| Part I：The TRAF3gene and protein expression and K-48ubiquitination level in endotoxin tolerance Kupffer cellsObjective: This study was to observe the changes of tumor necrosisreceptor associated factor3(TRAF3) gene and protein expression, K-48ubiquitination level, and the secretion of cytokines in endotoxin toleranceKupffer cells (KCs).Methods: The KCs were isolated by vitro collagenase digestion anddensity gradient centrifugation combined selective adherence to plastic.F4/80immunofluorescence staining was used to identify the purity of KCs.Trypan blue dye and swallowing ink experiment were used to judge theactivity and phagocytosis of KCs. The KCs morphological change wasdynamic observed over time. Then, KCs were randomly divided into twogroups: the endotoxin tolerance group (ETT Group) in which KCs was given10ng/ml LPS stimulation for24h to300ng/ml LPS stimulation and thenon-endotoxin tolerance group (NETT Group) in which KCs was directed to LPS stimulation (300ng/ml).The cells in the two groups were harvested at0,5,10,30,60min after treatment, respectively. Real-time fluorescentquantitative PCR (Q-PCR) was used to detect TRAF3mRNA expressionchanges. Western blotting and laser confocal was used to detect TRAF3change and K48ubiquitin protein (K48-Ub) level. ELISA method was usedto detect the secretion of IL-1, IL-10, and TNF-αintheculturesupernatant.Results:(1) Immunofluorescence staining indicated that F4/80molecules expressed strongly on the cell membrane. The purity of KCs wasmore than92.0%. Trypan blue staining showed the vitality of KCs was morethan98.5%. Swallowing ink test indicated that KCs consumed large amountsof carbon particles.(2) The TRAF3mRNA expression had no significantlydifference between ETT group and NETT group at the observation period(P>0.05). However, TRAF3protein expression in the ETT group wassignificantly higher than that in the NETT group (P<0.05).The K-48ubiquitination level in ETT group was significantly decreased comparedwith the NETT group (P<0.05).(3) The IL-1and TNF-αlevels in the cellculture supernatant in ETT group were significantly lower than those inNETT group (P<0.05). The secretion of IL-10in ETT group wassignificantly up-regulated compared with NETT group (P<0.05).Conclusion: The gene expression of TRAF3in has no obvious changewhen endotoxin tolerance happened. However, the protein expression ofTRAF3was up-regulated as a result of the inhibited K-48ubiquitination. Part II：Inhibiting TRAF3ubiquitination degradation induceendotoxin tolerance in Kupffer cellsObjective: Since cellular inhibitor of apoptosis proteins2（cIAP2） isspecificity ubiquitin E3-ligase for TRAF3, this study was to evaluate theeffects of cIAP2-shRNA on TRIF and MAPK signal pathway when thedegradation of TRAF3was suppressed.Methods: KCs were seeded into six-well at the density of3-4×106andrandomly divided into two groups: transfection group in which KCs wasstimulated with LPS (300ng/ml) after transfected with cIAP2-shRNAlentivirus plasmid for48h and the control group in which KCs wasstimulated with LPS (300ng/ml) transfected with control shRNA lentivirusplasmid for48h. The cells in the two groups were harvested at0,5,10,30,60min after the treatment, respectively. Laser confocal microscopy was usedto detect the protein expression of cIAP2, TRAF3and K-48Ub. Westernblotting was used to determine the protein expression of cIAP2, TRAF3,K-48Ub, JNK, p-JNK, p38, p-p38, TRIF and IRF3. ELISA method wasused to detect the secretion of IL-1, IL-10, and TNF-α in the culturesupernatant.Results:(1)The protein expressions of cIAP2and K-48Ub in thetransfection group were significantly lower than those in the control group ineach time point (P<0.05).(2) The protein expressions of TRAF3, TRIF and IRF3in transfection group were significantly higher than that of controlgroup (P<0.05).(3) The protein expression of JNK and p38between thesetwo groups had no significant difference (P>0.05). The protein expressionof p-p38and p-JNK in transfection group was much lower than these incontrol group (p<0.05).(4) The IL-1and TNF-αlevels in the cell culturesupernatant in transfection group were significantly lower than those incontrol group (P<0.05). The secretion of IL-10was significantly upregulatedcompared with control group (P<0.05).Conclusion: Inhibiting TRAF3ubiquitination degradation can induceendotoxin tolerance in Kupffer cellsPart III：The effect of down-regulated TRAF3expression onendotoxin tolerance in Kupffer cellsObjective: This study was to investigate the effect of TRAF3geneslience by shRNA on the activation of MAPK and TRIF signaling pathwaysand endotoxin tolerance in KCsMethods: KCs were seeded into six-well at the density of3-4×106andrandomly divided into three groups: transfection group in which KCs weretransfected with TRAF3-shRNA plasmids for24h, and then given smalldose of LPS (10ng/ml) pretreatment for another24h, at last given high dosesof LPS stimulation (300ng/ml); Endotoxin tolerance group (ETT group) inwhich KCs were transfected with control shRNA for24h, other treatment were same with transfection group; Non-endotoxin tolerance group in whichKCs were transfected with control shRNA for24h and stimulated with highdose of LPS stimulation (300ng/ml). The cells in the three groups wereharvested at0,30,60min, respectively. Western blotting was used to detectprotein expression of TRAF3, JNK, p-JNK, p38, p-p38, TRIF andIRF3.ELISA was used to detect the secretion of IL-1, IL-10, and TNF-α inthe culture supernatant.Results:(1) The protein expressions of TRIF and IRF3in transfectiongroup were significantly lower than these in the ETT group at various timepoints (P<0.05), but higher than these in NETT group (P <0.05).(2)TheJNK and p38protein expression in the three groups had no significantdifference.The protein expression of p-p38and p–JNK in tranfection groupwere significantly higher than these in ETT group (p<0.05), but lower thanNETT group (p <0.05).(3) The IL-1and TNF-αlevels in the cell culturesupernatant in transfection group were significantly higher than those in ETTgroup (P<0.05), but significantly lower than the NETT group.(4) Thesecretion of IL-10in transfection group was significantly down-regulatedcompared with control group (P<0.05), but up-regulated compared withNETT group (P<0.05).Conclusion: TRAF3was involved in endotoxin tolerance throughinhibiting k-48ubiquitination. Endotoxin tolerance was impaired but not completely eliminate when TRAF3gene was silenced. |
PDF Full Text Request