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Inhibitory Effect of N,N-Didesmethylgrossularine-1 on Inflammatory Cytokine Production in Lipopolysaccharide-Stimulated RAW 264.7 Cells  [PDF]
Taiko Oda,Jong-Soo Lee,Yuta Sato,Yasuaki Kabe,Satoshi Sakamoto,Hiroshi Handa,Remy E. P. Mangindaan,Michio Namikoshi
Marine Drugs , 2009, DOI: 10.3390/md7040589
Abstract: N,N-Didesmethylgrossularine-1 (DDMG-1), a compound with a rare α-carboline structure, was isolated from an Indonesian ascidian Polycarpa aurata as responsible for the observed inhibitory activity against TNF-α production in lipopolysaccharide-stimulated murine macrophage-like RAW264.7 cells. DDMG-1 inhibited the mRNA level of mTNF-α, IκB-α degradation, and binding of NF-κB to the target DNA site in LPS-stimulated RAW 264.7 cells. Moreover, DDMG-1 had an inhibitory effect on the production of IL-8, which is produced in CD14+-THP-1 cells stimulated by LPS. DDMG-1 is thus a promising drug candidate lead compound for the treatment of chronic inflammatory diseases, such as rheumatoid arthritis.
Inhibition of lipopolysaccharide-induced nitric oxide and prostaglandin E2 production by chloroform fraction of Cudrania tricuspidata in RAW 264.7 macrophages
Yang Gabsik,Lee Kyungjin,Lee Mihwa,Ham Inhye
BMC Complementary and Alternative Medicine , 2012, DOI: 10.1186/1472-6882-12-250
Abstract: Background Cudrania tricuspidata extract is an important traditional herbal remedy for tumors, inflammation, gastritis, and liver damage and is predominantly used in Korea, China, and Japan. However, the anti-inflammatory effects of the extract have not yet been conclusively proved. Methods In this study, we investigated the effects of the CHCl3 fraction (CTC) of a methanol extract of C. tricuspidata on the lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production in RAW 264.7 macrophage cells and mouse peritoneal macrophages, and the levels of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 in RAW 264.7 macrophage cells. Results We observed that the protein expression levels of inducible NO synthase and COX-2 enzymes were markedly inhibited by CTC in a concentration-dependent manner. In addition, CTC reduced the production of TNF-α, IL-1β, and IL-6 in the LPS-stimulated RAW 264.7 macrophage cells. Conclusions Our results show that the C. tricuspidata extract could modulate macrophage-mediated inflammatory functions such as the overproduction of cytokines, NO, and PGE2. The CTC was found to be the active fraction in this context.
Study of the Molecular Mechanism of Anti-inflammatory Activity of Bee venom in Lipopolysaccharide Stimulated RAW 264.7 Macrophages
PD Lam, PK Mandal, SY Hak, S-G Hwang
Tropical Journal of Pharmaceutical Research , 2010,
Abstract: Purpose: Bee venom (BV) is traditionally used in many inflammatory chronic conditions but its mechanism of action at molecular level is not fully understood. This study was undertaken to elucidate the mechanism of action of bee venom at the molecular level Methods: We used lipopolysaccharide (LPS) stimulation in Raw 264.7 macrophage (RM) cells and studied the effect of BV on cell proliferation, inflammation related protein expression by western blotting and RNA expression by reverse transcriptase polymerase chain reaction (RT-PCR). Results: Bee venom was toxic to RM cells above10 μg/ml but reduced the production of nitric oxide (NO) at 2–10 μg/ml in LPS stimulated RM cells by inhibiting the expression of inducible nitric oxide synthase (iNOS) and cyclooxigenase (COX)-2 via nuclear factor (NF)-κB. However, bee venom also induced the pro-inflammatory cytokine, interleukin (IL)-1β via p38 mitogen activated protein kinase (MAPK) which is known to stimulate inflammatory activity. Conclusion: It seems that NFκB and p38 MAPK signal pathways are involved in triggering the functional activation of LPS-stimulated macrophage. We suggest that some components of bee venom can cause inflammation by inducing IL-1β via p38 MAPK while others act as anti-inflammatory by suppressing iNOS and COX2 via NFκB.
Raw264.7 Cells Secrete Fibroblast Growth Stimulating Activity after Differentiation to Macrophages by Stimulation with Lipopolysaccharide  [PDF]
Jing-Yang Lai, Chung-Li Shu, Kazuhiro Morishita, Tomonaga Ichikawa, Yasuhisa Fukui
CellBio (CellBio) , 2014, DOI: 10.4236/cellbio.2014.33009
Raw264.7 cells are monocytic cells that can differentiate to activated macrophages after lipopoly-saccharide (LPS) stimulation. Here, we analyzed the factors secreted by Raw264.7 cells in response to LPS. The culture media of LPS-treated Raw264.7 cells was able to stimulate growth in MEF1F2 and NIH3T3 mouse fibroblast cell lines. We identified five secreted and LPS-induced chemokines, CCL2, CCL5, CCL12, CxCL2, and CxCL10, by microarray analysis and tested their stimulatory activity. We used commercially available bacterially expressed proteins, and found only CCL12, CxCL2 and CxCL10 stimulated growth in MEF1F2 and NIH3T3 cells. The saturation density of the cells was also increased. They were not able to stimulate growth in v-Src transformed MEF1F2 or SWAP-70 transformed NIH3T3 cells. We examined signaling pathways activated by these three factors. We found that ERK and p38 MAP kinase were activated and were required for the activity to stimulate the cell growth. Other pathways including phosophatidylinositol-3 kinase (PI3K), NFκB pathways were not activated. These results suggest that Raw264.7 cells secretes growth stimulation factors for fibroblasts when differentiated to macrophages implicating that fast growth of them is related to inflamation although the reason is still unclear.
Inhibition of Nitric Oxide (NO) Production in Lipopolysaccharide (LPS)-Activated Murine Macrophage RAW 264.7 Cells by the Norsesterterpene Peroxide, Epimuqubilin A  [PDF]
Sarot Cheenpracha,Eun-Jung Park,Bahman Rostama,John M. Pezzuto,Leng Chee Chang
Marine Drugs , 2010, DOI: 10.3390/md8030429
Abstract: Seven norsesterterpene peroxides: epimuqubilin A ( 1), muqubilone B ( 2), unnamed cyclic peroxide ester ( 3), epimuqubilin B ( 4), sigmosceptrellin A methyl ester ( 5), sigmosceptrellin A ( 6), and sigmosceptrellin B methyl ester ( 7), isolated from the marine sponge Latrunculia sp., were examined with regard to their effects on nitric oxide (NO) production in lipopolysaccharide (LPS)-activated murine macrophage RAW 264.7 cells. The results indicated epimuqubilin A ( 1) possessed potent NO inhibitory activity against lipopolysaccharide (LPS)-induced nitric oxide release with an IC 50 value of 7.4 μM, a level three times greater than the positive control, L-N G-monomethyl arginine citrate, followed by 6 (sigmosceptrellin A, IC 50 = 9.9 μM), whereas other compounds exhibited only modest activity (Table 1). These compounds did not show appreciable cytotoxicity at their IC 50 values for NO–inhibitory activity. The structure–activity upon NO inhibition could be summarized as follows: (1) a monocyclic carbon skeleton framework was essential for activity,(2) free acids gave higher activity, (3) the orientation of H 3-22 with an equatorial position increased activity, and (4) a bicyclic structure reduced activity. This is the first report of a norsesterterpene peroxide with NO–inhibitory activity. In addition, compounds 1– 7 were also evaluated for their inhibitory activities in the yeast glycogen synthase kinase-3βassay. In summary, several norsesterterpene peroxides showed novel biological activities of inhibition in NO production, suggesting that these might provide leads for anti-inflammatory or cancer chemopreventive agents.
p38 MAPK信号通路在LPS诱导RAW264.7细胞表达sTREM-1中的作用
Role of p38 MAPK Signaling Pathway in sTREM-1 Expression of RAW264.7 Cells Induced by Lipopolysaccharide

- , 2015, DOI: 10.7507/1671-6205.2015041
Abstract: 目的探讨p38丝裂原激活蛋白激酶(p38 MAPK)信号通路在脂多糖(LPS)诱导RAW264.7细胞表达可溶性髓样细胞触发受体1(sTREM-1)中的作用。 方法培养小鼠巨噬细胞株RAW264.7,采用相同浓度的LPS在不同时间诱导RAW264.7细胞,应用Western blot法分别检测p38 MAPK与磷酸化p38 MAPK (p-p38MAPK)蛋白表达水平,RT-PCR法检测p38 MAPK mRNA表达水平,酶联免疫吸附(ELISA)法检测细胞培养上清液中sTREM-1表达水平。用不同浓度p38 MAPK特异性抑制剂SB203580处理细胞,观察上述指标的变化。 结果LPS可呈时间依赖性诱导RAW264.7细胞p-p38 MAPK和p38 MAPK mRNA的表达;SB203580可呈浓度依赖性抑制p-p38 MAPK和p38 MAPK mRNA的表达;SB203580阻断p38 MAPK信号转导通路后,LPS对sTREM-1表达的诱导作用受到显著抑制,并且具有剂量依赖性。 结论LPS通过p38 MAPK信号通路诱导RAW264.7细胞表达sTREM-1。
ObjectiveTo investigate the role of the p38 MAPK signaling pathway in sTREM-1 expression of RAW264.7 cells induced by lipopolysaccharide (LPS). MethodsMacrophage cell line RAW264.7 cells were cultured in vitro and induced with the same concentration of LPS at different time. The protein expressions of p38 MAPK and phosphorylation of p38 MAPK(p-p38 MAPK) were detected by Western blot. The mRNA expression of p38 MAPK was detected by RT-PCR. The level of sTREM-1 was detected by enzyme linked immunosorbent assay method.The RAW264.7 cells were treated by SB203580 at different concentration,the changes of above indexes were observed. ResultsThe p-p38 MAPK and p38 MAPK mRNA could be inducted by LPS in a time-dependent manner. The p-p38 MAPK and p38 MAPK mRNA could be inhibited by SB203580. After SB203580 blocking p38 MAPK signal transduction pathway,the sTREM-1 expression was significantly inhibited in a dose dependent manner. ConclusionLPS can induce sTREM-1 expression in RAW264.7 cells by activating the p38 MAPK signaling pathway.
Zuonin B Inhibits Lipopolysaccharide-Induced Inflammation via Downregulation of the ERK1/2 and JNK Pathways in RAW264.7 Macrophages
Mee-Young Lee,Ji-Eun Yuk,Ok-Kyung Kwon,Sei-Ryang Oh,Hyeong-Kyu Lee,Kyung-Seop Ahn
Evidence-Based Complementary and Alternative Medicine , 2012, DOI: 10.1155/2012/728196
Abstract: We investigated whether Zuonin B exerts immunological effects on RAW264.7 cells. Zuonin B, isolated from flower buds of Daphne genkwa, suppressed the levels of nitric oxide and prostaglandin E2, as well as proinflammatory cytokines, such as tumor necrosis factor-α and interleukin-(IL-) 6, in lipopolysaccharide-stimulated macrophages. Moreover, the compound inhibited cyclooxygenase-2 and inducible nitric oxide synthase expression. Zuonin B attenuated NF-kappaB (NF-κB) activation via suppressing proteolysis of inhibitor kappa B-alpha (IκB-α) and p65 nuclear translocation as well as phosphorylation of extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinase. Additionally, IL-4 and IL-13 production in ConA-induced splenocytes was inhibited by Zuonin B. In conclusion, the anti-inflammatory effects of Zuonin B are attributable to the suppression of proinflammatory cytokines and mediators via blockage of NF-κB and AP-1 activation. Based on these findings, we propose that Zuonin B is potentially an effective functional chemical candidate for the prevention of inflammatory diseases.
Inhibition of nitric oxide production in lipopolysaccharide-activated RAW 264.7 macrophages by Jeju plant extracts
Eun-Jin Yang, , Eun-Young Yim, , Gwanpil Song, , Gi-Ok Kim, Chang-Gu Hyun
Interdisciplinary Toxicology , 2009, DOI: 10.2478/v10102-009-0022-2
Abstract: Nitric oxide (NO) produced in large amounts by inducible nitric oxide synthase (iNOS) is known to be responsible for the vasodilation and hypotension observed during septic shock and inflammation. Thus, inhibitors of iNOS may be useful candidates for the treatment of inflammatory diseases accompanied by the overproduction of NO. In this study, we prepared alcoholic extracts of Jeju plants and screened them for their inhibitory activity against NO production in lipopolysaccharide (LPS)-activated macrophages. Among the 260 kinds of plant extract tested, 122 extracts showed potent inhibitory activity towards NO production by more than 25% at a concentration of 100 μg/mL. Plants such as Malus sieboldii, Vaccinium oldhamii, Corylus hallaisanensis, Carpinus laxiflora, Styrax obassia, and Securinega suffruticosa showed the most potent inhibition (above 70%) at a concentration of 100 μg/mL. The cytotoxic effects of the plant extracts were determined by colorimetric MTT assays and most plant extracts exhibited only moderate cytotoxicity at 100 μg/mL. Therefore, these plants should be considered promising candidates for the further purification of bioactive compounds and would be useful for the treatment of inflammatory diseases accompanying overproduction of NO.
XH-14, a novel danshen methoxybenzo[b]furan derivative, exhibits anti-inflammatory properties in lipopolysaccharide-treated RAW 264.7 cells
Park Geun-Mook,Jun Jong-Gab,Kim Jin-Kyung
Journal of Inflammation , 2013, DOI: 10.1186/1476-9255-10-1
Abstract: Background XH-14 isolated from Salvia miltiorrhiza is a bioactive component and adenosine antagonist. In the present study, we evaluated anti-inflammatory properties of XH-14 in murine macrophages. Methods RAW 264.7 murine macrophage cell line was cultured with various concentrations of XH-14 in the absence or presence of lipopolysaccharide (LPS). LPS-induced release and mRNA expression of inflammatory mediators were examined by ELISA and real-time PCR. The modification of signal pathways involved in inflammatory reactions was determined by Western blotting analysis. Results XH-14 suppressed the generation of nitric oxide (NO) and prostaglandin E2, and the expression of inducible NO synthase and cyclooxygenase-2 induced by LPS. Similarly, XH-14 inhibited the release of pro-inflammatory cytokines induced by LPS in RAW 264.7 cells. The underlying mechanism of XH-14 on anti-inflammatory action was correlated with down-regulation of mitogen-activated protein kinase and activator protein-1 activation. Conclusions XH-14 inhibits the production of several inflammatory mediators and so might be useful for the treatment of various inflammatory diseases.
Development and characterisation study of liposomes-encapsulated piroxicam  [cached]
H.S. Chiong,M. Nazrul Hakim,M.R. Sulaiman,Z.A. Zakaria
International Journal of Drug Delivery , 2011,
Abstract: The objective of present work was to develop a novel liposomes-based drug delivery system for a lipophilic non-steroidal anti-inflammatory drug, piroxicam. The system was prepared using proliposomes method and optimised for different preparation parameters including type of proliposomes, concentration of drug, duration of hydration and type of particle size reduction treatment used. All prepared liposomal samples were extensively characterized for their drug-entrapment and size profile using various in-vitro techniques. Present work showed that the most optimum formulation (Pro-lipoTM Duo; 12mg piroxicam per gram Pro-lipoTM; 10 hours hydration time) produced highest amount of actual drug been entrapped in liposomes (800.4 mg/g Pro-lipoTM) with a satisfactory entrapment efficiency of 15.36%. This formulation had also produced liposomal samples with a homogenous (polydispersity index = 0.45) and small particle size (359.95nm). Extrusion technique was found to cause significant reduction in drug-entrapment and size profile of drug-loaded liposomes. A 4-weeks storage study showed that drug-entrapment and size profile of liposomal samples were stable in both refrigerated and room temperature. Electron microscopy revealed that prepared liposomal samples were spherical-shaped and showed concentric lamellae. In conclusion, present work successfully demonstrated a simple, reproducible and practical method of preparation for liposomes-encapsulated piroxicam. Keywords: Proliposomes; Liposomes; Piroxicam; Encapsulation; Particle size; Transmission electron microscopy
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