右美托咪定通过c-Fos/NLRP 3/caspase-1级联抑制LP S诱发的小胶质细胞炎症反应

目的探讨右美托咪定通过c-Fos/NLRP3/caspase-1级联抑制脂多糖(LPS)诱发的小胶质细胞炎症反应。方法选取新生的SD大鼠,取其小胶质细胞进行原代培养及分离纯化。采用LPS诱导建立小胶质细胞炎症模型。采用MTT法选取右美托咪定抑制LPS诱导小胶质细胞炎症反应的最适宜浓度。将细胞分为三组:对照组、LPS组和右美托咪定治疗组(D组)。采用实时定量PCR法测定细胞中IL-1β和TNF-α的mRNA表达量;采用ELISA法检测细胞上清液中IL-1β和TNF-α的含量;采用Western blot法检测原癌基因c-Fos、NLRP3和caspase-1的蛋白含量。结果与对照组比较,LPS组小胶质细胞中炎性因子IL-1β和TNF-α的mRNA表达量均明显升高(P0.05);D组IL-1β和TNF-α的mRNA表达量明显低于LPS组(P0.05)。与对照组比较,LPS组中小胶质细胞中炎性因子IL-1β和TNF-α的含量均明显增加(P0.05);而D组IL-1β和TNF-α的含量明显低于LPS组(P0.05)。与对照组比较,LPS组小胶质细胞中c-Fos、NLRP3和caspase-1的蛋白含量均明显增加(P0.01);而D组c-Fos、NLRP3和caspase-1的蛋白含量明显低于LPS组(P0.05)。结论右美托咪定可抑制LPS诱发的小胶质细胞中炎性因子IL-1β和TNF-α的mRNA表达量及蛋白含量,推测其作用机制可能与抑制c-Fos/NLRP3/caspase-1级联反应有关。     

激活PPAR-γ对脑缺血-再灌注损伤中炎症反应的影响

目的探究激活过氧化物酶体增殖物激活受体-γ(PPAR-γ)对脑缺血-再灌注损伤(CIRI)与脂多糖(LPS)诱导炎症反应的影响。方法在动物实验中,SD雄性大鼠40只,体重250～270 g,随机分成四组:假手术组(S组)、缺血-再灌注组(IR组)、二甲基亚砜(DMSO)+缺血-再灌注组(DIR组)和吡格列酮+缺血-再灌注组(PIR组)。其中S组进行假手术处理,IR组进行脑中动脉缺血(MCAO)建模处理,DIR组给予DMSO,随后进行MCAO造模处理。PIR组给予吡格列酮,随后进行MCAO造模处理。在细胞实验中,通过慢病毒转染BV2细胞使PPAR-γ过表达,将细胞培养孔随机分为三组:空白对照组(B组)、阴性对照组(L1组)、过表达组(L2组)。其中B组未做处理,L1组转染PPAR-γ阴性病毒,L2组转染PPAR-γ阳性病毒。之后将细胞培养孔随机分为四组:正常对照组(C组)、正常细胞+LPS处理组(L组)、阴性对照+LPS处理组(LL1组)和过表达+LPS处理组(LL2组)。其中C组未做处理,L组用LPS处理,LL1组转染阴性病毒并确定转染率后使用LPS处理,LL2组转染阳性病毒并确定转染率后用LPS处理。各组大鼠使用Zea Longa评分标准进行神经行为学评分,TTC染色比较脑梗死容积百分比大小,采用ELISA法检测四组大鼠和四组BV2细胞IL-1β和IL-18浓度,Western blot法检测PPAR-γ、活化的半胱天冬酶1(caspase-1)和NOD样受体家族3(NLRP3)蛋白相对含量。结果与IR组比较,PIR组大鼠行为学评分明显降低,脑梗死容积百分比明显减小,PPAR-γ的蛋白相对含量明显增高,活化的caspase-1、NLRP3蛋白相对含量明显降低,IL-1β和IL-18浓度明显降低(P0.05)。与B组比较,L2组的PPAR-γ蛋白相对含量明显增高。与C组比较,LL2组活化的caspase-1和NLRP3蛋白相对含量明显升高(P0.05),IL-1β和IL-18浓度明显升高(P0.05)。结论吡格列酮通过激活PPAR-γ减轻脑缺血-再灌注损伤,其机制是通过激活PPAR-γ,抑制NLRP3的表达,减轻炎症反应,进而使可能存在的caspase-1介导的细胞焦亡减少。     

异氟醚诱导的老龄小鼠术后认知功能障碍中海马神经炎性反应及机制

目的探讨异氟醚麻醉/手术后认知功能障碍的发生机制。方法 18个月龄雄性C57BL6小鼠随机分为对照组和麻醉/手术组(1.5%～2.0%异氟醚麻醉2 h+腹腔探查术);Morris水迷宫实验检测两组小鼠麻醉/手术前和麻醉/手术后3 d认知水平变化;蛋白免疫印迹(Western blot)法检测两组海马组织Nod样受体家族包含Pyrin结构域蛋白3(NLRP3)炎症小体相关蛋白[NLRP3、凋亡相关斑点样蛋白(ASC)、半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-1]、补体C1q结合蛋白(C1QBP)、兴奋性突触后蛋白(PSD95)的表达水平;免疫荧光(Immunofluorescence)法观察两组海马组织小胶质细胞中NLRP3炎症小体活化情况;酶联免疫吸附试验(ELISA)检测两组血清促炎因子白细胞介素(IL)-1b和脑源性神经营养因子(BDNF)的表达水平;投射电镜技术观察两组海马线粒体的形态变化;活性氧(ROS)检测试剂盒检测两组海马组织ROS表达水平。采用独立样本t检验或Mann-Withney检验分析两组间差异。结果 Morris水迷宫结果显示, 麻醉/手术组中小鼠逃避潜伏...     

电刺激对LPS诱导M1型小胶质细胞活化的影响

目的评价电刺激对LPS诱导M1型小胶质细胞活化的影响。方法将生长良好的BV2小胶质细胞采用随机数字表法分为3组(n=18):对照组(C组)、LPS组和LPS+电刺激组(LE组)。C组常规培养24 h, LPS组和LE组加入浓度为100 ng/ml的LPS培养基孵育24 h, LE组于LPS孵育前给予100 mV/mm的直流电刺激4 h。采用ELISA法检测上清液TNF-α和IL-1β浓度;免疫荧光染色法检测M1型小胶质细胞表面标志物CD32和诱导型一氧化氮合酶(iNOS)的表达水平;qRT-PCR法检测细胞CD32和iNOS的mRNA表达水平。结果与C组相比, LPS组和LE组上清液TNF-α和IL-1β浓度升高, 细胞CD32和iNOS及其mRNA表达上调(P<0.05);与LPS组相比, LE组上清液TNF-α和IL-1β浓度降低, 细胞CD32和iNOS及其mRNA表达下调(P<0.05)。结论电刺激可抑制LPS诱导M1型小胶质细胞活化, 从而减轻炎症反应。     

淀粉样蛋白A在人下肢闭塞性动脉硬化的表达及其激活Nod样受体家族包含pyrin结构域蛋白3炎性体的机制

目的 观察血清淀粉样蛋白A( SAA)、Nod样受体家族包含pyrin结构域蛋白3( NLRP3)、白细胞介素-1β(IL-1β)在人下肢闭塞性动脉硬化症(ASO)中的表达,并通过SAA激活NLRP3炎性体的体外实验分析在ASO中NLRP3炎性体激活的可能机制.方法 免疫组织化学方法检测SAA、NLRP3、IL-1β在ASO血管壁中的表达.SAA处理巨噬细胞,Western blot检测成熟型IL-1β和裂解型Caspase-1,检测细胞中IL-1β前体蛋白、Caspase-1前体蛋白的表达.RNA干扰技术下调单核细胞(THP-1)细胞中NLRP3的表达.免疫荧光技术检测SAA对人巨噬细胞中核因子(NF) -κB p65入核的影响.结果 ASO下肢血管壁中SAA、NLRP3、IL-1β的表达较正常动脉壁上调倍数分别为10.19 ±0.29、7.11 ±0.14、5.06 ±0.15,与正常动脉比较差异有统计学意义(P＜0.01),各蛋白表达呈现出共区域化;SAA处理巨噬细胞,SAA各剂量组(1、2、5、10 mg/L)较对照组比较,NLRP3上调倍数相应为(2.49±0.34、3.19 ±0.22、3.61 ±0.29、4.46±0.20,P＜0.01),Caspase-1p20上调倍数相应为(1.32 ±0.11、4.59±0.28、17.55±1.40、25.69±1.71,P＜0.01),IL-1β p17上调倍数相应为(1.24±0.09、2.48 ±0.22、15.59±0.96、23.57±0.80,P＜0.01),下调THP-1巨噬细胞中的NLRP3表达后,SAA诱导的IL-1β分泌明显减低.SAA可以上调人巨噬细胞中IL-1β前体蛋白的表达,而且被NF-κB特异性阻滞剂Bay1 1-7082所抑制,上调倍数分别为17.64 ±2.07、6.45±1.78,两组比较差异有统计学意义(P＜0.01).免疫荧光显示SAA促进巨噬细胞中NF-κB p65入核.结论 SAA在ASO血管壁中广泛沉积,并上调了NLRP3和IL-1β的表达.SAA可以激活NLRP3炎性体,引起IL-1β的分泌.SAA可以上调IL-1β前体蛋白的表达,从而起到NLRP3炎性体激活致IL-1β分泌的预启动功能.因此在ASO中SAA可能通过激活NLRP3炎性体而促进动脉硬化炎症的发生、发展.     

阿魏酸钠对大鼠脊髓损伤中NLRP3炎性小体的影响及机制

目的:探究阿魏酸钠(SF)对大鼠脊髓损伤(SCI)模型中NLRP3炎性小体的影响及机制。方法:体内研究采用压迫法建立大鼠脊髓损伤模型,雌性SD大鼠随机分成3组(n=12),Sham组不损伤脊髓,SCI+SF组按100mg/kg/d的剂量连续腹腔注射SF给药7d,SCI组给予等量的生理盐水溶液,术后第3天提取脊髓组织蛋白进行Western blot检测NLRP3和激活效应蛋白pro-caspase-1、P20、IL-1β以及自噬蛋白P62、LC3Ⅱ的表达,并对脊髓样本进行免疫荧光染色观察NLRP3变化,术后第7天对脊髓样本进行Nissl染色检测神经元存活情况。体外实验中对大鼠小胶质细胞(正常培养为对照组)采用LPS诱导NLRP3表达(LPS组),加入自噬阻断剂氯喹(CQ)(CQ组及LPS+CQ组)检测NLRP3激活效应以及溶酶体组织蛋白酶B(Cathepsin B,CTSB)活性,验证自噬与溶酶体功能对NLRP3激活的影响,再通过SF干预(LPS+CQ+SF组)带来的分子水平变化探讨其中的机制。结果:与Sham组相比,SCI组NLRP3表达升高并被激活,pro-caspase-1降解减少,活性产物P20、IL-1β增多,且自噬蛋白p62、LC3Ⅱ均在较高水平(P0.05);与SCI组比较,SCI+SF组NLRP3数量减少,激活减弱,P62、LC3Ⅱ水平下降(P0.05),Nissl染色显示前角神经元存活情况改善(P0.05)。与之相似,体外实验中,相比对照组,LPS组细胞NLRP3的表达上升(P0.05),但激活不显著,而LPS+CQ组NLRP3的水平更高,激活明显,pro-caspase-1降解,活性产物P20、IL-1β增多,此外,P62、LC3Ⅱ的水平异常升高,CTSB活性下降(P0.05),而相比LPS+CQ组,LPS+CQ+SF组中P62、LC3Ⅱ的水平出现同步下降,CTSB活性增强(P0.05),自噬流恢复。结论:阿魏酸钠通过保护自噬和溶酶体的功能,能够抑制NLRP3炎性小体的产生和激活,从而减轻SCI大鼠神经细胞损伤。     

脂氧素A4对LPS诱导小胶质细胞活化的影响及SIRT1/NF-κB信号通路在其中的作用

目的评价脂氧素A4(LXA4)对脂多糖(LPS)诱导小胶质细胞活化的影响及沉默信息调节因子1(SIRT1)/NF-κB信号通路在其中的作用。方法实验Ⅰ:取生长状态良好的小鼠BV2小胶质细胞, 采用随机数字表法分为4组(n=30):对照组(C组)、LXA4组、LPS组和LPS+LXA4组(LL1组)。实验Ⅱ:采用随机数字表法将小胶质细胞分为2组(n=30):LPS+LXA4组(LL2组)、LPS+LXA4+SIRT1抑制剂EX527组(LLE组)。C组正常培养;LXA4组和LPS组分别加入LXA4(终浓度100 nmol/L)、LPS(终浓度100 ng/ml)孵育24 h;LL1组和LL2组于LPS处理前1 h加入LXA4(终浓度100 nmol/L), 其余处理方法同LPS组;LLE组于LXA4处理前30 min加入EX527(终浓度为5 μmol/L), 其余处理方法同LL2组。采用qRT-PCR法检测诱导型一氧化氮合酶(iNOS)、CD32、精氨酸酶1(Arg-1)、CD206以及IL-1β、IL-6、TNF-α和IL-10的mRNA表达, ELISA法检测上清液IL-1β、IL...     

线粒体自噬对脂多糖诱导髓核细胞NOD样受体热蛋白结构域相关蛋白3炎症小体活化的实验研究

目的探讨线粒体自噬对脂多糖(LPS)诱导的髓核(NP)细胞NOD样受体热蛋白结构域相关蛋白3(NLRP3)炎症小体活化的影响。方法以大鼠椎间盘NP细胞为研究对象, 首先以LPS(200 μg/ml)分别干预髓核细胞0、24、48 h, 通过蛋白质印迹法(Western blot)检测LPS对NLRP3炎症小体活化和线粒体自噬的影响;通过免疫荧光观察LPS对线粒体自噬水平的影响;组间比较采用单因素方差分析。进一步利用线粒体自噬抑制剂Mdivi-1阻断线粒体自噬, 实验分组为对照组(con)、LPS(24 h)+Mdivi-1、LPS(24 h)。通过Western blot检测Mdivi-1对LPS诱导的NLRP3炎症小体活化的影响;通过免疫荧光评估各实验组Mito Tracker Red探针标记的线粒体与LC3B及NLRP3的细胞内共定位;通过免疫荧光评估各实验组Mito Tracker Red探针标记的线粒体与LC3B的细胞内共定位;通过免疫荧光检测分析Mdivi-1对LPS诱导的NP细胞线粒体膜电位(ΔΨm)及线粒体ROS的影响;组间比较采用t检验。结果与对照组比较, 随着LPS干...     

藁本内酯抑制星形胶质细胞中NF-κB依赖的细胞因子的表达

目的观察藁本内酯(LIG)对脂多糖(LPS)诱导的星形胶质细胞中促炎性细胞因子表达的抑制作用。方法将原代培养成功的星形胶质细胞随机分成四组,对照组(C组)、LPS组、LIG组、BAY组。C组未加入任何药物刺激细胞;LPS组加入浓度为1μg/ml的LPS刺激细胞6h;LIG组用浓度为50μM的LIG预孵育细胞30min后,再加入LPS刺激6h;BAY组用浓度为20μM的核转录因子(NF)-κB抑制剂BAY11-7082预孵育细胞30min后,再加入LPS刺激6h。采用Real-time PCR方法检测促炎性细胞因子白细胞介素(IL)-1β、肿瘤细胞坏死因子(TNF)-α、IL-6 mRNA的表达;采用Western blot方法检测pNF-κB p65的表达。结果与LPS组比较,C、LIG和BAY组IL-1β、TNF-α、IL-6mRNA的表达明显减少(P0.05)。与LPS组比较,C、LIG组pNF-κB p65蛋白的表达明显降低(P0.05)。结论藁本内酯可通过抑制原代培养的星形胶质细胞中NF-κB的活化来抑制LPS诱导的促炎症相关的细胞因子的表达。     

LPS对HepG-2细胞因子表达的影响

目的 探讨LPS对HepG-2细胞TGF-β1,VEGF,IL-6表达的影响.方法 用LPS刺激HepG-2细胞,RT-PCR和Western-blot方法检测TGF-β1,VEGF,IL-6基因的转录和蛋白的表达的情况,同时用HTA125阻断TLR4受体检测LPS对HepG-2细胞TGF-β1,VEGF,IL-6基因的转录和蛋白的表达的情况.结果 LPS刺激细胞后可以促进TGF-61、VEGF、IL-6基因的转录和蛋白的表达增加,抑制TLR4受体后LPS对细胞TGF-β1、VEGF、IL-6表达影响下降.结论 LPS可以通过TLR4受体来促进HepG-2细胞分泌TGF-131、VEGF、IL-6.     

异氟烷预处理改善内毒素诱导大鼠急性肺损伤和死亡率

背景异氟烷预处理对肺脏促炎症介质和多种内毒素诱导的急性肺损伤后生存率的作用鲜有系统报道。我们研究异氟烷预处理对脂多糖诱导的大鼠急性肺损伤的作用。方法雄性SD大鼠重250—300g,随机被分为4组：假手术组,即假手术-空白组（腹腔注射盐水）空白预处理（100％O2）;假手术．异氟烷组,假手术大鼠用异氟烷预处理;LPS组,即空白．LPS组,腹腔注射脂多糖,空白预处理;ISO—LAP组,腹腔注射脂多糖大鼠,异氟烷预处理。腹腔注射脂多糖,诱导内毒素血症,异氟烷预处理在注射内毒素前30分给予1．4％处理,然后观察动物6小时,监测动脉压、心率、血气。肺损伤程度通过肺湿／干比、伊文思蓝染料外渗、组织学检测评估。同时,检测肺脏NO、TNF-α、IL-1β、IL-6水平,另外,进行生存分析和肺iNOS（诱导NO合酶）基因表达检测。结果脂多糖诱导系统性低血压和重型肺损伤,以急性肺损伤程度增加、伴随肺功能的改变、肺组织NO、TNF-α、IL-1β、IL-6水平升高为证。异氧烷预处理减弱了系统性低血压和急性肺损伤发展,调节脂多糖诱导的肺脏硝酸盐／亚硝酸盐升高和促炎症介质释放,提高败血症大鼠生存率。iNOS表达可被脂多糖处理上调,被异氟烷预处理下调。结论异氟烷预处理可减弱肺脏促炎症介质释放,减少重度败血症大鼠死亡率,其保护作用可能由部分抑制iNOS—NO信号通路活性所介导.     

Wnt通路激活的结直肠癌细胞抑制肿瘤浸润性树突细胞成熟的机制研究

［摘要］ 目的 大部分肠癌存在Wnt通路激活,基础研究发现Wnt通路可影响肿瘤免疫,但其在结肠癌中的作用不明。本研究主要探讨Wnt通路激活对结肠癌免疫微环境的影响以期发现结直肠癌免疫治疗新靶点。方法 免疫荧光技术验证SW480和NCM460 Wnt通路状态;qPCR和Western blot技术对比Wnt通路激活的结肠癌细胞系SW480和正常肠上皮细胞NCM460 IL?1β表达水平的不同;分离人外周血单核细胞,用集落刺激因子（GM?CSF）和IL?4联合诱导形成未成熟树突细胞（iDC）,再加入IL?1β刺激诱导成熟树突细胞（mDC）,用流式细胞技术检测mDC细胞表面成熟分子表达量。结果 SW480细胞核表达beta?catenin,提示Wnt通路激活。使用Wnt通路抑制剂（ICG?001）处理SW480后IL?1β mRNA和蛋白表达水平均升高,且随浓度升高而升高,使用wnt通路激活剂（Licl）处理NCM460后IL?1βmRNA和蛋白表达水平均低于对照组,且随浓度升高而降低,且IL?1β蛋白表达量均与非磷酸化β?连环蛋白（active β?catenin）表达量呈负相关关系。IL?1β可促进iDC成熟,IL?1β 50 ng/mL组HLA?DR、CD86表达率分别为（69±3.62）%、（84.3±4.7）%,与RPMI组及TNF?a 10 ng/mL组相比,表达显著上调（P<0.001）。结论 Wnt通路激活的结直肠癌可能通过减少IL?1β的表达来阻碍肿瘤微环境中DC的成熟。     

The NLRP3 inflammasome in kidney disease and autoimmunity

The NLRP3 inflammasome is an intracellular platform that converts the pro‐inflammatory cytokines interleukin (IL)‐1β and IL‐18 to their active forms in response to ‘danger’ signals, which can be either host or pathogen derived, and mediates a form of inflammatory cell death called pyroptosis. This component of the innate immune system was initially discovered because of its role in rare autoinflammatory syndromes called cryopyrinopathies, but it has since been shown to mediate injurious inflammation in a broad range of diseases. Inflammasome activation occurs in both immune cells, primarily macrophages and dendritic cells, and in some intrinsic kidney cells such as the renal tubular epithelium. The NLRP3 inflammasome has been implicated in the pathogenesis of a number of renal conditions, including acute kidney injury, chronic kidney disease, diabetic nephropathy and crystal‐related nephropathy. The inflammasome also plays a role in autoimmune kidney disease, as IL‐1β and IL‐18 influence adaptive immunity through modulation of T helper cell subsets, skewing development in favour of Th17 and Th1 cells that are important in the development of autoimmunity. Both IL‐1 blockade and two recently identified specific NLRP3 inflammasome blockers, MCC950 and β‐hydroxybutyrate, have shown promise in the treatment of inflammasome‐mediated conditions. These targeted therapies have the potential to be of benefit in the growing number of kidney diseases in which the NLRP3 inflammasome has been implicated.     

Contrast induces kidney epithelial cell apoptosis through NRLP3 inflammasome pathway

Objective To investigate the effect of pyrin domain 3 (NLRP3) inflammasome in the process of contrast induced human kidney cell apoptosis. Methods Human kidney 2 (HK-2) cells were cultured in DMEM-F12 medium with 5% FBS. Cells were divided into control group, Contrast group (O group), NLRP3-siRNA+Iohexol group (si-NLRP3+O group), ASC-siRNA+Iohexol group (si-ASC+O group), and mannitol group (M group). Different concentrations of hypotonic contrast agent were added to HK-2 cell culture plates for 24, 48 and 72 h. Flow cytometry was used to detect apoptosis. NLRP3 and ASC mRNA expressions were detected by RT-PCR. The expressions of NLRP3, ASC, caspase-8/cleaved caspase-8, Bcl-2/Bax, caspase-1/cleaved caspase-1, and caspase-3/cleaved caspase-3 protein were detected by Western blot. The levels of interleukin (IL) 1β and IL-18 in supernatant were detected by ELISA. Results Compared with the control group, the rate of apoptotic cells, as well as the expressions of NLRP3, ASC and cleaved caspase-1 proteins were increased in HK-2 cells of contrast group. The expressions of NLRP3 and ASC mRNA in the contrast group also increased, so did IL-1β and IL-18 levels (all P＜0.05), suggesting that NLRP3 inflammasome in HK-2 cells was activated by contrast. Compared with the control group, the expressions of cleaved caspase-8, Bax and cleaved caspase-3 protein were increased, and the expression of anti-apoptotic protein Bcl-2 was decreased (all P＜0.05). Compared with the contrast group, the rate of apoptotic cells in the si-NLRP3+contrast group and si-ASC+contrast group was significantly decreased; the expression of cleaved caspase-1 was decreased; the expressions of Bax and cleaved caspase-3 were decreased, and Bcl-2 level was increased. The expressions of IL-1β and IL-18 in the supernatant of cells were decreased (all P＜0.05). Conclusion Contrast agent can activate the NLRP3 pathway in HK-2 cells and induce apoptosis, which could be reduced by blocking the NLRP3 pathway.     

The concept of the inflammasome and its rheumatologic implications

The inflammasome is a proteolytic complex that regulates IL1β and IL-18 secretion in macrophages and dendritic cells. Its plays a vital role in the control of the inflammatory and cellular responses to infectious and danger signals and is an essential part of the innate immune system. Four different inflammasomes have been identified so far, and the NLRP3-inflammasome has been the best-studied in relation to human disease. Activation of the NLRP3-inflammasome by microcrystals, such as monosodium urate (MSU) and basic calcium phosphate (BCP) crystals, leads to IL1β release, which in turn triggers local inflammation. Dysfunction of the NLRP3-inflammasome due to mutations of the NLRP3 gene is the cause of the auto-inflammatory syndrome CAPS. The symptoms and signs of inflammation in both conditions respond to IL1 blockade. IL1 inhibitors have also been used successfully in other idiopathic inflammatory diseases, suggesting that dysregulated inflammasome activity contributes to the pathogenesis of multiple diseases, but the precise underlying mechanisms remain to be identified.     

Targeting against the activity of the NLRP3 inflammasome is a potential therapy for rat testicular tissue cryopreservation and transplantation

The objective of the present experiment was to explore the role of NLRP3 inflammasome in the testicular tissue freezing, thawing and grafting; furthermore, the potential effect of a NLRP3 inhibitor on the function of testis transplant was explored. Tissues from male Wistar rats in pre-pubertal age were cryopreserved, thawed and auto-transplanted into the scrotum treated or not treated with the MCC950 (a NLRP3 inhibitor). After grafting, cryopreserved tissue was removed and analysed. Quantitative morphometric, immunohistochemical techniques and Western blotting were used to evaluate the survival of spermatogonia and the activation of the NLRP3 inflammasome after freezing/thawing/grafting. Moreover, serum IL-1β level was assessed with ELISA kits. The testicular transplants exhibited upregulated expression of the NLRP3 pathway meditors (NLRP3, IL-1β). In NLRP3 inhibition group, the rate of recovered grafts, the percentage of intact tubules and spermatogonial number were significantly higher than that in cryopreserved graft group. Moreover, serum concentration of IL-1β in NLRP3 inhibition group was significantly lower than that in cryopreserved graft group. Testicular tissue cryopreservation and transplantation exhibited upregulated expression of NLRP3 pathway and NLRP3 inflammasome blockade improves testicular graft function. These finding suggest that NLRP3 inflammasome is a therapeutic target for testicular tissue cryopreservation and transplantation.     

Bisphosphonate Induces Osteonecrosis of the Jaw in Diabetic Mice via NLRP3/Caspase‐1‐Dependent IL‐1β Mechanism

Diabetes mellitus is an established risk factor associated with bisphosphonate‐related osteonecrosis of the jaw (BRONJ). Sustained activation of Nod‐like receptor (NLR) family, pyrin domain‐containing protein 3 (NLRP3) inflammasome contributes to the persistent inflammation and impaired cutaneous wound healing in diabetic mice and human. We have recently demonstrated a compelling linkage between M1 macrophages and BRONJ conditions in both murine and human diseases. The aim of this study was to determine whether NLRP3 inflammasome activation is involved in BRONJ development in diabetic mice. We showed an increased incidence of delayed oral wound healing and bone necrosis of extraction sockets in db/db mice compared with those in nondiabetic db/+ controls, which correlated with an elevated expression of NLRP3, caspase‐1, and IL‐1β in macrophages residing at local wounds. Constitutively, bone marrow‐derived macrophages from db/db mice (db/db BMDMs) secrete a relatively higher level of IL‐1β than those from db/+ mice (db/+ BMDMs). Upon stimulation by NLRP3 activators, the secretion of IL‐1β by db/db BMDMs was 1.77‐fold higher than that by db/+ BMDMs (p < 0.001). Systemic treatment of mice with zoledronate (Zol), a nitrogen‐containing bisphosphonate, resulted in a 1.86‐ and 1.63‐fold increase in NLRP3/caspase‐1‐dependent IL‐1β secretion by db/+ and db/db BMDMs, respectively, compared with BMDMs derived from nontreated mice (p < 0.001). Importantly, systemic administration of pharmacological inhibitors of NLRP3 activation improved oral wound healing and suppressed BRONJ formation in db/db mice. Mechanistically, we showed that supplementation with intermediate metabolites of the mevalonate pathway, inhibitors of caspase‐1 and NLRP3 activation, an antagonist for P2X₇R, or a scavenger of reactive oxygen species (ROS), robustly abolished Zol‐enhanced IL‐1β release from macrophages in response to NLRP3 activation (p < 0.001). Our findings suggest that diabetes‐associated chronic inflammatory response may have contributed to impaired socket wound healing and rendered oral wound susceptible to the development of BRONJ via NLRP3 activation in macrophages. © 2015 American Society for Bone and Mineral Research.     

Isoflurane Preconditioning Reduces the Rat NR8383 Macrophage Injury Induced by Lipopolysaccharide and Interferon [gamma]

Background: Isoflurane exposure before an insult can reduce the insult-induced injury in various organs. This phenomenon is called isoflurane preconditioning. The authors hypothesize that isoflurane can precondition macrophages, cells that travel to all tissues and are important in the host defense and inflammation responses.

Methods: Rat NR8383 macrophages were pretreated with or without 1-3% isoflurane for 1 h at 30 min before they were incubated with or without 100 ng/ml lipopolysaccharide plus 50 U/ml interferon [gamma] for 24 h. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Flow cytometry was performed after cells were stained with annexin V and propidium iodide. Inducible nitric oxide synthase protein expression in macrophages was quantified by Western blotting.

Results: Lipopolysaccharide plus interferon [gamma] decreased cell viability by approximately 50%. This decrease was dose-dependently inhibited by aminoguanidine, an inducible nitric oxide synthase inhibitor. Lipopolysaccharide plus interferon [gamma] caused inducible nitric oxide synthase expression. This expression was inhibited by pretreatment with 2% but not 1% or 3% isoflurane. Isoflurane at 2% inhibited lipopolysaccharide plus interferon [gamma]-induced accumulation of nitrite, an oxidation product of nitric oxide. Pretreatment with 2% but not 1% or 3% isoflurane improved cell viability. Lipopolysaccharide plus interferon [gamma] increased the number of propidium iodide-positive staining cells. This increase was attenuated by 2% isoflurane pretreatment. The protective effect of 2% isoflurane was abolished by chelerythrine, calphostin C, or bisindolylmaleimide IX, protein kinase C inhibitors.     

NLRP3 inflammasome: A potential therapeutic target to minimize renal ischemia/reperfusion injury during transplantation

Renal transplantation is currently the best treatment option for patients with end-stage kidney disease. Ischemia/reperfusion injury (IRI), which is an inevitable event during renal transplantation, has a profound impact on the function of transplanted kidneys. It has been well demonstrated that innate immune system plays an important role in the process of renal IRI. As a critical component of innate immune system, Nod-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome has received great attention from scientific community over the past decade. The main function of NLRP3 inflammasome is mediating activation of caspase-1 and maturation of interleukin (IL)-1β and IL-18. In this review, we summarize the associated molecular signaling events about NLRP3 inflammasome in renal IRI, and highlight the possibility of targeting NLRP3 inflammasome to minimize renal IRI during transplantation.     

A Role of the Inflammasome in the Low Storage Capacity of the Abdominal Subcutaneous Adipose Tissue in Obese Adolescents

The innate immune cell sensor leucine-rich–containing family, pyrin domain containing 3 (NLRP3) inflammasome controls the activation of caspase-1, and the release of proinflammatory cytokines interleukin (IL)-1β and IL-18. The NLRP3 inflammasome is implicated in adipose tissue inflammation and the pathogenesis of insulin resistance. Herein, we tested the hypothesis that adipose tissue inflammation and NLRP3 inflammasome are linked to the downregulation of subcutaneous adipose tissue (SAT) adipogenesis/lipogenesis in obese adolescents with altered abdominal fat partitioning. We performed abdominal SAT biopsies on 58 obese adolescents and grouped them by MRI-derived visceral fat to visceral adipose tissue (VAT) plus SAT (VAT/VAT+SAT) ratio (cutoff 0.11). Adolescents with a high VAT/VAT+SAT ratio showed higher SAT macrophage infiltration and higher expression of the NLRP3 inflammasome–related genes (i.e., TLR4, NLRP3, IL1B, and CASP1). The increase in inflammation markers was paralleled by a decrease in genes related to insulin sensitivity (ADIPOQ, GLUT4, PPARG2, and SIRT1) and lipogenesis (SREBP1c, ACC, LPL, and FASN). Furthermore, SAT ceramide concentrations correlated with the expression of CASP1 and IL1B. Infiltration of macrophages and upregulation of the NLRP3 inflammasome together with the associated high ceramide content in the plasma and SAT of obese adolescents with a high VAT/VAT+SAT may contribute to the limited expansion of the subcutaneous abdominal adipose depot and the development of insulin resistance.