BACKGROUND & AIMS: :The anti-inflammatory potential of p38 mitogen-activated protein kinase (MAPK) inhibitors was coincidentally expanded to a dual inhibition of p38α MAPK and phosphodiesterase 4 (PDE4), and the potential benefits arising from the blockage of both inflammation-related enzymes were thoroughly investigated. The most promising compound, CBS-3595 (1), was successively evaluated in in vitro experiments as well as in ex vivo and in vivo preclinical studies after administration of 1 to rodents, dogs, and monkeys. The resulting data clearly indicated a potent suppression of tumor necrosis factor alpha release. For reconfirming the findings of the animal studies when administering 1 to healthy human volunteers, a phase I clinical trial was conducted. Apart from further information regarding the pharmacokinetic and pharmacodynamic characteristics of 1, it was demonstrated that dual inhibition of p38α MAPK and PDE4 is able to synergistically attenuate the excessive anti-inflammatory response.

背景与目标： : p38丝裂原活化蛋白激酶 (MAPK) 抑制剂的抗炎潜力被巧合地扩展为对p38α MAPK和磷酸二酯酶4 (PDE4) 的双重抑制，并且两种炎症相关酶的阻断所产生的潜在益处被彻底研究。在对啮齿动物，狗和猴子施用1之后，在体外实验以及体外和体内临床前研究中相继评估了最有希望的化合物CBS-3595 (1)。所得数据清楚地表明有效抑制了肿瘤坏死因子 α 的释放。为了在向健康的人类志愿者施用1时再次确认动物研究的结果，进行了I期临床试验。除了有关1的药代动力学和药效学特征的进一步信息外，还证明了p38α MAPK和PDE4的双重抑制能够协同减弱过度的抗炎反应。

BACKGROUND & AIMS: INTRODUCTION:Crohn's disease is an inflammatory bowel disease that has been debated to be associated with bacterial triggers such as Mycobacterium avium subspecies paratuberculosis (MAP). Standard treatment of Crohn's disease (CD) patients includes a family of immunomodulators and biologics such as Anti-Tumor Necrosis Factor alpha (Anti-TNFα). This cytokine in particular has been known to play vital roles in fighting microbial infections through formation and maintenance of granulomas. Areas covered: This perspective is focused on elucidating the negative effects of using Anti-TNFα therapeutic agents as a treatment option in CD patients who are more likely suspected to have MAP infection, and the role of other immunomodulators in MAP infection. Expert commentary: While treatment with Anti-TNFα is beneficial to reduce inflammation and to provide short term relief to the patients, it also compromises the immune system causing susceptibility to microbial infection. More than 50% of CD patients have shown no response to Anti-TNFα treatment which indicates a demand for introducing novel CD treatment in combination with antibiotics as a future CD treatment plan.

背景与目标： 简介: 克罗恩氏病是一种炎症性肠病，已被争论与细菌触发因素有关，例如鸟分枝杆菌亚种副结核杆菌 (MAP)。克罗恩病 (CD) 患者的标准治疗包括一系列免疫调节剂和生物制剂，例如抗肿瘤坏死因子 α (Anti-tnf α)。尤其是这种细胞因子在通过肉芽肿的形成和维持来对抗微生物感染中起着至关重要的作用。涵盖的领域: 该观点着重于阐明使用抗tnf α 治疗剂作为CD患者的治疗选择的负面影响，这些患者更可能怀疑患有MAP感染，以及其他免疫调节剂在MAP感染中的作用。专家评论: 虽然抗tnf α 治疗有利于减轻炎症并为患者提供短期缓解，但它也会损害免疫系统，导致对微生物感染的敏感性。超过50% 的CD患者对抗tnf α 治疗没有反应，这表明需要将新型CD治疗与抗生素结合作为未来的CD治疗计划。

BACKGROUND & AIMS: BACKGROUND:Endothelial cells are sensitive to changes in both blood components and mechanical stimuli. Endothelial cells may undergo phenotypic changes, such as changes in adhesion protein expression, under different shear stress conditions. Such changes may impact platelet and monocyte adhesion to endothelial cells. This phenomenon is linked to chronic vascular inflammation and the development of atherosclerosis. In the present study, we investigated the effects of ginkgolide B on platelet and monocyte adhesion to human umbilical vein endothelial cells (HUVECs) under different conditions of laminar shear stress. METHODS:Platelet and monocyte adhesion to endothelial cells was determined by the Bioflux 1000. HUVECs were incubated with ginkgolide B or aspirin for 12 h, and then TNFα was added for 2 h to induce the inflammatory response under conditions of 1 and 9 dyn/cm2 laminar shear stress. The protein expression was analyzed by Western blot. RESULTS:The number of platelets that adhered was greater under conditions of 1 dyn/cm2 than under conditions of 9 dyn/cm2 of laminar shear stress (74.8 ± 19.2 and 59.5 ± 15.1, respectively). Ginkgolide B reduced the tumor necrosis factor α (TNFα)-induced increase in platelet and monocyte adhesion to HUVECs at 1 and 9 dyn/cm2 of laminar shear stress. In TNFα-treated HUVECs, the number of monocytes that adhered was greater under conditions of 1 dyn/cm2 of laminar shear stress compared with 9 dyn/cm2 (29.1 ± 4.9 and 22.7 ± 3.7, respectively). Ginkgolide B inhibited the TNFα-induced expression of vascular cell adhesion molecule-1(VCAM-1), VE-cadherin, and Cx43 in HUVECs at 1 and 9 dyn/cm2. The expression of these proteins was not different between 1 and 9 dyn/cm2. CONCLUSIONS:Ginkgolide B suppressed platelet and monocyte adhesion under different conditions of laminar shear stress. Moreover, ginkgolide B reduced VCAM-1, VE-cadherin and Cx43 expression in TNFα-treated HUVECs under laminar shear stress. This suggested that ginkgolide B might shed light on the treatment of inflammation in atherosclerosis.

背景与目标：

BACKGROUND & AIMS: :The essential micronutrient zinc has long been known to be a functional component of diverse structural proteins and enzymes. More recently, important roles for free or loosely bound intracellular zinc as a signaling factor have been reported. Insufficient zinc intake was shown to exacerbate symptoms in mouse models of inflammation such as experimental colitis, while zinc supplementation was found to improve intestinal barrier function. Herein, we provide evidence that intracellular zinc is essential for maintaining intestinal epithelial integrity when cells are exposed to the inflammatory cytokine Tumor Necrosis Factor (TNF)α. Using the human intestinal Caco-2/TC7 cell line as an in vitro model, we demonstrate that depletion of intracellular zinc affects TNFα-triggered signaling by shifting intestinal cell fate from survival to death. The mechanism underlying this effect was investigated. We show that TNFα promotes a zinc-dependent survival pathway that includes modulation of gene expression of transcription factors and signaling proteins. We have identified multiple regulatory steps regulated by zinc availability which include the induction of cellular Inhibitor of APoptosis (cIAP2) mRNA, possibly through activation of Nuclear Factor-Kappa B (NF-κB), as both nuclear translocation of the p65 subunit of NF-κB and up-regulation of cIAP2 mRNA were impaired following zinc depletion. Moreover, X-linked inhibitor of apoptosis protein level was profoundly reduced by zinc depletion. Our results provide a possible molecular explanation for the clinical observation that zinc supplements ameliorate Crohn's disease symptoms and decrease intestinal permeability in experimental colitis.

背景与目标： : 必需微量营养素锌长期以来一直是多种结构蛋白和酶的功能成分。最近，已经报道了游离或松散结合的细胞内锌作为信号因子的重要作用。在小鼠炎症模型 (例如实验性结肠炎) 中，锌摄入不足会加剧症状，而补充锌可改善肠屏障功能。在本文中，我们提供了证据，当细胞暴露于炎性细胞因子肿瘤坏死因子 (TNF)α 时，细胞内锌对于维持肠上皮完整性至关重要。使用人肠Caco-2/TC7细胞系作为体外模型，我们证明了细胞内锌的耗竭通过将肠道细胞的命运从存活转移到死亡来影响tnf α 触发的信号传导。研究了这种作用的机制。我们表明tnf α 促进了锌依赖性的存活途径，包括转录因子和信号蛋白的基因表达的调节。我们已经确定了多个调节步骤受锌利用率的调节，包括诱导细胞凋亡抑制剂 (cIAP2) mRNA，可能是通过激活核因子 κ B (NF-κ B)，因为锌耗尽后NF-κ B p65亚基的核易位和cIAP2 mRNA的上调均受损。此外，锌耗竭使X连锁凋亡抑制剂蛋白水平大大降低。我们的结果为临床观察提供了可能的分子解释，即锌补充剂可改善实验性结肠炎的克罗恩病症状并降低肠通透性。

BACKGROUND & AIMS: :The mitogen-activated protein kinases (MAPKs) regulate a variety of cellular processes. The three main MAPK cascades are the extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinase (JNK), and p38 kinases. A typical MAPK cascade is composed of MAP3K-MAP2K-MAPK kinases that are held by scaffold proteins. Scaffolds function to assemble the protein tier and contribute to the specificity and efficacy of signal transmission. WD repeat domain 62 (WDR62) is a JNK scaffold protein, interacting with JNK, MKK7, and several MAP3Ks. The loss of WDR62 in human leads to microcephaly and pachygyria. Yet the role of WDR62 in cellular function is not fully studied. We used the CRISPR/Cas9 and short hairpin RNA approaches to establish a human breast cancer cell line MDA-MB-231 with WDR62 loss of function and studied the consequence to JNK signaling. In growing cells, WDR62 is responsible for the basal expression of c-Jun. In stressed cells, WDR62 specifically mediates TNFα-dependent JNK activation through the association with both the adaptor protein, TNF receptor-associated factor 2 (TRAF2), and the MAP3K protein, mixed lineage kinase 3. TNFα-dependent JNK activation is mediated by WDR62 in HCT116 and HeLa cell lines as well. MDA-MB-231 WDR62-knockout cells display increased resistance to TNFα-induced cell death. Collectively, WDR62 coordinates the TNFα receptor signaling pathway to JNK activation through association with multiple kinases and the adaptor protein TRAF2.

背景与目标： : 丝裂原活化蛋白激酶 (MAPKs) 调节多种细胞过程。三个主要的MAPK级联是细胞外信号调节激酶 (ERK)，c 6月N端激酶 (JNK) 和p38激酶。典型的MAPK级联反应由支架蛋白持有的MAP3K-MAP2K-MAPK激酶组成。支架的功能是组装蛋白质层，并有助于信号传递的特异性和有效性。WD重复结构域62 (WDR62) 是一种JNK支架蛋白，与JNK、MKK7和几个map3k相互作用。人类中WDR62的丢失会导致小头畸形和粗大症。然而，WDR62在细胞功能中的作用尚未得到充分研究。我们使用CRISPR/Cas9和短发夹RNA方法建立了具有WDR62功能丧失的人乳腺癌细胞系MDA-MB-231，并研究了JNK信号传导的后果。在生长的细胞中，WDR62负责c-6月的基础表达在应激细胞中，WDR62通过与衔接蛋白，TNF受体相关因子2 (TRAF2) 和MAP3K蛋白混合谱系激酶3结合，特异性介导TNF α 依赖性JNK激活。Tnf α 依赖性JNK激活也由HCT116和HeLa细胞系中的WDR62介导。MDA-MB-231 WDR62-knockout细胞显示对tnf α 诱导的细胞死亡的抗性增加。总的来说，WDR62通过与多种激酶和衔接蛋白traf2结合，协调tnf α 受体信号通路与JNK激活。

BACKGROUND & AIMS: :Tumor necrosis factor (TNF)-α is produced in brain in response to acute cerebral ischemia, and promotes neuronal apoptosis. Biologic TNF inhibitors (TNFIs), such as the etanercept, cannot be developed as new stroke treatments because these large molecule drugs do not cross the blood-brain barrier (BBB). A BBB-penetrating biologic TNFI was engineered by fusion of the type II human TNF receptor (TNFR) to each heavy chain of a genetically engineered chimeric monoclonal antibody (MAb) against the mouse transferrin receptor (TfR), designated as cTfRMAb-TNFR fusion protein. The cTfRMAb domain of the fusion protein acts as a molecular Trojan horse to deliver the fused TNFR across the BBB. Etanercept or the cTfRMAb-TNFR fusion protein (1 mg/kg) was administered intravenously in adult mice subjected to 1-hour reversible middle cerebral artery occlusion up to 90 minutes after the occlusion. Neuroprotection was assessed at 24 hours or 7 days after occlusion. The cTfRMAb-TNFR fusion protein treatment caused a significant 45%, 48%, 42%, and 54% reduction in hemispheric, cortical, and subcortical stroke volumes, and neural deficit, respectively. Intravenous etanercept had no therapeutic effect. Biologic TNFIs can be reengineered for BBB penetration, and the IgG-TNFR fusion protein is therapeutic after delayed intravenous administration in experimental stroke.

背景与目标： : 肿瘤坏死因子 (TNF)-α 在大脑中产生，以响应急性脑缺血，并促进神经元凋亡。生物肿瘤坏死因子抑制剂 (TNFIs)，如依那西普，不能作为新的中风治疗方法开发，因为这些大分子药物不会穿过血脑屏障 (BBB)。通过将II型人TNF受体 (TNFR) 融合到针对小鼠转铁蛋白受体 (TfR) 的基因工程嵌合单克隆抗体 (MAb) 的每个重链上，工程化了BBB穿透性生物TNFI，称为cTfRMAb-TNFR融合蛋白。融合蛋白的cTfRMAb结构域充当分子特洛伊木马，将融合的TNFR传递穿过BBB。依那西普或cTfRMAb-TNFR融合蛋白 (1  mg/kg) 在成年小鼠中静脉注射，该成年小鼠在闭塞后长达90  分钟的可逆性大脑中动脉闭塞1小时。在闭塞后24小时或7天评估神经保护作用。cTfRMAb-TNFR融合蛋白处理分别导致半球，皮质和皮质下中风体积以及神经缺陷的显着45%，48%，42% 和54% 减少。静脉注射依那西普无疗效。可以对生物TNFIs进行重组以进行BBB渗透，并且在实验性中风中延迟静脉内给药后，IgG-TNFR融合蛋白可以治疗。

BACKGROUND & AIMS: :The deprivation of zinc, caused by malnutrition or as a consequence of aging or disease, strongly affects immune cell functions, causing higher frequency of infections. Among other effects, an increased production of reactive oxygen species (ROS) and proinflammatory cytokines has been observed in zinc-deficient patients, but the underlying mechanisms were unknown. The aim of the current study was to define mechanisms explaining the increase in proinflammatory cytokine production during zinc deficiency, focusing on the role of epigenetic and redox-mediated mechanisms. Interleukin (IL)-1β and tumor necrosis factor (TNF)α production was increased in HL-60 cells under zinc deficiency. Analyses of the chromatin structure demonstrated that the elevated cytokine production was due to increased accessibilities of IL-1β and TNFα promoters in zinc-deficient cells. Moreover, the level of nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) oxidase-produced ROS was elevated under zinc deficiency, subsequently leading to p38 mitogen-activated protein kinase (MAPK) phosphorylation. The increased activation of p38 MAPK appeared to be necessary for posttranscriptional processes in IL-1β and TNFα synthesis. These data demonstrate that IL-1β and TNFα expression under zinc deficiency is regulated via epigenetic and redox-mediated mechanisms. Assuming an important role of zinc in proinflammatory cytokine regulation, this should encourage research in the use of zinc supplementation for treatment of inflammatory diseases.

背景与目标： : 由于营养不良或由于衰老或疾病而导致的锌剥夺强烈影响免疫细胞功能，导致更高的感染频率。除其他影响外，在缺锌患者中观察到活性氧 (ROS) 和促炎细胞因子的产生增加，但其潜在机制尚不清楚。当前研究的目的是定义解释锌缺乏期间促炎细胞因子产生增加的机制，重点是表观遗传和氧化还原介导机制的作用。缺锌条件下HL-60细胞中白细胞介素 (IL)-1β 和肿瘤坏死因子 (TNF)α 的产生增加。对染色质结构的分析表明，细胞因子产生的增加是由于缺锌细胞中IL-1β 和TNF α 启动子的可及性增加。此外，缺锌条件下烟酰胺腺嘌呤二核苷酸磷酸氧化酶 (NADPH) 氧化酶产生的ROS水平升高，随后导致p38丝裂原活化蛋白激酶 (MAPK) 磷酸化。p38 MAPK的激活增加似乎是IL-1β 和tnf α 合成中的转录后过程所必需的。这些数据证明了在锌缺乏下IL-1β 和tnf α 的表达是通过表观遗传和氧化还原介导的机制来调节的。假设锌的重要作用在促炎细胞因子调节中，这应该鼓励研究使用锌补充剂治疗炎症性疾病。

BACKGROUND & AIMS: OBJECTIVE:We previously demonstrated that the activation of transient receptor potential vanilloid 1 (TRPV1), a nociceptive ion channel receptor, by capsaicin led to the up-regulation of the osteoprotegerin (OPG)/receptor activator of nuclear factor kappa B ligand (RANKL) ratio in human periodontal ligament (HPDL) cells. Since TRPV1 is recognised as one of the thermo-sensitive cation channels, this study investigated the response of TRPV1 to thermal stimulation in HPDL cells. METHODS:HPDL cells were incubated at 45°C for thermal stimulation. The mRNA expression of OPG, RANKL, tumour necrosis factor α (TNFα), and interleukin-1 β (IL-1β) was determined by using RT-PCR. OPG secretion and RANKL protein expression were analysed by ELISA and Western blot analysis, respectively. The mechanisms of heat-induced TNFα expression were studied using several TRPV1 inhibitors. RESULTS:In contrast to capsaicin, thermal stimulation had no effect on OPG or RANKL expression. Interestingly, the mRNA expression of TNFα, but not IL-1β, was increased by heat. Using TRPV1 antagonists, we confirmed that TNFα up-regulation was mediated by TRPV1. Phospholipase C (PLC) was previously shown to be involved in capsaicin-induced OPG expression. However, we found that protein kinase C, not PLC, was required for heat-induced TNFα expression. Additionally, the use of cytochalasin D, an inhibitor of actin polymerisation, revealed that cytoskeleton rearrangement might be an important mechanism for cellular sensing of thermal stimuli. CONCLUSION:Our results indicate that TRPV1 plays a multi-functional role in HPDL cells depending on the stimuli. In response to heat, TRPV1 activation leads to the induction of TNFα expression.

背景与目标：

BACKGROUND & AIMS: :Transient Receptor Potential Melastatin-8 (TRPM8) reportedly plays a fundamental role in a variety of processes including cold sensation, thermoregulation, pain transduction and tumorigenesis. However, the role of TRPM8 in inflammation under cold conditions is not well known. Since cooling allows the convergence of primary injury and injury-induced inflammation, we hypothesized that the mechanism of the protective effects of cooling might be related to TRPM8. We therefore investigated the involvement of TRPM8 activation in the regulation of inflammatory cytokines. The results showed that TRPM8 expression in the mouse hypothalamus was upregulated when the ambient temperature decreased; simultaneously, tumor necrosis factor-alpha (TNFα) was downregulated. The inhibitory effect of TRPM8 on TNFα was mediated by nuclear factor kappa B (NFκB). Specifically, cold stress stimulated the expression of TRPM8, which promoted the interaction of TRPM8 and NFκB, thereby suppressing NFκB nuclear localization. This suppression consequently led to the inhibition of TNFα gene transcription. The present data suggest a possible theoretical foundation for the anti-inflammatory role of TRPM8 activation, providing an experimental basis that could contribute to the advancement of cooling therapy for trauma patients.

背景与目标： : 据报道，瞬时受体电位Melastatin-8 (TRPM8) 在各种过程中起着重要作用，包括冷感，体温调节，疼痛转导和肿瘤发生。然而，TRPM8在寒冷条件下炎症中的作用尚不清楚。由于冷却可以使原发性损伤和损伤引起的炎症趋于一致，因此我们假设冷却的保护作用机制可能与trpm8有关。因此，我们研究了TRPM8激活参与炎症细胞因子的调节。结果表明，当环境温度降低时，TRPM8在小鼠下丘脑中的表达上调; 同时，肿瘤坏死因子 α (tnf α) 下调。TRPM8对tnf α 的抑制作用由核因子 κ B (nf κ B) 介导。具体来说，冷应激刺激TRPM8的表达，从而促进TRPM8和nf κ b的相互作用，从而抑制nf κ b核定位。因此，这种抑制导致tnf α 基因转录的抑制。目前的数据为TRPM8激活的抗炎作用提供了可能的理论基础，为创伤患者的冷却疗法的发展提供了实验基础。

BACKGROUND & AIMS: :In cutaneous leishmaniasis, Leishmania amazonensis activates macrophage double-stranded, RNA-activated protein kinase R (PKR) to promote parasite growth. In our study, Leishmania major grew normally in RAW cells, RAW-expressing dominant-negative PKR (PKR-DN) cells, and macrophages of PKR-knockout mice, revealing that PKR is dispensable for L. major growth in macrophages. PKR activation in infected macrophages with poly I:C resulted in parasite death. Fifty percent of L. major-knockout lines for the ecotin-like serine peptidase inhibitor (ISP2; Δisp2/isp3), an inhibitor of neutrophil elastase (NE), died in RAW cells or macrophages from 129Sv mice, as a result of PKR activation. Inhibition of PKR or NE or neutralization of Toll-like receptor 4 or 2(TLR4 or TLR2) prevented the death of Δisp2/isp3. Δisp2/isp3 grew normally in RAW-PKR-DN cells or macrophages from 129Sv pkr(-/-), tlr2(-/-), trif(-/-), and myd88(-/-) mice, associating NE activity, PKR, and TLR responses with parasite death. Δisp2/isp3 increased the expression of mRNA for TNF-α by 2-fold and of interferon β (IFNβ) in a PKR-dependent manner. Antibodies to TNF-α reversed the 95% killing by Δisp2/isp3, whereas they grew normally in macrophages from IFN receptor-knockout mice. We propose that ISP2 prevents the activation of PKR via an NE-TLR4-TLR2 axis to control innate responses that contribute to the killing of L. major.-Faria, M. S., Calegari-Silva, T. C., de Carvalho Vivarini, A., Mottram, J. C., Lopes, U. G., Lima, A. P. C. A. Role of protein kinase R in the killing of Leishmania major by macrophages in response to neutrophil elastase and TLR4 via TNFα and IFNβ.

背景与目标： : 在皮肤利什曼病中，亚马逊利什曼原虫激活巨噬细胞双链RNA激活的蛋白激酶R (PKR) 以促进寄生虫的生长。在我们的研究中，利什曼原虫major在原始细胞，原始表达的显性负PKR (PKR-DN) 细胞和巨噬细胞中正常生长PKR-敲除小鼠，表明PKR对于巨噬细胞的L. major生长是必不可少的。poly I:C感染的巨噬细胞中的PKR激活导致寄生虫死亡。作为PKR激活的结果，50% 的ecotin样丝氨酸肽酶抑制剂 (ISP2; Δisp2/isp3) (一种中性粒细胞弹性蛋白酶 (NE) 的抑制剂) 的L. major敲除品系死于129Sv小鼠的原始细胞或巨噬细胞。抑制PKR或NE或中和Toll样受体4或2(TLR4或TLR2) 可防止 Δisp2/isp3死亡。Δisp2/isp3在来自129Sv PKR (-/-)，tlr2(-/-)，trif(-/-) 和myd88(-/-) 小鼠的RAW-pkr-DN细胞或巨噬细胞中正常生长，使NE活性，PKR和TLR反应与寄生虫死亡。Δ isp2/isp3以PKR依赖性方式使TNF-α 的mRNA表达增加2倍，干扰素 β (ifn β) 的mRNA表达增加2倍。TNF-α 抗体逆转了 Δ isp2/isp3对95% 的杀伤，而它们在来自IFN受体敲除小鼠的巨噬细胞中正常生长。我们建议ISP2通过NE-TLR4-TLR2轴阻止PKR的激活，以控制有助于杀死L的先天反应。少校。-法里亚，男S、，卡莱加里-席尔瓦，T。C.，德卡瓦略·维瓦里尼，A.，莫特拉姆，JC.，洛佩斯，美国。G.，利马，A。P。C.A。蛋白激酶R在巨噬细胞通过tnf α 和ifn β 响应中性粒细胞弹性蛋白酶和TLR4杀死利什曼原虫中的作用。

BACKGROUND & AIMS: :While apoptosis has been considered to be identical to programmed cell death, necroptosis, which is morphologically related to necrosis, has emerged as a novel type of programmed cell death. Necroptosis depends on two structurally related kinases, receptor-interacting serine-threonine kinase (RIPK)1 and RIPK3. RIPK1 is activated through oligomerization of upstream adaptor molecules such as Fas-associated protein with death domain (FADD) and TNF receptor-associated death domain (TRADD) that are triggered by TNFα or Fas ligand. Activated RIPK1 subsequently interacts with and activates RIPK3, resulting in necroptosis. However, contribution of oxidative stress to execution of necroptosis is still controversial. We found that a selective inhibitor for RIPK1, necrostatin-1 (Nec-1) significantly blocked TNFα-induced cell death and ROS accumulation in NF-κB activation-deficient cells. This suggests that these cells mostly died by necroptosis upon TNFα stimulation. Intriguingly, an antioxidant, butylated hydroxyanisole (BHA) blocked TNFα-induced necroptosis and ROS accumulation in NF-κB activation-deficient cells. However, Nec-1, but not BHA, inhibited TNFα-induced phosphorylation of RIPK1 in these cells, suggesting that ROS play a crucial role in execution of necroptosis downstream of RIPK1 activation. Structural and functional analyses using BHA related compounds revealed that both tert-butyl and hydroxy groups of BHA are crucial for its anti-necroptotic function. Together, these results suggest that TNFα-induced necroptosis is tightly associated with oxidative stress, and oxidative stress is induced downstream of RIPK1 activation.

背景与目标： : 虽然细胞凋亡被认为与程序性细胞死亡相同，但在形态上与坏死有关的坏死性坏死已成为一种新型的程序性细胞死亡。坏死性依赖于两种结构相关的激酶，受体相互作用的丝氨酸-苏氨酸激酶 (RIPK)1和ripk3。RIPK1通过由TNF α 或Fas配体触发的上游衔接子分子 (例如具有死亡结构域 (FADD) 和TNF受体相关死亡结构域 (TRADD) 的Fas相关蛋白) 的寡聚而被激活。激活的RIPK1随后与RIPK3相互作用并激活，导致坏死性。然而，氧化应激对坏死性坏死的影响仍然存在争议。我们发现RIPK1，necrostatin-1 (Nec-1) 的选择性抑制剂可显着阻止tnf α 诱导的NF-κ b活化缺陷细胞中的细胞死亡和ROS积累。这表明这些细胞在tnf α 刺激下大多死于坏死性。有趣的是，抗氧化剂丁基羟基茴香醚 (BHA) 阻止了tnf α 诱导的坏死和ROS在NF-κ b活化缺陷细胞中的积累。然而，Nec-1 (而不是BHA) 抑制了tnf α 诱导的这些细胞中RIPK1的磷酸化，这表明ROS在RIPK1激活下游的坏死性坏死中起着至关重要的作用。使用BHA相关化合物进行的结构和功能分析表明，BHA的叔丁基和羟基对于其抗坏死功能至关重要。总之，这些结果表明，tnf α 诱导的坏死性坏死与氧化应激密切相关，而氧化应激是在RIPK1激活的下游诱导的。

BACKGROUND & AIMS: :Pro-inflammatory cytokines secreted by macrophages and other cell types are implicated as intraovarian factors affecting different aspects of ovarian function including follicle and corpus luteum 'turnover', steroidogenesis and angiogenesis. Here, we compared granulosal (GC) and thecal (TC) expression of TNF, IL6 and their receptors (TNFRSF1A, TNFRSF1B and IL6R) during bovine antral follicle development; all five mRNA transcripts were detected in both GC and TC and statistically significant cell-type and follicle stage-related differences were evident. Since few studies have examined cytokine actions on TC steroidogenesis, we cultured TC under conditions that retain a non-luteinized 'follicular' phenotype and treated them with TNFα and IL6 under basal and LH-stimulated conditions. Both TNFα and IL6 suppressed androgen secretion concomitantly with CYP17A1 and LHCGR mRNA expression. In addition, TNFα reduced INSL3, HSD3B1 and NOS3 expression but increased NOS2 expression. IL6 also reduced LHCGR and STAR expression but did not affect HSD3B1, INSL3, NOS2 or NOS3 expression. As macrophages are a prominent source of these cytokines in vivo, we next co-cultured TC with macrophages and observed an abolition of LH-induced androgen production accompanied by a reduction in CYP17A1, INSL3, LHCGR, STAR, CYP11A1 and HSD3B1 expression. Exposure of TC to bacterial lipopolysaccharide also blocked LH-induced androgen secretion, an effect reduced by a toll-like receptor blocker (TAK242). Collectively, the results support an inhibitory action of macrophages on thecal androgen production, likely mediated by their secretion of pro-inflammatory cytokines that downregulate the expression of LHCGR, CYP17A1 and INSL3. Bovine theca interna cells can also detect and respond directly to lipopolysaccharide.

背景与目标： : 巨噬细胞和其他细胞类型分泌的促炎细胞因子被认为是影响卵巢功能不同方面的卵巢内因素，包括卵泡和黄体 “周转”，类固醇生成和血管生成。在这里，我们比较了牛窦卵泡发育过程中TNF，IL6及其受体 (TNFRSF1A，TNFRSF1B和IL6R) 的颗粒 (GC) 和thecal (TC) 表达; 在GC和TC中均检测到所有五个mRNA转录本，并且具有统计学意义的细胞类型和卵泡阶段相关差异。由于很少有研究检查细胞因子对TC类固醇生成的作用，因此我们在保留非黄体化 “卵泡” 表型的条件下培养TC，并在基础和LH刺激的条件下用tnf α 和IL6处理它们。Tnf α 和IL6均与CYP17A1和LHCGR mRNA表达同时抑制雄激素分泌。此外，tnf α 降低了INSL3，HSD3B1和NOS3的表达，但增加了NOS2的表达。IL6还降低了LHCGR和STAR的表达，但不影响HSD3B1，INSL3，NOS2或NOS3的表达。由于巨噬细胞是体内这些细胞因子的主要来源，我们接下来将TC与巨噬细胞共培养，并观察到LH诱导的雄激素产生的消除，伴随着CYP17A1，INSL3，LHCGR，STAR，CYP11A1和HSD3B1表达的降低。将TC暴露于细菌脂多糖中也会阻止LH诱导的雄激素分泌，这种作用被toll样受体阻滞剂 (TAK242) 降低。总的来说，结果支持巨噬细胞对鞘雄激素产生的抑制作用，可能是由其分泌促炎细胞因子介导的，这些细胞因子下调了LHCGR，CYP17A1和insl3的表达。牛卵泡膜细胞也可以检测脂多糖并直接响应。

BACKGROUND & AIMS: :TLR4 signalling through the MyD88 and TRIF-dependent pathways initiates translocation of the transcription factor NF-κB into the nucleus. In cell population studies using mathematical modeling and functional analyses, Cheng et al. suggested that LPS-driven activation of MyD88, in the absence of TRIF, impairs NF-κB translocation. We tested the model proposed by Cheng et al. using real-time single cell analysis in macrophages expressing EGFP-tagged p65 and a TNFα promoter-driven mCherry. Following LPS stimulation, cells lacking TRIF show a pattern of NF-κB dynamics that is unaltered from wild-type cells, but activation of the TNFα promoter is impaired. In macrophages lacking MyD88, there is minimal NF-κB translocation to the nucleus in response to LPS stimulation, and there is no activation of the TNFα promoter. These findings confirm that signalling through MyD88 is the primary driver for LPS-dependent NF-κB translocation to the nucleus. The pattern of NF-κB dynamics in TRIF-deficient cells does not, however, directly reflect the kinetics of TNFα promoter activation, supporting the concept that TRIF-dependent signalling plays an important role in the transcription of this cytokine.

背景与目标： : TLR4信号通过MyD88和TRIF依赖途径启动转录因子NF-κ b向细胞核的转运。在使用数学建模和功能分析的细胞群体研究中，Cheng等人。建议在没有TRIF的情况下，LPS驱动的MyD88激活会损害NF-κ b易位。我们测试了Cheng等人提出的模型。在表达EGFP标记的p65和tnf α 启动子驱动的mCherry的巨噬细胞中使用实时单细胞分析。LPS刺激后，缺乏TRIF的细胞显示出NF-κ b动力学模式，该模式与野生型细胞没有改变，但tnf α 启动子的激活受到损害。在缺乏MyD88的巨噬细胞中，响应LPS刺激，NF-κ b向细胞核的易位很小，并且tnf α 启动子没有激活。这些发现证实，通过MyD88的信号传导是LPS依赖性NF-κ b易位到细胞核的主要驱动因素。但是，TRIF缺陷细胞中NF-κ b动力学的模式并不直接反映tnf α 启动子激活的动力学，从而支持TRIF依赖性信号传导在该细胞因子的转录中起重要作用的概念。

BACKGROUND & AIMS: :TNFα is persistently elevated in many injury and disease conditions. Previous reports of cytotoxicity of TNFα for oligodendrocytes and their progenitors suggest that the poor endogenous remyelination in patients with traumatic injury or multiple sclerosis may be due in part to persistent inflammation. Understanding the effects of inflammatory cytokines on potential cell therapy candidates is therefore important for evaluating the feasibility of their use. In this study, we assessed the effects of long term exposure to TNFα on viability, proliferation, migration and TNFα receptor expression of cultured rat olfactory ensheathing cells (OECs) and Schwann cells (SCs). Although OECs and SCs transplanted into the CNS produce similar myelinating phenotypes, and might be expected to have similar therapeutic uses, we report that they have very different sensitivities to TNFα. OECs exhibited positive proliferative responses to TNFα over a much broader range of concentrations than SCs. Low TNFα concentrations increased proliferation and migration of both OECs and SCs, but SC number declined in the presence of 100 ng/ml or higher concentrations of TNFα. In contrast, OECs exhibited enhanced proliferation even at high TNFα concentrations (up to 1 µg/ml) and showed no evidence of TNF cytotoxicity even at 4 weeks post-treatment. Furthermore, while both OECs and SCs expressed TNFαR1 and TNFαR2, TNFα receptor levels were downregulated in OECs after exposure to100 ng/ml TNFα for 5-7 days, but were either elevated or unchanged in SCs. These results imply that OECs may be a more suitable cell therapy candidate if transplanted into areas with persistent inflammation.

背景与目标： : tnf α 在许多损伤和疾病情况下持续升高。先前关于tnf α 对少突胶质细胞及其祖细胞的细胞毒性的报道表明，创伤性损伤或多发性硬化症患者的内源性髓鞘再生不良可能部分归因于持续的炎症。因此，了解炎性细胞因子对潜在细胞治疗候选者的影响对于评估其使用的可行性非常重要。在这项研究中，我们评估了长期暴露于tnf α 对培养的大鼠嗅鞘细胞 (OECs) 和雪旺细胞 (SCs) 的活力，增殖，迁移和tnf α 受体表达的影响。尽管移植到CNS中的OECs和SCs产生相似的髓鞘表型，并且可能具有相似的治疗用途，但我们报告它们对tnf α 的敏感性非常不同。OECs在比SCs更宽的浓度范围内对tnf α 表现出阳性增殖反应。低tnf α 浓度增加了OECs和SCs的增殖和迁移，但在100 ng/ml或更高浓度的tnf α 存在下SC数下降。相反，即使在高TNF α 浓度 (高达1 µ g/ml) 下，OECs也显示出增强的增殖，并且即使在治疗后4周也没有显示出TNF细胞毒性的证据。此外，尽管OECs和SCs均表达tnf αr1和tnf αr2，但暴露于100 ng/ml tnf α 5-7天后，OECs中的tnf α 受体水平下调，但SCs中的tnf α 受体水平升高或保持不变。这些结果表明，如果将OECs移植到具有持续炎症的区域中，则OECs可能是更合适的细胞治疗候选者。

BACKGROUND & AIMS: BACKGROUND:The role of peripheral tumor necrosis factor alpha (TNFα) in inflammatory bowel disease (IBD) is well established, but its central nervous system (CNS) effects are not understood. Thrombin, another mediator of inflammation in IBD, has been implicated in CNS vagal neuron apoptosis in the dorsal motor nucleus of the vagus (DMV). This study evaluates DMV TNFα exposure, characterizes effects of TNFα on DMV neurons, and identifies a relationship between DMV TNFα and thrombin in IBD. METHODS:2,4,6-Trinitrobenzene sulfonic acid was administered via enema to induce colonic inflammation in rats. TNFα in serum, cerebrospinal fluid (CSF), and DMV tissues were determined by ELISA and DMV TNFα expression by quantitative reverse transcription PCR (RT-PCR). TNFα was administered into the fourth intracerebral ventricle (4 V) adjacent to the DMV, with and without blockade of TNF receptor 1 (TNFR1) and the thrombin receptor proteinase-activated receptor 1 (PAR1). Immunofluorescence was used to evaluate microglial activation (Cd11b) and prothrombin presence in DMV sections. Apoptosis was examined using terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) and activated caspase-3 immunofluorescence. RESULTS:IBD is associated with increased TNFα protein in serum, CSF, and DMV tissue; DMV TNFα transcription is also increased. TNFα (4 V) caused a 54 % increase in microglial activation, a 27 % increase in DMV prothrombin protein, and a 31 % increase in vagal neuron apoptosis by TUNEL. There was a 52 % increase in activated caspase-3 immunofluorescence in TNFα-treated animals (p < 0.05). All effects of 4 V TNFα were prevented by TNFR1 blockade. TNFα-induced apoptosis was prevented by PAR1 blockade. CONCLUSIONS:IBD is associated with DMV exposure to TNFα, causing excess DMV prothrombin and vagal apoptosis.

背景与目标：