Carnosic acid protects against ROS/RNS-induced protein damage and upregulates HO-1 expression in RAW264.7 macrophages

The overproduction of reactive oxygen species (ROS) and reactive nitrogen species (RNS) is a deleterious process that can be an important mediator of damage to cell structures including protein, lipid and DNA, and consequently leads to various disease states and senescence. In the present study, the protective effects of carnosic acid (CA) on ROS/RNS-induced protein damages were examined. CA dose-dependently decreased the fragmentation, carbonylation, and nitration of bovine serum albumin (BSA) in AAPH and Hemin/nitrite/H₂O₂ incubation systems, respectively. Moreover, CA effectively attenuated protein carbonylations in the radical-treated RAW264.7 cells. Furthermore, after pretreatments of RAW264.7 macrophages with CA, the generation of ROS and NO induced by AAPH and H₂O₂ or LPS were significantly suppressed, and heme oxygenase-1 (HO-1) protein expression was time- and dose-dependently upregulated. Hence, our results indicated that CA might be beneficial for cellular proteins in oxidative stress or inflammation conditions by alleviating ROS/RNS generation and inducing the expression of antioxidant enzyme HO-1.     

Linarin down-regulates phagocytosis,pro-inflammatory cytokine production,and activation marker expression in RAW264.7 macrophages

Food Science and Biotechnology - Plant-extracted flavonoid glycosides have been reported to be bioactive compounds with pleiotropic functions, including antioxidant, anti-inflammatory, and...     

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.     

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.     

龙眼蛋白对C57BL/6小鼠及RAW264.7巨噬细胞的炎症因子的影响研究

目的 探讨龙眼蛋白对C57BL/6小鼠及RAW264.7巨噬细胞的炎症因子影响及作用机制。方法 采用反向色谱-质谱法(reverse-phase liquid chromatography-mass spectrometry,RPLC-MS)鉴定龙眼蛋白的组成。采用100 mg/(kg·d)和200 mg/(kg·d)龙眼蛋白腹腔注射C57BL/6小鼠,酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)测定小鼠血液炎症因子的表达,苏木精-伊红(hematoxylin-eosin,HE)染色法染色分析肺部炎症病理反应;用0.2、0.4、0.6 mg/mL龙眼蛋白处理小鼠RAW264.7巨噬细胞,噻唑蓝[3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide,MTT]法测定细胞活力,实时荧光定量聚合酶链式反应法(real-time quantitative polymerase chain reaction,Q-PCR)和ELISA检测炎症因子表达,蛋白质免疫印迹法(w...     

Anti-inflammatory and anti-genotoxic activity of branched chain amino acids (BCAA) in lipopolysaccharide (LPS) stimulated RAW 264.7 macrophages

Food Science and Biotechnology - The purpose of this study was to evaluate the anti-inflammatory and anti-genotoxic activity of branched-chain amino acids (BCAAs) in lipopolysaccharide (LPS)...     

Anti-inflammatory activity of ethanolic extract from skipjack tuna (Katsuwonus pelamis) heart in LPS-induced RAW 264.7 cells and mouse ear edema model

Food Science and Biotechnology - This study investigated the anti-inflammatory activity of the ethanolic extract (THEE) obtained from the heart of skipjack tuna using lipopolysaccharide...     

Dimethoxycurcumin, a synthetic curcumin analogue with higher metabolic stability, inhibits NO production, inducible NO synthase expression and NF-kappaB activation in RAW264.7 macrophages activated with LPS

Excess production of nitric oxide (NO) by inducible NO synthase (iNOS) in activated macrophages is linked to acute and chronic inflammation. Thus, it would be valuable to develop inhibitors of NO and/or iNOS for potential therapeutic use. We investigated whether dimethoxycurcumin (DiMC), a synthetic curcumin analogue with higher metabolic stability over curcumin, could inhibit NO production and iNOS expression in activated macrophages. RAW264.7 macrophages were activated with lipopolysaccharide (LPS) in the absence or presence of DiMC, which contains four methoxy groups at two aromatic rings, curcumin containing two, bis-demethoxycurcumin (BDMC) containing none, or tetrahydrocurcumin (THC) containing two but lacking conjugated double bonds in the central seven-carbon chain. NO production, iNOS expression and NF-kappaB activity were examined. DiMC, curcumin and BDMC inhibited NO production, iNOS expression and NF-kappaB activation, with DiMC being the most effective, followed by curcumin and BDMC. THC failed to inhibit NO production, iNOS expression and NF-kappaB activation. Our results suggest that DiMC inhibits NO production, iNOS expression and NF-kappaB activation in LPS-activated macrophages, which may be due not only to the conjugated double bonds but also the increased number of methoxy groups.     

Polygonum viviparum L. inhibits the lipopolysaccharide‐induced inflammatory response in RAW264.7 macrophages through haem oxygenase‐1 induction and activation of the Nrf2 pathway

BACKGROUND: Polygonum viviparum L. (PV) is a member of the family Polygonaceae and is widely distributed in high‐elevation areas. It is used as a folk remedy to treat inflammation‐related diseases. This study was focused on the anti‐inflammatory response of PV against lipopolysaccharide (LPS)‐induced inflammation in RAW264.7 macrophages. RESULTS: Treatment with PV did not cause cytotoxicity at 0–50 µg mL^?1 in RAW264.7 macrophages, and the IC₅₀ value was 270 µg mL^?1. PV inhibited LPS‐stimulated nitric oxide (NO), prostaglandin (PG)E₂, interleukin (IL)‐1β and tumour necrosis factor (TNF)‐α release and inducible NO synthase (iNOS) and cyclooxygenase (COX)‐2 protein expression. In addition, PV suppressed the LPS‐induced p65 expression of nuclear factor (NF)‐κB, which is associated with the inhibition of IκB‐α degradation. These results suggest that, among mechanisms of the anti‐inflammatory response, PV inhibits the production of NO and these cytokines by down‐regulating iNOS and COX‐2 gene expression. Furthermore, PV can induce haem oxygenase (HO)‐1 protein expression through nuclear factor E2‐related factor 2 (Nrf2) activation. A specific inhibitor of HO‐1, zinc(II) protoporphyrin IX, inhibited the suppression of iNOS and COX‐2 expression by PV. CONCLUSION: These results suggest that PV possesses anti‐inflammatory actions in macrophages and works through a novel mechanism involving Nrf2 actions and HO‐1. Thus PV could be considered for application as a potential therapeutic approach for inflammation‐associated disorders. © 2012 Society of Chemical Industry     

两种方法提取佛手渣多糖及其对巨噬细胞RAW264.7免疫调节活性的研究

本文以提取精油和黄酮后的佛手渣为原料,分别采用连续相变萃取(CPE)法和热水浸提法(HWE)法提取佛手多糖,在单因素实验的基础上,以佛手多糖提取率为指标,通过响应面优化得出两种方法的最佳提取工艺,并利用RAW264.7巨噬细胞模型评价佛手多糖的免疫活性,采用MTT法、中性红比色法和Griess法分别检测佛手多糖对RAW264.7细胞的生长、吞噬活性和释放NO能力的影响。结果表明,CPE法提取佛手多糖的最佳工艺条件为:温度99 ℃,时间4.5 h,流速66 L/h,提取率为16.09%±0.12%,得率为21.31%±0.13%;HWE法提取佛手多糖的最佳工艺条件为:95 ℃,时间6.0 h,液料比63 mL/g,提取率为15.43%±0.21%,得率为20.22%±0.16%;与HWE法相比,CPE法将佛手多糖提取率提高了4.28%(P<0.05),得率提高了5.39%(P<0.05),且提取时间缩短了1/4。佛手多糖在浓度低于500 μg/mL时对RAW264.7巨噬细胞无明显毒性作用,且可以促进NO的分泌以及提高吞噬活性。上述结果说明,CPE法适合佛手多糖的提取,优于传统的HWE法,且佛手多糖具有免疫类保健食品和药品开发的潜力。     

溪黄草常压回流提取物与减压回流提取物抗氧化性的比较

本文对比了常压回流提取法与减压提取法得到的溪黄草不同溶剂提取物总酚,黄酮,单宁提取率及抗氧化性强弱。实验结果表明:溪黄草提取物抗氧化性及总酚、黄酮、单宁提取率均受提取溶剂极性影响。在相同溶剂提取条件下,溪黄草提取物抗氧化性及总酚、黄酮、单宁提取率均受不同提取方法影响。常压回流提取物的抗氧化性高于减压回流提取物的抗氧化性。溪黄草中除总酚、黄酮、单宁外,还有其他物质会对抗氧化性有贡献。料液比的恒定及提取溶剂的选择是溪黄草有效成分提取的关键所在。高温不仅不会破坏溪黄草有效成分,还能增强有效成分的溶出。     

Lotus (Nelumbo nucifera Gaertn) plumule polysaccharide protects the spleen and liver from spontaneous inflammation in non-obese diabetic mice by modulating pro-/anti-inflammatory cytokine gene expression

A novel lotus plumule polysaccharide (LPPS) was administered to non-obese diabetic (NOD) mice for 15 weeks to evaluate the protective effects of LPPS on type 1 diabetes. After the 15-week feeding experiment, tumour necrosis factor (TNF)-α, interleukin (IL)-6 and IL-10 expressions in the spleen, liver and kidney of the experimental mice were measured using enzyme-linked immunosorbent assay or real-time quantitative polymerase chain reaction assay. The results showed that LPPS significantly (p < 0.05) decreased the absolute weights of the enlarged spleens in the NOD mice in a dose-dependent manner, inhibited pro-inflammatory TNF-α and IL-6 cytokine production and decreased the secretion ratio of IL-6/IL-10 in splenocyte cultures. LPPS markedly decreased the relative expression of pro-/anti-inflammatory cytokine genes (TNF-α/IL-10 and IL-6/IL-10) in the livers of NOD mice. Our results suggest that LPPS protected the spleen and liver from spontaneous inflammation in NOD mice by modulating pro-/anti-inflammatory cytokine gene expression.     

Nitric oxide (NO) production in mammalian non-tumorigenic epithelial cells of the small intestine and macrophages induced by individual strains of lactobacilli and bifidobacteria

Nitric oxide (NO) affects multiple gastrointestinal functions, including mucosal inflammation and antimicrobial activity. The aim of this study was to screen the ability of probiotic bacteria to stimulate NO production in porcine intestinal epithelial cells and macrophages in the presence and absence of interferon gamma (INF-γ). Production of NO in intestinal epithelium was stimulated by individual strains of lactobacilli without INF-γ priming. While none of the tested bifidobacteria were capable of inducing NO production, most constitutively secreted NO. Most tested strains induced a significant increase in NO production compared with the control cells in the macrophage cell line 3D4/21. Results support the protective role of the individual strains of the genera Lactobacillus and Bifidobacterium and may lead to new approaches for manipulating and regulating immune responses at the mucosal surfaces of the gastrointestinal tract.     

The production of menaquinones (vitamin K2) by intestinal bacteria and their role in maintaining coagulation homeostasis.

Vitamin K is an essential cofactor necessary for the production of clotting factors II, VII, IX, and X in humans and has recently been found to be an essential factor for many other proteins in the body. There are two sources of this essential vitamin, including vitamin K1, or phylloquinone which is primarily found in green leafy vegetables and vitamin K2 or menaquinone which is synthesized by certain intestinal bacteria. The precise contribution of the bacterially synthesized menaquinone to overall vitamin K requirements in man is unknown. This paper reviews the available literature regarding the production and liberation of menaquinones from bacteria, the animal experiments which have been done to examine the absorption of menaquinones and the indirect and direct evidence in humans regarding utilization of menaquinones. The preponderance of the evidence suggests that bacterially synthesized menaquinones, particularly in the ileum can and do play a significant role in contributing to vitamin K requirements in humans to prevent clinically significant coagulopathy, especially during periods of episodic dietary lack of the vitamin.