原花青素对RAW264.7细胞膜相关前列腺素E2合成酶-1表达的影响

目的 探讨原花青素对RAW264.7细胞膜相关前列腺素E2合成酶-1(mPGES-1)表达的影响.方法 酶免疫测定法(EIA)检测原花青素对PGE2生成的影响,逆转录聚合酶链反应(RT-PCR)检测mPGES-1mRNA的表达,Western blotting检测mPGES-1蛋白的表达.结果 脂多糖(LPS)可以促进RAW264.7细胞PGE2的生成同时上调mPGES-1mRNA和蛋白的表达,而原花青素(4、20mg·L-1)下调LPS诱导的RAW264.7细胞mPGE-1mRNA和蛋白的表达,从而抑制LPS诱导的RAW264.7细胞PGE2的生成.结论 原花青素在mRNA和蛋白水平抑制LPS诱导的RAW264.7细胞mPGES-1表达从而减少PGE2的合成,这可能是原花青素抗炎的机制之一.     

原花青素对RAW264.7细胞膜相关前列腺素E2合成酶-1表达的影响

目的探讨原花青素对RAW264.7细胞膜相关前列腺素E2合成酶-1(mPGES-1)表达的影响。方法酶免疫测定法(EIA)检测原花青素对PGE2生成的影响,逆转录聚合酶链反应(RT-PCR)检测mPGES-1mRNA的表达,Western blotting检测mPGES-1蛋白的表达。结果脂多糖(LPS)可以促进RAW264.7细胞PGE2的生成同时上调mPGES-1mRNA和蛋白的表达,而原花青素(4、20 mg.L-1)下调LPS诱导的RAW264.7细胞mPGES-1mRNA和蛋白的表达,从而抑制LPS诱导的RAW264.7细胞PGE2的生成。结论原花青素在mRNA和蛋白水平抑制LPS诱导的RAW264.7细胞mPGES-1表达从而减少PGE2的合成,这可能是原花青素抗炎的机制之一。     

苹果多糖的制备及对脂多糖诱导RAW 264.7细胞凋亡的影响

摘 要 目的：观察苹果多糖（AP）对脂多糖（LPS）诱导RAW 264.7细胞凋亡的影响及其机制。方法: 采用水提醇沉法从苹果果渣中提取苹果多糖,并测定其糖含量和分子量。采用流式细胞仪考察AP对LPS诱导RAW 264.7细胞凋亡的作用。采用Western Blot法考察AP对LPS诱导RAW 264.7细胞中Bcl 2、Bax蛋白表达的影响。结果: 经水提醇沉法提取纯化得到苹果多糖的糖含量为91.3%,重均分子量为184613 Da。流式细胞仪检测结果显示,LPS诱导RAW 264.7细胞凋亡明显高于正常对照组（P<0.01）;与LPS组比较,浓度为0.5 mg·mL^-1的AP作用72 h后RAW 264.7细胞凋亡率显著降低（P<0.01）,浓度为1.0 mg·mL^-1的AP作用48,72 h后RAW 264.7细胞凋亡率显著降低（P<0.01）。Western Blot检测结果显示,与正常对照组比较,LPS诱导RAW 264.7细胞中的Bcl 2蛋白在48,72 h表达明显升高,差异具有统计学意义（P<0.01）;与LPS组比较,浓度为1 mg·mL^-1的AP作用后,升高了LPS诱导的RAW 264.7细胞Bcl 2蛋白的表达,降低其Bax蛋白的表达,差异具有统计学意义（P<0.01）。结论:AP抑制LPS诱导RAW 264.7细胞凋亡,其机制可能是通过提高RAW 264.7细胞中Bcl 2蛋白的表达,降低其Bax蛋白表达。     

芦荟大黄素对LPS诱导的RAW264.7细胞NO生成及iNOS表达的影响

目的观察芦荟大黄素(aloe-emodin)对脂多糖(LPS)诱导的RAW264.7细胞一氧化氮(NO)生成及诱生型一氧化氮合酶(iNOS)mRNA表达的作用。方法采用LPS诱导的RAW264.7细胞株建立细胞炎症反应模型。采用Griess试剂法测定NO释放量;采用硝普钠释放NO法测定NO自由基含量的变化;采用反转录聚合酶链反应(RT-PCR)分析iNOS mRNA表达改变。结果芦荟大黄素在0.69～2.50mg·L-1剂量范围内可抑制LPS诱导的RAW264.7细胞NO的释放,并呈剂量和时间依赖关系;芦荟大黄素在0.63～5.00mg·L-1剂量范围内可下调LPS诱导的RAW264.7细胞iNOS mRNA含量;而此范围内芦荟大黄素无直接清除NO自由基作用,不影响iNOS活性。结论芦荟大黄素可明显降低LPS诱导的RAW264.7细胞NO释放,呈时间和剂量依赖关系,此作用并非通过捕捉NO或抑制iNOS活性来实现,而是通过抑制iNOS mRNA表达发挥作用的。     

山萘酚对脂多糖诱导RAW264.7细胞中COX-2和iNOS表达及产物生成的影响

目的探讨山萘酚对脂多糖(lipopolysaccharides,LPS)诱导RAW 264.7细胞COX-2及iNOS表达的影响。方法四氮唑盐法(monote-trazolium test,MTT)检测山奈酚对RAW264.7细胞生长增殖的影响,放射免疫测定法(RIA)检测山萘酚对PGE2和NO生成的影响,免疫印迹法(western blotting)检测COX-2及iNOS蛋白的表达。结果山萘酚抑制LPS诱导的RAW 264.7细胞PGE2和NO的生成,同时下调LPS诱导的RAW 264.7细胞COX-2及iNOS蛋白的表达。结论山萘酚抑制2个诱导酶COX-2和iNOS的表达,从而减少炎性产物PGE2和NO的生成,这可能是山萘酚抗炎的机制之一。     

染料木素通过TIPE 2负性调控Akt活性促进脂多糖活化的巨噬细胞凋亡

目的探究染料木素(genistein,GEN)对脂多糖(lipopolysaccharide,LPS)活化的RAW264.7细胞凋亡的影响及其可能的药理学作用机制。方法GEN预孵育RAW264.7细胞或慢病毒介导的肿瘤坏死因子α诱导蛋白8样分子2(tumor necrosis factor-α-induced protein 8-like 2,TIPE 2)过表达细胞2 h,再与LPS共孵育24 h,采用CCK 8试剂盒检测细胞活力,Annexin V-FITC/PI试剂盒检测细胞凋亡水平,qRT-PCR检测TNF-α、IL-6、caspase-8、caspase-3和TIPE 2 mRNA,Western blot检测iNOS、COX-2、caspase-8、caspase-3、TIPE 2、Akt和p-Akt蛋白表达。结果LPS促进RAW264.7细胞TNF-α、IL-6、iNOS、COX-2合成;GEN抑制LPS活化的RAW264.7细胞活力,凋亡细胞增多,并上调caspase-8、caspase-3、TIPE 2 mRNA及蛋白表达;TIPE 2过表达上调活化RAW264.7细胞caspase-8、caspase-3 mRNA及蛋白表达,减少Akt磷酸化,且与GEN具有协同作用。结论GEN可能通过上调TIPE 2抑制Akt活性,激活外源性凋亡途径,促进LPS活化的RAW264.7细胞凋亡。     

当归A_3活性部位对小鼠巨噬细胞环氧化酶-2活性及基因表达的影响

目的研究当归A3活性部位对小鼠巨噬细胞RAW264.7环氧化酶-2(cyclooxygenase-2,COX-2)活性及基因表达的影响。方法采用酶联免疫法(enzyme-line immu-nosorbnent assay,ELISA)检测前列腺素E2(prostaglandin E2,PGE2)产量及COX-2活性,采用RT-PCR和Western blot法检测COX-2 mRNA及蛋白表达水平。结果 A3(20、40、80mg.L-1)剂量依赖性地抑制LPS诱导的RAW264.7细胞PGE2产量、COX-2活性、COX-2 mRNA及蛋白表达水平增高。结论 A3能够直接抑制PGE2产量,此作用可能与抑制COX-2基因表达有关。     

基于Keap1/Nrf2/HO-1通路汉黄芩素7-O-β-D-乙基葡萄糖醛酸苷体外抗氧化应激作用研究

目的 探讨汉黄芩素 7-O-β-D-乙基葡萄糖醛酸苷（WODE）对脂多糖（LPS）诱导的小鼠巨噬细胞（RAW264.7）的抗氧化应激作用及机制。方法 MTS法检测WODE（2.5、5.0、10.0、20.0、40.0、80.0、160.0 μmol·L^-1）对RAW264.7细胞活力的影响；体外培养RAW264.7细胞，WODE（10、20、40 μmol·L^-1）或地塞米松（1 μmol·L^-1，阳性药）预处理1 h，再给予LPS刺激24 h（造模过程不再加药），对照组不加 LPS 和受试物，模型组只给予 LPS 刺激；荧光探针检测胞内活性氧（ROS）水平；Griess反应测定细胞上清液中 NO 生成量；ELISA 检测细胞上清液中肿瘤坏死因子-α（TNF-α）和白细胞介素-6（IL-6）的分泌；实时荧光定量 PCR（qRT-PCR）法检测细胞内诱导型一氧化氮合成酶（iNOS）、环氧化酶-2（COX-2）、白细胞介素-1β（IL-1β）、醌氧化还原酶1（NQO-1）、超氧化物歧化酶-1（SOD-1）的mRNA表达水平；Western blotting法检测细胞核因子E2相关因子2（Nrf2）和血红素加氧酶-（1 HO-1）蛋白表达水平；免疫荧光染色法检测细胞内Kelch ECH相关蛋白（1 Keap1）表达水平。结果 与对照组比较，WODE浓度小于40 μmol·L^-1时，细胞存活率没有明显变化；浓度大于80 μmol·L^-1时，细胞存活率下降，但未见统计学差异。与模型组比较，WODE 10、20、40 μmol·L^-1组 ROS水平显著降低（P＜0.01）；20、40 μmol·L^-1 组 NO 释放显著降低（P＜0.05、0.01）；40 μmol·L^-1 组 iNOS mRNA 表 达 水 平 显 著 降 低（P＜0.01）；10、20、40 μmol·L^-1 组 COX-2 和20、40 μmol·L^-1组IL-1β mRNA表达水平显著降低（P＜0.05、0.01）；10、20、40 μmol·L^-1组TNF-α和IL-6的释放受到显著抑制（P＜0.01）；10、20、40 μmol·L^-1组NQO-1 mRNA表达水平显著升高（P＜0.01），40 μmol·L^-1组SOD-1 mRNA表达水平显著升高（P＜0.01）；10、20、40 μmol·L^-1组Keap1蛋白表达水平显著降低（P＜0.01）；10、20、40 μmol·L^-1组HO-1蛋白和mRNA表达水平显著提高（P＜0.05、0.01）；20、40 μmol·L^-1 组 Nrf2 蛋白和 40 μmol·L^-1 组 Nrf2 mRNA 表达水平显著增加（P＜0.01）。结论 WODE 对 LPS 诱导的RAW264.7细胞的氧化应激具有抑制作用，其作用机制可能与调控Keap1/Nrf2/HO-1信号通路相关。     

EPA、DHA对脂多糖诱导大鼠系膜细胞增殖及PPARγ表达的影响

目的 观察二十碳五烯酸（EPA）和二十二碳六烯酸（DHA）对脂多糖（LPS）诱导大鼠肾小球系膜细胞（GMCs）增殖及过氧化物酶体增殖物激活受体γ（PPARγ）表达的影响。方法 用脂多糖(15 mg·L^-1)刺激大鼠肾小球系膜细胞,二十碳五烯酸和二十二碳六烯酸(10、100 μmol·L^-1)分别培养48 h后,采用噻唑蓝（MTT）方法检测肾小球系膜细胞的增殖情况;采用实时定量PCR方法检测过氧化物酶体增殖物激活受体γ mRNA的表达;采用Western Blot方法检测过氧化物酶体增殖物激活受体γ 蛋白的表达。结果 二十碳五烯酸和二十二碳六烯酸显著抑制脂多糖诱导的肾小球系膜细胞增殖,增加过氧化物酶体增殖物激活受体γ mRNA和蛋白的表达。结论 二十碳五烯酸和二十二碳六烯酸对受损肾小球系膜细胞的保护作用可能与抑制肾小球系膜细胞增殖,激活潜在的抗炎因子过氧化物酶体增殖物激活受体γ有关。     

二妙散对巨噬细胞分化的调控作用研究

目的 研究二妙散对脂多糖（lipopolysaccharide,LPS）诱导巨噬细胞分化的影响。方法 噻唑蓝（methyl thiazolyltetrazolium,MTT）法测定二妙散水煎液对细胞活力的影响;将LPS （100 ng·mL^-1）及不同浓度（0.1,1.0,10 mg·mL^-1）的二妙散水煎液共同作用RAW264.7细胞后,酶联免疫吸附法（enzyme-linked immune sorbent assay,ELISA）检测上清液中肿瘤坏死因子-α（tumor necrosis factor-α,TNF-α）、白介素-10（interleukin 10,IL-10）的含量,实时荧光定量PCR （Real-timefluorescent quantitative PCR,qPCR）检测RAW264.7细胞中白介素-1β（Interleukin 1β,IL-1β）,IL-6的mRNA水平,Westernblot检测RAW264.7细胞NOS2的蛋白表达情况。结果 二妙散水煎液在0.01~100 mg·mL^-1内对RAW264.7细胞无明显毒性。与LPS诱导的模型组相比,二妙散水煎液能明显降低细胞上清液中TNF-α的含量（P<0.05）、升高IL-10的含量（P<0.05）,且可显著抑制RAW264.7细胞中M1分化标记物IL-1β、IL-6的mRNA水平（P<0.05）及NOS2的蛋白表达（P<0.05）。结论 二妙散可通过抑制巨噬细胞向M1促炎方向分化,从而发挥抗炎作用。     

Inhibitory effects of 2,3,5,4'-tetrahydroxystilbene-2-O-beta-D-glucoside on experimental inflammation and cyclooxygenase 2 activity

The inhibitory effects of 2,3,5,4'-tetrahydroxystilbene-2-O-beta-d-glucoside (THSG), extracted from the roots of Polygonum multiflorum Thunb, on inflammatory activity in animal models and cyclooxygenase-2 (COX-2) activity in lipopolysaccharide (LPS)-induced mouse RAW264.7 macrophage cells were investigated. The carrageenin (CGN)-induced rat paw oedema model and dimethylbenzene-induced mouse ear oedema model were prepared; MTT assay, semi-quantitative RT-PCR, Western blot and ELISA were adopted. THSG 2.3, 4.6 and 9.2 mg kg(- 1) by oral administration inhibited mouse ear oedema and the percentage of inhibition of THSG 9.2 mg kg(- 1) is 87%. THSG 3.2, 6.4 and 12.8 mg kg(- 1) by oral administration dose-dependently inhibited rat paw oedema and the percentage of inhibition of THSG 12.8 mg kg(- 1) is 56% at 6 h. Indomethacin 13 and 9 mg kg(- 1) showed 90% and 57% inhibition in the same animal models, respectively. LPS 1 microg ml(- 1) significantly up-regulated prostaglandin E(2) (PGE(2)) production (inducing COX-2 activity) by 35% (exogenous arachidonic acid, AA), which was dose-dependently decreased by THSG 1, 10, and 100 micromol L(- 1) and the percentage of inhibition of THSG 10 micromol L(- 1) was 40%. NS-398 10 micromol L(- 1) decreased PGE(2) production by 42%. THSG 1, 10, 100 micromol L(- 1) was shown to markedly inhibit the LPS-induced COX-2 protein and mRNA expression in RAW264.7 cells (P < 0.05) but had no effect on COX-1 protein and mRNA (P>0.05). In summary, the data showed that THSG possessed an anti-inflammatory effect, which was perhaps related to the inhibition of COX-2 enzyme activity and expression in RAW264.7 macrophage cells.     

穿心莲内酯对巨噬细胞环氧化酶2及其产物表达的影响

目的研究穿心莲内酯对巨噬细胞环氧化酶2(COX-2)表达及其主要产物前列腺素E2(PGE2)生成的影响。方法取生长良好的小鼠巨噬细胞RAW264.7,加入不同浓度的穿心莲内酯(终浓度1、10、50μmol/L)进行预干预,1h后再加入脂多糖(LPS,终浓度1μg/mL)刺激,并设空白组和穿心莲内酯单独作用组作为对照组。取培养18h细胞上清,用Elisa法检测PGE2生成量;取培养24h细胞,提取总蛋白,用WesternBlot法检测穿心莲内酯对COX-2蛋白表达的影响。结果 LPS可以显著诱导RAW264.7细胞COX-2表达和PGE2的生成,与对照组比较P<0.01;穿心莲内酯预干预可以抑制LPS诱导的COX-2蛋白表达,下调PGE2的生成。结论穿心莲内酯可通过降低LPS诱导的巨噬细胞COX-2表达和PGE2生成,发挥抗炎作用,这可能是其抗炎的作用机制之一。     

蟛蜞菊内酯对脂多糖诱导RAW264.7细胞环氧化酶2、NO及TNF-α的影响

目的:研究蟛蜞菊内酯对脂多糖(lipopo-lysaccharide,LPS)诱导RAW264.7巨噬细胞环氧化酶2(COX-2)、NO及TNF-α的作用。方法:ELISA方法检测0.2、2、20μmol/L不同浓度蟛蜞菊内酯对终浓度为10μg/mL LPS诱导RAW264.7细胞产生TNF-α、NO及前列腺素E2(PGE2)的影响,Western blot方法检测蟛蜞菊内酯对LPS诱导COX-2酶蛋白表达的影响。结果:LPS能够明显诱导小鼠RAW264.7细胞产生的COX-2酶蛋白,蟛蜞菊内酯低中高3个浓度均能抑制LPS诱导产生的COX-2酶蛋白表达。PGE2可以被LPS诱导增加,与空白组比有显著差异。蟛蜞菊内酯低中高3个浓度均能抑制LPS诱导产生的PGE2、NO和TNF-α,呈现剂量依赖性。结论:蟛蜞菊内酯抗炎的作用机制可能为抑制COX-2的蛋白表达,进而抑制PGE2的生成,也可能与抑制NO和TNF-α生成有关。     

八肽胆囊收缩素抑制LPS诱导的RAW264.7细胞AP-1活性及IL-6表达

目的研究八肽胆囊收缩素(CCK-8)对LPS诱导RAW264.7细胞IL-6表达的影响及相关机制。方法用ELISA及RT-PCR法检测RAW264.7细胞IL-6蛋白及mR-NA表达;用EMSA方法检测RAW264.7细胞AP-1 DNA结合活性。结果①LPS可时间依赖性的诱导RAW264.7细胞IL-6蛋白及mRNA表达;②10-10 mol.L-1 CCK-8对LPS诱导的RAW264.7细胞IL-6表达无明显影响;10-8、10-6 mol.L-1 CCK-8浓度依赖性地抑制了LPS诱导的RAW264.7细胞IL-6表达;③10-10 mol.L-1 CCK-8未影响LPS诱导的AP-1活性,10-8、10-6 mol.L-1 CCK-8浓度依赖性地抑制了LPS诱导的AP-1活性。结论 CCK-8通过抑制AP-1 DNA结合活性而抑制了LPS诱导的RAW264.7细胞IL-6表达,这可能是CCK-8发挥抗炎作用的信号转导机制之一。     

Lycorine inhibits lipopolysaccharide-induced iNOS and COX-2 up-regulation in RAW264.7 cells through suppressing P38 and STATs activation and increases the survival rate of mice after LPS challenge

As a natural alkaloid extracted from Amaryllidaceae, lycorine shows various biological effects on tumor cells. Here we show that lycorine dose-dependently inhibited the LPS-induced up-regulation of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein level in RAW264.7 cells. Besides, it also inhibited NO, PGE(2), TNF-α and IL-6 release from LPS-treated RAW264.7 cells. RT-PCR experiments showed that lycorine suppressed LPS-induced iNOS but not COX-2 gene expression. Moreover, lycorine decreased LPS-induced mortality in mice. Mechanistically, LPS-induced activation of P38 and STATs pathways was suppressed significantly by lycorine. In addition, lycorine did not interfere with the phosphorylation of ERK1/2, JNK1/2 and NF-κB pathways. In conclusion, lycorine inhibits LPS-induced production of pro-inflammatory mediators and increases the survival rate of mice after LPS challenge, suggesting that lycorine could play an anti-inflammatory role in response to LPS.     

Polygonum cuspidatum, compared with baicalin and berberine, inhibits inducible nitric oxide synthase and cyclooxygenase-2 gene expressions in RAW 264.7 macrophages

Polygonum cuspidatum water extract (PCWE) was shown to be a potent inhibitor of lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). PCWE was compared to baicalin isolated from Scutellaria baicalensis Georgi and berberine of Coptidis rhizoma and Phellodendri cortex, for their effects on LPS-induced nitric oxide (NO) production and iNOS and COX-2 gene expressions in RAW 264.7 macrophages. Both PCWE and the compounds inhibited LPS-induced NO production in a concentration-dependent manner without a cytotoxicity. The decrease in NO production was in parallel with the inhibition of LPS-induced iNOS gene expression by PCWE and the compounds. In contrast, iNOS enzyme activity was not inhibited by PCWE and two agents. In addition, only PCWE inhibited LPS-induced prostaglandin E2 (PGE2) production and COX-2 gene expression without affecting COX-2 enzyme activity, while baicalin or berberine did not. Furthermore, N-nitro-L-arginine (NLA) and N-nitro-L-arginine methyl ester (L-NAME) pretreatment enhanced LPS-induced iNOS protein expression, which was inhibited by these PCWE and two agents, although LPS-induced COX-2 protein expression was not affected by NLA and L-NAME. PCWE inhibited PGE2 production and COX-2 protein expression in NLA/LPS or L-NAME/LPS-co-treated RAW 264.7 cell, however, baicalin or berberine did not. From the results, it was concluded that co-treatment with NOS inhibitors and PCWE effectively blocks acute production of NO and inhibits expression of iNOS and COX-2 genes.     

COX-2 as possible target for the inhibition of PGE2 production by Rikko-san in activated macrophage

We have previously reported that Rikko-san (RKS) inhibited the lipopolysaccharide (LPS)-stimulated prostaglandin (PG) E2 in mouse macrophage-like RAW264.7 cells without affecting the expression of cyclooxygenase (COX)-2. Here RKS inhibition of the enzyme activity of both COX-1 and COX-2 proteins was investigated. Western blot analysis showed that RKS did not significantly change the S-nitrosylated COX-2 protein level. On the other hand, RKS inhibited the PG production catalyzed by purified COX-2, more effectively than that catalyzed by purified COX-1. These results suggest that RKS inhibits the PGE2 production by selectively inhibiting the COX-2 activity in activated macrophages.     

Wogonin, baicalin, and baicalein inhibition of inducible nitric oxide synthase and cyclooxygenase-2 gene expressions induced by nitric oxide synthase inhibitors and lipopolysaccharide

We previously reported that oroxylin A, a polyphenolic compound, was a potent inhibitor of lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). In the present study, three oroxylin A structurally related polyphenols isolated from the Chinese herb Huang Qui, namely baicalin, baicalein, and wogonin, were examined for their effects on LPS-induced nitric oxide (NO) production and iNOS and COX-2 gene expressions in RAW 264.7 macrophages. The results indicated that these three polyphenolic compounds inhibited LPS-induced NO production in a concentration-dependent manner without a notable cytotoxic effect on these cells. The decrease in NO production was in parallel with the inhibition by these polyphenolic compounds of LPS-induced iNOS gene expression. However, these three compounds did not directly affect iNOS enzyme activity. In addition, wogonin, but not baicalin or baicalein, inhibited LPS-induced prostaglandin E2 (PGE2) production and COX-2 gene expression without affecting COX-2 enzyme activity. Furthermore, N-nitro-L-arginine (NLA) and N-nitro-L-arginine methyl ester (L-NAME) pretreatment enhanced LPS-induced iNOS (but not COX-2) protein expression, which was inhibited by these three polyphenolic compounds. Wogonin, but not baicalin or baicalein, similarly inhibited PGE2 production and COX-2 protein expression in NLA/LPS or L-NAME/LPS-co-treated RAW 264.7 cells. These results indicated that co-treatment with NOS inhibitors and polyphenolic compounds such as wogonin effectively blocks acute production of NO and, at the same time, inhibits expression of iNOS and COX-2 genes.