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。     

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。     

知母皂苷通过调节NF-κB-iNOS-NO信号通路抑制LPS诱导的RAW264.7细胞功能

目的研究知母皂苷(SAaB)对脂多糖(LPS)诱导的RAW264.7细胞功能的影响,以及对NF-κB-诱导型一氧化氮合酶(iNOS)-NO信号通路的调节。方法以LPS刺激RAW264.7细胞构建体外炎症细胞模型,采用Griess法和ELISA法分别检测SAaB干预下,RAW264.7炎症细胞中的NO、iNOS、肿瘤坏死因子α(TNF-α)和白介素-6(IL-6)的含量;Western blot检测RAW264.7细胞NF-κB p65蛋白的表达水平。结果 RAW264.7细胞在LPS(10 mg·L~(-1))诱导下,NO、iNOS、TNF-α、IL-6的含量以及NF-κB p65蛋白表达水平与正常对照组比较均明显增高。SAaB(0.3、3、30 mg·L~(-1))可明显降低RAW264.7炎症细胞中NO、iNOS、TNF-α和IL-6的含量(P<0.01),且明显下调NF-κB p65的蛋白表达水平(P<0.01)。结论 SAaB可通过调节NF-κB-iNOS-NO信号通路,抑制LPS诱导的RAW264.7细胞功能。     

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。     

白木香叶提取物对LPS诱导RAW264.7巨噬细胞炎症因子的影响

目的 采用LPS刺激巨噬细胞RAW264.7,建立体外的炎症模型,探讨白木香叶提取物抗炎活性和作用机制.方法 采用MTT法检测白木香叶提取物( ASPE)对RAW264.7细胞的毒性作用;采用ELISA法检测ASPE对IL-6表达情况;采用Western blotting检测iNOS和COX-2蛋白表达情况,结果 ASPE能抑制LPS刺激RAW264.7所产生的炎症反应,其作用机制可能与其抑制iNOS和COX-2蛋白表达有关.结论 白木香叶提取物有抗炎活性.     

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.     

中介素通过激活AMPK信号通路对脂多糖诱导巨噬细胞极化的影响

目的探讨中介素(intermedin,IMD)对脂多糖(lipopolysaccharide,LPS)诱导小鼠单核巨噬细胞系RAW 264.7极化的影响及其作用机制。方法RAW 264.7细胞随机分为对照组、LPS组、LPS+IMD组、LPS+IMD+CC(AMPK抑制剂Compound C)组。Real time-PCR法检测TNF-α、CD86、iNOS、Arg^-1、CD206 mRNA表达,Western blot法检测p-AMPK、AMPK、TNF-α、IL-6和IL^-10蛋白表达,流式细胞术检测巨噬细胞亚型,ELISA法检测培养基上清IL-6和TNF-α浓度。结果与对照组及LPS组比较,IMD处理可增加AMPK磷酸化水平,增加p-AMPK/AMPK比值;与对照组相比,LPS诱导可导致巨噬细胞发生M1极化,M1型标志分子CD86、TNF-α及iNOS mRNA表达升高,M2型标志分子CD206、Arg^-1 mRNA表达降低,上调促炎因子TNF-α、IL-6表达,降低抑炎因子IL^-10表达,使M1型细胞数量增加,细胞上清中TNF-α、IL-6分泌增加;而IMD处理可抑制LPS诱导的M1极化,AMPK抑制剂Compound C组处理可在一定程度上拮抗这一作用。结论IMD通过激活AMPK信号通路抑制LPS诱导的巨噬细胞M1型极化。     

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。     

铁皮石斛多糖对RAW264.7细胞分泌TNF-α的影响

目的研究铁皮石斛多糖(polysaccharides from Den-drobium officinale,DOP)对小鼠巨噬细胞系RAW264.7细胞分泌TNF-α的影响并探讨其作用机制。方法 MTT法检测细胞活性;ELISA法检测TNF-α的分泌水平;反转录聚合酶链反应(RT-PCR)检测TNF-αmRNA表达量;Western blot技术检测胞质I-κBα蛋白的表达量。结果 DOP在20～160mg·L-1范围内明显促进RAW264.7细胞增殖,无细胞毒性;与空白对照组比较,DOP剂量依赖性地促进TNF-α的分泌;上调TNF-αmRNA表达;Western blot结果显示DOP能明显降低胞质内I-κBα蛋白水平。结论 DOP能增强RAW264.7细胞分泌TNF-α,其作用机制可能与降低胞质内I-κBα蛋白水平,活化核转录因子NF-κB,诱导TNF-αmRNA的表达有关。     

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。     

A limited series of synthetic tetrahydroisoquinoline alkaloids reduce inflammatory gene iNOS via inhibition of p-STAT-1 and suppress HMGB1 secretion in LPS-treated mice lung tissue

Tetrahydroisoquinoline alkaloids (THIs) have shown to increase survival and beneficial effect on animal model of sepsis, partly due to heme oxygenase-1 (HO-1) induction. Here, we aimed to compare a limited series of synthesized THIs on HO-1 induction and inhibitory effect of iNOS and COX-2 expression in lipopolysaccharide (LPS)-activated RAW264.7 cells. To the end, most promising compound (THI-61) was tested whether this compound reduces iNOS protein expression and inflammatory markers (HMGB1, TNF-α) in LPS-treated mice lung tissue. The results indicated that N-carbonyl substituted THI seem to affect HO-1 induction depending on which functional group is attached to C1 position. All compounds that reduce LPS-activated NF-κB-luciferase activity showed to preferential inhibition of iNOS/NO but not COX-2/PGE₂ that was partly related to inhibition of STAT-1 phosphorylation. In particular, THI-61 induced translocation of Nrf2 from cytosol into the nucleus by an increased Nrf2-ARE binding activity, and reduced IL-1β production in LPS-activated RAW264.7 cells. The reduced expression of iNOS/NO by THI-61 was reversed by siHO-1RNA-transfection. In LPS-treated mice, THI-61 significantly reduced iNOS protein in lung tissues, and HMGB1 and TNF-α levels in the BALF. We concluded that 1) lipophilic moiety of 1C substituent is much more important in N-carbonyl substituted THI for induction of HO-1, 2) newly synthesized THI-61 may be beneficial for treatment of lung injury.     

Cordycepin inhibits lipopolysaccharide-induced inflammation by the suppression of NF-kappaB through Akt and p38 inhibition in RAW 264.7 macrophage cells

Cordyceps militaris, a caterpillar-grown traditional medicinal mushroom, produces an important bioactive compound, cordycepin (3'-deoxyadenosine). Cordycepin is reported to possess many pharmacological activities including immunological stimulating, anti-cancer, anti-virus and anti-infection activities. The molecular mechanisms of cordycepin on pharmacological and biochemical actions of macrophages in inflammation have not been clearly elucidated yet. In the present study, we tested the role of cordycepin on the anti-inflammation cascades in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. In LPS-activated macrophage, nitric oxide (NO) production was inhibited by butanol fraction of C. militaris and the major component of C. militaris butanol faction was identified as cordycepin by high performance liquid chromatography. To investigate the mechanism by which cordycepin inhibits NO production and inducible nitric oxide synthase (iNOS) expression, we examined the activation of Akt and MAP kinases in LPS-activated macrophage. Cordycepin markedly inhibited the phosphorylation of Akt and p38 in dose-dependent manners in LPS-activated macrophage. Moreover, cordycepin suppressed tumor necrosis factor (TNF-alpha) expression, IkappaB alpha phosphorylation, and translocation of nuclear factor-kappaB (NF-kappaB). The expressions of cycloxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were significantly decreased in RAW 264.7 cell by cordycepin. Taken together, these results suggest that cordycepin inhibits the production of NO production by down-regulation of iNOS and COX-2 gene expression via the suppression of NF-kappaB activation, Akt and p38 phosphorylation. Thus, cordycepin may provide a potential therapeutic approach for inflammation-associated disorders.     

Inducible nitric oxide synthase inhibitors of Chinese herbs III. Rheum palmatum

In this paper, the effects of bioactive compounds of Rheum palmatum L. on the inhibition of NO production from RAW 264.7 cells were explored. Seven main anthraquinone derivatives were isolated from the root of R. palmatum, and of these, emodin and rhein significantly inhibited nitrite production from lipopolysaccharide (LPS)-activated RAW 264.7 cells. The IC(50) values for inhibition of nitrite production by emodin and rhein were 60.7 and 67.3 microM, respectively. After iNOS enzyme activity was stimulated by LPS for 12 h, treatment with emodin or rhein at 20 microg/ml for 18 h did not significantly inhibit NO production. The data show that the inhibitory activity of emodin and rhein is not due to direct inhibition of iNOS enzyme activity. However, expression of iNOS and the COX-2 protein was inhibited by emodin in LPS-activated RAW 264.7 cells, and PGE(2) production was reduced. Rhein also inhibited LPS-induced iNOS protein expression, but not COX-2 or PGE(2) production. On the other hand, inhibition effects on NO production from RAW 264.7 cells were enhanced and cytotoxic effects decreased by co-treatment with emodin and rhein. In conclusion, emodin and rhein are major iNOS inhibitors of R. palmatum and may possibly serve as bioactive substances for anti-inflammation effects.     

人参总皂苷在巨噬细胞RAW264.7中降低脂多糖诱导的炎症和氧化应激

目的: 通过脂多糖诱导巨噬细胞RAW264.7建立炎症和氧化应激模型,探讨人参总皂苷的抗炎和抗氧化效果以及对脂多糖诱导的巨噬细胞RAW264.7自噬水平的影响。方法: 培养RAW264.7细胞,给予脂多糖刺激并加入不同浓度人参总皂苷,使用硝酸还原酶法检测细胞内一氧化氮水平;ELISA法检测小鼠肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)的分泌;DCFH-DA荧光探针法检测人参总皂苷处理后活性氧的变化;超氧化物阴离子荧光探针(dihydroethidium,DHE)检测细胞内超氧化物阴离子水平;吖啶橙染色法检测自噬体的形成;Western Blot法检测COX-2、NF-κB、Nrf2、HO-1、GCLC、GCLM、Beclin1、ATG7和LC3的蛋白表达。结果: 与脂多糖模型组相比,随着TGS的浓度增加,细胞一氧化氮水平、TNF-α的分泌降低,细胞内活性氧和超氧阴离子的含量降低,巨噬细胞RAW264.7酸性自噬体的数量增加;Nrf2、HO-1、GCLC、GCLM、Beclin1、ATG7及LC3的蛋白表达提高,COX-2、NF-_KB的蛋白表达降低。结论: 在RAW264.7巨噬细胞中,人参总皂苷可通过NF-κB、Nrf2/ARE通路以及提高自噬水平来降低脂多糖引起的炎症反应和氧化损伤。     

3‑Bromo‑5‑(ethoxymethyl)‑1,2‑benzenediol inhibits LPS-induced pro-inflammatory responses by preventing ROS production and downregulating NF-κB in vitro and in a zebrafish model

The anti-inflammatory effects of 3‑bromo‑5‑(ethoxymethyl)‑1,2‑benzenediol (BEMB) from Polysiphonia morrowii were evaluated in lipopolysaccharide (LPS)-induced RAW264.7 cells and zebrafish embryo. BEMB showed anti-inflammatory effects by inhibiting the production of nitric oxide (NO) and reactive oxygen species (ROS), and the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in the LPS-activated RAW264.7 cells and zebrafish embryo without cytotoxicity. Moreover, BEMB suppressed the protein and mRNA expression levels of nuclear factor (NF)-κB (p65 and inhibitor of NF-κB [IκB]-A) in RAW264.7 cells and zebrafish embryo, respectively. Collectively, the results of this study indicate that BEMB suppressed the production of pro-inflammatory mediators such as NO, iNOS, and COX-2 as well as their regulation in LPS-induced RAW264.7 cells and zebrafish embryos by inhibiting ROS production and NF-κB expression. Therefore, this study suggests that BEMB could be a potential anti-inflammatory agent for the treatment of inflammatory diseases.     

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.     

豆豉姜活性成分9,9’-O-二-(E)-阿魏酰基-内消旋-5,5’-二甲氧基开环异落叶松树脂酚(LCA)抗炎作用研究

目的对傣药豆豉姜活性成分LCA在动物及细胞水平上的抗炎作用进行评价。方法采用耳肿胀和棉球肉芽肿模型进行LCA抗炎的体内药效学评价。采用脂多糖(LPS)诱导的小鼠单核巨噬细胞(RAW264.7),通过检测其一氧化氮(NO)、肿瘤坏死因子-α(TNF-α)的分泌量以及诱导型一氧化氮合酶(iNOS)、环氧合酶-2(COX-2)蛋白的表达水平,以评价LCA的体外抗炎活性。结果在小鼠耳肿胀的实验中,LCA组的耳肿胀度明显低于模型组,且高浓度组对小鼠耳肿胀的抑制率高于阳性药组;在大鼠棉球肉芽肿实验中,LCA高剂量组显著减轻了棉球肉芽肿的湿重与干重。此外,LCA显著减少LPS诱导的RAW264.7细胞中NO、TNF-α的分泌,降低iNOS、COX-2蛋白的表达。结论LCA具有显著的体内外抗炎作用,值得进一步深入研究。