半胱氨酰白三烯受体拮抗剂对脑缺血的保护作用

炎症是影响脑缺血急性损伤严重程度以及慢性期恢复的主要因素之一,而半胱氨酰白三烯是体内最重要的炎症介质之一,通过半胱氨酰白三烯受体(CysLT1R和CysLT2R)调节脑缺血后炎症等病变。目前,已证实CysLT1R拮抗剂对脑缺血动物模型急性和亚急性/慢性损伤的保护作用,其神经元保护作用可能是间接通过调节胶质细胞,对星形胶质细胞增殖及胶质疤痕形成则有明确的抑制作用。CysLT2R拮抗剂还有待深入研究,但可能与神经元和星形胶质细胞损伤、小胶质细胞激活等有密切关系。CysLT1R和Cys-LT2R拮抗剂是抗脑缺血损伤潜在的新型治疗药物。     

白三烯受体拮抗剂ONO-1078对内皮素-1诱导的大鼠局灶性脑缺血的保护作用

目的观察白三烯受体拮抗剂ONO-1078对内皮素-1诱导的大鼠局灶性脑缺血的保护作用。方法向大脑中动脉附近微量缓慢注射内皮素-1(120 pmol,6 μL,>6 min),诱导大鼠局灶性脑缺血模型,在注射内皮素-1前1 h ip ONO-1078(0.1 mg·kg^-1)。观察神经症状、脑水肿程度、脑梗死体积、纹状体和皮层的存活神经元数的变化。结果 脑内微量注射内皮素-1引起动物出现明显神经症状、脑梗死、脑水肿及皮层和纹状体的存活神经元减少。预先ip ONO-1078显著抑制脑水肿,减小脑梗死体积,增加纹状体和皮层的存活神经元数,可减轻神经症状,但无显著意义。结论ONO-1078对内皮素-1诱导的脑缺血损伤有保护作用,白三烯参与了脑缺血后的组织损伤过程。     

Nuclear translocation of cysteinyl leukotriene receptor 1 is involved in oxygen-glucose deprivation-induced damage to endothelial cells

Aim:

Cysteinyl leukotriene receptor 1 (CysLT₁ receptor) is located in epithelial cells, and translocates from the plasma membrane to the nucleus in a ligand-dependent manner. Here, we investigated whether CysLT₁ receptors translocated to the nucleus in endothelial cells after ischemic insult in vitro and whether it was involved in ischemic injury to endothelial cells.

Methods:

EA.hy926 cell line, derived from human umbilical vein endothelial cells, was subjected to oxygen-glucose deprivation (OGD). The expression and distribution of CysLT₁ receptors were detected by immunofluorescent staining, immunogold labeling and immunoblotting analyses. Cell viability was evaluated using MTT reduction assay. Necrosis and apoptosis were determined by double fluorescent staining with propidium iodide and Hoechst 33342.

Results:

CysLT₁ receptors were primarily distributed in the cytoplasm and nucleus in EA.hy926 cells, and few was found in the cell membrane. OGD induced the translocation of CysLT₁ receptors from the cytoplasm to the nucleus in a time-depen dent manner, with a peak reached at 6 h. OGD-induced nuclear translocation of CysLT₁ receptors was inhibited by pretreatment with the CysLT₁ receptor antagonist pranlukast (10 μmol/L), or by preincubation with NLS-pep, a peptide corresponding to the nuclear localization sequence of CysLT₁ receptor (10 μg/mL). However, zileuton, an inhibitor of 5-lipoxygenase that was a key enzyme in cysteinyl leukotriene generation, did not inhibit the nuclear translocation of CysLT₁ receptors. Moreover, preincubation with NLS-pep (0.4 μg/mL) significantly ameliorated OGD-induced cell viability reduction and necrosis.

Conclusion:

CysLT₁ receptors in endothelial cells translocate to the nucleus in a ligand-independent manner after ischemic insult in vitro, and it is involved in the ischemic injury.     

白三烯受体拮抗剂0N0-1078对大鼠局灶性脑缺血的神经保护作用(英文)

目的:观察白三烯受体拮抗剂ONO-1078(pran-lukast)对大鼠局灶性脑缺血是否具有神经保护作用.方法:以大脑中动脉阻塞30分钟诱导局灶性脑缺血,并再灌注24小时.ONO-1078(0.003-1.0mg·kg~(-1))或生理盐水(1 mL·kg~(-1))在脑缺血前30分钟和缺血后2小时各腹腔注射1次.脑缺血24小时后,测定神经症状评分、脑梗死体积、神经元密度(皮质、海马和纹状体)、脑水肿以及小血管周围白蛋白渗出.结果:ONO-1078轻度改善神经症状;但显著减少脑梗死体积和神经元缺失,呈钟型量效关系,以 0.01-0.3 mg·kg~(-1)作用最明显;0.01-1.0 mg·kg~(-1)可减轻缺血半球面积增大以及白蛋白渗出.结论:ONO-1078可保护大鼠局灶性脑缺血,可能部分由于抑制了脑水肿.本研究提示白三烯受体拮抗剂可能代表一类治疗急性脑缺血新药.     

半胱氨酰白三烯受体拮抗剂pranlukast对小鼠局灶性脑缺血的治疗作用

目的　观察半胱氨酰白三烯受体拮抗剂pranlukast(ONO 10 78)在小鼠局灶性脑缺血后的治疗作用。方法　采用大脑中动脉阻塞造成小鼠持续性局灶性脑缺血 ,缺血后1、6、2 4h分别给小鼠腹腔注射 pranlukast或依达拉奉 ,观察药物对缺血 2 4、4 8h后的神经功能缺损症状 ,4 8h后的脑梗死体积、两侧大脑半球比值、神经元密度的影响。结果　Pranlukast 0 1、0 2mg·kg-1及依达拉奉 3、10mg·kg-1均能减轻神经症状、减小脑梗死体积、降低缺血侧 /非缺血侧大脑半球比值、减轻海马CA1区、皮层和纹状体的神经元密度降低。结论　Pranlukast脑缺血后给药对脑损伤有治疗作用 ,提示有治疗缺血性脑卒中的临床前景。     

Inhibition of ex vivo neutrophil activation by oral LY293111, a novel leukotriene B4 receptor antagonist

1 The effects of orally administered LY293111 on ex vivo neutrophil Mac-1 upregulation were determined in a total of 24 healthy male subjects within three study periods.
2 In the first period, eight volunteers received 60  mg LY293111 or placebo three times daily in 22 total doses over 8 days followed by a 1 week follow-up. The average ex vivo Mac-1 response of the LY293111 group was 56% of the pre-dose control (95% confidence interval (CI) 44.3 to 67.9%; P <0.01). The inhibitory effect was maximum at the end of dosing and had disappeared by day 14.
3 In the second period, eight subjects received 120  mg LY293111 or placebo three times daily in 22 total doses over 8 days followed by a 1 week follow-up. The average response of the LY293111 group was 70% of the pre-dose control (95% CI 59.7 to 81.0%; P <0.01). The inhibitory effect was maximum the day following the initial dose and continued throughout the dosing period.
4 In the third period, eight subjects received 200  mg LY293111 or placebo twice daily in 15 total doses over 8 days followed by a 1 week follow-up. Mac-1 upregulation was 64% of pre-dose levels (95% CI 53.8 to 75.1%; P <0.01) over the course of the study period. The inhibition had disappeared 2 days following the final dose. Alternate neutrophil stimulation by fMLP was not inhibited.
5 No statistically significant inhibition was observed for placebo-treated subjects.
6 No statistically significant differences were apparent between the active dose regimens.
7 The results indicate that orally administered LY293111 is pharmacologically active in humans. Results from this study may be useful in determining dose selection for efficacy trials.     

Inhibition of synovial LTB4 production by a specific leukotriene biosynthesis inhibitor,MK-0591, in a rabbit model of joint inflammation

The effect of a leukotriene biosynthesis inhibitor (MK-0591) on LTB₄ synthesis was examined in a rabbit model of joint inflammation. Intra-articular (ia) injection of human recombinant interleukin-1β (rlL-1β, 50–400 ng/joint) resulted in dose-dependent infiltration of leukocytes into the synovium but produced only background levels of LTB₄ over a time course of 14–16 h, suggesting that the leukocytes were not activated with respect to LTB₄ metabolism. The influx of leukocytes in the synovial space was not inhibited by MK-0591. Injection of A23187 (100 nmol/joint, ia) in addition to rlL-1β elicited significant LTB₄ release in the synovial fluid. MK-0591 given iv 30 min before A23187 significantly inhibited the release of LTB₄ (ED₅₀ = 0.3 mg/kg), without affecting the number of leukocytes in the synovium. In rabbit whole blood challenged with A23187 in vitro, MK-0591 also potently inhibited LTB₄ synthesis (IC₅₀ = 126 ± 29 nM). The in vivo inhibitory action of MK-0591 was in good agreement with the plasma levels of the compound (0.62 μM in the group treated with MK-0591 at 3 mg/kg) and its biochemical efficacy in vitro (100% inhibition of LTB₄ synthesis). The effect of MK-0591 was comparable to that of another leukotriene biosynthesis inhibitor, MK-886. Indomethacin at dose up to 3 mg/kg iv did not affect the production of LTB₄ in the joint. The present study demonstrates that MK-0591 inhibits LTB₄ biosynthesis with high potency in the rabbit synovium, a microenvironment rich in protein and leukocytes. It would be of interest to determine whether such an action would be beneficial in the management of rheumatoid arthritis or other inflammatory conditions in humans. ©1993 Wiley-Liss, Inc.     

Inhibition of microglial activity alters spinal wide dynamic range neuron discharge and reduces microglial Toll‐like receptor 4 expression in neuropathic rats

It is believed that neuropathic pain results from aberrant neuronal discharges although some evidence suggests that the activation of glia cells contributes to pain after an injury to the nervous system. This study aimed to evaluate the role of microglial activation on the hyper‐responsiveness of wide dynamic range neurons (WDR) and Toll‐like receptor 4 (TLR4) expressions in a chronic constriction injury (CCI) model of neuropathic pain in rats. Adult male Wistar rats (230 ± 30 g) underwent surgery for induction of CCI neuropathy. Six days after surgery, administration of minocycline (10, 20, and 40 mg/kg, i.p.) was initiated and continued until day 14. After administration of the last dose of minocycline or saline, a behavioral test was conducted, then animals were sacrificed and lumbar segments of the spinal cord were collected for Western blot analysis of TLR4 expression. The electrophysiological properties of WDR neurons were investigated by single unit recordings in separate groups. The findings showed that after CCI, in parallel with thermal hyperalgesia, the expression of TLR4 in the spinal cord and the evoked response of the WDR neurons to electrical, mechanical, and thermal stimulation significantly increased. Post‐injury administration of minocycline effectively decreased thermal hyperalgesia, TLR4 expression, and hyper‐responsiveness of WDR neurons in CCI rats. The results of this study indicate that post‐injury, repeated administration of minocycline attenuated neuropathic pain by suppressing microglia activation and reducing WDR neuron hyper‐responsiveness. This study confirms that post‐injury modulation of microglial activity is a new strategy for treating neuropathic pain.     

红景天苷介导TLR4调控小胶质细胞激活对小鼠抑郁样行为的改善作用

目的 探讨红景天苷对脂多糖（LPS）诱导炎症抑郁模型小鼠的作用及机制。方法 48只Balb/c小鼠随机分为对照组、模型组、盐酸氟西汀（阳性药,20 mg/kg）组、红景天苷（25 mg/kg）组,每组12只。各组连续ig给药14 d后,除对照组外,其余各组ip 1 mg/kg LPS连续7 d制备抑郁症模型。以糖水偏好实验、旷场实验、悬尾实验和强迫游泳实验检测小鼠抑郁样症状;结束后取脑组织与血液检测肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）水平;Iba1免疫荧光染色检测脑组织小胶质细胞激活;Western blotting法检测脑组织TLR4信号通路蛋白TLR4、NLRP3、cleaved-Caspase-1/Caspase-1表达。结果 与模型组比较,红景天苷与盐酸氟西汀预防给药能够显著上调小鼠糖水摄取率（P<0.05）,增加小鼠穿格评分（P<0.05）,下调悬尾与强迫游泳不动时间（P<0.05）;显著逆转LPS造成的TNF-α、IL-1β水平上调（P<0.05）;明显抑制小胶质细胞激活,显著降低Iba1荧光强度（P<0.05）;显著降低脑组织中TLR4、NLRP3与cleaved-Caspase-1/Caspase-1蛋白表达（P<0.05）。结论 ip LPS成功构建了炎症诱导的抑郁模型,红景天苷通过TLR4信号通路调控小胶质细胞激活,从而改善小鼠抑郁样行为。     

Neuroprotective effect of ONO-1078, a leukotriene receptor antagonist, on transient global cerebral ischemia in rats

AIM: To determine whether ONO-1078 {pranlukast, 4-oxo-8-[p-(4-phenylbutyloxy)benzoyl-amono]-2-(tetrazol-5-yl)-4H-1-benzopyran hemihydrate}, a potent leukotriene receptor antagonist, possesses a neuroprotective effect on global cerebral ischemia in rats, and to explore its possible mechanism of action. METHODS: Transient global cerebral ischemia was induced by four-vessel occlusion for 10 min and followed by 72-h reperfusion. ONO-1078 (0.03-0.3 mg/kg) and edaravone (MCI-186, 3-methyl-1-phenyl-2-pyrazolin-5-one, a neuroprotective agent) 10 mg/kg were ip injected 30 min before ischemia and 1 h after reperfusion, and once a day afterward. Neurological outcome was evaluated before ischemia and 24, 48, 72 h after reperfusion. Neuron density, the expressions of N-methyl-Daspartate (NMDA) receptor subunit proteins (NR1, NR2A, NA2B) and vascular cell adhesion molecule 1(VCAM-1) in the cerebral cortex and hippocampus were measured at 72 h after reperfusion. RESULTS: ONO-1078 (0.1, 0.3 mg/kg) and edaravone (10 mg/     

Downregulation of steroid hormone receptor expression and activation of cell signal transduction pathways induced by a chiral nonylphenol isomer in mouse sertoli TM4 cells

Nonylphenols (NPs) are considered as important environmental toxicants and potential endocrine disrupting compounds which can disrupt male reproductive system. 4‐[1‐Ethyl‐1‐methylhexy] phenol (4‐NP₆₅) is one of the main isomers of technical nonylphenol mixtures. In the present study, effect of NPs was evaluated from an isomer‐specific viewpoint using 4‐NP₆₅. Decreased mRNA expression levels of estrogen receptor (ER)‐α, ER‐β, androgen receptor (AR) and progesterone receptor (PR) were observed in the cells exposed to 4‐NP₆₅ for 24 h. Furthermore, 4‐NP₆₅ treatment evoked significant decrease in protein expression levels of ER‐α and ER‐β. Levels of mullerian inhibiting substance and transferrin were found to change significantly in 4‐NP₆₅ challenged cells. Additionally, JNK1/2‐MAPK pathway was activated due to 4‐NP₆₅ exposure, but not ERK1/2 and p38‐MAPK pathways. Meanwhile, 4‐NP₆₅ increased the p‐Akt level and showed no effects on the Akt level which indicated that Akt pathway was activated by 4‐NP₆₅. In conclusion, these findings have shown that 4‐NP₆₅ exposure affected expression of cell receptors and cell signaling pathways in Sertoli TM4 cells. We proposed that molecular mechanism of reproductive damage in Sertoli cells induced by NPs may be mediated by cell receptors and/or cell signal transduction pathways, and that the effects were dependent on the side chain of NP isomers. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 469–476, 2017.     

Glucagon-like peptide-1 receptor agonist Exendin-4 improves neurological outcomes by attenuating TBI- induced inflammatory responses and MAPK activation in rats

Traumatic brain injury (TBI) can be exacerbated and prolonged for months or even years by chronic inflammatory processes with long-term consequences on neurodegeneration and neurological impairment. However, there are no clear pharmacological therapies of benefit to manage neurological dysfunctions, which, relating to the molecular mechanisms underlying the behavioral deficits after TBI, have yet to be fully identified. Recently, a glucagon-like peptide 1 (GLP-1) agonist, Exendin-4, was approved not only for the treatment of type 2 diabetes mellitus, but it also played a neurotrophic role in various CNS neurological diseases. In this study, we evaluated the neuroprotective effects of Exendin-4 on neurological outcome, cerebral blood flow, neurodegeneration, and inflammatory responses by utilizing a cortical contusion impact injury (CCI) model in rats. We found that TBI rats displayed neurological impairments, neurodegeneration, reduction of cerebral blood flow, and inflammatory responses, while Exendin-4 promoted neurological, cognitive, and cerebral blood flow recovery and attenuated neural degeneration and inflammatory cytokines after TBI. Furthermore, Exendin-4 treatment significantly diminished the TBI-induced overexpression of TNFα and IL-1β, as well as phosphorylation of p38 and ERK1/2. These data suggest a strong beneficial action of the glucagon-like peptide-1 receptor agonist Exendin-4 in improving neurological outcomes by attenuating inflammatory responses induced by traumatic brain injury, which is of therapeutic potential for TBI.     

脂氧素A_4对大鼠局部永久性脑缺血的保护作用

目的探讨脂氧素A4(lipoxinA4,LXA4)对大鼠局部永久性脑缺血损伤的保护作用。方法健康成年SD♂大鼠,体重200～250g,随机分为4组:假手术组(Sham),缺血模型组(Model),LXA410ng治疗组,LXA4100ng治疗组。用改良线栓法制备大鼠右侧大脑中动脉永久性缺血模型。Sham组插入栓线仅约10mm。大鼠于放线栓成功后10min内给予右侧侧脑室内注射等容量的LXA4(5μl)或生理盐水(5μl)。缺血24h后,评价神经功能缺损情况;2,3,5-氯化三苯四唑(TTC)染色观察脑梗死范围;分光光度计法测定缺血侧脑皮质丙二醛(MDA)含量和髓过氧化物酶(MPO)活性;ELISA法检测缺血侧皮质肿瘤坏死因子α(TNF-α)和白介素1β(IL-1β)的含量;HE染色观察脑组织病理学改变。结果LXA410ng、100ng能明显改善神经功能损伤症状,减少大鼠局部永久性缺血后脑梗死体积百分比,降低脑组织MPO活性以及MDA、TNF-α和IL-1β的含量,减轻脑组织病理学损伤,且LXA4100ng组在改善大鼠神经功能及抑制皮质TNF-α的表达上明显优于10ng组。结论LXA4对大鼠局部永久性脑缺血损伤有一定保护作用。     

二烯丙基二硫下调cyclin D1和CDK4的表达诱导人鼻咽癌CNE2细胞G_1期阻滞

目的研究二烯丙基二硫(DADS)对人鼻咽癌CNE2细胞的影响及其分子机制。方法采用MTT法检测DADS对CNE2细胞增殖抑制作用;流式细胞术分析DADS对CNE2细胞周期分布的影响;运用RT-PCR和Western blot方法分析DADS作用CNE2细胞后,cyclin D1和CDK4的表达变化。结果 MTT结果显示,不同浓度DADS(90、140、240、400μmol·L-1)处理CNE2细胞48h后,生长抑制率分别为4.0%、13.8%、25.8%、51.2%;流式细胞术分析显示,DADS阻滞CNE2细胞于G1期,并呈浓度依赖性;RT-PCR和Western blot结果表明,细胞周期调控基因cyclinD1、CDK4表达下调。结论 DADS对CNE2细胞的增殖抑制作用与其阻滞细胞G1期有关,并且可能是通过抑制cyclin D1、CDK4的表达使CNE2细胞阻滞于G1期。     

Synergism between interleukin (IL)-17 and toll-like receptor 2 and 4 signals to induce IL-8 expression in cystic fibrosis airway epithelial cells

Cystic fibrosis (CF) is the most common lethal inherited disorder and is caused by mutations in the gene encoding the CF transmembrane regulator (CFTR). The CF lung expresses a profound proinflammatory phenotype that appears to be related to a constitutive hypersecretion of interleukin (IL)-8 from airway epithelial cells in response to microbial infection. Since overproduction of IL-8 in CF contributes to massive bronchial infiltrates of neutrophils, identification of the pathways underlying IL-8 induction could provide novel drug targets for treatment of neutrophil-dominated inflammatory diseases such as CF. Here, we show that IL-17A synergistically increases IL-8 production induced by a toll-like receptor (TLR) 2 agonist, peptidoglycan (PGN), or TLR4 agonist, lipopolysaccharide (LPS), in a human CF bronchial epithelial cell line (CFBE41o-). A strong synergism was also observed in primary human CF bronchial epithelial cells, but not in human non-CF cell lines and primary cells. Notably, despite the induction of nuclear factor-κB and MAP kinases during TLR2 or TLR4 activation in CFBE41o-, IL-17A-dependent synergism appears to be the result of enhanced PGN- or LPS-induced phosphorylation of p38. Taken together, these studies provide evidence that IL-17A is a critical factor in increasing IL-8 expression in bacteria-infected CF airways via a pathway that regulates p38 phosphorylation.     

The toll like receptor 4‐myeloid differentiation factor 88 pathway is essential for particulate matter‐induced activation of CD4‐positive cells

Asian sand dust (ASD), a type of particulate matter (PM) found in Asia, can be transported to East Asia. We recently found that acute splenic inflammation is induced by ASD in mouse models. In this study, we examined the effect of sub‐chronic ASD exposure on mouse immune cells. Mice were intratracheally administered ASD once every 2 weeks for 8 weeks and killed 24 hours after the final administration. Wild‐type (WT) mice showed increased cell viability after ASD administration. In contrast, ASD administration induced splenocyte activation in toll‐like receptor (TLR)2^?/?, but not TLR4^?/? mice. Furthermore, concanavalin A‐induced interleukin‐2 production increased after ASD administration in WT and TLR2^?/? mice, but not in TLR4^?/? or myeloid differentiation factor (MyD)88^?/? mice. Immunoblotting demonstrated that nuclear factor κB (NF‐κB) was activated in WT mice, but not in TLR4^?/? or MyD88^?/? mice. The NF‐κB‐dependent gene products CDK2 and intercellular cell adhesion molecule‐1 were upregulated upon ASD administration in WT mice, but not in TLR4^?/? or MyD88^?/? mice. Furthermore, the particles themselves, rather than particle constituents, activated NF‐κB in CD4‐positive cells through the TLR4 or MyD88 pathway. Taken together, these results indicate that particle‐induced splenic inflammation occurs via TLR4‐MyD88 signaling.     

Toll-like receptor 3 agonist Poly I:C protects against simulated cerebral ischemia in vitro and in vivo

Aim:

To examine the neuroprotective effects of the Toll-like receptor 3 (TLR3) agonist Poly I:C in acute ischemic models in vitro and in vivo.

Methods:

Primary astrocyte cultures subjected to oxygen-glucose deprivation (OGD) were used as an in vitro simulated ischemic model. Poly I:C was administrated 2 h before OGD. Cell toxicity was measured using MTT assay and LDH leakage assay. The levels of TNFα, IL-6 and interferon-β (IFNβ) in the media were measured using ELISA. Toll/interleukin receptor domain-containing adaptor-inducing IFNβ (TRIF) protein levels were detected using Western blot analysis. A mouse middle cerebral artery occlusion (MCAO) model was u sed for in vivo study. The animals were administered Poly I:C (0.3 mg/kg, im) 2 h before MCAO, and examined with neurological deficit scoring and TTC staining. The levels of TNFα and IL-6 in ischemic brain were measured using ELISA.

Results:

Pretreatment with Poly I:C (10 and 20 μg/mL) markedly attenuated OGD-induced astrocyte injury, and significantly raised the cell viability and reduced the LDH leakage. Poly I:C significantly upregulated TRIF expression accompanied by increased downstream IFNβ production. Moreover, Poly I:C significantly suppressed the pro-inflammatory cytokines TNFα and IL-6 production. In mice subjected to MCAO, administration of Poly I:C significantly attenuated the neurological deficits, reduced infarction volume, and suppressed the increased levels of TNFα and IL-6 in the ischemic striatum and cortex.

Conclusion:

Poly I:C pretreatment exerts neuroprotective and anti-inflammatory effects in the simulated cerebral ischemia models, and the neuroprotection is at least in part due to the activation of the TLR3-TRIF pathway.