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ObjectiveTo explore the mechanism of Buyang Huanwutang in regulating macrophage polarization based on the Toll-like receptor 4 （TLR4） / nuclear factor-κB （NF-κB） / nucleotide-binding oligomerization domain-like receptor 3 （NLRP3） pathway. MethodRAW264.7 macrophages were intervened with lipopolysaccharide （LPS） of different concentrations （0， 1.25， 2.5， 5， 10， 20， 40， and 80 mg·L-1） for 24 hours. Cell Counting Kit-8 （CCK-8） assay was used to determine the cell viability of RAW264.7 macrophages. The optimal concentration was chosen to establish an in vitro inflammation model induced by LPS. Cells were divided into a blank group （20% blank serum）， a model group （20% blank serum + 10 mg·L-1 LPS）， a model control group （20% FBS + 10 mg·L-1 LPS）， low-， medium-， and high-dose （5%， 10%， and 20%） Buyang Huanwutang-containing serum groups， a high-dose （20%） Buyang Huanwutang combined with NLRP3 inhibitor MCC950 （50 μmol·L-1） group， a high-dose （20%） Buyang Huanwutang combined with reactive oxygen species （ROS） inhibitor NAC （10 μmol·L-1） group， and a high-dose （20%） Buyang Huanwutang combined with NF-κB inhibitor PDTC （10 μmol·L-1） group. Enzyme-linked immunosorbent assay （ELISA） was used to detect the expression of interleukin-1β （IL-1β）， interleukin-18 （IL-18）， and tumor necrosis factor-α （TNF-α） in RAW264.7 macrophages. Flow cytometry was employed to measure ROS levels in macrophages. Western blot was used to determine the protein expression of M1-type macrophage-related factors inducible nitric oxide synthase （iNOS） and TNF-α， M2-type macrophage-related factors arginase-1 （Arg-1） and interleukin-10 （IL-10）， as well as the proteins in the TLR4/NF-κB/NLRP3 pathway. ResultCCK-8 results indicated that under 10 mg·L-1 LPS stimulation， RAW264.7 macrophages exhibited the highest cell viability （P<0.01）. Compared with the blank group， the model group showed significantly increased levels of IL-1β， IL-18， and TNF-α （P<0.05，P<0.01）， increased ROS expression （P<0.05，P<0.01）， increased protein expression of M1-type macrophage factors iNOS and TNF-α （P<0.01）， decreased protein expression of M2-type macrophage factors Arg-1 and IL-10 （P<0.05，P<0.01）， and upregulated expression levels of TLR4， myeloid differentiation factor 88 （MyD88）， phosphorylated inhibitor of NF-κB （p-IκB）/NF-κB inhibitor （IκB）， phosphorylated NF-κB （p-NF-κB） p65/NF-κB p65， NLRP3， apoptosis-associated speck-like protein containing a CARD （ASC）， and pro-Caspase-1 （P<0.05， P<0.01）. Compared with the model group， all Buyang Huanwutang-treated groups and inhibitor groups significantly reduced levels of IL-1β， IL-18， and TNF-α （P<0.01）， suppressed the expression of inflammatory factors in RAW264.7 macrophages， decreased cellular ROS expression levels （P<0.01）， downregulated M1-type macrophages iNOS and TNF-α protein expression （P<0.01）， upregulated M2-type macrophages Arg-1 and IL-10 protein expression （P<0.01）， and lowered protein expression levels of TLR4， MyD88， p-IκB/IκB， p-NF-κB p65/NF-κB p65， NLRP3， ASC， and pro-Caspase-1 （P<0.05， P<0.01）. ConclusionBuyang Huanwutang can improve macrophage inflammation， potentially by reducing macrophage ROS levels， inhibiting RAW264.7 macrophage polarization， and downregulating the protein expression levels of the TLR4/NF-κB/NLRP3 pathway.
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Aim To investigate the effects of Kudino- side D on lipid accumulation induced by oxidized low density lipoprotein ( ox-LDL) and inflammation induced by lipopolysaccharide ( LPS ) in RAW264.7 cells.Methods Foam cells were established by incubating the RAW264.7 cells with ox-LDL.The concentration of lipid droplets in the cells was observed by oil red staining, and the level of total cholesterol (TC) in cells was measured by enzyme method.The gene and protein expressions of scavenger receptors CD36 and SR-A1, ATP binding cassette transporters A1 and Gl ( ABCA1 and ABCGI) were detected by RT-qPCR and Western blot, respectively.The expressions of inter- leukin-6 (IL-6), interleukin-1 (3 (IL-ip), monocyte chemoattractant protein-1 (MCP-1 ) and tumor necrosis factor-a (TNF-a) were detected by ELISA and RT-qPCR.The protein expressions of mTOR and p-mTOR were detected by Western blot.Results Compared with model group, the high dose of Kudinoside D decreased the content of TC and down-regulated the gene and protein expression of SR-A induced by ox-LDL.Meanwhile Kudinoside D also decreased the levels of IL-ip and MCP-1 and down-regulated the protein expression of p-mTOR induced by LPS.Conclusions Kudinoside D may reduce the intracellular TC content by down-regulating the gene and protein expression of SR-A1.Kudinoside D may play an anti-inflammatory role through mTOR pathway.
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Objective:To establish a method for evaluating the biological activity of water extract lyophilized powder of Qingjin Huatantang based on the phagocytic and secretory functions of macrophages， and to control the quality of this formula from the biological activity level. Method:The phagocytic and inflammation models of RAW264.7 macrophages were established， the inhibition rates of water extract lyophilized powder of Qingjin Huatantang on interleukin-6 （IL-6） secretion and phagocytic index of neutral red of RAW264.7 macrophages were chosen as indicators to investigate the biological activity of Qingjin Huatantang， and the biological limit was searched. Result:The optimal inoculation density of RAW264.7 macrophages was 3×10<sup>5</sup> pcs/mL， and the concentration of lipopolysaccharide （LPS） was 1 mg·L<sup>-1</sup> after treatment for 24 h. When the concentration was 500 mg·L<sup>-1</sup>， water extract lyophilized powder of Qingjin Huatantang had no toxicity and no obvious promotion effect on the proliferation of RAW264.7 macrophages， and at this concentration， the phagocytosis of RAW264.7 macrophages for neutral red was significantly promoted， the phagocytic index was >113%. In addition， the lyophilized powder had a significant and stable inhibitory effect on IL-6 secretion of RAW264.7 macrophages induced by LPS， the inhibitory rate was >45%. Conclusion:Combined with the anti-inflammatory and immunomodulatory effects of Qingjin Huatantang， this study establishes an <italic>in vitro </italic>biological limit method for evaluating the quality of water extract of Qingjin Huatantang based on the phagocytic and secretory functions of RAW264.7 macrophages， and 500 mg·L<sup>-1</sup> was confirmed as the limit concentration. Under the limit concentration， Qingjin Huatantang water extract can significantly promote the phagocytic index of macrophages or significantly inhibit the secretion of IL-6 of RAW264.7 macrophages induced by LPS， which can be judged as qualified.
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Objective To investigate the effects of crude Fomes officinalis polysaccharide ( FOPS ) and its purified constituent ( FOPS-a) on mitogen-activated protein kinases ( MAPK) signaling pathway and se-cretion of tumor necrosis factor-α (TNF-α) in RAW264. 7 macrophages. Methods RAW264. 7 macrophages were treated with FOPS and FOPS-a respectively at different concentrations (50, 100, 200 μg/ml) for 24 h. RT-qPCR and Western blot methods were respectively used to detect the expression at mRNA level and the phos-phorylated proteins of p38, c-Jun NH2-terminal kinase ( JNK ) and extracellular signal-regulated kinase ( ERK) . Changes in the phosphorylation of these proteins and TNF-α secretion were respectively detected by Western blot and ELISA after treating the macrophages with MAPK-specific inhibitors ( PD98059, SP600125, SB203580). Results Compared with the blank control group, different concentrations of FOPS and FOPS-a could significantly increase the expression at mRNA level and the phosphorylation of p38 and ERK (P<0. 05). Three inhibitors of MAPK could markedly decrease the phosphorylation of ERK1/2 and p38 and TNF-αsecretion that were induced by FOPS and FOPS-a (P<0. 05). Conclusions FOPS and FOPS-a might have immunomodu-latory effects on RAW264. 7 macrophages through activating ERK1/2 and p38 MAPK signal transduction pathways.
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OBJECTIVE: To study the effect of serum containing Jieduquyuziyin-prescription (JP) on signal pathway of interleukin-1 receptor-associated kinase 1 (IRAK1) in mononuclear macrophages of mice stimulated by lipopolysaccharide (LPS), and to explore the effect of Jieduquyuziyin-prescription on IRAK1 and NF-κB inflammatory signaling pathways, which providing a good theoretical support for its anti-inflammatory clinical medication. METHODS: In this study, mice mononuclear macrophages cultured in vitro were randomly divided into blank group, LPS group, JP serum group, blank serum group, LPS plus JP serum group, LPS plus blank serum group, IRAK1 inhibitor group, inhibitor plus LPS group, inhibitor plus JP serum group and inhibitor plus blank serum group. After intervention for 24 h, the activity of JP on macrophages was tested by CCK8 method. The IRAK1 expression in macrophages was tested by immunofluorescence chemical staining. The content of TNF-α in the supernatant of the cells was detected by ELISA. The mRNA expressions of IRAK1, NF-κB, TNF-α and IL-6 were detected by RT-PCR. The protein expressions of IRAK1, p-IRAK1 and NF-κB were detected by Western-blot. The LC-MS was used to detect the active ingredients in JP serum. RESULTS: The results show that 2.5% of JP serum is the optimal concentration. Jieduquyuziyin-prescription could down-regulate the expression of TNF-α and IL-6 and inhibit the expression of IRAK1 and activate NF-κB(P<0.05). Paeoniflorin and ferulic acid were detected in the JP serum. CONCLUSION: Jieduquyuziyin-prescription can inhibit the expression of IRAK1 and NF-κB in mouse monocyte-macrophage cells after LPS stimulation and provide a good theoretical support for its anti-inflammatory clinical medication.
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Severe acute pancreatitis (SAP) is characterized by both local and systemic inflammatory responses. This study was designed to develop a site-specific delivery strategy for SAP therapy using celastrol (CLT). First, murine RAW264.7 cells were used as a model of macrophage cell line, cell membranes were obtained by emptying intracellular contents via hypotonic lysing, mechanical membrane disruption, and differential centrifugation. Poly(ethylene glycol) methyl ether-block-poly(lactic-co-glycolic acid) (PEG-PLGA) nanoparticles (NPs) were then prepared by sonication. With the collected membrane materials, macrophage membrane coated PEG-PLGA NPs (RNPs) were then prepared by extrusion through a 400 nm polycarbonate membrane. Biodistribution study in rats with SAP showed RNPs selectively accumulated in the inflamed pancreatic tissues. Compared with CLT loaded NPs, CLT loaded RNPs were proven to effectively attenuate local pancreatic inflammation and systemic inflammation in rats with SAP.
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Objective To investigate the effects of high glucose and lysophosphatidylcholine (LPC) on the immune function of in vitro cultured macrophages during Nocardia farcinica infection. Meth-ods RAW264.7 macrophages were cultured in vitro under different conditions as follows: routine culture (control group),50 mmol/L glucose (high glucose group),10 mg/L LPC(LPC groupⅠ),25 mg/L LPC (LPC groupⅡ) and 50 mmol/L glucose+25 mg/L LPC(high glucose and LPC group). The activity of mac-rophages in each group was tested after 6,12,24 and 36 h of culture. After 24 h of culture, macrophages were collected from every group and co-cultured with Nocardia farcinica. Dynamic phagocytosis rates were detected at 1,2,3,4,5 and 6 h after co-culture. Toxic effects of Nocardia farcinica on macrophages and concentrations of IL-10 and TNF-α were measured at 1,3 and 6 h after co-culture. Results Macrophages in all four experimental groups showed decreased activity as compared with those in the control group (P<0.01). Phagocytosis of Nocardia farcinica by macrophages was also reduced by high glucose and LPC. Phagocytosis rates of high glucose group and LPC groupⅡ at 1 and 2 h,LPC groupⅠat 1,2 and 3 h,and high glucose and LPC group at 1,2,3 and 4 h after co-culture were significantly lower than that of the con-trol group (P<0.05 or P<0.01). Compared with the control group, significantly reduced toxic effects on macrophages caused by Nocardia farcinica was observed in the experimental groups (P<0.05 or P<0.01). Compared with the control group,LPC groupsⅠand Ⅱ and high glucose and LPC group had decreased se-cretion of IL-10 at 3 h,and high glucose group and LPC groupⅠhad decreased secretion of TNF-α at 1 h(P<0.05). Conclusion Culture macrophages under the conditions of high glucose and LPC would reduce their activity and impair their ability to phagocytose Nocardia farcinica. Moreover, high glucose and LPC might have impacts on the toxic effects of Nocardia farcinica on macrophages and the secretion of IL-10 and TNF-α.
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Objective Inflammation is a defensive reaction of body , but excessive inflammatory response can lead to physi-cal injury.The aim of this study was to explore the effects of luteolin on the secretion of inflammatory cytokines from lipopolysaccharide (LPS) and interferon-g(IFN-γ) activated RAW264.7 cells. Methods RAW264.7 cells were divided into 5 groups: control group (without any medicine), M1 group (polarized M1 cells activated by final concentration of 10 ng/mL LPS+20 ng/mL IFN-γ), M1+5L group (simultaneous activation of LPS and IFN-γplus final concentration of 5μmol/L luteolin), M1+10L group(simultaneous activa-tion of LPS and IFN-γplus 10μmol/L luteolin), M1+20L group(simultaneous activation of LPS and IFN-γplus 20μmol/L luteolin). The cell morphological transformation was observed by laser confocal microscope ;the mRNA levels of iNOS , IL-1βand IL-6 were test-ed by real-time quantitative PCR respectively;the secretion levels of TNF-αand IL-6 in culture supernatant were detected by ELISA;the changes of p-STAT3 (ser727) protein pathways were examined by western blot. Results Cellular morphology of activated RAW 264.7 cells changed obviously .Compared with the control group , the mRNA levels of iNOS, IL-1βand IL-6 decreased significantly in the other 4 groups(P<0.05).The iNOS level in M1+20L group significantly de-creased compared with M1 group[(29.52±3.07) vs (98.91±10.65), P<0.01].As to IL-1βlevel, it decreased significantly in M1+10L group(78.38±8.65) and M1+20L group(41.59±6.80) compared with M1 group(110.69±4.12)(P<0.05).While the IL-6 levels decreased significantly in M1+5L group(177.51±19.28), M1+10L group (106.14±5.63), M1+20L group(27.15±1.26), compared with M1 group(394.10±33.47)(P<0.05).LPS+IFN-γcould induce in-creased p-STAT3 (ser727) expression in M1 phenotype of RAW264.7 cells which was proved by its significant increase in M 1 group, M1+5L group and M1+10L group compared with control group (P<0.05).In comparison to M1 group, p-STAT3-ser expression in M1 phenotype downregulated in M1+5L group, M1+10L group, M1+20L group(P<0.05), along with dose-dependent characteristic.Com-pared with control group, the levels of IL-6 and TNF-αincreased significantly in M1 group, M1+5L group and M1+10L group.Com-pared with M1 group, the levels of IL-6 and TNF-αdecreased significantly in M1+5L group, M1+10L group and M1+20L group(P<0.05) , in which IL-6 showed concentration independence and TNF-αshowed no concentration independence . Conclusion Luteolin inhibits the secretion of pro-inflammatory cytokines through the down-regulation of p-STAT3 so as to exert anti-inflammatory effects .
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AIM To study the in vitro and in vivo anti-inflammatory effects of extract from Daphniphyllum calycinum Benth and its mechanism of action.METHODS LPS-induced mouse RAW264.7 macrophage and mouse endotoxemia model were used to observe the influences of D.calycinum extract on the levels of NO,TNF-α,IL-1β and IL-10.The toxicity of D.calycinum extract on RAW264.7 macrophage was determined by MTT assay.Fifty female BALB/c mice were administered with D.calycinum extract by gavage.Fourteen days later,the mice were intraperitoneally injected with LPS,the levels of NO,TNF-α,IL-1 β and IL-10 in serum were determined by Griess and ELISA kit.The protein expressions of NO and TNF-α in mouse liver were studied by immunohistochemistry method.RESULTS D.calycinum extract could significantly inhibit the release of NO,TNF-α,IL-1 β and IL-10.Immunohistochemistry results showed that the extract could inhibit the protein expressions of iNOS and TNF-α.CONCLUSION D.calycinum extract has a significant anti-inflammatory effect,and its mechanism may be related to decreasing the release of inflammatory factors.
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AIM To study the in vitro and in vivo anti-inflammatory effects of extract from Daphniphyllum calycinum Benth and its mechanism of action.METHODS LPS-induced mouse RAW264.7 macrophage and mouse endotoxemia model were used to observe the influences of D.calycinum extract on the levels of NO,TNF-α,IL-1β and IL-10.The toxicity of D.calycinum extract on RAW264.7 macrophage was determined by MTT assay.Fifty female BALB/c mice were administered with D.calycinum extract by gavage.Fourteen days later,the mice were intraperitoneally injected with LPS,the levels of NO,TNF-α,IL-1 β and IL-10 in serum were determined by Griess and ELISA kit.The protein expressions of NO and TNF-α in mouse liver were studied by immunohistochemistry method.RESULTS D.calycinum extract could significantly inhibit the release of NO,TNF-α,IL-1 β and IL-10.Immunohistochemistry results showed that the extract could inhibit the protein expressions of iNOS and TNF-α.CONCLUSION D.calycinum extract has a significant anti-inflammatory effect,and its mechanism may be related to decreasing the release of inflammatory factors.
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An ultra performance liquid chromatography tandem quadrupole time-of-flight mass spectrometric method was developed for rapid analysis of glycerophospholipids in RAW264.7 macrophage. The modified Bligh-Dyer was applied to extract glycerophospholipids from RAW264.7 macrophage. The target compounds, detected by mass spectrometry in ESI+ and ESI- mode, were separated by gradient elution with mobile phase (A) water (containing 10mmol·L-1 ammonium acetate and 0.25% acetic acid) and (B) acetonitrile/isopropanol (1:1) (containing 10mmol·L-1 ammonium acetate and 0.25% acetic acid). A total of 82 glycerophospholipids including 57 phosphatidylcholines (PCs), 21 phosphatidylethanolamines (PEs), three phosphatidylglycerols (PGs) and one phosphatidylinositol (PI) were deduced. The UHPLC-QTOF/MS method is rapid, simple and credible for targeting analysis of glycerophospholipids of RAW264.7 macrophage.
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Objective To determine the effect of lipoxins (LX) A4 and its agonist (BML-111) on the survival of RAW264.7 macrophage cells and TLR4/NF-κB signaling pathway. Methods RAW264.7 cells were treated with different concentrations of LPS, then the effect of LX A4 and BML-111 on the survival rate of these cells was observed. Cytotoxicity were detected by CCK-8 method and RT-PCR was used to detect the TLR4 and TRAF6 mRNA. The protein levels of TLR4 and pNF-κB p65 in RAW264.7 were determined by Western Blot. Results The survival rates of macrophage treated with LPS for 6 h in 1 000 ng/mL LPS in LX A4 group and BML-111 group were significantly higher than that in control group (P 0.05). Conclusion LX A4 and BML-111 could inhibit the cytotoxicity of LPS on the RAW264.7 macrophage cells through the inhibition of the activation of TLR4/NF-κB signaling pathway, then reduce the inflammation. And this stable BML-111 may appear as another promising treatment for IBD disease.
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ABSTRACT:Objective To observe the effect of curcumin on RAW264.7 macrophages induced with LPS and IFNγ(M1)and the mechanisms involved.Methods Curcumin of different concentrations (6.25 μmol/L,12.5μmol/L and 25 μmol/L)was used to treat RAW264.7 macrophages induced with LPS and IFNγ(M1)for 12 h,and RAW264.7 macrophages induced with LPS and IFNγ(M1)were incubated with 20μmol/L GW9662 and 25 μmol/L curcumin for 12 h.Using Real-time PCR,ELISA and Western blotting analysis,we examined the expressions of IL-1β,IL-6,PPARγand phenotype markers M2 (KLF4,FIZZ1,and MGL1 )and the expressions of KLF4 and FIZZ1 when PPARγwas inhibited.Results Curcumin of different concentrations all could inhibit the expressions of IL-1βand IL-6 in RAW264.7 macrophages induced with LPS and IFNγ(M1).Curcumin of different concentra-tions could upregulate the expression of M2 markers (KLF4,FIZZ1 and MGL1)and PPARγin RAW264.7 macro-phages induced with LPS and IFNγ(M1).When M1 macrophages were incubated with curcumin and GW9662,the expression of the M2 phenotype markers was reduced.Conclusion Curcumin polarized the M1 phenotype macro-phages derived from RAW264.7 macrophages to become M2 phenotype through activating PPARγ.