Activation of acid-sensing ion channels （ASICs） plays an important role in neuroinflammation. Macrophage recruitment to the sites of inflammation is an essential step in host defense. ASIC1 and ASIC3 have been repor...Activation of acid-sensing ion channels （ASICs） plays an important role in neuroinflammation. Macrophage recruitment to the sites of inflammation is an essential step in host defense. ASIC1 and ASIC3 have been reported to mediate the endocytosis and maturation of bone marrow derived macrophages. However, the expression and inflammation-related functions of ASICs in RAW 264.7 cells, another common macrophage, are still elusive. In the present study, we first demonstrated the presence of ASIC 1, ASIC2a and ASIC3 in RAW 264.7 macrophage cell line by using reverse transcriptase polymerase chain reaction （RT-PCR）, Western blotting and immunofluorescence experiments. The non-specific ASICs inhibitor amiloride and specific homomeric ASICla blocker PcTxl reduced the production of iNOS and COX-2 by LPS-induced activating RAW 264.7 cells. Furthermore, not only amiloride but also PcTxl inhibited the migration and LPS-induced apoptosis of RAW 264.7 cells. Taken together, our findings suggest that ASICs promote the inflammatory response and apoptosis of RAW 264.7 cells, and ASICs may serve as a potential novel target for immunological disease therapy.展开更多
Objective: To compare the inhibitory effects of acetone extracts from the stem bark of three Acacia species(Acacia dealbata, Acacia ferruginea and Acacia leucophloea) on nitric oxide production.Methods: The lipopolysa...Objective: To compare the inhibitory effects of acetone extracts from the stem bark of three Acacia species(Acacia dealbata, Acacia ferruginea and Acacia leucophloea) on nitric oxide production.Methods: The lipopolysaccharide(LPS)-stimulated RAW 264.7 macrophage cells were used to investigate the regulatory effect of acetone extracts of three Acacia stem barks on nitric oxide production and the expression of inducible nitric oxide synthase,cyclooxygenase-2 and tumor necrosis factor-a. Further, the phenolic profile of acetone extracts from the Acacia barks was determined by liquid chromatography-mass spectrometry/mass spectrometry analysis.Results: All the three extracts significantly decreased LPS-induced NO production as well as the expression of inducible nitric oxide synthase, cyclooxygenase-2 and tumor necrosis factor-a in a concentration dependent manner(25, 50 and 75 mg/m L). In the liquid chromatography-mass spectrometry/mass spectrometry analysis, acetone extract of Acacia ferruginea bark revealed the presence of 12 different phenolic components including quercetin, catechin, ellagic acid and rosmanol. However, Acacia dealbata and Acacia leucophloea barks each contained 6 different phenolic components.Conclusions: The acetone extracts of three Acacia species effectively inhibited the NO production in LPS-stimulated RAW 264.7 cells and the presence of different phenolic components in the bark extracts might be responsible for reducing the NO level in cells.展开更多
Diaporisoindole B(DPB),an isoprenylisoindole alkaloid isolated from the mangrove endophytic fungus Diaporthe sp.SYSU-HQ3,has been proved to inhibit the production of nitric oxide(NO)in lipopolysaccharide(LPS)-challeng...Diaporisoindole B(DPB),an isoprenylisoindole alkaloid isolated from the mangrove endophytic fungus Diaporthe sp.SYSU-HQ3,has been proved to inhibit the production of nitric oxide(NO)in lipopolysaccharide(LPS)-challenged RAW 264.7 mouse macrophages,showing potent anti-inflammatory effects.In this study,we further investigated the anti-inflammatory effects of DPB and explored the possible mechanisms in LPS-challenged RAW 264.7 mouse macrophages.The results showed that DPB(3.125,6.2,12.5 and 25μM)could significantly reduce LPS-induced levels of PGE2,and inhibit the expressions of i NOS and COX-2 in a dose-dependent manner.In addition,DPB also inhibited LPS-induced production of inflammatory cytokines,including TNF-α,IL-1β,IL-6.Moreover,we further investigated signal transduction mechanisms by which DPB exerted anti-inflammatory effects.DPB could affect LPS-mediated nuclear factor kappa B(NF-κB)signaling pathway activation via down-regulating the upstream myeloid differentiation protein 88(MyD88)at the protein level.Additionally,DPB also strongly inhibited the phosphorylation of mitogen-activated protein kinases(MAPKs),including extracellular signal-regulated kinase(ERK)1/2,c-Jun N-terminal kinase(JNK)and p38.Therefore,DPB might exert anti-inflammatory effects by suppressing NF-κB activation and MAPKs pathways via down-regulating MyD88 in RAW 264.7 cells.展开更多
Objective:To study the protective effect of paeonol on LPS-induced inflammatory injury of macrophage RAW264.7 cells and its mechanism.Methods:Macrophage RAW264.7 cells were cultured and divided into blank group,LPS(1...Objective:To study the protective effect of paeonol on LPS-induced inflammatory injury of macrophage RAW264.7 cells and its mechanism.Methods:Macrophage RAW264.7 cells were cultured and divided into blank group,LPS(1μg/mL)group,paeonol(240μmol/mL)group and TAK242(10μmol/mL)group.The cell activity was detected by CCK8 method,the cell morphology was observed by inverted microscope,the contents of GSH and MDA in cell culture medium were determined by colorimetry,the mitochondrial membrane potential was detected by JC-1 method,the expression distribution of F4/80 and p-NF-κB protein was detected by immunofluorescence method,and the expression of TLR4/MAPK/NF-κB related pathway protein was detected by Western blotting.Results:Compared with the blank group,the cell viability induced by 1μg/mL LPS was 0.4972±0.061(P<0.01),which was close to the half inhibition rate.Compared with LPS group,the expression of p-NF-κB protein in 240μmol/mL paeonol pretreated cell group was down-regulated most significantly(P<0.01),and the expression of TLR4 protein was inhibited most significantly in 10μmol/mL TAK242 pretreated cell group.Compared with LPS group(P<0.01),the cell morphology of paeonol group recovered.Decrease MDA content and increase GSH content in cell culture medium(P<0.01),In the results of mitochondrial membrane potential,the red light of paeonol group was significantly enhanced and the green light was significantly weakened(P<0.001).The expression distribution of F4/80 and p-NF-κB protein in paeonol group decreased significantly(P<0.01),and the expressions of TLR4,p-IκB,p-p38,p-JNK and p-NF-κB protein were down-regulated(P<0.05).Conclusion:Paeonol can improve the inflammatory injury of RAW264.7 cells induced by LPS,and its mechanism may be related to TLR4/MAPK/NF-κB pathway.展开更多
Objective To investigate in vitro cytotoxicity and oxidative stress response induced by multiwalled carbon nanotubes （MWCNTs）. Methods Cultured macrophages （murine RAW264.7 cells） and alveolar epithelium cells typ...Objective To investigate in vitro cytotoxicity and oxidative stress response induced by multiwalled carbon nanotubes （MWCNTs）. Methods Cultured macrophages （murine RAW264.7 cells） and alveolar epithelium cells type II （human A549 lung cells） were exposed to the blank control, DNA salt control, and the MWCNTs suspensions at 2.5, 10, 25, and 100 ug/mL for 24 h. Each treatment was evaluated by cell viability, cytotoxicity and oxidative stress. Results Overall, both cell lines had similar patterns in response to the cytotoxicity and oxidative stress of MWCNTs. DNA salt treatment showed no change compared to the blank control. In both cell lines, significant changes at the doses of 25 and 100 ug/mL treatments were found in cell viabilities, cytotoxicity, and oxidative stress indexes. The reactive oxygen species （ROS） generation was also found to be significantly higher at the dose of 10 ug/mL treatment, whereas no change was seen in most of the indexes. The ROS generation in both cell lines went up in minutes, reached the climax within an hour and faded down after several hours. Conclusion Exposure to MWCNTs resulted in a dose-dependent cytotoxicity in cultured RAW264.7 cells and A549 cells, that was closely correlated to the increased oxidative stress.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
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
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 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
Macrophages play an important role in peripheral nerve regeneration,but the specific mechanism of regeneration is still unclear.Our preliminary findings indicated that neutrophil peptide 1 is an innate immune peptide ...Macrophages play an important role in peripheral nerve regeneration,but the specific mechanism of regeneration is still unclear.Our preliminary findings indicated that neutrophil peptide 1 is an innate immune peptide closely involved in peripheral nerve regeneration.However,the mechanism by which neutrophil peptide 1 enhances nerve regeneration remains unclear.This study was designed to investigate the relationship between neutrophil peptide 1 and macrophages in vivo and in vitro in peripheral nerve crush injury.The functions of RAW 264.7 cells we re elucidated by Cell Counting Kit-8 assay,flow cytometry,migration assays,phagocytosis assays,immunohistochemistry and enzyme-linked immunosorbent assay.Axonal debris phagocytosis was observed using the CUBIC(Clear,Unobstructed Brain/Body Imaging Cocktails and Computational analysis)optical clearing technique during Wallerian degeneration.Macrophage inflammatory factor expression in different polarization states was detected using a protein chip.The results showed that neutrophil peptide 1 promoted the prolife ration,migration and phagocytosis of macrophages,and CD206 expression on the surfa ce of macrophages,indicating M2 polarization.The axonal debris clearance rate during Wallerian degeneration was enhanced after neutrophil peptide 1 intervention.Neutrophil peptide 1 also downregulated inflammatory factors interleukin-1α,-6,-12,and tumor necrosis factor-αin invo and in vitro.Thus,the results suggest that neutrophil peptide 1 activates macrophages and accelerates Wallerian degeneration,which may be one mechanism by which neutrophil peptide 1 enhances peripheral nerve regeneration.展开更多
Previous studies have suggested that polypeptides extracted from milk, soybean, fish, eggs, and meat possess potential anti-inflammatory effects. To date, few studies have reported the anti-inflammatory function of st...Previous studies have suggested that polypeptides extracted from milk, soybean, fish, eggs, and meat possess potential anti-inflammatory effects. To date, few studies have reported the anti-inflammatory function of sturgeon peptides and their underlying mechanisms are unknown. The current study was therefore to determine the anti-inflammatory potential of sturgeon peptides with lipopolysaccharide (LPS)-induced RAW264.7 inflammatory model. Pepsin hydrolysate (PeH) was purified by ultrafiltration and Sephadex G-15 gel filtration chromatography. PeH significantly reduced the inflammatory mediator (NO) and inflammatory cytokines (IL-6, TNF-α and IL-1β) expression in a dose-dependent manner. Moreover, the purified sturgeon peptide (F2) possessed strong antioxidant potential and effectively inhibited DPPH and ABTS free radicals. F2 significantly suppressed the expression of MAPK, IκBα, and NF-κB p65, indicating that F2 exerted anti-inflammatory influence by the inhibition of MAPK and NF-κB pathways.展开更多
In the present study, 3-methylcarbazole and 1-methoxy-3-methylcarbazole were isolated from the culture of Streptomyces sp. LJK109, endophyte of Alpinia galanga Swartz. 3-methylcarbazole, a carbazole derivative, has be...In the present study, 3-methylcarbazole and 1-methoxy-3-methylcarbazole were isolated from the culture of Streptomyces sp. LJK109, endophyte of Alpinia galanga Swartz. 3-methylcarbazole, a carbazole derivative, has been found to be highly potent as anti-inflammatory agent. The immunomodulatory activity of these agents in toll like receptor (TLR)-activated RAW 264.7 macrophages induced by lipopolysaccharide (LPS), Poly(I:C), and pam3CSK was investigated by assessing nitric oxide (NO) and pro-inflammatory cytokines. The 3-methylcarbazoles dose-dependently suppressed the release of NO, PGE2, TNF-α, IL-1β, IL-6 and IL-10 in LPS- and pam3CSK-activated macrophages but not in Poly(I:C)-activated macrophages. Our results suggest that 3-methylcarbazoles can be further developed as a promising anti-inflammatory remedy.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 investigate which life stage of the parasite has the ability to stimulate release of pro- or anti-inflammatory mediators from macrophages.Methods:The human macrophage/ monocyte cell line THP-1,the mouse m...Objective:To investigate which life stage of the parasite has the ability to stimulate release of pro- or anti-inflammatory mediators from macrophages.Methods:The human macrophage/ monocyte cell line THP-1,the mouse macrophage cell line RAW 264.7 and naive peritoneal macrophages（PM） from the rodent host Mastomys coucha（M.coucha） were incubated at 37℃in 5%CO<sub>2</sub> atmosphere with extracts of microfilariae（Mf）,third stage infective larvae（L<sub>3</sub>） and adult worms（Ad） of Brugia malayi.After 48 hr post exposure,IL-1β,IL-6,TNF-α,IL-10 and nitric oxide（NO） in cell-free supernatants were estimated.Results:Extracts of all the life stages of the parasite were capable of stimulating pro-（IL-1β,IL-6 and TNF-α） and anti-inflammatory （IL-10） cytokines in both the cell lines and peritoneal macrophages of M.coucha.Mf was the strongest stimulator of pro-inflammatory cytokines followed by L<sub>3</sub> and Ad;however,Ad was a strong stimulator of IL-10 release.Mf was found to have potential to modulate LPS-induced NO release in RAW cells.Ad-induced NO release was concentration dependent with maximum at 20μg/mL in both RAW and PMs.Conclusions:The results show that parasites at all life stages were capable of stimulating pro-（IL-1β,IL-6 and TNF-α） and anti-inflammatory（IL-10） cytokines and NO release from macrophages of susceptible host M.coucha,human and mouse macrophage cell lines.Mf can suppress the LPS-induced NO release in RAW cells.The findings also show that the two cell lines may provide a convenient in vitro system for assaying parasite-induced inflammatory mediator release.展开更多
基金This work was supported by grants from the National Natural science Foundation of China （No. 81473199）, and the Fundamental Research Funds for the Central Universities （No, 015TS 125）.
文摘Activation of acid-sensing ion channels （ASICs） plays an important role in neuroinflammation. Macrophage recruitment to the sites of inflammation is an essential step in host defense. ASIC1 and ASIC3 have been reported to mediate the endocytosis and maturation of bone marrow derived macrophages. However, the expression and inflammation-related functions of ASICs in RAW 264.7 cells, another common macrophage, are still elusive. In the present study, we first demonstrated the presence of ASIC 1, ASIC2a and ASIC3 in RAW 264.7 macrophage cell line by using reverse transcriptase polymerase chain reaction （RT-PCR）, Western blotting and immunofluorescence experiments. The non-specific ASICs inhibitor amiloride and specific homomeric ASICla blocker PcTxl reduced the production of iNOS and COX-2 by LPS-induced activating RAW 264.7 cells. Furthermore, not only amiloride but also PcTxl inhibited the migration and LPS-induced apoptosis of RAW 264.7 cells. Taken together, our findings suggest that ASICs promote the inflammatory response and apoptosis of RAW 264.7 cells, and ASICs may serve as a potential novel target for immunological disease therapy.
基金Supported in part by the Ministry of Trade,Industry and Energy,Korea Institute for Advancement of Technology(KIAT)through the Inter-ER Cooperation Project(Project No.R0000474)
文摘Objective: To compare the inhibitory effects of acetone extracts from the stem bark of three Acacia species(Acacia dealbata, Acacia ferruginea and Acacia leucophloea) on nitric oxide production.Methods: The lipopolysaccharide(LPS)-stimulated RAW 264.7 macrophage cells were used to investigate the regulatory effect of acetone extracts of three Acacia stem barks on nitric oxide production and the expression of inducible nitric oxide synthase,cyclooxygenase-2 and tumor necrosis factor-a. Further, the phenolic profile of acetone extracts from the Acacia barks was determined by liquid chromatography-mass spectrometry/mass spectrometry analysis.Results: All the three extracts significantly decreased LPS-induced NO production as well as the expression of inducible nitric oxide synthase, cyclooxygenase-2 and tumor necrosis factor-a in a concentration dependent manner(25, 50 and 75 mg/m L). In the liquid chromatography-mass spectrometry/mass spectrometry analysis, acetone extract of Acacia ferruginea bark revealed the presence of 12 different phenolic components including quercetin, catechin, ellagic acid and rosmanol. However, Acacia dealbata and Acacia leucophloea barks each contained 6 different phenolic components.Conclusions: The acetone extracts of three Acacia species effectively inhibited the NO production in LPS-stimulated RAW 264.7 cells and the presence of different phenolic components in the bark extracts might be responsible for reducing the NO level in cells.
基金National Natural Science Foundation of China(Grant No.41906033)the Natural Science Foundation of Guangdong Province of China(Grant No.2019A1515012084)。
文摘Diaporisoindole B(DPB),an isoprenylisoindole alkaloid isolated from the mangrove endophytic fungus Diaporthe sp.SYSU-HQ3,has been proved to inhibit the production of nitric oxide(NO)in lipopolysaccharide(LPS)-challenged RAW 264.7 mouse macrophages,showing potent anti-inflammatory effects.In this study,we further investigated the anti-inflammatory effects of DPB and explored the possible mechanisms in LPS-challenged RAW 264.7 mouse macrophages.The results showed that DPB(3.125,6.2,12.5 and 25μM)could significantly reduce LPS-induced levels of PGE2,and inhibit the expressions of i NOS and COX-2 in a dose-dependent manner.In addition,DPB also inhibited LPS-induced production of inflammatory cytokines,including TNF-α,IL-1β,IL-6.Moreover,we further investigated signal transduction mechanisms by which DPB exerted anti-inflammatory effects.DPB could affect LPS-mediated nuclear factor kappa B(NF-κB)signaling pathway activation via down-regulating the upstream myeloid differentiation protein 88(MyD88)at the protein level.Additionally,DPB also strongly inhibited the phosphorylation of mitogen-activated protein kinases(MAPKs),including extracellular signal-regulated kinase(ERK)1/2,c-Jun N-terminal kinase(JNK)and p38.Therefore,DPB might exert anti-inflammatory effects by suppressing NF-κB activation and MAPKs pathways via down-regulating MyD88 in RAW 264.7 cells.
文摘Objective:To study the protective effect of paeonol on LPS-induced inflammatory injury of macrophage RAW264.7 cells and its mechanism.Methods:Macrophage RAW264.7 cells were cultured and divided into blank group,LPS(1μg/mL)group,paeonol(240μmol/mL)group and TAK242(10μmol/mL)group.The cell activity was detected by CCK8 method,the cell morphology was observed by inverted microscope,the contents of GSH and MDA in cell culture medium were determined by colorimetry,the mitochondrial membrane potential was detected by JC-1 method,the expression distribution of F4/80 and p-NF-κB protein was detected by immunofluorescence method,and the expression of TLR4/MAPK/NF-κB related pathway protein was detected by Western blotting.Results:Compared with the blank group,the cell viability induced by 1μg/mL LPS was 0.4972±0.061(P<0.01),which was close to the half inhibition rate.Compared with LPS group,the expression of p-NF-κB protein in 240μmol/mL paeonol pretreated cell group was down-regulated most significantly(P<0.01),and the expression of TLR4 protein was inhibited most significantly in 10μmol/mL TAK242 pretreated cell group.Compared with LPS group(P<0.01),the cell morphology of paeonol group recovered.Decrease MDA content and increase GSH content in cell culture medium(P<0.01),In the results of mitochondrial membrane potential,the red light of paeonol group was significantly enhanced and the green light was significantly weakened(P<0.001).The expression distribution of F4/80 and p-NF-κB protein in paeonol group decreased significantly(P<0.01),and the expressions of TLR4,p-IκB,p-p38,p-JNK and p-NF-κB protein were down-regulated(P<0.05).Conclusion:Paeonol can improve the inflammatory injury of RAW264.7 cells induced by LPS,and its mechanism may be related to TLR4/MAPK/NF-κB pathway.
基金supported partly by a grant from Shanghai Science and Technology Committee International Collaboration Program (Project No. 055207078)
文摘Objective To investigate in vitro cytotoxicity and oxidative stress response induced by multiwalled carbon nanotubes （MWCNTs）. Methods Cultured macrophages （murine RAW264.7 cells） and alveolar epithelium cells type II （human A549 lung cells） were exposed to the blank control, DNA salt control, and the MWCNTs suspensions at 2.5, 10, 25, and 100 ug/mL for 24 h. Each treatment was evaluated by cell viability, cytotoxicity and oxidative stress. Results Overall, both cell lines had similar patterns in response to the cytotoxicity and oxidative stress of MWCNTs. DNA salt treatment showed no change compared to the blank control. In both cell lines, significant changes at the doses of 25 and 100 ug/mL treatments were found in cell viabilities, cytotoxicity, and oxidative stress indexes. The reactive oxygen species （ROS） generation was also found to be significantly higher at the dose of 10 ug/mL treatment, whereas no change was seen in most of the indexes. The ROS generation in both cell lines went up in minutes, reached the climax within an hour and faded down after several hours. Conclusion Exposure to MWCNTs resulted in a dose-dependent cytotoxicity in cultured RAW264.7 cells and A549 cells, that was closely correlated to the increased oxidative stress.
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 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.
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.
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 the National Natural Science Foundation of China,No.32371048(to YK)the Peking University People’s Hospital Research and Development Funds,No.RDX2021-01(to YK)the Natural Science Foundation of Beijing,No.7222198(to NH)。
文摘Macrophages play an important role in peripheral nerve regeneration,but the specific mechanism of regeneration is still unclear.Our preliminary findings indicated that neutrophil peptide 1 is an innate immune peptide closely involved in peripheral nerve regeneration.However,the mechanism by which neutrophil peptide 1 enhances nerve regeneration remains unclear.This study was designed to investigate the relationship between neutrophil peptide 1 and macrophages in vivo and in vitro in peripheral nerve crush injury.The functions of RAW 264.7 cells we re elucidated by Cell Counting Kit-8 assay,flow cytometry,migration assays,phagocytosis assays,immunohistochemistry and enzyme-linked immunosorbent assay.Axonal debris phagocytosis was observed using the CUBIC(Clear,Unobstructed Brain/Body Imaging Cocktails and Computational analysis)optical clearing technique during Wallerian degeneration.Macrophage inflammatory factor expression in different polarization states was detected using a protein chip.The results showed that neutrophil peptide 1 promoted the prolife ration,migration and phagocytosis of macrophages,and CD206 expression on the surfa ce of macrophages,indicating M2 polarization.The axonal debris clearance rate during Wallerian degeneration was enhanced after neutrophil peptide 1 intervention.Neutrophil peptide 1 also downregulated inflammatory factors interleukin-1α,-6,-12,and tumor necrosis factor-αin invo and in vitro.Thus,the results suggest that neutrophil peptide 1 activates macrophages and accelerates Wallerian degeneration,which may be one mechanism by which neutrophil peptide 1 enhances peripheral nerve regeneration.
文摘Previous studies have suggested that polypeptides extracted from milk, soybean, fish, eggs, and meat possess potential anti-inflammatory effects. To date, few studies have reported the anti-inflammatory function of sturgeon peptides and their underlying mechanisms are unknown. The current study was therefore to determine the anti-inflammatory potential of sturgeon peptides with lipopolysaccharide (LPS)-induced RAW264.7 inflammatory model. Pepsin hydrolysate (PeH) was purified by ultrafiltration and Sephadex G-15 gel filtration chromatography. PeH significantly reduced the inflammatory mediator (NO) and inflammatory cytokines (IL-6, TNF-α and IL-1β) expression in a dose-dependent manner. Moreover, the purified sturgeon peptide (F2) possessed strong antioxidant potential and effectively inhibited DPPH and ABTS free radicals. F2 significantly suppressed the expression of MAPK, IκBα, and NF-κB p65, indicating that F2 exerted anti-inflammatory influence by the inhibition of MAPK and NF-κB pathways.
文摘In the present study, 3-methylcarbazole and 1-methoxy-3-methylcarbazole were isolated from the culture of Streptomyces sp. LJK109, endophyte of Alpinia galanga Swartz. 3-methylcarbazole, a carbazole derivative, has been found to be highly potent as anti-inflammatory agent. The immunomodulatory activity of these agents in toll like receptor (TLR)-activated RAW 264.7 macrophages induced by lipopolysaccharide (LPS), Poly(I:C), and pam3CSK was investigated by assessing nitric oxide (NO) and pro-inflammatory cytokines. The 3-methylcarbazoles dose-dependently suppressed the release of NO, PGE2, TNF-α, IL-1β, IL-6 and IL-10 in LPS- and pam3CSK-activated macrophages but not in Poly(I:C)-activated macrophages. Our results suggest that 3-methylcarbazoles can be further developed as a promising anti-inflammatory remedy.
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.
基金supported by a grant Indian Council of Medical Research, New Delhi and SP/SO/B-46/2000 from the Department of Science and Technology,New DelhiUGC Senior Research Fellowship support to SKV
文摘Objective:To investigate which life stage of the parasite has the ability to stimulate release of pro- or anti-inflammatory mediators from macrophages.Methods:The human macrophage/ monocyte cell line THP-1,the mouse macrophage cell line RAW 264.7 and naive peritoneal macrophages（PM） from the rodent host Mastomys coucha（M.coucha） were incubated at 37℃in 5%CO<sub>2</sub> atmosphere with extracts of microfilariae（Mf）,third stage infective larvae（L<sub>3</sub>） and adult worms（Ad） of Brugia malayi.After 48 hr post exposure,IL-1β,IL-6,TNF-α,IL-10 and nitric oxide（NO） in cell-free supernatants were estimated.Results:Extracts of all the life stages of the parasite were capable of stimulating pro-（IL-1β,IL-6 and TNF-α） and anti-inflammatory （IL-10） cytokines in both the cell lines and peritoneal macrophages of M.coucha.Mf was the strongest stimulator of pro-inflammatory cytokines followed by L<sub>3</sub> and Ad;however,Ad was a strong stimulator of IL-10 release.Mf was found to have potential to modulate LPS-induced NO release in RAW cells.Ad-induced NO release was concentration dependent with maximum at 20μg/mL in both RAW and PMs.Conclusions:The results show that parasites at all life stages were capable of stimulating pro-（IL-1β,IL-6 and TNF-α） and anti-inflammatory（IL-10） cytokines and NO release from macrophages of susceptible host M.coucha,human and mouse macrophage cell lines.Mf can suppress the LPS-induced NO release in RAW cells.The findings also show that the two cell lines may provide a convenient in vitro system for assaying parasite-induced inflammatory mediator release.
