依托咪酯通过miR-146a 调节TLR4 通路对内毒素急性肺损伤小鼠炎症 反应的保护作用研究

目的:观察依托咪酯对内毒素急性肺损伤小鼠炎症反应的影响,并探讨其作用机制。方法:雄性成年BLAB/c小鼠60只,随机分为空白对照组、模型组、依托咪酯组,每组20只。模型组及药物干预组应用腹腔注射脂多糖(LPS,20 mg/kg)制作急性肺损伤小鼠模型,空白对照组给予等体积生理盐水腹腔注射。药物干预组于造模后0.5 h给予依托咪酯2.5 mg/kg。空白对照组及模型组给予等体积生理200μl/只。于给药后6 h检测小鼠血清中的内毒素水平,BALF中TNF-α、IL-6和IL-1β的含量;RT-PCR检测miR-146a、MyD88、IRAK1、IRF5、TRAF6 mRNA在肺组织中的表达;Western blot检测TLR4、MyD88、TRAF6、IRAK1、IRF5蛋白在肺组织中的表达。结果:模型组、依托咪酯组与空白对照组相比,血清内毒素含量、TNF-α、IL-6和IL-1β含量、miR-146a表达水平、TLR4、MyD88、IRAK1、IRF5、TRAF6 mRNA表达水平及TLR4、MyD88、IRAK1、IRF5、TRAF6蛋白表达均明显升高,差异具有统计学意义(P0.05);依托咪酯组与模型组比较,小鼠血清内毒素含量、TNF-α、IL-6和IL-1β含量、TLR4、MyD88、IRAK1、IRF5、TRAF6 mRNA表达水平及TLR4、MyD88、IRAK1、IRF5、TRAF6蛋白表达水平均明显下降,差异具有统计学意义(P0.05)。结论:依托咪酯能够通过增加miR-146a的含量调节TLR4通路对内毒素急性肺损伤小鼠炎症反应的保护作用。     

TREM-1促进巨噬细胞的迁移并参与脂多糖诱导的小鼠心肌功能障碍

目的探讨髓系细胞触发受体-l(triggering receptor expressed oil myeloid cells-1,TREM-1)对脂多糖(lipopolysaccharide,LPS)诱导的小鼠心肌功能障碍的作用以及对巨噬细胞RAW264.7迁移的影响。方法将健康C57BL/6J雄性小鼠随机分为对照组、LPS组、LPS+LP17组、LPS+TAK-242组并进行相应处理,6 h后超声心电图检测小鼠心功能指标,12 h取心肌组织HE染色观察病理改变,免疫组化染色观察巨噬细胞表面分子F4/80阳性表达,ELISA检测血清中炎症因子TNF-α、IL-6、IL-1β及IFN-γ的表达情况,实时荧光定量PCR检测TREM-1、BNP以及炎症因子TNF-α、IL-6、IL-1β、IFN-γ的mRNA水平;培养巨噬细胞RAW264.7,将细胞随机分为正常组、LPS组、LP17+LPS组、TAK-242+LPS组并进行对应处理,实时荧光定量PCR检测TREM-1与TLR4的mRNA水平,ELISA检测细胞培养液中炎症因子TNF-α、IL-6、IL-1β及IFN-γ的表达情况,Transwell小室观测RAW264.7细胞迁移情况,蛋白免疫印迹检测RAW264.7中TLR4/NF-κB信号通路相关蛋白水平。结果与对照组相比,超声心电图检测结果显示LPS组小鼠的EF与FS下降,LVD增大,IVS减小,LVPW变薄,变化均显著(P0.05),实时荧光定量PCR结果显示TREM-1 mRNA与BNP mRNA表达水平上升(P0.05),免疫组化染色法显示F4/80有大量阳性表达(P0.01),ELISA检测显示炎症因子TNF-α、IL-6、IL-1β、IFN-γ的含量增加(P0.05),实时荧光定量PCR检测也表明这些炎性因子mRNA水平升高(P0.05);与LPS组小鼠相比,注射TREM-1抑制剂LP17使EF、FS上升,有效抑制了LVD增大、IVS减小以及LVPW变薄的现象(P0.05),TREM-1 mRNA与BNP mRNA表达水平均降低(P0.05),心肌损伤减轻,F4/80阳性表达减少(P0.01),炎症因子水平也明显下降(P0.05),与注射TLR4抑制剂TAK-242的效果一致。与对照组相比,LPS诱导RAW264.7细胞后,细胞中TREM-1 mRNA与TLR4 mRNA水平升高(P0.05),炎症因子TNF-α、IL-6、IL-1β、IFN-γ的含量增加(P0.05),细胞大量迁移(P0.01),细胞中TLR4、MyD88、NF-κBp65、p-IκBα蛋白水平升高(P0.05);与LPS组相比,TREM-1抑制剂LP17预处理后再给予LPS诱导,细胞中TREM-1 mRNA与TLR4 mRNA水平降低(P0.05),炎症因子的含量下降(P0.05),RAW264.7细胞迁移被抑制(P0.01),TLR4、MyD88、NF-κBp65、p-IκBα蛋白水平也明显降低(P0.05)。结论给小鼠注射TREM-1抑制剂可有效减轻LPS诱导的心肌功能障碍,抑制RAW264.7细胞TREM-1表达可抑制细胞的迁移,TLR4/NF-κB途径可能参与了这一过程。     

TLR4/MyD88/AMPK通路参与调节高脂饮食诱导的肥胖小鼠脂肪组织炎症和细胞凋亡北大核心CSCD

目的:探讨Toll样受体4(TLR4)基因敲除对高脂饮食诱导的肥胖小鼠脂肪组织炎症和细胞凋亡的影响。方法:雄性C57BL/6J和TLR4基因敲除小鼠随机分为正常对照组(NC)、肥胖组(OB)、TLR4基因敲除组(TK)、TLR4基因敲除肥胖组(TO),NC组给予正常饲料喂养,其他组给予高脂饲料喂养。16周后取血检测各组小鼠空腹血糖(FPG)、总胆固醇(TC)、三酰甘油(TG)、低密度脂蛋白(LDL)、高密度脂蛋白(HDL)和游离脂肪酸(FFA)水平;HE染色观察各组小鼠脂肪细胞形态变化;Western blot检测TLR4、磷酸化腺苷酸活化蛋白激酶α(p-AMPKα)、髓样分化因子88(MyD88)、Bax和Bcl-2蛋白表达;ELISA检测小鼠血清单细胞趋化因子-1(MCP-1)、IL-6、TNF-α、IL-10水平。结果:与NC组相比,OB组小鼠脂肪细胞明显增大,FPG、TC、TG、LDL、FFA、MCP-1、IL-6和TNF-α水平明显升高(P<0.01),HDL和IL-10水平明显降低(P<0.01,P<0.05),TLR4、MyD88和Bax表达明显增加(P<0.01),p-AMPKα和Bcl-2表达明显减少(P<0.05);与OB组相比,TO组小鼠上述检测指标表达明显逆转。结论:TLR4基因敲除可通过MyD88依赖的AMPK缓解高脂饮食诱导的肥胖小鼠脂肪组织慢性炎症和细胞凋亡。     

卵巢癌细胞株SKOV3对THP-1细胞TLR信号通路的作用

卵巢癌是目前死亡率最高的妇科生殖道肿瘤,Toll样受体在组织损伤修复、组织损伤诱导炎症及肿瘤进展中发挥重要作用,其中单核-巨噬细胞中的TLR在炎症与肿瘤间发挥桥梁作用。本研究利用THP-1和卵巢癌细胞系SK-OV-3共培养体系及抗TLR1、TLR2、TLR6抗体中和实验,荧光定量PCR法检测细胞中促炎症细胞因子(IL-1β、IL-6、IL-8、TNF-α)mRNA表达水平;利用western blot检测MyD88、TRAF6、TANK、NF-κB和P-NF-κB的表达水平。结果显示THP-1与SK-OV-3共培养组较单独培养组,TLR信号通路蛋白MyD88、TRAF6、TANK及P-NF-κB表达上调,IL-1β、IL-8及TNF-α促炎症细胞因子的mRNA表达水平显著增加(Fold=4.27,Fold=4.92,Fold=3.08,P0.05),而IL-6 mRNA表达水平无显著改变。抗体中和实验显示,anti-TLR1/anti-TLR2/anti-TLR6处理组THP-1中促炎症细胞因子IL-1β(Fold=0.43,Fold=0.38,Fold=0.44)、IL-8(Fold=0.43,Fold=0.48,Fold=0.42)、TNF-a(Fold=0.23,Fold=0.21,Fold=0.23)mRNA表达水平较未加单抗共培养组显著下调,差异有统计学意义(P0.05);IL-6 mRNA表达水平则无显著改变。anti-TLR1/anti-TLR2/anti-TLR6处理组TLR信号通路蛋白MyD88、TRAF6、TANK、NF-κB、P-NF-κB表达水平较未加单抗共培养组下调。以上结果提示卵巢癌生长微环境中,TLR1/TLR2/TLR6介导的信号通路活化可诱导THP-1中促炎症细胞因子表达上调,在卵巢癌的发生、发展中发挥一定作用。     

TLR2基因敲除下调高脂饮食诱导的肥胖小鼠脂肪组织炎症因子表达和细胞凋亡

目的研究Toll样受体2(TLR2)基因敲除对高脂饮食诱导的肥胖小鼠脂肪组织炎症因子表达和细胞凋亡的影响。方法雄性C57BL6小鼠和TLR2基因敲除小鼠分为:正常对照组、肥胖组、TLR2基因敲除组和TLR2基因敲除肥胖组,并给予普通饮食或高脂饮食喂养。16周后检测各组小鼠空腹血糖、甘油三酯、总胆固醇、低密度脂蛋白、高密度脂蛋白和游离脂肪酸水平。分光光度计法检测小鼠脂肪组织Caspase3活性,Western blot检测Bcl2、Bax、MyD88和p65NFκB蛋白相对表达量。荧光实时定量PCR检测小鼠脂肪组织IL-6和TNF-αmRNA相对表达量。结果与正常对照组相比,肥胖组小鼠空腹血糖、甘油三酯、总胆固醇、低密度脂蛋白、高密度脂蛋白水平降低和游离脂肪酸水平升高;脂肪组织Caspase3活性、Bax蛋白、MyD88蛋白、p65NFκB蛋白、IL-6 mRNA和TNF-αmRNA相对表达量升高,Bcl2蛋白相对表达量减少。与肥胖组相比,基因敲除肥胖组小鼠空腹血糖、甘油三酯、总胆固醇、低密度脂蛋白、高密度脂蛋白水平降低和游离脂肪酸水平降低;脂肪组织Bcl2蛋白相对表达量增加,Caspase3活性、Bax蛋白、MyD88蛋白、p65NFκB蛋蛋白、IL-6 mRNA和TNF-αmRNA相对表达量减少。结论 TLR2基因敲除下调了高脂饮食诱导的肥胖小鼠脂肪组织炎症因子的表达和细胞凋亡。     

贞芪扶正胶囊介导TLR4/MyD88/NF-κB信号通路抑制慢性湿疹小鼠炎症反应

目的 探讨贞芪扶正胶囊对慢性湿疹小鼠炎症反应的抑制作用.方法 SPF级C57BL/6小鼠30只,按随机数字表法随机分为对照组(control组)、模型组(CAD组)与贞芪扶正胶囊组(ZQFZ组).观察各组小鼠双耳肿胀程度、双耳变应评分;HE染色观察病灶处皮肤病理学变化;ELISA法检测血清炎性因子TNF-α、IL-6及IL-1β水平;RT-PCR检测皮肤中TNF-α、IL-6及IL-1β mRNA表达;Western blot检测TLR4、MyD88及NF-κB蛋白表达;RT-PCR检测皮肤中TLR4、MyD88及NF-κB mRNA表达.结果 ZQFZ可以显著改善小鼠双耳肿胀程度,降低小鼠双耳变应评分(P＜0.05);减轻小鼠皮肤炎症浸润并降低血浆及皮肤中TNF-α、IL-6及IL-1β蛋白与mRNA表达(P＜0.05);同时ZQFZ还可显著下调小鼠皮肤组织中TLR4、MyD88、NF-κB蛋白与mRNA表达(P＜0.05).结论 贞芪扶正胶囊可以通过抑制炎性反应达到治疗慢性湿疹作用,其作用机制可能与其调控TLR4/MyD88/NF-κB信号通路有关.     

沉默MyD88抑制NF-κB p65活化对TNF-α诱导的心肌细胞凋亡和炎症损伤的保护作用

为探究沉默髓样分化因子88(myeloid differentiation factor 88,MyD88)抑制NF-κB p65活化对TNF-α诱导的心肌细胞凋亡和炎症损伤的影响,采用TNF-α处理心肌细胞,将其随机分为4组:Control组、TNF-α组、TNF-α+shR NA-NC组和TNF-α+sh-MyD88组。Western blotting检测心肌细胞MyD88蛋白表达水平及NF-κB p65磷酸化情况; ELISA检测细胞培养上清液肌酸激酶同工酶MB(creatine kinase MB,CK-MB)、肌红蛋白(myohemoglobin,Mb)和肌钙蛋白Ⅰ(cardiac troponinⅠ,cTnⅠ)及IL-1β、iNOS、IL-6、IL-10水平; FACS检测心肌细胞凋亡情况; 5-溴脱氧尿嘧啶核苷(5-bromo-2'-deoxyuridine,BrdU)染色检测心肌细胞增殖情况; RT-PCR检测Ki67、Bcl-2、Bax、c-Myc mRNA表达水平。结果表明,与TNF-α+shR NA-NC组比较,TNF-α+sh-MyD88组MyD88蛋白表达水平显著降低(P0.05); CK-MB、Mb和cTnⅠ水平显著降低(均P0.05);心肌细胞凋亡率显著降低(P0.05); BrdU染色阳性细胞数显著增多(P0.05); Ki67、c-Myc mRNA水平和Bcl-2 mRNA/Bax mRNA比值显著升高(均P0.05); IL-1β、iNOS、IL-6水平显著降低(均P0.05),IL-10水平显著升高(P0.05); p-p65/p65比值显著降低(P0.05)。以上结果说明,沉默MyD88抑制NF-κB p65的活化对TNF-α诱导的心肌细胞损伤具有保护作用。     

miR-218-5p靶向HMGB1参与脂多糖诱导的足细胞损伤

目的探究miR-218-5p、高迁移率蛋白B1(HMGB1)在脂多糖(LPS)诱导的小鼠足细胞损伤中的作用。方法将小鼠足细胞分为4组:正常对照组、LPS诱导组、HMGB1 shRNA+LPS组、miR-218-5p mimics+LPS组。Western blot和Real-time PCR分别检测各组HMGB1、TLR4、MyD88、TNF-α、IL-6、synaptopodin、ZO-1蛋白和mRNA表达水平;免疫荧光检测synaptopodin、ZO-1的表达和定位;Transwell检测足细胞迁移能力;双荧光素酶报告基因验证miR-218-5p和HMGB1的关系。结果 LPS刺激后,HMGB1、TLR4、MyD88表达明显升高,炎性因子TNF-α、IL-6表达升高,而足细胞标记蛋白synaptopodin、ZO-1表达降低,同时足细胞迁移能力明显增强。沉默HMGB1或过表达miR-218-5p可降低TLR4、MyD88,TNF-α、IL-6表达,增加synaptopodin、ZO-1表达,抑制足细胞迁移能力,缓解足细胞损伤。荧光素酶报告基因检测提示miR-218-5p可直接作用...     

LXR-ABCA1/ABCG1通路对BCG感染的巨噬细胞凋亡和炎性反应的调控作用

目的 揭示LXR-ABCA1/ABCG1通路对BCG感染后巨噬细胞凋亡和炎性反应的调控作用。方法 采用T0901317预处理RAW264.7巨噬细胞2 h和BCG感染24 h,设置4个实验组：对照组、T0901317组、BCG感染组和T0901317+BCG感染组。采用Western blot方法检测凋亡相关蛋白cleaved-Caspase3、cleaved-Caspase8、cleaved-Caspase9和TLR信号通路相关蛋白TLR2、TLR4、MyD88、TRAF6、p-NF-κB p65、IRF3、TBK1的表达,采用ELISA方法检测细胞培养上清中促炎细胞因子TNF-α、IL-6、IL-1β的含量。结果 与对照组比较,BCG感染组凋亡相关蛋白cleaved-Caspase3、cleaved-Caspase8、cleaved-Caspase9及依赖MyD88途径蛋白TLR2、TLR4、MyD88、TRAF6、p-NF-κBp65的表达均上调（P<0.01）,促炎细胞因子TNF-α、IL-6、IL-1β表达水平上升（P<0.01）;与BCG感染组相比,T09013...     

四妙勇安汤含药血清对巨噬细胞TLR4/MyD88信号通路及其下游炎症因子的影响

目的研究四妙勇安汤含药血清对LPS诱导的小鼠腹腔巨噬细胞Toll样受体4(TLR4)、髓样分化因子88(MyD88)信号通路及TNF-α、IL-6等炎症因子表达的影响,初步探讨其抗动脉粥样硬化的作用机制。方法采用大、中、小剂量四妙勇安汤水煎剂灌胃干预清洁级SD大鼠,制备含药血清;经MTT法观察含药血清对细胞增殖的影响后,选择剂量浓度为20%的含药血清干预体外培养的经LPS活化的巨噬细胞,采用ELISA法检测细胞上清液中TNF-α及IL-6含量;荧光定量PCR及Westernblot法检测细胞TLR4、MyD88的mRNA及蛋白的表达变化。结果用LPS刺激细胞后,引起TLR4、MyD88、TNF-α及IL-6的高表达,与正常血清对照组相比,差异具有统计学意义(P0.01);而用四妙勇安汤含药血清干预后,能显著抑制各项指标的高表达,与模型组相比,差异具有统计学意义(P0.05或P0.01)。结论四妙勇安汤含药血清能够有效抑制LPS诱导的巨噬细胞炎症反应,其机制可能与调控TLR4/MyD88信号转导通路有关,这可能是该方防治动脉粥样硬化及免疫炎症性疾病作用的机制之一。     

脂多糖预致敏的人骨髓间充质干细胞促炎机制研究

目的探索脂多糖（LPS）预致敏的人骨髓间充质干细胞（MSC）产生促炎功能的免疫调节机制。方法采用Real-time PCR和免疫荧光法检测MSC被预致敏前后TLR4信号通路相关分子（如TLR4、MyD88、TRAF6等）的表达水平,以及NF-κB的入核情况。通过Real-time PCR比较MSC被致敏前后促炎因子（IL-1β、IL-6、MIP-2、TNF-α）和Th1/Th2型细胞因子及其受体的表达差异。结果与未致敏的MSC相比,LPS预致敏的MSC中TLR4表达升高,NF-κB入核增加,促炎性因子IL-1β、IL-6、MIP-2、TNF-α表达升高,提示LPS预致敏可以激活MSC中的TLR4信号通路,并且诱导MSC中Th1型细胞因子及其受体表达升高,而Th2型细胞因子及其受体表达无变化或减少。结论MSC被LPS预致敏后TLR4信号通路激活,Th1型细胞因子及受体表达上调,从而诱导MSC分化成促炎表型。     

小檗碱和育亨宾对内毒素血症小鼠脾脏TLR4信号通路中相关基因表达的影响

目的: 观察小檗碱和α₂肾上腺素能受体拮抗剂育亨宾对内毒素血症小鼠脾脏Toll样受体4(TLR4)信号通路84种基因表达的影响,并初步探讨其作用机制。方法: 雄性BALB/c小鼠随机分为对照组、脂多糖(LPS)组、小檗碱+LPS组、小檗碱+育亨宾+LPS组、育亨宾+LPS组、小檗碱组、小檗碱+育亨宾组和育亨宾组。分别用蒸馏水、小檗碱(50 mg/kg)、小檗碱+育亨宾(50 mg/kg+2 mg/kg) 和育亨宾(2 mg/kg)灌胃,每天1次,连续3 d,第3 d灌胃1 h后,腹腔注射LPS(20 mg/kg)或生理盐水。腹腔注射1 h后,用RT² Profiler^TM PCR Array分析技术检测小鼠脾脏TLR4信号通路84种基因mRNA的表达;用Western blotting分析小鼠脾脏TLR4信号通路的抑制分子细胞因子信号抑制物(SOCS)1、SOCS3和白细胞介素-1受体相关激酶(IRAK)-M蛋白的表达。结果: LPS可上调小鼠脾脏TLR4信号转导通路中相关炎症因子的mRNA表达,包括CXCL10、TNF-α、IL-1α、IL-1β、IL-6、IFN-γ和IFN-β。小檗碱能显著下调下调髓样分化因子(MyD88)依赖信号通路下游TNF-α、IL-1α、IL-1β和IL-6 mRNA的表达,也能MyD88非依赖信号通路下游基因IFN-β和CXCL10 mRNA的表达(P<0.05)。育亨宾能显著下调内毒素血症小鼠脾脏IL-1α、IL-1β 和IFN-β mRNA的表达(P<0.05),但对TNF-α、IL-6和CXCL10 mRNA表达的下调作用与LPS组相比没有显著差异(P>0.05)。小檗碱与育亨宾合剂能显著下调内毒素血症小鼠脾脏IFN-β和CXCL10 mRNA的表达,但不能显著下调内毒素血症小鼠脾脏IL-1α、IL-1β、TNF-α 和IL-6 mRNA的表达。LPS攻击后1 h,小檗碱和(或)育亨宾均不能增强内毒素血症小鼠脾脏SOCS1、SOCS3和IRAK-M蛋白的表达。结论: 小檗碱和育亨宾均能抑制LPS诱导的MyD88依赖和非依赖信号通路下游部分基因的表达,这种抑制作用的机制与SOCS1、SOCS3和IRAK-M蛋白无关。     

黄芪总皂苷对脂多糖诱导的BV2小胶质细胞的抗炎机制

目的:探讨黄芪总皂苷(TAS)对脂多糖(LPS)诱导的BV2小胶质细胞炎症损伤的抗炎作用机制.方法:用CCK-8法筛选出对细胞活力无抑制的药物浓度;用浓度为1 mg/L的LPS刺激BV2细胞24 h,建立细胞炎症模型;实验分为正常组、LPS组、高剂量(75 mg/L)TAS组和低剂量(50 mg/L)TAS组;应用流式...     

Potential autocrine regulation of interleukin-33/ST2 signaling of dendritic cells in allergic inflammation

This study identified a novel phenomenon that dendritic cells (DCs) produced interleukin (IL)-33 via Toll-like receptor (TLR)-mediated innate pathway. Mouse bone marrow–derived DCs were treated with or without microbial pathogens or recombinant murine IL-33. IL-33 mRNA and protein were found to be expressed by DCs and largely induced by several microbial pathogens, highly by lipopolysaccharide (LPS) and flagellin. Using two mouse models of topical challenge by LPS and flagellin and experimental allergic conjunctivitis, IL-33-producing DCs were observed in ocular mucosal surface and the draining cervical lymph nodes in vivo. The increased expression levels of myeloid differentiation primary-response protein 88 (MyD88), nuclear factor (NF)-κB1, NF-κB2, and RelA accompanied by NF-κB p65 nuclear translocation were observed in DCs exposed to flagellin. IL-33 induction by flagellin was significantly blocked by TLR5 antibody or NF-κB inhibitor quinazoline and diminished in DCs from MyD88 knockout mice. IL-33 stimulated the expression of DC maturation markers, CD40 and CD80, and proallergic cytokines and chemokines, OX40L, IL-4, IL-5, IL-13, CCL17 (C-C motif chemokine ligand 17), TNF-α (tumor necrosis factor-α), and IL-1β. This stimulatory effect of IL-33 in DCs was significantly blocked by ST2 antibody or soluble ST2. Our findings demonstrate that DCs produce IL-33 via TLR/NF-κB signaling pathways, suggesting a molecular mechanism by which local allergic inflammatory response may be amplified by DC-produced IL-33 through potential autocrine regulation.     

绿茶多酚通过抑制TLR4通路减轻蛛网膜下腔出血大鼠早期脑损伤

目的探讨绿茶多酚通过抑制TLR4通路对蛛网膜下腔出血大鼠早期脑损伤的影响。方法建立大鼠蛛网膜下腔出血模型,随机分为模型组、绿茶多酚组、TAK-242(TLR4抑制剂)组、绿茶多酚+TAK-242组,每组12只;另取12只大鼠设为假手术组。药物处理后,对所有大鼠进行神经功能缺损评分,检测各组大鼠脑组织含水量,采用Evans蓝外渗实验评定血脑屏障通透性,采用苏木精-伊红(HE)染色检测各组大鼠脑皮层神经元受损情况,采用酶联免疫吸附法(ELISA)检测血清中白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)水平,采用蛋白免疫印迹法检测脑组织中TLR4通路蛋白TLR4、MyD88、NF-κB p65表达情况。结果与假手术组相比,模型组大鼠皮层结构紊乱疏松,神经元出现变性、坏死,数目减少,细胞核固缩、深染等病理改变,脑组织及神经元受损,神经功能缺损评分、脑组织含水量、Evans蓝渗出量、血清中IL-6及TNF-α水平、脑组织TLR4、MyD88及NF-κB p65蛋白表达均明显升高(P<0.05);与模型组相比,绿茶多酚组、TAK-242组、绿茶多酚+TAK-242组大鼠脑皮层神经元损伤减轻,神经功能缺损评分、脑组织含水量、Evans蓝渗出量、血清中IL-6及TNF-α水平、脑组织TLR4、MyD88及NF-κB p65蛋白表达均降低(P<0.05);与绿茶多酚组及TAK-242组分别相比,绿茶多酚+TAK-242组大鼠脑组织及神经元病理损伤进一步减轻,神经功能缺损评分、脑组织含水量、Evans蓝渗出量、血清中IL-6及TNF-α水平、脑组织TLR4、MyD88及NF-κB p65蛋白表达均降低(P<0.05)。结论绿茶多酚可通过抑制TLR4通路减轻蛛网膜下腔出血大鼠早期脑损伤。     

视黄酸通过TLR4/NF-κB通路对脂多糖诱导血管炎症与氧化应激的影响

目的 研究视黄酸(RA)对脂多糖(LPS)诱导的血管炎症与氧化应激的保护作用并探讨其作用机制。 方法 SD大鼠给予RA和TLR4抑制剂连续口腔灌胃2周后,除对照组外,LPS组、RA 3 mg/kg组、RA 15 mg/kg组和TLR4抑制剂组（TAK-242, 3 mg/kg）,通过腹腔注射LPS (10 mg/kg)以建立血管炎症模型。血管张力测定系统检测血管舒张功能,硝酸还原酶法检测大鼠血清中一氧化氮（NO）的含量,ELISA法检测血清中IL-18、IL-1β、TNF-α、IL-6和GSH-px的水平,WST-1法和TBA法分别检测血清中超氧化物歧化酶（SOD）活力、丙二醛（MDA）,DHE荧光探针检测血管活性氧(ROS)的水平,免疫组化法检测血管 NF-κB p65的表达,免疫印迹法检测血管TLR4、eNOS和p-eNOS的表达。 结果 与LPS组相比,RA能够改善血管舒张功能,增加p-eNOS和NO的水平,减少血清炎症因子IL-18、IL-1β、TNF-α、IL-6的含量,降低血清中MDA和ROS的生成,升高血清中SOD的生成和GSH-px的释放量,并且下调血管TLR4和NF-κB p65的表达水平。另外,RA对LPS诱导的血管炎症与氧化应激的影响与TLR4抑制剂作用相似。 结论 RA对LPS诱导的血管炎症与氧化应激具有抑制作用,其可能通过TLR4/NF-κB p65信号通路发挥作用。     

Toll-like receptor 2 mediates inflammatory cytokine induction but not sensitization for liver injury by Propioni- bacterium acnes

Recognition of Gram-positive bacteria by Toll-like receptor 2 (TLR2) induces activation of proinflammatory pathways. In mice, sensitization with the Gram-positive Propionibacterium acnes followed by a challenge with the TLR4 ligand, lipopolysaccharide (LPS), results in fulminant hepatic failure. Here, we investigated the role of TLR2 in liver sensitization to LPS-induced injury. Stimulation of Chinese hamster ovary cells and peritoneal macrophages with heat-killed P. acnes required expression of TLR2 but not of TLR4, suggesting that P. acnes was a TLR2 ligand. Cell activation by P. acnes was myeloid differentiation primary-response protein 88 (MyD88)-dependent, and it was augmented by coexpression of CD14 in mouse peritoneal macrophages. In vitro, P. acnes behaved as a TLR2 ligand and induced TLR4 hetero- and TLR2 homotolerance in peritoneal macrophages. In vivo priming of wild-type mice with P. acnes, but not with the selective TLR2 ligands peptidoglycan and lipotheicoic acid, resulted in hepatocyte necrosis, hyperelevated serum levels of tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-6, interferon-gamma (IFN-gamma), and IL-12 (p40/p70), and increased RNA expression of proinflammatory cytokines (IL-12p40, IL-1alpha, IL-6, IL-1beta, IL-18, IFN-gamma) in the liver after a LPS challenge. Furthermore, P. acnes priming sensitized TLR2-deficient (TLR2-/-) but not MyD88-/- mice to LPS-induced injury, evidenced by hepatocyte necrosis, increased levels of serum TNF-alpha, IFN-gamma, IL-6, and liver proinflammatory cytokine mRNA expression. IFN-gamma, a cytokine sensitizing to endotoxin, was induced by P. acnes in splenocytes of TLR2-/- and TLR9-/- but not MyD88-/- mice. These results suggest that although P. acnes triggers TLR2-mediated cell activation, TLR2-independent but MyD88-dependent mechanisms mediate in vivo sensitization by P. acnes for LPS-induced liver injury.     

Resveratrol protects BV2 mouse microglial cells against LPS-induced inflammatory injury by altering the miR-146a-5p/TRAF6/NF-κB axis

Abstract

Objective: To investigate the role of miR-146a-5p in the effects of resveratrol (RSV) on inflammatory response in BV2 mouse microglial cells.

Materials and methods: BV2 cells were pretreated by RSV and stimulated with lipopolysaccharide (LPS). Cell Viability was checked using a MTT assay. Real-Time PCR was performed to detect the levels of pro-inflammatory cytokines (tumor necrosisfactor-α－TNF-α, interleukin-1β－IL-1β and interleukin-6 － IL-6) and miR-146a-5p expression. Western blot was used to analyze the protein expression of TNF receptor associated factor 6 (TRAF6) and phospho-nuclear factor kappa B (pNF-κB). Gain-of-function and loss-of-function analysis of miR-146a-5p was performed using transfection of miR-146a-5p mimic and miR-146a-5p inhibitor, respectively.

Results: Pretreatment with RSV significantly and dose dependently inhibited LPS-induced production of TNF-α, IL-1β and IL-6 in BV2 cells. MiR-146a-5p was significantly upregulated after LPS treatment, and further increased in RSV and LPS-co-treated cells. MiR-146a-5p overexpression via miR-146a-5p mimic transfection downregulated the mRNA level of TNF-α, IL-1β and IL-6, as well as abrogated the protein expression of TRAF6 and pNF-κB in BV2 cells exposed to LPS. More importantly, the reducion of TNF-α, IL-1β and IL-6 level by RSV were reversed by miR-146a-5p silence via miR-146a-5p inhibitor transfection. Furthermore, silencing miR-146a-5p attenuated the inhibitory effect of RSV on the TRAF6/NF-κB pathway which was activated after induction with LPS. Conclusions: RSV can suppress LPS-induced inflammatory injury via modulating the miR-146a-5p/TRAF6/NF-κB axis in BV2 mouse microglial cells.     

Gx‐50 reduces β‐amyloid‐induced TNF‐α, IL‐1β, NO,and PGE2 expression and inhibits NF‐κB signaling in a mouse model of Alzheimer's disease

Chronic inflammation, which is regulated by overactivated microglia in the brain, accelerates the occurrence and development of Alzheimer's disease (AD). Gx‐50 has been investigated as a novel drug for the treatment of AD in our previous studies. Here, we investigated whether gx‐50 possesses anti‐inflammatory effects in primary rat microglia and a mouse model of AD, amyloid precursor protein (APP) Tg mice. The expression of TNF‐α, IL‐1β, NO, prostaglandin E2, and the expression of iNOS and COX2 were inhibited by gx‐50 in amyloid β (Aβ) treated rat microglia; additionally, microglial activation and the expression of IL‐1β, iNOS, and COX2 were also significantly suppressed by gx‐50 in APP⁺ transgenic mice. Furthermore, gx‐50 inhibited the activation of NF‐κB and MAPK cascades in vitro and in vivo in APP‐Tg mice. Moreover, the expression of TLR4 and its downstream signaling proteins MyD88 and tumor necrosis factor receptor associated factor 6 (TRAF6) was reduced by gx‐50 in vitro and in vivo. Interestingly, silencing of TLR4 reduced Aβ‐induced upregulation of IL‐1β and TRAF6 to levels similar to gx‐50 inhibition; moreover, overexpression of TLR4 increased the expression of MyD88 and TRAF6, which was significantly reduced by gx‐50. These findings provide strong evidence that gx‐50 has anti‐inflammatory effects against Aβ‐triggered microglial overactivation via a mechanism that involves the TLR4‐mediated NF‐κBB/MAPK signaling cascade.     

青蒿素减轻LPS诱导的肠上皮细胞屏障功能损伤的实验研究

目的:探究青蒿素对脂多糖(lipopolysaccharide,LPS)诱导的大鼠肠上皮IEC-6细胞屏障功能损伤的影响。方法:体外培养IEC-6细胞,随机分为5组:对照组、LPS(100 mg/L)组和LPS+青蒿素(30、50和100μmol/L)组,MTT法检测各组细胞毒性变化,ELISA检测各组细胞分泌炎性因子TNF-α、IL-1β和IL-6水平的变化,电阻仪检测肠上皮细胞跨上皮电阻(TER),酶标仪检测单层细胞对辣根过氧化物酶(HRP)的通透性,RT-qPCR和Western blot检测各组细胞紧密连接蛋白(ZO-1、claudin-1和occludin)以及TLR4/My D88/NF-κB mRNA和蛋白表达的变化。结果:LPS与青蒿素在本实验浓度范围对IEC-6细胞均无毒性。与对照组相比,LPS处理下,细胞分泌TNF-α、IL-1β和IL-6水平以及TLR4/My D88/NF-κB的mRNA和蛋白表达明显增加,ZO-1、claudin-1和occludin的mRNA和蛋白表达降低。而青蒿素干预下,细胞分泌TNF-α、IL-1β和IL-6水平以及TLR4/My D88/NF-κB mRNA和蛋白表达明显降低,ZO-1、claudin-1和occludin的mRNA和蛋白表达升高(P0.05),均呈现浓度依赖性。结论:青蒿素可能通过抑制TLR4/My D88/NF-κB通路减轻LPS诱导的肠上皮细胞屏障功能损伤。