Caspase-1抑制剂对大鼠血管平滑肌细胞钙化的初步影响

目的:研究Caspase-1抑制剂zVAD对大鼠血管平滑肌细胞钙化的影响。方法:体外分离、培养大鼠血管平滑肌细胞,加入β-磷酸甘油酯(β-GP)诱导细胞发生钙化;在细胞中加入Caspase-1抑制剂zVAD或DMSO对照,按照不同时间点收取细胞进行半定量PCR,检测骨保护素(OPG)/核因子κB受体活化因子配体(RANKL)的表达变化;通过茜素红钙化染色,直观观察zVAD对血管平滑肌细胞钙化的影响。结果:β-GP加入细胞后,可见细胞内OPG/RANKL的mRNA表达水平逐步增加,加入zVAD后增加趋势减缓;茜素红钙化染色显示,zVAD可抑制血管平滑肌细胞钙化。结论:分离、体外培养了大鼠的血管平滑肌细胞,并且发现在钙化过程中OPG/RANKL表达增加,而Caspase-1抑制剂zVAD可有效抑制OPG/RANKL的表达,提示炎症小体可能通过OPG/RANKL诱导动脉钙化的产生。     

二甲双胍对华法令诱导大鼠动脉钙化的影响及其机制初探

目的:研究二甲双胍(metformin,MET)对华法令(Warfarin,WFN)诱导大鼠动脉钙化的影响及其机制。方法:将28只SD大鼠随机分为正常对照组、8周(W)钙化组、8W钙化+8W MET 100 mg/kg治疗组、8W钙化+8W MET 200 mg/kg治疗组。采用Von Kossa染色法检测胸主动脉组织中钙结节;邻甲酚肽络合酮比色法测定颈总动脉组织中钙沉积含量;免疫组化染色检测血管壁中成骨基因Runx2及血管平滑肌标志物α-SMA的表达;Western Blot检测血管壁中Runx2及自噬标志物LC3II的表达。结果:WFN干预8 W后,大鼠动脉中钙沉积含量显著增加(P0.01),MET治疗组主动脉钙含量与钙化组相比均显著降低(P0.01)。Von Kossa染色可见钙化组(WFN组)动脉壁中层黑色连续钙盐沉积条带,而进行MET治疗后,黑色条带明显减少,对照组未见黑色条带。免疫组化显示钙化组血管壁Runx2表达阳性,棕褐色染色较深,而α-SMA表达则显著下降,基本未见棕褐色染色沉积;MET治疗后能够逆转上述趋势。Western Blot显示钙化组血管壁Runx2表达明显上升,MET治疗后Runx2表达被抑制。此外,钙化过程中伴随着自噬标志物LC3II表达轻度上升;随着MET浓度升高,血管壁中自噬水平呈剂量依赖性显著升高。结论:二甲双胍能够有效抑制大鼠动脉钙化,减轻血管平滑肌细胞由收缩表型向成骨样表型转换,其机制可能与诱导自噬有关。     

补镁对大鼠血管钙化的影响

目的:在大鼠血管钙化模型上,观察外源性补充硫酸镁对大鼠血管钙化的影响,以探讨硫酸镁在血管钙化中作用及机制。方法:用维生素D3加尼古丁诱导大鼠血管钙化,von Kossa染色、钙含量测定及碱性磷酸酶活性测定判断血管钙化程度;用半定量RT-PCR方法测定血管钙化标志分子骨桥蛋白mRNA水平;用生物化学方法测定血管一氧化氮(NO)、超氧化物歧化酶(SOD)和丙二醛(MDA)含量。结果:钙化组大鼠血压升高,收缩压较对照组高27%(P<0.05);血管von Kossa染色见血管中膜弹性纤维间可见大量棕黑色颗粒沉积,主动脉钙含量及碱性磷酸酶活性分别较对照高3.9倍和3.4倍(P<0.01),钙化血管组织骨桥蛋白mRNA表达上调40%(P<0.01),血管钙化后可加重血管组织过氧化损伤;而诱导钙化后外源性补充硫酸镁可减轻血管钙化程度,与钙化组比较,低、高剂量硫酸镁组均明显缓解上述指标的变化。结论:诱导血管钙化后外源性补充硫酸镁可以减轻大鼠血管钙化和血管损伤程度。     

华法令诱导大鼠体内主动脉的钙化作用

目的:研究华法令对大鼠体内主动脉的钙化作用。方法:采用华法令(3 mg/g饲料)的饲料喂养诱导大鼠体内动脉钙化。实验分3组(n=6),对照组、6 W钙化组、12 W钙化组。Von Kossa染色观察钙结节形成,邻甲酚酞络合酮比色法定量测定钙沉积含量。采用TUNEL染色法检测大鼠主动脉组织内血管平滑肌细胞(Vascular Smooth Muscle Cell,VSMC)凋亡。Western blot法检测大鼠主动脉组织中生长抑制特异蛋白6(Growth Arrest Specific Gene 6 Protein,Gas 6)蛋白表达水平。结果:钙化组钙结节、钙沉积含量及VSMC凋亡均高于对照组(P0.01)有显著差异。钙化形成与凋亡表达呈显著正相关(R2=0.8853,P0.0001);钙化组Gas 6蛋白表达量较对照组下调(P0.01)有显著差异。结论:华法令通过下调Gas 6蛋白诱导大鼠体内主动脉钙化形成。     

动脉钙化发生机制研究进展

动脉钙化是指钙盐沉积在动脉壁组织的一种病理改变,会减少主动脉和支动脉的弹性,改变心血管系统的血液流动力学,导致高血压、主动脉瓣狭窄、心脏肥厚、心肌和下肢缺血、充血性心力衰竭等严重心脑血管疾病发生。动脉钙化在老年人群中是一种常见的疾病。早期研究发现尿毒症患者体内磷酸钙沉积的抑制剂——焦磷酸盐水平升高,故有学者认为钙磷被动地沉积于血管壁是引起血管钙化的主要原因。近年来的研究发现,血管钙化并非简单地由于磷酸钙晶体被动地沉积于血管壁,而是一个与骨发育相似的主动的、可预防和可逆转的高度可调控的生物学过程。动脉钙化的发生受多因素共同调控,但其确切机制尚不清楚,最近发现炎症小体也以某种未知的机制参与钙化调控过程。     

黄芪甲苷减轻大鼠脑缺血再灌损伤并抑制NF-κB磷酸化及NLRP3炎症小体活化

本文旨在探讨黄芪甲苷(astragaloside IV, AST-IV)在大鼠脑缺血再灌注损伤中的保护作用及其抗炎机制。采用改良线栓法制备大鼠大脑中动脉栓塞脑缺血再灌注模型,以神经功能缺失评分和脑梗死体积综合评价AST-IV抗脑缺血再灌注损伤的作用,Western blot检测脑组织中NLRP3、ASC、pro-Caspase-1、Caspase-1、pro-IL-1β、IL-1β、pro-IL-18、IL-18、磷酸化NF-κB和总NF-κB蛋白表达水平。结果显示,与模型组比较,AST-IV能降低大鼠神经功能缺失评分,减少脑梗死体积,降低脑组织中NLRP3、Caspase-1、pro-IL-1β、IL-1β、pro-IL-18和IL-18蛋白水平,并抑制磷酸化NF-κB蛋白表达。以上结果提示,AST-IV具有抗脑缺血再灌注损伤作用,其机制可能与抑制NF-κB蛋白磷酸化以及抑制NLRP3炎症小体活化有关。     

牙龈卟啉单胞菌感染通过激活NLRP3小体诱导牙周膜细胞炎症反应及凋亡效应

目的观察牙龈卟啉单胞菌感染通过激活含NLR家族PYRIN域蛋白3(NLRP3)小体诱导人牙周膜细胞(hPDLCs)炎症反应及凋亡的效应。方法取健康前磨牙样本并分离培养hPDLCs,分为牙龈卟啉单胞菌感染的感染组和常规处理的对照组,检测细胞中NLRP3小体[NLRP3、凋亡相关斑点样蛋白(ASC)、含半胱氨酸的天冬氨酸蛋白水解酶(Caspase)-1]、凋亡基因[自杀相关因子(Fas)、Fas配体(FasL)、B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关x蛋白(Bax)、Caspase-3]的表达量及培养基中炎症细胞因子[白细胞介素(IL)-1β、IL-18、肿瘤坏死因子-α(TNF-α)]的含量。结果感染组hPDLCs中NLRP3、ASC、Caspase-1、Fas、FasL、Bax、Caspase-3的表达量及培养基中IL-1β、IL-18、TNF-α的含量明显高于对照组,细胞中Bcl-2的表达量明显低于对照组。结论牙龈卟啉单胞菌感染能够诱导hPDLCs的炎症反应及凋亡且该作用与NLRP3小体的激活有关。     

炎症促进大鼠动脉粥样硬化初期内皮功能病变机制研究

目的:观察炎症因素诱导大鼠动脉粥样硬化发病过程中对血管内皮细胞的影响。方法:实验分为单纯高脂对照组和炎症组,分别腹腔注射给予无菌医用液体石蜡和酵母多糖(Zym,20mg/kg,1次/3天)。所有大鼠均喂食含3%胆固醇的高脂饲料,共8周。透射电镜观察主动脉超微结构;应用定量PCR法测定腹主动脉组织中诱导型一氧化氮合酶(iNOS)mRNA、血管细胞粘附分子(VCAM)-1 mRNA、以及基质金属蛋白酶7(MMP7)mRNA的表达。结果:炎症组可见游走于内膜下层的平滑肌细胞和和吞噬脂质颗粒的单核细胞,单纯高脂对照组仅见内皮细胞损伤和退行性变,未见内膜下层形成泡沫细胞,AS样病变较炎症组轻。与对照组相比,炎症组动脉壁iNOS mRNA表达降低,VCAM-1 mRNA及MMP7 mRNA标大量显著升高。结论:炎症刺激能够损伤动脉血管内皮细胞,诱导炎症因子释放增加,促进动脉粥样硬化的发生。     

槐果碱抑制糖尿病大鼠肾损伤、NLRP3炎性小体和NF-κB通路活化

目的 观察槐果碱（sophocarpine, SC）对糖尿病大鼠肾损伤的影响,并探讨其相关机制。方法 选用SD大鼠,以高脂饮食12周和链脲佐菌素处理制作糖尿病模型,灌胃给予槐果碱治疗12周。PAS染色以及纤连蛋白（fibronectin, FN）和Cleaved Caspase-3免疫组织化学染色评估肾组织损伤,ELISA检测肾组织中IL-1β及IL-6水平,Western blot检测肾组织中NLRP3、凋亡相关斑点样蛋白（apoptosis-associated speck-like protein containing CARD, ASC）、Cleaved Caspase-1和NF-κB p65蛋白表达水平。结果 糖尿病大鼠肾重指数、尿蛋白排泄水平、血清尿酸水平、肾小球系膜基质扩张系数均较对照大鼠明显升高,肾组织中FN、Cleaved Caspase-3、IL-1β、IL-6、NLRP3、ASC、Cleaved Caspase-1和NF-κB p65蛋白水平均较对照大鼠明显上调,槐果碱干预能明显抑制糖尿病大鼠上述指标变化。结论 槐果碱可能通过抑制NLRP3炎性小体和NF-κB通路...     

NLRP3炎症小体表达与慢性阻塞性肺疾病合并肺癌的相关性研究

目的:分析核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎症小体表达与慢性阻塞性肺疾病(COPD)合并肺癌的相关性。方法:选取2015年1月-2018年2月我院收治的COPD合并肺癌患者62例作为实验组及同期88例COPD患者作为对照组。酶联免疫吸附法(ELISA)检测两组患者外周血IL-1β、IL-18浓度,免疫组化法检测两组患者术后肺病理组织中Caspase-1、ASC、NLRP3、IL-1β、IL-18蛋白相对表达量,并比较不同病理特征下患者术后肺病理组织中NLRP3、ASC、Caspase-1、IL-1β、IL-18蛋白相对表达量的差异,并分析其与COPD合并肺癌的相关性。结果:实验组患者外周血IL-1β、IL-18水平均明显高于对照组,差异具有统计学意义(P0.05);实验组患者术后肺病理组织中NLRP3、ASC、Caspase-1、IL-1β、IL-18蛋白相对表达量均明显高于对照组,差异均具有统计学意义(P0.05);中低分化、临床分期Ⅲ期、淋巴结有转移的急性加重期COPD合并肺癌患者术后肺病理组织中NLRP3、ASC、Caspase-1、IL-1β、IL-18蛋白相对表达量高于高分化、临床分期Ⅰ-Ⅱ期、淋巴结无转移的稳定期COPD合并肺癌患者,差异具有统计学意义(P0.05)。经Spearman秩相关性分析发现,患者术后肺病理组织中NLRP3、ASC、Caspase-1、IL-1β、IL-18蛋白相对表达量与COPD合并肺癌患者病情严重程度、淋巴结转移情况、分化程度以及病情所处时期均呈正相关(r0,P0.05)。结论:NLRP3炎症小体通路可能参与了COPD合并肺癌的发展过程,其释放的细胞因子IL-1β、IL-18水平升高可能与患者持续炎症有关,并进一步导致机体免疫病理损伤,促进疾病进展。     

Crystal structure of NALP3 protein pyrin domain (PYD) and its implications in inflammasome assembly

NALP3 inflammasome, composed of the three proteins NALP3, ASC, and Caspase-1, is a macromolecular complex responsible for the innate immune response against infection with bacterial and viral pathogens. Formation of the inflammasome can lead to the activation of inflammatory caspases, such as Caspase-1, which then activate pro-inflammatory cytokines by proteolytic cleavage. The assembly of the NALP3 inflammasome depends on the protein-interacting domain known as the death domain superfamily. NALP3 inflammasome is assembled via a pyrin domain (PYD)/PYD interaction between ASC and NALP3 and a caspase recruitment domain/caspase recruitment domain interaction between ASC and Caspase-1. As a first step toward elucidating the molecular mechanisms of inflammatory caspase activation by formation of inflammasome, we report the crystal structure of the PYD from NALP3 at 1.7-Å resolution. Although NALP3 PYD has the canonical six-helical bundle structural fold similar to other PYDs, the high resolution structure reveals the possible biologically important homodimeric interface and the dynamic properties of the fold. Comparison with other PYD structures shows both similarities and differences that may be functionally relevant. Structural and sequence analyses further implicate conserved surface residues in NALP3 PYD for ASC interaction and inflammasome assembly. The most interesting aspect of the structure was the unexpected disulfide bond between Cys-8 and Cys-108, which might be important for regulation of the activity of NALP3 by redox potential.     

Reconstituted NALP1 inflammasome reveals two-step mechanism of caspase-1 activation

Interleukin (IL)-1beta maturation is accomplished by caspase-1-mediated proteolysis, an essential element of innate immunity. NLRs constitute a recently recognized family of caspase-1-activating proteins, which contain a nucleotide-binding oligomerization domain and leucine-rich repeat (LRR) domains and which assemble into multiprotein complexes to create caspase-1-activating platforms called "inflammasomes." Using purified recombinant proteins, we have reconstituted the NALP1 inflammasome and have characterized the requirements for inflammasome assembly and caspase-1 activation. Oligomerization of NALP1 and activation of caspase-1 occur via a two-step mechanism, requiring microbial product, muramyl-dipeptide, a component of peptidoglycan, followed by ribonucleoside triphosphates. Caspase-1 activation by NALP1 does not require but is enhanced by adaptor protein ASC. The findings provide the biochemical basis for understanding how inflammasome assembly and function are regulated, and shed light on NALP1 as a direct sensor of bacterial components in host defense against pathogens.     

Thioredoxin-interacting protein mediates NALP3 inflammasome activation in podocytes during diabetic nephropathy

Numerous studies have shown that the NALP3 inflammasome plays an important role in various immune and inflammatory diseases. However, whether the NALP3 inflammasome is involved in the pathogenesis of diabetic nephropathy (DN) is unclear. In our study, we confirmed that high glucose (HG) concentrations induced NALP3 inflammasome activation both in vivo and in vitro. Blocking NALP3 inflammasome activation by NALP3/ASC shRNA and caspase-1 inhibition prevented IL-1β production and eventually attenuated podocyte and glomerular injury under HG conditions. We also found that thioredoxin (TRX)-interacting protein (TXNIP), which is a pro-oxidative stress and pro-inflammatory factor, activated NALP3 inflammasome by interacting with NALP3 in HG-exposed podocytes. Knocking down TXNIP impeded NALP3 inflammasome activation and alleviated podocyte injury caused by HG. In summary, the NALP3 inflammasome mediates podocyte and glomerular injury in DN, moreover, TXNIP participates in the formation and activation of the NALP3 inflammasome in podocytes during DN, which represents a novel mechanism of podocyte and glomerular injury under diabetic conditions.     

1,25-dihydroxyvitamin D3 receptor is upregulated in aortic smooth muscle cells during hypervitaminosis D

Several studies have demonstrated that excess of vitamin D3 is toxic particularly to vascular tissues. A notable pathological feature is arterial calcification. The nature of the toxic metabolite in hypervitaminosis D and the pathogenesis of arterial calcification are not clearly understood. The present study was undertaken to explore whether arterial calcification is a sequel of increased calcium uptake by arterial smooth muscle mediated by up regulation of vitamin D receptor in the cells in response to elevated circulating levels of vitamin D3 in serum. The experimental study was performed in 20 New Zealand white female rabbits aged 6 months. Animals in the test group were injected 10,000 IU of cholecalciferol intramuscularly twice a week for one month. Six control animals were given intra-muscular injections of plain cottonseed oil. Animals were sacrificed and aortas were examined for pathological lesions, 1,25-dihyroxyvitamin D3 (1,25(OH)2 D3) receptor levels and 45Ca uptake in smooth muscle cells. Serum samples collected at intervals were assayed for levels of 25-OH-D3 and calcium. The results showed that in animals given injections of cholecalciferol, serum levels of 25-OH-D3 were elevated. In four of these animals calcification and aneurysmal changes were seen in the aorta. Histological lesions comprised of fragmentation of elastic fibers as well as extensive loss of elastic layers. 1,25(OH)2 D3 receptor levels were up regulated and 45Ca uptake enhanced in aortas of animals which were given excessive vitamin D3. The evidences gathered suggest that excess vitamin D is arteriotoxic and that the vitamin induces arterial calcification through up regulation of 1,25(OH)2D3 receptor and increased calcium uptake in smooth muscle cells of the arteries.     

Innate immunity functional gene polymorphisms and gout susceptibility

Gout is a common autoinflammatory disease characterized with elevated serum urate and recurrent attacks of intra-articular crystal deposition of monosodium urate. Accumulating evidence has demonstrated that MSU crystal-induced inflammation is a paradigm of innate immunity and the TLRs, NALP3 inflammasome and IL1R pathways are involved in gout development. Innate immunity components containing TLR2, TLR4, CD14, NALP3, ASC, Caspase-1 and CARD-8 are essential in the development of gouty inflammation. Recent studies suggest that innate immunity component gene functional mutations contribute to the development of autoinflammatory diseases including hereditary periodic fever syndrome, arthritis as well as inflammatory bowel disease. Taking into account these genetic findings, we would like to propose a novel hypothesis that the gene functional mutations might make innate immunity components as attractive susceptibility candidates and genetic markers for gout. Further clinical genetic studies need to be performed to confirm the role of innate immunity in the etiology of gout.     

Selenium Attenuates Staphylococcus aureus Mastitis in Mice by Inhibiting the Activation of the NALP3 Inflammasome and NF-κB/MAPK Pathway

Mastitis is one of the most important diseases affecting the dairy industry in the world, and it also poses a great threat to human food safety. In this study, we explored whether selenium can inhibit the activation of the NALP3 inflammasome and NF-κB/MAPK pathway to achieve anti-inflammatory effects. Sixty BALB/c female mice were randomly divided into three groups according to diets of different selenium concentrations (high, normal, and low). After 90 days, mice fed the same selenium concentration were randomly divided into two smaller groups, one of which was inoculated with Staphylococcus aureus and the other injected with saline as a control. Through histopathologic examination staining, western blot, qPCR, and ELISA, the results showed that with increasing selenium concentrations, the expression levels of IL-1β, TNF-α, NALP3, caspase-1, and ASC were decreased in mouse mammary tissue. Therefore, this study revealed that selenium can attenuate S. aureus mastitis by inhibiting the activation of the NALP3 inflammasome and NF-κB/MAPK pathway.

     

AIM2/ASC triggers caspase-8-dependent apoptosis in Francisella-infected caspase-1-deficient macrophages

The inflammasome is a signalling platform leading to caspase-1 activation. Caspase-1 causes pyroptosis, a necrotic-like cell death. AIM2 is an inflammasome sensor for cytosolic DNA. The adaptor molecule ASC mediates AIM2-dependent caspase-1 activation. To date, no function besides caspase-1 activation has been ascribed to the AIM2/ASC complex. Here, by comparing the effect of gene inactivation at different levels of the inflammasome pathway, we uncovered a novel cell death pathway activated in an AIM2/ASC-dependent manner. Francisella tularensis, the agent of tularaemia, triggers AIM2/ASC-dependent caspase-3-mediated apoptosis in caspase-1-deficient macrophages. We further show that AIM2 engagement leads to ASC-dependent, caspase-1-independent activation of caspase-8 and caspase-9 and that caspase-1-independent death is reverted upon caspase-8 inhibition. Caspase-8 interacts with ASC and active caspase-8 specifically colocalizes with the AIM2/ASC speck thus identifying the AIM2/ASC complex as a novel caspase-8 activation platform. Furthermore, we demonstrate that caspase-1-independent apoptosis requires the activation of caspase-9 and of the intrinsic pathway in a typical type II cell manner. Finally, we identify the AIM2/ASC-dependent caspase-1-independent pathway as an innate immune mechanism able to restrict bacterial replication in vitro and control IFN-γ levels in vivo in Casp1(KO) mice. This work underscores the crosstalk between inflammasome components and the apoptotic machinery and highlights the versatility of the pathway, which can switch from pyroptosis to apoptosis.     

Lipopolysaccharide Primes the NALP3 Inflammasome by Inhibiting Its Ubiquitination and Degradation Mediated by the SCFFBXL2 E3 Ligase

The inflammasome is a multiprotein complex that augments the proinflammatory response by increasing the generation and cellular release of key cytokines. Specifically, the NALP3 inflammasome requires two-step signaling, priming and activation, to be functional to release the proinflammatory cytokines IL-1β and IL-18. The priming process, through unknown mechanisms, increases the protein levels of NALP3 and pro-IL-1β in cells. Here we show that LPS increases the NALP3 protein lifespan without significantly altering steady-state mRNA in human cells. LPS exposure reduces the ubiquitin-mediated proteasomal processing of NALP3 by inducing levels of an E3 ligase component, FBXO3, which targets FBXL2. The latter is an endogenous mediator of NALP3 degradation. FBXL2 recognizes Trp-73 within NALP3 for interaction and targets Lys-689 within NALP3 for ubiquitin ligation and degradation. A unique small molecule inhibitor of FBXO3 restores FBXL2 levels, resulting in decreased NALP3 protein levels in cells and, thereby, reducing the release of IL-1β and IL-18 in human inflammatory cells after NALP3 activation. Our findings uncover NALP3 as a molecular target for FBXL2 and suggest that therapeutic targeting of the inflammasome may serve as a platform for preclinical intervention.