缺血后处理对大鼠局灶性脑缺血再灌注损伤时TLR4信号通路表达的影响

目的：观察缺血后处理对大鼠局灶性脑缺血再灌注损伤后TLR4通路表达的影响。方法：成年健康雄性SD大鼠110只,随机分为假手术组（sham组）（n=10）、缺血再灌注组（I／R组）和后处理组（IP组）,后两组又依据缺血再灌注6h、12h、24h、48h、72h不同的时间点再分五个亚组。对各组行神经行为学评分,脑组织梗死体积测量,TUNEL技术检测神经细胞凋亡的情况,免疫组织化学技术观察各组大鼠脑组织TLR4、NF—KB和TNF—a蛋白的表达,原位杂交方法检测各组大鼠脑组织TLR4mRNA、NF-KBmRNA的表达。结果：缺血后处理可下调TLR4、NF-KB、TNF-a细胞炎性因子的表达,抑制细胞凋亡、减少脑梗死体积,改善神经行为。结论：后处理可通过抑制TLR4信号通路表达,减少脑梗死体积,改善神经功能。     

体外模拟脑缺血再灌注条件下BV-2细胞TLR4信号途径对TLR2表达的影响

研究证实,TLR4和TLR2有可能作为重要炎性受体介导脑缺血再灌注炎性损伤.然而目前还不清楚在此过程中TLR2和TLR4受体之间是否存在着交叉对话可能.本研究首先利用针对TLR4基因的RNA干扰技术阻断体外模拟脑I/R条件下BV-2细胞TLR4信号传导途径,观察此时TLR2受体表达变化,初步探讨TLR4信号途径对TLR2表达的影响.然后利用NF-κB抑制剂PDTC阻断NF-κB活性来观察体外模拟脑I/R条件下BV-2细胞TLR2和TLR4受体表达变化,进一步阐明NF-κB在TLR2和TLR4交叉对话中的作用.结果表明：（1）体外模拟脑I/R条件下阻断BV-2细胞TLR4信号传导途径可以明显抑制TLR2和NF-κB表达的上调;（2）PDTC预处理后在体外模拟脑I/R条件下BV-2细胞TLR2和TLR4的表达均下调.结果提示,脑I/R损伤中TLR4受体激活后有可能通过NF-κB的介导,进一步影响TLR2的表达,从而导致炎症反应的链式放大,两者协同加重了脑损伤的过程.     

昼夜节律对大鼠局灶性脑缺血再灌注损伤的影响

目的观察昼夜节律对大鼠局灶性脑缺血再灌注损伤的影响。方法将大鼠置人工光(6:00-18:00)暗(18:00-6:00)环境中,适应性饲养4周。然后将大鼠随机分为6:00组、12:00组、18:00组和24:00组,分别在相应的时间点采用线栓法制备局灶性脑缺血再灌注损伤大鼠模型。再灌注24 h后,观察昼夜节律对模型大鼠神经损伤症状,脑梗死范围,脑组织超氧化物歧化酶(SOD)活性和MDA含量,脑细胞内游离Ca2+浓度,血清中内皮素(ET)和血管内皮细胞生长因子(VEGF),以及脑组织病理变化的影响。结果各组大鼠均具有明显的神经损伤症状,其中24:00组大鼠的神经损伤症状明显轻于6:00组;6:00、12:00组大鼠脑梗死范围明显大于18:00、24:00组;18:00、24:00组大鼠SOD活性均显著高于6:00、12:00组,MDA含量均显著低于6:00组;24:00组大鼠脑细胞内游离Ca2+浓度显著低于6:00和12:00组,18:00组大鼠脑细胞内游离Ca2+浓度显著低于6:00;18:00、24:00组大鼠血清ET含量显著低于6:00组大鼠;24:00组大鼠血清VEGF含量显著低于6:00、12:00组;18:00、24:00组大鼠脑组织病理变化明显轻于6:00、12:00组。结论昼夜节律对大鼠脑缺血再灌注损伤具有显著影响。     

莫诺苷对大鼠局灶性脑缺血再灌注损伤神经功能的影响

目的观察中药山茱萸的有效成分莫诺苷对大鼠局灶性脑缺血再灌注后神经功能的影响。方法Wistar雄性大鼠随机分为假手术组、模型组、莫诺苷小剂量组（30 mg/kg）、莫诺苷中剂量组（90 mg/kg）、莫诺苷大剂量组（270 mg/kg）、维生素E（VE）（35 mg/kg）,采用线栓法制作大鼠局灶性脑缺血再灌注模型,缺血30 min后再灌注3 d,应用Zea Longa法、爬网格、平行木、吊绳、Ludmila Belayev 12分评分法,观察莫诺苷对神经功能缺损的改善作用。结果与模型组比较,莫诺苷给药组（小、中、大剂量）Zea Longa法评分均差异极显著（P〈0.01）;与模型组比较,莫诺苷给药组（小、中、大剂量）吊绳法评分均差异极显著（P〈0.01）;Ludmila Belayev 12分评分法评分与模型组比较,莫诺苷大剂量组差异极显著（P〈0.001）,中剂量组差异极显著（P〈0.01）。结论莫诺苷对局灶性脑缺血再灌注模型大鼠有改善行为学评分的作用。     

N-乙酰半胱氨酸对大鼠肝脏缺血再灌注损伤NF-κB和ICAM-1表达的影响

目的:探讨大鼠肝脏缺血再灌注损伤NF-κB和ICAM-1表达情况及NAC的保护作用机制.方法:45只雄性SD大鼠随机分成三组:假手术组(Sham组,n=5);缺血再灌注损伤组(I/R 组,n=20)缺血60min后分别再灌注1、3、6、12h;N-乙酰半胱氨酸组(NAC组,n=20):先自阴茎背静脉给大鼠注射溶于生理盐水的NAC,20min后再按I/R组处理.在各规定的再灌注时间点,分别采用western-blot和免疫组化方法测定肝组织中NF-κB和ICAM-1的表达.结果:I/R组和NAC组再灌注1、3、6、12h后,NF-k B的表达均明显高于Sham组(p＜0.01),于再灌注3h达到高峰;ICAM-1的表达均明显高于Sham组(p＜0.01),于再灌注6h达到高峰.NAC组再灌注1、3、6h与VR组相同时间点比较:NF-k B和ICAM-1的表达均低于I/R组(p＜0.05).NAC组再灌注12h与I/R组相同时间点比较:NF-K B和ICAM一1的表达虽然在数值上有所减少,但统计学上无差异(p＞0.05).结论:大鼠肝脏缺血再灌注后NF-κB和ICAM-1表达增加,NAC可抑制NF-k B激活,减少ICAM-1表达减轻大鼠肝脏缺血再灌注损伤.     

N-乙酰半胱氨酸对大鼠肝脏缺血再灌注损伤NF-κ B和ICAM-1表达的影响

目的：探讨大鼠肝脏缺血再灌注损伤NF-κB和ICAM-1表达情况及NAC的保护作用机制。方法：45只雄性SD大鼠随机分成三组：假手术组（Sham组,n=5）;缺血再灌注损伤组（I/R组,n=20）缺血60min后分别再灌注1、3、6、12h;N-乙酰半胱氨酸组（NAC组,n=20）：先自阴茎背静脉给大鼠注射溶于生理盐水的NAC,20min后再按I/R组处理。在各规定的再灌注时间点,分别采用western-blot和免疫组化方法测定肝组织中NF-κB和ICAM-1的表达。结果：I/R组和NAC组再灌注1、3、6、12h后,NF-κB的表达均明显高于Sham组（p〈0.01）,于再灌注3h达到高峰;ICAM-1的表达均明显高于Sham组（p〈0.01）,于再灌注6h达到高峰。NAC组再灌注1、3、6h与I/R组相同时间点比较：NF-κB和ICAM-1的表达均低于I/R组（p〈0.05）。NAC组再灌注12h与I/R组相同时间点比较：NF-κB和ICAM-1的表达虽然在数值上有所减少,但统计学上无差异（p〉0.05）。结论：大鼠肝脏缺血再灌注后NF-κB和ICAM-1表达增加,NAC可抑制NF-κB激活,减少ICAM-1表达减轻大鼠肝脏缺血再灌注损伤。     

缺血后处理对大鼠再灌注损伤肺细胞凋亡的影响

目的：探讨缺血后处理对再灌注损伤肺细胞凋亡的影响。方法：健康雄性sD大鼠24只,随机分为对照组（C组）、缺血／再灌注组（I／n组）和缺血后处理组（IPostC组）（n=8）。对比观察各组血清中丙二醛（MDA）、超氧化物歧化酶（SOD）、髓过氧化物酶（MPO）活力及含量变化,原位缺口末端标记法（TUNEL）检测肺组织细胞凋亡情况,免疫组化及RT-PCR法检测肺组织中Bax、Bcl一2蛋白和基因的表达。结果：I／R组与c组相比,MDA含量、MPO活力明显升高,SOD活力明显下降（均P〈0．01）,肺组织原位细胞凋亡检测示I／R组凋亡指数（AI）（39．03±3．46）显著高于C组（2．88±0．34）,Bcl-2／Bax比值在蛋白和基因水平明显降低（均P〈0．01）;IPostC组与I／R组相比MDA含量显著降低（P〈0．05）,MPO活力显著降低（P〈0．01）,SOD活性升高（P〈0．01）,AI为8．03±0．88显著低于L／R组,并能明显升高Bcl-2／Bax比值（均P〈0．01）。结论：缺血后处理通过减轻脂质过氧化反应及中性粒细胞聚集,降低Bax／Bel．2比值,使肺组织细胞凋亡减少,从而有效地减轻肺缺血／再灌注损伤。     

白藜芦醇对大鼠局灶性脑缺血再灌注的治疗作用

目的:观察白藜芦醇对大鼠局灶性脑缺血再灌注损伤的治疗作用及可能的机制。方法:将SD大鼠随机分为2组:对照组(n=16),白藜芦醇组(n=16)。对照组再灌注即刻腹腔给予0.5 ml生理盐水,白藜芦醇组再灌注即刻腹腔给予20 mg/kg白藜芦醇。再灌注22小时后,进行神经功能学评分、脑梗死容积测定,用分光光度仪测定脑组织溶浆中SOD、MDA和MPO的含量。结果:再灌注22小时后,白藜芦醇治疗组可以改善大鼠神经功能学评分和降低脑梗死面积(P<0.05),同时可以增加脑组织溶浆中SOD的活性,降低MDA和MPO的含量。结论:白藜芦醇通过减轻白细胞的浸润、提高自由基的清除率对大鼠局灶性脑缺血再灌注损伤发挥治疗作用。     

Ucf-101对大鼠局灶性脑缺血再灌注后Caspase-3蛋白表达的影响

目的观察Ucf—101对大鼠脑缺血再灌注后神经元caspase-3蛋白表达及细胞凋亡的影响,研究其对缺血性脑损伤是否具有保护作用。方法将36只雄性WiStar大鼠随机分为3组：假手术组、缺血组及Ucf—101组,采用线栓法建立大鼠右侧大脑中动脉闭塞（middle cerebral artery occlusion,MCAO）2h再灌注模型,于再灌注后6h和24h断头取脑,采用TTC法测梗死体积,TUNEL法原位标记DNA片段,检测TUNEL阳性细胞的变化,免疫组化法观察脑皮质神经元caspase-3的表达。结果脑缺血再灌注后不同时间点（6h、24h）,Ucf-101组与缺血组相比梗死体积明显缩小,有显著性差异（P〈0．05）;假手术组未见梗死现象。缺血组TUNEL阳性细胞数较假手术组明显增多（P〈0．05）,脑皮质caspase-3的表达较假手术组亦显著增强（P〈0．05）,给予Ucf-101处理后,TUNEL阳性细胞数较缺血组明显减少（P〈0．05）,caspase-3的表达较缺血组亦明显减弱（P〈0．05）。结论Ucf-101能有效地抑制脑缺血再灌注损伤,下调脑皮质神经元Caspase-3蛋白的表达,抑制神经元的凋亡,发挥神经保护作用。     

粪菌移植对小鼠实验性结肠炎 TLR4信号通路及肠黏膜屏障的影响

目的 探讨粪菌移植(FMT)对溃疡性结肠炎(UC)小鼠肠黏膜屏障的影响及可能机制。 方法 小鼠饮用2.0%葡聚糖硫酸钠(DSS)溶液构建小鼠UC模型;50只成年雄性C57BL/6J小鼠,随机留取10只取粪便(这10只不参与后续的实验),其余40只称重、编号,随机分为空白对照组(Con组)、DSS模型对照组(Model组)、美沙拉嗪组(Model+5ASA组)和粪菌液组(Model+FMT组),每组10只,Con组和Model组均给予0.9% NaCl溶液灌肠,给药组分别给予美沙拉嗪、粪便滤液灌肠;评估疾病活动指数(DAI)、各组结肠组织病理情况,用透射电镜检测各组小鼠的结肠黏膜上皮细胞结构的变化情况,ELISA检测各组血清内毒素、炎症因子TNFα水平变化,免疫组化法检测结肠组织Toll样受体4(TLR4)及核因子κB(NFκB)的表达变化,Western blot检测各组ZO1蛋白表达。 结果 与Model组相比,粪菌移植明显改善小鼠的DAI指数和结肠组织的病理损伤,结肠上皮细胞间隙增宽程度减轻,腺上皮细胞间连接较紧密,结肠黏膜上皮细胞微绒毛完整,排列整齐,内毒素、TNFα的含量明显下降,TLR4及NFκB在结肠组织的表达明显下降,ZO1蛋白表达明显升高,促进结肠黏膜屏障的修复,差异具有统计学意义(t=7.954 3,P结论 FMT可减少内毒素及炎症因子的产生,改善结肠炎症,TLR4NFκB信号通路可能是FMT修复结肠黏膜屏障功能的机制之一。     

TLR4mAb 对急性溃疡性结肠炎模型大鼠TLR4、TNF-alpha、IL-1beta 表达的影响

目的：探讨Toll 样受体4 单克隆抗体（TLR4mA）对溃疡性结肠炎（UC）模型大鼠肠TLR4、TNF-alpha、IL-1beta表达的影响及其对 UC 的可能作用机制。方法：30只SD 大鼠随机分为正常对照组、模型组、TLR4mAb 干预组。模型组及TLR4mAb 干预组采用三硝 基苯磺酸（TNBs）法造模,TLR4mAb 干预组给予TLR4mAb10 ug 腹腔注射,正常对照组及模型组以生理盐水代替TLR4mAb 腹 腔注射,剂量及频次相同,第8 天处死全部大鼠。分别进行疾病活动指数（DAI）评分及组织病理学（HPS）评分,采用免疫组化法检 测结肠组织TLR4 的原位表达,酶联免疫吸附试验（ELISA 法）检测血清肿瘤坏死因子-alpha（TNF-alpha）、白细胞介素-1beta（IL-1beta）的浓 度。结果：模型组DAI及HPS 评分均明显高于正常对照组（P<0.05）,TLR4mAb 干预组较模型组有所缓解（P<0.05）。TLR4、TNF- alpha、IL-1beta在模型组表达均明显高于正常对照组,差异有统计学意义（P<0.05）,在TLR4mAb 干预组的表达均明显低于模型组,差异 有统计学意义（P<0.05）。结论：急性溃疡性结肠炎的发病与异常免疫反应有关。TLR4mAb 可影响肠黏膜TLR4 及下游炎症因子 TNF-alpha、IL-1beta的表达,减轻急性溃疡性结肠炎大鼠的肠道炎症反应。     

Detailed qualitative dynamic knowledge representation using a BioNetGen model of TLR-4 signaling and preconditioning

INTRODUCTION: Intracellular signaling/synthetic pathways are being increasingly extensively characterized. However, while these pathways can be displayed in static diagrams, in reality they exist with a degree of dynamic complexity that is responsible for heterogeneous cellular behavior. Multiple parallel pathways exist and interact concurrently, limiting the ability to integrate the various identified mechanisms into a cohesive whole. Computational methods have been suggested as a means of concatenating this knowledge to aid in the understanding of overall system dynamics. Since the eventual goal of biomedical research is the identification and development of therapeutic modalities, computational representation must have sufficient detail to facilitate this 'engineering' process. Adding to the challenge, this type of representation must occur in a perpetual state of incomplete knowledge. We present a modeling approach to address this challenge that is both detailed and qualitative. This approach is termed 'dynamic knowledge representation,' and is intended to be an integrated component of the iterative cycle of scientific discovery. METHODS: BioNetGen (BNG), a software platform for modeling intracellular signaling pathways, was used to model the toll-like receptor 4 (TLR-4) signal transduction cascade. The informational basis of the model was a series of reference papers on modulation of (TLR-4) signaling, and some specific primary research papers to aid in the characterization of specific mechanistic steps in the pathway. This model was detailed with respect to the components of the pathway represented, but qualitative with respect to the specific reaction coefficients utilized to execute the reactions. Responsiveness to simulated lipopolysaccharide (LPS) administration was measured by tumor necrosis factor (TNF) production. Simulation runs included evaluation of initial dose-dependent response to LPS administration at 10, 100, 1000 and 10,000, and a subsequent examination of preconditioning behavior with increasing LPS at 10, 100, 1000 and 10,000 and a secondary dose of LPS at 10,000 administered at approximately 27h of simulated time. Simulations of 'knockout' versions of the model allowed further examination of the interactions within the signaling cascade. RESULTS: The model demonstrated a dose-dependent TNF response curve to increasing stimulus by LPS. Preconditioning simulations demonstrated a similar dose-dependency of preconditioning doses leading to attenuation of response to subsequent LPS challenge - a 'tolerance' dynamic. These responses match dynamics reported in the literature. Furthermore, the simulated 'knockout' results suggested the existence and need for dual negative feedback control mechanisms, represented by the zinc ring-finger protein A20 and inhibitor kappa B proteins (IkappaB), in order for both effective attenuation of the initial stimulus signal and subsequent preconditioned 'tolerant' behavior. CONCLUSIONS: We present an example of detailed, qualitative dynamic knowledge representation using the TLR-4 signaling pathway, its control mechanisms and overall behavior with respect to preconditioning. The intent of this approach is to demonstrate a method of translating the extensive mechanistic knowledge being generated at the basic science level into an executable framework that can provide a means of 'conceptual model verification.' This allows for both the 'checking' of the dynamic consequences of a mechanistic hypothesis and the creation of a modular component of an overall model directed at the engineering goal of biomedical research. It is hoped that this paper will increase the use of knowledge representation and communication in this fashion, and facilitate the concatenation and integration of community-wide knowledge.     

Toll 样受体4在小于胎龄儿生后发生胰岛素抵抗的作用研究

目的:探讨Toll样受体4在小于胎龄儿生后发生胰岛素抵抗的作用。方法:建立动物模型,分为小于胎龄儿追赶生长组(S1组)、小于胎龄儿无追赶生长组(S2组)、适于胎龄儿组(AGA组)。生后4周和12周取血、肝脏和脂肪组织,检测血糖、胰岛素、甘油三酯、游离脂肪酸和总胆固醇,计算胰岛素抵抗指数(Homeostasis model assessment for insulin resistance index,HOMA-IR);ELISA法检测血清白介素-6、肿瘤坏死因子-α;实时定量RT-PCR法检测相同体质量肝脏和脂肪组织中Toll样受体4、髓细胞样分化因子88、核因子κB、肿瘤坏死因子-α和白介素-6 mRNA的表达。结果:与适于胎龄儿组和小于胎龄儿无追赶生长组相比,小于胎龄儿追赶生长组随年龄增长血糖、血清胰岛素、游离脂肪酸、甘油三酯和HOMA-IR逐渐增高(P0.05),相同体质量肝脏和脂肪组织中Toll样受体4、髓细胞样分化因子88、核因子κB、肿瘤坏死因子-α和白介素-6 mRNA表达量也逐渐升高(P0.05);肝脏和脂肪组织中Toll样受体4信号通路与胰岛素抵抗指数HOMA-IR呈显著正相关(P0.05),脂肪组织中的相关性显著高于肝脏组织(P0.05)。结论:SGA生后追赶生长者随年龄增长出现糖脂代谢异常;肝脏和脂肪组织Toll样受体4信号途径激活,诱发以TNF-α和IL-6为炎性介质的慢性炎症,促进胰岛素抵抗发生发展;脂肪组织在胰岛素抵抗发生发展中作用更强。     

乳酸杆菌脂磷壁酸抑制脂多糖诱导的大鼠肝脏Kupffer 细胞TLR4 通路 的激活

目的：探讨保加利亚乳酸杆菌脂磷壁酸（Lipoteichoic Acid of ,LBG-LTA）对大鼠肝脏Kupffer细胞 Toll样受体4（Toll-like receptor 4,TLR4）信号通路的作用。方法：雄性健康Wistar大鼠10 只（2 月龄,体重250～300 g）处死后,分 离培养肝脏Kupffer 细胞;培养LBG,并提取制备LBG-LTA;Kupffer 细胞,在有或无LBG-LTA（0.1、1、10 ug/mL）预处理的情况 下,给予脂多糖（lipopolysaccharide,LPS,1 EU/mL）刺激后,Western blot 检测各孔Kupffer细胞的TLR4、TANK 结合激酶1（TANK binding kinase-1,TBK1）及核中的核因子B（nuclear factor-kB,NF-kB）水平,酶联免疫吸附法检测各孔培养上清中的肿瘤坏死因子 alpha（tumor necrosis factor-alpha,TNF-alpha）和白介素1beta（interleukin-1beta,IL-1beta）。结果：分离的Kupffer 细胞经不同浓度LBG-LTA 预处理 后,其在LPS刺激下所表达的TLR4、TBK1、核中NF- kB的水平及生成的TNF-alpha和IL-1茁明显低于无LBG-LTA预处理情况下的 LPS 孔（P<0.05）,且LBG-LTA 的作用呈浓度依赖性。结论：LBG-LTA以浓度依赖的方式抑制了LPS 诱导下大鼠Kupffer细胞 TLR4 通路的激活。     

姜黄素下调脊髓Toll样受体4及下游细胞因子减弱大鼠神经病理性痛

观察鞘内注射姜黄素对坐骨神经慢性压迫性损伤(CCI)大鼠痛阈和脊髓组织Toll样受体4(TLR4)及TNF-α、IL-1β和IL-10表达的影响．鞘内置管的120只大鼠随机均分为4组：假手术组(Sham),CCI组,溶剂对照组(SC),姜黄素治疗组(Cur,100 μg/天),建立CCI大鼠疼痛模型,术后第1、3、7、10和14天鞘内给药并测定痛阈,第3、7天取腰段脊髓第4～6节段(L4～L6)以Real-time PCR与Western blotting方法检测TLR4、HMGB1 mRNA和蛋白质的表达,ELISA法观察脊髓组织中TNF-α、IL-1β及IL-10表达变化．与Sham组相比,CCI组大鼠机械性痛阈与热痛阈显著降低(均P<0.05),同时脊髓组织TLR4、HMGB1 mRNA和蛋白质的表达明显增加(均P<0.05),TNF-α、IL-1β与IL-10的含量也明显升高(均P<0.05);鞘内注射姜黄素明显降低脊髓TLR4、高迁移率族蛋白1(HMGB1),TNF-α和IL-1β的表达,显著升高脊髓IL-10的表达,同时明显改善CCI大鼠疼痛行为(P<0.05)．姜黄素减轻神经病理性疼痛可能与下调TLR4途径促炎症因子表达有关,抑制TLR4途径有望成为治疗神经病理性疼痛的新策略．     

PA-MSHA引起注射局部急性炎症及白细胞介素17、肿瘤坏死因子α和Toll样受体4的表达改变

为研究皮下注射铜绿假单胞菌甘露糖敏感血凝菌毛株(Pseudomonas aeruginosa mannose sensitive hemagglutinin,PA-MSHA)注射液后局部组织的病理学改变及白细胞介素17(interleukin 17,IL-17)、肿瘤坏死因子α(tumor necrosis factorα,TNF-α)和Toll样受体4(Toll-like receptor 4,TLR4)表达情况,将30只小鼠随机分为两组,分别皮下注射PA-MSHA和生理盐水,在注射后6、12、24、48及72h取注射局部组织,苏木精-伊红(hematoxylin-eosin,HE)染色后观察病理形态改变,并用免疫组化染色和图像分析检测IL-17、TNF-α和TLR4表达水平。结果显示,小鼠皮下注射PA-MSHA后,在给药部位引起以中性粒细胞浸润为主的急性炎症;局部炎症灶中IL-17、TNF-α和TLR4表达在注射后6h升高,24h达到高峰,然后逐渐减低,至72h实验组与对照组无差异;3种炎症因子的表达与炎症反应程度相平行。结果表明,皮下注射PA-MSHA能引起局部组织急性炎症反应,且能改变局部免疫状态。     

Glycyrrhizin inhibits lipopolysaccharide-induced inflammatory response by reducing TLR4 recruitment into lipid rafts in RAW264.7 cells

Background

The aim of this study was to investigate the effect of glycyrrhizin on LPS-induced endotoxemia in mice and clarify the possible mechanism.

Methods

An LPS-induced endotoxemia mouse model was used to confirm the anti-inflammatory activity of glycyrrhizin in vivo. In vitro, RAW264.7 cells were stimulated with LPS in the presence or absence of glycyrrhizin. The expression of cytokines was determined by ELISA. Toll-like receptor 4 (TLR4) was determined by Western blot analysis. Nuclear factor-kB (NF-κB) and Interferon regulatory factor 3 (IRF3) activation were detected by Western blotting and luciferase assay. Lipid raft staining was detected by immunocytochemistry.

Results

In vivo, the results showed that glycyrrhizin can improve survival during lethal endotoxemia. In vitro, glycyrrhizin dose-dependently inhibited the expression of TNF-α, IL-6, IL-1β and RANTES in LPS-stimulated RAW264.7 cells. Western blot analysis showed that glycyrrhizin suppressed LPS-induced NF-κB and IRF3 activation. However, glycyrrhizin did not inhibit NF-κB and IRF3 activation induced by MyD88-dependent (MyD88, IKKβ) or TRIF-dependent (TRIF, TBK1) downstream signaling components. Moreover, glycyrrhizin did not affect the expression of TLR4 and CD14 induced by LPS. Significantly, we found that glycyrrhizin decreased the levels of cholesterol of lipid rafts and inhibited translocation of TLR4 to lipid rafts. Moreover, glycyrrhizin activated ABCA1, which could induce cholesterol efflux from lipid rafts.

Conclusion

Glycyrrhizin exerts an anti-inflammatory property by disrupting lipid rafts and inhibiting translocation of TLR4 to lipid rafts, thereby attenuating LPS-mediated inflammatory response.

General significance

Learning the anti-inflammatory mechanism of glycyrrhizin is crucial for the anti-inflammatory drug development.