NLRP3炎性小体研究新进展

NLRP3炎性小体是一种分子量约为700Kda的大分子多蛋白复合体,能被多种病原相关的分子模式或损伤相关的分子模式活化,对固有免疫系统免疫功能的发挥具有极其重要的作用。但如果其被过度激活则可通过活化的半胱天冬酶-1持续地将pro-IL-1β和pro-IL-18剪切为成熟的IL-1β和IL-18,进而激活下游信号转导通路,产生大量的炎性介质,引起机体发生严重的炎症反应,最终促进多种炎症性疾病的发生与发展,如Muckle—wells综合征、2型糖尿病、非酒精性脂肪肝、动脉粥样硬化、炎症性肠病和阿尔兹海默病等。因此,对NLRP3炎性小体进行深入的研究不仅有助于阐释固有免疫系统如何有效地发挥其免疫功能,而且作为系列炎症反应的核心,NLRP3炎性小体：还可能成为多种炎症性疾病防治的新靶点。我们就NLRP3炎性小体的结构与功能,激活与调控,分布与疾病的近期研究作一综：违。     

NLRP3 炎性小体研究新进展

NLRP3 炎性小体是一种分子量约为700Kda 的大分子多蛋白复合体,能被多种病原相关的分子模式或损伤相关的分子模式 活化,对固有免疫系统免疫功能的发挥具有极其重要的作用。但如果其被过度激活则可通过活化的半胱天冬酶-1 持续地将 pro-IL-1茁和pro-IL-18 剪切为成熟的IL-1茁和IL-18,进而激活下游信号转导通路,产生大量的炎性介质,引起机体发生严重的炎 症反应,最终促进多种炎症性疾病的发生与发展,如Muckle-Wells综合征、2 型糖尿病、非酒精性脂肪肝、动脉粥样硬化、炎症性肠 病和阿尔兹海默病等。因此,对NLRP3 炎性小体进行深入的研究不仅有助于阐释固有免疫系统如何有效地发挥其免疫功能,而 且作为系列炎症反应的核心,NLRP3 炎性小体还可能成为多种炎症性疾病防治的新靶点。我们就NLRP3 炎性小体的结构与功 能,激活与调控,分布与疾病的近期研究作一综述。     

CUL1基因对MPP+诱导的SH-SY5Y细胞存活和NLRP3炎症体通路的影响

摘要 目的：探究Cullin1（CUL1）基因对1-甲基-4-苯基吡啶离子（MPP+）诱导的SH-SY5Y细胞存活和核苷酸结合寡聚化结构域样受体3（NLRP3）炎症体通路的影响。方法：（1）将SH-SY5Y细胞分为NC组、NC-sh组、CUL1-sh组、NC-OE组和CUL1-OE组。使用Lipofectamine 2000试剂对细胞转染相应的慢病毒。（2）将SH-SY5Y细胞分为Control组、MPP+组和MPP++CUL1-OE组。MPP+组和MPP++CUL1-OE组细胞使用1 mmol/L的MPP+处理48 h,Control组细胞正常培养。通过MTT法检测细胞增殖,通过Annexin V-FITC/PI双染色法和TUNEL染色法检测细胞凋亡,通过qRT-PCR检测CUL1的mRNA水平,通过Western blot检测CUL1、NLRP3、凋亡相关斑点样蛋白（ASC）、cleaved caspase-1、白细胞介素（IL）-1β和IL-18蛋白水平。通过ELISA法检测细胞培养上清液中IL-1β和IL-18水平。结果：（1）与NC组和NC-sh组比较,CUL1-sh组CUL1的mRNA和蛋白相对表达量降低,相对细胞活力降低,Annexin V-FITC/PI阳性率和TUNEL阳性率升高,NLRP3、ASC、cleaved caspase-1、IL-1β和IL-18蛋白相对表达量以及细胞培养上清液中IL-1β和IL-18水平升高（P<0.05）。与NC组和NC-OE组比较,CUL1-OE组CUL1的mRNA和蛋白相对表达量升高,相对细胞活力升高,Annexin V-FITC/PI阳性率和TUNEL阳性率降低,NLRP3、ASC、cleaved caspase-1、IL-1β和IL-18蛋白相对表达量以及细胞培养上清液中IL-1β和IL-18水平降低（P<0.05）。（2）与Control组比较,MPP+组CUL1的mRNA和蛋白相对表达量降低,相对细胞活力降低,Annexin V-FITC/PI阳性率和TUNEL阳性率升高,NLRP3、ASC、cleaved caspase-1、IL-1β和IL-18蛋白相对表达量以及细胞培养上清液中IL-1β和IL-18水平升高（P<0.05）。与MPP+组比较,MPP++CUL1-OE组CUL1的mRNA和蛋白相对表达量升高,相对细胞活力升高,Annexin V-FITC/PI阳性率和TUNEL阳性率降低,NLRP3、ASC、cleaved caspase-1、IL-1β和IL-18蛋白相对表达量以及细胞培养上清液中IL-1β和IL-18水平降低（P<0.05）。结论：CUL1可能通过抑制NLRP3炎症体激活促进MPP+诱导的SH-SY5Y细胞存活。     

NLRP1炎性体

核苷酸结合寡聚化结构域样受体蛋白1(NLRP1)炎性体是NLRP1在识别胞内病原相关分子模式(PAMP)后与凋亡相关斑点样蛋白(ASC)以及半胱天冬氨酸酶(Caspase-1、Caspase-5)前体等分子结合形成的蛋白复合物,活化后促进IL-1β、IL-18、IL-33等炎症因子的成熟和释放,在先天性免疫中发挥重要作用。本文主要介绍了NLRP1炎性体的组成、激活机制、信号通路、负向调控及生物学功能,综述了NLRP1炎性体在炭疽、弓形虫病、泛发型白癜风、肠炎、牛皮癣等疾病中作用的研究进展。     

NLRP3炎症小体与心脏骤停复苏后心肌缺血再灌注损伤的研究进展

心脏骤停已成为世界范围内的重大公共卫生问题,及时有效的心肺复苏可以挽救生命,改善心脏骤停患者的预后。尽管心肺复苏技术取得了进步,但与心脏骤停相关的死亡率仍然很高。NLRP3炎症小体是细胞内多种蛋白质构成的复合物,在先天免疫中起着重要作用。组织损伤后,NLRP3炎症小体激活产生大量细胞因子如白细胞介素(IL)-1β和IL-18,最终导致炎症性细胞死亡(细胞焦亡)。虽然适度的炎症反应有利于损伤组织愈合,但过度的NLRP3炎症小体激活会产生不利影响。NLRP3炎症小体在大量心血管疾病(cardiovascular diseases, CVDs)中发挥关键作用。心脏骤停和复苏后缺血再灌注损伤(I/R损伤)可以通过各种信号通路激活NLRP3炎症小体。抑制NLRP3炎症小体活性可以改善心脏骤停和复苏后缺血再灌注损伤。该文将讨论NLRP3炎症小体在心脏骤停和复苏过程中对细胞损伤的作用,同时针对抑制NLRP3炎症小体激活,改善心脏骤停和复苏后缺血再灌注损伤的治疗方法进行系统阐述。     

右美托咪定上调HIF-1α抑制NLRP3炎性体的激活减轻糖尿病小鼠心肌缺血再灌注损伤

摘要 目的：探讨右美托咪定（DEX）对糖尿病小鼠心肌缺血再灌注损伤的保护作用及可能分子机制。方法：将60只8周龄的SPF级C57BL小鼠高脂喂养6周,第7周通过腹腔注射链脲佐菌素（STZ）45 mg/kg/天,1次/天,连续5天,建立2型糖尿病模型,建模后采用随机数字表法分为假手术组（sham组）、缺血再灌注组（I/R组）、缺氧诱导因子-1α（HIF-1α）抑制剂2ME2组（2ME2组）、DEX组、DEX+2ME2组（DM组）,每组12只。假手术组仅切开皮肤后缝合,其余四组开胸结扎冠状动脉左前降支,缺血60 min后松开结扎线结,再灌注120 min建立缺血再灌注损伤模型。于再灌注120 min时抽取小鼠腹主动脉血,ELISA检测血清肌钙蛋白I（cTnI）、白介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）的浓度,随后处死小鼠,分离左心室,HE染色观察心肌组织形态结构,Western blot检测HIF-1α、nod样受体蛋白3（NLRP3）的表达量,再灌注24 h时超声心动图评估心功能。结果：与sham组相比,I/R组、2ME2组、DEX组、DM组cTnI、IL-1β,TNF-α浓度明显升高,心肌组织结构紊乱,心肌纤维断裂增加,心肌细胞明显肿胀,炎性细胞浸润增加,心肌组织NLRP3表达量显著增加,每搏量（SV）、射血分数（EF）%、短轴缩短率（FS）%明显下降（P＜0.05）;与I/R组相比,DEX组、DM组HIF-1α表达量明显增加,NLRP3表达明显降低,cTnI、IL-1β,TNF-α浓度明显下降,心肌组织结构明显改善,炎性细胞浸润明显减少,SV,EF、FS明显升高（P＜0.05）;与DEX组相比,DM组HIF-1α表达量明显降低,NLRP3表达量明显增加,cTnI、IL-1β、TNF-α浓度明显增加,心肌组织结构紊乱,心肌纤维断裂增加,心肌细胞明显肿胀,炎性细胞浸润增加,SV,EF、FS明显降低（P＜0.05）。结论：DEX可能通过上调心肌组织HIF-1α的表达,抑制NLRP3炎性体的激活,减轻心脏炎症反应,改善糖尿病小鼠心肌缺血再灌注损伤。     

细胞焦亡在创伤性脑损伤中的作用

创伤性脑损(traumatic brain injury, TBI)是全球脑损伤患者死亡和致残的主要原因。创伤导致的不受控的内源性介质释放作为危险信号被危险相关分子模式(damage associated molecular patterns,DAMPs)感知,触发炎性体(inflammasome)蛋白复合物组装。炎性体是TBI后的关键细胞内多蛋白信号感知平台,炎性体组装可以诱导含半胱氨酸的天冬氨酸蛋白水解酶-1(cysteinyl aspartate specific proteinase-1, caspase-1)活化促使白细胞介素(interleukin, IL)-1β和IL-18的成熟和释放,启动细胞焦亡。近年来越来越多的证据表明,炎症小体主要是NLRP3、NLRP1和AIM2介导的细胞焦亡,参与TBI后组织损伤和功能障碍。本文简要总结目前关于细胞焦亡在TBI中作用的研究进展。     

国外研究信息

<正>白细胞介素1受体相关激酶1旁路引发和直接连接Toll样受体至快速核苷酸结合寡聚化结构域样受体含热蛋白结构域3炎性体激活致病性感染和组织损伤引发炎性体的组装,这种胞质蛋白复合物激活半胱天冬酶-1(caspase-1),促进白细胞介素1β前体(pro-IL-1β)和pro-IL-18的切割成熟,还能调节caspase-1依赖的程序性细胞死亡(pyroptosis)。众所周知,微生物识别Toll样受体(TLR)主要是诱导激活caspase-1,但已知的TLR功能仅限于上调炎性体的作用。感染致命微生物后,TLR和核苷酸结合寡聚化结构域样受体(NLR)有可能同时     

NLRP3/caspase-1/IL-1β信号通路在缺氧缺血性脑损伤的作用

新生儿缺氧缺血性脑损伤(hypoxic-ischemic brain damage,HIBD)是导致新生儿死亡和婴幼儿伤残的主要原因,发病机制包括细胞能量代谢衰竭、血管源性脑水肿、兴奋性氨基酸神经毒性、氧化应激、炎症反应等的交互作用,其中炎症反应是重要的病理生理过程,涉及NLRP3(NOD-like receptor thermal protein domain associated protein 3)/caspase-1/IL-1β信号通路的激活,促进白细胞介素-1β(interleukin-1β,IL-1β)等炎症因子成熟与释放,诱导神经细胞焦亡。本文就NLRP3/caspase-1/IL-1β信号通路的概述、激活途径、介导HIBD的作用机制及HIBD治疗的相关新进展等方面进行综述,以期为HIBD的神经保护策略提供新思路。     

NLRP3炎性体与代谢性疾病的研究进展

代谢性疾病是由体内氨基酸、葡萄糖和脂质代谢紊乱引起的一类疾病,慢性炎症反应是其重要特征之一.Nod样受体蛋白3(Nod-like receptor protein 3,NLRP3)炎性体是位于细胞内的一种蛋白质复合体,主要功能为活化半胱氨酸天冬氨酸蛋白酶1(caspase-1)以间接调控白介素1β(IL-1β)、IL-18和IL-33等的成熟和分泌.NLRP3炎性体是炎性体相关研究的热点,多种内源性或外源性危险信号通过激活这一蛋白质复合体上调炎性因子的表达水平,从而促进多种代谢性疾病的发生发展.本文对NLRP3炎性体的结构、功能、调节以及在代谢性疾病中的作用做一综述,以期为代谢性疾病的防治提供新靶点.     

The NLRP3 inflammasome: activation and regulation

The NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome is a cytoplasmic supramolecular complex that is activated in response to cellular perturbations triggered by infection and sterile injury. Assembly of the NLRP3 inflammasome leads to activation of caspase-1, which induces the maturation and release of interleukin-1β (IL-1β) and IL-18, as well as cleavage of gasdermin D (GSDMD), which promotes a lytic form of cell death. Production of IL-1β via NLRP3 can contribute to the pathogenesis of inflammatory disease, whereas aberrant IL-1β secretion through inherited NLRP3 mutations causes autoinflammatory disorders. In this review, we discuss recent developments in the structure of the NLRP3 inflammasome, and the cellular processes and signaling events controlling its assembly and activation.     

Inflammasome activation and IL-1β/IL-18 processing are influenced by distinct pathways in microglia

Microglia are important innate immune effectors against invading CNS pathogens, such as Staphylococcus aureus (S. aureus), a common etiological agent of brain abscesses typified by widespread inflammation and necrosis. The NLRP3 inflammasome is a protein complex involved in IL-1β and IL-18 processing following exposure to both pathogen- and danger-associated molecular patterns. Although previous studies from our laboratory have established that IL-1β is a major cytokine product of S. aureus-activated microglia and is pivotal for eliciting protective anti-bacterial immunity during brain abscess development, the molecular machinery responsible for cytokine release remains to be determined. Therefore, the functional role of the NLRP3 inflammasome and its adaptor protein apoptosis-associated speck-like protein (ASC) in eliciting IL-1β and IL-18 release was examined in primary microglia. Interestingly, we found that IL-1β, but not IL-18 production, was significantly attenuated in both NLRP3 and ASC knockout microglia following exposure to live S. aureus. NLRP3 inflammasome activation was partially dependent on autocrine/paracrine ATP release and α- and γ-hemolysins produced by live bacteria. A cathepsin B inhibitor attenuated IL-β release from NLRP3 and ASC knockout microglia, demonstrating the existence of alternative inflammasome-independent mechanisms for IL-1β processing. In contrast, microglial IL-18 secretion occurred independently of cathepsin B and inflammasome action. Collectively, these results demonstrate that microglial IL-1β processing is regulated by multiple pathways and diverges from mechanisms utilized for IL-18 cleavage. Understanding the molecular events that regulate IL-1β production is important for modulating this potent proinflammatory cytokine during CNS disease.     

Leptospira interrogans infection leads to IL-1β and IL-18 secretion from a human macrophage cell line through reactive oxygen species and cathepsin B mediated-NLRP3 inflammasome activation

Leptospirosis is a worldwide zoonosis caused by spirochetes from the genus Leptospira. Although there is a large diversity of clinical signs and symptoms, a severe inflammatory response is common to all leptospirosis patients. The mechanism of IL-1β secretion during Leptospira infection has been previously studied in mouse macrophages. However, the outcome of Leptospira infection is very different in human and murine macrophages, and the mechanisms responsible for IL-1β secretion in human macrophages had not been investigated. This study therefore examines the effects of Leptospira interrogans infection on inflammasome activation and proinflammatory cytokine expression in human macrophages. Increased mRNA and protein expression of NLRP3 was observed by real time RT-PCR and flow cytometry at 1 h after co-cultivation. Enzyme-linked immunosorbent assay (ELISA) determination showed that IL-1β and IL-18 are released in the culture supernatants at 1 h after cultivation. The inhibition assay showed that glybenclamide (a K⁺ efflux inhibitor that blocks NLRP3 inflammasome activation) and N-benzyloxycarbony-Val-Ala-Asp (O-methyl)-fluoromethylketone (Z-VAD-FMK; a caspase-1 inhibitor) and NLRP3 depletion with siRNAs reduced the levels of IL-1β and IL-18 release. Moreover, the levels of IL-1β and IL-18 production decreased in CA-074 (a cathepsin B inhibitor) and NAC (an anti-oxidant) pretreated human macrophages, compared to untreated controls. This study suggests that L. interrogans infection leads to reactive oxygen species (ROS)- and cathepsin B-dependent NLRP3 inflammasome activation, which subsequently mediates caspase-1 activation and IL-1β and IL-18 release.     

Vascular endothelial cells senescence is associated with NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation via reactive oxygen species (ROS)/thioredoxin-interacting protein (TXNIP) pathway

Endothelial dysfunction caused by endothelial cells senescence and chronic inflammation is tightly linked to the development of cardiovascular diseases. NLRP3 (NOD-like receptor family pyrin domain-containing3) inflammasome plays a central role in inflammatory response that is associated with diverse inflammatory diseases. This study explores the effects and possible mechanisms of NLRP3 inflammasome in endothelial cells senescence. Results show an increment of pro-inflammatory cytokine interleukin (IL) −1β secretion and caspase-1 activation during the senescence of endothelial cells induced by bleomycin. Moreover, secreted IL-1β promoted endothelial cells senescence through up-regulation of p53/p21 protein expression. NLRP3 inflammasome was found to mediate IL-1β secretion through the production of ROS (reactive oxygen species) during the senescence of endothelial cells. Furthermore, the association of TXNIP (thioredoxin-interacting protein) with NLRP3 induced by ROS promoted NLRP3 inflammasome activation in senescent endothelial cells. In addition, the expressions of NLRP3 inflammasome related genes, ASC (apoptosis associated speck-like protein containing a CARD), TXNIP, cleaved caspase-1 and IL-1β, were also increased in vitro and in vivo studies. These findings indicate that endothelial senescence could be mediated through ROS and NLRP3 inflammasome signaling pathways, suggesting a potential target for the prevention of endothelial senescence-related cardiovascular diseases.     

MicroRNA-133a-1 regulates inflammasome activation through uncoupling protein-2

Inflammasomes are multimeric protein complexes involved in the processing of IL-1β through Caspase-1 cleavage. NLRP3 is the most widely studied inflammasome, which has been shown to respond to a large number of both endogenous and exogenous stimuli. Although studies have begun to define basic pathways for the activation of inflammasome and have been instrumental in identifying therapeutics for inflammasome related disorders; understanding the inflammasome activation at the molecular level is still incomplete. Recent functional studies indicate that microRNAs (miRs) regulate molecular pathways and can lead to diseased states when hampered or overexpressed. Mechanisms involving the miRNA regulatory network in the activation of inflammasome and IL-1β processing is yet unknown. This report investigates the involvement of miR-133a-1 in the activation of inflammasome (NLRP3) and IL-1β production. miR-133a-1 is known to target the mitochondrial uncoupling protein 2 (UCP2). The role of UCP2 in inflammasome activation has remained elusive. To understand the role of miR-133a-1 in regulating inflammasome activation, we either overexpressed or suppressed miR-133a-1 in differentiated THP1 cells that express the NLRP3 inflammasome. Levels of Caspase-1 and IL-1β were analyzed by Western blot analysis. For the first time, we showed that overexpression of miR-133a-1 increases Caspase-1 p10 and IL-1β p17 cleavage, concurrently suppressing mitochondrial uncoupling protein 2 (UCP2). Surprisingly, our results demonstrated that miR-133A-1 controls inflammasome activation without affecting the basal expression of the individual inflammasome components NLRP3 and ASC or its immediate downstream targets proIL-1β and pro-Caspase-1. To confirm the involvement of UCP2 in the regulation of inflammasome activation, Caspase-1 p10 and IL-1β p17 cleavage in UCP2 of overexpressed and silenced THP1 cells were studied. Suppression of UCP2 by siRNA enhanced the inflammasome activity stimulated by H₂O₂ and, conversely, overexpression of UCP2 decreased the inflammasome activation. Collectively, these studies suggest that miR-133a-1 suppresses inflammasome activation via the suppression of UCP2.     

NLRP3 Inflammasome Is Involved in Q-VD-OPH Induced Necroptosis Following Cerebral Ischemia-Reperfusion Injury

Necroptosis is a manner of caspase-independent cell death,which accounts for delayed ischemic cerebral injury, and can be used as a novel tool to expand the treatment time window in ischemic cerebral injury. Q-VD-OPH, a novel pan caspase inhibitor, has been identified as an inducer of necroptosis. In this study, we determined the optimal dose of Q-VD-OPH, which induces necroptosis in rats by the middle cerebral artery occlusion, followed by reperfusion. Furthermore, we report that the NLRP3 inflammasome is involved in necroptosis, with levels of NLRP3 inflammasome proteins as well as inflammatory cytokines, such as IL-1β, being elevated. We also demonstrated that NLRP3 was not only expressed in microglia and vascular endothelial cell, but also in neurons when necroptosis is induced with Q-VD-OPH. Inhibition of NLRP3 by glyburide strongly suppressed the expression of NLRP3 inflammasome proteins and IL-1β, and markedly reduced brain tissue damage. Our findings provide evidence that pretreatment with Q-VD-OPH suppresses apoptosis and induces necroptosis in the cerebral ischemia-reperfusion model. We also identified that the NLRP3 inflammasome plays an important role in neuronal necroptosis, and that NLRP3 inflammasome deficiency reduces brain tissue damage after cerebral ischemia-reperfusion injury in rats.     

Measles virus V protein inhibits NLRP3 inflammasome-mediated interleukin-1β secretion

Inflammasomes are cytosolic protein complexes that stimulate the activation of caspase-1, which in turn induces the secretion of the inflammatory cytokines Interleukin-1β (IL-1β) and IL-18. Recent studies have indicated that the inflammasome known as the NOD-like-receptor-family, pyrin domain-containing 3 (NLRP3) inflammasome recognizes several RNA viruses, including the influenza and encephalomyocarditis viruses, whereas the retinoic acid-inducible gene I (RIG-I) inflammasome may detect vesicular stomatitis virus. We demonstrate that measles virus (MV) infection induces caspase-1-dependent IL-1β secretion in the human macrophage-like cell line THP-1. Gene knockdown experiments indicated that IL-1β secretion in MV-infected THP-1 cells was mediated by the NLRP3 inflammasome but not the RIG-I inflammasome. MV produces the nonstructural V protein, which has been shown to antagonize host innate immune responses. The recombinant MV lacking the V protein induced more IL-1β than the parental virus. THP-1 cells stably expressing the V protein suppressed NLRP3 inflammasome-mediated IL-1β secretion. Furthermore, coimmunoprecipitation assays revealed that the V protein interacts with NLRP3 through its carboxyl-terminal domain. NLRP3 was located in cytoplasmic granular structures in THP-1 cells stably expressing the V protein, but upon inflammasome activation, NLRP3 was redistributed to the perinuclear region, where it colocalized with the V protein. These results indicate that the V protein of MV suppresses NLRP3 inflammasome-mediated IL-1β secretion by directly or indirectly interacting with NLRP3.     

Critical role of nucleotide-binding oligomerization domain-like receptor 3 in vascular repair

Vascular remodeling characterized by hyperproliferative neointima formation is an unfavorable repair process that is triggered by vascular damage. This process is characterized by an increased local inflammatory and proliferative response that critically involves the pro-inflammatory cytokine interleukin-1β (IL-1β). IL-1β is expressed and cytosolically retained as a procytokine that requires additional processing prior to exerting its pro-inflammatory function. Maturation and release of pro IL-1β is governed by a cytosolic protein scaffold that is known as the inflammasome.Here we show that NLRP3 (NOD-like receptor family, pryin domain containing 3), an important activating component of the inflammasome, is involved in neointima formation after vascular injury. NLRP3 deficiency itself does not affect the functional cardiovascular phenotype and does not alter peripheral differential blood counts. However, neointima development following wire injury of the carotid artery was significantly decreased in NLRP3-deficient mice as compared to wild-type controls. In all, NLRP3 plays a non-redundant role in vascular damage mediated neointima formation.Our data establish NLRP3 as a key player in the response to vascular damage, which could open new avenues to therapeutic intervention.