The Effect Of HMGB1A-box On LPS/TLR4Pathway In SW480and THP-1Cells

Background and objective:To study the influence of HMGB1-A box high-expressing on the LPS/TLR4signaling pathways of SW480cells and THP-1cells. So, we conduct this study toinvestigate the effect of HMGB1-A box on the LPS/TLR4signaling pathway andprovide theoretical and experimental evidence for targeting in IBD.Method:â… . The influence of LPS on HMGB1expression in SW480cellsCultivated SW480cells to the logarithmic phase, we gave differentconcentration of LPS and collected cells respectively at12h,24h （Repeated3times independently）Groups were divided as follows:â‘ Control group: SW480cells add0ng/ml LPS;â‘¡Experiment group1: SW480cells add100ng/ml LPS;â‘¢Experiment group2: SW480cells add1000ng/ml LPS;â‘£Experiment group3: SW480cells add10000ug/ml LPS;â…¡. Constructing and identifying HMGB1-Abox high-expressing cells1. Constructing and identifying the HMGB1-A box recombinant expressionplasmidâ‘´Searched for HMGB1-A box gene sequences in the GeneBank, and givengene synthesis by the company.⑵Constructed, amplified and extracted the recombinant plasmid after thesynthesis of HMGB1-A box gene were inserted into pEGFP-N1vector. Thenidentifying the HMGB1-A box recombinant expression plasmid withdual-endonuclease digestion and sequencing.2. Expression of HMGB1-A box recombinant eukaryotic plasmid in SW480cells SW480cells were transfected with pEGFP-N1-HMGB1-A box using FuGENE6Transfection Reagent. We observed the presence of green fluorescence andfluorescence intensity respectively, and collected the cells， then identied theexpression of HMGB1-Abox.（Repeated experiment3times independently）.â…¢. Influence of the high HMGB1-A box expression and EP on the HMGB1,TLR4/LPS signaling pathways and the secretion of cytokines of SW480intestinalepithelial cells:Groups were divided as follows:1Control group: SW480cells2EP group：SW480cells+EP3Blank plasmid group: SW480cells were transfected with pEGFP-N1（blankplasmid）.4A box plasmid group:SW480cells were transfected with pEGFP-N1-HMGB1–AboxFour groups were divided into LPS and no LPS groups （EP5mM, pre-treatmentof1h early; LPS1ug/ml,24h）. We collected the cells and cells culture supernatantafter24h.（Repeated experiment3times independently）.â…£. Influence of the high HMGB1-A box expression and EP on the HMGB1,TLR4/LPS signaling pathways and the secretion of cytokines in THP-1cells:SW480cells and THP1cells were co-culture in Transwell system. Groupswere divided as follows:1Control group: SW480cells+THP-12EP group: SW480cells+EP+THP-13Blank plasmid group: SW480cells were transfected with pEGFP-N1（blankplasmid）+THP-1.4Abox plasmid group: SW480cells were transfected with pEGFP-N1-HMGB1-Abox+THP-1Four groups were divided into LPS and no LPS groups （EP5mM,pre-treatment:1h; LPS1ug/ml,24h）. We also collected the cells and cells culturesupernatant after24h.（Repeated experiment3times independently）.â…¤.Assessment: â‘ The mRNA expression of HMGB1,TLR4and MyD88mRNA in SW480cellsand THP-1cells were determined by Real Time-qPCR.â‘¡The protein expression of HMGB1, TLR4, MyD88protein and pNF-κB p65in SW480cells and THP-1cells were determined by Western Blot.â‘¢The level of HMGB1,TNF-α, IL-6and IFN-γ in cell culture supernatant weredetermined by ELISA.Results:â… .Influence of LPS on HMGB1expression in SW480cells:1.Different concentration of LPS was treated to SW480cells for12h, HMGB1mRNA expression has increased in all groups,but the LPS concentration of100ng/mlgroup compared with control group, there are statistically significant （P <0.05）;different concentrations of LPS was treated to SW480cells for24h, HMGB1mRNAexpression has increased in all groups, but1000ng/ml group of HMGB1mRNAexpression compared with control group, with statistical difference （P <0.01）, whilethere were no statistical difference among other groups （P>0.05）.2. SW480cells with LPS treatment on different time, the HMGB1mRNAexpressions of the24h group were higher than the12h group, but there werestatistically significant difference only between1000ng/ml group and control group（P <0.05）. Thus, we chose SW480cells with the time of LPS treatment lasting24hand the concentration of1000ng/ml.â…¡. Construction and identification of high HMGB1-A box expression ofeukaryotic plasmid cell lines:1. Construction and identification of the HMGB1-A box recombinant expressionplasmidThe pEGFP-N1-HMGB1-A box recombinant plasmid was successfullyconstrusted. It was confirmed by sequencing and restriction enzyme digestion, andtwo bands in the agarose gel by enzyme digestion were observed. The molecular wasconsistent with expected size. The inserted HMGB1-A box fragments was consistentwith the published data （Gen-Bank Accession: NM₀02128.4）.2. Expression of HMGB1-A box recombinant eukaryotic plasmid in SW480 cellsSW480cells transfected with HMGB1-A box recombinant plasmid appearedgreen fluorescence under the fluorescent microscope. HMGB1-A box mRNAexpressions in cells were higher than no-transfection cells （P <0.01）. This resultssuggested the SW480cells were successfully transfected.â…¢. Influence of the high HMGB1-A box expression and EP on the HMGB1,TLR4/LPS signaling pathways and the secretion of cytokines in SW480intestinalepithelial cells:1. HMGB1mRNAand protein expression in each group:There were not significant differences of the HMGB1mRNA and proteinexpression among the groups without LPS treatment （P>0.05）. In the LPS treatmentgroups, the HMGB1mRNA and protein expression in EP group was significantlylower than the control group, empty plasmid group and A box transfection group （P<0.05）, but there were not statistical differences among the control group, emptyplasmid group and A box transfection group （P>0.05）; All LPS treatment groups butEP group were significantly higher than the corresponding group without LPStreatment （P <0.05，P <0.01）.2. TLR4mRNAand protein expression in each group:There were not significant differences of the TLR4mRNA and proteinexpression among the groups without LPS treatment （P>0.05）. In the LPS treatmentgroups, the TLR4mRNA and protein expression in EP group was significantly lowerthan the control group, empty plasmid group and A box transfection group （P <0.05）,but there were not statistical differences among the control group, empty plasmidgroup and A box transfection group （P>0.05）; All LPS treatment groups but EP groupwere significantly higher than the corresponding group without LPS treatment （P<0.05，P <0.01）.3. MYD88mRNAand protein expression in each group:There were not significant differences of the MYD88mRNA and proteinexpression among the groups without LPS treatment （P>0.05）. In the LPS treatmentgroups, the MYD88mRNA and protein expression in EP group was significantly lower than the control group, empty plasmid group and A box transfection group （P<0.05）, but there were not statistical differences among the control group, emptyplasmid group and A box transfection group （P>0.05）; All LPS treatment groups butEP group were significantly higher than the corresponding group without LPStreatment （P <0.05，P <0.01）.4. pNF-κB p65protein expression in each group:There were not significant differences of the p-NF-κB p65protein expressionamong the groups without LPS treatment （P>0.05）. In the LPS treatment groups, thep-NF-κB p65protein expression in EP group was significantly lower than the controlgroup, empty plasmid group and A box transfection group （P <0.05）, but there werenot statistical differences between the control group, empty plasmid group and A boxtransfection group （P>0.05）; All LPS treatment groups but EP group weresignificantly higher than the corresponding group without LPS treatment （P <0.05，P<0.01）.5. The cytokines level of HMGB1、IL-1β、TNF-α and IL-6in cell culturesupernatant of SW480cellsâ‘´There were not significant differences of the HMGB1level among the groupswithout LPS treatment （P>0.05）；In the LPS treatment groups,HMGB1level in EPgroup was significantly lower than the control group, empty plasmid group and A boxtransfection group （P<0.05）, but there were not statistical differences among thecontrol group, empty plasmid group and A box transfection group （P>0.05）; All LPStreatment groups but EP group were significantly higher than the corresponding groupwithout LPS treatment （P <0.05，P <0.01）.⑵There were not significant differences of the IL-1β、TNF-α and IL-6levelamong the groups without LPS treatment （P>0.05）; In the groups with LPStreatment, IL-1β、TNF-α and IL-6level in EP group was significantly lower than thecontrol group, empty plasmid group and A box transfection group （P <0.05）, but therewere not statistical differences among the control group, empty plasmid group and Abox transfection group （P>0.05）; All LPS treatment groups but EP group were significantly higher than the corresponding group without LPS treatment （P <0.05，P<0.01）.â…£. Influence of the high HMGB1-A box expression and EP on the HMGB1,TLR4/LPS signaling pathways and the secretion of cytokines in THP-1cells:1.HMGB1mRNAand protein expression in each group:Co-cultured with SW480cells, there were no significant differences of theHMGB1mRNA and protein expressions in THP-1cells among the groups withoutLPS treatment （P>0.05）.In the groups with LPS treatment, HMGB1mRNAexpressions of THP-1cells in the EP group were significantly lower than the controlgroup and empty plasmid group （P <0.05）.Additionally,A box transfection group wassignificantly lower than the empty plasmid group （P<0.05）. HMGB1proteinexpression in THP-1cells in the EP group and A box-transfection group weresignificantly lower than the control group and empty plasmid group （P <0.05）. AllLPS treatment groups but EP group and A box transfection group were significantlyhigher than the corresponding group without LPS treatment （P <0.05, P <0.01）.2.TLR4mRNAand protein expression in each group:Co-cultured with SW480cells, there were no significant differences of the TLR4mRNA and protein expressions in THP-1cells among the groups without LPStreatment （P>0.05）. In the LPS treatment groups,TLR4mRNA expressions in THP-1cells in the EP group and A box transfection group were significantly lower than thecontrol group and empty plasmid group （P<0.05）. TLR4protein expressions inTHP-1cells in the EP group were significantly lower than the control group andempty plasmid group （P <0.01）, and A box transfection group was significantly lowerthan the empty plasmid group （P<0.05）. All LPS treatment groups but EP group and Abox transfection group were significantly higher than the corresponding groupwithout LPS treatment （P <0.05, P <0.01）.3.MYD88mRNAand protein expression in each group:Co-cultured with SW480cells, there were no significant differences of theMYD88mRNA and protein expressions in THP-1cells among the groups withoutLPS treatment （P>0.05）. In the LPS treatment groups, MYD88mRNA and proteinexpressions in THP-1cells in the EP group and A box transfection group were significantly lower than the control group and empty plasmid group （P<0.05）. AllLPS treatment groups but EP group and A box transfection group were significantlyhigher than the corresponding group without LPS treatment （P <0.05, P <0.01）.4.pNF-κB p65protein expression in each group:Co-cultured with SW480cells, there were no significant differences of thep-NF-κB p65protein expression in THP-1cells were not significant difference amongthe groups without LPS treatment （P>0.05）. In the LPS treatment groups p-NF-κBp65protein expression in THP-1cells in the EP group and A box transfection groupwere significantly lower than the control group and empty plasmid group （P<0.01）.p-NF-κB p65protein expressions of THP-1cells in the A box transfection group weresignificantly lower than the empty plasmid group （P<0.05）. All LPS treatment groupsbut EP group and A box transfection group were significantly higher than thecorresponding group without LPS treatment （P <0.05, P <0.01）5.The cytokines level of HMGB1、IL-1β、TNF-α and IL-6in cell culturesupernatant of THP-1cells（1）Co-cultured with SW480cells, there were no significant difference ofHMGB1level in THP-1cells among the groups without LPS treatment （P>0.05）; Inthe LPS treatment groups, HMGB1level in the EP group and A box transfectiongroup was significantly lower than the control group and empty plasmid group（P<0.05,0.01）; All LPS treatment groups but EP group and A box transfection groupwere significantly higher than the corresponding group without LPS treatment （P<0.05, P <0.01）（2）Co-cultured with SW480cells，there were no significant difference ofIL-1β、TNF-α and IL-6level in THP-1cells among the groups without LPS treatment（P>0.05）; In the LPS treatment groups, IL-1β、TNF-α and IL-6level in the EP groupand A box transfection group was significantly lower than the control group andempty plasmid group （P <0.01）; All LPS treatment groups but EP group and A boxtransfection group were significantly higher than the corresponding group withoutLPS treatment （P <0.05, P <0.01）Conclusion: 1.LPS can activate TLR4signaling pathway， up-regulate the expression ofHMGB1in SW480and THP-1cells, and promote secretion of pro-inflammatorycytokine.2.In the co-culture system using HMGB1-A box highly expressied SW480celland THP-1cell,HMGB1-A box can inhibite the expression and secretion ofHMGB1in THP-1cell, as well the LPS/TLR4signaling pathway and the secretionof pro-inflammatory cytokines.3. EP can inhibit the HMGB1expression and secretion in SW480and THP-1cells,as well the LPS/TLR4signaling pathway and the secretion of pro-inflammatorycytokines.