苜草素对巨噬细胞吞噬活性和NO、IL-6、TNF-α分泌的影响

目的研究苜草素对小鼠巨噬细胞RAW264.7吞噬活性和NO、IL-6、TNF-α的影响,探讨苜草素对巨噬细胞免疫功能的影响机制。方法以不同浓度的苜草素和10 g/ml的脂多糖(LPS)分别处理巨噬细胞RAW264.7,采用中性红法测定巨噬细胞的吞噬活性,ELISA检测TNF-α分泌量。以不同浓度的苜草素处理LPS诱导的巨噬细胞RAW264.7,采用Griess分析法测定NO含量,ELISA检测IL-6分泌量,半定量RT-PCR方法测定巨噬细胞中iNOS、IL-6、TNF-αmRNA水平。结果苜草素在0.4、0.6、0.8和1.0 mg/ml时能显著提高巨噬细胞RAW264.7的吞噬活性(P<0.05);在0.6、0.8和1.0 mg/ml时显著增加TNF-α的分泌量(P<0.01),显著降低LPS诱导的巨噬细胞NO产生量(P<0.05);在0.2、0.4、0.6、0.8和1.0 mg/ml时显著降低LPS诱导的巨噬细胞IL-6的产生量(P<0.01)。苜草素可抑制LPS诱导的iNOS mRNA和IL-6 mRNA的表达,增加TNF-αmRNA的表达。结论苜草素可以提高巨噬细胞RAW264.7的吞噬活性,在转录水平上影响小鼠巨噬细胞iNOS、IL-6、TNF-α的基因表达,对巨噬细胞免疫功能起调节作用。     

高温及内毒素复合因素对IL-1 βmRNA表达影响

目的 探讨高温和内毒素(LPS)复合因素对RAW 264.7细胞IL-1β mRNA表达的影响.方法 采用体外培养的巨噬细胞系RAW 264.7细胞,分为37,40℃对照组,37,40℃LPS组.用RT-PCR方法检测RAW 264.7细胞IL-1 βmRNA表达.结果 RAW264.7细胞在37和40℃加或不加LPS条件下随着时间的变化IL-1 βmRNA表达各不相同,在37℃条件下,LPS组随着时间的不断延长,IL-1 βmRNA表达不断增强;40 ℃条件下对照组IL-1βmRNA表达亦表现出不断增强,而LPS组IL-1 βmRNA表达表现为先增强后减弱.结论 高温和内毒素复合因素能够下调RAW 264.7细胞IL-1 βmRNA表达,考虑其原因与高温和内毒素复合因素诱导产生的热休克蛋白及该条件下RAW 264.7细胞的大量凋亡有关.     

巨噬细胞IL-1β mRNA在高温和内毒素复合影响下的表达

目的 研究高温和内毒素(LPS)双重因素对巨噬细胞IL-1β mRNA表达的影响.方法 采用体外培养的巨噬细胞系RAW264.7细胞,分为常温(37℃)对照组,高温(40℃)对照组,37℃ LPS组,38℃ LPS组,39℃ LPS组和40℃ LPS组.在含95%O2和5%CO2条件下培养1、2、3 h后,TRIZOL提取细胞总RNA,核酸蛋白分析仪检测RNA的浓度和纯度,琼脂糖凝胶电泳分析RNA的完整性,RT-PCR方法检测巨噬细胞IL-1β mRNA的表达.结果 在提取的总RNA无降解、无蛋白质污染的前提下,温度升高和内毒素都能引起IL-1β mRNA表达上调,内毒素在38℃时对IL-1β mRNA表达的上调作用最大,但高温和内毒素复合影响可下调RAW264.7细胞IL-1β mRNA表达.结论 单纯的高温或内毒素均能上调RAW264.7细胞IL-1β mRNA表达,但高温复合内毒素却下调IL-1β mRNA表达.     

染料木黄酮对脂多糖诱导的RAW264.7细胞炎症因子、腺苷酸激活蛋白激酶磷酸化的影响

目的研究染料木黄酮(genistein,Gen)对脂多糖(LPS)诱导的巨噬细胞炎症因子产生和腺苷酸激活蛋白激酶(AMPK)磷酸化的影响。方法体外培养RAW 264.7小鼠单核巨噬细胞,加入1、5、10、50、100 mol/L的Gen共同培养,MTT法检测其对细胞活性的影响。将RAW264.7细胞随机分为4组:空白对照组不加任何药物,模型组加入终浓度为1 g/ml的LPS进行刺激,Gen高、低剂量组分别加入终浓度为10、5 mol/L的Gen预处理1h后,再加入终浓度为1 g/ml的LPS刺激,24h后收取细胞上清用酶联免疫(ELISA)方法检测TNF-α、IL-6含量,Westernblot检测AMPKα蛋白及其磷酸化的表达水平。结果 100 mol/L的Gen对体外培养RAW264.7细胞的增殖有影响,而其他浓度则无。LPS处理的RAW 264.7细胞与对照组比较,TNF-α、IL-6生成显著增多(P<0.01)、AMPKα磷酸化水平显著降低(P<0.01),而AMPKα总蛋白表达水平无差异(P>0.05)。Gen干预可减少LPS诱导的TNF-α、IL-6生成,上调AMPKα磷酸化水平的表达,与LPS组相比,差异均有统计学意义(P<0.01)。结论 Gen能抑制LPS诱导巨噬细胞的炎症反应,减少TNF-α、IL-6生成,这可能与Gen促进AMPKα磷酸化,从而激活AMPKα有关。     

氨基胍对内毒素活化小鼠巨噬细胞L-arginine转运及CAT-2表达影响的实验研究

目的:通过氨基胍(AG)干预内毒素活化小鼠巨噬细胞RAW264.7模型,观察NO的产生、左旋精氨酸(L-Arg)转运以及碱性氨基酸转运体-2(CAT-2)mRNA的表达,旨在研究氨基胍对L-Arg跨膜转运及CAT-2表达的影响.方法:实验分为四组,即对照组,LPS对照组,AG组,AG+LPS组.接种RAW264.7细胞...     

亚甲蓝对LPS诱导巨噬细胞炎性反应的影响

目的观察亚甲蓝对脂多糖(LPS)诱导小鼠RAW246.7巨噬细胞炎性反应因子表达和细胞NF-κB活性的影响,以初步探讨亚甲蓝在LPS诱导巨噬细胞炎性反应中的作用。方法体外培养小鼠巨噬细胞系RAW264.7细胞,利用脂多糖(LPS)刺激RAW264.7细胞建立体外内毒素炎性反应模型。进行实验分组:Ⅰ组(对照组):完全无血清培养基孵育;Ⅱ组(模型组):在无血清培养基中加入浓度为1 g/m L的LPS进行孵育;Ⅲ组(亚甲蓝组):完全无血清培养基中加入亚甲蓝20μmol/L,2 h后加入1 g/m L的LPS孵育,细胞培养24 h后:1实时荧光定量聚合酶链反应(real-time PCR)观察IL-6、IL-8、MCP-1 m RNA的表达;2酶联免疫吸附法(ELISA)检测细胞培养上清液中IL-6、IL-8、MCP-1蛋白水平;3荧光素酶报告实验(luciferase activity assay)检测细胞中NF-κB的活性。结果LPS刺激巨噬细胞后,细胞中炎性反应因子m RNA和蛋白表达增高,而亚甲蓝预处理能够减弱LPS对炎性反应因子的诱导作用;LPS可以诱导巨噬细胞NF-κB活化,亚甲蓝能够抑制NF-κB的激活。结论亚甲蓝可以通过抑制NF-κB的激活调控LPS诱导的炎性反应。     

毛蕊异黄酮对脂多糖和γ干扰素诱导的RAW264.7细胞产生一氧化氮的影响

目的探讨毛蕊异黄酮对脂多糖(LPS)和γ干扰素(INF-γ)诱导的RAW264.7分泌一氧化氮(NO)的影响。方法LPS和INF-γ共同刺激生长良好的RAW264.7细胞,并用不同浓度(25、50、100μmol/L)毛蕊异黄酮进行干预;MTT法检测毛蕊异黄酮对RAW264.7细胞的毒性作用;硝酸还原酶法检测细胞上清液中NO含量;实时定量PCR法检测细胞中诱生型一氧化氮合酶(iNOS)mRNA的表达水平。结果 125、50、100μmol/L浓度的毛蕊异黄酮对RAW264.7细胞无毒性作用(P0.05);2LPS和INF-γ共同刺激RAW264.7细胞后,细胞上清液中NO的含量明显增加(P0.01);毛蕊异黄酮进行干预后,NO的含量明显下降,并呈现出浓度依赖性(P0.01);3用LPS和INF-γ共同刺激RAW264.7细胞后,细胞中iNOS mRNA的表达水平显著升高(P0.01);毛蕊异黄酮进行干预后,iNOS mRNA表达水平显著降低,并呈现出浓度依赖性(P0.01)。结论毛蕊异黄酮能抑制LPS和INF-γ诱导的RAW264.7细胞产生NO,其抗肿瘤作用可能与此有关。     

辐射诱导的小鼠肺泡上皮细胞死亡通过干扰素基因刺激因子通路促进RAW细胞M1型极化研究

目的:探讨细胞中重要的DNA感受器干扰素基因刺激因子(STING)在小鼠单核巨噬细胞(RAW264.7)细胞活化中的作用。方法:收集⁶⁰Co γ射线照射后小鼠肺泡上皮细胞(MLE-12)的培养上清,对RAW264.7细胞进行刺激,使用蛋白免疫印迹(Western Blot)检测刺激后RAW264.7细胞中STING蛋白的表达,使用酶联免疫吸附测定(ELISA)法检测处理后RAW264.7细胞经典激活的巨噬细胞(M1)型和非经典激活的巨噬细胞(M2)型相关细胞因子的表达水平。结果:Western Blot检测结果显示,刺激后RAW264.7细胞中STING蛋白的表达水平显著上调;ELISA检测结果表明,刺激后RAW264.7细胞上清中的肿瘤坏死因子α(TNF-α)和白细胞介素-1(IL-1)显著增加;脱氧核糖核酸酶I(DNase I)处理能够抑制RAW264.7细胞中STING通路的激活。ELISA检测结果表明,DNase I能够抑制RAW264.7细胞向M1型极化。结论:辐射诱导的MLE-12细胞死亡能够释放DNA,从而激活RAW264.7细胞中的环磷酸鸟苷-腺苷...     

白藜芦醇对LPS刺激RAW264.7细胞炎症因子影响研究

[目的]观察白藜芦醇对脂多糖刺激小鼠RAW264.7细胞炎症因子的影响.[方法]体外培养小鼠RAW264.7细胞,脂多糖刺激小鼠RAW264.7细胞分泌细胞因子,用不同浓度白藜芦醇进行干预,ELISA法检测培养上清液中IL-10和PGE2含量.[结果]白藜芦醇可抑制脂多糖刺激小鼠RAW264.7细胞释放PGE2,促进抗炎因子IL-10表达,并呈一定量效关系,其影响细胞因子释放作用与细胞毒作用无关.[结论]自藜芦醇呈浓度依赖性地抑制脂多糖刺激小鼠RAW264.7细胞释放PGE2,促进IL-10表达.     

不同照射方式对IL-4诱导的小鼠RAW264.7细胞极化的影响

目的探讨不同照射方式对白细胞介素4(IL-4)诱导的RAW264.7细胞向M2型巨噬细胞极化的影响。方法以IL-4刺激RAW264.7细胞诱导其向M2型极化,在IL-4诱导细胞前、后分别进行不同剂量的照射,采用Westernblot检测不同刺激条件下RAW264.7细胞YM-1和Arg-1的表达水平,采用流式细胞术(FCM)检测不同刺激条件下RAW264.7细胞表面CD206和CD209的表达。结果不同的照射方式均上调IL-4诱导的RAW264.7细胞YM-1和Arg-1的蛋白表达水平,在IL-4诱导细胞后进行照射比在IL-4诱导细胞前进行照射显著上调细胞YM-1和Arg-1的蛋白表达;照射能抑制IL-4诱导下细胞表面CD206和CD209的表达。结论不同的照射方式促进IL-4诱导的RAW264.7细胞向M2型巨噬细胞极化,但抑制RAW264.7细胞的功能分子CD206和CD209的表达。     

Mume Fructus water extract inhibits pro-inflammatory mediators in lipopolysaccharide-stimulated macrophages

Mume Fructus (Family Rosaceae) is used as a traditional drug and health food in Asian countries. However, its therapeutic mechanisms and effects on macrophage-mediated inflammation remain unknown. In this study we examined the effect of Mume Fructus water extract (MFWE) on pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. The investigation focused on whether MFWE inhibited nitric oxide (NO) and prostaglandin (PG) E2 productions, as well as the expressions of inducible NO synthase (iNOS), cyclooxygenase (COX)-2, interleukin (IL)-6, nuclear factor-kappaB (NF-kappaB), and mitogen-activated protein kinases (MAPKs) in LPS-stimulated RAW 264.7 cells. We found that MFWE inhibited LPS-induced NO, PGE(2), and IL-6 productions as well as the expressions of iNOS and COX-2. Furthermore, MFWE suppressed the LPS-induced phosphorylations of p38 MAPK and extracellular signal-regulated kinase MAPK, as well as IkappaBalpha degradation and NF-kappaB activation. These results suggest that MFWE has inhibitory effects on LPS-induced PGE2, NO, and IL-6 production, as well as the expressions of iNOS and COX-2 in the murine macrophage. These inhibitory effects occur through blockades on the phosphorylation of MAPKs following IkappaBalpha degradation and NF-kappaB activation.     

介孔纳米SiO2致小鼠巨噬细胞株RAW264.7细胞毒性与氧化损伤

[目的]探讨介孔纳米SiO2对小鼠巨噬细胞株RAW264.7细胞的毒性和氧化损伤作用。[方法]设4个浓度组（2.5、10、50、100I.tg／mL）和生理盐水对照组,染毒24h后,用四甲基偶氮唑盐（MTT）方法观察细胞毒性,并根据染毒后细胞的总蛋白（TP）、乳酸脱氢酶（LDH）、一氧化氮（NO）、谷胱甘肽（GSH）、超氧化物歧化酶（SOD）和丙二醛（MDA）的变化情况分析介孔纳米SiO2的细胞毒性和氧化损伤作用。[结果]10、50和100μg／mL浓度组细胞存活率随着染毒浓度的升高而降低,差异有统计学意义;50、100μg／mL浓度组细胞及其上清液中TP、LDH、NO和MDA含量随染毒浓度增加而升高,而GSH和SOD含量降低;而且与细胞发生融解破碎、间隙加大等形态学改变情况相符。[结论]介孔纳米SiO2对体外培养巨噬细胞株RAW264.7具有细胞毒性和氧化损伤作用,且随剂量的加大而增强。     

Synergistic anti-inflammatory activity of omega-3 lipid and rofecoxib pretreatment on macrophage proinflammatory cytokine production occurs via divergent NF-kappaB activation

Omega-3 lipid pretreatment significantly decreases TNF-alpha production in LPS-stimulated Mphis; however, this response is only a partial inhibition, suggesting that other nonsubstrate- (lipid) dependent mechanisms are involved. The cyclooxygenase (COX)-2 enzyme is principally responsible for lipid metabolism; thus, a selective COX-2 inhibitor (Rofecoxib) would clarify if it is an omega-3 lipid direct effect or a COX-2 enzyme-associated modulated reduction in TNF-alpha. Moreover, potential synergy between omega-3 lipids and selective COX-2 inhibition is postulated. HYPOTHESIS: Through divergent regulatory mechanisms, omega-3 lipids in combination with Rofecoxib will synergistically decrease the LPS-stimulated Mphi inflammatory response. METHODS: RAW 264.7 cells were pretreated with omega-3 lipids, Rofecoxib, or combination treatment and then washed and exposed to LPS. Supernatants were collected for ELISA, total proteins were obtained to determine COX-2 protein expression by Western blot, and nuclear extracts were isolated to determine NF-kappaB activation by electromobility shift assay. RESULTS: TNF-alpha and PGE2 production was significantly decreased with omega-3 and Rofecoxib pretreatment, and with combination treatment a further decrease in TNF-alpha production was observed. COX-2 protein expression was demonstrated to increase in omega-3, Rofecoxib, and combination groups stimulated with LPS. No alteration in NF-kappaB activation was observed with Rofecoxib or combination pretreatment compared with LPS-stimulated control cells. Repletion of prostaglandin (PGE2) in the Mphi model significantly decreased TNF-alpha in all groups. CONCLUSIONS: Omega-3 lipids and Rofecoxib independently decrease TNF-alpha and PGE2 production in LPS-stimulated Mphi, yet in combination a synergistic reduction in TNF-alpha production is observed. Although the anti-inflammatory effects observed from omega-3 lipids are known to occur partially through decreasing NF-kappaB activation, we demonstrated that Rofecoxib or even a combination of omega-3 and Rofecoxib does not alter NF-kappaB activation, as seen with omega-3 lipids alone. These data support that combination treatment may result in decreased Mphi inflammation, yet this occurs via divergent mechanisms.     

Differential effects of organosulfur compounds from garlic oil on nitric oxide and prostaglandin E2 in stimulated macrophages

OBJECTIVE: We investigated the inhibition of nitric oxide (NO) and prostaglandin E2 (PGE2) production by the garlic oil derivatives, diallyl sulfide (DAS), diallyl disulfide (DADS), and allyl methyl sulfide (AMS), in lipopolysaccharide (LPS)-activated RAW 264.7 cells. METHODS: Cells were treated with LPS (330 ng/mL) and various concentrations of DAS, DADS, and AMS. NO and PGE2 released into the medium and expressions of inducible NO synthase and cyclooxygenase-2 protein were measured. RESULTS: All three compounds suppressed stimulated NO production, among which AMS exhibited the least inhibition. Western blot analysis showed that DAS and DADS, but not AMS, inhibited the corresponding inducible NO synthase expression. An in vitro study showed that all three compounds possess NO clearance activity, and that DADS and AMS were more effective than DAS. On the contrary, only DAS inhibited activated PGE2 production and cyclooxygenase-2 protein expression. CONCLUSIONS: The garlic derivatives, DAS, DADS, and AMS, differentially regulated the production of NO and PGE2 in stimulated macrophages. DAS decreased stimulated NO and PGE2 production by inhibiting inducible NO synthase and cyclooxygenase-2 expressions, and its enzyme inhibiting and NO clearance activity may also partly contribute to the suppression of NO. DADS inhibited activated NO production by decreasing inducible NO synthase expression and by directly clearing NO, whereas AMS suppressed NO mainly through its direct NO clearance activity. Further, neither DADS nor AMS showed any inhibitory effect on stimulated PGE2 production.     

Hexane fraction from Laminaria japonica exerts anti-inflammatory effects on lipopolysaccharide-stimulated RAW 264.7 macrophages via inhibiting NF-kappaB pathway

Purpose

Laminaria japonica is a representative marine brown alga used as a culinary item in East Asia. L. japonica extract was shown to exert various biological activities; however, its anti-inflammatory activity has not been reported. The aim of this study is to investigate the molecular mechanisms underlying its anti-inflammatory action.

Methods

Anti-inflammatory mechanisms of L. japonica n-hexane fraction (LHF) were assessed using lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. An anti-inflammatory compound isolated from LHF by reverse-phase chromatography was identified using nuclear magnetic resonance (NMR) spectroscopy.

Results

Our results indicate that LHF significantly inhibited LPS-stimulated nitric oxide (NO) and prostaglandin E₂ (PGE₂) secretion in a dose-dependent manner and suppressed the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) with no cytotoxicity. As results, levels of pro-inflammatory cytokines were significantly reduced by pretreatment of LHF in LPS-stimulated RAW 264.7 cells. Treatment of LHF strongly suppressed nuclear factor-κB (NF-κB) promoter-driven expression and nuclear translocation of NF-κB by preventing proteolytic degradation of inhibitor of κB (IκB)-α in LPS-stimulated RAW 264.7 cells. Moreover, LHF inhibited the phosphorylation of Akt and mitogen-activated protein kinase (MAPK) in LPS-stimulated RAW 264.7 cells. One of the anti-inflammatory compounds was isolated from LHF and identified as fucoxanthin.

Conclusions

These results indicate that the LHF-mediated inhibition of NO and PGE₂ secretion in LPS-stimulated macrophages is regulated by NF-κB inactivation through inhibition of IκB-α, MAPKs, and Akt phosphorylation. LHF may be considered as a functional food candidate for the prevention or treatment of inflammatory diseases.     

Luteolin and chrysin differentially inhibit cyclooxygenase-2 expression and scavenge reactive oxygen species but similarly inhibit prostaglandin-E2 formation in RAW 264.7 cells

Inflammation and oxidative stress are associated with cancer, atherosclerosis, and other chronic diseases. Dietary flavonoids have been reported to possess antiinflammatory and antioxidant properties, but their mechanisms of action and structure-activity relations have not been fully investigated. We hypothesized that differences in antioxidant activity between the structurally similar flavones, luteolin and chrysin (differing only in B-ring hydroxylation patterns), would differentially affect inflammation-associated Cox-2 expression and PGE2 formation. Pretreatment of RAW 264.7 macrophage-like cells with 25, 50, or 100 micromol/L concentrations of luteolin inhibited lipopolysaccharide (LPS)-induced Cox-2 protein expression (P < 0.0001). Chrysin pretreatment did not reduce LPS-induced Cox-2 protein expression at any level tested. Conversely, both luteolin and chrysin completely suppressed LPS-induced PGE2 formation (P < 0.001). Luteolin, but not chrysin, inhibited xanthine/xanthine oxidase-generated superoxide formation at 100 micromol/L in a cell-free system (P < 0.001). Although both luteolin and chrysin reduced LPS-induced hydroxyl radical formation relative to the positive control (P < 0.001), luteolin was superior to chrysin (P = 0.003). In summary, luteolin and chrysin suppressed PGE2 formation equally well, despite differential effects on Cox-2 protein expression and on superoxide and hydroxyl radical scavenging. These data indicate that flavones may display similar antiinflammatory activity via different mechanisms.     

肺炎衣原体感染对巨噬细胞内ox-LDL源性脂质蓄积的影响

目的 研究肺炎衣原体(Chlamydia pneumoniae,Cpn)感染对氧化低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)处理RAW264.7后细胞内脂质蓄积的影响.方法 Cpn在Hep-2细胞内增殖,用RAW264.7进行实验.Giemsa染色检测RAW264.7内Cpn感染情况,油红O染色检测细胞内脂质摄取情况,RT-PCR检测ABCA1mRNA表达情况.结果 油红O染色见RAW264.7内脂质摄取增加,RT-PCR检测ABCA1mRNA的表达增加.结论 Cpn感染促使RAW264.7内ox-LDL源性脂质蓄积增加,在此过程中ABCA1mRNA的表达增加,为进一步阐明肺炎衣原体感染和动脉粥样硬化关系提供了实验依据.