The Effects Of Low Phospholipid Transfer Protein Expression On Inflammatory Factors Expression And Its Potentialmechanism In Macrophage

ObjectiveInflammation plays key roles in pathogenesis and development of various humandiseases. As a type of immune cell, macrophage is critical in inflammation progress.Nuclear factor kappa-B(NFκB) is one of the most important transcription factors ininflammatory signal pathways, and its activation triggers the expression of manyinflammatory cytpkines including interleukin and tumor necrosis factor α (TNF-α).Phospholipid transfer protein(PLTP) is reported toparticipant inflammation process by itsneutralizing bacterial Lip-opolysaccharid(LPS). Lethal endotoxemia cause higherpro-inflammatory cytokines expression, more severe organs damage, and lowersurvival rate in systematic PLTP deficient mice(PLTP-/-). The high expressedinflammatory cytokines are target genes of NFκB and wide expressed in macrophages,suggested that this phenomenon might be closed to the NFκB activation in macrophage.We use macrophage models to investigate the effects of low PLTP expression oninflammatory factors and its potential molecular mechanism.Methods1Mice breeding, heterozygote mice(PLTP+/-)were used to generate thehomozygote)and wild type mice(WT) offsprings. Eight male PLTP-/-or WTlittermates were used in this study.2Eight-week age og mice were sacrificed and induced peritoneal derivedmacrophage(PMD) and isolate bone marrow cells were harvested respectively.The bone marrow derived macrophages (BMDM) were obtained after induced by L cell medium including colony stimulating factor for ten days.3RAW264.7and Human umbilical vein epithelial cell(HUVEC) werecultured and divide into three groups and signed as blank, Control siRNA andPLTP siRNA respectively.4WT and PLTP-/-BMDM were stimulated with200ng/ml LPS in differenttimes(0.5h,1h,2h) respectively.5PLTP knockdown RAW264.7were stimulated with200ng/ml LPS indifferent times(0.5h,1h,2h) respectively.6PLTP knockdown HUVEC were stimulated with20ng/ml TNF-α indifferent times(0.5h,1h,2h)respectively.7The detection of NFκB inflammatory signal pathway correlated proteins:NFκB active subunit p65、inhibition subunit IκB-α、Signal transducers and activatorsof transcription3(STAT3) and phosphorylated protein pSTAT3、transform growth factoractivated kinase1(TAK1) and phosphorylated protein pTAK1, nuclear internal proteinsH3and cytoplasm protein glyceraldehyde-3-phosphatedehydrogenase(GAPDH) weredetermined by Western Blot.8The detection of correlated inflammatory factors:Interleukin-6(IL-6), interleukin-10(IL-10), tumor necrosis factor-α(TNF-α)and interferon-γ(IFN-γ) level were determined by real-time quantitative PCR.9IL-6IL-10TNF-α and IFN-γ level were etermined by enzyme-linkedimmunosorbent assay (ELISA) kit.10Immunofluorescent staining detects nuclear protein p65.Result1LPS induced pro-inflammatory cytokines expression was increased inPLTP deficient macrophage:(1) PLTP-/-peritoneal macrophage inflammatory factors IL-10、IFN-γ、TNF-αand IL-6are higher than WT.(P<0.01)(2) PLTP-/-BMDM inflammatory factors are higher than WT: IL-10、TNF-α andIL-6are higher than WT by100.7℅(P<0.05), IFN-γ is not obviouslydifferent(P>0.05), no statistical significance.(3) PLTP-/-BMDM inflammatory factors are higher than WT: IL-10and TNF-αare higher than WT by198.5℅(P<0.05), IL-6is higher than WT by23.4℅(P<0.05), IFN-γ is not obviously different(P>0.05), no statistical significance.2PLTP deficiency enhanced nuclear translocation of NFκB in BMDM andLPS induced NFκB activation was enhanced in PLTP knockdown RAW264.7(1)The content of BMDM nuclear p65, PLTP-/-VS WT increases30.5℅(P<0.05).(2) The content of BMDM cytoplasm protein IκB-α, PLTP-/-VS WT reduces33.6℅(P<0.05).(3) The content of PLTP knockdown RAW264.7nuclear p65, PLTP siRNA VSControl siRNA increases30.5℅(P<0.05).(4) The content of PLTP knockdown RAW264.7cytoplasm protein IκB-α, PLTPsiRNA VS Control siRNA reduces43℅(P<0.05).3TNFα induced NFκB activation was enhanced in PLTP knockdownHUVEC.(1) The content of PLTP konckdown HUVEC nuclear p65, PLTP siRNA VSControl siRNA p65increases54℅(P<0.05).(2) The content of PLTP knockdown HUVEC cytoplasm protein IκB-α, PLTPsiRNA VS Control siRNA reduces351.2℅(P<0.05).4PLTP knockdown enhanced NFκB activation via TAK1.(1) The content of PLTP konckdown RAW264.7pTAK1, PLTP siRNA VSControl siRNA increases150℅(P<0.05).(2) The content of PLTP konckdown HUVEC pTAK1, PLTP siRNA VSControl siRNA increases1010.3℅(P=0.05).5Macrophage derived PLTP did not activate ABCA1-JAK2-STAT3pathway.In the presence of PLTP, pSTAT3level is not different obviously, provingglibenclamide does not influence pSTAT3and explaining PLTP does notactivate ABCA1-pSTAT3.6LPS induced WT and PLTP-/-BMDM, LPS induced PLTP knockdownRAW264.7and TNF-α induced PLTP konckdown RAW264.7were detected byimmunofluorescent staining；(1) The content of BMDM nuclear p65, PLTP-/-VS WT increases obviously.(2) The content of PLTP konckdown RAW264.7nuclear p65, PLTP siRNAVS Control siRNA increases obviously.(3) The content of PLTP konckdown HUVEC nuclear p65, PLTP siRNA VS Control siRNA increases obviously.Conclusion(1) LPS induced inflammatory factors expression were enhanced in PLTPdeficient peritoneal macrophage as well as BMDM(2) PLTP deficient increased the nuclear level of active NFκB unit p65.(3)The enhancing of nuclear level of active NFκB unit p65might berelative with TAK1phosphorylation in RAW264.7and HUVEC.