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
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 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
Skin aging and most age-related diseases are associated with a low-grade chronic inflammation. The nucleoside adenosine, a potent endogenous anti-inflammatory agent, is deeply involved in inflammatory diseases and, by...Skin aging and most age-related diseases are associated with a low-grade chronic inflammation. The nucleoside adenosine, a potent endogenous anti-inflammatory agent, is deeply involved in inflammatory diseases and, by interaction with the adenosine A2 receptor (A2AR) it immediately promotes a mechanism of defence against the inflammatory damage. The aim of our study was to investigate whether polydeoxyribonucleotide (PDRN), a mixture of deoxyribonucleotides polymers of different lengths that like adenosine, binds the A2A receptor, can reduce the inflammatory state in the macrophage cell line. RAW264.7, murine macrophage cells, were incubated with PDRN in the presence and in the absence of lipopolysaccaride (LPS), which was the major component of the outer membrane of gram-negative bacteria and which acted as a strong macrophage activator. We assessed the production of nitric oxide and the secretion of inflammatory mediators (i.e., TNF-α, IL-10, IL-12 and VEGF-A). Our data showed that PDRN produced a significant decrease of inflammation in macrophages pre-stimulated with LPS, assessed in terms of the nitric oxide content (p 2A receptor, contributed to a great extent towards reducing inflammation.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
Macrophages show significant heterogeneity in function and phenotype, which could shift into different populations of cells in response to exposure to various micro-environmental signals. These changes, also termed as...Macrophages show significant heterogeneity in function and phenotype, which could shift into different populations of cells in response to exposure to various micro-environmental signals. These changes, also termed as macrophage polarization, of which play an important role in the pathogenesis of many diseases. Numerous studies have proved that Hesperidin(HDN), a traditional Chinese medicine, extracted from fruit peels of the genus citrus, play key roles in anti-inflammation, anti-tumor, anti-oxidant and so on. However, the role of HDN in macrophage polarization has never been reported. Additional, because of its poor water solubility and bioavailability. Our laboratory had synthesized many hesperidin derivatives. Among them, hesperidin derivatives-12(HDND-12) has better water solubility and bioavailability. So, we evaluated the role of HDND-12 in macrophage polarization in the present study. The results showed that the expression of Arginase-1(Arg-1), interleukin-10(IL-10), transforming growth factor β(TGF-β) were up-regulated by HDND-12, whereas the expression of inducible Nitric Oxide Synthase(iNOS) was down-regulated in LPS-and IFN-γ-treated(M1) RAW264.7 cells. Moreover, the expression of p-JAK2 and p-STAT3 were significantly decreased after stimulation with HDND-12 in M1-like macrophages. More importantly, when we taken AG490(inhibitor of JAK2/STAT3 signaling), the protein levels of iNOS were significantly reduced in AG490 stimulation group compare with control in LPS, IFN-γ and HDND-12 stimulation cells. Taken together, these findings indicated that HDND-12 could prevent polarization toward M1-like macrophages, at least in part, through modulating JAK2/STAT3 pathway.展开更多
OBJECTIVE: To investigate the effect of Young Yum pill (YYP) on inflammatory mediators in cultured RAW 264.7 cells and elucidate the nuclear factor- kappa B (NF-κB)-related mechanism behind the action. METHODS: YYP w...OBJECTIVE: To investigate the effect of Young Yum pill (YYP) on inflammatory mediators in cultured RAW 264.7 cells and elucidate the nuclear factor- kappa B (NF-κB)-related mechanism behind the action. METHODS: YYP was extracted with 95% ethanol Lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages were used to evaluate the effect of YYP on inflammatory mediators. Production of nitric oxide (NO) and prostaglandin E2 (PGE2) were measured by Griess test and enzyme-linked immunosorbent assay, respectively. The levels of genes and proteins involved in the generation of inflammatory mediators were examined using real-time polymerase chain reaction and Western blotting, respectively. RESULTS: YYP dose-dependently suppressed LPS-induced production of NO, PGE2 and tumor necrosis factor-α(TNF-α), and elevation of mRNA and protein levels of inducible NO synthase and cyclooxygenase- 2 in RAW 264.7 macrophages. These observations were associated with decreased NF-κB p65 phosphorylation and nuclear localization, enhanced Akt (protein kinase B) phosphorylation, as well as reduced inhibitor of κB (IκB)α degradation and IκB kinase α/β phosphorylation. CONCLUSION: The present study demonstrated an inhibitory effect of YYP on the NF-κB-regulated inflammatory mediators NO, PGE2 and TNF-α in LPS-stimulated RAW 264.7 macrophages, providing a pharmacological basis for the use of YYP in treating inflammatory disorders.展开更多
Lipopolysaccharides(LPS) contamination in herbal crude polysaccharides is inevitable. The present study was performed to explore the effect of polymyxin B on abolishing the influence of LPS contamination in mononuclea...Lipopolysaccharides(LPS) contamination in herbal crude polysaccharides is inevitable. The present study was performed to explore the effect of polymyxin B on abolishing the influence of LPS contamination in mononuclear cells. LPS was pretreated with polymyxin B sulfate(PB) at different concentrations for 1, 5 or 24 h, and then used to stimulate RAW264.7 and mouse peritoneal macrophages(MPMs). The nitric oxide(NO) and tumor necrosis factor-α(TNF-α) in cell culture supernatant, as the indications of cell response, were assayed. Bupleurum chinensis polysaccharides(BCPs) with trace amount contamination of LPS was treated with PB. 30 μg·mL^(–1) of PB, treating LPS(10 and 1000 ng·mL^(–1) in stimulating RAW264.7 and MPMs respectively) at 37 ℃ for 24 h, successfully abolished the stimulating effect of LPS on the cells. When the cells were stimulated with LPS, BCPs further promoted NO production. However, pretreated with PB, BCPs showed a suppression of NO production in MPMs and no change in RAW264.7. In the in vitro experiments, LPS contamination in polysaccharide might bring a great interference in assessing the activity of drug. Pretreatment with PB(30 μg·mL^(–1)) at 37 °C for 24 h was sufficient to abolish the effects of LPS contamination(10 and 1 000 ng·mL^(–1)).展开更多
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 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.
文摘Skin aging and most age-related diseases are associated with a low-grade chronic inflammation. The nucleoside adenosine, a potent endogenous anti-inflammatory agent, is deeply involved in inflammatory diseases and, by interaction with the adenosine A2 receptor (A2AR) it immediately promotes a mechanism of defence against the inflammatory damage. The aim of our study was to investigate whether polydeoxyribonucleotide (PDRN), a mixture of deoxyribonucleotides polymers of different lengths that like adenosine, binds the A2A receptor, can reduce the inflammatory state in the macrophage cell line. RAW264.7, murine macrophage cells, were incubated with PDRN in the presence and in the absence of lipopolysaccaride (LPS), which was the major component of the outer membrane of gram-negative bacteria and which acted as a strong macrophage activator. We assessed the production of nitric oxide and the secretion of inflammatory mediators (i.e., TNF-α, IL-10, IL-12 and VEGF-A). Our data showed that PDRN produced a significant decrease of inflammation in macrophages pre-stimulated with LPS, assessed in terms of the nitric oxide content (p 2A receptor, contributed to a great extent towards reducing inflammation.
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.
基金supported by the Key Fund Project of Anhui Education Department(No.KJ2016A364)National Natural Science Foundation of China(No.81473268)
文摘Macrophages show significant heterogeneity in function and phenotype, which could shift into different populations of cells in response to exposure to various micro-environmental signals. These changes, also termed as macrophage polarization, of which play an important role in the pathogenesis of many diseases. Numerous studies have proved that Hesperidin(HDN), a traditional Chinese medicine, extracted from fruit peels of the genus citrus, play key roles in anti-inflammation, anti-tumor, anti-oxidant and so on. However, the role of HDN in macrophage polarization has never been reported. Additional, because of its poor water solubility and bioavailability. Our laboratory had synthesized many hesperidin derivatives. Among them, hesperidin derivatives-12(HDND-12) has better water solubility and bioavailability. So, we evaluated the role of HDND-12 in macrophage polarization in the present study. The results showed that the expression of Arginase-1(Arg-1), interleukin-10(IL-10), transforming growth factor β(TGF-β) were up-regulated by HDND-12, whereas the expression of inducible Nitric Oxide Synthase(iNOS) was down-regulated in LPS-and IFN-γ-treated(M1) RAW264.7 cells. Moreover, the expression of p-JAK2 and p-STAT3 were significantly decreased after stimulation with HDND-12 in M1-like macrophages. More importantly, when we taken AG490(inhibitor of JAK2/STAT3 signaling), the protein levels of iNOS were significantly reduced in AG490 stimulation group compare with control in LPS, IFN-γ and HDND-12 stimulation cells. Taken together, these findings indicated that HDND-12 could prevent polarization toward M1-like macrophages, at least in part, through modulating JAK2/STAT3 pathway.
基金Supported by Wai Yuen Tong Medicine Company Limited,Innovation and Technology Fund(UIT/315)General Research Fund(GRF:12125116)of Hong KongFRG1/16-17/048 and FRG2/16-17/033 from the Hong Kong Baptist University
文摘OBJECTIVE: To investigate the effect of Young Yum pill (YYP) on inflammatory mediators in cultured RAW 264.7 cells and elucidate the nuclear factor- kappa B (NF-κB)-related mechanism behind the action. METHODS: YYP was extracted with 95% ethanol Lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages were used to evaluate the effect of YYP on inflammatory mediators. Production of nitric oxide (NO) and prostaglandin E2 (PGE2) were measured by Griess test and enzyme-linked immunosorbent assay, respectively. The levels of genes and proteins involved in the generation of inflammatory mediators were examined using real-time polymerase chain reaction and Western blotting, respectively. RESULTS: YYP dose-dependently suppressed LPS-induced production of NO, PGE2 and tumor necrosis factor-α(TNF-α), and elevation of mRNA and protein levels of inducible NO synthase and cyclooxygenase- 2 in RAW 264.7 macrophages. These observations were associated with decreased NF-κB p65 phosphorylation and nuclear localization, enhanced Akt (protein kinase B) phosphorylation, as well as reduced inhibitor of κB (IκB)α degradation and IκB kinase α/β phosphorylation. CONCLUSION: The present study demonstrated an inhibitory effect of YYP on the NF-κB-regulated inflammatory mediators NO, PGE2 and TNF-α in LPS-stimulated RAW 264.7 macrophages, providing a pharmacological basis for the use of YYP in treating inflammatory disorders.
基金supported by the National Natural Science Foundation of China(Nos.81330089,30925042,and 81274165)the State Key Program for New Drugs from the Ministry of Science and Technology,China(No.2012ZX09301001-003)the Science and Technology Commission of Shanghai Municipality(Nos.10XD1405900 and 12JC1400800)
文摘Lipopolysaccharides(LPS) contamination in herbal crude polysaccharides is inevitable. The present study was performed to explore the effect of polymyxin B on abolishing the influence of LPS contamination in mononuclear cells. LPS was pretreated with polymyxin B sulfate(PB) at different concentrations for 1, 5 or 24 h, and then used to stimulate RAW264.7 and mouse peritoneal macrophages(MPMs). The nitric oxide(NO) and tumor necrosis factor-α(TNF-α) in cell culture supernatant, as the indications of cell response, were assayed. Bupleurum chinensis polysaccharides(BCPs) with trace amount contamination of LPS was treated with PB. 30 μg·mL^(–1) of PB, treating LPS(10 and 1000 ng·mL^(–1) in stimulating RAW264.7 and MPMs respectively) at 37 ℃ for 24 h, successfully abolished the stimulating effect of LPS on the cells. When the cells were stimulated with LPS, BCPs further promoted NO production. However, pretreated with PB, BCPs showed a suppression of NO production in MPMs and no change in RAW264.7. In the in vitro experiments, LPS contamination in polysaccharide might bring a great interference in assessing the activity of drug. Pretreatment with PB(30 μg·mL^(–1)) at 37 °C for 24 h was sufficient to abolish the effects of LPS contamination(10 and 1 000 ng·mL^(–1)).
