黄芪甲苷Ⅳ对内毒素诱导RAW264.7细胞损伤的保护作用及机制研究

目的:研究黄芪皂苷Ⅳ对LPS诱导的巨噬细胞RAW264.7损伤的保护作用及机制.方法:测定细胞活力判断心肌细胞损伤的程度.TNF-α,IL-1β,IL-6,IL-10的释放以及NF-кB蛋白、Akt和磷酸化Akt表达用以研究作用机制.结果:黄芪皂苷Ⅳ对LPS引起的RAW264.7细胞损伤具有显著的抑制作用,1,3和10μM黄芪皂苷Ⅳ可显著降低LPS诱导的RAW264.7细胞TNF-α,IL-1β和IL-6的生成,促进IL-10的释放.黄芪皂苷Ⅳ剂量依赖性地增加了由LPS刺激而引起的RAW264.7细胞NF-kB蛋白的表达,抑制了LPS所致的p-Akt蛋白表达的升高.结论:黄芪皂苷Ⅳ可通过Akt-NF-kB途径调控促炎因子和抗炎因子表达的失衡,有效发挥对LPS诱导巨噬细胞RAW264.7损伤的保护作用.     

过氧化氢刺激RAW264.7巨噬细胞促炎细胞因子和趋化因子基因表达

免疫反应细胞经呼吸瀑布作用产生的活性氧是巨噬细胞促炎细胞因子和趋化因子表达的信号分子,但目前缺乏过氧化氢(H2O2)刺激巨噬细胞表达促炎细胞因子和趋化因子的直接证据．本研究以离体培养的小鼠RAW264.7巨噬细胞为研究体系,探讨外源H2O2对RAW264.7巨噬细胞促炎因子和趋化因子基因表达和生成的影响．MTT法结合实时荧光定量PCR(qRT-PCR)、酶联免疫吸附试验(ELISA)结果显示,RAW264.7细胞在H2O2浓度低于40 μmol/L时不影响RAW264.7细胞的增殖活力．20 μmol/L和40 μmol/L H2O2显著增强RAW264.7细胞TNF-α、IL-1β、MCP-1和MIP-2基因转录和蛋白质生成,并存在剂量依赖效应;而200 U/mL过氧化氢酶预处理则可减弱由H2O2刺激的TNF-α、IL-1β、MCP-1和MIP-2基因表达和蛋白生成．这些结果提示,H2O2是刺激巨噬细胞促炎因子和趋化因子表达或生成的重要因子,对机体炎症反应的发生具有重要作用．     

二氢青蒿素对超高分子量聚乙烯颗粒诱导的巨噬细胞源性炎性因子释放的影响

目的:研究二氢青蒿素(Dihydroartemisinin,DHA)对超高分子量聚乙烯(Ultra highmolecularweightpolyethylene,UHMWPE)颗粒诱导的小鼠巨噬细胞系RAW264.7细胞源性炎性因子释放的影响。方法:建立UHMWPE颗粒诱导的小鼠巨噬细胞系RAW264.7细胞源性炎性因子释放模型;施加不同浓度的二氢青蒿素观察药物对细胞的影响,酶联免疫分析法(Enzyme-linked immuno sorbent assay,ELISA)检测细胞培养液上清中TNF-α,IL-1β,IL-6和IL-10含量,MTT法检测细胞毒性反应。结果:酶联免疫分析方法结果表明,二氢青蒿素可以显著抑制由UHMWPE颗粒诱导的小鼠RAW264.7细胞促炎细胞因子TNF-α,IL-1和IL-6的表达,并显著促进抗炎因子IL-10的释放,其效应具有剂量依赖性。结论:二氢青蒿素具有显著的抗炎作用,可以抑制UHMWPE颗粒诱导的巨噬细胞炎症反应,其在预防人工关节置换术后假体无菌性松动的药物治疗方面具有潜在的作用。     

余甘子不同溶剂提取物抗炎活性的研究

本试验旨在筛选出余甘子不同溶剂提取物的最佳抗炎活性部位。试验以水、乙醇、乙酸乙酯和石油醚为提取溶剂得到余甘子不同溶剂提取物(水提取物分成三个组分:水(1)组分分子量小于6000;水(2)组分分子量在6000到10000之间;水(3)组分分子量大于10000),采用LPS诱导RAW264.7巨噬细胞建立炎症模型;以NO分泌量和细胞因子(TNF-α、IL-1β、IL-6)释放量为指标,筛选出余甘子不同溶剂提取物最佳抗炎活性部位。余甘子不同溶剂提取物在浓度25~400μg/m L之间对细胞无明显细胞毒性(P0.05)。与模型组相比,水(1)、水(2)和乙醇提取物能显著抑制LPS诱导巨噬细胞分泌NO(P0.05)。在细胞因子实验中,乙醇提取物极显著抑制LPS诱导RAW264.7巨噬细胞分泌IL-1β和TNF-α(P0.01);乙酸乙酯提取物抑制LPS诱导RAW264.7巨噬细胞分泌IL-6的效果最佳。综合评价得出余甘子乙醇提取物作为筛选出的最佳抗炎活性部位,其极显著抑制LPS诱导RAW264.7巨噬细胞分泌NO和细胞因子(TNF-α、IL-1β、IL-6),可以作为后续分离鉴定抗炎活性物质单体的活性部位。     

表没食子儿茶素-3-没食子酸酯抑制TNF-α和IL-β在小鼠皮肤烫伤修复期间的表达

肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和白细胞介素-1β(interleukin-1β,IL-1β)在创伤修复中起着至关重要的作用.本研究利用小鼠皮肤深II度烫伤模型,采用逆转录聚合酶链反应(RT-PCR)和酶联免疫吸附试验(ELISA)检测烫伤部位组织Tnf-αmRNA和Il-1βmRNA的表达水平以及TNF-α和IL-1β的含量,探讨表没食子儿茶素-3-没食子酸酯(EGCG)对小鼠皮肤烫伤修复期间TNF-α和IL-1β表达的影响.结果显示,用0.2 mg/g EGCG膏剂涂敷烫伤皮肤,处理12 h可致组织Tnf-αmRNA表达水平和TNF-α含量下降,处理24 h可致组织Il-1βmRNA表达水平和IL-1β含量下降.上述结果提示,0.2 mg/g EGCG处理能抑制烫伤组织TNF-α和IL-1β的表达,减弱创伤组织的炎症反应,有助于创伤组织的修复.     

银杏叶提取物对内毒素诱导巨噬细胞核因子-κB活化及炎性细胞因子表达的调节

探讨银杏叶提取物（EGb761）对内毒素（LPS）诱导RAW264．7细胞核因子-κB（NF-κB）活化及炎性细胞因子基因表达的调节,为银杏叶提取物的临床运用提供理论依据．分别用LPS或EGb761＋LPS处理体外培养的小鼠巨噬细胞系RAW264．7细胞,采用蛋白质印迹分析检测细胞中NF-κB活性,用逆转录-聚合酶链反应（RT-PCR）和酶联免疫吸附法（ELISA）检测细胞中TNF-α、IL-1β、IL-6 mRNA和蛋白的表达．研究结果表明LPS组NF-κB活性和TNF-α、IL-1β、IL-6含量在刺激后2～12h明显高于正常对照组,而EGb761＋LPS组NF-κB活性和TNF-α、IL-1β、IL-6含量均显著低于LPS组．结果提示LPS可诱导RAW264．7细胞NF-κB活化,导致TNF-α、IL-1β、IL-6基因表达增强,而EGb761能抑制NF-κB活化而调节TNF-α、IL-1β、IL-6基因的表达．     

EV-A71感染小鼠巨噬细胞诱导促炎细胞因子和趋化因子反应

为初步探索EV-A71在小鼠巨噬细胞中的复制情况和抗病毒的固有免疫应答,本文以小鼠巨噬细胞系RAW264.7为细胞模型,通过建立EV-A71绝对定量qPCR方法检测EV-A71病毒载量;EV-A71和紫外灭活的EV-A71感染RAW264.7,不同时间点提取总RNA,RT-qPCR检测促炎细胞因子、趋化因子和模式识别受体的mRNA表达变化水平。本研究成功建立了EV-A71的绝对定量qPCR检测方法,并发现EV-A71感染RAW264.7后随着感染时间的延长,EV-A71病毒载量呈递减趋势;EV-A71和紫外灭活的EV-A71可以引起IL-1β、IL-6、TNF-α促炎细胞因子和IP-10、MCP-1、MIP-1α趋化因子反应,上调TLR2、TLR1、TLR6、MDA5和RIG-I mRNA表达。研究结果显示,EV-A71在小鼠巨噬细胞中具有较低水平的复制,同时产生促炎细胞因子和趋化因子反应。     

灵芝菌丝体冻干粉对脂多糖诱导的小鼠巨噬细胞相关因子分泌及蛋白表达的影响

通过体外培养小鼠巨噬细胞RAW264.7,脂多糖(LPS)诱导刺激,考察了不同浓度灵芝菌丝体冻干粉(FDPGLM)处理后细胞活力、NO和IL‐6水平、Toll样受体4(TLR4)和i NOS m RNA表达、IκBa和磷酸化核转录因子κB(NF‐κB)p65蛋白表达之间的差异。结果显示:相对于LPS诱导来说,FDPGLM能以剂量依赖的方式显著抑制LPS诱导引起的NO和IL‐6水平上升(P0.01),显著下调TLR4 mRNA表达(P0.01),并显著抑制IκBa蛋白降解和NF‐κB p65蛋白磷酸化(P0.01),由此推测在LPS诱导的RAW264.7细胞中,FDPGLM可能经由TLR4/NF‐κB信号途径抑制促炎基因的激活并抑制促炎细胞因子比如IL‐6的分泌,提示FDPGLM在抗炎药理作用上的应用前景。     

连翘脂素对LPS诱导RAW264.7细胞炎症反应的影响

为研究连翘脂素的抗炎效应及其抗炎机制,以地塞米松作为阳性对照,建立脂多糖(LPS)诱导小鼠巨噬细胞RAW264.7炎症模型,检测炎症因子的释放及相关蛋白和mRNA的表达,以期提高对连翘脂素抗炎作用的全面认识并为连翘脂素临床开发提供有力的科学依据。实验采用Griess法检测细胞上清液中NO含量,ELISA法检测TNF-α和IL-6的含量,Westernblot法检测iNOS、COX-2蛋白的表达,RT-qPCR法检测iNOS、COX-2mRNA的表达。与LPS组比较,连翘脂素组和地塞米松组可以明显降低LPS诱导的RAW264.7细胞释放NO、TNF-α和IL-6的量,并呈现浓度依赖关系。Westrenblot和RT-qPCR结果显示连翘脂素能抑制LPS诱导的iNOS、COX-2的蛋白表达以及mRNA的表达,并呈浓度依赖关系。实验研究表明连翘脂素能够明显抑制LPS诱导的RAW264.7细胞炎症因子的释放,iNOS、COX-2蛋白及mRNA的表达从而抑制炎症反应。     

黄芩苷对脂多糖诱导的巨噬细胞NF-κB活化和炎症因子表达的影响

目的:研究黄芩苷对脂多糖(LPS)诱导小鼠巨噬细胞核因子κB(NF-κB)及肿瘤坏死因子α(TNF-α)、白介素6(IL-6)表达的影响.方法:分别用LPS(终浓度1μgomL-1)和LPs+黄芩苷(终浓度10,50,100μmol moloL-1)处理生长良好的小鼠巨噬细胞RAW264.7,用RT-PCR法和Elisa法检测细胞及其上清液中TNF-α、IL-6 mRNA和蛋白的表达变化,用Western Blot法检测细胞核内NF-κB p65蛋白含量变化.结果:LPS刺激RAW264.7细胞可导致NF-κB激活,上调TNF-α、IL-6表达;黄芩苷预处理能降低LPS诱导的NF-κB出活化和TNF-α、IL-6表达.结论:黄芩苷可通过抑制NF-κB活化,下调LPS诱导的巨噬细胞TNF-α、IL-6的生成,发挥抗炎作用.这可能是其抗动脉粥样硬化的作用机制之一.     

表没食子儿茶素-3-没食子酸酯抑制TNF-a和IL-1β在小鼠皮肤烫伤修复期间的表达

肿瘤坏死因子-a(tumor necrosis factor-a,TNF-a)和白细胞介素-1β(interleukin, IL-1β)在创伤修复中起着至关重要的作用.本研究利用小鼠皮肤深II度烫伤模型,采用逆转录聚合酶链反应(RT-PCR)和酶联免疫吸附试验(ELISA)检测烫伤部位组织Tnf-a mRNA和Il-1β mRNA的表达水平以及TNF-a和IL-1β的含量,以探讨表没食子儿茶素-3-没食子酸酯(EGCG)对小鼠皮肤烫伤修复期间TNF-a和IL-1β表达的影响.结果显示,用0.2 mg/g EGCG膏剂涂敷烫伤皮肤,处理12 h可致组织Tnf-a mRNA表达水平和TNF-a含量下降,处理24 h可致组织Il-1β mRNA表达水平和IL-1β含量下降.上述结果提示0.2 mg/g EGCG处理能抑制烫伤组织TNF-a和IL-1β的表达,减弱创伤组织的炎症反应,有助于创伤组织的修复.     

Dipyrithione inhibits lipopolysaccharide-induced iNOS and COX-2 up-regulation in macrophages and protects against endotoxic shock in mice

Dipyrithione (PTS2) possesses anti-bacterial and anti-fungal activity. In the present study, we found that PTS2 dose-dependently inhibited the LPS-induced up-regulation of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein level in RAW264.7 cells. RT-PCR experiments showed that PTS2 suppressed LPS-induced iNOS but not COX-2 expression at the mRNA level. As expected, PTS2 prevented NO secretion in RAW264.7 cells. Furthermore, PTS2 administration significantly decreased LPS-induced mortality in mice. Mechanistically, PTS2 decreased expression and phosphorylation of STAT1, but did not interfere with the MAPK and NF-kappaB pathways. In conclusion, PTS2 protects mice against endotoxic shock and inhibits LPS-induced production of pro-inflammatory mediators, suggesting that PTS2 could play an anti-inflammatory role in response to LPS.     

Astaxanthin exerts anti-inflammatory and antioxidant effects in macrophages in NRF2-dependent and independent manners

Although anti-inflammatory effects of astaxanthin (ASTX) have been suggested, the underlying mechanisms have not been fully understood. Particularly, the modulatory action of ASTX in the interplay between nuclear factor E2-related factor 2 (NRF2) and nuclear factor κB (NFκB) to exert its anti-inflammatory effect in macrophages is unknown. The effect of ASTX on mRNA and protein expression of pro-inflammatory and antioxidant genes and/or cellular reactive oxygen species (ROS) accumulation were determined in RAW 264.7 macrophages, bone marrow-derived macrophages (BMDM) from wild-type (WT) and Nrf2-deficient mice, and/or splenocytes and peritoneal macrophages of obese mice fed ASTX. The effect of ASTX on M1 and M2 macrophage polarization was evaluated in BMDM. ASTX significantly decreased LPS-induced mRNA expression of interleukin 6 (Il-6) and Il-1β by inhibiting nuclear translocation of NFκB p65; and attenuated LPS-induced ROS with an increase in NRF2 nuclear translocation, concomitantly decreasing NADPH oxidase 2 expression in RAW 264.7 macrophages. In BMDM of WT and Nrf2-deficient mice, ASTX decreased basal and LPS-induced ROS accumulation. The induction of Il-6 mRNA by LPS was repressed by ASTX in both types of BMDM while Il-1β mRNA was decreased only in WT BMDM. Furthermore, ASTX consumption lowered LPS sensitivity of splenocytes in obese mice. ASTX decreased M1 polarization of BMDM while increasing M2 polarization. ASTX exerts its anti-inflammatory effect by inhibiting nuclear translocation of NFκB p65 and by preventing ROS accumulation in NRF2-dependent and -independent mechanisms. Thus, ASTX is an agent with anti-inflammatory and antioxidant properties that may be used for the prevention of inflammatory conditions.     

Salicortin suppresses lipopolysaccharide-stimulated inflammatory responses via blockade of NF-κB and JNK activation in RAW 264.7 macrophages

We isolated the phenolic glucoside salicortin from a Populus euramericana bark extract, and examined its ability to suppress inflammatory responses as well as the molecular mechanisms underlying these abilities, using lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Salicortin inhibited iNOS expression and the subsequent production of NO in a dose-dependent manner in the LPS-stimulated RAW 264.7 cells. Salicortin significantly suppressed LPS-induced signal cascades of NF-κB activation, such as IKK activation, IκBα phosphorylation and p65 phosphorylation in RAW 264.7 cells. In addition, salicortin inhibited the LPS-induced activation of JNK, but not ERK or p38 MAPK. Furthermore, salicortin significantly inhibited production of pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6 in the LPS-stimulated RAW 264.7 cells. These findings suggest that salicortin may show its anti-inflammatory activity by suppressing the LPS-induced expression of pro-inflammatory mediators through inhibition of NF-κB and JNK MAPK signaling cascades in macrophages. [BMB Reports 2014; 47(6): 318-323]     

(S)-tetrahydroisoquinoline alkaloid inhibits LPS-induced arachidonic acid release through downregulation of cPLA2 expression

Sepsis, a systemic inflammatory response syndrome, remains a potentially lethal condition. (S)-1-α-Naphthylmethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (CKD712) is noted as a drug candidate for sepsis. Many studies have demonstrated its significant anti-inflammatory effects. Here we first examined whether CKD712 inhibits lipopolysaccharide (LPS)-induced arachidonic acid (AA) release in the RAW 264.7 mouse monocyte cell line, and subsequently, its inhibitory mechanisms. CKD712 reversed LPS-associated morphological changes in the RAW 264.7 cells, and inhibited LPS-induced release of AA in a concentrationdependent manner. The inhibition was apparently due to the diminished expression of a cytosolic form of phospholipase A₂ (cPLA₂) by CKD712, resulting from reduced NF-κB activation. Furthermore, CKD712 inhibited the activation of ERK1/2 and SAP/JNK, but not of p38 MAPK. CKD712 had no effect on the activity or phosphorylation of cPLA₂ and on calcium influx. Our results collectively suggest that CKD712 inhibits LPS-induced AA release through the inhibition of a MAPKs/NF-κB pathway leading to reduced cPLA₂ expression in RAW 264.7 cells.