Glucosamine inhibits LPS-induced COX-2 and iNOS expression in mouse macrophage cells (RAW 264.7) by inhibition of p38-MAP kinase and transcription factor NF-kappaB

Glucosamine supplements are very promising nonsteroidal anti-inflammatory agents widely used for the treatment of arthritis in animals and humans. In this study, we have proposed the molecular mechanism underlying the anti-inflammatory properties of glucosamine hydrochloride (GLN) using mouse macrophage cell line (RAW 264.7). Treatment with GLN inhibited LPS-stimulated nitric oxide (NO) production. Western blotting and RT-PCR analysis showed that GLN treatment decreased LPS-induced inducible nitric-oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein and mRNA expression in RAW 264.7 cells, respectively. To further elucidate the mechanism of inhibitory effect of GLN, we studied the LPS-induced phosphorylation of mitogen-activated protein kinases (pp44/42 and pp38). Our results clearly indicated that GLN treatment resulted in a reduction of pp38, whereas activation of p44/42 was not affected. In addition, LPS-induced activation of nuclear factor-kappaB (NF-kappaB) DNA binding suggests an inhibitory effect of GLN. These results indicate that GLN suppresses the LPS-induced production of NO, expression of iNOS and COX-2 by inhibiting NF-kappaB activation and phosphorylation of p38 MAP kinase.     

Anti-inflammatory activity of Chrysanthemum zawadskii var. latilobum leaf extract through haem oxygenase-1 induction

Chrysanthemum zawadskii var. latilobum (CZ) has been widely used as a tea in Korea. In this study, we investigated the involvement of anti-inflammatory haem oxygenase-1 (HO-1) in the inhibitory activity of a CZ leaf extracts on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. At the highest concentration of CZ leaf extract (50 μg/mL), NO production was 10% of that in LPS-stimulated macrophages, whereas iNOS protein expression was 5% of that in LPS-stimulated macrophages. CZ leaf extract also inhibited iNOS mRNA expression in a concentration-dependant manner, and at 50 μg/mL, iNOS mRNA expression was approximately 15% of that in LPS-stimulated cells. The CZ leaf extract induced HO-1 expression in a dose-dependent manner, and blocking HO-1 activity abolished the anti-inflammatory effect of CZ leaf extract. These results suggest that CZ leaf extract has a potent anti-inflammatory activity in RAW264.7 cells through the induction of HO-1.     

桑葚提取物体外抗炎作用及机制的研究

本文采用脂多糖(LPS)刺激小鼠腹腔巨噬细胞RAW264.7,建立细胞体外的炎症模型,研究桑葚提取物对LPS诱导巨噬细胞RAW264.7分泌功能的影响及其作用机制。实验用1μg/mL LPS刺激RAW264.7细胞,在不同浓度样品的干预下,用MTT法检测不同浓度的样品对RAW264.7细胞的作用;用Griess法检测细胞液中NO的含量;用酶联免疫吸附法(ELISA)检测细胞液中PGE2含量;用免疫印迹法(Western Blot)和RT-PCR法检测桑葚提取物对细胞iNOS和COX-2表达的影响;用HPLC法检测桑葚提取物中白藜芦醇的含量。结果表明桑葚提取物浓度在0.5~2 mg/mL范围内对细胞生长无明显影响;在1~2 mg/mL范围内能有效抑制NO和PGE2的分泌并能有效抑制iNOS和COX-2的表达;桑葚提取物中白藜芦醇的含量为107.44±0.48μg/g。这表明桑葚提取物抑制炎症相关因子表达量,从而减弱促炎症反应,发挥抗炎功效,其抗炎活性可能与桑葚中含有较高的白藜芦醇相关。     

Evaluation of the anti-inflammatory effects of phloretin and phlorizin in lipopolysaccharide-stimulated mouse macrophages

Many reports suggest that phloretin and phlorizin have antioxidant properties and can inhibit glucose transportation, the anti-inflammatory effects and mechanism of phloretin and phlorizin remain unclear. This study aims to evaluate the anti-inflammatory effects of phloretin and phlorizin in LPS-stimulated murine RAW264.7 macrophages. RAW264.7 cells were pretreated with various concentrations of phloretin or phlorizin (3–100 μM) and cell inflammatory responses were induced with LPS. Pretreated with 10 μM phloretin significantly inhibited the levels of NO, PGE₂, IL-6, TNF-α, iNOS and COX-2. Furthermore, it was demonstrated that phloretin suppressed the nuclear translocation of NF-κB subunit p65 proteins, and decreased phosphorylation in MAPK pathways. Surprisingly, phlorizin did not suppress the inflammatory response in LPS-stimulated RAW264.7 cells. These results suggest that phloretin has an anti-inflammatory effect that reduces levels of proinflammatory cytokines and mediators in RAW264.7 cells.     

蒲公英糖蛋白体外抗炎作用及对NF-κB信号通路的调控

探讨蒲公英糖蛋白(glycoprotein from Taraxacum,TG)对由脂多糖引起的RAW264.7细胞炎症的抗炎效果,并阐明其活性的基本分子机制。利用脂多糖刺激RAW264.7细胞,建立体外炎症模型,采用噻唑蓝比色法检测TG对RAW264.7细胞的增殖毒性,Griess试剂法检测了一氧化氮(nitric oxide,NO)的分泌情况,反转录聚合酶链式反应检测炎症细胞因子——白细胞介素-6(interleukin 6,IL-6)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)m RNA以及诱导型一氧化氮合酶(inducible nitric oxide synthase,i NOS)m RNA的表达水平,酶联免疫吸附测定法测定IL-6和TNF-α的分泌量,Western blot检测P-IκB-α蛋白的表达水平,用以研究TG对核转录因子-κB(nuclear factor-kappa B,NF-κB)信号转导通路的抑制作用。结果表明:TG能够显著甚至极显著地抑制NO的分泌,IL-6、TNF-α、i NOS的m RNA的表达,IL-6和TNF-α的分泌(P0.05、P0.01)。TG高度显著上调了IκB-α的蛋白表达(P0.001),并显著下调了P-IκB-α的蛋白表达(P0.05),且与TG质量浓度成正比。其中在TG质量浓度为250、500、1 000μg/m L时对TNF-α分泌量的抑制率分别为28.6%、65.4%、89.3%,对IL-6分泌量的抑制率分别为32.3%、54.1%、85.7%。TG间接抑制了NF-κB信号转导途径,有显著的体外抗炎效果且抗炎效果与TG的质量浓度呈剂量依赖性。     

Antioxidant and anti-inflammatory properties of taiwanese yam (Dioscorea japonica Thunb. var. pseudojaponica (Hayata) Yamam.) and its reference compounds

Dioscorea japonica Thunb. var. pseudojaponica (DP) is consumed as food and widely used in traditional Chinese medicine in Taiwan. The aims of this study are to investigate the antioxidant and anti-inflammatory effects of ethanol extract of DP (EDP) and its reference compounds. Fingerprint chromatogram from HPLC indicated that EDP contains gallic acid and vanillic acid. EDP was evaluated for its antioxidant effects and LPS-induced nitrite oxide (NO) production in RAW264.7 cells. EDP decreased the LPS-induced NO production and expressions of iNOS and COX-2 in RAW264.7 cells. In-vivo anti-inflammatory activities of EDP were assessed in mouse paw oedema induced by λ-carrageenan (Carr). We investigated the antioxidant mechanism of EDP via studies of the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) and the levels of malondialdehyde (MDA) in the oedematous paw. The results showed that EDP might be a natural antioxidant and anti-inflammatory agent.     

羊栖菜多酚通过核转录因子-κB/丝裂原活化蛋白激酶通路缓解脂多糖诱导的RAW264.7细胞炎症反应

目的：研究羊栖菜多酚对脂多糖（lipopolysaccharide,LPS）诱导小鼠单核巨噬细胞白血病细胞RAW264.7细胞炎症反应的影响。方法：噻唑蓝法测定细胞活力;Griess法测定一氧化氮（NO）水平;实时荧光定量聚合酶链式反应测定白细胞介素（interleukin,IL）-6、IL-1β、肿瘤坏死因子（tumor necrosis factor,TNF）-α、环氧合酶2（cyclooxygenase-2,COX-2）和诱导型一氧化氮合酶（inducible nitric oxide synthase,iNOS）的基因相对表达量;流式细胞术测定细胞吞噬能力;蛋白免疫印迹法测定信号通路丝裂原活化蛋白激酶（mitogen-activated protein kinases,MAPKs）和核转录因子（nuclear factor,NF）-κB信号通路关键蛋白表达水平。结果：羊栖菜多酚对RAW264.7细胞的安全质量浓度范围为0～160 μg/mL。与LPS组相比,羊栖菜多酚剂量依赖性降低巨噬细胞吞噬能力并抑制NO的生成。同时,羊栖菜多酚下调促炎细胞因子（IL-1β、IL-6、TNF-α）和炎症诱导酶（iNOS、COX-2）的mRNA水平,且作用效果与给药剂量及LPS刺激时间相关。这些炎性介质的表达与羊栖菜多酚抑制p38 MAPK和NF-κB p65的激活有关。结论：羊栖菜多酚可通过减弱p38 MAPK和NF-κB p65信号通路的激活水平,抑制下游炎症介质的转录表达,从而缓解LPS诱导的巨噬细胞炎症反应。     

Anti-inflammatory effect of the extract from fermented <Emphasis Type="Italic">Asterina pectinifera</Emphasis> with <Emphasis Type="Italic">Cordyceps militaris</Emphasis> mycelia in LPS-induced RAW264.7 macrophages

In our previous work, Asterina pectinifera was fermented with Cordyceps militaris mycelia to improve its bioactivities and was reported to have strong antioxidant activities. The aim of the current study was to investigate its anti-inflammatory effect and mechanisms of action. In this study, we observed the inhibitory effect of the extract from fermented A. pectinifera with C. militaris mycelia (FACM) on nitric oxide (NO) production and its molecular mechanism in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. FACM could decrease LPS-induced NO production. Western blot analysis showed that FACM could down-regulate LPS-induced expression of inducible NO synthase without affecting cyclooxygenase-2. Moreover, FACM exhibited anti-inflammatory activity in LPS-induced RAW264.7 mouse macrophage cells through proinflammatory mediators including TNF-α and IL-6 via nuclear factor kappa B pathway. FACM inhibited LPS-induced phosphorylation of extracellular-signal-regulated kinase expression. Our results suggest that FACM may be a potential candidate for inflammation therapy by attenuating the generation of cytokines, production of NO, and generation of ROS in RAW264.7 cells.     

4 种迷迭香化合物对脂多糖诱导的RAW264.7细胞氧化应激和炎症的抑制作用

目的：研究4 种迷迭香化合物（鼠尾草酸、鼠尾草酚、迷迭香酚和迷迭香酸）对脂多糖（lipopolysaccharide,LPS）诱导的RAW264.7细胞氧化应激和炎症反应的抑制作用。方法：以LPS诱导RAW264.7细胞构建炎症模型,通过比色法、实时荧光定量聚合酶链式反应、Western blot等方法表征迷迭香化合物对活性氧（reactive oxygen species,ROS）、炎症因子以及相关酶类的影响。结果：上述4 种迷迭香化合物对LPS诱导的细胞内ROS相对含量、丙二醛（malonic dialdehyde,MDA）含量、NO释放量和肿瘤坏死因子α（tumor necrosis factor α,TNF-α）、白细胞介素1β（interleukin 1β,IL-1β）、IL-6 mRNA相对表达量,以及诱导型一氧化氮合酶（inducible nitric oxide synthase,iNOS）和环氧合酶2（cyclooxygenase 2,COX-2）蛋白相对表达量的升高有显著抑制作用,对超氧化物歧化酶（superoxide dismutase,SOD）和过氧化氢酶（catalase,CAT）活力有显著保护作用,且具有浓度依赖效应。质量浓度10 μg/mL的鼠尾草酸对LPS诱导的ROS相对含量、NO释放量、TNF-α mRNA相对表达量、MDA含量升高的抑制作用和对SOD和CAT活力的保护作用更好,质量浓度10 μg/mL的迷迭香酚对LPS诱导的IL-1β、IL-6 mRNA相对表达量、iNOS和COX-2蛋白相对表达量升高的抑制作用更好。结论：4 种迷迭香化合物均具有一定的抗氧化和抗炎作用,能缓解炎症反应,其中水溶性迷迭香酸有效质量浓度高于其他3 种。     

Inhibitory effects of new varieties of bitter melon on lipopolysaccharide-stimulated inflammatory response in RAW 264.7 cells

Bitter melon is widely used as a food and a traditional herbal medicine for the treatment of diabetes; it also possesses antioxidant, anti-tumour, anti-bacterial, anti-obesity, and anti-hypertension activities. The aim of this work was to study the in vitro anti-inflammatory effects of different solvent extracts of the seeds and fruits of new varieties of bitter melon. Of the 30 bitter melon extracts tested, the ethyl acetate extract of the fruit of Momordica charantia MDS72 (EAEF-MCMDS72) exhibited the strongest anti-inflammatory activity. EAEF-MCMDS72 significantly inhibited the production of NO, IL-6, PGE₂, TNF-α, and MCP-1 in lipopolysaccharide (LPS)-treated RAW 264.7 cells. The expression levels of the iNOS and COX-2 proteins in LPS-stimulated RAW 264.7 cells were inhibited by EAEF-MCMDS72. Real-time RT-PCR analysis indicated that the mRNA expression levels of iNOS, COX-2, IL-6, and TNF-α were suppressed by EAEF-MCMDS72. Moreover, four known triterpene compounds including (23E)-cucurbita-5,23,25-triene-3β,7β-diol (1), (23E)-25-methoxycucurbit-23-ene-3β,7β-diol (2), (23E)-5β,19-epoxycucurbita-6,23-diene-3β,25-diol (3), and 3,7-dioxo-23,24,25,26,27-pentanorcucurbit-5-en-22-oic acid (4) were isolated from the fruit of M. charantia MDS72. Compounds 2, 3, 4 suppressed the LPS-induced NO production in RAW 264.7 cells. Furthermore, the present study may contribute to our understanding of the anti-inflammatory effects of EAEF-MCMDS72 and its triterpene compounds.     

重组黑芝免疫调节蛋白对小鼠巨噬细胞RAW264.7向M1型分化的影响

真菌免疫调节蛋白具有免疫调节和抗肿瘤功效。巨噬细胞在肿瘤免疫治疗中发挥重要的作用,M1型巨噬细胞具有很强的肿瘤杀伤能力和抗原递呈能力。本研究通过真菌免疫调节蛋白在体外对小鼠巨噬细胞RAW264.7经典活化M1型的影响探究其抗肿瘤的作用机制。利用重组黑芝免疫调节蛋白（recombinant Ganoderma atrum immunomodulatory protein,rFIP-gat）干预RAW264.7细胞,用中性红吞噬实验测定细胞吞噬能力,一氧化氮（nitric oxide,NO）检测试剂盒测定NO浓度,采用实时荧光定量聚合酶链式反应测定肿瘤坏死因子-α（tumor necrosis factor α,TNF-α）、诱导型一氧化氮合酶（inducible nitric oxide synthase,iNOS）的mRNA相对表达量。结果表明,rFIP-gat以剂量依赖的方式增强RAW264.7细胞的吞噬能力,促进其产生NO,并提高iNOS和TNF-α基因的转录。因此,rFIP-gat可能具有诱导RAW264.7细胞向M1型巨噬细胞分化的作用。     

Antioxidant and anti-inflammatory properties of Dichondra repens Forst. and its reference compounds

Dichondra repens (DR) is the main constituent in herbal beverages and consumed daily as a nutrition supplement for the liver in Taiwan. This study investigated the antioxidant and anti-inflammatory effects of D. repens ethanol extract (EDR) and its reference compounds ex vivo and in vivo. Fingerprint chromatograms (from HPLC) indicated that EDR contained vanillin, umbelliferone and scopoletin. EDR was evaluated for its antioxidant effects and LPS-induced NO production in RAW 264.7 cells. EDR decreased the LPS-induced NO production and expressions of iNOS and COX-2 in RAW 264.7 cells. In vivo anti-inflammatory activities of EDR were assessed in mouse paw oedema, induced by λ-carrageenan (Carr). We investigate the antioxidant mechanism of EDR via studies of the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) in the liver and the levels of malondialdehyde (MDA) and nitrite oxide (NO) in the oedematous paw. Serum NO and TNF-α were also measured. EDR exerts anti-inflammatory effects by suppressing TNF-α, NO, and might be related to the decrement of the level of MDA in the oedema paw via increasing the activities of CAT, SOD and GPx in the liver. The results show that EDR might be a natural antioxidant and anti-inflammatory agent.     

Anti-inflammatory effects of Punica granatum Linne in vitro and in vivo

Inflammation can cause various physical dysfunctions. Punica granatum Linne (pomegranate), a high phenolic content fruit, is widely used as an antipyretic analgesic in Chinese culture. Pomegranate has shown potential nitric oxide (NO) inhibition in LPS-induced RAW 264.7 macrophage cells. Moreover, pomegranate (100 mg/kg) significantly decreased carrageenan-induced mice paw edema for 1, 3, 4, and 5 h. Therefore, column chromatography combined with in vitro bioassay-guided fractionation was used to isolate the active anti-inflammatory components from the pomegranate. Punicalagin (1), punicalin (2), strictinin A (3), and granatin B (4) were obtained with yields of 0.093%, 0.015%, 0.003%, and 0.013%, respectively. All these hydrolysable tannins inhibited NO production and iNOS expression in RAW 264.7 cells. Among them, 4 showed the strongest iNOS and COX-2 inhibitory effects, and exhibited these effects in the inhibition of paw swelling and the PGE₂ level in carrageenan-induced mice. Taken together, we suggest that 4 could be used as a standard marker for the anti-inflammatory effect of pomegranate.     

红汁乳菇中愈创木烷型倍半萜化合物的抗炎活性

以新鲜红汁乳菇子实体为原料,采用有机溶剂浸提提取、乙酸乙酯萃取、硅胶柱层析得到单体化合物,通过紫外-可见光谱、高效液相色谱、质谱和核磁共振波谱分析鉴定单体化合物的结构,利用脂多糖（lipopolysaccharide,LPS）诱导RAW264.7巨噬细胞炎症模型,通过聚合酶链式反应和免疫印迹法分析单体化合物对炎症因子mRNA和蛋白相对表达水平的影响,以及对丝裂原活化蛋白激酶（mitogen-activated protein kinase,MAPK）炎症信号通路的作用。结果：从红汁乳菇中提取出具有较强抗炎活性的单体化合物,经鉴定为愈创木烷型倍半萜类化合物;与模型组相比,该倍半萜能极显著降低脂多糖刺激巨噬细胞中白细胞介素-6（interleukin,IL）-6、IL-1β、肿瘤坏死因子-α（tumor necrosis factor-α,TNF-α）和一氧化氮合酶（inducible nitric oxide synthase,iNOS）的mRNA相对表达水平（P＜0.01）;该倍半萜能降低LPS刺激的巨噬细胞中炎症因子环氧化酶-2（cyclooxygenase-2,COX-2）、IL-1β、IL-6、iNOS和TNF-α蛋白相对表达水平,且呈现一定的浓度依赖性;该倍半萜能降低磷酸化的细胞外信号激酶（p44/42）、p38 MAPK（简称p38蛋白）和c-Jun氨基端激酶（c-Jun N-terminal kinase,JNK）3 种蛋白激酶的磷酸化水平,从而抑制MAPK炎症通路的活性。