The Molecular Mechanism Research Of Activation Of NLRP3Inflammasome Pathway By Free Fatty Acid In Kupffer Cells

Objective：①To investigate the effectiveness, simplity and stablilityof modified method of isolating Kupffer cells (KCs), which depent onperfusing by PBS, digestiving by collagenase in vitro, centrifugating bydiscontinuous gradient and selective adherencing.②To study the role ofNLRP3inflammasome on stimulating KCs inflammation by free fatty acid(FFA).③To investigate role of NLRP3inflammasome on stimulation KCsinflammation by LPS.④The cultured KCs were stimulated by FFAfollowed by LPS to discuss the role of FFA-NLRP3inflammasome andIL-1pathway on stimulation KCs inflammation by LPS.Methods：①The mice were randomly divided into3groups accordingto the specific steps: perfusing by PBS without in situ perfusion collagenaseplus gradient centrifugation without Percoll liquids (modified method Agroup); perfusing by PBS without collagenase in situ perfusion plus gradientcentrifugation with Percoll liquids (B group); perfusion in situ withcollagenase plus gradient centrifugation with Percoll liquids (C group). Immunostaining with F4/80antibody and using trypan blue dye test wereused to determine cell viability. Swallowing ink experiments determine cellpurity and function. The survival time and morphological changes wereobserved to investigate the effectiveness and simply of different methods.②KCs isolated from mouse were randomly divided into5groups: culturedin medium only was control group; FFA group with0.2mmol/l; FFA groupwith0.3mmol/l; FFA group with0.4mmol/l; FFA group with0.5mmol/l.NLRP3、 Caspase-1、ASC protein was detected by Western blot,NLRP3、 Caspase-1、ASC mRNA was detected by real-time fluorescentquantitative PCR, IL-1、 IL-18were detected by ELISA. To studyinflammatory role of FFA (0.35mM) on KCs, the cells isolated from mousewere randomly divided into5groups: control group; FFA group (0.35mM);FFA+Control shRNA plasmid group, transfected with Control shRNAplasmid before treatment with FFA (0.35mM); FFA+NLRP3shRNAplasmid group, treatment with FFA after transfected with NLRP3shRNAplasmid. NLRP3、Caspase-1、ASC and IL-1、IL-18were detected asablove.③We randomly divided KCs isolated from mouse into4groups:cultured in medium only; LPS group with o.5ug/ml; LPS group with1ug/ml;LPS group with2ug/ml and control group. NLRP3、 Caspase-1、 ASCprotein was detected by Western blot, NLRP3、 Caspase-1、 ASC mRNAwas detected by real-time fluorescent quantitative PCR, IL-1、IL-18weredetected by ELISA. To study the role of NLRP3on inflammation of KCs stimulated by LPS, KCs were randomly divided into4groups: control group;LPS group (1ug/ml); LPS+Control shRNA plasmid group, treatment withLPS (1ug/ml) after transfected with Control shRNA plasmid; LPS+NLRP3shRNA plasmid group, treatment with LPS after transfected with NLRP3shRNA plasmid. NLRP3、Caspase-1、ASC and IL-1、IL-18were detectedas ablove.④KCs isolated from mouse were randomly divided into6groups:LPS group with1ug/ml; FFA group with0.35mM; LPS+FFA group, treatedwith FFA(0.35mM) followed by LPS (1ug/ml); LPS+FFA+Control shRNAplasmid group, treatment with FFA and LPS as ablove after transfected withControl shRNA plasmid; LPS+FFA+NLRP3shRNA plasmid group,treatment with FFA and LPS as ablove after transfected with NLRP3shRNA plasmid; control group, cultured in medium only. NLRP3、Caspase-1、ASC and IL-1、IL-18were detected as ablove.Results：①KCs isolated freshly were just quasi-circular, cells wereharvested at one hour after inoculation with high purity, but the cell yield isrelatively low. KCs were harvested with relatively high yield after2hinoculation. Iuminescent fluorescence in the328nm was not observed inKCs. KCs were seen to swallow many ink particles by in vitro experiments.2d and3d after cultivation, cells showed star or irregular in shape, adheringand fully extending. KCs were cultured for4w still alive.Immunofluorescence showed that the isolated cells were KCs. Trypan bluestaining was showed that the vitality of cells in each group were about 90%(P＞0.05). Perfusion by PBS, collagenase digestion in vitro,discontinuous gradient centrifugation and selective adherence was aeffectiveness, simply and stablility of modified method of isolating KCs.②eight hours after transfection with NLRP3shRNA plasmid, the transfectionefficiency was85%under fluorescent microscope identification. FFA withvarious concentrations stimulated cells significantly inflammation of KCs,along with the increase concentration of FFA, the current efficiency ofinflammation was significant (P＜0.001). NLRP3gene silencing withNLRP3shRNA can effectively inhibit the inflammation of KCs. Caspase-1and ASC expression in FFA group and FFA+Control shRNA group weresignificantly higher compared with the control group (P＜0.001). NLRP3gene silencing can reduce FFA-induced caspase-1, ASC, IL-and IL-18expression (P＜0.001). The expression of IL-and IL-18was increased incell culture medium by different concentration FFA stimulation (P＜0.001).③LPS with different concentrations stimulated cells significantlyinflammation of KCs, the higher concentration of LPS, the more significantof inflammation in KCs. Compared with the control group, IL-and IL-18expression in LPS with different concentration group were significantlyhigher (P＜0.001). However, the difference of expression of caspase-1,ASC and NLRP3were not significant in all groups (P＞0.05). NLRP3genesilencing can not reduce LPS-induced the expression of IL-and IL-18(P＞0.05).④Compared with the control group, FFA and LPS stimulated cells significantly inflammation of KCs. Pretreatment with FFA significantlyenhanced the production of LPS-induced IL-and IL-18(P＜0.001).NLRP3gene silencing with NLRP3shRNA can effectively inhibit theinflammation of KCs treated with FFA followed by LPS. NLRP3, caspase-1,ASC, IL-and IL-18expression in FFA+LPS group and FFA+LPS+Control shRNA group were significantly higher (P＜0.001). NLRP3gene silencing can reduce FFA+LPS-induced caspase-1, ASC, IL-andIL-18expression (P＜0.001).Conclusion：(1). The modified method of isolating KCs by perfusionof PBS plus collagenase digestion in vitro plus discontinuous gradientcentrifugation and selective adherence was effective and simple.(2) FFAcan stimulate cultured KCs inflammation with expression of IL-and IL-18by the pathway of NLRP3inflammasome.(3) LPS can stimulate culturedKCs inflammation with expression of IL-1and IL-18, but thisinflammation undepended on the pathway of NLRP3inflammasome.(4)FFA enhanced the inflammation of KCs stimulate by the LPS and thiseffect may be partially depented on the NLRP3inflammasome whichpromoted the expression of IL-1.