Objective To examine the effect of neuropeptide Y （NPY） on TGF-β1 production in RAW264.7 macrophages. Methods Enzyme linked immunosorbent assay （ELISA） was used to detect TGF-β1 production. Cell counting kit 8 ...Objective To examine the effect of neuropeptide Y （NPY） on TGF-β1 production in RAW264.7 macrophages. Methods Enzyme linked immunosorbent assay （ELISA） was used to detect TGF-β1 production. Cell counting kit 8 （CCK-8） was used to assay the viability of RAW264.7 cells. Western blot was used to detect the phosphorylation of PI3K p85. Results NPY treatment could promote TGF-β1 production and rapid phosphorylation of PI3K p85 in RAW264.7 cells via Y1 receptor. The elevated TGF-β 1 production induced by NPY could be abolished by wortrnannin pretreatment. Conclusion NPY may elicit TGF-β production in RAW264.7 cells via Y1 receptor, and the activated PI3K pathway may account for this effect.展开更多
Two immunomodulatory polysaccharides(Vp2a-Ⅱ and Vp3) were isolated and identified from Apocynum venetum L. flowers, and their innate immune-stimulating functions and working mechanisms were evaluated in RAW264.7 cell...Two immunomodulatory polysaccharides(Vp2a-Ⅱ and Vp3) were isolated and identified from Apocynum venetum L. flowers, and their innate immune-stimulating functions and working mechanisms were evaluated in RAW264.7 cells. Both the level of released nitric oxide(NO) and expression of inducible nitric oxide synthase(iNOS) m RNA were significantly enhanced in the RAW264.7 macrophages cells treated by Vp2a-Ⅱ and Vp3. Vp2a-Ⅱ(100–800 μg/m L) and Vp3(400 μg/mL) could significantly increase the phagocytic activity of RAW264.7 cells and the secretion and m RNA expression of TNF-α and IL-6 in a concentrationdependent manner through affecting mitogen-activated protein kinase(MAPK) activity and nuclear factor κB(NF-κB) nuclear translocation. Vp2a-Ⅱ might activate the MAPK signaling pathways and induce the nuclear translocation of NF-κB p65, whilst Vp3 likely activated the NF-κB and MAPK signaling pathways without influencing the p38 MAPK route.展开更多
Objective:To explore the anti-inflammatory effect of the traditional Chinese medicine Zhikang capsule(ZKC)on lipopolysaccharide(LPS)-induced RAW264.7 cells.Methods:Safe concentrations of ZKC(0.175,0.35,and 0.7 mg/mL)w...Objective:To explore the anti-inflammatory effect of the traditional Chinese medicine Zhikang capsule(ZKC)on lipopolysaccharide(LPS)-induced RAW264.7 cells.Methods:Safe concentrations of ZKC(0.175,0.35,and 0.7 mg/mL)were used after the half-maximal inhibitory concentration(IC_(50))of RAW264.7 cells was calculated through the CCK-8 assay.In addition,the optimal intervention duration of ZKC(0.7 mg/mL)on RAW264.7 cells was determined to be 6 h,since all proinflammatory mediators[tumor necrosis factor-alpha(TNF-α),interleukin-1 beta(IL-1β),inteleukin-6(IL-6),cyclooxygenase-2(COX-2),inducible nitric oxide synthase(iNOS),and monocyte chemotactic protein-1(MCP-1)]had a decreasing tendency and relatively down-regulated mRNA expression levels as compared with other durations(4,8,and 12 h).RAW264.7 cells were pretreated with ZKC at various concentrations(0.175,0.35 and 0.7 mg/mL)for 6 h and then stimulated with LPS(1 μg/mL)for an additional 12 h.Results:In terms of inflammation,ZKC could reverse LPS-induced upregulation of TNF-α,IL-1β,IL-6,COX-2,iNOS,and MCP-1 at both the mRNA and protein levels in RAW264.7 cells in a dose-dependent manner.In terms of the NF-κB signaling pathway,ZKC could reduce phosphorylated p65 and promote M2 polarization of RAW264.7 cells under LPS stimulation in a dose-dependent manner.Moreover,ZKC exhibited a protective effect on macrophages from apoptosis.Conclusion:ZKC exhibited obvious antiinflammatory and anti-apoptotic effects on LPS-induced RAW264.7 cells at the cellular level,and a weakened NF-κB signaling pathway may be a potential significant target.展开更多
OBJECTIVE The emerging role of chronic inflammation is the major degenerative diseases of modern society such as periodontitis,atherosclerosis,rheumatoid arthritis,Parkinson′s disease and even cancer.Eight components...OBJECTIVE The emerging role of chronic inflammation is the major degenerative diseases of modern society such as periodontitis,atherosclerosis,rheumatoid arthritis,Parkinson′s disease and even cancer.Eight components were isolated from Derris laxiflora Benth.,In this study,we found these compounds from Derris laxiflora Benth suppress lipopolysaccharide-induced inflammatory response in murine macrophage(RAW 264.7)cells.METHODS RAW 264.7cells were cultured in DMEM media supplemented with 10%(V/V)heated-inactivated FBS,penicillin 100U·mL-1 and streptomycin 100μg·mL-1.The cells were incubated at 37℃in a humidified atmosphere of 5%CO2in air.RAW264.7cells were seeded in a 24-well plate at a density of 2×105 mL-1 and then incubated with or without LPS(100ng·mL-1)in the absence or presence of compounds for 24 h.Effects of these isolates on NO production were measured indirectly by analysis of nitrite levels using the Griess reaction.Quercetin was used as a positive control.RESULTS ight components were isolated from Derris laxiflora Benth.,including three new pterocarpans 7,6′-dihydroxy-3′-methoxypterocarpan(1),derrispisatin(2),derriscoumaronochromone(3)and three new flavonoids cis-3,4′-dihydroxy-5,7-dimethoxyflavan(4),derriflavanone B(5),iso-lupinenol(6)as well as two known ones,lonchocarpol A(7)and lonchocarpol D(8).The structures of these new compounds were determined by analysis of their spectroscopic data.Raw264.7 cells were treated with the compounds from Derris laxiflora Benth for 24 h.Among them,compounds 5,7 and 8 significantly suppressed the NO production in LPS-treated RAW264.7 cells with IC50 values<10μg·mL-1.CONCLUSION In this study,we found that compounds from Derris laxiflora Benth suppresses lipopolysaccharide-induced inflammatory response in murine Raw264.7 cells.展开更多
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 ...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.展开更多
Objective:To investigate the bone-resorbing effect of demethylbelamcandaquinone B(Dmcq B)extracted from Marantodes pumilum var.alata on osteoclast differentiation in RAW264.7 cells.Methods:RAW264.7 macrophages were di...Objective:To investigate the bone-resorbing effect of demethylbelamcandaquinone B(Dmcq B)extracted from Marantodes pumilum var.alata on osteoclast differentiation in RAW264.7 cells.Methods:RAW264.7 macrophages were differentiated using RANKL into osteoclast-like cells.Then,they were treated with 10μg/mL Marantodes pumilum var.alata crude aqueous extract,5μg/mL dichloromethane fraction,and 0.6μg/mL Dmcq B and 0.06μg/mL estradiol.Tartrate-resistant acid phosphatase 5b(TRACP 5b)as an osteoclast phenotypic marker was determined by TRACP staining and TRACP 5b colometric assay,and bone-resorbing pits were examined.The gene expression of pro-inflammatory cytokines(TNF-αand IL-6)was measured.Moreover,the protein expressions of pro-inflammatory cytokines(TNF-αand IL-6)and estrogen receptors were evaluated.Results:Marantodes pumilum var.alata crude aqueous extract and Dmcq B inhibited RANKL-stimulated osteoclast differentiation as evidenced by size reduction of giant multinucleated osteoclast cells,decreased TRACP 5b activity as well as the subsiding of resorbed pit area compared with normal control.In addition,they reduced the gene and protein expressions of TNF-αand IL-6.Marantodes pumilum var.alata,Dmcq B,and estradiol treatments increased the protein expressions of estrogen receptors alpha and beta in osteoclasts.Conclusions:Marantodes pumilum var.alata and its active compound,Dmcq B can inhibit osteoclast differentiation.展开更多
Background: Hyperbaric Oxygen (HBO) Therapy improves the outcome of various types of sugery, such as postoperative bone grafts, fixation of jaw fractures, and osteoplasty of jaw deformities. Therefore, it is important...Background: Hyperbaric Oxygen (HBO) Therapy improves the outcome of various types of sugery, such as postoperative bone grafts, fixation of jaw fractures, and osteoplasty of jaw deformities. Therefore, it is important to determine the effects of HBO on bone regeneration. The purpose of this study was to clarify the influence of a hyperbaric oxygen environment on bone regeneration at the osteoclastogenic cytokines level. Sample & Methods: RAW264.7 cells were stimulated under the various conditions by using a hyperbaric oxygen chamber. We evaluated the ability of the RAW264.7 macrophages to proliferate, differentiate and produce various osteoclastogenic cyto-kines. Results: A hyperbaric oxygen (HBO) environment and high concentration oxygen (HCO) environment increase cellular proliferation in a time-dependent manner. On the other hand, a HCO environment and a hyperbaric with room air (HBA) environment enhanced the differentiation of RAW264.7 cells. In addition, NFATc1 and c-Fos were increased by both the HBA environment and HCO environment. However, the effects of HBA and HCO environments were in contrast with each other with regard to RANK, TNF-α, C-FMS and TRAP. Conclusions: It was suggested that a HBO environment influenced the bone regeneration by altering osteoclastogenic cytokines level, and that a HCO environment and HBA environment acted independently on the proliferation and differentiation of macrophages and the secretion of osteoclastogenic cytokines, and that they acted in concert in a hyperbaric oxygen environment to induce conditions favoring regeneration.展开更多
背景:溶质载体家族1成员5(solute carrier family 1 member 5,SLC1A5)在多种疾病中发挥了潜在作用,但确切作用机制尚不清楚。构建稳定的SLC1A5过表达和敲低细胞模型可为深入研究SLC1A5在疾病中的确切作用机制以及发现潜在治疗靶点提供...背景:溶质载体家族1成员5(solute carrier family 1 member 5,SLC1A5)在多种疾病中发挥了潜在作用,但确切作用机制尚不清楚。构建稳定的SLC1A5过表达和敲低细胞模型可为深入研究SLC1A5在疾病中的确切作用机制以及发现潜在治疗靶点提供有力的实验工具。目的:构建小鼠SLC1A5过表达和敲低的慢病毒载体,以建立稳定转染的RAW264.7细胞株,为深入探讨SLC1A5在炎症中的作用提供实验基础。方法:根据SLC1A5基因序列设计合成引物并使用聚合酶链反应扩增该基因片段。将目的基因定向接入经Age I/Nhe I酶切的载体质粒GV492中构建重组慢病毒质粒,对阳性克隆进一步筛选后测序比对结果;pHelper1.0质粒载体、pHelper2.0质粒载体、目的质粒载体与293T细胞共同培养并转染,获得慢病毒原液进行包装和滴度测定;在此基础上,通过体外培养RAW264.7细胞,确定嘌呤霉素工作质量浓度;不同滴度的慢病毒分别与RAW264.7细胞共同培养,根据荧光强度确定转染效率;用嘌呤霉素挑选出稳定转染细胞,实时荧光定量聚合酶链反应和蛋白免疫印迹方法检测稳定转染细胞株的SLC1A5基因和蛋白表达水平。结果与结论:(1)测序序列与目的序列一致提示重组慢病毒载体构建成功;(2)过表达SLC1A5慢病毒的滴度为1×10~9 TU/mL,敲低SLC1A5慢病毒的滴度为3×10~9 TU/mL;(3)确定RAW264.7细胞嘌呤霉素工作质量浓度为3μg/mL;(4)过表达/敲低SLC1A5慢病毒转染RAW264.7细胞的最佳条件皆为HiTransG P转染增强液且感染复数值等于50;(5)过表达SLC1A5稳转细胞株中SLC1A5基因和蛋白的表达量明显上调,而敲低SLC1A5稳转细胞株中SLC1A5基因和蛋白的表达量显著下调。结果表明,成功构建了小鼠SLC1A5过表达和敲低的慢病毒载体并获得稳定转染的RAW264.7细胞株。展开更多
Objective To investigate the anti-oxidant and anti-inflammatory effects of ethanol extract of Polygala sibirica L.var megalopha Fr.(EEP)on RAW264.7 mouse macrophages.Methods RAW264.7 cells were pretreated with 0–200&...Objective To investigate the anti-oxidant and anti-inflammatory effects of ethanol extract of Polygala sibirica L.var megalopha Fr.(EEP)on RAW264.7 mouse macrophages.Methods RAW264.7 cells were pretreated with 0–200µg/mL EEP or vehicle for 2 h prior to exposure to 1µg/mL lipopolysaccharide(LPS)for 24 h.Nitric oxide(NO)and prostaglandin(PGE2)production were determined by Griess reagent and enzyme-linked immunosorbent assay(ELISA),respectively.The mRNA levels of inducible nitric oxide synthase(iNOS),cyclooxygenase-2(COX-2),tumor necrosis factorα(TNF-α),interleukin-1beta(IL-1β),and IL-6 were determined using reverse transcription polymerase chain reaction(RT-PCR).Western blot assay was used to determine the protein expressions of iNOS,COX-2,phosphorylation of extracellular regulated protein kinases(ERK1/2),c-Jun N-terminal kinase(JNK),inhibitory subunit of nuclear factor Kappa B alpha(IκB-α)and p38.Immunofluorescence was used to observe the nuclear expression of nuclear factor-κB p65(NF-κB p65).Additionally,the anti-oxidant potential of EEP was evaluated by reactive oxygen species(ROS)production and the activities of catalase(CAT)and superoxide dismutase(SOD).The 2,2-diphenyl-1-picrylhydrazyl(DPPH),hydroxyl(OH),superoxide anion(O2−)radical and nitrite scavenging activity were also measured.Results The total polyphenol and flavonoid contents of EEP were 23.50±2.16 mg gallic acid equivalent/100 g and 43.78±3.81 mg rutin equivalent/100 g.With EEP treatment(100 and 150µg/mL),there was a notable decrease in NO and PGE2 production induced by LPS in RAW264.7 cells by downregulation of iNOS and COX-2 mRNA and protein expressions(P<0.01 or P<0.05).Furthermore,with EEP treatment(150µg/mL),there was a decrease in the mRNA expression levels of TNF-α,IL-1βand IL-6,as well as in the phosphorylation of ERK,JNK and p38 mitogen-activated protein kinase(MAPK,P<0.01 or P<0.05),by blocking the nuclear translocation of NF-κB p65 in LPS-stimulated cells.In addition,EEP(100 and 150µg/mL)led to an increase in the anti-oxidant enzymes activity of SOD and CAT,with a concomitant decrease in ROS production(P<0.01 or P<0.05).EEP also indicated the DPPH,OH,O2−radical and nitrite scavenging activity.Conclusion EEP inhibited inflammatory responses in activated macrophages through blocking MAPK/NF-κB pathway and protected against oxidative stress.展开更多
The saponin ginsenoside Rk1 is a major compound isolated from ginseng.Ginsenoside Rk1 has been reported to have anti-inflammatory and anti-tumor properties and to be involved in the regulation of metabolism.However,th...The saponin ginsenoside Rk1 is a major compound isolated from ginseng.Ginsenoside Rk1 has been reported to have anti-inflammatory and anti-tumor properties and to be involved in the regulation of metabolism.However,the effect and mechanism of anti-inflammatory action of ginsenoside Rk1 has not been fully clarified.We investigated whether ginsenoside Rk1 could suppress the inflammatory response in lipopolysaccharide-stimulated RAW264.7 macrophages and to explore its mechanism of the action.RAW264.7 cells were treated with LPS(1 μg×mL^(–1))in the absence or the presence of Ginsenoside Rk1(10,20,and 40 μmol×L^(–1)).Then the inflammatory factors were tested with Griess reagents,ELISA,and RT-PCR.The proteins were analyzed by Western blotting.Ginsenoside Rk1 inhibited lipopolysaccharide-induced expression of nitric oxide(NO),interleukin(IL)-6,IL-1β,tumor necrosis factor(TNF)-α,and monocyte chemotactic protein(MCP)-1.Ginsenoside Rk1 inhibited the lipopolysaccharide-stimulated phosphorylation of NF-κB and janus kinase(Jak)2 and signal transducer and activator of transcription(Stat)3 at Ser727 and Tyr705.These data suggested that ginsenoside Rk1 could inhibit expression of inflammatory mediators and suppress inflammation further by blocking activation of NF-κB and the Jak2/Stat3 pathway in LPS-stimulated RAW264.7 cells.展开更多
Marine fungi are important members of the marine microbiome,which have been paid growing attention by scientists in recent years.The secondary metabolites of marine fungi have been reported to contain rich and diverse...Marine fungi are important members of the marine microbiome,which have been paid growing attention by scientists in recent years.The secondary metabolites of marine fungi have been reported to contain rich and diverse compounds with novel structures(Chen et al.,2019).Aspergillus terreus,the higher level marine fungus of the Aspergillus genus(family of Trichocomaceae,order of Eurotiales,class of Euro・tiomycetes,phylum of Ascomycota).展开更多
B-3 exopolysaccharide is extracted from the Antarctic psychrophilic bacterium Psychrobacter sp. B-3. We have previously shown that it activates macrophages and affects their immunoregulatory activities. To determine w...B-3 exopolysaccharide is extracted from the Antarctic psychrophilic bacterium Psychrobacter sp. B-3. We have previously shown that it activates macrophages and affects their immunoregulatory activities. To determine what genes are affected during this process, we detected the genes differentially expressed in cells of RAW264.7 macrophages treated with B-3 exopolysaccharide by transcriptomic analysis. B-3 exopolysaccharide treatment caused differential expression of 420 genes, of which 178 were up-regulated and 242 were down-regulated. These genes were shown to be involved in many aspects of cell function, mainly metabolism and immunity. Genes were enriched in multiple immune-related pathways, and the most significantly enriched genes were involved in antigen processing and presentation pathways. The pathway in which differentially expressed genes were the most significantly enriched was the metabolic pathway; specifically, the expression of many metabolic enzyme genes was altered by B-3 exopolysaccharide treatment. Additionally, the genes involved in metabolisms of amino acids, carbohydrates, lipids and nucleotides, varied to certain degrees. B-3 exopolysaccharide, therefore, appears to directly affect the immune function of RAW264.7 macrophages as an immunostimulant, or to indirectly change intracellular metabolism. This is the first study to determine the effect of an Antarctic psychrophilic bacterial exopolysaccharide on RAW264.7 macrophages. Our findings provide an important reference for research into the regulation of macrophage immune function by different polysaccharides.展开更多
文摘Objective To examine the effect of neuropeptide Y （NPY） on TGF-β1 production in RAW264.7 macrophages. Methods Enzyme linked immunosorbent assay （ELISA） was used to detect TGF-β1 production. Cell counting kit 8 （CCK-8） was used to assay the viability of RAW264.7 cells. Western blot was used to detect the phosphorylation of PI3K p85. Results NPY treatment could promote TGF-β1 production and rapid phosphorylation of PI3K p85 in RAW264.7 cells via Y1 receptor. The elevated TGF-β 1 production induced by NPY could be abolished by wortrnannin pretreatment. Conclusion NPY may elicit TGF-β production in RAW264.7 cells via Y1 receptor, and the activated PI3K pathway may account for this effect.
基金supported by Research on Precision Nutrition and Health Food,Department of Science and Technology of Henan Province(CXJD2021006)。
文摘Two immunomodulatory polysaccharides(Vp2a-Ⅱ and Vp3) were isolated and identified from Apocynum venetum L. flowers, and their innate immune-stimulating functions and working mechanisms were evaluated in RAW264.7 cells. Both the level of released nitric oxide(NO) and expression of inducible nitric oxide synthase(iNOS) m RNA were significantly enhanced in the RAW264.7 macrophages cells treated by Vp2a-Ⅱ and Vp3. Vp2a-Ⅱ(100–800 μg/m L) and Vp3(400 μg/mL) could significantly increase the phagocytic activity of RAW264.7 cells and the secretion and m RNA expression of TNF-α and IL-6 in a concentrationdependent manner through affecting mitogen-activated protein kinase(MAPK) activity and nuclear factor κB(NF-κB) nuclear translocation. Vp2a-Ⅱ might activate the MAPK signaling pathways and induce the nuclear translocation of NF-κB p65, whilst Vp3 likely activated the NF-κB and MAPK signaling pathways without influencing the p38 MAPK route.
文摘Objective:To explore the anti-inflammatory effect of the traditional Chinese medicine Zhikang capsule(ZKC)on lipopolysaccharide(LPS)-induced RAW264.7 cells.Methods:Safe concentrations of ZKC(0.175,0.35,and 0.7 mg/mL)were used after the half-maximal inhibitory concentration(IC_(50))of RAW264.7 cells was calculated through the CCK-8 assay.In addition,the optimal intervention duration of ZKC(0.7 mg/mL)on RAW264.7 cells was determined to be 6 h,since all proinflammatory mediators[tumor necrosis factor-alpha(TNF-α),interleukin-1 beta(IL-1β),inteleukin-6(IL-6),cyclooxygenase-2(COX-2),inducible nitric oxide synthase(iNOS),and monocyte chemotactic protein-1(MCP-1)]had a decreasing tendency and relatively down-regulated mRNA expression levels as compared with other durations(4,8,and 12 h).RAW264.7 cells were pretreated with ZKC at various concentrations(0.175,0.35 and 0.7 mg/mL)for 6 h and then stimulated with LPS(1 μg/mL)for an additional 12 h.Results:In terms of inflammation,ZKC could reverse LPS-induced upregulation of TNF-α,IL-1β,IL-6,COX-2,iNOS,and MCP-1 at both the mRNA and protein levels in RAW264.7 cells in a dose-dependent manner.In terms of the NF-κB signaling pathway,ZKC could reduce phosphorylated p65 and promote M2 polarization of RAW264.7 cells under LPS stimulation in a dose-dependent manner.Moreover,ZKC exhibited a protective effect on macrophages from apoptosis.Conclusion:ZKC exhibited obvious antiinflammatory and anti-apoptotic effects on LPS-induced RAW264.7 cells at the cellular level,and a weakened NF-κB signaling pathway may be a potential significant target.
基金The project supported by Department of Industrial Technology,Ministry of Economic Affairs,Chinese TaipeiMedical and Pharmaceutical Industry Technology and Development Center
文摘OBJECTIVE The emerging role of chronic inflammation is the major degenerative diseases of modern society such as periodontitis,atherosclerosis,rheumatoid arthritis,Parkinson′s disease and even cancer.Eight components were isolated from Derris laxiflora Benth.,In this study,we found these compounds from Derris laxiflora Benth suppress lipopolysaccharide-induced inflammatory response in murine macrophage(RAW 264.7)cells.METHODS RAW 264.7cells were cultured in DMEM media supplemented with 10%(V/V)heated-inactivated FBS,penicillin 100U·mL-1 and streptomycin 100μg·mL-1.The cells were incubated at 37℃in a humidified atmosphere of 5%CO2in air.RAW264.7cells were seeded in a 24-well plate at a density of 2×105 mL-1 and then incubated with or without LPS(100ng·mL-1)in the absence or presence of compounds for 24 h.Effects of these isolates on NO production were measured indirectly by analysis of nitrite levels using the Griess reaction.Quercetin was used as a positive control.RESULTS ight components were isolated from Derris laxiflora Benth.,including three new pterocarpans 7,6′-dihydroxy-3′-methoxypterocarpan(1),derrispisatin(2),derriscoumaronochromone(3)and three new flavonoids cis-3,4′-dihydroxy-5,7-dimethoxyflavan(4),derriflavanone B(5),iso-lupinenol(6)as well as two known ones,lonchocarpol A(7)and lonchocarpol D(8).The structures of these new compounds were determined by analysis of their spectroscopic data.Raw264.7 cells were treated with the compounds from Derris laxiflora Benth for 24 h.Among them,compounds 5,7 and 8 significantly suppressed the NO production in LPS-treated RAW264.7 cells with IC50 values<10μg·mL-1.CONCLUSION In this study,we found that compounds from Derris laxiflora Benth suppresses lipopolysaccharide-induced inflammatory response in murine Raw264.7 cells.
文摘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.
基金supported by NKEA Research Grant Scheme(NRGS)(NH1113D018-2)。
文摘Objective:To investigate the bone-resorbing effect of demethylbelamcandaquinone B(Dmcq B)extracted from Marantodes pumilum var.alata on osteoclast differentiation in RAW264.7 cells.Methods:RAW264.7 macrophages were differentiated using RANKL into osteoclast-like cells.Then,they were treated with 10μg/mL Marantodes pumilum var.alata crude aqueous extract,5μg/mL dichloromethane fraction,and 0.6μg/mL Dmcq B and 0.06μg/mL estradiol.Tartrate-resistant acid phosphatase 5b(TRACP 5b)as an osteoclast phenotypic marker was determined by TRACP staining and TRACP 5b colometric assay,and bone-resorbing pits were examined.The gene expression of pro-inflammatory cytokines(TNF-αand IL-6)was measured.Moreover,the protein expressions of pro-inflammatory cytokines(TNF-αand IL-6)and estrogen receptors were evaluated.Results:Marantodes pumilum var.alata crude aqueous extract and Dmcq B inhibited RANKL-stimulated osteoclast differentiation as evidenced by size reduction of giant multinucleated osteoclast cells,decreased TRACP 5b activity as well as the subsiding of resorbed pit area compared with normal control.In addition,they reduced the gene and protein expressions of TNF-αand IL-6.Marantodes pumilum var.alata,Dmcq B,and estradiol treatments increased the protein expressions of estrogen receptors alpha and beta in osteoclasts.Conclusions:Marantodes pumilum var.alata and its active compound,Dmcq B can inhibit osteoclast differentiation.
文摘Background: Hyperbaric Oxygen (HBO) Therapy improves the outcome of various types of sugery, such as postoperative bone grafts, fixation of jaw fractures, and osteoplasty of jaw deformities. Therefore, it is important to determine the effects of HBO on bone regeneration. The purpose of this study was to clarify the influence of a hyperbaric oxygen environment on bone regeneration at the osteoclastogenic cytokines level. Sample & Methods: RAW264.7 cells were stimulated under the various conditions by using a hyperbaric oxygen chamber. We evaluated the ability of the RAW264.7 macrophages to proliferate, differentiate and produce various osteoclastogenic cyto-kines. Results: A hyperbaric oxygen (HBO) environment and high concentration oxygen (HCO) environment increase cellular proliferation in a time-dependent manner. On the other hand, a HCO environment and a hyperbaric with room air (HBA) environment enhanced the differentiation of RAW264.7 cells. In addition, NFATc1 and c-Fos were increased by both the HBA environment and HCO environment. However, the effects of HBA and HCO environments were in contrast with each other with regard to RANK, TNF-α, C-FMS and TRAP. Conclusions: It was suggested that a HBO environment influenced the bone regeneration by altering osteoclastogenic cytokines level, and that a HCO environment and HBA environment acted independently on the proliferation and differentiation of macrophages and the secretion of osteoclastogenic cytokines, and that they acted in concert in a hyperbaric oxygen environment to induce conditions favoring regeneration.
文摘Objective To investigate the anti-oxidant and anti-inflammatory effects of ethanol extract of Polygala sibirica L.var megalopha Fr.(EEP)on RAW264.7 mouse macrophages.Methods RAW264.7 cells were pretreated with 0–200µg/mL EEP or vehicle for 2 h prior to exposure to 1µg/mL lipopolysaccharide(LPS)for 24 h.Nitric oxide(NO)and prostaglandin(PGE2)production were determined by Griess reagent and enzyme-linked immunosorbent assay(ELISA),respectively.The mRNA levels of inducible nitric oxide synthase(iNOS),cyclooxygenase-2(COX-2),tumor necrosis factorα(TNF-α),interleukin-1beta(IL-1β),and IL-6 were determined using reverse transcription polymerase chain reaction(RT-PCR).Western blot assay was used to determine the protein expressions of iNOS,COX-2,phosphorylation of extracellular regulated protein kinases(ERK1/2),c-Jun N-terminal kinase(JNK),inhibitory subunit of nuclear factor Kappa B alpha(IκB-α)and p38.Immunofluorescence was used to observe the nuclear expression of nuclear factor-κB p65(NF-κB p65).Additionally,the anti-oxidant potential of EEP was evaluated by reactive oxygen species(ROS)production and the activities of catalase(CAT)and superoxide dismutase(SOD).The 2,2-diphenyl-1-picrylhydrazyl(DPPH),hydroxyl(OH),superoxide anion(O2−)radical and nitrite scavenging activity were also measured.Results The total polyphenol and flavonoid contents of EEP were 23.50±2.16 mg gallic acid equivalent/100 g and 43.78±3.81 mg rutin equivalent/100 g.With EEP treatment(100 and 150µg/mL),there was a notable decrease in NO and PGE2 production induced by LPS in RAW264.7 cells by downregulation of iNOS and COX-2 mRNA and protein expressions(P<0.01 or P<0.05).Furthermore,with EEP treatment(150µg/mL),there was a decrease in the mRNA expression levels of TNF-α,IL-1βand IL-6,as well as in the phosphorylation of ERK,JNK and p38 mitogen-activated protein kinase(MAPK,P<0.01 or P<0.05),by blocking the nuclear translocation of NF-κB p65 in LPS-stimulated cells.In addition,EEP(100 and 150µg/mL)led to an increase in the anti-oxidant enzymes activity of SOD and CAT,with a concomitant decrease in ROS production(P<0.01 or P<0.05).EEP also indicated the DPPH,OH,O2−radical and nitrite scavenging activity.Conclusion EEP inhibited inflammatory responses in activated macrophages through blocking MAPK/NF-κB pathway and protected against oxidative stress.
基金supported by the National Natural Science Foundation of China(Nos.81173369,81303253,81530097,and 81222051)the National Key Technology R&D Program “New Drug Innovation” of China(Nos.2012ZX09301002-002-002 and 2012ZX09304-005)+1 种基金the Natural Science Foundation of Beijing,China(No.132210)the Doctoral Scientific Fund Project of the Ministry of Education of China(No.20120001110105)
文摘The saponin ginsenoside Rk1 is a major compound isolated from ginseng.Ginsenoside Rk1 has been reported to have anti-inflammatory and anti-tumor properties and to be involved in the regulation of metabolism.However,the effect and mechanism of anti-inflammatory action of ginsenoside Rk1 has not been fully clarified.We investigated whether ginsenoside Rk1 could suppress the inflammatory response in lipopolysaccharide-stimulated RAW264.7 macrophages and to explore its mechanism of the action.RAW264.7 cells were treated with LPS(1 μg×mL^(–1))in the absence or the presence of Ginsenoside Rk1(10,20,and 40 μmol×L^(–1)).Then the inflammatory factors were tested with Griess reagents,ELISA,and RT-PCR.The proteins were analyzed by Western blotting.Ginsenoside Rk1 inhibited lipopolysaccharide-induced expression of nitric oxide(NO),interleukin(IL)-6,IL-1β,tumor necrosis factor(TNF)-α,and monocyte chemotactic protein(MCP)-1.Ginsenoside Rk1 inhibited the lipopolysaccharide-stimulated phosphorylation of NF-κB and janus kinase(Jak)2 and signal transducer and activator of transcription(Stat)3 at Ser727 and Tyr705.These data suggested that ginsenoside Rk1 could inhibit expression of inflammatory mediators and suppress inflammation further by blocking activation of NF-κB and the Jak2/Stat3 pathway in LPS-stimulated RAW264.7 cells.
基金This work was supported by the Natural Science Foundation of Guangdong Province(No.2018A030307046)the Basic Research Project of Shenzhen Science and Technology Innovation Commission(No.JCYJ20190813105005619)+2 种基金the Shenzhen Dapeng New District Industrial Development Fund(No.KY20180203)the Shenzhen Dapeng New District Scientific and Technological Research and Development Fund(No.KJYF202001-07)the the Innovation and Development Project about Marine Economy Demostration of Zhanjiang City(No.XM-202008-01B1),China.
文摘Marine fungi are important members of the marine microbiome,which have been paid growing attention by scientists in recent years.The secondary metabolites of marine fungi have been reported to contain rich and diverse compounds with novel structures(Chen et al.,2019).Aspergillus terreus,the higher level marine fungus of the Aspergillus genus(family of Trichocomaceae,order of Eurotiales,class of Euro・tiomycetes,phylum of Ascomycota).
基金The Important National Science&Technology Specific Projects under contract No.2011ZX8001-003the National Natural Science Fundation of China under contract No.40706053Chinese Polar Environment Comprehensive Investigation&Assessment Programs under contract No.CHINARE2017-01-05
文摘B-3 exopolysaccharide is extracted from the Antarctic psychrophilic bacterium Psychrobacter sp. B-3. We have previously shown that it activates macrophages and affects their immunoregulatory activities. To determine what genes are affected during this process, we detected the genes differentially expressed in cells of RAW264.7 macrophages treated with B-3 exopolysaccharide by transcriptomic analysis. B-3 exopolysaccharide treatment caused differential expression of 420 genes, of which 178 were up-regulated and 242 were down-regulated. These genes were shown to be involved in many aspects of cell function, mainly metabolism and immunity. Genes were enriched in multiple immune-related pathways, and the most significantly enriched genes were involved in antigen processing and presentation pathways. The pathway in which differentially expressed genes were the most significantly enriched was the metabolic pathway; specifically, the expression of many metabolic enzyme genes was altered by B-3 exopolysaccharide treatment. Additionally, the genes involved in metabolisms of amino acids, carbohydrates, lipids and nucleotides, varied to certain degrees. B-3 exopolysaccharide, therefore, appears to directly affect the immune function of RAW264.7 macrophages as an immunostimulant, or to indirectly change intracellular metabolism. This is the first study to determine the effect of an Antarctic psychrophilic bacterial exopolysaccharide on RAW264.7 macrophages. Our findings provide an important reference for research into the regulation of macrophage immune function by different polysaccharides.
