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为探讨肝X受体激动剂对NLRP3配体ATP诱导小鼠巨噬细胞炎性复合体活化的影响,应用荧光定量PCR方法测定肝X受体激动剂对LPS诱导的Pro-IL-1β、NLRP3等基因表达的影响,并用免疫蛋白印迹的方法检测Caspase-1的活化片段。结果证明,肝X受体(LXRs)激动剂能抑制NLRP3炎性体的活化。LXRs激动剂T0901317和GW3965显著抑制LPS联合ATP诱导的Caspase-1剪切成熟和IL-1β分泌,并且LXRs激动剂对NLRP3炎性体的抑制作用,部分源于其抑制NLRP3和IL-1β的基因转录。 相似文献
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为了探究槐花散对肠上皮细胞炎性损伤的保护作用及其机制，本试验用细胞内毒素脂多糖(LPS)诱导肠上皮细胞系IEC-6损伤模型，首先通过CCK8法筛选槐花散水提液的作用浓度，继而通过蛋白免疫印迹法检测NOD样受体蛋白3(NLRP3)炎性小体主要成分NLRP3、ASC和caspase-1及其下游白细胞介素-18(IL-18)蛋白的表达水平，综合评价槐花散对NLRP3炎性小体介导的肠上皮细胞炎性损伤的作用。结果显示，不同浓度的槐花散皆能显著抑制LPS诱导的IEC-6细胞炎性小体激活，并降低下游IL-18炎性因子表达(P<0.05)。结果表明，槐花散可能通过抑制炎性小体激活以保护肠上皮细胞抵抗LPS诱导的细胞炎性损伤。 相似文献
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本研究通过建立脂多糖(LPS)诱导大鼠小肠隐窝上皮细胞(IEC-6)炎性损伤模型,测定乳酸脱氢酶(LDH)释放率和细胞划痕愈合率,以评估橙皮苷对LPS诱导的IEC-6细胞炎性损伤的保护作用;通过Western blot法测定NLRP3、Caspase-1、IL-1β、p-NF-κB p65、NF-κB p65、p-IκB和IκB等蛋白的表达及炎性因子TNF-α的分泌水平,以探讨橙皮苷对IEC-6细胞炎性损伤的保护作用机制。结果表明,橙皮苷显著抑制LPS诱导的IEC-6细胞Caspase-1和IκB的激活,抑制炎性因子TNF-α的分泌,进而抑制细胞活性的降低以及迁移能力下降,从而保护IEC-6细胞免受LPS诱导的细胞炎性损伤。 相似文献
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《中国兽医学报》2020,(3)
以野生型(WT)小鼠和钙调磷酸酶调节因子1(RCAN1)基因缺失(RCAN1~(-/-))小鼠骨髓来源巨噬细胞为模型,探究RCAN1对沙门菌诱导的炎性体活化的影响。用沙门菌和沙门菌鞭毛蛋白处理细胞诱导炎性体活化后,采用ELISA方法检测细胞培养上清中IL-1β和TNF-α的分泌,采用Western blot方法检测caspase-1、IL-1β的剪切成熟以及ASC的寡聚化,分析RCAN1对炎性体活化影响。结果显示,RCAN1能够显著促进沙门菌诱导的IL-1β的分泌,但对TNF-α的分泌无影;能够促进caspase-1和IL-1β前体的剪切与成熟、促进ASC寡聚化。沙门菌鞭毛蛋白能够诱导NLRC4炎性体活化。鞭毛蛋白处理后,RCAN1能够促进IL-1β的分泌,但不影响TNF-α的分泌。结果表明,RCAN1能够促进沙门菌诱导的NLRC4炎性体活化信号。 相似文献
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甲型流感病毒(influenza A virus,IAV)对全人类的健康构成了持续性威胁,感染时会引发炎症性疾病。NOD样受体蛋白3(NOD-like receptor protein 3,NLRP3)炎性小体是一种天然免疫系统传感器,与甲型流感病毒感染引发的炎症反应相关。在宿主细胞被甲型流感病毒感染时,能够激活NLRP3炎性小体,促使pro-IL-1β和pro-IL-18剪切成熟的IL-1β和IL-18并分泌至胞外,进而引发炎症反应。研究表明,NLRP3炎性小体对于在甲型流感病毒感染期间诱导先天性免疫应答至关重要,并且由甲型流感病毒感染引发过度激活的NLRP3炎性小体与细胞因子风暴和不受控制的炎症反应有关。本文回顾了甲型流感病毒与NLRP3炎性小体之间关系的研究进展,并讨论了NLRP3炎性小体在甲型流感病毒致病机理中的保护作用和致病作用。 相似文献
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为研究布鲁氏菌LPS对巨噬细胞中NLRP3炎症小体的影响,本试验提取布鲁氏菌2308、RB51和△WbkA的LPS,以不同浓度与小鼠巨噬细胞相互作用,荧光定量PCR检测其对NLRP3、ASC、Caspase1、IL1-β、IL18转录水平的影响。结果显示RB51LPS和△WbkA LPS上调NLRP3炎症小体相关基因的转录水平,且呈浓度依赖性,而浓度对2308LPS调节NLRP3炎症小体相关基因转录的作用不大;且同一浓度下,RB51LPS和2308LPS比△WbkA LPS更好的调节Caspase1、IL1-β、IL18的转录水平。 相似文献
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旨在探讨牛分枝杆菌减毒株卡介苗(Bacillus Calmette-Guérin,BCG)感染人单核巨噬细胞THP-1细胞后PERK/ATF4/CHOP通路对NLRP3炎性小体的调控作用。在BCG单独感染或与PERK小干扰RNA共处理THP-1细胞后,采用qRT-PCR和Western blot方法检测NLRP3炎性小体相关分子和PERK/ATF4/CHOP通路标志性分子在mRNA、蛋白水平的表达;在BCG单独感染或与PERK抑制剂GSK2656157共处理THP-1细胞后,分别采用qRT-PCR和Western blot方法检测NLRP3炎性小体相关分子和PERK/ATF4/CHOP通路标志性分子在mRNA、蛋白水平的表达,采用ELISA方法检测白细胞介素-1β(interleukin-1β,IL-1β)和白细胞介素-18(interleukin-18,IL-18)的释放量,采用CCK-8方法检测THP-1细胞活率,采用免疫荧光检测NLRP3与ASC的共定位。结果表明：在BCG单独感染THP-1细胞不同时间后,PERK、NLRP3、ASC和Caspase-1在蛋白水平的表达均随感染时间延长而升高,且在24 h达到最高(P<0.001),IL-1β和IL-18的释放随时间递增,24 h达到最高(P<0.001)。在BCG单独感染或与PERK小干扰RNA共处理THP-1细胞24 h后,与未感染对照组相比,siNC+BCG感染组NLRP3、ASC、Caspase-1、PERK、ATF4、CHOP分子的mRNA和蛋白表达均显著(P<0.05)或极显著(P<0.001)上调,而siPERK+BCG感染组与siNC+BCG感染组相比,NLRP3等关键分子的mRNA和蛋白表达均显著(P<0.05)或极显著下调(P<0.01,P<0.001);在BCG单独感染或与GSK2656157共同作用THP-1细胞24 h后,与未感染对照组相比,BCG感染组NLRP3、ASC、Caspase-1、PERK、ATF4、CHOP分子的mRNA和蛋白表达均显著(P<0.05)或极显著(P<0.01)上调,IL-1β和IL-18的释放极显著增加(P<0.001),细胞活率极显著下调(P<0.001),而BCG+GSK2656157感染组与BCG单独感染组相比,上述分子的mRNA和蛋白表达均显著(P<0.05)或极显著下调(P<0.01),IL-1β和IL-18的释放显著(P<0.05)或极显著(P<0.01)减少,细胞活率显著上调(P<0.05),免疫荧光的结果显示NLRP3与ASC存在共定位,且GSK2656157可以极显著抑制BCG感染引起的NLRP3和ASC的表达上调(P<0.001)。以上研究结果表明,PERK/ATF4/CHOP通路对BCG感染巨噬细胞后NLRP3炎性小体的活化具有调控作用。 相似文献
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Jeeyoung Kim Huijeong Ahn Heung-Myong Woo Eunsong Lee Geun-Shik Lee 《Veterinary research communications》2014,38(3):193-200
Inflammasomes, which are intracellular sensors of endogenous or exogenous danger signals, activate caspase-1, resulting in interleukin (IL)-1β maturation. Although most studies on inflammasomes have been performed in human and/or mouse-derived macrophages, porcine inflammasome activation has not been elucidated even though pigs are considered one of the best animal models for translational and preclinical investigations. In this study, we optimized detection of porcine IL-1β secretion, which is the most well established indicator of inflammasome activation, and compared inflammasome activation between miniature and domestic pigs as well as between porcine and murine macrophages. In our results, anti-sera against murine IL-1β had higher affinity to porcine IL-1β than anti-sera against human IL-1β, even though the amino acid sequence of porcine IL-1β was more similar to that of human IL-1β. In addition, there was no significant difference in inflammasome activation between miniature and domestic pigs. Furthermore, well established inflammasome triggers (ATP, nigericin, and crystals) in humans and mice had similar effects on porcine NLRP3 inflammasome activation. We further elucidated the upstream signaling pathway of porcine inflammasome activation using pharmacological inhibitors. Similar to the mechanisms of inflammasome activation in humans and mice, potassium efflux and reactive oxygen species generation were confirmed as key pathways in porcine inflammasome activation. Thus, inflammasome activation in pigs is not different from that in humans or mice. 相似文献
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为探究金针菇多糖(FVP)和发酵金针菇多糖(FFVP)对小鼠单核巨噬细胞(RAW264.7)炎症反应的影响与机制,以脂多糖(LPS)构建RAW264.7炎症模型,设置CON组(正常培养基)、LPS组(正常培养基+1μg/mL LPS)、FVP组(正常培养基+1μg/mL LPS+25、50或100μg/mL FVP)和FFVP组(正常培养基+1μg/mL LPS+25、50或100μg/mL FFVP),通过测定RAW264.7的细胞活力、吞噬能力、活性氧(ROS)和一氧化氮(NO)含量以及炎症因子白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、白细胞介素-18(IL-18)和肿瘤坏死因子-α(TNF-α)的含量与mRNA相对表达量,比较FVP和FFVP抑制巨噬细胞炎症反应的作用;以核转录因子-κB(NF-κB)抑制剂BAY11-7082处理RAW264.7,通过Western blot检测磷酸化核转录因子-κB抑制蛋白α(p-IκBα)、NOD样受体家族含pyrin结构域蛋白3(NLRP3)、半胱天冬蛋白酶-1(Caspase-1)和IL-1β的蛋白相对表达量,探究FVP和FFVP对LPS诱导的巨噬细胞炎症反应可能的作用机制。结果表明:FVP和FFVP均能抑制LPS引起的RAW264.7 ROS和NO含量升高,浓度为100μg/mL时,两者差异显著(P<0.05);相比LPS组,FVP和FFVP分别使RAW264.7的吞噬能力提升37.65%和47.06%;FVP和FFVP降低炎症因子IL-1β、IL-6、IL-18和TNF-α的含量及mRNA相对表达量,并呈剂量依赖性。Western blot结果表明以BAY11-7082与FVP或FFVP同时处理,RAW264.7的p-IκBα、NLRP3、Caspase-1和IL-1β的蛋白相对表达量显著降低(P<0.05),说明FVP/FFVP可通过降低IκBα的磷酸化抑制NLRP3信号通路的激活。综上可知,FVP和FFVP均能增强RAW264.7的细胞活力和吞噬能力,降低ROS和NO含量,通过抑制NF-κB-NLRP3信号通路的激活抑制巨噬细胞炎症反应;相同浓度下,FFVP的抗炎效果优于FVP。 相似文献
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试验旨在研究布鲁氏菌侵染宿主细胞过程中AIR对由布鲁氏菌感染引发的炎症反应的影响。本试验分别对膜融合蛋白Tecpr1中AIR结构域进行干扰(I-A)、过表达(O-A)、干扰后回补(OA-IA),建立羊种布鲁氏菌16M侵染细胞的模型。以二氯荧光素(DCFH-DA)为探针,利用激光共聚焦显微镜观察 ROS产生的变化及各组细胞线粒体分布的动态变化;通过qRT-PCR技术检测16M侵染宿主细胞引起NLRP3、 ASC和Caspase-1表达量的变化;通过ELISA方法检测IL-18、IL-1β和Caspase-1炎性因子表达量的变化。结果表明,16M能以时间依赖性方式诱导RAW264.7细胞产生ROS,I-A组和 O-A组线粒体异常集聚程度高;qRT-PCR及ELISA检测结果表明,16M侵染宿主细胞能够引起不同处理组细胞中炎性相关基因NLRP3、ASC和Caspase-1及炎性因子IL-18、IL-1β和Caspase-1表达量的变化。AIR缺失后,ROS 释放量发生变化,线粒体出现异常集聚,且AIR与炎性小体的活化、炎症反应的发生密切相关。 相似文献
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XU Ya-fang CHEN Chuang-fu WANG Hao FU Qiang SHI Hui-jun SUN Zhi-hua ZHANG Hui GUO Fei 《中国畜牧兽医》2016,43(6):1437-1445
In order to investigate the effect of AIR on inflammatory reaction infected by Brucellamelitensis (16M), the AIR domain of Tecpr1 gene of murine macrophages RAW264.7 were knocked down (I-A), overexpressed (O-A) and reversed (OA-IA). Using the chlorine fluorescein (DCFH-DA) as a probe, we detected the variation of ROS production and mitochondria distribution by confocal laser scanning microscopy. We observed the expression changes of NLRP3, ASC and Caspase-1 by qRT-PCR and the expression changes of IL-18,IL-1β and Caspase-1 in host cells by ELISA. The results showed that 16M could stimulate RAW264.7 cells to produce ROS by time-dependent pathway, and I-A group and O-A group showed more abnormal accumulation of mitochondrial. The results of qRT-PCR and ELISA suggested that it had effect on the expression levels of NLRP3, ASC,Caspase-1 and IL-18, IL-1β and Caspase-1 in cells of different groups. Those results indicated that with AIR gene deletion, the release amount of ROS changed, mitochondrial clustered abnormally, and AIR was closely related to the activation of inflammasomes and induction of inflammatory reactions. 相似文献
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Huijeong Ahn Jeongeun Kim Sungkyun Kwon Pyeung-Hyeun Kim Hyuk Moo Kwon Eunsong Lee Geun-Shik Lee 《Veterinary research communications》2018,42(4):265-273
Pigs are an important livestock and serve as a large animal model due to physiological and anatomical similarities with humans. Thus, components of the porcine immune system such as inflammasomes need to be characterized for disease control, vaccination, and translational research purposes. Previously, we and others elucidated porcine nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family Pyrin domain containing 3 (NLRP3) inflammasome activation. However, until now, porcine NLR family caspase recruitment domain (CARD)-containing 4 (NLRC4) and absent in melanoma 2 (AIM2) inflammasomes have been not well studied. In this study, we treated well defined NLRC4 and AIM2 inflammasome triggers to porcine peripheral blood mononuclear cells (PBMCs) and murine bone-marrow derived macrophages (BMDMs) and observed interleukin (IL)-1β maturation as a readout of inflammasome activation. NLRC4 (flagellin) and AIM2 (dsDNA) triggers led to IL-1β secretion in both porcine PBMCs and mice macrophages. In addition, porcine and mouse NLRC4 and AIM2 inflammasomes responded differently to NLRP3 inhibitors. Bacterial inflammasome triggers, Salmonella enterica serovar Typhimurium, Listeria monocytogenes, and Escherichia coli, also induced IL-1β secretion in porcine PBMCs. Taken together, we suggest that known triggers of NLRC4 and AIM2 inflammasomes in mice induce IL-1β secretion in porcine PBMCs. 相似文献
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Mari Spildrejorde Stephen J. Curtis Belinda L. Curtis Ronald Sluyter 《Veterinary immunology and immunopathology》2014,157(1-2):105-110
Binding of extracellular adenosine 5′-triphosphate (ATP) or lipopolysaccharide (LPS) to the damage-associated molecular pattern receptor P2X7 or the pathogen-associated molecular pattern receptor Toll-like receptor (TLR)4, respectively, can induce the release of the pleiotropic cytokine interleukin (IL)-1β in humans and mice. However, the release of IL-1β in dogs remains poorly defined. Using a canine IL-1β enzyme-linked immunosorbent assay, this study investigated whether ATP or LPS could induce IL-1β release in a canine blood-based assay. Short-term incubations (30 min) with ATP induced IL-1β release in LPS-primed canine blood, and this process could be near-completely impaired by the P2X7 antagonist, A438079. In contrast, ATP failed to induce IL-1β release from blood not primed with LPS. ATP-induced IL-1β release was observed with LPS-primed blood from eight different pedigrees or cross breeds. Long-term incubations (24 h) with LPS induced IL-1β release in canine blood in a concentration-dependent manner. This process was not altered by co-incubation with A438079. LPS-induced IL-1β release was observed with blood from 10 different pedigrees or cross breeds. These results demonstrate that both extracellular ATP and LPS can induce IL-1β release in dogs, and that ATP- but not LPS-induced IL-1β release in blood is dependent on P2X7 activation. These findings support the role of both P2X7 and TLR4 in IL-1β release in dogs. 相似文献
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Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) that mediate first line of host defence to pathogens. TLR agonists are potent immunostimulatory agents that help to prime a robust adaptive immune response. In the present study, adjuvant potential of Poly I:C and lipopolysaccharide (LPS) were evaluated with live Newcastle disease virus (NDV) vaccine. Cornish chickens were immunized with live Newcastle disease virus (NDV) vaccine (R2B-mesogenic strain) adjuvanted either with Poly I:C (TLR3 agonist) or LPS-TLR4 agonist and both. Humoral Immune response to ND vaccine was evaluated through haemagglutination inhibition (HI) test and ELISA, while the cellular immune response (CMI) was quantified by lymphocyte transformation test (LTT). IL-1β cytokine mRNA levels in spleen tissue were also quantified by real time PCR. The results suggest that TLR3 and TLR4 agonists are an efficient immune-stimulators separately, as LPS co-administered group has shown significantly higher serum titre on second week post-immunization and Poly I:C group on third week post-immunization both by HI and ELISA (P?<?0.01), however, the combined administration of both LPS and Poly I:C did not give any complementary effect on serum titre. There were no significant differences in stimulation indices (SI) and IL-1β cytokine levels between groups at different intervals post-immunization. Hence, TLR agonists LPS followed by Poly I:C could be used as adjuvant to enhance the immune response to NDV vaccine in chicken.
相似文献19.
【目的】验证黑种草子提取物对小鼠酒精性肝损伤的修复作用并探究其机制,为其临床开发研究提供理论依据。【方法】将56只3周龄昆明小鼠随机均分为7组：空白对照组,模型组,黑种草子提取物高、中、低剂量组,黑种草子粉剂组及水飞蓟宾阳性对照组,每组8只。除空白对照组外,其余各组均以10 mL/kg 60%酒精灌胃,每天1次,连续15 d。第16天,黑种草子提取物高、中、低剂量组分别给予8、4、2 mL/kg黑种草子提取物,每天1次,连续10 d后处死小鼠。计算各组小鼠肝脏指数;检测血清中谷丙转氨酶（alanine aminotransferase,ALT）、谷草转氨酶（aspartate aminotransferase,AST）、过氧化氢酶（catalase,CAT）、超氧化物歧化酶（superoxide dismutase,SOD）和谷胱甘肽过氧化物酶（glutathione peroxidase,GSH-Px）活力;制备肝脏石蜡切片观察其病理变化;Western blotting检测NOD样受体热蛋白结构域相关蛋白3（NOD-like receptor thermal protein domain associated protein 3,NLRP3）、含半胱氨酸的天冬氨酸蛋白水解酶1（cysteinyl aspartate specific proteinase 1,Caspase-1）和白细胞介素1β（interleukin-1β,IL-1β）的蛋白表达水平。【结果】与空白对照组相比,模型组小鼠肝脏指数显著上升（P<0.05）,肝细胞出现明显颗粒变性,水泡变性,核溶解,界限区分不清,肝索消失;血清ALT、AST活力显著上升（P<0.05）,SOD、CAT、GSH-Px活力显著下降（P<0.05）,说明肝损伤模型建立成功,且肝脏组织中NLRP3、Caspase-1和IL-1β蛋白表达水平显著升高（P<0.05）。与模型组相比,黑种草子提取物各剂量组血清ALT、AST活力均呈下降趋势,SOD、CAT、GSH-Px活力均呈上升趋势,其中高剂量组上述各指标均呈显著差异（P<0.05）,肝脏组织结构明显改善,NLRP3、Caspase-1和IL-1β蛋白表达水平均显著下降（P<0.05）。【结论】高剂量（8 mL/kg）黑种草子提取物可显著提高肝损伤小鼠的抗氧化水平,且可以通过抑制NLRP3炎性小体的表达而减少IL-1β的合成,从而改善肝脏功能和修复受损的肝脏组织结构。 相似文献