AIM: To investigate whether induction of tolerance of mice to lipopolysaccharide (LPS) was able to inhibit apoptotic reaction in terms of characteristic DNA fragmentation and protect mice from lethal effect. METHODS: ...AIM: To investigate whether induction of tolerance of mice to lipopolysaccharide (LPS) was able to inhibit apoptotic reaction in terms of characteristic DNA fragmentation and protect mice from lethal effect. METHODS: Experimental groups of mice were pretreated with non-lethal amount of LPS (0.05 μg). Both control and experimental groups simultaneously were challenged with LPS plus D-GaIN for 6-7 h. The evaluations of both DNA fragmentations from the livers and the protection efficacy against lethality to mice through induction of tolerance to LPS were conducted. RESULTS: In the naive mice challenge with LPS plus D-GaIN resulted in complete death in 24 h, whereas a characteristic apoptotic DNA fragmentation was exclusively seen in the livers of mice receiving LPS in combination with D-GaIN. The mortality in the affected mice was closely correlated to the onset of DNA fragmentation. By contrast, in the mice pre-exposed to LPS, both lethal effect and apoptotic DNA fragmentation were suppressed when challenged with LPS/D-GalN. In addition to LPS, the induction of mouse tolerance to TNF also enabled mice to cross-react against death and apoptotic DNA fragmentation when challenged with TNF and/or LPS in the presence of D-GaIN. Moreover, this protection effect by LPS could last up to 24 h. TNFR1 rather than TNFR2 played a dual role in signaling pathway of either induction of tolerance to LPS for the protection of mice from mortality or inducing morbidity leading to the death of mice. CONCLUSION: The mortality of D-GalN-treated mice in response to LPS was exceedingly correlated to the onset of apoptosis in the liver, which can be effectively suppressed by brief exposure of mice to a minute amount of LPS. The induced tolerance status was mediated not only by LPS but also by TNF. The developed tolerance to either LPS or TNF can be reciprocally cross-reacted between LPS and TNF challenges, whereas the signaling of induction of tolerance and promotion of apoptosis was through TNFR1, rather than TNFR2.展开更多
Objective： The aim of the study was to investigate the effect of lipopolysaccharide （LPS） on the expression of nuclear factor kappa B （NF-κB） in 4-（methylitrosamino）-1-（3-pyridyl）-1-butanone （NNK）-mediated...Objective： The aim of the study was to investigate the effect of lipopolysaccharide （LPS） on the expression of nuclear factor kappa B （NF-κB） in 4-（methylitrosamino）-1-（3-pyridyl）-1-butanone （NNK）-mediated primary mouse peritoneal macrophages in vitro. Methods： The activity of peritoneal rnacrophages treated with different concentrations of LPS was detected by MTT assay in rider to find the optimal concentration. Peritoneal macrophages were also treated with NNK （100-500 μM）, with or without LPS for 9 h. The expression of NF-κB was demonstrated via immunocytochemistry （ICC） and Western- blot, respectively. Results： The concentration of LPS at 25 μg/mL was found to be the optimal concentration to improve the activity of peritoneal macrophages （P 〈 0.01）. Simultaneously, LPS （25 μg/mL） increased the expression of NF-κB in both the nucleus and cytoplasm and facilitated transfer of NF-κB to the nucleus. NNK treatment significantly inhibited the expression of NF-κB in a concentration-dependent manner, among the LPS-stimulated or unstimulated peritoneal macrophages, especially when cotreated with LPS （25 μg/mL, P 〈 0.01 ）. Furthermore, NNK treatment （500 μM） with LPS yielded a significant decrease in NF-κB translocation to nucleus and inhibited the expression of NF-κB （P 〈 0.005）. Conclusion： LPS enhances the suppression of NF-κB expression in NNK-mediated mouse peritoneal macrophages, which may provide a theoretical basis for the inhibition of cancer.展开更多
In order to analyze the mechanism of immunomodulation by LPS on murine peritoneal suppressor macrophages, we have, using RNase protection assay,checked the changes of mRNA expression pattern of several cytokine genes ...In order to analyze the mechanism of immunomodulation by LPS on murine peritoneal suppressor macrophages, we have, using RNase protection assay,checked the changes of mRNA expression pattern of several cytokine genes during the immuno-modulation.It has been found that, after treating peritoneal suppressor macrophages with LPS, mRNAs of IL-12 p35, IL-12 p40,IL-6 and IFN-γ are newly appeared, while those of IL-1α, IL-1β and IL-1Ra are increased and those of other cytokines, like TGF-β1 and MIF are not changed at all.It seems certain that those cytokines, whose expression is increased by LPS stimulation, may be responsible for the functional changes of suppressor macrophages during immuno-modulation. Among these changes, the appearance of IL-12 mRNA may play a critical role, and, in this regard, the synergetic effect between IFN-γ and LPS on the increase of IL-12 p35 and IL-12 p40 mRNA expression is an interesting finding.展开更多
Lipopolysaccharide (LPS) was found to induce significant hepatocytotoxicity against cultured mouse hepatocytes. Degeneration and necrosis of cultured hepatocytes and decrease of hepatocyte viability were prominent. T...Lipopolysaccharide (LPS) was found to induce significant hepatocytotoxicity against cultured mouse hepatocytes. Degeneration and necrosis of cultured hepatocytes and decrease of hepatocyte viability were prominent. The aspartate transferase level and 3H-TdR release in the medium were significantly increased after treatment, and the degree of these changes paralleled with LPS concentration. Various other parameters showed no significant difference between the hepatocytes cultured alone and those cocultured with nonparenchymal liver cells. However, if the nonparenchymal liver cells were isolated from mice which had been pretreated with D-galactosamine (GalN) not only was the hepatocyotoxicity induced by LPS enhanced, but the cells also showed certain cytotoxicity against cultured hepatocytes even without LPS, These results suggest that nonparenchymal liver cells might promote LPL-induced hepatocyte injury.展开更多
In this investigation,the effects of PE,EtOAc,and BuOH fractions from Zanthoxylum bungeanum on cholesterol accumulation induced by sterols and LPS were determined in vitro and in vivo.HepG2 cells induced by 25-hydroxy...In this investigation,the effects of PE,EtOAc,and BuOH fractions from Zanthoxylum bungeanum on cholesterol accumulation induced by sterols and LPS were determined in vitro and in vivo.HepG2 cells induced by 25-hydroxychoelsterol and cholesterol were employed as cell model.After treatment with PE,EtOAC,or BuOH fractions,cellular total cholesterol and apolipoprotein B secretion were significantly reduced.In addition,compared with control group,expressions of SREBP2,HMGCR,and ACAT decreased,while CYP27A1,ABCA1,and LDLR levels increased.Cholesterol accumulation was also induced in C57BL/6 mice by LPS and the mice were used as the animal model.Determination of serum TNF-α level and hepatic mRNA expression of TNF-α,IL-6,iNOS,COX-2 revealed that EtOAc and BuOH fractions had anti-inflammatory effects.Furthermore,hepatic total cholesterol was reduced,accompanied by the elevation of LXR-α and ABCA1 gene expression in BuOH fraction treated mice.Since EtOAc and BuOH fractions were found active,bioassay-guided isolation was performed and β-sitosterol,eudesmin,sesamin and syringaresinol-β-D-glucoside were isolated from the fractions.展开更多
Ginsenosides, the main active constituents of Panax ginseng Meyer （P. ginseng）, have potential therapeutic effects. All tested ginsenosides except gin- senoside F1 have previously been reported in inflammation studi...Ginsenosides, the main active constituents of Panax ginseng Meyer （P. ginseng）, have potential therapeutic effects. All tested ginsenosides except gin- senoside F1 have previously been reported in inflammation studies using the RAW 264.7 macrophage cell line. We ex- amined the anti-inflammatory effects of single sugar moiety ginsenosides such as compound K （CK）, Rh2, Rhl, and F1 that were isolated from P. ginseng through in silico docking studies. We investigated their biological activity predictions, including absorption, distribution, metabolism, excretion, and toxicity and PASS properties, on the suppression of NF- κB, followed by in vitro validation in lipopolysaccharide （LPS）-stimulated RAW 264.7 macrophage cells. The molecular docking results of our study showed that all treated ginsenosides are non-toxic and may be drug-like molecules. The molecular binding interactions of these ginsenosides with the active residues of NF-κB noticeably support their anti-inflammatory activity. CK and Rhl sig- nificantly reduced the production of nitric oxide, cyclooxy- genase-2 （COX-2）, and pro-inflammatory cytokines such as prostaglandin E2 and tumor necrosis factor alpha （TNF-α） in a dose-dependent manner. Real-time PCR and Western blot analyses further confirmed that protopanaxadiols （PPDs） and protopanaxatriols （PPTs） inhibitory effects may have been due to the down-regulation of TNF-α, inducible nitric oxide synthase, COX2, nuclear factor kappa B （NF-κB）, and I kappa B kinase. The expression of co-stimulatory molecules such as ROS was also inhibited by CK and Rhl in a dose- dependent manner. Furthermore, activation of NF-κB in LPS-stimulated RAW 264.7 macrophages was significantly suppressed by CK and Rhl. Taken together, these results provide evidence that PPD- and PPT-type ginsenosides in- cluding CK and Rhl may exhibit strong anti-inflammatory effects by inhibiting pro-inflammatory mediators through down-regulation of NF-κB.展开更多
Objective: To investigate the dose and time-dep endent effects of lipopolysaccharide (LPS) on cytoskeletal F-acitn and G-actin reorganizations by visualizing their distribution and measuring their contents in human um...Objective: To investigate the dose and time-dep endent effects of lipopolysaccharide (LPS) on cytoskeletal F-acitn and G-actin reorganizations by visualizing their distribution and measuring their contents in human umbilical vein endothelial cell line ECV-304. Methods: F-actin was labeled with rhodamine-phalloidin and G -actin with deoxyribonuclease I (DNase I)conjugated with fluorescein isothiocya nate (FITC). Contents of cytoskeletal proteins were obtained by flow cytometry. Results: F-actin was mainly distributed peripherally in endoth elial cells under normal conditions. LPS stimulation caused the formation of str ess fibers and filopodia. G-actin was normally seen in perinuclear and nuclear areas in control ECV-304 cells. Under LPS stimulation,G-actin dots appeared i n the cytoplasmic region. The actin disorganization was accompanied by the time - and dose- dependent decrease in F-actin pool and increase in G-actin pool . Conclusions: LPS can induce characteristic morphological altera tions of actin cytoskeleton and formation of intercellular gap in endothelial ce lls,accompanied by changes in F-actin and G-actin pools.展开更多
This study aimed to comprehend the largely unknown role of voltage-gated potassium channel 1.3 （Kvl.3） in the phagocytic function of macrophages. We found that blocking of the Kv 1.3 channel with 100 pmol L 1 Sticho...This study aimed to comprehend the largely unknown role of voltage-gated potassium channel 1.3 （Kvl.3） in the phagocytic function of macrophages. We found that blocking of the Kv 1.3 channel with 100 pmol L 1 Stichodactyla helianthus neurotoxin （ShK） enhanced the phagocytic capacities of both resting and lipopolysaccharide （LPS）-stimulated RAW264.7 macrophages in the chicken erythrocyte system. In the fluorescein isothiocyanate （FITC）-labeled Escherichia coli k-12 system, ShK increased the phagocytic capacities of resting RAW264.7 cells, but not of the LPS-stimulated cells, as LPS alone stimulated almost satu- rated phagocytosis of the macrophages. ShK increased the nitric oxide （NO） production in LPS-activated cells, but not in rest- ing RAW264.7 cells. There was no effect of ShK alone on the cytokine secretions in resting RAW264.7 cells, but it suppressed IL-113 secretion in LPS-stimulated RAW264.7 cells. At a concentration of 100 pmol L 1, ShK did not affect the viability of the tested cells. Kv 1.3 was expressed in RAW264.7 cells; this expression was downregulated by LPS, but significantly upregulat- ed by disrupting caveolin-dependent endocytosis with filipin III. In addition, cytochalasin D, an inhibitor of actin polymeriza- tion, did not affect the Kvl.3 expression. Thus, blocking of the Kvl.3 channel enhances the phagocytic capacity and NO pro- duction of this cell line. Our results suggest that Kv 1.3 channel serves as a negative regulator of phagocytosis in macrophages and can therefore be a potential target in the treatment of macrophage dysfunction.展开更多
基金Supported by a fellowship (to Zhou B) from Max-Planck-Society, Germany, and partially supported by the National Key Basic ResearchDevelopment Program (973 Program) of China, No. 2002CB513006 (to Zhou B)
文摘AIM: To investigate whether induction of tolerance of mice to lipopolysaccharide (LPS) was able to inhibit apoptotic reaction in terms of characteristic DNA fragmentation and protect mice from lethal effect. METHODS: Experimental groups of mice were pretreated with non-lethal amount of LPS (0.05 μg). Both control and experimental groups simultaneously were challenged with LPS plus D-GaIN for 6-7 h. The evaluations of both DNA fragmentations from the livers and the protection efficacy against lethality to mice through induction of tolerance to LPS were conducted. RESULTS: In the naive mice challenge with LPS plus D-GaIN resulted in complete death in 24 h, whereas a characteristic apoptotic DNA fragmentation was exclusively seen in the livers of mice receiving LPS in combination with D-GaIN. The mortality in the affected mice was closely correlated to the onset of DNA fragmentation. By contrast, in the mice pre-exposed to LPS, both lethal effect and apoptotic DNA fragmentation were suppressed when challenged with LPS/D-GalN. In addition to LPS, the induction of mouse tolerance to TNF also enabled mice to cross-react against death and apoptotic DNA fragmentation when challenged with TNF and/or LPS in the presence of D-GaIN. Moreover, this protection effect by LPS could last up to 24 h. TNFR1 rather than TNFR2 played a dual role in signaling pathway of either induction of tolerance to LPS for the protection of mice from mortality or inducing morbidity leading to the death of mice. CONCLUSION: The mortality of D-GalN-treated mice in response to LPS was exceedingly correlated to the onset of apoptosis in the liver, which can be effectively suppressed by brief exposure of mice to a minute amount of LPS. The induced tolerance status was mediated not only by LPS but also by TNF. The developed tolerance to either LPS or TNF can be reciprocally cross-reacted between LPS and TNF challenges, whereas the signaling of induction of tolerance and promotion of apoptosis was through TNFR1, rather than TNFR2.
文摘Objective： The aim of the study was to investigate the effect of lipopolysaccharide （LPS） on the expression of nuclear factor kappa B （NF-κB） in 4-（methylitrosamino）-1-（3-pyridyl）-1-butanone （NNK）-mediated primary mouse peritoneal macrophages in vitro. Methods： The activity of peritoneal rnacrophages treated with different concentrations of LPS was detected by MTT assay in rider to find the optimal concentration. Peritoneal macrophages were also treated with NNK （100-500 μM）, with or without LPS for 9 h. The expression of NF-κB was demonstrated via immunocytochemistry （ICC） and Western- blot, respectively. Results： The concentration of LPS at 25 μg/mL was found to be the optimal concentration to improve the activity of peritoneal macrophages （P 〈 0.01）. Simultaneously, LPS （25 μg/mL） increased the expression of NF-κB in both the nucleus and cytoplasm and facilitated transfer of NF-κB to the nucleus. NNK treatment significantly inhibited the expression of NF-κB in a concentration-dependent manner, among the LPS-stimulated or unstimulated peritoneal macrophages, especially when cotreated with LPS （25 μg/mL, P 〈 0.01 ）. Furthermore, NNK treatment （500 μM） with LPS yielded a significant decrease in NF-κB translocation to nucleus and inhibited the expression of NF-κB （P 〈 0.005）. Conclusion： LPS enhances the suppression of NF-κB expression in NNK-mediated mouse peritoneal macrophages, which may provide a theoretical basis for the inhibition of cancer.
文摘In order to analyze the mechanism of immunomodulation by LPS on murine peritoneal suppressor macrophages, we have, using RNase protection assay,checked the changes of mRNA expression pattern of several cytokine genes during the immuno-modulation.It has been found that, after treating peritoneal suppressor macrophages with LPS, mRNAs of IL-12 p35, IL-12 p40,IL-6 and IFN-γ are newly appeared, while those of IL-1α, IL-1β and IL-1Ra are increased and those of other cytokines, like TGF-β1 and MIF are not changed at all.It seems certain that those cytokines, whose expression is increased by LPS stimulation, may be responsible for the functional changes of suppressor macrophages during immuno-modulation. Among these changes, the appearance of IL-12 mRNA may play a critical role, and, in this regard, the synergetic effect between IFN-γ and LPS on the increase of IL-12 p35 and IL-12 p40 mRNA expression is an interesting finding.
文摘Lipopolysaccharide (LPS) was found to induce significant hepatocytotoxicity against cultured mouse hepatocytes. Degeneration and necrosis of cultured hepatocytes and decrease of hepatocyte viability were prominent. The aspartate transferase level and 3H-TdR release in the medium were significantly increased after treatment, and the degree of these changes paralleled with LPS concentration. Various other parameters showed no significant difference between the hepatocytes cultured alone and those cocultured with nonparenchymal liver cells. However, if the nonparenchymal liver cells were isolated from mice which had been pretreated with D-galactosamine (GalN) not only was the hepatocyotoxicity induced by LPS enhanced, but the cells also showed certain cytotoxicity against cultured hepatocytes even without LPS, These results suggest that nonparenchymal liver cells might promote LPL-induced hepatocyte injury.
基金National Science and Technology Major Project(Grant No. 2012ZX09501001-003)
文摘In this investigation,the effects of PE,EtOAc,and BuOH fractions from Zanthoxylum bungeanum on cholesterol accumulation induced by sterols and LPS were determined in vitro and in vivo.HepG2 cells induced by 25-hydroxychoelsterol and cholesterol were employed as cell model.After treatment with PE,EtOAC,or BuOH fractions,cellular total cholesterol and apolipoprotein B secretion were significantly reduced.In addition,compared with control group,expressions of SREBP2,HMGCR,and ACAT decreased,while CYP27A1,ABCA1,and LDLR levels increased.Cholesterol accumulation was also induced in C57BL/6 mice by LPS and the mice were used as the animal model.Determination of serum TNF-α level and hepatic mRNA expression of TNF-α,IL-6,iNOS,COX-2 revealed that EtOAc and BuOH fractions had anti-inflammatory effects.Furthermore,hepatic total cholesterol was reduced,accompanied by the elevation of LXR-α and ABCA1 gene expression in BuOH fraction treated mice.Since EtOAc and BuOH fractions were found active,bioassay-guided isolation was performed and β-sitosterol,eudesmin,sesamin and syringaresinol-β-D-glucoside were isolated from the fractions.
基金supported by a post-doctoral fellowship grant from the Kyung Hee University in 20120351
文摘Ginsenosides, the main active constituents of Panax ginseng Meyer （P. ginseng）, have potential therapeutic effects. All tested ginsenosides except gin- senoside F1 have previously been reported in inflammation studies using the RAW 264.7 macrophage cell line. We ex- amined the anti-inflammatory effects of single sugar moiety ginsenosides such as compound K （CK）, Rh2, Rhl, and F1 that were isolated from P. ginseng through in silico docking studies. We investigated their biological activity predictions, including absorption, distribution, metabolism, excretion, and toxicity and PASS properties, on the suppression of NF- κB, followed by in vitro validation in lipopolysaccharide （LPS）-stimulated RAW 264.7 macrophage cells. The molecular docking results of our study showed that all treated ginsenosides are non-toxic and may be drug-like molecules. The molecular binding interactions of these ginsenosides with the active residues of NF-κB noticeably support their anti-inflammatory activity. CK and Rhl sig- nificantly reduced the production of nitric oxide, cyclooxy- genase-2 （COX-2）, and pro-inflammatory cytokines such as prostaglandin E2 and tumor necrosis factor alpha （TNF-α） in a dose-dependent manner. Real-time PCR and Western blot analyses further confirmed that protopanaxadiols （PPDs） and protopanaxatriols （PPTs） inhibitory effects may have been due to the down-regulation of TNF-α, inducible nitric oxide synthase, COX2, nuclear factor kappa B （NF-κB）, and I kappa B kinase. The expression of co-stimulatory molecules such as ROS was also inhibited by CK and Rhl in a dose- dependent manner. Furthermore, activation of NF-κB in LPS-stimulated RAW 264.7 macrophages was significantly suppressed by CK and Rhl. Taken together, these results provide evidence that PPD- and PPT-type ginsenosides in- cluding CK and Rhl may exhibit strong anti-inflammatory effects by inhibiting pro-inflammatory mediators through down-regulation of NF-κB.
文摘Objective: To investigate the dose and time-dep endent effects of lipopolysaccharide (LPS) on cytoskeletal F-acitn and G-actin reorganizations by visualizing their distribution and measuring their contents in human umbilical vein endothelial cell line ECV-304. Methods: F-actin was labeled with rhodamine-phalloidin and G -actin with deoxyribonuclease I (DNase I)conjugated with fluorescein isothiocya nate (FITC). Contents of cytoskeletal proteins were obtained by flow cytometry. Results: F-actin was mainly distributed peripherally in endoth elial cells under normal conditions. LPS stimulation caused the formation of str ess fibers and filopodia. G-actin was normally seen in perinuclear and nuclear areas in control ECV-304 cells. Under LPS stimulation,G-actin dots appeared i n the cytoplasmic region. The actin disorganization was accompanied by the time - and dose- dependent decrease in F-actin pool and increase in G-actin pool . Conclusions: LPS can induce characteristic morphological altera tions of actin cytoskeleton and formation of intercellular gap in endothelial ce lls,accompanied by changes in F-actin and G-actin pools.
基金supported by the National Key Basic Research Program of China(2011CB93350)National Natural Science Foundation of China(31171088,31471126,81470540,81300139)
文摘This study aimed to comprehend the largely unknown role of voltage-gated potassium channel 1.3 （Kvl.3） in the phagocytic function of macrophages. We found that blocking of the Kv 1.3 channel with 100 pmol L 1 Stichodactyla helianthus neurotoxin （ShK） enhanced the phagocytic capacities of both resting and lipopolysaccharide （LPS）-stimulated RAW264.7 macrophages in the chicken erythrocyte system. In the fluorescein isothiocyanate （FITC）-labeled Escherichia coli k-12 system, ShK increased the phagocytic capacities of resting RAW264.7 cells, but not of the LPS-stimulated cells, as LPS alone stimulated almost satu- rated phagocytosis of the macrophages. ShK increased the nitric oxide （NO） production in LPS-activated cells, but not in rest- ing RAW264.7 cells. There was no effect of ShK alone on the cytokine secretions in resting RAW264.7 cells, but it suppressed IL-113 secretion in LPS-stimulated RAW264.7 cells. At a concentration of 100 pmol L 1, ShK did not affect the viability of the tested cells. Kv 1.3 was expressed in RAW264.7 cells; this expression was downregulated by LPS, but significantly upregulat- ed by disrupting caveolin-dependent endocytosis with filipin III. In addition, cytochalasin D, an inhibitor of actin polymeriza- tion, did not affect the Kvl.3 expression. Thus, blocking of the Kvl.3 channel enhances the phagocytic capacity and NO pro- duction of this cell line. Our results suggest that Kv 1.3 channel serves as a negative regulator of phagocytosis in macrophages and can therefore be a potential target in the treatment of macrophage dysfunction.
