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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 study the effect of nickel-titanium stent(NTS) and consequent anti-allergy dexamethasone therapy on macrophage cells reactivity to lipopolysaccharide (LPS) from gram-negative bacterium .Methods The macrophage cell line Raw 264 .7 and dexamethasone-pretreated Raw264 .7 were co-cultured with NTS for 4 days ,and stimulated with LPS for 24 hours .The surface marker CD molecules of CD80 ,CD86 and FasL were detected with flowcytometr method ,the supernant cytokine production of proinflammatory cytokines IL-6 and TNF-αwas valued with ELISA method ,and intracellular inflammatory signal pathway acti-vation of NF-κB ,GAS ,ISRE and STAT3 was checked with signal molecule specific promoter lunciferase analysis .Results The stent pre-treatment improved LPS-mediated CD80 expression ,suppressed FasL production ,decreased IL-6 secretion and NF-κB ac-tivation ,the results have statistical significance (P<0 .05) .The dexamethasone treatment improved stent-mediated up-regulated ex-pression of CD80 ,FasL and TNF-α,and suppressed the activation of intracellular inflammatory signal pathway of NF-κB ,ISRE and STAT3 ,the results have statistical significance(P<0 .05) .Conclusion NTS inhibit macrophage cells Raw264 .7 react to TLR4 ag-onist LPS ,and dexamethasone treatment improved the function .
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@#ObjectiveTo investigate the effect of Puerarin on expression of inductible nitric oxide synthase (iNOS) in RAW264.7 macrophage cells induced by lipopolysaccharide （LPS）. MethodsThe protein and mRNA levels of lipopolysaccharide (LPS)-induced iNOS was determined in RAW264.7 macrophage cells using Western blotting and RT-PCR respectively. ResultsPuerarin inhibited expression of iNOS concentration dependently (P<0.01). ConclusionPuerarin may reduce iNOS level, result in prevention of inflammation.
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BACKGROUND: Secretory phospholipase A2 (sPLA2) are a group of extracellular enzymes that release fatty acids at the sn-2 position of phospholipids. Group IIA sPLA2 (sPLA2-IIA) has been detected in the inflammatory fluids, and its plasma level increases in the inflammatory disease. This study examined the effect of sPLA2-IIA on mouse macropahges in order to investigate the potential mechanism of sPLA2-induced inflammation. MATERIALS AND METHODS: Wild type PLA2 and mutant H48Q PLA2 were purified from HEK293 cells transfected with the corresponding plasmids, and the PLA2 activities were measured using 1-palmitoyl-2-[1- (14) C]linoleoyl-3-phosphatidylethanolamine as substrates. The TNF-alpha and IL-6 released in the supernatants were determined by ELISA. In addition, the TNF-alpha and IL-6 mRNA were analyzed by RT-PCR. RESULTS: sPLA2-IIA stimulated the production of TNF-alpha and IL-6 in a dose- and time-dependent manner. In addition, the effect of sPLA2-IIA on cytokine production from the macrophage was found to be associated with the accumulation of their specific mRNA. The mRNA levels of TNF-alpha and IL-6 peaked at 2 and 6 hours in a time-dependent manner, respectively. CONCLUSION: In conclusion, the production of proinflammatory cytokine might be mediated by the binding of sPLA2-IIA to the receptors.