HSP72过度表达对脂多糖刺激的巨噬细胞中NF-kB活性的影响

目的研究过度表达的热休克蛋白72(heat shock protein,HSP72)对脂多糖(1ipopolysaccharide、LPS)刺激的巨噬细胞中NF-KB活性的影响。方法构建pCD-hsp72重组质粒,转染小鼠巨噬细胞RAW264．7,G418筛选稳定转染hsp72基因的细胞,Westem印迹检测转染细胞HSP72的表达水平及LPS刺激后RAW264．7细胞内HSP72的表达水平、LPS刺激后转染细胞中IKBot的含量和胞核中NF-KB的含量的变化。结果构建pCD-hsp72重组质粒并转入巨噬细胞,获得稳定转染hsp72基因的细胞株,LPS刺激细胞内HSP72表达增加；HSP72可通过减少LPS刺激的巨噬细胞中IkBα的降解而抑制NF-kB活性。结论HSP72能抑制LPS激活的巨噬细胞中NF-kB的活化。     

汉黄芩素对LPS 和ATP 联合诱导的巨噬细胞炎症反应的抑制作用

目的:探究汉黄芩素对巨噬细胞炎症反应的抑制作用。方法:设正常对照组,LPS和ATP联合刺激组(LPS刺激4 h后加ATP刺激30 min),汉黄芩素干预组(刺激的同时加入汉黄芩素)。利用免疫荧光法观察各组细胞NF-κB的核定位,荧光定量PCR法检测各组细胞IL-1β、IL-18、IL-6、TNF-α、NLRP3及Caspase-1的mRNA水平,流式细胞术检测各组细胞活性氧(ROS)水平,酶联免疫吸附法检测胞外IL-6和TNF-α水平。结果:与LPS和ATP刺激组相比,汉黄芩素干预组细胞胞内NF-κB核定位明显减少,胞内IL-1β、IL-18、IL-6、TNF-α、NLRP3和Caspase-1的mRNA,胞内ROS及胞外IL-6和TNF-α水平都显著降低(P0. 01)。结论:汉黄芩素可能通过减少巨噬细胞ROS的产生,降低NF-κB的核定位,进而弱化NF-κB调控的炎症相关分子的基因转录来干预巨噬细胞的炎症反应。     

黄芩苷对NLRP3炎症小体活化和细胞焦亡的抑制作用及其机制研究

目的以LPS致敏的小鼠巨噬细胞(J774A.1或者腹腔巨噬细胞)作为细胞模型,探讨黄芩苷对ATP诱导的炎症小体活化和细胞焦亡的影响及其机制。方法 C57BL/6小鼠腹腔注射30 g·L-1巯基乙酸盐培养基诱导大量腹腔巨噬细胞产生;利用LPS致敏巨噬细胞,再以ATP激活NLRP3炎症小体;采用免疫印迹法检测细胞裂解物和培养上清液中IL-1β、caspase-1、HMGB1等蛋白的表达水平;以基于微球的免疫测定法(CBA)检测细胞培养上清液中IL-1β的水平;碘化丙锭(PI)染色法检测黄芩苷对LPS+ATP诱导的细胞焦亡的影响。结果黄芩苷处理能剂量依赖性地抑制LPS+ATP刺激下巨噬细胞中caspase-1的活化,成熟IL-1β(Mr17 000)和HMGB1的释放;同时,黄芩苷也能明显抑制LPS+ATP诱导的小鼠巨噬细胞发生焦亡。腺苷酸环化酶抑制剂MDL12330A和蛋白激酶A(PKA)抑制剂H89可以明显逆转黄芩苷对ATP诱导的细胞焦亡的抑制作用。结论黄芩苷可能通过影响巨噬细胞中PKA的活性,进而抑制NLRP3炎症小体活化与细胞焦亡。     

HSP72过度表达对脂多糖刺激的巨噬细胞中NF-κB活性的影响

目的研究过度表达的热休克蛋白72(heat shock protein,HSP72)对脂多糖(lipopolysaccha-ride,LPS)刺激的巨噬细胞中NF-κB活性的影响.方法构建pCD-hsp72重组质粒,转染小鼠巨噬细胞RAW264.7,G418筛选稳定转染hsp72基因的细胞,Western印迹检测转染细胞HSP72的表达水平及LPS刺激后RAW264.7细胞内HSP72的表达水平、LPS刺激后转染细胞中IκBα的含量和胞核中NF-κB的含量的变化.结果构建pCD-hsp72重组质粒并转入巨噬细胞,获得稳定转染hsp72基因的细胞株,LPS刺激细胞内HSP72表达增加;HSP72可通过减少LPS刺激的巨噬细胞中IκBα的降解而抑制NF-κB活性.结论HSP72能抑制LPS激活的巨噬细胞中NF-κB的活化.     

结直肠癌细胞来源的外泌体miR-34a对M2型巨噬细胞极化的影响北大核心CSCD

目的:探究结直肠癌来源的外泌体对巨噬细胞极化的影响及其作用机制。方法:建立THP-1来源的M2型巨噬细胞体外诱导分化模型,利用流式细胞染色实验检测细胞分化效率;通过与结直肠癌细胞共培养、结直肠癌细胞外泌体干预M2型巨噬细胞分化,利用实时荧光定量PCR实验检测ARG1、IL-10和NLRP3 mRNA的表达水平。结果:体外诱导分化的M2型THP-1巨噬细胞CD163阳性细胞比例显著升高;与对照组相比,与结直肠癌细胞HCT8和HCT116共培养的M2型巨噬细胞ARG1、IL-10表达水平显著升高,NLRP3表达水平下降。与转染阴性对照microRNA(miR-NC)组相比,转染miR-34a inhibitor的HCT8和HCT116共培养或外泌体刺激下,M2型巨噬细胞ARG1、IL-10表达水平下降,NLRP3表达水平升高;与miR-NC组相比,转染miR-34a inhibitor的THP-1细胞ARG1、IL-10表达水平显著下降,NLRP3表达水平显著升高。结论:结直肠癌细胞来源的外泌体miR-34a可以抑制巨噬细胞NLRP3的表达,促进巨噬细胞M2型极化。     

皮质酮抑制LPS诱导的小鼠巨噬细胞NLRP3表达及与XO的关系

目的:探讨皮质酮(CORT)对脂多糖(LPS)诱导的小鼠巨噬细胞核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)表达的抑制作用及与黄嘌呤氧化酶(XO)的关系。方法:用LPS构建小鼠巨噬细胞RAW 264.7的炎症模型。运用不同浓度(0~900μg/L)的CORT处理巨噬细胞后提取细胞总蛋白,Western blot法检测细胞NLRP3和caspase-1的蛋白水平;按处理因素分组为:对照组、LPS组、LPS+CORT组和LPS+别嘌呤醇(allopurinol)组,分别于0、0.5、1、1.5和2 h提取细胞成分,运用real-time PCR和Western blot法检测细胞NLRP3和XO的m RNA和蛋白水平。结果:高于700μg/L的CORT可显著抑制LPS诱导的巨噬细胞中NLRP3的表达及caspase-1的活化(P0.05);与LPS组相比,LPS+CORT在下调LPS诱导的巨噬细胞NLRP3表达的同时抑制XO的表达(P0.05),LPS+allopurinol组巨噬细胞NLRP3的表达减少(P0.05)。结论:较高浓度的CORT能抑制LPS诱导的小鼠巨噬细胞NLRP3的表达,而CORT的抑制作用可能与其下调XO的表达水平有关。     

脂多糖诱导的小鼠腹腔巨噬细胞基因表达谱分析

目的　利用基因芯片技术分析脂多糖 (LPS)活化的小鼠腹腔巨噬细胞基因表达谱 ,以更全面地了解LPS诱导的巨噬细胞反应。方法　以未刺激的和用 1mg/LLPS刺激的小鼠腹腔巨噬细胞制备3 3 P标记的cDNA探针 ,分别与含有 1176个已知基因的小鼠cDNA芯片杂交。结果　活化组和未刺激组间的 2倍差异表达基因为 118个 ,3倍差异表达基因为 6 9个 ,其中 4 4个上调 ,2 5个下调。转录因子、细胞内信号调节蛋白、炎症细胞因子和细胞凋亡相关基因的转录发生明显的调节变化。结论提供了LPS活化的巨噬细胞综合基因表达信息 ,并筛选出一些新的可能与LPS活化相关的基因。     

Ficolin通过激活补体加重poly(I:C)联合LPS诱导的急性肺脏损伤北大核心CSCD

目的:探究纤维胶凝素(ficolin)在poly(I:C)联合LPS刺激诱导的急性肺损伤中的作用及机制。方法:6~8周龄SPF级C57BL/6小鼠和ficolin基因敲除小鼠分别随机分组,连续2 d滴鼻50μg/d poly(I:C),然后50μg/d滴鼻LPS 1 d,构建poly(I:C)和LPS联合刺激诱导的急性肺损伤小鼠模型,单独刺激或滴鼻PBS为对照组;HE染色检测肺组织病理变化;流式细胞术检测每组小鼠肺组织中性粒细胞和巨噬细胞的比例变化;Western blot检测每组小鼠肺组织和RAW264.7细胞中C3的表达变化和活化情况。结果:成功建立了poly(I:C)联合LPS刺激诱导的急性肺损伤小鼠模型。联合刺激组小鼠肺脏病理损伤加重,肺泡融合和弥漫性损伤,大量炎症细胞浸润;其中中性粒细胞和间质巨噬细胞比例显著提高,肺泡巨噬细胞比例显著降低。联合刺激增加小鼠肺组织和RAW264.7细胞中补体C3的表达和酶解活化,而敲除ficolin可明显降低联合刺激诱导的C3活化,并减少间质巨噬细胞募集和肺泡巨噬细胞耗竭,改善急性肺损伤。结论:Ficolin可通过激活补体加重poly(I:C)联合LPS刺激介导的肺脏炎症和急性肺损伤,是重症肺炎潜在的治疗靶点。     

Aire 对巨噬细胞极化影响的研究

目的:研究自身免疫调节因子(Aire)对巨噬细胞极化的影响。方法:分别用LPS、IL-4 以及LPS 联合免疫复合物刺激小鼠单核巨噬细胞系RAW264郾7 细胞、稳定表达GFP-Aire 的RAW264.7 细胞(A33-3) 细胞和稳定表达GFP 的RAW264.7 细胞(C1-6),使其向M1(LPS)、M2a(IL-4)和M2b(LPS 联合免疫复合物)型巨噬细胞极化。通过Real-time PCR 检测各组细胞中M1 型巨噬细胞特征分子IL-1、iNOS 和IL-6,M2a 型特征分子Arg-1 和M2b 型特征分子IL-10 的表达水平,研究Aire 对各种类型巨噬细胞极化的影响。结果:LPS 在0.5 g/ ml 浓度时,RAW264.7 细胞中M1 型巨噬细胞产物IL-1 、iNOS和IL-6 基因表达量最高;而IL-4 以及LPS 联合免疫复合物的刺激作用有显著的剂量依赖性,都在浓度最高时RAW264.7 细胞中Arg1(M2a)和IL-10(M2b)基因表达量最高。LPS 刺激后,A33-3 细胞中IL-1 和iNOS 表达水平明显高于C1-6 细胞,IL-6 则相反;IL-4 及LPS 联合免疫复合物刺激后,A33-3 细胞中Arg1 和IL-10 的表达水平明显低于C1-6 细胞。结论:Aire 可能促进巨噬细胞向M1 极化,同时抑制其向M2a 和M2b 极化。     

雪胆乙素通过抑制小鼠巨噬细胞中AMPK活性抑制NLRP3炎症小体活化和细胞焦亡

目的 本研究旨在探索雪胆乙素对ATP诱导的炎症小体活化和细胞焦亡的影响,并探讨其潜在的作用机制。方法利用第一信号（LPS）刺激巨噬细胞,再以第二信号（ATP）激活NLRP3炎症小体;采用Western blot检测caspase-1、GSDMD等蛋白的表达水平;ELISA检测细胞培养上清液中IL-1β的水平;免疫荧光染色检测雪胆乙素对LPS+ATP诱导的ASC斑点形成的影响;碘化丙锭（PI）染色法检测细胞死亡。结果 我们发现雪胆乙素处理能够抑制ATP诱导的巨噬细胞中ASC斑点的形成,并抑制LPS+ATP刺激下巨噬细胞中caspase-1的切割及活化;进一步抑制成熟IL-1β释放至培养液上清中;同时,雪胆乙素也能够明显抑制巨噬细胞中GSDMD-NT的形成以及细胞焦亡。另外,雪胆乙素能够抑制AMPK的磷酸化,而AMPK的激动剂AICAR促进成熟IL-1β的释放,以及可以明显逆转雪胆乙素对ATP诱导的细胞焦亡的抑制作用。结论 雪胆乙素可能通过抑制巨噬细胞中AMPK的活性而抑制NLRP3炎症小体活化与细胞焦亡。     

Negative regulation of NLRP3 inflammasome by SIRT1 in vascular endothelial cells

NLRP3 inflammasome not only functions as a critical effector in innate immunity, but also triggers the production of proinflammatory cytokines involved in inflammation-associated diseases. Sirtuin 1 (SIRT1) plays an important role in the regulation of cellular inflammation. However, whether the activation of NLRP3 inflammasome is regulated by SIRT1 remains unknown. In this study, we investigated the regulatory effect of SIRT1 on NLRP3 inflammasome and the underlying mechanisms. We found that lipopolysaccharide (LPS) and adenosine triphosphate (ATP)-induced the activation of NLRP3 inflammasome in human umbilical vein endothelial cells (HUVECs). Activation of SIRT1 inhibited NLRP3 inflammasome activation and subsequent caspase-1 cleavage as well as interleukin (IL)-1β secretion, whereas SIRT1 knockdown obviously enhanced the activation of NLRP3 inflammasome in HUVECs. Importantly, gene silencing of SIRT1 abrogated the inhibitory effect of SIRT1 activator on NLRP3 inflammasome formation and IL-1β production in HUVECs stimulated with LPS plus ATP. Further study indicated that cluster of differentiation 40 (CD40) may be involved in the regulation of NLRP3 inflammasome by SIRT1. In vivo studies indicated that implantation of the periarterial carotid collar increased the arterial expression levels of CD40 and CD40 Ligand (CD40L), but inhibited arterial SIRT1 expression in the rabbits. Moreover, treatment with SIRT1 activator decreased CD40 and CD40L levels in collared arteries. Meanwhile, serum IL-1β level, the marker of inflammasome activation, was also inhibited by SIRT1 activation. Taken together, these findings revealed a novel regulatory mechanism of NLRP3 inflammasome by SIRT1, which may be related to suppression of CD40.     

MD-2 regulates LPS-induced NLRP3 inflammasome activation and IL-1beta secretion by a MyD88/NF-κB-dependent pathway in alveolar macrophages cell line

Myeloid differentiation protein 2 (MD-2) is required in the recognition of lipopolysaccharide (LPS) by toll-like receptor 4 (TLR4), and participates in LPS-induced alveolar macrophage (AM) inflammation during acute lung injury (ALI). Activation of the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome aggravates inflammation in LPS-induced ALI. However, there is currently little known about the relationship between MD-2 signaling and the NLRP3 inflammasome. This study showed that NLRP3 expression, IL-1beta (IL-1β) secretion, and pyroptosis were up-regulated after LPS stimulation in the NR8383 AM cell-line. MD-2 gene knock-down reduced LPS-induced mRNA and protein expression of NLRP3 and IL-1β secretion in NR8383 cells, and inhibited the MyD88/NF-κB signaling pathway. Conversely, over-expression of MD-2 not only heightened NLRP3, MyD88, and NF-κB p65 protein expression, it also aggravated the LPS-induced inflammatory response. Furthermore, the NF-κB inhibitor SN50 had a beneficial role in decreasing NLRP3 and caspase-1 mRNA and protein expression. The observations suggest that MD-2 helps to regulate LPS-induced NLRP3 inflammasome activation and the inflammatory response in NR8383 cells, and likely does so by affecting MyD88/NF-κB signaling.     

Inhibition of NLRP3 inflammasome: a new protective mechanism of cinnamaldehyde in endotoxin poisoning of mice

Context: Cinnamaldehyde (CA) has a protective effect in endotoxin poisoning of mice, but there is no direct evidence for the protective effect of CA through inhibition of NLRP3 inflammasome activation in endotoxin poisoning of mice.

Objective: We aimed to investigate the protective mechanism of CA in endotoxin poisoned mice through NLRP3 inflammasome.

Materials and methods: First, we evaluated the anti-inflammatory effect of CA in phorbol-12-myristate acetate–differentiated THP-1 cells through the NLRP3 inflammasome. Second, in a mouse model of lipopolysaccharide (LPS)-induced endotoxin poisoning, CA was administrated for 5 d (once a day) before the 15?mg/kg LPS challenge. Then, the levels of IL-1β in serum were measured, and the effect of CA on the NLRP3 inflammasome activation and the expression of cathepsin B and P2X7R proteins in lung were explored.

Results: In vitro, CA decreased the levels of p20, pro-IL-1β and IL-1β in cell culture supernatants, as well as the expression of NLRP3 and IL-1β mRNA in cells. In vivo, CA decreased IL-1β production in serum. Furthermore, CA suppressed LPS-induced NLRP3, p20, Pro-IL-1β, P2X7 receptor (P2X7R) and cathepsin B protein expression in lung, as well as the expression of NLRP3 and IL-1β mRNA.

Conclusions: CA has a protective effect in the endotoxin poisoned mice through the inhibition of NLRP3 inflammasome activation. Furthermore, CA suppresses the NLRP3 inflammasome activation by inhibiting the expression of cathepsin B and P2X7R protein expression. CA can be considered as a potential therapeutic candidate for diseases involved in endotoxin poisoning such as sepsis.     

外源性硫化氢对肝细胞NLRP3炎症小体的影响

目的:研究外源性硫化氢(H_2S)对人肝细胞NLRP3炎症小体的影响。方法:采用不同浓度的脂多糖(LPS)诱导人肝细胞L02和SMMC-7721建立炎症模型,Western blot检测细胞中NLRP3炎症小体的表达并结合细胞毒性实验(MTT法)确定合适的LPS浓度。细胞分为4组:对照组用普通培养基培养18.5 h;LPS组用普通培养基培养0.5 h后,再用100μg/L LPS刺激18 h;LPS+H_2S组和H_2S组用200μmol/L硫氢化钠(Na HS)刺激0.5 h后,再分别用100μg/L LPS和普通培养基培养18 h。各组处理后分别收集细胞,Western blot检测细胞中NLRP3和caspase-1的蛋白表达量。结果:与对照组相比,LPS组细胞内NLRP3和caspase-1的表达增加(P0.05),H_2S组细胞内的NLRP3和caspase-1表达无明显变化;与LPS组相比,LPS+H_2S组细胞内的NLRP3和caspase-1表达减少(P0.05)。结论:外源性H_2S可抑制人肝细胞中NLRP3炎症小体的表达。     

阿托伐他汀通过下调NLRP1炎性体表达抑制IL-1β和IL-18的释放

 目的:探讨核苷酸结合寡聚化结构域样受体蛋白1(NLRP1)炎性体在阿托伐他汀抑制THP-1巨噬细胞白细胞介素-1β(IL-1β)和白细胞介素-18(IL-18)分泌中的作用。方法:用10 μg/L脂多糖诱导THP-1巨噬细胞分泌IL-1β和IL-18,以不同浓度阿托伐他汀(1、10和20 μmol/L)孵育细胞24 h,或以10 μmol/L阿托伐他汀处理细胞不同时间(12、24和48 h),或转染NLRP1 siRNA以沉默细胞内NLRP1的表达。采用RT-PCR检测细胞内NLRP1炎性体mRNA的表达,Western blot检测细胞内NLRP1炎性体蛋白的表达,ELISA检测细胞上清液中IL-1β和IL-18的含量。结果:阿托伐他汀可抑制THP-1巨噬细胞NLRP1炎性体mRNA和蛋白的表达,且这种效应呈浓度和时间依赖性。转染NLRP1 siRNA后,THP-1巨噬细胞NLRP1的蛋白表达明显下降,且阿托伐他汀对IL-1β和IL-18分泌的抑制作用明显增强。结论:阿托伐他汀通过抑制NLRP1炎性体表达减少巨噬细胞IL-1β和IL-18的释放,发挥抗炎作用,进而延缓动脉粥样硬化进展。     

Oxidized mitochondrial DNA activates the NLRP3 inflammasome during apoptosis

We report that in the presence of signal 1 (NF-κB), the NLRP3 inflammasome was activated by mitochondrial apoptotic signaling that licensed production of interleukin-1β (IL-1β). NLRP3 secondary signal activators such as ATP induced mitochondrial dysfunction and apoptosis, resulting in release of oxidized mitochondrial DNA (mtDNA) into the cytosol, where it bound to and activated the NLRP3 inflammasome. The antiapoptotic protein Bcl-2 inversely regulated mitochondrial dysfunction and NLRP3 inflammasome activation. Mitochondrial DNA directly induced NLRP3 inflammasome activation, because macrophages lacking mtDNA had severely attenuated IL-1β production, yet still underwent apoptosis. Both binding of oxidized mtDNA to the NLRP3 inflammasome and IL-1β secretion could be competitively inhibited by the oxidized nucleoside 8-OH-dG. Thus, our data reveal that oxidized mtDNA released during programmed cell death causes activation of the NLRP3 inflammasome. These results provide a missing link between apoptosis and inflammasome activation, via binding of cytosolic oxidized mtDNA to the NLRP3 inflammasome.     

Dysregulated production of interleukin-1β upon activation of the NLRP3 inflammasome in patients with familial Mediterranean fever

Familial Mediterranean fever (FMF) is caused by mutations in pyrin, a protein expressed in innate immune cells that interacts with caspase-1 and other inflammasome components to regulate interleukin (IL)-1β maturation. Since NLRP3 inflammasome represents major source of IL-1β, we studied its protein expression and function in FMF. We isolated peripheral white blood cells (WBCs) from 20 symptoms-free FMF patients and 21 healthy individuals. Intracellular protein expression of NLRP3, caspase-1, IL-1β at baseline and after LPS/ATP sequential treatment for NLRP3 activation was assessed by immunoblotting. Secreted IL-1β was quantified by ELISA. THP-1 cells were transfected with wild-type or mutant pyrin and IL-1β secretion was measured. FMF WBCs exhibited lower NLRP3 and active caspase-1 protein expression compared to healthy individuals, and LPS/ATP treatment resulted in significantly lower intracellular IL-1β levels in FMF patients. Likewise, LPS/ATP induced caspase-1-dependent IL-1β release at significantly lower amounts in the FMF group (1182 ± 192 versus 2134 ± 245 pg/mL in controls, p = 0.004). Consistently, THP-1 cells transfected with FMF-associated M694V mutant pyrin displayed lower LPS/ATP-induced IL-1β compared with wild-type pyrin-transfected cells. FMF WBCs demonstrate reduced NLRP3-mediated IL-1β production. Additional studies are needed to define whether this finding represents a compensatory mechanism to control inflammation or is directly linked to disease pathogenesis.