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.     

Dietary lutein modulates inducible nitric oxide synthase (iNOS) gene and protein expression in mouse macrophage cells (RAW 264.7)

Lutein is an oxycarotenoid primarily found in dark-green leafy vegetables such as spinach and kale. Other dietary sources which contain moderate amounts of lutein include corn, egg yolks, and fruits like oranges and kiwi. Although a number of in vivo studies have demonstrated the anti-inflammatory effect of lutein, its in vitro anti-inflammatory molecular mechanism of action is unknown. In this study, we have investigated the in vitro anti-inflammatory effect of lutein using LPS-stimulated mouse macrophage cell line (RAW 264.7). The inhibition of LPS-stimulated nitric oxide (NO) was measured and the expression of inducible NO synthase (iNOS) was assessed at the mRNA and protein levels in mouse macrophage cells after treatment with lutein. Lutein decreased the LPS-induced NO production by 50% compared to LPS alone. Real-time PCR analysis showed a 1.9-fold reduction in iNOS expression at the mRNA level. Western blotting revealed that lutein decreased LPS-induced iNOS expression at the protein level by 72.5%. The results of this study suggest the anti-inflammatory properties of lutein demonstrated by the decrease in the expression of iNOS at the mRNA and protein levels in RAW 264.7 mouse macrophage cells.     

Anti-inflammatory and antioxidant activities of red pepper (Capsicum annuum L.) stalk extracts: Comparison of pericarp and placenta extracts

Red pepper stalks, an agricultural waste product, are casually discarded at landfills where they oxidize and harm the local ecology. To our knowledge, no feasible method has been designed to evaluate the bioavailability of red pepper stalks. Therefore, the present study focused on the stalk waste investigating its superoxide dismutase (SOD) activity and inhibitory effect on nitric oxide (NO) production in macrophages. Interestingly, by comparison with red pepper pericarp and placenta extracts, the stalks were found to have higher phenolic, flavonoid, and capsaicin contents. Chromatographic data showed differences in the amounts of phenolic compounds in various parts of red pepper. At a concentration of 100 μg/mL, the stalk, placenta, and pericarp had SOD activities of 52.3%, 7.4%, and 10.4%, respectively. A very high level of chlorogenic acid (3.82 mg/g d w) and p-coumaric acid (2.98 mg/g d w) gave the stalks of red peppers the most powerful antioxidant activity among the three tested parts. The high phenolic and SOD activity-containing stalks also had the most significant NO inhibitory effect (53.5%) in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. These results demonstrated that there is an opportunity to develop a new anti-inflammatory agent from agricultural wastes.     

Suppressive effect of carotenoid extract of Dunaliella salina alga on production of LPS-stimulated pro-inflammatory mediators in RAW264.7 cells via NF-κB and JNK inactivation

The carotenoid extract from Dunaliella salina was used to evaluate the suppressive effects on lipopolysaccharide (LPS)-induced pro-inflammatory mediators in RAW264.7 cells. The extract composed all-trans forms of α-carotene (28.8 mg/g extract), β-carotene (471.1 mg/g extract), lutein (7.1 mg/g extract) and zeaxanthin (7.2 mg/g extract), 13- or 13′-cis-β-carotene (12.1 mg/g extract), 9- or 9′-cis-α-carotene (19.1 mg/g extract) and 9- or 9′-cis-β-carotene (440.3 mg/g extract) dose-dependently reduced the production of interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α), the protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and the secretion of nitric oxide (NO) and prostaglandin E₂ (PGE₂) in LPS-activated RAW264.7 cells. Its attenuation of LPS-induced inflammatory responses was closely related to inhibition of the nuclear NF-κB p50 subunit translocation by blocking inhibitor of κBα (IκB) phosphorylation and degradation correlated with suppressing IκB kinase (IKK) α/β phosphorylation, as well as down-regulation of the c-Jun NH₂-terminal kinase (JNK) activation.     

Anti-inflammatory activity of ethanolic extract of Sargassum sagamianum in RAW 264.7 cells

Aim of the study is to evaluate the antiinflammatory effects of ethanolic extract of the marine brown alga Sargassum sagamianum collected from Yeonhwari coast of Korea. Ethanolic extract of S. sagamianum (SA-E extract) inhibited expression of nitric oxide (NO) and cytokines (IL-6, IL-1β, and TNF-α) as well as inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 in lipopolysaccharide (LPS)-induced RAW 264.7 cells without affecting cell viability. In addition, the expression of nuclear factor (NF)-κB p65 was suppressed by SA-E extract. Furthermore, the rate of formation of edema in the mouse ear was reduced by 46% at the highest dose tested (250 mg/kg) compared to that in the control. This study suggests that SA-E extract exerts potent inhibitory effects on LPS-induced expression of inflammatory mediators such as NO, iNOS, COX-2, and cytokines in macrophages through suppression of the NF-κB p65 pathway. SA-E extract might have potential clinical applications as an anti-inflammatory agent.     

红松松仁多糖对脂多糖诱导RAW264.7细胞炎症反应的抑制作用

目的:探讨红松松仁多糖PNP40c-1对脂多糖(Lipopolysaccharides,LPS)诱导RAW264.7细胞炎症反应的抑制作用及可能机制。方法:以LPS刺激RAW264.7细胞诱导炎症模型,分别采用MTT法、比色法、酶联免疫法、RT-PCR和Western Blot等方法,比较LPS诱导前后巨噬细胞的细胞活力、吞噬能力、一氧化氮(nitric oxide,NO)/一氧化氮合酶(nitric oxide synthase,iNOS)和细胞因子表达的变化;同时对Nrf2/HO-1信号通路中关键蛋白核因子E2相关因子2(nuclear factor(erythroid-derived 2)-like 2 protein,Nrf2)和血红素氧化酶-1(heme oxygenase-1,HO-1)的mRNA和蛋白表达水平进行研究,以探讨PNP40c-1的具体作用机制。结果:在LPS诱导RAW264.7细胞的炎症反应中,PNP40c-1以剂量依赖性显著提高巨噬细胞的吞噬能力(P<0.05),抑制LPS诱导的NO和iNOS过量表达;PNP40c-1还可通过Nrf2/HO-1信号通路调节炎症因子如肿瘤坏死因子(tumor necrosis factor-α,TNF-α),白介素-1β(interleukin-1β,IL-1β)、白介素-6(IL-6)的分泌,缓解炎症反应。结论:红松松仁多糖PNP40c-1对LPS诱导的炎症反应具有一定的抑制作用,其作用机制与调节Nrf2/HO-1信号通路有关。     

Suppression of inducible nitric oxide synthase expression and amelioration of lipopolysaccharide-induced liver injury by polyphenolic compounds in Eucalyptus globulus leaf extract

Eucalyptus leaf extract (ELE) is rich in hydrolyzable tannins. We examined the effects of ELE and its constituents on lipopolysaccharide (LPS)-induced liver injury in mice. Mice fed a diet supplemented with 1% ELE were intraperitoneally administered LPS. Six hours later, the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were significantly lower in the ELE-supplemented mice than in the controls; LPS-induced hepatic inducible nitric oxide synthase (iNOS) expression was also suppressed. ELE lowered LPS-stimulated iNOS expression in cultured RAW 264.7 macrophages. Furthermore, the aglycones of hydrolyzable tannins, gallic acid (GA) and ellagic acid (EA), inhibited iNOS induction to a greater extent than did ELE (15-fold higher). When mice were fed a 1% GA or EA diet, the increase in the serum ALT and AST activities and hepatic iNOS expression in response to the LPS challenge were significantly attenuated. Thus, hydrolyzable tannins in ELE ameliorate LPS-induced liver injury.     

海南无刺蜂蜂蜜中多酚类物质成分分析及其抗氧化、抗炎活性评价

以海南无刺蜂蜂蜜及其多酚类提取物为研究对象,分别测定分析无刺蜂蜂蜜的理化成分和多酚物质,研究其抗氧化和抗炎活性。通过液相色谱-串联四极杆飞行时间质谱分析多酚类成分,并采用福林-酚法和硝酸铝比色法测定多酚类提取物总酚酸和总黄酮含量。采用1,1-二苯基-2-三硝基苯肼（1,1-diphenyl-2-picrylhydrazyl,DPPH）自由基、2,2’-联氮-双-（3-乙基苯并噻唑啉-6-磺酸）二铵盐自由基（2,2’-azinobis-(3-ethylbenzthiazoline-6-sulphonate) radical,ABTS＋·）清除实验和铁离子还原力实验评价多酚类提取物的体外抗氧化活性,并采用细菌脂多糖（lipopolysaccharides,LPS）诱导的RAW 264.7细胞体外炎症模型,探究多酚类提取物的抗炎活性。结果显示,无刺蜂蜂蜜水分质量分数为（26.3±0.1）%,pH 3.7±0.2,蛋白质含量为（628.2±9.0）mg/kg,淀粉酶值为（19.6±0.2）mL/（g·h）,总糖质量分数为（46.7±6.0）%,包括果糖（21.0±3.7）%、葡萄糖（24.9±2.2）%、蔗糖（0.8±0.1）%;多酚类提取物的总酚酸和总黄酮含量分别为（96.6±0.4）μg CAE/g和（16.1±0.3）μg QE/g;多酚类提取物的DPPH自由基和ABTS＋?清除能力IC50值分别为（435.1±0.4）、（423.0±0.3）μg/mL,铁离子还原力为（0.6±0.02）μmol Trolox/g;在体外抗炎实验中,多酚类提取物能显著抑制由LPS诱导的RAW 264.7细胞NO的释放,并显著抑制促炎症基因iNOS、IL-1β、IL-6和MCP-1的表达,显著增强抗氧化基因HO-1的表达,并呈剂量相关性。综上所述,海南无刺蜂蜂蜜营养成分丰富,富含酚酸和黄酮类物质,且具有很强的抗氧化和抗炎能力,具有良好的开发利用价值。     

Characteristics and in vitro anti-inflammatory activities of protein extracts from pre-germinated black soybean [Glycine max (L.)] treated with high hydrostatic pressure

We investigated the influence of high hydrostatic pressure (HHP) and pre-germination on black soybean protein and peptide characteristics. Black soybean was germinated (2 or 4 d) and subjected to HHP (0.1–150 MPa; 24 h), followed by preparation of extracts and testing for anti-inflammatory activity. The highest soluble protein content (815.85 mg/g) was in soybeans subjected to HHP (100 MPa) after germination (4 d). Germination and HHP caused the degradation of high molecular weight proteins; we detected the highest content of < 3 and < 10 kDa peptides (60.46 and 88.47 mg) and free amino acids (65.70 mg) in extracts from pre-germinated soybeans that received HHP (150 MPa). Pre-germinated (4 d) soybean extracts treated with HHP (150 MPa) significantly inhibited LPS-induced expression of inflammatory markers, nitric oxide (25.01%), TNF-α (76.78%), IL-1β (58.99%), and IL-6 (84.48%) by RAW 264.7 macrophages. These results suggest that peptides derived from black soybeans subjected to HHP and pre-germination may mediate anti-inflammatory activity.     

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.     

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.     

酱香型白酒中吡嗪类物质体外抗炎作用研究

为研究酱香型白酒中吡嗪类物质的体外抗炎活性及其作用机制,采用脂多糖（LPS）诱导RAW264.7细胞建立炎症模型,并通过酶联免疫吸附试验（ELISA）测定细胞上清液中一氧化氮（NO）、白细胞介素-6（IL-6）及肿瘤坏死因子-α（TNF-α）的含量,进而检测诱导型一氧化氮合酶（iNOS）、环氧化酶-2（COX-2）、血红素氧合酶-1（HO-1）、白细胞介素-1（IL-1）、IL-6、TNF-α的信使核糖核酸（mRNA）的表达及对花生四烯酸代谢通路中与生成前列腺素E2（PGE2）有关的关键酶的影响。结果表明,2,3,5-三甲基吡嗪（BQ-5）可抑制细胞上清液中NO上升,抑制磷脂酶A2（PLA2）及COX-2活性,下调LPS诱导的iNOS、COX-2、IL-1、IL-6及TNF-α的mRNA的表达。由此可见,酱香型白酒中含有的吡嗪类物质具有一定的抗炎活性,其中BQ-5可减少炎症介质（IL-1、IL-6、TNF-α）的表达,抑制iNOS基因的表达从而抑制NO的产生,抑制花生四烯酸代谢途径中PGE2合成的关键酶而发挥抗炎作用。