荔枝果肉乙酸乙酯提取物对巨噬细胞分泌促炎递质前列腺素E_2和一氧化氮的影响

目的研究荔枝果肉乙酸乙酯提取物对巨噬细胞J774分泌促炎递质前列腺素E(2PGE2)和一氧化氮(NO)的影响,初步探讨其诱导炎症性"上火"反应的机制。方法用不同浓度的荔枝乙酸乙酯提取物处理巨噬细胞J774,分别采用紫外分光光度法和Griess试剂法检测PGE2和NO含量;半定量RT-PCR检测环加氧酶(COX-2)和诱导型一氧化氮合酶(iNOS)的表达。结果与空白对照组相比,荔枝乙酸乙酯提取物在100-900μg/ml的浓度范围内可显著提高巨噬细胞J774上清PGE2、NO的含量(P<0.05),900μg/ml提取物能显著提高COX-2和iNOS的mRNA表达水平(P<0.05)。结论荔枝果肉乙酸乙酯提取物能够诱导巨噬细胞J774分泌促炎介质,从而引起炎症性"上火"反应。     

葫芦素B对脂多糖诱导小鼠肺泡巨噬细胞炎症反应的影响

目的了解葫芦素B(CuB)对脂多糖(LPS)诱导小鼠肺泡巨噬细胞炎症反应的影响及其分子机制。方法将小鼠肺泡巨噬细胞分为对照组、LPS组和LPS+不同浓度CuB组,对照组不作任何处理,LPS组用1μg/m L LPS诱导,另外3组加入不同浓度CuB使浓度分别为1、2.5和5μM,培养2 h后用1μg/m L LPS诱导。采用CCK-8法检测细胞活性,ELISA法检测细胞因子(TNF-α、IL-1β、IL-6、PGE2)水平,Griess Reagent法检测NO浓度,WB法检测i NOS、COX-2、Nrf2和HO-1的蛋白表达水平。结果分组培养24 h后各组细胞活性差异无统计学意义(P0.05),本实验浓度下的LPS和CuB对细胞无明显毒性。LPS组细胞上清液中细胞因子和NO水平均高于对照组(P0.05);经CuB处理的LPS组细胞上清液中炎症因子浓度较LPS组明显降低(均P0.05),并呈剂量依赖性。LPS刺激后,细胞中i NOS、COX-2、Nrf-2和HO-1表达量均增加,CuB可抑制i NOS和COX-2的表达,促进Nrf-2和HO-1的表达。结论 CuB能抑制LPS诱导后巨噬细胞炎症因子的释放,作用机制可能是抑制i NOS和COX-2表达,促进Nrf-2和HO-1表达。     

盐酸小檗碱对LPS诱导的小鼠RAW264.7细胞PGE_2、COX-2表达的影响

目的研究盐酸小檗碱对脂多糖(LPS)诱导的小鼠RAW264.7巨噬细胞生长增殖的影响,探讨盐酸小檗碱的抗炎作用机理。方法MTT法测定盐酸小檗碱对LPS诱导的小鼠RAW264.7巨噬细胞生长增殖的影响;酶联免疫法(ELA)检测盐酸小檗碱对PGE2生成的影响;逆转录聚合酶链反应(RT-PCR)检测COX-2 mRNA的表达;蛋白印迹法(Western Blotting)检测COX-2的蛋白表达。结果盐酸小檗碱对LPS诱导的小鼠RAW264.7巨噬细胞有生长增殖抑制作用;盐酸小檗碱抑制LPS诱导的小鼠RAW264.7巨噬细胞PGE2的生成;但是对COX-2 mRNA及COX-2蛋白表达均无明显影响。结论盐酸小檗碱对LPS诱导的小鼠RAW264.7巨噬细胞有生长增殖抑制作用,盐酸小檗碱不是通过抑制COX-2生成的途径来影响PGE2的生成的。     

花色苷对ApoE基因缺陷小鼠炎症信号转导的影响

目的:探讨黑米皮(BRF)花色苷(ANTH)对ApoE基因(ApoE-/-)缺陷小鼠动脉粥样硬化(AS)斑块形成及炎症信号转导的影响。方法:将45只雄性ApoE-/-小鼠随机分为三组:阳性对照组(A组)、花色苷提取后黑米皮组(B组)和黑米皮花色苷组(C组);15只正常小鼠为阴性对照组(D组)。B组和C组分别加入黑米皮花色苷提取物及5%花色苷提取后的黑米皮,饲养20w,取血后处死动物,测定主动脉脂质斑块大小和血液各型NOS水平及NO水平,Western-blot法检测血管壁内ICAM-1及NF-κB表达。结果:C组的主动脉脂质斑块面积大小明显低于A组和B组;C组总一氧化氮合酶(tNOS)水平明显高于A组和B组;C组血清中iNOS水平略有降低,但差异不显著;C组血清cNOS和NO水平显著升高;C组COX-2mRNA、ICAM-1及NF-κB蛋白质表达下降。结论:黑米皮花色苷是黑米皮抗AS的活性成分,其作用机制与抑制NF-κB介导的炎性因子iNOS、COX-2表达及促进血管舒张因子NO生成有关。     

花色苷通过腺苷一磷酸激活蛋白激酶减少肝脏HepG2细胞的脂肪积聚

目的探讨花色苷单体矢车菊素-3-葡萄糖苷(Cy-3-g)对HepG2细胞脂肪含量的影响以及可能机制。方法HepG2细胞分别用1、10、100μmol/LCy-3-g处理1h,设置对照,检测腺苷一磷酸激活蛋白激酶(AMPK)及其磷酸化水平、肉碱脂酰转移酶-I(CPT-I)、乙酰辅酶A羧化酶(ACC)磷酸化水平、AMPK活性、甘油三酯含量以及脂肪酸氧化水平。结果与对照组相比,显著上调CPT-I蛋白的表达水平,显著增强AMPK的活性,显著促进AMPK蛋白磷酸化,但对AMPK蛋白表达水平无影响,显著减少HepG2细胞内的甘油三酯含量,10、100μmol/LCy-3-g能显著促进ACC的磷酸化,100μmol/LCy-3-g能显著促进HepG2细胞脂肪酸氧化。结论花色苷Cy-3-g可以减少HepG2细胞脂肪积聚;其机制可能与调节HepG2细胞AMPK-ACC-CPT-I信号通路,促进脂肪酸氧化有关。     

花色苷抗炎抗肿瘤相关信号通路研究

目的 用基因芯片观察花色苷矢车菊素-3-葡萄苷(C3G)作用人白血病单核细胞细胞株(TI-W-1)源性巨噬细胞前后相关信号通路上基因表达的差异.方法 100 μmol/L的C3G作用于THP-1源性巨噬细胞12h,用信号转导通路发现者基因芯片研究C3G对THP-1源性巨噬细胞基因表达谱的影响,并利用半定量逆转录-聚合酶链反应(RT-PCR)法对筛选出的部分基因进行验证.结果 C3G作用后有7条信号通路中的15个基因发生改变,其中表达上调的基因有5个,包括肿瘤基因p53(TP53)、核因子KB抑制因子a(NFKBIA)、视黄醇结合蛋白基因2(RBP2)等;表达下调的基因有10个,包括FBJ鼠科骨肉瘤病毒癌基因同源物(V-fos)、诱导型一氧化氮合酶(NOS2A)、前列腺素内过氧化物合酶2(PTGS2)等,这些基因编码的宿主蛋白均为肿瘤免疫、炎症反应相关的信号转导通路中的关键点.半定量RT-PCR结果显示,经C3G干预后,NOS2A和FTGS2的表达水平下调了58%和54%.结论花色苷类植物化合物可能通过促有丝分裂、果蝇无翼基因同源物(WNT)、p53、磷脂酶C、核因子KB(NFKB)、Janus激酶/信号转导和转录活化因子(JAK/STAT)和维甲酸等信号转导通路发挥其抗炎、抗肿瘤功能.     

维生素C对染镍肺泡巨噬细胞iNOS的影响

目的 探讨镍在大鼠肺泡巨噬细胞诱导型一氧化氮合成酶(iNOS)基因诱导表达中的作用，以及维生素C对巨噬细胞iNOS诱导表达的影响及其机制。方法 采用体外细胞培养法测定在维生素C(25，50，100μmol／L)作用下，体外染镍肺泡巨噬细胞的存活率及活力。同时观察细胞内一氧化氮(nitric oxide，NO)和活性氧的产生，以及一氧化氮合酶(nitric oxide synthase，NOS)活力的变化。以逆转录-聚合酶链反应(RT—PCR)法测定iNOS mRNA的表达。结果 在体外染镍肺泡巨噬细胞中加入不同浓度的维生素C后可提高该细胞的存活率和活力，可减少NO和活性氧的产生，降低NOS和活性，还可抑制镍诱导的iNOS mRNA的表达水平。结论 镍可通过诱导大鼠肺泡巨噬细胞．NOS基因表达，产生大量NO，导致细胞脂质过氧化，而维生素C可通过抑制iNOS mRNA的表达，拮抗镍对肺泡巨噬细胞的细胞毒作用。     

补肾强筋胶囊含药血清对IL-1β刺激的人原代软骨细胞的影响

目的研究补肾强筋胶囊含药血清对IL-1β刺激的人原代软骨细胞的影响。方法采用IL-1β(10 ng/mL)刺激人原代软骨细胞,并且加入不同浓度的补肾强筋胶囊含药血清处理24 h。然后用格式试剂法和酶联免疫法分别检测软骨细胞上清液中NO和PGE2水平,采用RT-q PCR检测软骨细胞COX-2、iNOS、MMP-1、MMP-13、TIMP-1 mRNA表达。结果补肾强筋胶囊含药血清可明显降低软骨细胞NO和PGE2水平,并显著降低COX-2、iNOS、MMP-1、MMP-13 mRNA表达,而对TIMP-1 mRNA表达无明显影响。结论补肾强筋胶囊含药血清对IL-1β刺激的人原代软骨细胞有一定保护作用,其作用机制与降低COX-2、iNOS、MMP-1、MMP-13 mRNA表达有关。     

花色苷对THP-1细胞中ABCG1表达的影响及机制分析

目的研究花色苷矢车菊素-3-O-β-葡萄糖苷(C3G)对人巨噬细胞THP-1中ABCG1表达的影响,并分析相关作用机制。方法以C3G干预培养诱导分化的THP-1细胞,荧光实时定量RT-PCR检测ATP结合盒式转运蛋白G1(ABCG1)以及其直接调节因子肝脏X受体α(liver X receptorα,LXRα)的mRNA表达,时间分辨荧光共振能量转移(time-resolved fluorescence resonance energy transfer,TR-FRET)方法检测C3G与LXRα配体结合域的结合能力。结果 100μmol/L C3G处理THP-1细胞16 h显著增加了ABCG1 mRNA的表达(为对照组的1.98倍,P<0.05);尽管在TR-FRET试验中,C3G并未呈现典型的配体结合曲线,而50μmol/L和100μmol/L C3G显著增加了THP-1细胞中LXRαmRNA的表达(分别为对照组的2.21倍和2.83倍,P<0.05)。结论 C3G促进了ABCG1表达,该机制可能通过增加核受体LXRαmRNA表达来实现。     

雷公藤内酯醇对LPS诱导小鼠腹腔巨噬细胞分泌炎症相关因子的影响

目的:探讨雷公藤内酯醇对LPS诱导小鼠腹腔巨噬细胞分泌炎症相关因子的影响。方法:分离纯化小鼠腹腔巨噬细胞,设置PBS组(PBS+生理盐水)、LPS组(LPS终浓度为10μg/ml+生理盐水)及LPS+雷公藤内酯醇组(LPS终浓度为10μg/ml,雷公藤内酯醇终浓度为10μg/ml);共培养24h后Griess试剂比色法及ELISA法分别用于检测培养上清中一氧化氮(NO)及白细胞介素-8(IL-8)含量;RT-PCR法检测细胞IL-8 m RNA表达量。结果:10μg/ml雷公藤内酯醇可显著下调LPS诱导的巨噬细胞NO及IL-8的产生(P0.05),抑制率分别为80.94%和44.19%;RT-PCR结果显示,10μg/ml雷公藤内酯醇可显著下调LPS诱导的巨噬细胞IL-8 m RNA的表达。结论:雷公藤内酯醇可能通过抑制LPS诱导的巨噬细胞中NO及IL-8的表达达到抗炎作用。     

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.     

Anti-inflammatory effect of enzymatic hydrolysates from Styela clava flesh tissue in lipopolysaccharide-stimulated RAW 264.7 macrophages and in vivo zebrafish model

BACKGROUND/OBJECTIVES

In this study, potential anti-inflammatory effect of enzymatic hydrolysates from Styela clava flesh tissue was assessed via nitric oxide (NO) production in lipopolysaccahride (LPS) induced RAW 264.7 macrophages and in vivo zebrafish model.

MATERIALS/METHODS

We investigated the ability of enzymatic hydrolysates from Styela clava flesh tissue to inhibit LPS-induced expression of pro-inflammatory mediators in RAW 264.7 macrophages, and the molecular mechanism through which this inhibition occurred. In addition, we evaluated anti-inflammatory effect of enzymatic hydrolysates against a LPS-exposed in in vivo zebrafish model.

RESULTS

Among the enzymatic hydrolysates, Protamex-proteolytic hydrolysate exhibited the highest NO inhibitory effect and was fractionated into three ranges of molecular weight by using ultrafiltration (UF) membranes (MWCO 5 kDa and 10 kDa). The above 10 kDa fraction down-regulated LPS-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), thereby reducing production of NO and prostaglandin E₂ (PGE₂) in LPS-activated RAW 264.7 macrophages. The above 10 kDa fraction suppressed LPS-induced production of pro-inflammatory cytokines, including interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α. In addition, the above 10 kDa fraction inhibited LPS-induced phosphorylation of extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinase (JNK), and p38. Furthermore, NO production in live zebrafish induced by LPS was reduced by addition of the above 10 kDa fraction from S. clava enzymatic hydrolysate.

CONCLUSION

The results of this study suggested that hydrolysates derived from S. clava flesh tissue would be new anti-inflammation materials in functional resources.     

维生素C对镍诱导的肺泡巨噬细胞氧化损伤的保护作用

目的:研究维生素C(VC)拮抗Ni2O3的细胞氧化损伤的作用及对大鼠肺泡巨噬细胞iNOS诱导表达的影响。方法:采用体外细胞培养法测定在VC(25,50,100μmol/L)作用下,体外染镍肺泡巨噬细胞的死亡率及活力。同时观察细胞内丙二醛(MDA)、一氧化氮(nitricoxide,NO)和活性氧(ROS)的产生;超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)、诱导型一氧化氮合酶(nitricoxidesynthase,iNOS)活力的变化以及应用RT-PCR法测定iNOSmRNA的表达。结果:在体外染镍肺泡巨噬细胞中加入不同浓度的VC后可降低该细胞的死亡率并提高细胞的活力,可减少MDA、NO和活性氧的产生,降低NOS的活性,提高SOD、GSH-Px、CAT的活性,iNOSmRNA表达也较镍组降低。结论:镍可致细胞脂质过氧化,VC可通过提高抗氧化酶的活性,抑制iNOSmRNA的表达,减少活性氧及NO的产生,拮抗镍对肺泡巨噬细胞的氧化损伤。     

Spirulina and C-phycocyanin reduce cytotoxicity and inflammation-related genes expression of microglial cells

Abstract

Objective

Our aim was to investigate the effects of Spirulina on BV-2 microglial cell cytotoxicity and inflammatory genes expression.

Methods

BV-2 microglial cells were treated with lipopolysaccharide (LPS) (1 µg/ml) and various concentrations of Spirulina platensis water extract or its active component (C-phycocyanin (C-PC)) for 24 hours. Cytotoxicity (lactate dehydrogenase (LDH) release) and expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) mRNAs were assayed.

Results

LPS increased LDH production and up-regulated expression of iNOS, COX-2, TNF-α, and IL-6 by BV-2 microglial cells. However, Spirulina platensis water extract and C-PC significantly reduced LPS-induced LDH release, and expression of iNOS, COX-2, TNF-α, and IL-6 mRNAs.

Conclusion

Spirulina can reduce the cytotoxicity and inhibit expression of inflammation-related genes of LPS-stimulated BV-2 microglial cells.