The Molecular Mechanism Study Of The Lipopolysaccharide Triggered Inflammation Suppressed By Phospholipid Transfer Protein

Object In lethal endoxemia,accumulated lipopolysaccharide(LPS)in circulation is the primary cause of the inflammatory cytokine secretion,systemic inflammatory response,multiple organ failure,and subsequently,the individual death.To improve the treatment effect of the endoxemia,the LPS neutralization is necessary to suppress the existing inflammatory response.Phospholipid transfer protein(PLTP)is one of the key plasma proteins in lipoprotein metabolism regulation.Higher plasma activity of PLTP was observed in acute or chronic inflammatory diseases such as sepsis and periodontitis,while the exact role remains unknown.Recent reports indicated that PLTP could attenuate the pro-inflammatory effects of LPS in vitro,suggesting that PLTP may display anti-inflammatory effects induced by LPS in vivo.In this study,the effects and mechanism of PLTP in lethal endotoxemia mouse model were investigated.Meterial and Method Animals:PLTP-/-and PLTP-Tg were kindly obtained from Dr.Jiang.Animals were on a homogenous C57BL/6 background(9 generation backcrosses).Experimental animals were housed in a temperature and humidity controlled room with a 12/12 h light-dark cycle.8-10week-age of male mice(N=10)were used in this study.All experiments were approved by the laboratory animals’ ethical committee of Taishan Medical University and followed national guidelines for the care and use of animals.LPS injection:LPS was suspended in endotoxin-free,0.15 M sodium chloride and handled following reported method.The LPS was injected intraperitoneally into mice(5mg/kg body weight for PLTP-/-,and 6mg/kg body weight for WT or PLTP-Tg;single doses).Survival rate:After 9 days of LPS injection of the survival rates were calculated.Plasma lipid analysis:Isolate the plasma,using fast protein liquid chromatography (FPLC)to get the results of lipid profile.Cell culture,transfection and PLTP-LPS incubation:Collect the Peritoneal derived macrophages(PDM)and Bone marrow derived macrophages(BMDM),cultivate mousederived RAW264.7 cells.The PLTPsi RNA as well as the control group si RNA used in the RAW264.7 transfection experiments were 150pmol/L.All serum used in this experiment is Fetal bovine serum without LPS.In LPS and PLTP co-incubation experiment,the final volume of LPS,10-20μL concentrated cultured media and D-Hanks buffer solution is 50μl,the three for co-incubation,the experimental conditions is 37°C,60 min.The control group is the mixture of the LPS and D-Hanks buffer solution.RNA extraction and real-time PCR(Real-Time polymerase chain reaction,RTRCR):Extract total RNA,reverse transcription and RT-PCR to detect the contents of the m RNA of the main inflammationary factor.Protein separation,protein electrophoresis and Western blot(WB):Use RIPA cell lysis buffer and protease inhibitor mixture tablet to isolate protein completely.Use WB to determine the content of the apolipoprotein AI(Apo AI),p65 and IκB-α.ELISA:We use ELISA method to detect the content of Tumor necrosis factor-α(TNF-α),Interleukin-6(IL-6)and Interferon-γ(IFN-γ),Interleukin-1β(IL-1β)in plasma or cell culture medium,use the multifunctional enzyme label to read the absorption.Cell surface receptor analysis:BMDM was stained with 1μg/ml TLR4-MD-2 complex antibody for 60 min on ice,washed with ice cold PBS for 3 times and then analyzed on Flow Cytometer(BD FACS Calibur).PLTP activity detection:Before the test,we prepare the donor and acceptor respectively.Mix donor(3μL),receptor(3μL)and plasma(up to 5μL)or concentrated culture medium(3-10μL)and use TNE(10m Mol/LTris,0.15mol/L sodium chloride,2m Mol/L EDTA,p H=7.4)to adjust the final volume to 100μL,the mixture were incubated at 37°C,in 96-well black plate for 70 min.We use multifunctional enzyme label to test(excitation wavelength 465 nm,emission wavelength 535nm),we read the result on the point of 0,10,20,30,40,50,60,70 min respectively.The unit is expressed with pmol/μL/min.Statistical analysis:All the experiments were repeated three or four times and Graph Pad Prism software was used for statistical analysis.Data were typically expressed as mean±S.D.Data between two groups were analyzed by Student’s t test.Survival rates were analyzed by the Kaplan-Meier method and compared using the x2 test.A statistically significant difference was assumed at P≤0.05.The results 1.PLTP significantly improved the model of fatal endoxemia9 days survival rate(1)Plasma PLTP activity and HDL were determined in PLTP transgenic mice(PLTPTg),wild type mice(WT),and PLTP-/-prior to LPS injection.Plasma PLTP activity was almost absent in PLTP-/-,while moderately enhanced in PLTP-Tg compared with WT.The cholesterol level and the apo AI level of HDL were decreased both in PLTP-Tg and PLTP-/-compared with WT.(2)Twenty-four hours after 6mg/kg LPS injection(5mg/kg in PLTP-/-),HDL cholesterol(HDL-C)levels were not changed.Nine days after LPS injection,the survival rates were 90%,60%,and10% in PLTP-Tg,WT,and PLTP-/-,respectively.2.PLTP reduced the levels of inflammatory cytokines in fatal endoxemia PLTP could suppress LPS induced TNF-α and IL-6 expression,however,the levels of IFN-γ and IL-1β were not changed.3.PLTP inhibited the content of macrophage inflammatory cytokines transcription levels that induced by LPS(1)PDM or BMDM from WT or PLTP-/-were treated with LPS(200ng/m L)for12h.PLTP deficiency dramatically enhanced m RNA expression of TNF-α,IL-6,IFN-γ,and IL-1β induced by LPS in PDM.(2)PLTP deficiency significantly enhanced m RNA expression of TNF-α,IL-6,and IL-1β.(3)The expression of IL-6 and TNF-α were decreased in PLTP-Tg 8h after LPS stimulation,while the expression of IFN-γ and IL-1β were not changed.4.PLTP reduced the expression of macrophage inflammatory cytokine levels induced by LPS(1)The levels of TNF-α,IL-6,and IFN-γ in cultured media were determined.Similar to m RNA results,PLTP deficiency significantly enhanced TNF-α and IL-6 in cultured media,while no change was found in IFN-γ.(2)We also conducted the same assay in PLTP-Tg BMDM.PLTP-Tg BMDM released less pro-inflammatory cytokines in 8h after LPS stimulation.5.Lack of PLTP raised the NF-κB pathway activation induced by LPS(1)We evaluated the role of PLTP in LPS induced NF-κB activation in PLTP-/-BMDM and PLTP knockdown RAW264.7 by western blot(WB).Nuclear p65 level was significantly enhanced in PLTP-/-BMDM after LPS stimulation.Consistently,cytoplasmic IκB-α degradation in PLTP-/-BMDM was enhanced in a time-dependent manner after LPS treatment.(2)We also evaluated the NF-κB activation in PLTP knockdown RAW264.7 induced by LPS.PLTP knockdown enhanced the nuclear content of p65 stimulated by LPS dramatically.6.Macrophage-derived PLTP abated the expression of TNF-α induced by LPS,not by changing the number and the activity of TLR4.(1)No difference was observed between WT and PLTP-/-,which indicated that PLTP expression could not affect the distribution and function of TLR4.(2)The concentrated cultured medium(CCM)of macrophage were harvested for co-incubation with LPS before the treatment of BMDM.PLTP-/-CCM treated LPS could induce higher level of TNF-α compared with WT CCM treated LPS.(3)We also determined the phospholipid transfer activity of CCM.PLTP activity was higher in CCM from WT BMDM compared with PLTP-/-BMDM.7.In extracellular PLTP combined with LPS to form the large molecular weight low cell toxic compounds(1)We determined pro-inflammatory effects of LPS pre-incubated with active or inactive recombinant PLTP(r PLTP).Compared with heat-inactived r PLTP treated LPS or albumin treated LPS,active r PLTP treated LPS could induce lower TNF-α and IL-6expression in PLTP-/-BMDM.(2)We also conducted the similar experiment in WT BMDM.r PLTP could repress LPS induced TNF-α and IL-6 expression,while the levels of cytokines were much lower compared with the assay in PLTP-/-BMDM.(3)We separated the co-incubated mixture of PLTP and LPS with native gel.Most of active PLTP and 56°C treated PLTP were consumed and large size complex were stacked in the borderline of running gel,while no obvious consumption of the albumin or the 95°C denatured PLTP were observed.(4)The FITC labelled LPS(FITC-LPS)was employed in co-incubation system.Active PLTP could concentrate more FITC-LPS than albumin or 95°C treated PLTP.The co-incubated mixture of PLTP and FITC-LPS was determined by WB.A purchased PLTP(p PLTP)was loaded as positive control.Active PLTP collocated with LPS and formed large size complex,while 95°C treated PLTP could not bind to LPS in the co-incubation system.(5)RAW264.7 cells were treated with pre-incubated complex of r PLTP/LPS for cell viability determination.r PLTP(0.5μg/m L)neutralized LPS and increased the cell viability challenged by different concentrations of LPS(0.2μg/m L and 2.0μg/m L,respectively), while no cell protection was observed in heat-treated PLTP groups.Conclusion(1)PLTP can improve the individual survival rates in endoxemia;(2)The macrophages-derived PLTP reduce the expression of inflammatory cytokines induced by LPS,by inhibiting the NF-kappa B activation;(3)PLTP can combine with LPS directly to form the large molecular weight low cell toxic compound.