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。     

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。     

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。     

原花青素对脂多糖诱导RAW2647细胞COX-2酶活性、mRNA及蛋白表达的影响

目的探讨原花青素对脂多糖(LPS)诱导小鼠巨噬细胞株RAW264.7细胞COX-2酶活性及蛋白表达的影响。方法放射免疫法检测COX-2酶活性，RT-PCR检测COX-2 mRNA表达，Western blotting检测COX-2蛋白表达。结果原花青素(0.8，4和20 mg·L^-1)不影响LPS诱导RAW264.7细胞COX-2酶活性，可下调LPS诱导RAW264.7细胞COX-2 mRNA表达；原花青素(4和20 mg·L^-1)下调LPS诱导RAW264.7细胞COX-2蛋白表达。结论原花青素不影响LPS诱导RAW2647细胞COX-2酶活性，但对LPS诱导RAW264.7细胞COX-2 mRNA及蛋白表达抑制作用明显。     

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.     

穿心莲内酯对脂多糖诱导RAW264.7细胞炎症反应的抑制作用

目的探讨穿心莲内酯对脂多糖诱导RAW264.7细胞炎症反应的抑制作用及其作用机制。方法采用噻唑蓝比色(MTT)法分析穿心莲内酯对RAW264.7细胞活力的影响。通过脂多糖处理RAW264.7细胞24 h建立细胞炎症模型,造模前1 h用穿心莲内酯2.5、5、10、20μmol/L预处理。荧光定量PCR法检测RAW264.7细胞内相关抗氧化应激酶基因和i NOS水平。穿心莲内酯单独处理RAW264.7细胞24 h,Western blotting法检测Keap1/Nrf2/HO-1信号通路相关蛋白和Keap1、Nrf2、HO-1蛋白水平。免疫荧光检测转录因子Nrf2在胞质及核内的分布情况。结果与对照组比较,穿心莲内酯剂量相关性地抑制RAW264.7细胞活力,差异具有统计学意义(P0.05、0.01、0.001)。穿心莲内酯显著抑制脂多糖诱导RAW264.7细胞的i NOS水平(P0.001),增加相关抗氧化酶基因HO-1、NQO1 m RNA水平。穿心莲内酯抑制Keap1表达,增加Nrf2和HO-1蛋白水平的表达。结论穿心莲内酯可抑制脂多糖诱导的炎症反应,其作用机制可能与激活Keap1/Nrf2/HO-1信号通路从而调控抗氧化酶HO-1、NQO1 m RNA表达水平相关。     

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。     

苹果多酚对脂多糖刺激的RAW264.7细胞COX-2/PGE2和iNOS/NO表达的研究

目的 研究苹果多酚(APE)对脂多糖(LPS)诱发的RAW264.7细胞炎症COX-2/PGE2和iNOS/NO表达的抑制作用.方法 采用APE干预LPS刺激的小鼠单核/巨噬细胞RAW264.7,然后用Griess Reagent法和ELISA法检测细胞分泌的一氧化氮(NO)和前列腺素E2(PGE2)的水平,并且采用RT-qPCR和Western blotting技术检测APE对诱导型一氧化氮合酶(iNOS)和环氧化酶-2(COX-2)水平的影响以及核转录因子(NF-κB)的蛋白表达.结果 APE能显著抑制LPS刺激的RAW264.7细胞中NO、PGE2的含量,下调iNOS及COX-2的表达及NF-κB的磷酸化.结论 APE通过下调NF-κB的活性及iNOS与COX-2表达而发挥抗炎作用.     

赤芍801对小鼠巨噬细胞COX-1和COX-2活性、mRNA及蛋白表达的影响

目的研究赤芍801(propyl gallate,PrG)对环氧酶(cy-c looxygenase,COX)活性、mRNA及蛋白表达的影响。方法采用基于小鼠腹腔巨噬细胞的COX-1和COX-2体外筛选模型,用钙离子导入剂(calc ium ionophore A23187)短时刺激小鼠腹腔巨噬细胞,测定培养上清液中的6酮--前列腺素F1α(6-keto-PGFlα)的量反映COX-1活性;用脂多糖(lipopolysac-charide,LPS)长时间刺激细胞,测定培养上清液中的前列腺素E2(PGE2)的量反映COX-2活性。半定量RT-PCR法检测PrG对LPS刺激的Raw小鼠巨噬细胞COX-1、COX-2 mRNA表达的影响。W estern b lot法检测PrG对LPS刺激的Raw小鼠巨噬细胞COX-1、COX-2蛋白表达的影响。结果PrG体外10,5μmol.L-1浓度下不影响6-keto-PGFαl生成(P>0.05),而在1,0.5,0.1,0.05μmol.L-1浓度下则诱导6-keto-PGF1α生成(P<0.01),并呈较好的剂量依赖性。PrG体外1,10μmol.L-1可抑制PGE2生成(P<0.05)。PrG不同浓度对LPS刺激的Raw小鼠巨噬细胞COX-1、COX-2 mR-NA表达无明显影响。PrG(100,10,1μmol.L-1)浓度下可抑制COX-2蛋白表达,但对COX-1蛋白表达无影响。结论在0.05~10μmol.L-1浓度范围内,PrG低浓度时促进6-keto-PGF1α生成,高浓度时抑制PGE2生成,提示其高浓度时抑制COX-2,低浓度时激活COX-1;不同浓度PrG对COX-1、COX-2 mRNA表达均无明显影响。PrG(1~100μmol.L-1)浓度下可抑制COX-2蛋白表达,但对COX-1蛋白表达无影响。     

穿琥宁抗炎作用的血红素加氧酶-1的信号转导机制

目的初步探讨穿琥宁抗炎作用的信号转导机制。方法用WST-8细胞计数试剂盒检测穿琥宁的细胞毒性;用IN Cell Analyzer 2000活细胞成像系统及组成型增强表达绿色荧光蛋白偶联NF-κBP65(EGFP-NF-κBP65)融合蛋白的CHO细胞和EGFP偶联促分裂原活化蛋白激酶APk2(EGFP-MAPK-APk2)融合蛋白的BHK细胞观察穿琥宁对白细胞介素1β(IL-1β)或肿瘤坏死因子α(TNF-α)诱导NF-κBP65核转位及脂多糖(LPS)诱导的P38MAPK下游分子MAPK-APk2核转位的影响;Western印迹法检测穿琥宁和血红素加氧酶-1(HO-1)特异性抑制剂锌原卟啉(ZnPP)对RAW264.7细胞HO-1、诱导型一氧化氮合酶(iNOS)和环氧化酶2(COX-2)表达的影响;Griess反应和ELISA法测定穿琥宁对RAW264.7细胞一氧化氮(NO)和前列腺素E2(PGE2)生成的影响。结果穿琥宁3～100μmol·L-1作用24 h对RAW264.7细胞无细胞毒性。穿琥宁不能抑制由IL-1β诱导的NF-κBP65核转位和LPS诱导的MAPK-APk2核转位,对TNF-α诱导的NF-κBP65核转位有较弱的抑制作用,抑制率为20%,无明显浓度效应关系。穿琥宁3,10,30和100μmol·L-1作用4 h能诱导RAW264.7细胞HO-1表达,穿琥宁100μmol· L-1作用6 h显著抑制IL-1β诱导的COX-2表达和PGE2产生,作用12 h抑制iNOS表达和NO产生。HO-1活性抑制剂ZnPP能部分逆转穿琥宁的上述作用。结论穿琥宁的抗炎作用可能主要通过HO-1信号转导,继而抑制iNOS和COX-2的表达及NO和PGE2的产生。对NF-κB信号传导系统的调节作用尚不清楚。     

Extract from Nandina domestica inhibits lipopolysaccharide-induced cyclooxygenase-2 expression in human pulmonary epithelial A549 cells

Extract from fruits of Nandina domestica THUNBERG (NDE) has been used to improve cough and breathing difficulty in Japan for many years. To explore whether NDE may alleviate respiratory inflammation, we investigated its effect on expression of cyclooxygenase-2 (COX-2) and production of prostaglandin E? (PGE?) in human pulmonary epithelial A549 cells in culture. Treatment with lipopolysaccharide (LPS; 6?μg/mL) resulted in an increase of COX-2 expression and PGE? production in A549 cells. Both the LPS-induced COX-2 expression and PGE? production were significantly inhibited by NDE (1-10?μg/mL) in a concentration-dependent manner. NDE did not affect COX-1 expression nor COX activity. These results suggest that NDE downregulates LPS-induced COX-2 expression and inhibits PGE? production in pulmonary epithelial cells. Furthermore, higenamine and nantenine, two major constituents responsible for tracheal relaxing effect of NDE, did not mimic the inhibitory effect of NDE on LPS-induced COX-2 expression in A549 cells. To identify active constituent(s) of NDE responsible for the anti-inflammatory effect, NDE was introduced in a polyaromatic absorbent resin column and stepwise eluted to yield water fraction, 20% methanol fraction, 40% methanol fraction, 99.8% methanol fraction, and 99.5% acetone fraction. However, none of these five fractions alone inhibited LPS-induced COX-2 expression. On the other hand, exclusion of water fraction from NDE abolished the inhibitory effect of NDE on LPS-induced COX-2 expression. These results suggest that constituent(s) present in water fraction is required but not sufficient for the anti-inflammatory activity of NDE, which may result from interactions among multiple constituents.     

Inhibition of LPS-induced NO and PGE2 production by asiatic acid via NF-kappa B inactivation in RAW 264.7 macrophages: possible involvement of the IKK and MAPK pathways

In the present study, we investigated the effect of asiatic acid (the aglycon of asiaticoside) and asiaticoside isolated from the leaves of Centella asiatica (Umbelliferae) on LPS-induced NO and PGE(2) production in RAW 264.7 macrophage cells. Asiatic acid more potently inhibited LPS-induced NO and PGE(2) production than asiaticoside. Consistent with these observations, the protein and mRNA expression levels of inducible iNOS and COX-2 enzymes were inhibited by asiatic acid in a concentration-dependent manner. In addition, asiatic acid dose-dependently reduced the production of IL-6, IL-1 beta and TNF-alpha in LPS-stimulated RAW 264.7 macrophage cells. Furthermore, asiatic acid inhibited the NF-kappaB activation induced by LPS, and this was associated with the abrogation of I kappa B-alpha degradation and with subsequent decreases in nuclear p65 and p50 protein levels. Moreover, the phosphorylations of IKK, p38, ERK1/2, and JNK in LPS-stimulated RAW 264.7 cells were suppressed by asiatic acid in a dose-dependent manner. These results suggest that the anti-inflammatory properties of asiatic acid might be the results from the inhibition of iNOS, COX-2, IL-6, IL-1 beta, and TNF-alpha expressions through the down-regulation of NF-kappaB activation via suppression of IKK and MAP kinase (p38, ERK1/2, and JNK) phosphorylation in RAW 264.7 cells.     

Ethyl acetate extract from Angelica Dahuricae Radix inhibits lipopolysaccharide-induced production of nitric oxide, prostaglandin E2 and tumor necrosis factor-alphavia mitogen-activated protein kinases and nuclear factor-kappaB in macrophages.

Angelica dahurica (Umbelliferae) has been used to treat headache of common cold, supraorbital neuralgia, painful swelling on the body, nasal stuffiness, leukorrhea and arthralgia due to wind-dampness in Korean traditional medicine. It is also claimed to be effective in the treatment of acne, erythema, headache, toothache, sinusitis, colds and flu. The present study focused whether the ethyl acetate extract from Angelica Dahuricae Radix (EAAD) inhibits production of nitric oxide (NO), prostaglandin E(2) (PGE(2)) and tumor necrosis factor (TNF)-alpha, as well as expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor-kappaB (NF-kappaB) and mitogen-activated protein kinases (MAPKs) in lipopolysaccharide (LPS)-stimulated macrophages. EAAD inhibited LPS-induced NO, PGE(2) and TNF-alpha production as well as expression of iNOS and COX-2 in RAW 264.7 cells. EAAD inhibited LPS-induced TNF-alpha production in THP-1 cells. Furthermore, EAAD suppressed LPS-induced phosphorylation of p38 MAPK and extracellular-signal regulated kinases 1/2 (ERK1/2), I-kappaBalpha degradation, and NF-kappaB activation in RAW 264.7 cells. These results suggest that EAAD has the inhibitory effects on LPS-induced TNF-alpha, NO and PGE(2) production, and expression of iNOS and COX-2 in macrophage through blockade in the phosphorylation of MAPKs, following I-kappaBalpha degradation and NF-kappaB activation.     

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.     

In vitro modulation of LPS/calcimycin induced inflammatory and allergic mediators by pure compounds of Andrographis paniculata (King of bitters) extract

The aim of the current study is to probe the anti-inflammatory/anti-allergic potential of seven phytoconstituents (andrographolide, neoandrographolide, isoandrographolide, andrograpanin, 14-deoxy-11,12-didehydroandrographolide, 7-O-methylwogonin and skullcapflavone-I) isolated from Andrographis paniculata (King of bitters) on the production of key inflammatory/allergic mediators (NO, PGE(2), IL-1 beta, IL-6, LTB(4), TXB(2) and histamine). The results demonstrated that andrographolide, isoandrographolide, 7-O-methylwogonin and skullcapflavone-I significantly inhibited LPS stimulated NO and PGE(2) release in J774A.1 macrophages. Andrographolide, isoandrographolide and 7-O-methylwogonin showed considerable inhibition of IL-1 beta production in LPS elicited macrophages. LPS induced IL-6 production was significantly inhibited by andrographolide, isoandrographolide and skullcapflavone-I in a concentration dependent manner. The results revealed that andrographolide, isoandrographolide and skullcapflavone-I significantly decreased TXB(2) release in A23187 activated HL-60 promyelocytic cells. Furthermore, the anti-allergic properties of the phytoconstituents was investigated on A23187 induced LTB(4) production (HL-60 cells) and histamine release (RBL-2H3 basophilic cells). The results showed that only skullcapflavone-I and 7-O-methylwogonin showed marked inhibitory effect on LTB(4) production, however, only 7-O-methylwogonin exerted dose-dependent inhibition towards histamine release. Therefore, this study indicates that some of these phytoconstituents exhibit potent anti-inflammatory/anti-allergic effects by modulating different inflammatory/allergic mediators. Hence, these phytoconstituents might provide useful phytomedical treatment against variety of inflammatory and allergic disorders.     

Effects of flavonoids on prostaglandin E2 production and on COX-2 and mPGES-1 expressions in activated macrophages

Prostaglandin E2 (PGE2) has a central role in inflammation and both cyclooxygenase-2 (COX-2) and prostaglandin E synthases are critical enzymes in its synthesis. In inflammation, bacterial products and cytokines enhance the expression of COX-2 and inducible microsomal prostaglandin E synthase-1 (mPGES-1) which are functionally coupled to result in increased PGE2 formation in macrophages and tissue cells. In the present study, we systematically investigated the effects of 26 naturally occurring flavonoids on PGE2 production and on COX-2 and mPGES-1 expression in activated macrophages. Twelve flavonoids, i.e., flavone, luteolin-7-glucoside, kaempferol, isorhamnetin, morin, quercetin, naringenin, taxifolin, pelargonidin, daidzein, genistein, and genistin effectively inhibited lipopolysaccharide (LPS)-induced PGE2 production. Four flavonoids (flavone, isorhamnetin, daidzein, and genistein) inhibited significantly LPS-induced COX-2 expression, while mPGES-1 expression was downregulated by kaempferol and isorhamnetin. The present study characterizes the effects of flavonoids on PGE2 production and on COX-2 and mPGES-1 expression in activated macrophages. The results add to our knowledge of the anti-inflammatory actions of flavonoids and introduce kaempferol and isorhamnetin as compounds capable of downregulating the expression of mPGES-1.     

JNK inhibitor SP600125 reduces COX-2 expression by attenuating mRNA in activated murine J774 macrophages

Inducible prostaglandin synthase (cyclooxygenase-2, COX-2) is highly expressed in inflammation. The signaling mechanisms involved in the up-regulation of COX-2 are not known in detail. In the present study we investigated the role of c-Jun NH2-terminal kinase (JNK), a member of the mitogen-activated protein kinase (MAPK) family in COX-2 expression and prostaglandin (PG) E2 production in murine J774 macrophages activated by bacterial lipopolysaccharide (LPS). LPS caused a transient activation of JNK which was followed by increased COX-2 expression. Anthra(1,9-cd)pyrazol-6(2H)-one (SP600125), an inhibitor of JNK, inhibited phosphorylation of c-Jun with an IC50 of 5-10 microM. At the same concentrations SP600125 suppressed also LPS-induced COX-2 protein levels and PGE2 production. SP600125 did not alter LPS-induced COX-2 mRNA levels when measured 3 h after addition of LPS, whereas mRNA levels were significantly reduced in SP600125-treated cells when measured 24 h after addition of LPS. LPS-induced COX-2 mRNA levels reduced faster in cells treated with SP600125 than in control cells. Cycloheximide (that is known to activate JNK) enhanced COX-2 expression and its effect was inhibited by SP600125. The present results suggest that JNK pathway is involved in the up-regulation of COX-2 expression possibly by a mechanism related to the stability of COX-2 mRNA.