NLRP3 inflammasome

Neuroscience Letters 600 (2015) 182–187

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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet

Research article

Umbelliferone ameliorates cerebral ischemia–reperfusion injury via upregulating the PPAR gamma expression and suppressing TXNIP/NLRP3 inflammasome Xiangxiang Wang, Ruipeng Li, Xuan Wang, Qiang Fu ∗∗ , Shiping Ma ∗ Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing 210009, PR China

h i g h l i g h t s • Umbelliferone notably ameliorates cerebral ischemia–reperfusion injury. • Umbelliferone suppresses expression of NLRP3 inflammasome. • Umbelliferone attenuates cerebral ischemia–reperfusion injury via regulating PPAR␥.

a r t i c l e

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Article history: Received 3 May 2015 Received in revised form 5 June 2015 Accepted 7 June 2015 Available online 10 June 2015 Keywords: Umbelliferone Cerebral ischemia–reperfusion TXNIP NLRP3 inflammasome PPAR-␥

a b s t r a c t Umbelliferone (UMB), a natural antioxidant belonging to coumarin derivatives, is able to cross the bloodbrain barrier and protect neuronal cells from death. Here we aimed to investigate the effects of UMB in a rat model of focal cerebral ischemia induced by middle cerebral artery occlusion/reperfusion (MCAO/R). Pretreatment with UMB (15 and 30 mg/kg) for 7 consecutive days ameliorated the neurological outcomes, infarct volume and brain edema in brains of MCAO rats. Our results provided evidence that UMB significantly protected neuronal cells against cerebral ischemia reperfusion-induced injury. Furthermore, UMB treatment could inhibited the level of oxidative stress and the production of inflammatory cytokines in brain tissues of MCAO rats. In addition, UMB significantly upregulated the expression of peroxisome proliferator-activated receptor-␥ (PPAR-␥), which exhibited neuroprotective effects in neurodegenerative disease. UMB treatment also suppressed NLRP3 inflammasome activation via reducing expression of Thiredoxin-interactive protein (TXNIP). These results suggest that UMB may have beneficial effects for neuroprotection against focal cerebral ischemic partly through the inhibition of TXNIP/NLRP3 inflammasome and activation of PPAR-␥. © 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction: Ischemic stroke is the leading cause of brain injury which is characterized as high incidence, high morbidity and high mortality [1]. Ischemic stroke can induce many damaging effects, such as disruption of blood-brain barrier, brain edema, which cause brain injury [2]. Prevention of ischemia/reperfusion injury is becoming an important issue in Ischemic stroke treatment. The mechanism of ischemic injury is complex, the currently acknowledged theories include excitotoxicity, cailcium overload, and oxidative stress, inflammation, eventually leading to apoptosis or necrosis [3].

∗ Corresponding author. ∗∗ Corresponding author. E-mail address: [email protected] (S. Ma). http://dx.doi.org/10.1016/j.neulet.2015.06.016 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.

The brain is an organ predisposed to oxidative stress. Reactive oxygen species (ROS) working in concert with an inflammatory process, may play an important role in the pathogenesis of Ischemic stroke. Although oxidative stress, inflammation and apoptosis occur together in Ischemic stroke, oxidative stress seems to be a potential initiation [4]. Recently it is shown that thioredoxininteracting protein (TXNIP) activation is a key event linking oxidative stress to inflammation and apoptosis in neurons [5]. In response to ROS released from mitochondria, TXNIP dissociates from the complex and rapidly binds to NLRP3 (Nod-like receptor family, pyrin domain containing 3) inflammasome. TXNIP then induces inflammasomes activation, which results in maturation and secretion of IL-1␤ and IL-18 [6]. The mature IL-1␤ and IL-18 are needed for the processing of inflammation and apoptosis [7]. Umbelliferone (UMB), a member of coumarin derivatives, widely exists in plants with anti-oxidant and free radical

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scavenging properties [8]. UMB has been reported to exhibit neuroprotective effects in MPTP-induced mouse model and cross the blood-brain barrier, indicating that UMB has the potential to protect neuron from apoptosis [9]. UMB positively regulates peroxisome proliferator-activated receptor-␥ (PPAR-␥) activity and this action is involved in amelioration of metabolic disorders and alcohol-induced fatty liver [10–12]. PPAR-␥, belonging to the nuclear hormone receptor superfamily, has been certified as an important regulator of apoptosis, oxidative stress and inflammation [13–15], and also plays a significant role in the neuroprotection against cerebral ischemia–reperfusion injury [16]. However, few studies have discussed the neuroprotective effects of UMB in Ischemic stroke. The present study is aimed to elucidate the mechanism through which UMB ameliorated ischemia/reperfusion injury by inhibition of inflammasome activation. 2. Materials and methods 2.1. Materials Umbelliferone (purity ≥ 98%) (Nanjing Zelang Medical Technology Co., Ltd., China), 2,3,5-triphenyltetrazolium chloride (TTC) (Amresco, USA), SOD and MDA assay kits (Nanjing Jiancheng Bioengineering Institute, China), IL-1␤ and IL-18 ELISA kits (Shenzhen Dakewe Biotech Co., Ltd., China), Bicinchoninic acid (BCA) kit (Beyotime Institute of Biotechnology Co., Ltd., Shanghai, China). Antibodies used in Western blot analysis: anti-caspase-1 (ab108362) were purchased from Abcam (Cambridge, MA, USA); anti-NLRP3 (NBP2-12446) and anti-TXNIP (NBP1-54578) were purchased from Novus Biologicals (Littleton, CO, USA); anti-PPAR␥ (sc-271392), anti-␤-actin (sc-130656) and anti-rabbit IgG (HRP) (sc-45101) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). 2.2. Experiment animals Male rats of Sprague–Dawley with body weight ranging from 220 to 270 g were procured from Comparative Medicine Center, Yangzhou University (Yangzhou, China) and maintained under standard conditions (temperature: 25 ± 1 ◦ C, air humidity: 60–65%) with a 12 h light/12 h dark cycle. Water and standard chow were provided ad libitum during the whole experiment procedure. All experimental procedures were carried out strictly in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985). 2.3. Surgeries and drug administration Rats were randomly divided into sham, MCAO, UMB-treated groups. UMB (15 and 30 mg/kg) vehicle was administered by intraperitoneal injection for 7 days before MCAO. 10% dimethyl sulph oxide was used as a vehicle solution for the intraperitonial administration of Umbelliferone [17,18]. Rats were anesthetized with intraperitoneal injection of chloral hydrate (350 mg/kg). With the right common carotid artery (CCA) exposed by a short incision, the internal carotid artery (ICA) and external carotid artery (ECA) were separated out. With ECA being ligated, CCA and ICA being clamped by bulldog clamp, a 3-0 monofilament nylon suture with the tip rounded was inserted from the CCA into the ICA up to 18–20 mm. Body temperatures were maintained at 37 ± 0.5 ◦ C by a heating equipment. Rats were subjected to 2 h occlusion, and 22 h reperfusion. Neurologic deficit, brain water content (BWC), infarct volume and biochemical analysis were evaluated after 22 h of reperfusion.

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2.4. Neurologic deficit evaluation A blinded investigator evaluated sensorimotor scores after 22 h of reperfusion. The neurological functions were scored referring to the Zea-Longa neurological deficit score [19], which was a 5-point scale as follows: score 0, no neurologic deficit; score 1, failure to extend forepaw fully; score 2, failure to walk straight and circling to one side; score 3, falling to one side; score 4, no spontaneous motor activity. 2.5. Infarct volume measurement After 22 h of reperfusion, animals were killed by decapitation and the brain was stored immediately at −20 ◦ C for 30 min. Frozen brain was cut into five coronal sections. The brain slices were put into 2% 2,3,5-triphenyltetrazolium chloride (TTC) solution at 37 ◦ C for 30 min, under the conditions of protection from light. The measured slices were photographed, and the area of ischemic brain injury was calculated by a blinded observer with Image J software, expressed as infarct area percentage (%). 2.6. Brain water content(BWC) After 22 h of reperfusion, after animals were decapitated and the brain was removed immediately, the wet brain weight (WW) was weighed with an electronic balance and placed in 120 ◦ C dry oven, with dry weight (DW) to be obtained after 24 h. BWC (%) = (WW − DW)/WW × 100%. 2.7. Nissl staining After anesthetization with intraperitoneal injection of chloral hydrate (350 mg/kg), rats were perfused through the left ventricle with 100 ml cold saline (0.9%), followed by 100 ml cold paraformaldehyde (4%). Brains were removed quickly, fixated with 10% formalin at room temperature, and sectioned into coronal slices with a cryostat microtome at −20 ◦ C. The sections were stained with tolridine blue and examined with microscope. To observe neuronal survival in the CA1 region, neurons with round and palely stained nuclei were considered as surviving cells, which were indicated by arrows in the picture. Data were expressed as the number of surviving cells/field. 2.8. Determination of superoxide dismutase (SOD), malondialdehyde (MDA) in brain tissues Rat hippocampus was separated out and homogenized with normal saline (w:v 1:9) on ice. The homogenate was centrifuged at 2500–3000 rpm for 10 min, and 10% supernatant was collected with an flnnpipette. Protein concentration of the supernatant was measured by BCA kit. SOD activities and MDA content were measured according to the instructions of test kits. 2.9. Measurement of IL-1ˇ and IL-18 production The levels of IL-1␤ and IL-18 in hippocampus were determined by ELISA assays, according to the instructions from the manufacturer and quantified with a microplate reader at 450 nm. The data were shown as picogram per milliliter (pg/ml). 2.10. Western blot analysis Rat hippocampus was separated out and homogenized with icecold lysis buffer containing protein inhibitor. The homogenate was centrifuged at 12,000 rpm for 20 min at 4 ◦ C. Protein concentration

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Fig. 1. (A) Effects of UMB on neurological deficit scores (A), brain water content (B), TTC staining (C) and infract volume (D) in MCAO rats. Each data was presented as mean ± SEM (n ≥ 3). #P < 0.05, ##P < 0.01 vs Sham; *p < 0.05, **p < 0.01 vs MCAO.

of the supernatant was measured by bicinchoninic acid (BCA) protein assay kit on the basis of the manufacturer’s instructions. The western blot procedure was performed as previously described [18]. The corresponding quantifications represents values were obtained as a ratio of the target to control. 2.11. Statistical analysis All data were analyzed by the GraphPad Prism 5.0 software. Results presented in the paper were shown as the mean ± SEM and implemented by a one-way analysis of variance (ANOVA), followed by Tukey’s test for inter-group comparisons. Value of p ≤ 0.05 was regarded to be significantly different. 3. Result 3.1. Effects of UMB on the neurological defect scores and brain water content in rats As shown in Fig. 1A, there were no neurological deficit in Sham group, and the neurological function scores of MCAO group were significantly higher than Sham group. Compared with the model, the scores in groups pretreated with UMB (15 and 30 mg/kg) showed a significantly decreasing trend [F(3,36) = 33.86, p < 0.01]. The BWC is measured after 22 h of reperfusion with data presented in Fig. 1B. The BWC was significantly increased in MCAO group compared with the sham and significantly decreased in UMBpretreated (15 and 30 mg/kg) rats [F(3,8) = 4.55, p < 0.05].

tion appeared, but infarct volume in UMB groups were significantly reduced compared with MCAO group (P < 0.05 or P < 0.01). 3.3. Nissl staining Nissl staining was carried out in hippocampus CA1 region. The data revealed that the number of positive cells in MCAO group was significantly reduced (Fig. 2) [F(3,8) = 17.29, p < 0.01]. The number of surviving cells was significantly reversed by the pretreatment with UMB (15 and 30 mg/kg). The arrow represented the surviving neuronal cells. 3.4. Effects on SOD and MDA SOD activities (Fig. 3A) were significantly reduced in MCAO group [F(3,8) = 28.27, p < 0.01]. Conversely, MDA levels (Fig. 3B) were significantly elevated[F(3,16) = 30.68, p < 0.01]. Pretreatment with UMB could increase SOD activities, but reduce MDA levels in brain tissues. 3.5. Effects on IL1ˇ and IL-18 Protein levels (Fig. 4A,B) of interleukins-1␤ (IL1␤), and interleukins-18 (IL-18) were significantly elevated in MCAO group; pretreatment with UMB (15 and 30 mg/kg) significantly attenuated the production of these cytokines [IL1␤: F(3,8) = 16.38, p < 0.01; IL18: F(3,8) = 14.53, p < 0.01]. 3.6. UMB suppressed the expression of NLRP3 inflammasome

3.2. UMB reduced the infarct volume in rats Effect of UMB on the infarct volume in rats was presented in Fig. 1C,D. After 22 h of reperfusion, a significant cerebral infarc-

NLRP3 inflammasome is a multiple protein complex, which activated caspase-1 and created a platform for maturation of IL-1␤ and IL-18. UMB attenuated NLRP3 and cleaved caspase-1 expres-

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Fig. 2. Neuronal damage was assessed by Nissl staining in the hippocampus CA1 region. Each data was presented as mean ± SEM (n ≥ 3). #P < 0.05 vs Sham; *p < 0.05 vs MCAO.

Fig. 3. Effects of UMB treatment on MDA (A) and SOD (B). Each data was presented as mean ± SEM (n ≥ 3). # P < 0.05, ##P < 0.01 vs Sham; *p < 0.05, **p < 0.01 vs MCAO.

Fig. 4. IL-1␤ (A) and IL-18 (B) were determined by ELISA assays. The expression of NLRP3 (C), cleaved caspase-1 (D) were detected by Western blot. Each data was presented as mean ± SEM (n ≥ 3). #P < 0.05, ##P < 0.01 vs Sham; *p < 0.05, **p < 0.01 vs MCAO.

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Fig. 5. Western blot analysis, UMB attenuated the expression of TXNIP; Each data was presented as mean ± SEM (n ≥ 3). #P < 0.05, ##P < 0.01 vs Sham; *p < 0.05, **p < 0.01 vs MCAO.

Fig. 6. Effects of UMB treatment on the expression of PPAR-␥. Each data was presented as mean ± SEM (n ≥ 3). #P < 0.05 vs Sham.

sion (Fig. 4C,D). These results indicated that UMB suppressed NLRP3 inflammasome activation and inflammatory response. 3.7. UMB inhibited TXNIP induction After cerebral ischemia–reperfusion, TXNIP was significantly increased in rat brain tissue (Fig. 5); pretreatment with UMB reduced TXNIP expression, indicating the inhibitory role of UMB on TXNIP activation.

found that UMB pretreatment improved neurological outcomes, reduced infarct volume and brain edema. The mechanism of the neuroprotective effects of UMB in the brain tissue of rats after MCAO was discussed. Our results showed that UMB treatment alleviated brain injury through the inhibition of the level of IL-1␤ and IL-18 in brain tissues, which resulted in protection from neuronal damage, as indicated by an increase in nissl-stained positive cells in the brain. Furthermore, our data indicate that UMB inhibited the inflammatory cytokines production through the inhibition of TXNIP/NLRP3 inflammasome activation. In addition, UMB treatment significantly upregulated the expression of PPAR-␥. These results demonstrated that the UMB has beneficial effects in ischemic reperfusion-induced brain injury, at least in part, through the upregulate the expression of PPAR-␥ and inhibition of TXNIP/NLRP3 inflammasome. The brain is prone to the damage induced by oxidative stress. Previous studies showed that oxidative stress caused by overproduction of ROS plays a key role in the pathogenesis of Ischemic stroke [20,21]. MCAO results in the reduced levels of the antioxidant system including superoxide dismutase (SOD) and increased level of lipid peroxidation such as MDA, subsequently leading to the damage of cellular membrane, protein and DNA and ultimately inducing neuronal death. In our study, UMB could increase SOD activities and reduced MDA levels in brain tissues of MCAO rats, which is consistent with the previous studies. More and more studies have demonstrated that inflammation is an important contributor to the pathophysiology of vascular diseases, especially stroke [22]. Recent studies have highlighted the role of NLRP3 inflammasome, a member of NLR family, which activated caspase-1 and served as a platform for maturation of IL-1␤ and IL-18. The NLRP3 inflammasome is one of the most recently identified protein complex implicated in the development of brain injury induced by ischemia/reperfusion [23]. Moreover, recent studies showed that oxidative stress-induced cell death is regulated by inflammsome activation, for which TXNIP is required. TXNIP is the endogenous inhibitor and regulator of thioredoxin, which could rapidly bind to NLRP3 inflammasome, triggering assembly and oligomerization of the inflammasome. In addition, TXNIP expression is upregulated in brain tissue of MCAO rats. In the present study, we found that UMB treatment significantly inhibited oxidative stress and suppressed inflammasome activation by blocking TXNIP, resulting in inhibition of inflammatory cytokines secretion and the inflammation response. PPAR-␥ was constitutively expressed in the brain [24], which had been regarded as a significant pharmacological target for central nervous system (CNS) disease [25]. PPAR␥ agonist could alleviate the harmful effects of oxidative stress and inflammation during brain ischemia, indicating the neuroprotective role of PPAR␥ in ischemic stroke [26]. In addition, a recent paper has found PPAR-␥ binding sites in the promoter regions of a member of the NLRP family, indicating the correlation between PPAR-␥ and NLRP family of protein [27]. In agreement with these studies, we found that UMB upregulated the expression of PPAR-␥ and significantly alleviated oxidative stress and inflammation in MCAO rats.

3.8. UMB increased the level of PPAR- in MCAO rats The expression of PPAR-␥ was measured by Western blot. PPAR-␥ was seldom expressed in Sham animals, and was almost invariably in MCAO (Fig. 6). Pretreatment with UMB greatly upregulated the expression of PPAR-␥. 4. Discussion In this study, we evaluated the effects of pretreatment with UMB in a rat model of cerebral ischemic-reperfusion for the first time and

5. Conclusions Our results provide evidence that the chronic exposure to MCAO induced cerebral damage through oxidative stress and inflammatory response. Pretreatment with UMB exhibited neuroprotective effects in ischemia/reperfusion-induced brain injury. This effect of UMB may be partly related with inhibition of NLRP3 inflammasome in the brain and upregulation of PPAR-␥ expression, suggesting the prevention effect of UMB for the Ischemic stroke.

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Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments This study is supported by the Fundamental Research Funds for the Central Universities (Program No. JKZD2013009) and the project in our department is funded by Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References [1] B. Owens, Stroke, Nature 510 (2014) S5. [2] T.C. Wu, J.C. Grotta, Stroke treatment and prevention five new things, Neurology 75 (2010) 16–21. [3] A. Michael Moskowitz, Eng H. Lo, Costantino Iadecola, The science of stroke: mechanisms in search of treatments, Neuron 67 (2010) 181–198. [4] J. Tschopp, Mitochondria sovereign of inflammation? Eur. J. Immunol. 41 (2011) 1196–1202. [5] R. Zhou, A. Tardivel, B. Thorens, I. Choi, J. Tschopp, Thioredoxin-interacting protein links oxidative stress to inflammasome activation, Nat. Immunol. 11 (2010) 136–140. [6] D.Y.W. Fann, S.Y. Lee, S. Manzanero, P. Chunduri, C.G. Sobey, T.V. Arumugam, Pathogenesis of acute stroke and the role of inflammasomes, Ageing Res. Rev. 12 (2013) 941–966. [7] R. Zhou, A.S. Yazdi, P. Menu, J. Tschopp, A role for mitochondria in NLRP3 inflammasome activation, Nature 469 (2011) 221–225. [8] D. Zhao, M.N. Islam, B.R. Ahn, H.A. Jung, B.W. Kim, J.S. Choi, In vitro antioxidant and anti-inflammatory activities of Angelica decursiva, Arch. Pharm. Res. 35 (2012) 179–192. [9] S.R. Subramaniam, E.M. Ellis, Neuroprotective effects of umbelliferone and esculetin in a mouse model of Parkinson’s disease, J. Neurosci. Res. 91 (2013) 453–461. [10] Jarinyaporn Naowaboot, et al., Umbelliferone increases the expression of Adipocyte-specific genes in 3 T3-L1 Adipocyte, Phytother. Res. 28 (11) (2014) 1671–1675. [11] B. Ramesh, K.V. Pugalendi, Antihyperglycemic effect of umbelliferone in streptozotocin-diabetic rats, J. Med. Food 9 (2006) 562–566. [12] Myung-Joo Kim, et al., Dietary umbelliferone attenuates alcohol-induced fatty liver via regulation of PPAR␣ and SREBP-1c in rats, Alcohol 48 (2014) 707–715.

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