratrol and albendazole; no young adult worms were found in the brain and the amount of hemorrhage was significantly reduced.
Fig. 9. Possible mechanisms of the anti-inflammatory and protective pathways of resveratrol in mice infected with Angiostrongylus cantonensis. We investigated the potential function of sirtuin-1 in regulating angiostrongyliasis meningoencephalitis in resveratrol-treated mice. NF-κB inhibition could downregulate the production of pro-inflammatory factors (IFN-γ, IL-5, IL-33, TNF-α, and MMP-9) in A. cantonensis-infected mice. By contrast, NF-κB inhibition could upregulate the anti-inflammatory factor IL-10. Blocking the NF-κB signaling by resveratrol could reduce claudin-5 degradation and eosinophil production in angiostrongyliasis meningoencephalitis. Therefore, we suggest that this parasitic infection disrupts three layers of BBB to leukocyte infiltration by MMP-9 activities during infection. These processes are associated with inflammatory response in BBB destruction caused by A. cantonensis. The severity of A. cantonensis-induced meningoencephalitis in resveratrol-treated mice were considerably reduced.
washed two times with double-distilled water (containing 2.5% Triton X-100) for 30 min. The gel was subsequently incubated in reaction buffer (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 10 mM CaCl2, 0.02% Brij-35, and 0.01% NaN3) at 37 °C for 18 h. The gel was stained with 0.25% Coomassie Brilliant Blue R-250 (Sigma, USA) for 1 h and then
2.9. Gelatin zymography CSF was separated on 7.5% SDS-PAGE with 0.1% gelatin (Sigma, MO, USA). Electrophoresis was performed in running buffer (25 mM Tris, 250 mM glycine, and 1% SDS) at room temperature. Each gel was 81
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destained in 15% methanol/7.5% acetic acid. Gelatinase activity was visualized as clear bands against a blue-stained background. Quantitative analysis was performed using a computer-assisted imaging densitometer system (UN-SCAN-IT™ gel Version 5.1, Silk Scientific, USA).
higher in mice treated with albendazole alone, resveratrol alone, or resveratrol-albendazole co-treatment than in untreated A. cantonensisinfected mice (Fig. 3).
2.10. Evaluation of BBB permeability
Gelatin zymography measures the activity of MMP-9, which migrates electrophoretically as 94 kDa species. This metalloproteinase activity was significantly increased in CSF and brain of mice infected with A. cantonensis. MMP-9 activity was significantly reduced in mice treated with resveratrol alone, albendazole alone, or resveratrol-albendazole (Fig. 4).
3.4. Influence of resveratrol on MMP-9 activity
BBB permeability was assessed based on the concentrations of Evans blue in brains. Mice were injected with 2% (wt/vol) Evans blue dye (5 mL/kg body weight; Sigma, St. Louis, MO, USA) in saline via the tail vein. After 2 h of circulation, the mice were anesthetized and transcardially perfused with saline to remove intravascular dye. The brains were weighed and homogenized in 50% trichloroacetic acid solution. The homogenates were centrifuged at 12,000g for 10 min, and the supernatants were collected. Each supernatant was measured at 620 nm for absorbance to calculate the Evans blue concentrations by using a spectrophotometer (Hitachi U3000, Tokyo, Japan).
3.5. Influence of resveratrol on BBB permeability BBB disruption was estimated through Evans blue extravasation in mouse model. Concentration of Evans blue is an indicator of BBB leakage during A. cantonensis infection in mice. BBB permeability was significantly increased in A. cantonensis-infected mice compared with that in uninfected mice. Furthermore, BBB permeability was significantly attenuated in mice treated with resveratrol, albendazole, or resveratrol-albendazole (Fig. 5).
2.11. Eosinophil counts in CSF CSF samples were mixed with Unopette buffer (Vacutainer System, Becton Dickinson, Franklin Lakes, NJ, USA) containing 2% acetic acid. The mixture was placed in a hemocytometer cell counting chamber (Paul Marienfeld, Lauda-Koenigshofen, Germany) to count the eosinophils.
3.6. Influence of resveratrol on claudin-5 degradation Claudin-5 degradation in CSF is an indicator of damaged tight junction in BBB during A. cantonensis infection in mice. Western blot analysis indicated that the protein levels of claudin-5 increased significantly in CSF of infected mice compared with those in uninfected controls. The levels of protein claudin-5 decreased significantly after treatment with resveratrol alone, albendazole alone, or resveratrol-albendazole (Fig. 6).
2.12. Statistical analysis Results among different mouse groups were compared by performing non-parametric Kruskal–Wallis test followed by post-testing using Dunn’s multiple comparison of means. All results were presented as mean ± standard deviation (S.D.). P values less than 0.05 indicated statistical significance.
3.7. Influence of resveratrol on eosinophil counts
3. Results
The amount of CSF eosinophils significantly increased in mice infected with A. cantonensis compared with that in uninfected controls. Treatment with resveratrol alone, albendazole alone, or resveratrol-albendazole significantly reduced eosinophil counts, whereas DMSO treatment exerted no effect on eosinophil count (Fig. 7).
3.1. Influence of resveratrol on sirtuin-1 The effect of resveratrol was investigated in a murine angiostrongyliasis model. Western blot analysis showed that protein levels of sirtuin-1 decreased significantly in the brains of A. cantonensis-infected mice compared with those in the brains of uninfected controls. Furthermore, the protein levels of sirtuin-1 increased significantly after treatment with resveratrol alone, albendazole alone, or resveratrol-albendazole (Fig. 1).
3.8. Pathological observation of brain surface Light microscopic examination revealed that the brain of uninfected mice showed normal blood vessel, whereas the brain of untreated infected mice showed hemorrhage of enlarged blood vessels and young adult worms. Treatment with resveratrol alone significantly reduced the amount of hemorrhage. Furthermore, treatment with albendazole alone or resveratrol-albendazole significantly reduced the number of young adult worms and amount of hemorrhage (Fig. 8).
3.2. Influence of resveratrol on p65 Western blot analysis showed that the protein levels of p-p65 and p65 increased significantly in the brain of A. cantonensis-infected mice compared with those in uninfected controls. The protein levels of p-p65 and p65 decreased significantly after treatment with resveratrol alone, albendazole alone, or resveratrol-albendazole (Fig. 2).
4. Discussion Sirtuin-1, which can be activated by resveratrol, is highly expressed in the cortex, hippocampus, cerebellum, and hypothalamus (Ramadori et al., 2008). Activated sirtuin-1 is essential in the anti-inflammatory effects of resveratrol, and such role of sirtuin-1 may be partially attributed to inhibition of NF-κB (Busch et al., 2012). The NF-κB family of transcription factors plays a central role in inflammation and immunity. By inhibiting NF-κB, resveratrol downregulates the activation of immune cells and the subsequent synthesis and release of pro-inflammatory mediators. In vitro studies have shown that sirtuin-1 limits inflammation by inhibiting NF-κB and the transcription factor of NF-κB involved in expression of proinflammatory cytokines, such as IFN-γ and TNF-α (Yeung et al., 2004; Gao and Ye, 2008). P65 is an important subunit of an activated NF-κB dimer. We postulated that through
3.3. Influence of resveratrol on cytokines To determine the production of cytokines and their possible role in mouse resistance to A. cantonensis, we used sandwich ELISAs to assay IFN-γ, IL-5, IL-10, IL-33, and TNF-α in CSF. The concentrations of IFN-γ, IL-5, IL-33, and TNF-α were significantly higher in A. cantonensis-infected mice than in uninfected mice, where low levels of these cytokines were found. Compared with the corresponding concentrations of these cytokines in untreated infected mice, those in mice treated with albendazole alone, resveratrol alone, or resveratrol-albendazole were significantly reduced. By contrast, IL-10 concentration was significantly 82
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In conclusion, A. cantonensis infection in mice leads to overproduction of pro-inflammatory factors and causes meningoencephalitis, which is prevented by resveratrol treatment. The anti-inflammatory effects of resveratrol contribute to the improvement of meningoencephalitis in A. cantonensis-infected mice. The treatment with sirtuin-1 agonist, given in a therapeutic window of time after A. cantonensis infection.
reduced p-p65 activity, sirtuin-1 agonist is a therapeutic option that can improve the inflammatory factors and subsequently the angiostrongyliasis meningoencephalitis. To determine the anti-inflammatory role of sirtuin-1 beyond mouse brains, we investigated the proinflammatory, as well as the anti-inflammatory cytokine profiles, in mouse model. Our observation that the levels of proinflammatory cytokines (IFN-γ, IL-5, IL-33, and TNF-α) are lower while those of the anti-inflammatory cytokine IL-10 are higher in resveratrol-treated animals than in wild-type meningoencephalitis animals is consistent with the trend in immunomodulatory function. Our result demonstrated that the sirtuin-1 activator resveratrol suppressed IL-5 production and reduced the eosinophilic inflammatory cell infiltration in mouse model of A. cantonensis-induced meningoencephalitis. The reduced IL-5 level may explain the inhibition of eosinophilic inflammatory cell infiltration. These findings demonstrate a link between sirtuin-1 and cytokines in inflammatory responses during A. cantonensis infection and meningoencephalitis, strongly suggestive of the anti-inflammatory function of sirtuin-1. Sirtuin-1 activation is a key process in neuroprotective action of resveratrol (Ramadori et al., 2008; Feng et al., 2013). Our results reveal that overexpression of CNS sirtuin-1 in mice with meningoencephalitis was significantly reduced in the brain surface and reduced the infiltration of eosinophils into the subarachnoid space. These results demonstrate that sirtuin-1 is a potent anti-neuroinflammatory agent and strongly indicate its potential usefulness as therapeutic intervention for meningoencephalitis afflicting the CNS. Specifically, data showing that sirtuin-1 reduces eosinophil infiltration within the subarachnoid space in meningoencephalitis mouse model suggest a mechanism wherein diminishing the persistent eosinophil infiltration might reduce ongoing neuronal damage. Evan blue dye is widely used to detect BBB leakage as this dye conjugates with serum albumin to form a large molecular complex incapable of crossing an intact BBB under normal physiological circumstances (Wolman et al., 1981). We therefore investigated the beneficial effects of resveratrol in protecting the integrity of the BBB in angiostrongyliasis meningoencephalitis. Our study showed that Evan blue leakage was markedly increased in mice with meningoencephalitis compared with that in control mice, and this phenomenon was associated with increased degradation of the tight junction protein claudin5. Evan blue content in the brain was significantly reduced in resveratrol-treated mice compared with that in untreated mice displaying meningoencephalitis. Moreover, in experimental autoimmune encephalomyelitis mouse model, resveratrol exhibits protective effects in an experimental autoimmune encephalomyelitis therapy; such effect was attained through the anti-inflammation and antioxidant activities of resveratrol, in which it protects the tight junction proteins in the basement membrane to improve BBB integrity (Wang et al., 2016). These results collectively indicated that resveratrol could prevent disruption of BBB in meningoencephalitis mouse model. In this study, sirtuin-1 expression was upregulated in resveratroltreated mice. The results clearly indicate that sirtuin-1 overexpression improves the pathological processes of meningoencephalitis. Therefore, we propose a possible mechanism (Fig. 9) by which resveratrol relieves A. cantonensis-induced meningoencephalitis through sirtuin-1 activation. Meningoencephalitis is often accompanied by BBB disruption and crossing of plasma components, such as eosinophils, through the compromised BBB, aggravating inflammation and increasing the production of neurotoxins that can ultimately result in neuron death. Resveratrol downregulates the synthesis of pro-inflammatory mediators and the subsequent activation of eosinophils through NF-κB inhibition. Moreover, resveratrol inhibits the activation of MMP-9, significantly reducing leakage of Evans blue and degradation of tight junction protein, consequently improving the outcomes of meningoencephalitis in mice. Sirtuin-1 overexpression improves meningoencephalitis processes possibly by reducing the activation of pro-inflammatory cytokines, proteolytic enzyme MMP-9, and eosinophil infiltration.
Acknowledgements We wish to thank Ping-Sung Chiu of the Department of Parasitology, Chung Shan Medical University for providing an invaluable assistance in the conduct of this study. This work was supported by the Intramural Research Program of Chung Shan Medical University Hospital, Taichung, Taiwan (CSH-2016-C-021) and in part by the Ministry of Science and Technology, Taiwan (Grant No. MOST 103-2320-B-040023-MY3). References Bradamante, S., Barenghi, L., Villa, A., 2004. Cardiovascular protective effects of resveratrol. Cardiovasc. Drug Rev. 22, 169–188. Busch, F., Mobasheri, A., Shayan, P., Lueders, C., Stahlmann, R., Shakibaei, M., 2012. Resveratrol modulates interleukin-1β-induced phosphatidylinositol 3-kinase and nuclear factor (B signaling pathways in human tenocytes. J. Biol. Chem. 287, 38050–38063. Chen, K.M., Lai, S.C., 2007. Biochemical and pathological evaluation of albendazole/ thalidomide co-therapy against eosinophilic meningitis or meningoencephalitis induced by Angiostrongylus cantonensis. J. Antimicrob. Chemother. 59, 264–276. Chiu, P.S., Lai, S.C., 2013. Matrix metalloproteinase-9 leads to claudin-5 degradation via the NF-κB pathway in BALB/c mice with eosinophilic meningoencephalitis caused by Angiostrongylus cantonensis. PLoS One 8, e53370. Chiu, P.S., Lai, S.C., 2014. Matrix metalloproteinase-9 leads to blood-brain barrier leakage in mice with eosinophilic meningoencephalitis caused by Angiostrongylus cantonensis. Acta Trop. 140, 141–150. Donmez, G., Arun, A., Chung, C.Y., McLean, P.J., Lindquist, S., Guarente, L., 2012. SIRT1 protects against alpha-synuclein aggregation by activating molecular chaperones. J. Neurosci. 32, 124–132. Feng, X., Liang, N., Zhu, D., Gao, Q., Peng, L., Dong, H., Yue, Q., Liu, H., Bao, L., Zhang, J., Hao, J., Gao, Y., Yu, X., Sun, J., 2013. Resveratrol inhibits β-amyloid-induced neuronal apoptosis through regulation of SIRT1-ROCK1 signaling pathway. PLoS One 8, e59888. Foti Cuzzola, V., Ciurleo, R., Giacoppo, S., Marino, S., Bramanti, P., 2011. Role of resveratrol and its analogues in the treatment of neurodegenerative diseases: focus on recent discoveries. CNS Neurol. Disord. Drug Targets 10, 849–862. Gao, Z., Ye, J., 2008. Inhibition of transcriptional activity of c-JUN by SIRT1. Biochem. Biophys. Res. Commun. 376, 793–796. Glozak, M.A., Sengupta, N., Zhang, X., Seto, E., 2005. Acetylation and deacetylation of non-histone proteins. Gene 363, 15–23. Hou, R.F., Tu, W.C., Lee, H.H., Chen, K.M., Chou, H.L., Lai, S.C., 2004. Elevation of plasminogen activators in cerebrospinal fluid of mice with eosinophilic meningitis caused by Angiostrongylus cantonensis. Int. J. Parasitol. 34, 1355–1364. Hsu, W.Y., Chen, J.Y., Chien, C.T., Chi, C.S., Han, N.T., 1990. Eosinophilic meningitis caused by Angiostrongylus cantonensis. Pediatr. Infect. Dis. J. 9, 443–445. Jeong, H., Cohen, D.E., Cui, L., Supinski, A., Savas, J.N., Mazzulli, J.R., Yates J.R.3rd. Bordone, L., Guarente, L., Krainc, D., 2011. SIRT1 mediates neuroprotection from mutant huntingtin by activation of the TORC1 and CREB transcriptional pathway. Nat. Med. 18, 159–165. Mackerras, M.J., Sandars, D.F., 1954. Lifehistory of the rat lung-worm and its migration through the brain of its host. Nature 173, 956–957. Meng, Z., Li, J., Zhao, H., Liu, H., Zhang, G., Wang, L., Hu, H.E., Li, D.I., Liu, M., Bi, F., Wang, X., Tian, G., Liu, Q., Buren, B., 2015. Resveratrol relieves ischemia-induced oxidative stress in the hippocampus by activating SIRT1. Exp. Ther. Med. 10, 525–530. Milne, J.C., Denu, J.M., 2008. The Sirtuin family: therapeutic targets to treat diseases of aging. Curr. Opin. Chem. Biol. 12, 11–17. Nakata, R., Takahashi, S., Inoue, H., 2012. Recent advances in the study on resveratrol. Biol. Pharm. Bull. 35, 273–279. Parsons, J.C., Grieve, R.B., 1990. Effect of egg dosage and host genotype on liver trapping in murine larval toxocariasis. J. Parasitol. 76, 53–58. Ramadori, G., Lee, C.E., Bookout, A.L., Lee, S., Williams, K.W., Anderson, J., Elmquist, J.K., Coppari, R., 2008. Brain SIRT1: anatomical distribution and regulation by energy availability. J. Neurosci. 28, 9989–9996. Shyu, L.Y., Hu, M.E., Chou, C.H., Chen, K.M., Chiu, P.S., Lai, S.C., 2015. Fibronectin changes in eosinophilic meningitis with blood-CSF barrier disruption. Exp. Parasitol. 151–152, 73–79. Sugaya, H., Yoshimura, K., 1988. T-cell-dependent eosinophilia in the cerebrospinal fluid of the mouse infected with Angiostrongylus cantonensis. Parasite Immunol. 10, 127–138. Tseng, Y.K., Tu, W.C., Lee, H.H., Chen, K.M., Chou, H.L., Lai, S.C., 2004. Ultrastructural
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Resveratrol relieves Angiostrongylus cantonensis – Induced meningoencephalitis by activating sirtuin-1 An-Chih Chena,1, Ling-Yuh Shyub,1, Yue-Loong Hsinc, Ke-Min Chenb, Shih-Chan Laib, a b c
MARK ⁎
Department of Neurology, Chung-Shan Medical University Hospital, Taichung 402, Taiwan Department of Parasitology, Chung Shan Medical University, Taichung 402, Taiwan Neurology Department, Chung Shan Medical University, Taichung, Taiwan
A R T I C L E I N F O
A B S T R A C T
Keywords: Angiostrongylus cantonensis Meningoencephalitis Resveratrol Sirtuin-1
Resveratrol, a natural herbal compound found in high levels in grapes and red wine, is frequently used as activator of sirtuin-1. This study investigated the potential function of sirtuin-1 in regulating angiostrongyliasis meningoencephalitis in resveratrol-treated mice. Mice were subjected to meningoencephalitis to study the protective effect of resveratrol against meningoencephalitis and investigate the effects of sirtuin-1 activation on brain. Results demonstrated that sirtuin-1 level decreased in mice with meningoencephalitis and significantly increased in resveratrol-treated mice. Moreover, resveratrol treatment significantly reduced eosinophil counts, p65, Interferon-γ, interleukin (IL)-5, IL-33, and tumor necrosis factor-α levels, matrix metalloproteinase-9 activity, claudin-5 degradation, and blood–brain barrier permeability. By contrast, the anti-inflammatory factor IL10 was significantly increased in resveratrol-treated mice. Resveratrol treatment was partially beneficial in controlling the pathological processes of angiostrongyliasis meningoencephalitis. The results demonstrate the neuroprotective and anti-inflammatory effects of resveratrol against Angiostrongylus cantonensis-induced eosinophilic meningoencephalitis in mice. Treatment with sirtuin-1 agonist was given within a therapeutic window after A. cantonensis infection.
1. Introduction Angiostrongylus cantonensis (rat lungworm) is a parasitic nematode that causes severe dysfunction of the central nervous system (CNS) and induces eosinophilic meningitis or meningoencephalitis (Mackerras and Sandars, 1954; Hsu et al., 1990). The levels of pro-inflammatory biomarkers, such as interleukin (IL)-1β, IL-5, tumor necrosis factor (TNF)α, and matrix metalloproteinase (MMP)-9, are significantly increased in A. cantonensis-infected mice (Chen and Lai, 2007). Blood–CSF barrier dysfunction is associated with degradation of tight junction proteins and extracellular matrix as a result of MMP-9 activation in angiostrongyliasis meningoencephalitis (Chiu and Lai, 2013; Shyu et al., 2015). When infected with A. cantonensis, mice manifest marked systemic and CSF eosinophilia, reaching a peak level at around day 20. Eosinophil is cytotoxic in A. cantonensis infection, and a prominent infiltration of eosinophils occurs around the worm surface (Sugaya and Yoshimura, 1988; Tseng et al., 2004). Moreover, MMP-9 leads to blood–brain barrier (BBB) leakage in mice with A. cantonensis-induced eosinophilic meningoencephalitis (Chiu and Lai, 2014). Protein acetylation is a post-translational modification and an ⁎
1
important transcriptional regulatory mechanism. This process is controlled by histone acetyltransferases and histone deacetylases (Glozak et al., 2005). Sirtuin-1 is a member of sirtuins, which constitute the class III histone deacetylases (Milne and Denu, 2008). The neuroprotective role of sirtuin-1 has been demonstrated in models of various neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease (Jeong et al., 2011; Donmez et al., 2012). Resveratrol (3,5,4ʹ-trihydroxy-trans-stilbene), a natural agonist of sirtuin-1, is a naturally occurring phytoalexin that is present in skin of red grapes and a component of red wine (Nakata et al., 2012). Resveratrol acts as an activator of sirtuin-1 and demonstrates neuroprotective effects in various models (Bradamante et al., 2004; Meng et al., 2015). Accumulated evidence has documented that resveratrol exerts anti-neuroinflammation action by downregulating various pro-inflammatory biomarkers, such as IL-1β and TNF-α in neurodegenerative diseases (Foti Cuzzola et al., 2011). Moreover, resveratrol treatment is partially beneficial in controlling pathologic processes in Chagas disease (Wan et al., 2016). Therefore, we hypothesized that resveratrol can attenuate inflammation by upregulating sirtuin-1 in mice with eosinophilic meningoencephalitis.
Corresponding author at: Department of Parasitology, Chung Shan Medical University, 110, Section 1, Chien-Kuo North Road, Taichung 402, Taiwan. E-mail address: [email protected] (S.-C. Lai). An-Chih Chen and Ling-Yuh Shyu contributed equally to this work and both are first author.
http://dx.doi.org/10.1016/j.actatropica.2017.05.023 Received 17 January 2017; Received in revised form 17 May 2017; Accepted 19 May 2017 Available online 22 May 2017 0001-706X/ © 2017 Elsevier B.V. All rights reserved.
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Fig. 1. Effects of resveratrol on sirtuin-1 levels. (a) Time-course studies on sirtuin-1 levels in mouse brain. β-actin was used as loading control. (b) Quantification and normalization to β-actin showed that sirtuin-1 level was significantly reduced (*P < 0.05) on days 15, 20, and 25 after Angiostrongylus cantonensis infection. (c) Influence of resveratrol treatment on sirtuin-1 after A. cantonensis infection. (d) Quantification and normalization to βactin showed that sirtuin-1 level significantly increased (#P < 0.05) in A. cantonensis-infected mice (Infected) compared with that in uninfected controls. Mice treated with resveratrol (Res) alone, albendazole (ABZ) alone, or resveratrol-albendazole (Res + ABZ) significantly increased (*P < 0.05) the sirtuin-1 level compared with that in untreated A. cantonensis-infected mice. Bars represent the mean ± S.D. of three independent experiments performed in duplicate.
Fig. 2. Effects of resveratrol treatment on p65 levels in mouse brain. Protein levels of p-p65 (a) and p65 (c). β-actin was used as loading control. Quantification and normalization to β-actin showed that p-p65 (b) and p65 (d) were significantly increased (#P < 0.05) in Angiostrongylus cantonensisinfected mice (Infected) compared with that in uninfected controls. Treatment with resveratrol (Res) alone, albendazole (ABZ) alone, or resveratrol-albendazole (Res + ABZ) significantly reduced the levels of p-p65 and p65 (*P < 0.05) compared with those in untreated A. cantonensis-infected mice. Bars represent the mean ± S.D. of three independent experiments performed in duplicate.
2. Materials and methods
The present study investigated the deacetylation mechanisms during A. cantonensis infection. We aimed to determine whether treatment with sirtuin-1 agonist will be beneficial in improving the inflammatory response in angiostrongyliasis meningoencephalitis, providing clinical evaluation of the therapeutic strategy against A. cantonensis-induced eosinophilic meningoencephalitis.
2.1. Experimental animals Male BALB/c mice (specific pathogen-free grade and 5-week old) were purchased from the National Laboratory Animal Center, Taipei, Taiwan. The mice were maintained in an alternating 12h–12 h light77
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Fig. 3. Effects of resveratrol on the levels of IFN-γ, IL-5, IL-10, IL-33, and TNF-α. Concentrations of (a) IFN-γ, (b) IL-5, (c) IL-10, (d) IL-33, and (e) TNF-α were significantly increased (#P < 0.05) in Angiostrongylus cantonensis-infected mice (Infected) compared with those in uninfected controls. Concentrations of IFN-γ, IL-5, IL-33, and TNF-α in mice treated with resveratrol (Res) alone, albendazole (ABZ) alone, or resveratrol-albendazole (Res + ABZ) were significantly lower (*P < 0.05) than those in untreated A. cantonensis-infected mice. By contrast, IL-10 was significantly increased in mice treated with resveratrol alone (Res), albendazole alone (ABZ), or resveratrol-albendazole (Res + ABZ) compared with that in meningoencephalitis mice. Bars represent the mean ± S.D. of three independent experiments performed in duplicate.
dark cycle and provided with Purina Laboratory Chow and water ad libitum. The mice were kept in our laboratory for more than one week prior to experimental infection. All procedures involving animal use and care were approved by the Institutional Animal Care and Use Committee of Chung-Shan Medical University, Taiwan and performed in accordance with the institutional guidelines for animal experiments.
purchased from Sigma (St. Louis, MO, USA). Goat anti-mouse albumin polyclonal antibody was purchased from Bethyl Laboratories (Montgomer, USA). Horseradish peroxidase (HRP)-conjugated antirabbit IgG, HRP-conjugated anti-goat IgG, and HRP-conjugated antimouse IgG were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).
2.2. Antibodies
2.3. Larval preparation
The anti-mouse monoclonal antibody p-p65 and p65 generated in rabbit was purchased from Cell Signaling Technology (Beverly, MA). Goat anti-mouse claudin-5 polyclonal antibody and rabbit anti-mouse sirtuin-1 polyclonal antibody were purchased from Santa Cruz Biotechnology (CA, USA). Anti-mouse β-actin monoclonal antibody was
Infective larvae (L3) of A. cantonensis were originally isolated from wild giant African snails (Achatina fulica). The snails were bred for several months and then infected with A. cantonensis L1 by cycling through rats (definitive host) in Wufeng Experimental Farm (Taichung, Taiwan). Infective larvae (L3) were recovered by using the method 78
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Fig. 4. Effects of resveratrol on activity of matrix metalloproteinase (MMP)-9. MMP-9 bands (94 kDa) were detected in mouse (a) brains and (b) CSF. Quantitative analysis showed that MMP-9 levels were significantly increased (#P < 0.05) in Angiostrongylus cantonensis-infected mice (Infected) compared with those in uninfected controls. Mice treated with resveratrol (Res) alone, albendazole (ABZ) alone, or resveratrol-albendazole (Res + ABZ) showed significantly lower (*P < 0.05) MMP-9 than the untreated A. cantonensis-infected mice. Bars represent the mean ± S.D. of three independent experiments performed in duplicate.
2.5. Treatment of animals
developed by Parsons and Grieve (1990) with modification. The snail shells were crushed and then the tissues were homogenized with pepsin-HCl solution (pH of 1–2, 500 IU pepsin/g tissue) and digested through agitation at 37 °C in a water bath for 2 h. The sediments were clarified through series of washing with double-distilled water to clearly see the larvae, which were counted under a microscope. The morphological criteria of A. cantonensis L3 were confirmed according to the method described by Hou et al. (2004).
A total of 72 mice were randomly divided into six groups (12 mice/ group). Food and water were withheld for 12 h before infection. The uninfected control mice were orally administered with distilled water only on day 10 PI. All other groups were infected with 30 A. cantonensis larvae, including the untreated infected control mice, which were orally treated with distilled water only on day 10 PI. The experimental mice were separately treated with DMSO alone (10 mg/kg/day), resveratrol alone (10 mg/kg/day) albendazole alone (10 mg/kg/day), and albendazole-resveratrol (10 mg each/kg/day) for 7 consecutive days starting on day 10 PI. All groups were sacrificed on day 22 PI; the brains and CSF of the mice were collected for biochemical analysis.
2.4. Animal infection A total of 30 male BALB/c mice were randomly allocated into six groups (control, D5, D10, D15, D20, and D25), each group contains five mice. Food and water were withheld for 12 h before infection. The mice in five experimental groups (D5, D10, D15, D20, and D25) were infected with 30 A. cantonensis larvae via oral inoculation and then sacrificed on days 5, 10, 15, 20, or 25 post-inoculation (PI). Control mice received water only and sacrificed on day 25 PI. Brains of the mice were quickly removed and frozen at −80 °C.
2.6. CSF collection The mice were anesthetized intraperitoneally with urethane (1.25 g/kg). Each mouse was placed in a stationary instrument where the body was laid 135° relative to the head. The skin on the neck was shaved and swabbed three times with 70% ethanol. The subcutaneous tissues and muscles were separated. A capillary tube was inserted 79
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Fig. 7. Effects of resveratrol on eosinophil counts. Eosinophil counts significantly increased (#P < 0.05) in Angiostrongylus cantonensis-infected mice (Infected) compared with those in uninfected controls. Mice treated with resveratrol (Res) alone, albendazole (ABZ) alone, or resveratrol-albendazole (Res + ABZ) showed significantly lower (*P < 0.05) eosinophil than the untreated A. cantonensis-infected mice. Bars represent the mean ± S.D. of three independent experiments performed in duplicate.
Fig. 5. Effects of resveratrol on BBB permeability. Mouse BBB permeability was detected by performing extravasation of Evans blue during Angiostrongylus cantonensis infection. Evans blue dye units were significantly increased (#P < 0.05) in A. cantonensis-infected mice (Infected) compared with those in uninfected controls. Mice treated with resveratrol (Res) alone, albendazole (ABZ) alone, or resveratrol-albendazole (Res + ABZ) showed significantly lower (*P < 0.05) Evans blue dye units than the untreated A. cantonensisinfected mice. Bars represent the mean ± S.D. of three independent experiments performed in duplicate.
through the dura mater into cisterna magna and then CSF was poured into the capillary tube. The CSF was subsequently injected into a 0.5 mL Eppendorf tube and centrifuged at 3000g for 5 min at 4 °C. The supernatants were collected in a new 0.5 mL Eppendorf tube and then frozen at −80 °C.
2.7. Western blot analysis The mouse brains were homogenized with RIPA lysis buffer (150 mM sodium chloride, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS, and 50 mM Tris, pH 7.5) that contains a protease inhibitor cocktail (Sigma, MO, USA). The homogenates were centrifuged at 12,000g for 10 min at 4 °C. Protein concentration was determined by using protein assay kits (Bio-Rad, USA). The homogenates were mixed with an equal volume of sample buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 5% 2-mercaptoethanol, and 0.05% bromophenol blue) and boiled at 95 °C for 5 min. The mixtures were electrophoretically separated on SDS-polyacrylamide gels (PAGE) and then transferred onto polyvinyl difluoride membranes. The membranes were blocked with 5% non-fat milk in PBS (containing 0.1% Tween 20 and PBST) for 1 h at room temperature and then incubated with primary antibodies at 37 °C for 1 h. Subsequently, the membranes were washed three times with PBST followed by incubation with HRP-conjugated secondary antibody (1:10,000 dilution) at 37 °C for 1 h. Reactive protein bands were visualized by performing enhanced chemiluminescence (Amersham, Little Chalfont, Bucks, UK). Bands were quantitatively analyzed using a computer-assisted imaging densitometer system (UN-SCAN-ITTM gel Version 5.1, Silk Scientific, USA). Densitometric scanning result was presented as the ratio of the band density of target protein to that of the loading control (β-actin or albumin).
Fig. 6. Effects of resveratrol on claudin-5 levels in CSF. (a) Protein bands of claudin-5 obtained from mice CSF. Albumin was used as loading control. (b) Claudin-5 level was significantly increased (#P < 0.05) in Angiostrongylus cantonensis-infected mice (Infected) compared with that in uninfected controls. Mice treated with resveratrol (Res) alone, albendazole (ABZ) alone, or resveratrol-albendazole (Res + ABZ) showed a significantly lower (*P < 0.05) claudin-5 level than the untreated A. cantonensis-infected mice. Bars represent the mean ± S.D. of three independent experiments performed in duplicate.
2.8. Cytokine assay Production of cytokines (IFN-γ, IL-5, IL-10, IL-33, and TNF-α) in CSF was assayed using commercial enzyme-linked immunosorbent assay (ELISA) kits (Biosource, Camarillo, CA, USA) according to the manufacturer’s instructions. CSF samples for cytokine assays were pooled given that the amounts of CSF harvested from each mouse were frequently insufficient for individual assays.
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Fig. 8. Light microscopic observations. (a) Brain of uninfected mice; no Angiostrongylus cantonensis nor hemorrhage was observed in the brain. (b) Brain of untreated infected mice; young adult worms (arrowheads) and hemorrhage were evident. (c) Brain of DMSO-treated mice; young adult worms (arrowheads) and hemorrhage can be observed. (d) Brain of resveratrol-treated mice; the brain shows a significantly reduced amount of hemorrhage. Arrowheads indicate young adult worms. (e) Brain of albendazole-treated mice; no young adult worms were found in the brain and the amount of hemorrhage was significantly reduced. (f) Brain of mice treated with the combination of resveratrol and albendazole; no young adult worms were found in the brain and the amount of hemorrhage was significantly reduced.
Fig. 9. Possible mechanisms of the anti-inflammatory and protective pathways of resveratrol in mice infected with Angiostrongylus cantonensis. We investigated the potential function of sirtuin-1 in regulating angiostrongyliasis meningoencephalitis in resveratrol-treated mice. NF-κB inhibition could downregulate the production of pro-inflammatory factors (IFN-γ, IL-5, IL-33, TNF-α, and MMP-9) in A. cantonensis-infected mice. By contrast, NF-κB inhibition could upregulate the anti-inflammatory factor IL-10. Blocking the NF-κB signaling by resveratrol could reduce claudin-5 degradation and eosinophil production in angiostrongyliasis meningoencephalitis. Therefore, we suggest that this parasitic infection disrupts three layers of BBB to leukocyte infiltration by MMP-9 activities during infection. These processes are associated with inflammatory response in BBB destruction caused by A. cantonensis. The severity of A. cantonensis-induced meningoencephalitis in resveratrol-treated mice were considerably reduced.
washed two times with double-distilled water (containing 2.5% Triton X-100) for 30 min. The gel was subsequently incubated in reaction buffer (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 10 mM CaCl2, 0.02% Brij-35, and 0.01% NaN3) at 37 °C for 18 h. The gel was stained with 0.25% Coomassie Brilliant Blue R-250 (Sigma, USA) for 1 h and then
2.9. Gelatin zymography CSF was separated on 7.5% SDS-PAGE with 0.1% gelatin (Sigma, MO, USA). Electrophoresis was performed in running buffer (25 mM Tris, 250 mM glycine, and 1% SDS) at room temperature. Each gel was 81
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destained in 15% methanol/7.5% acetic acid. Gelatinase activity was visualized as clear bands against a blue-stained background. Quantitative analysis was performed using a computer-assisted imaging densitometer system (UN-SCAN-IT™ gel Version 5.1, Silk Scientific, USA).
higher in mice treated with albendazole alone, resveratrol alone, or resveratrol-albendazole co-treatment than in untreated A. cantonensisinfected mice (Fig. 3).
2.10. Evaluation of BBB permeability
Gelatin zymography measures the activity of MMP-9, which migrates electrophoretically as 94 kDa species. This metalloproteinase activity was significantly increased in CSF and brain of mice infected with A. cantonensis. MMP-9 activity was significantly reduced in mice treated with resveratrol alone, albendazole alone, or resveratrol-albendazole (Fig. 4).
3.4. Influence of resveratrol on MMP-9 activity
BBB permeability was assessed based on the concentrations of Evans blue in brains. Mice were injected with 2% (wt/vol) Evans blue dye (5 mL/kg body weight; Sigma, St. Louis, MO, USA) in saline via the tail vein. After 2 h of circulation, the mice were anesthetized and transcardially perfused with saline to remove intravascular dye. The brains were weighed and homogenized in 50% trichloroacetic acid solution. The homogenates were centrifuged at 12,000g for 10 min, and the supernatants were collected. Each supernatant was measured at 620 nm for absorbance to calculate the Evans blue concentrations by using a spectrophotometer (Hitachi U3000, Tokyo, Japan).
3.5. Influence of resveratrol on BBB permeability BBB disruption was estimated through Evans blue extravasation in mouse model. Concentration of Evans blue is an indicator of BBB leakage during A. cantonensis infection in mice. BBB permeability was significantly increased in A. cantonensis-infected mice compared with that in uninfected mice. Furthermore, BBB permeability was significantly attenuated in mice treated with resveratrol, albendazole, or resveratrol-albendazole (Fig. 5).
2.11. Eosinophil counts in CSF CSF samples were mixed with Unopette buffer (Vacutainer System, Becton Dickinson, Franklin Lakes, NJ, USA) containing 2% acetic acid. The mixture was placed in a hemocytometer cell counting chamber (Paul Marienfeld, Lauda-Koenigshofen, Germany) to count the eosinophils.
3.6. Influence of resveratrol on claudin-5 degradation Claudin-5 degradation in CSF is an indicator of damaged tight junction in BBB during A. cantonensis infection in mice. Western blot analysis indicated that the protein levels of claudin-5 increased significantly in CSF of infected mice compared with those in uninfected controls. The levels of protein claudin-5 decreased significantly after treatment with resveratrol alone, albendazole alone, or resveratrol-albendazole (Fig. 6).
2.12. Statistical analysis Results among different mouse groups were compared by performing non-parametric Kruskal–Wallis test followed by post-testing using Dunn’s multiple comparison of means. All results were presented as mean ± standard deviation (S.D.). P values less than 0.05 indicated statistical significance.
3.7. Influence of resveratrol on eosinophil counts
3. Results
The amount of CSF eosinophils significantly increased in mice infected with A. cantonensis compared with that in uninfected controls. Treatment with resveratrol alone, albendazole alone, or resveratrol-albendazole significantly reduced eosinophil counts, whereas DMSO treatment exerted no effect on eosinophil count (Fig. 7).
3.1. Influence of resveratrol on sirtuin-1 The effect of resveratrol was investigated in a murine angiostrongyliasis model. Western blot analysis showed that protein levels of sirtuin-1 decreased significantly in the brains of A. cantonensis-infected mice compared with those in the brains of uninfected controls. Furthermore, the protein levels of sirtuin-1 increased significantly after treatment with resveratrol alone, albendazole alone, or resveratrol-albendazole (Fig. 1).
3.8. Pathological observation of brain surface Light microscopic examination revealed that the brain of uninfected mice showed normal blood vessel, whereas the brain of untreated infected mice showed hemorrhage of enlarged blood vessels and young adult worms. Treatment with resveratrol alone significantly reduced the amount of hemorrhage. Furthermore, treatment with albendazole alone or resveratrol-albendazole significantly reduced the number of young adult worms and amount of hemorrhage (Fig. 8).
3.2. Influence of resveratrol on p65 Western blot analysis showed that the protein levels of p-p65 and p65 increased significantly in the brain of A. cantonensis-infected mice compared with those in uninfected controls. The protein levels of p-p65 and p65 decreased significantly after treatment with resveratrol alone, albendazole alone, or resveratrol-albendazole (Fig. 2).
4. Discussion Sirtuin-1, which can be activated by resveratrol, is highly expressed in the cortex, hippocampus, cerebellum, and hypothalamus (Ramadori et al., 2008). Activated sirtuin-1 is essential in the anti-inflammatory effects of resveratrol, and such role of sirtuin-1 may be partially attributed to inhibition of NF-κB (Busch et al., 2012). The NF-κB family of transcription factors plays a central role in inflammation and immunity. By inhibiting NF-κB, resveratrol downregulates the activation of immune cells and the subsequent synthesis and release of pro-inflammatory mediators. In vitro studies have shown that sirtuin-1 limits inflammation by inhibiting NF-κB and the transcription factor of NF-κB involved in expression of proinflammatory cytokines, such as IFN-γ and TNF-α (Yeung et al., 2004; Gao and Ye, 2008). P65 is an important subunit of an activated NF-κB dimer. We postulated that through
3.3. Influence of resveratrol on cytokines To determine the production of cytokines and their possible role in mouse resistance to A. cantonensis, we used sandwich ELISAs to assay IFN-γ, IL-5, IL-10, IL-33, and TNF-α in CSF. The concentrations of IFN-γ, IL-5, IL-33, and TNF-α were significantly higher in A. cantonensis-infected mice than in uninfected mice, where low levels of these cytokines were found. Compared with the corresponding concentrations of these cytokines in untreated infected mice, those in mice treated with albendazole alone, resveratrol alone, or resveratrol-albendazole were significantly reduced. By contrast, IL-10 concentration was significantly 82
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In conclusion, A. cantonensis infection in mice leads to overproduction of pro-inflammatory factors and causes meningoencephalitis, which is prevented by resveratrol treatment. The anti-inflammatory effects of resveratrol contribute to the improvement of meningoencephalitis in A. cantonensis-infected mice. The treatment with sirtuin-1 agonist, given in a therapeutic window of time after A. cantonensis infection.
reduced p-p65 activity, sirtuin-1 agonist is a therapeutic option that can improve the inflammatory factors and subsequently the angiostrongyliasis meningoencephalitis. To determine the anti-inflammatory role of sirtuin-1 beyond mouse brains, we investigated the proinflammatory, as well as the anti-inflammatory cytokine profiles, in mouse model. Our observation that the levels of proinflammatory cytokines (IFN-γ, IL-5, IL-33, and TNF-α) are lower while those of the anti-inflammatory cytokine IL-10 are higher in resveratrol-treated animals than in wild-type meningoencephalitis animals is consistent with the trend in immunomodulatory function. Our result demonstrated that the sirtuin-1 activator resveratrol suppressed IL-5 production and reduced the eosinophilic inflammatory cell infiltration in mouse model of A. cantonensis-induced meningoencephalitis. The reduced IL-5 level may explain the inhibition of eosinophilic inflammatory cell infiltration. These findings demonstrate a link between sirtuin-1 and cytokines in inflammatory responses during A. cantonensis infection and meningoencephalitis, strongly suggestive of the anti-inflammatory function of sirtuin-1. Sirtuin-1 activation is a key process in neuroprotective action of resveratrol (Ramadori et al., 2008; Feng et al., 2013). Our results reveal that overexpression of CNS sirtuin-1 in mice with meningoencephalitis was significantly reduced in the brain surface and reduced the infiltration of eosinophils into the subarachnoid space. These results demonstrate that sirtuin-1 is a potent anti-neuroinflammatory agent and strongly indicate its potential usefulness as therapeutic intervention for meningoencephalitis afflicting the CNS. Specifically, data showing that sirtuin-1 reduces eosinophil infiltration within the subarachnoid space in meningoencephalitis mouse model suggest a mechanism wherein diminishing the persistent eosinophil infiltration might reduce ongoing neuronal damage. Evan blue dye is widely used to detect BBB leakage as this dye conjugates with serum albumin to form a large molecular complex incapable of crossing an intact BBB under normal physiological circumstances (Wolman et al., 1981). We therefore investigated the beneficial effects of resveratrol in protecting the integrity of the BBB in angiostrongyliasis meningoencephalitis. Our study showed that Evan blue leakage was markedly increased in mice with meningoencephalitis compared with that in control mice, and this phenomenon was associated with increased degradation of the tight junction protein claudin5. Evan blue content in the brain was significantly reduced in resveratrol-treated mice compared with that in untreated mice displaying meningoencephalitis. Moreover, in experimental autoimmune encephalomyelitis mouse model, resveratrol exhibits protective effects in an experimental autoimmune encephalomyelitis therapy; such effect was attained through the anti-inflammation and antioxidant activities of resveratrol, in which it protects the tight junction proteins in the basement membrane to improve BBB integrity (Wang et al., 2016). These results collectively indicated that resveratrol could prevent disruption of BBB in meningoencephalitis mouse model. In this study, sirtuin-1 expression was upregulated in resveratroltreated mice. The results clearly indicate that sirtuin-1 overexpression improves the pathological processes of meningoencephalitis. Therefore, we propose a possible mechanism (Fig. 9) by which resveratrol relieves A. cantonensis-induced meningoencephalitis through sirtuin-1 activation. Meningoencephalitis is often accompanied by BBB disruption and crossing of plasma components, such as eosinophils, through the compromised BBB, aggravating inflammation and increasing the production of neurotoxins that can ultimately result in neuron death. Resveratrol downregulates the synthesis of pro-inflammatory mediators and the subsequent activation of eosinophils through NF-κB inhibition. Moreover, resveratrol inhibits the activation of MMP-9, significantly reducing leakage of Evans blue and degradation of tight junction protein, consequently improving the outcomes of meningoencephalitis in mice. Sirtuin-1 overexpression improves meningoencephalitis processes possibly by reducing the activation of pro-inflammatory cytokines, proteolytic enzyme MMP-9, and eosinophil infiltration.
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