A new compound W026B alleviates ischemic brain injury through inhibiting the production of inflammatory cytokines in pMCAO and tMCAO, and enhances the beneficial effect of tPA 
Ye Zhang1, Danping Zheng1, Mengyang Shui1, Ye Liu2, Xiaoyan Liu1*, Yinye Wang1*          
1. Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China
2. Beijing Honghui New Medical Technology Co.Ltd.; Beijing Daxing Biological Medicine Industry Base, Beijing 102600, China 
 
 
Abstract: Neruoprotection is considered as one of important therapeutic approaches for ischemic stroke. Inflammation plays an important role in the pathogenesis of ischemic stroke, and the inhibition of inflammation in the ischemic brain tissue may provide neuroprotective effect. In this study, we observed the influence of permanent middle cerebral artery occlussion (pMCAO) and transient MCAO (tMCAO) on NF-κB level and production of several inflammatory cytokines in injured hemisphere in mice, investigated the regulative effect of a new compound W026B on these influences in the two MCAO models. In pMCAO model, 10 μg/kg and 100 μg/kg of W026B (i.v.) significantly reduced infarct volumes, 100 μg/kg of W026B significantly decreased neurologic deficit scores and brain water contents, and 10 μg/kg and 100 μg/kg of W026B reduced Evans blue exudation from ischemic brain tissue. The level of NF-κB was elevated by 17.6 times in injured hemisphere, and the levels of TNF-α, IL-1β and IL-17 were elevated by 2.3 times, 2.2 times and 3.8 times compared with the sham operation group, respectively, 100 μg/kg of W026B significantly reduced these inflammatory cytokines. In tMCAO model, the elevation of NF-κB, TNF-α, IL-1β and IL-17 was 2.3 times, 1.4 times, 1.5 times and 1.4 times compared with the sham operation group, respectively. Moreover, 100 μg/kg of W026B significantly decreased the levels of these inflammatory cytokines. In embolic MCAO mice model, W026B alone significantly reduced infarct volumes, and combined application with tPA further reduced infarct volume. In conclusion, W026B displayed significant protecive effect on three brain ischemia models. It could protect brain against injury induced by ischmia and ischemia-reperfusion through inhibiting the production of NF-κB, TNF-α, IL-1β and IL-17. These results suggest that W026B has a value for further study.  
Keywords: Ischemia-reperfusion; NF-κB; Proinflammatory cytokines;W026B  
CLC number: R962                Document code: A                 Article ID: 10031057(2018)1067511
 
 
 
1. Introduction
Neuroprotection is considered as one of important therapeutic approaches for ischemic stroke. In recent decades, hundreds of clinical trials of neuroprotective agents have failed in the pre-clinical phase[1]. Despite these tremendous efforts, there remains a lack of effective neuroprotective agents clinically. Therefore, it is urgentlynecessary to develop clinically effective neuroprotecive agents for acute ischemic stroke[2].
Inflammation and immune responses have emerged as important elements in the onset and progression of stroke[3]. Experimentally and clinically, the brain responds to ischemic injury with an acute and prolonged inflammatory process, characterized by rapid activation of microglia, production of proinflammatory mediators, and infiltration of various types of inflammatorycells into the ischemic brain tissue. These cellular events collaboratively contribute to ischemic brain injury[4]. Administration of a specific iNOS inhibitor or a COX-2 inhibitor after ischemic insult shows a partial protective effect in mice, with recovery in neurological deficit and a reduction in infarct volume[5]. Several compoundsprotect brain from ischemia reperfusion (I-R) by reducing NF-κB activation, NO and cytokine production[6,7]. Apparently, the inhibition of inflammation in the ischemic brain tissue may provide neuroprotective effect.
There have been several stroke models established in rodents to simulate ischemic stroke in humans, includingintraluminal filament occlusion model, thromboembolic stroke model and so on[8]. Because of the complexity of human stroke, it is hard to mimic all aspects of human ischemic injury in one animal model. The permanent and transient middle cerebral artery occlusion (pMCAO and tMCAO) models are the most frequently used, with the advantages of convenience and well-reproducible infarcts. Embolic middle cerebral artery occlusion (eMCAO) is the model that most clinically mimics human ischemic stroke, which can determine the effect of thrombolytic agents and further evaluate the combinative effect of neuroprotective agents.
W026B is a newly synthesized compound. In this study, we investigated the effect of W026B on above-mentioned three models to evaluate its effectiveness, and assess its influence on NF-κB and several pro-inflammatory cytokines in pMCAO and tMCAO mouse models to preliminary reveal the mechanism of its protective effect.
2. Material and methods
2.1. Chemicals and agents
W026B[9] (purity: 95.4%) was provided by Beijing Honghui New Medical Technology Co. Ltd. (Beijing, China). Edaravone (EDA) injection was obtained from Simcere (Nanjing, China). Butylphthalide (NBP) injectionwas purchased from Cspc Phamaceutical Group Company Ltd. Human recombinant tissue type plasminogen activator (tPA) injection was provided by Beijing Honghui New Medical Technology Co.; Ltd. (Beijing, China).
2.2. Animals
Male Kunming mice (25–30 g) were obtained from the Department of Laboratory Animal Science, Peking University. The protocol of animal experiment was approved by the Ethics Committee of Animal Experiments of the Peking University Health Science Center (permit number: LA2013-69).
2.3. pMCAO model in mice
Mice were randomly assigned to the sham operation group, vehicle group, W026B-treated group, EDA-treatedand NBP-treated groups. The pMCAO model was prepared according to the previous method[10]. Vehicle, W026B or NBP was administered 1.5 h after occlusion (i.v.), EDA was administered twice (i.v.) after the occlusion of MCA and 1.5 h after occlusion, respectively. Body temperature was maintained at 37±0.5 °C on a temperatur-regulable operating board during surgery.
2.4. tMCAO model in mice
The animals were grouped as above. The filament was introduced into ICA through ECA to occlude MCA. At 1.5 h after occlusion, the filament was withdrawn, the ECA was ligatured, and the CCA left unfastened. Vehicle or W026B was immediately administered after the ending of occlusion (i.v.), and EDA was administered (i.v.) after the occlusion of MCA and the ending of occlusion, respectively. Body temperature was maintained at 37±0.5 °C. 
2.5. eMCAO Preparation of embolus
The preparation of clots was performed with previouslydescribed method[11] with minor modifications. Blood donor mice were anesthetized with chloride hydrate (500 mg/kg, i.p.). Fresh arterial blood was collected from the heart by a 1-mL syringe and then injected into PE0402 tubule (ID: 0.2 mm, OD: 0.4 mm, Anlai Technology CO.; LTD.). The collected samples were allowed to stand at room temperature overnight and subsequently stored at 4 °C. The clots were then flushed out from the tubule and cut into 1.0±0.3 cm embolus. A single clot of 10 mm was transferred to the catheter filled with normal saline.
The right CCA, right ICA and right ECA were exposed.The right CCA and ICA were temporarily clamped using microvascular clips, and eMCAO was induced by injecting embolus into ICA as descried by Tauheed et al[12]. Successful embolization and reperfusion by tPA was monitored by laser doppler flowmetry (LDF), relative regional cerebral blood flow (rCBF) was detected with LDF (Peri-Flux System 5000) throughout the surgery. Mice were randomly divided into five groups as follows: sham, vehicle, tPA, W026B and W026B+tPA. Vehicle or W026B was administered (i.v.) 1.5 h after embolic occlusion, and tPA was administered (i.v.) 1 h after embolic occlusion.
2.6. Neurobehavioral evaluation
Neurologic deficit scores were blindly evaluated 24 h after reperfusion according to an expanded seven-point scale evaluation criterion reported previously[10].
2.7. Quantification of infarction volume and assessment of brain water contents
Mice were transcardially perfused with NS to removeblood in the tissue under anesthesia after neurobehaviorevaluation. The brains were collected, coronally sectioned into five slices at 2-mm intervals, and then stained by immersion in 1% TTC according to previous reports[10]. The sections were photographed, and the normal and infarcted areas were analyzed using Adobe Photoshop CC.
The brain water content was determined according to a previously described method[13]. Brain water content was calculated by taking the difference betweenthe wet weight and dry weight.
2.8. Measurement of Evans blue exudation
pMCAO model was prepared as described above, each group of animals received Evans blue (80 mg/kg, i.v.) 3 h before sacrificed, and then were transcardially perfused with NS to remove the intravascular dye under anesthesia. Brains were collected, and the left and right hemisphere were weighed, respectively. The dye was extracted in 1.0 mL of formamide at 45 °C for 72 h[14]. The supernatants were analyzed by measuring the absorbance at 620 nm by microplate reader.
2.9. Western blotting
Animal experiments were performed asdescribed above. Proteins in nucleus were extracted from injured cerebral hemispheres with a nuclear and cytoplasmic extraction kit (CW0199B, CWBIO). Western blotting analysis was performed as described previously[7]. Antibodies used in the present study included rabbit NF-κB p65 polyclonal antibody (1:500, Santa Cruz) and mouse anti-Histone H1 monoclonal antibody (1:500, Santa Cruz). The immunoactive bands were analyzed with a ChemiDoc XRS System (Bio-Rad).
2.10. ELISA assay
Animal treatment, MCAO model preparation and brain tissue collection were performed as described above. Levels of TNF-α, IL-1β and IL-17 in the supernatant of homogenates were detected by ELISA.
2.11. Statistical analysis
All data were expressed as mean ± SEM. Statistical evaluation was performed using one-way analysis of variance. Significant differences between groups were assessed by Tukey’s test, and P<0.05 was considered statistically significant.
3. Results
3.1. W026B protects brain against injury induced by both pMCAO and tMCAO
In pMCAO model, the percent of infarct volume of model group was 45.8%±5.1%. 10 μg/kg (21 nmol/kg), 20 μg/kg (42 nmol/kg) and 100 μg/kg (210 nmol/kg) of W026B reduced the percent of infarct volume to 24.9%±2.8%, 23.1%±5.4% and 20.8%±2.9%, respectively(Fig. 1A, B). In addition, 210 nmol/kg of W026B significantly reduced the neurological deficit scores and brain water content (Fig. 1C, D). Two positive controls, EDA ((3 mg/kg equal to 17.2 μmol/kg) ×2 times) and NBP (50 mg/kg equal to 257 μmol/kg), showed similar effects on the infarct volume.
In tMCAO, 10 μg/kg and 100 μg/kg of W026B reduced the percent of infarct volume from 33.9%±1.0% to 25.9%±0.9% and 18.5%±1.7%, respectively (Fig. 2A, B).These doses of W026B also significantly decreased brain water content (Fig. 2C).
 3.2. W026B decreased exudation amount of Evans blue in pMCAO
The dye amounts in the injured cerebral hemispheres of the model group were significantly increased (12.20±1.80 mg/g, Fig. 3). W026B significantly and dose-dependently reduced the dye amount in theinjured hemisphere. The dye amounts for 100 μg/kg of W026B group was 4.46±1.80 mg/g. The dye amounts for EDA (3 mg/kg i.v.;2 times) and NBP (50 mg/kg) groups was 5.89±1.40 mg/g and 6.08±1.90 mg/g, respectively.
3.3. W026B lowered the levels of nuclear p65 subunit of NF-κB in injured hemisphere both in pMCAO and in tMCAO
The nuclear level of p65 subunit of NF-κB was elevated in injured hemisphere in both pMCAO and tMCAO model with about 17.6-fold and 2.3-foldincrease, respectively, compared with ipsilateral hemisphere of the sham operation group. W026B strongly lowereded the elevated p65 expression level (from 17.60±4.77 to 2.69±0.86 in pMCAO and from 4.71±0.13 to 1.49±0.17 in tMCAO), nearly close to the value of the sham operation (Fig. 4A and Fig. 5A).
3.4. W026B reduced the levels of TNF-α in injured hemisphare in both pMCAO and in tMCAO
TNF-α level was significantly increased in injured hemisphere in pMCAO (from 42.80±2.68 pg/mg to 99.30±6.72 pg/mg). 10 μg/kg and 100 μg/kg of W026B strongtly lowered the TNF-α level to 60.50±1.35 pg/mg and 55.00±3.22 pg/mg, respectively (Fig. 4B). Similarly, in tMCAO, the TNF-α level in insulted hemisphere waselevated from 166.83±9.64 pg/mg to 228.08±23.47 pg/mg. 10 μg/kg and 100 μg/kg of W026B reduced the TNF-α levelfrom 228.08±23.47 pg/mg to 174.76±7.24 pg/mg and 197.06±10.41 pg/mg, respectively, though no significant difference was found in low dose of W026B (Fig. 5B).
3.5. W026B decreased IL-1β level in injured hemisphare both in pMCAO and in tMCAO
In pMCAO, the IL-1β level of injured hemisphere in the model group was significantly elevated compared with the sham operation group from 159.80±7.00 pg/mg to 345.40±22.80 pg/mg. Moreover, 100 μg/kg of W026B evidently reduced the IL-1β level from 345.40±22.80 pg/mg to 204.90±27.80 pg/mg, and 10 μg/kg of W026B also reduced the IL-1β level from 345.40±22.80 pg/mg to 251.40±38.00 pg/mg, though no significant difference was observed (Fig. 4C). In tMCAO, the IL-1β level was 142.88±11.25 pg/mg in the sham group, it was significantly increased to 217.76±11.68 pg/mg in the tMCAO insulted group, and 100 μg/kg and 10 μg/kg of W026B reduced the levels of IL-1β to 153.21±9.87 pg/mg and 187.74±8.95 pg/kg, respectively (Fig. 5C).
 
 
 
Figure 1. Efficacy of W026B (i.v.) in the pMCAO models. (A) Representative of TTC-stained brain slices after 24 h ischemia. The white region shows the infarct size, while the red region shows the viable tissue. (B) cerebral infarct volume. (C) Brain water content.(D) Neurological deficit scores. *P<0.05, **P<0.01 compared with vehicle groups. ^^P<0.05 compared with sham groups. Data were expressed as mean±SEM.

 

Figure 2. Efficacy of W026B (i.v.) in the tMCAO models. (A)Representative of TTC-stained brain slices at 24 h after reperfusion. The white region shows the infarct size, while the red region shows the viable tissue. (B)Cerebral infarct volume. (C) Brain water content. *P<0.05, **P<0.01, ***P<0.001 compared with vehicle groups. ^^^P<0.001 compared with sham groups. Data were expressed as mean±SEM.

  
 
Figure 3. Effect of W026B (i.v.) on Evans blue exudation in mice subjected to pMCAO (mean±SEM). *P<0.05, **P<0.01 compared with vehicle groups. ^^P<0.01 compared with sham groups. 

 

 
Figure 4. Effect of W026B (i.v.) on the expression of (A) NF-κB p65, (B) TNF-α, (C) IL-1β and (D) IL-17A in mice subjected to pMCAO (mean±SEM). **P<0.01 compared with vehicle groups. ^^P<0.01 compared with sham groups.

 

 
Figure 5. Effect of W026B (i.v.) on the expression of (A) NF-κB p65, (B) TNF-α, (C) IL-1β and (D) IL-17A in mice subjected to tMCAO (mean±SEM). *P<0.05, **P<0.01 compared with vehicle groups. ^P<0.05, ^^P<0.01, ^^^P<0.001 compared with sham groups. 

 

3.6. W026B supressed IL-17A level in injured hemisphare both in pMCAO and in tMCAO
IL-17 levels in pMCAO and tMCAO model groups were both significantly increased, with a 3.8-fold and 1.4-fold increase compared with the sham operation group, respectively. In pMCAO, 10 μg/kg and 100 μg/kg of W026B all powerfully supressed the IL-17 level by 72.8% and 84.4%, respectively (Fig. 4D). In tMCAO, however, these two doses only slightly supressed the elevated IL-17 level by 8.9% and 13.1%, respectively (Fig. 5D).
3.7. The effects of W026B and tPA single or combined use on the cerebral injury induced by eMCAO
In embolic model group (vehicle treated group), cerebral blood flow (CBF) significant declined, and CBF remained at low level throughout the monitoring period of about 2 h (Fig. 6A, C). In tPA-treated group, CBF was gradually increased within 30–60 min after the injection of tPA (Fig. 6B, D), indicating that the embolic MCAO model as established.
 
 
Figure 6. CBF monitoring during embolus occlusion. (A) Representative of LDF recordings in Vehicle-treated mice subjected to eMCAO. (B) Representative of LDF recordings in tPA-treated mice subjected to eMCAO. (C) Representative of LDF recordings in W026B-treated mice subjected to eMCAO. (D) Representative of LDF recordings in W026B+tPA-treated mice subjected to eMCAO.  
 
As shown in Figure 7, the infarct volume for eMCAO model reached up to 47.9%±1.2%, theinfarct volume for tPA-treated group was decreased to 23.6%±2.3%, and infarct volume for the W026B-treated group was 26.5%±2.4%, and the combined use of the two agents further reduced the infarct volume to 16.5%±2.0%. 
 
 
 
Figure 7. Efficacy of W026B (i.v.) in the eMCAO models. (A) Representative of TTC-stained brain slices at 24 h after embolusinjection. The white region shows the infarct size, while the red region shows the viable tissue. (B) cerebral infarct volume. ***P<0.001compared with vehicle groups. ^P<0.05. Data were expressed as mean±SEM. 
  
 
4. Discussion 
W026B is a newly synthesized compound based on w007B[7]. This study showed that its effective doses are far smaller than two positive controls (Fig. 1, Fig. 2), suggesting that W026B is a potent neuroprotectant in cerebral ischemic and I-R injury.
Furthermore, we found that W026B significantly reduced the exudation of Evans blue (Fig. 3), suggesting that it could inhibit the inflammatory response during ischemia, thereby protecting BBB. In addition, W026B showed a potently protective effect on embolic occlusionmodel, and it enhanced the protective effect of tPA thrombolysis (Fig. 7), indicating that W026B could suppress the side reaction of tPA thrombolysis, such as BBB injury induced by reperfusion.
Inflammation is one of important pathophysiological mechanisms underlying the cerebral ischemia and I-R injury. Many inflammation mediators, including TNF-α, IL-1β, IL-17 and NO, are released from macrophages, microglial cells and astrocytes during stroke, leading to damage ischemic neuron[3], and the initiation of an inflammatory response may result in disruption of BBB. Inflammation mediators are significantly elevated in the plasma of stroke patient, thus inflammation is considered as a risk factor for ischemic stroke[15]. W007 has potent anti-inflammatory effect in cerebral I-R rats. We believed that W026B might have such effect. Therefore, we investigated the influence of W026B on inflammatory response in pMCAO and tMCAO models, respectively.
NF-κB is one of crucial regulator of inflammatory response after cerebral injury, and it plays a key role in the initiation of inflammation[16] and is involved in I-R injury[17]. It has been demonstrated that NF-κB is activated after MCAO, as revealed by the nuclear translocation of the NF-κB subunit p65[18]. We first detected NF-κB (p65 subunit) level in nucleus and found that W026B suppressed NF-κB (p65) level by 84.7% in pMCAO (Fig. 4A) and 68.4% in tMCAO (Fig. 5A), suggesting that W026B could almost block NF-κB activation, leading to inhibited expressions of diverse inflammation cytokines or mediators.
TNF-α is a well-known cytokine involved in the inflammatory response elicited in the region of cerebralischemia, and levels of TNF-α may remain elevated in the affected brain tissue for at least 24 h after ischemic insult[19]. Therefore, we observed the influence of W026B on the TNF-α level in ischemic or I-R brain tissue. The result showed that W026B (100 μg/kg) almost abolished TNF-α elevation induced by pMCAO (Fig. 4B) and tMCAO (Fig. 5B), suggestingthat W026B exerted it anti-inflammatory action by blocking TNF-α generation during brain ischemia and I-R.
IL-1β is a proinflammatory cytokine. Its expression is a powerful stimulus for leukocyte recruitment to the CNS, and experimentally induced elevations of IL-1β levels in the brain cause disruptions in BBB[20]. In this study, 100 µg/kg of W026B almost completely blocked the elevation of IL-1β level induced by pMCAO and significantly inhibited the elevation of IL-1β level induced by tMCAO, indicating that W026Bcould also inhibit the inflammatory response via suppressing the generation of IL-1β.
IL-17 is a mediator of communication between immune cells and tissue. The numbers of IL-17-expressing blood mononuclear cells are elevated as early as 1 h after the ischemic insult[21] in rat MCAO model. IL-17 is believed to have a particular role in the delayed phase of the post infarct inflammatory cascade. Increased numbers of IL-17-expressing cells can be found in the peripheral blood of patients who suffered from an ischemic stroke compared with healthy individuals[22]. IL-17A has been implicated in the pathogenesis of ischemic brain injury[23], and cerebral I-R significantly increases the levels of IL-17A in the penumbral area[24]. An IL-17A-blocking antibody decreases infarct size and improves neurologic outcome in the murine model[20]. Therefore, we would like to know whether W026B could influence IL-17A in brain injury induced by ischemic or I-R. The results showed that 10 µg/kgand 100 µg/kg of W026B powerfully suppressed the level of IL-17A in pMCAO (Fig. 4D), and significantly reduced level of IL-17A was also observed in tMCAO (Fig. 5D). Apparently, this effect was likely an important mechanism for W026B to inhibit inflammatory response and reduce brain injury.
TNF-α, IL-1β and IL-17A are all implicated in neuroinflammation of ischemic injury, they stimulate their own receptors and activate signal pathways, resulting in neuroinflammatory injury, and there is interactive regulation between cytokines. W026B potently lowered levels of these three cytokines. However, it remains unclear whether W026B inhibited all three cytokines, respectively, or only inhibited one or two of them, and thereby influenced other cytokines.The other possibility was that W026B blocked the activation of NF-κB, thus inhibiting the expressions of these proinflammatory cytokines. The detail mechanismof W026B on inflammatory response during cerebral ischemia and I-R remains to be elucidated.
In conclusion, W026B has potently protective effect on ischemia or I-R induced brain injury, and theprotective effect may be partly attributed to its inhibitory effect on the generation of proinflammatory cytokines in injured brain tissue.
Acknowledgements
This work was supported by National Natural Science Foundation of China (Grant No. 81573333, 81503060).
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新化合物W026B通过抑制炎性细胞因子的产生减轻缺血性脑损伤, tPA合用作用增强
张烨1, 郑丹萍1, 水梦洋1, 刘晔2, 刘晓岩1*, 王银叶1*
1. 北京大学医学部 药学院 分子与细胞药理学系,北京 100191
2. 北京红惠新医药科技有限公司, 北京102600       
摘要: 神经保护被认为是缺血性脑卒中的重要治疗方法之一。炎症在缺血性脑卒中的发病机制中起着重要作用,抑制缺血性脑组织的炎症反应可能起到神经保护作用。本研究观察了新化合物W026B对小鼠永久性大脑中动脉阻塞模(pMCAO)和短暂性大脑中动脉阻塞模型(tMCAO)的保护作用和对缺血侧脑组织NF-κB和一些炎性细胞因子表达的影响。在pMCAO模型中静脉注射W026B 10 µg/kg100 µg/kg可明显减少小鼠缺血脑梗塞体积, 100 µg/kg W026B显著降低神经功能缺陷评分和脑含水量; 10 µg/kg 100 µg/kg W026B可减少血管内伊文思蓝渗出至缺血脑组织。与假手术组相比, 缺血脑组织中细胞核内的NF-κB水平升高了17.6, 组织中的TNF-αIL-1β IL-17分别升高了2.3倍、2.2倍和3.8, 100 µg/kg W026B可显著减少这些炎性细胞因子的水平。在tMCAO模型中, 缺血组织中的NF-κBTNF-αIL-1β IL-17的也分别升高2.3倍、1.4倍、1.5倍和1.4, 100 µg/kg W026B也可显著减少这些炎性细胞因子的产生。在小鼠血栓栓塞eMCAO模型中W026B单独应用也可检索小脑梗死体积, tPA联合应用作用增强。总之, W026B在三种脑缺血模型上均具有明显的保护作用, 这些保护作用与其减少细胞核中的NF-κB的含量, 减少TNF-αIL-1β L-17的产生有关。这些结果提示W026B可能具有进一步研究的价值。 
关键词: 脑缺血再灌注; NF-κB; 炎症因子; W026B

   

Received: 2018-03-25,Revised: 2018-06-25, Accepted: 2018-09-10.
Foundation items: National Natural Science Foundation of China (Grant No. 81573333, 81503060).
*Corresponding author. Tel.: +86-010-82802652, E-mail: wangyinye@bjmu.edu.cn; liuyan@bjmu.edu.cn