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
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: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
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 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
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.
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 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.
