Mechanism Of Epidermal Growth Factor Receptor On The Release Of TNF-alpha Induced By HMGB1 In Endotoxemia

Endotoxemia is one of systemic inflammatory response syndrome(SIRS)in clinical.Severe trauma,burns,ischemia and necrosis of gastrointestinal mucosa,poor immunity and even mechanical ventilation,all of these are likely to induce endotoxemia.In addition,endotoxemia is more common in peroperative period.Once it is not well treated in time,endotoxemia patients will suffer from severe sepsis,which will result in a series of reactions such as septic shock,acute heart failure,acute lung injury(ALI),acute kidney injury,multiple organ dysfunction syndromes(MODS)and multiple organ failure(MOF)at last.It is always the leading cause of mortality in intensive care unit(ICU).Scientists still focus on researching the pathogenesis of endotoxemia and sepsis for the purpose of finding suitable therapeutic methods.High mobility group box 1 protein(HMGB1),which is thought to be a most important "late" inflammatory cytokine,is an important biomarker in prognosis of sepsis by evaluating its concentration in peripheral blood.Endotoxin,also knowned as lipopolysaccharide(LPS),is the major cause of Endotoxemia.It not only can bind to pattern recognition receptor(PRR)such as Toll like recptor(TLR)to produce tumor necrosis factor alpha(TNF-?),interleukin(IL)-6,IL-?,IL-8 and so on,but also can regulate translocation of HMGB1 from nucleus to extracellular.The extracellular HMGB1 can activate MAPK,PI3K/Akt,NFr?B signaling pathway by interacting with receptor for advanced glycation end product(RAGE),TLR2 and TLR4,and produce inflammatory cytokines through signaling cascade.Necrosis cells resulted from ischemia,hypoxia,trauma tissue can also directly release HMGB1 that lead to sterile SIRS.The mechanism of HMGB1 in inflammation has been a hot topic.In recent years,in addition to HMGB1,the epidermal growth factor receptor(EGFR)signaling pathway involved in regulation of inflammation also has gradually been discovered and taken seriously.EGFR,one of the human epidermal growth factor receptors(HER),possess tyrosine kinase activity.In addition to EGFR,its family members also include HER2,HER3 and HER4.LPS has been found to bind TLR4 and promote release of tumor necrosis factor-a-converting enzyme(TACE),which crack transforming growth factor-a(TGF-a)to release.TGF-?,one of EGFR ligands,can bind to EGFR and promote EGFR to form homodimer or heterodimer and lead to autophosphorylation.HER2 is an orphan receptor,which can not bind to any ligand.It involves in EGFR signaling pathway by combining EGFR together to form heterodimer.It is reported that heterodimer is more effective than homodimer in signaling transduction.Phosphorylation of EGFR activates downstream signaling pathways including MAPK,PI3K/Akt pathway,and further activates NF?B or specifcity protein-1 transcription factors,finally up-regulate the release of IL-8,IL-1 and other cytokines.We can see that EGFR and HER2 have much in common in mediating inflammation.Both of them can interact with TLR,and activate the MAPK,PI3K/Akt,NF?B pathway.But there is no definite evidence whether the activated EGFR can mediate the release of TNF-a.Additionally,whether EGFR is involved in inflammatory recation induced by HMGB1 is still unknown.In our early study,we found that EGFR inhibitor PD 168393 or Erlotinib could significantly reduce the expression level of TNF-a in LPS-induced primary myocardial cells.This confirmed that indeed EGFR is involved in the production of TNF-a in inflammation.Similarily,EGFR inhibitor PD168393 or Erlotinib could significantly reduce the expression level of TNF-a in multiple types of cells induced by HMGB1.These findings provide sufficient evidence to support our later study.In this study,two EGFR inhibitors PD 168393 or Erlotinib was preconditioned in THP1 cell line and primary bone marrow-derived macrophages(BMM).To investigate two major problems:the effect of EGFR on TNF-a expression induced by HMGB1 and the mechanism of EGFR involved in TNF-a induced by HMGB1.Methods1.Cytotoxicity and viability testThe cytotoxicity of different concentrations of EGFR inhibitor PD 168393 and Erlotinib was tested with cell counting kit-8(CCK-8),then selected the non-cytotoxic concentration for this research.The cell viability 'was tested at 24h and 48h after HMGB1 administration with CCK-8.2.GroupsTHP1 or BMM cells were divided into six groups:Control group(Ctrl),Erlotinib group(Er,20?M),PD168393 group(PD,10?M),HMGB1 group(H1,1?g/ml),HMGB1+Erlotinib group(H1+Er)and HMGB1+PD168393 group(H1+PD).The cells were pretreated for 30min with different inhibitors before stimulated by HMGB1.In addition,we set a new group,which was administrated with boiled HMGB1 to exclude the interference of LPS.3.Silenced EGFR and HER2 through transfecting siRNAPD 168393 is an irreversible selective inhibitor,which inhibits both HER2 and EGFR.We silenced the mRNA expression of EGFR and HER2 with siRNA before stimulated with HMGB1.In order to figure out whether HER2 was involved in the expression of TNF-a,we used siRNA to silence the mRNA expression of HER2 and EGFR.Seven groups were set:Control group(Ctrl),Lipo2000 transfection reagent control group(Lipo2000),the negative control group(NC),HMGB1 group,HMGB1 + si-EGFR group,HMGB1 + si-HER2 groups.HMGB-1 was administered after cell transfection for 24-48 hours.4.Detection the expression of TNF-a in supernatant with Enzyme-Linked Immunosorbent Assay(ELISA)Cell supernatants were collected to test the expression of TNF-a with ELISA.5.Detection of mRNA expression of EGFR and HER2 with PCR The total RNA was extracted and reverse transcripted,then mRNA expression of EGFR and HER2 was tested by quantitative real-time polymerase chain reaction(QRT-PCR).6.Detected the expression of phosphorylated proteins ERK1/2 and P38 with western blot Cells were collected and washed twice with PBS and detected the expression of cell phosphorylation protein ERK1/2 and P38 by western blot.7.Statistical analysisData analysis was performed by SPSS 19.0 software.Data were expressed as mean ± standard deviation(S.D.).Single factor at two different time-points were assessed by using repeat measurement.Comparation between two groups was tested by independent samples t test and comparation among multiple groups were tested by one way ANVOA.If equal variance assumed,we chose LSD t test for post hoc multiple comparisons,if not,then we used Dunnett's t test.P values<0.05 was considered statistically significant.Figures were made by using GraphPad Prism 5.0 drawing software.Results1.Expession of phosphorylated protein of EGFR stimulated by HMGB1 in THP1 cellsTHP1 cells were deivided into control group and HMGB1 group.Cells of HMGB1 group was stimulated by HMGB1 in the concentration of 1?g/ml for 4 hours,while control group was administratered with equal-volume of 0.9%normal salt.Result showed that expession of phosphorylated protein of EGFR was significantly increased in HMGB1 group compared with control group(P<0.05).2.Cytotoxicity of different concentrations of PD168393 and ErlotinibFor PD168393,compared with the control group and the other concentration groups,cell viability significantly decreased in the highest concentration of 20 ?M(P<0.05);for erlotinib,Compared with control and other groups,cell viability significantly decreased in concentrations of 50?M and 1000?M(P<0.05).According to the result,we chose the concentration of 10?M in PD168393 and 20?M in Erlotinib for next experiments.3.Cell viability in HMGB1-stimulated THP1 cellsTHP1 cells were stimulated with HMGB1in the concentration of 1?g/ml after pretreated with Erlotinib and PD168393 for 30min,and cultured for 24 hours or 48hours.CCK-8 results demonstrated a significant difference of cell viability at 24 hours and 48 hours(P<0.001).There was interaction effect between culture time and different groups(P<0.001).HMGB1 improve cell viability with the prolongation of stimulation time(P<0.05).To compare different groups at the same time,cell viability of HMGB1 group significantly increased at 24hours and 48hours compared with control group(P<0.01).Compared with HMGB1 group,cell viability of HMGB1+Erlotinib group decreased statistically significant at 24hours and 48hours(P<0.01);It had no differences between HMGB1 group and HMGB1+PD168393 group at 24 hours(P>0.05),but has significant difference at 48 hours(P<0.01).4.Effect of EGFR inhibitors on the expression of TNF-a induced by HMGB1After THP1 cells stimulated with 4 hours,the level of TNF-a in control group was(14.85±8.52)pg/ml,the level of TNF-a in HMGB-1 group released significantly(P<0.01)with(4192.931688.41)pg/ml.Both of two EGFR inhibitors could inhibit the release of TNF-a(P<0.01).So that the concentration levels of TNF-a were reduced to(474.78 ± 25.43)pg/ml(Erlotinib),(1333.48 ± 105.85)pg/ml(PD168393).PD168393 inhibited the generation of TNF-? stimulated by HMGB-1,which was more effective than the use of Erlotinib(P<0.01).Compared with HMGB1 group,the expression of TNF-a after stimulated with boiled HMGB1 was significantly lower((177.96 ± 14.50)pg/ml,P<0.01),which confirmed that the generation of TNF-a caused by HMGB-1 mainly dued to HMGB1 itself,not LPS.In BMM cells,the results were similar with THP1 cells.The levels of TNF-a increased from(14.1414±21)pg/ml to(1728.41±265.35)pg/ml after HMGB1 stimulation.The difference was statistically significant(P<0.01).The use of Inhibitors could reduce the generation of TNF-a approximately 50%,which was statistically significant(P<0.01).But the inhibitory potency of two inhibitors was no significant difference(P>0.05).5.Effect of siRNA on the expression levels of TNF-a induced by HMGB1THP1 cells were transfection with small interfering RNA(siRNA)of EGFR or HER2 for 24 to 48 hours and then tested the inhibition ratio of EGFR or HER2 mRNA expression.THP1 cell was stimulated with HMGB1 for 4 hours if transfection inhibition ratio was up to 50-60%.The expression of TNF-? in HMGB1 group,HMGB1+si-EGFR group and HMGB1+si-HER2 group increased compared with the control group,which were statistically significant(P<0.001).In HMGB1?si-EGFR group and HMGB1+si-HER2 group,the expression of TNF-? were inhibited compared with HMGB1 group,which was statistically significant(P<0.05).The generation of TNF-? in HMGB1+si-EGFR group and HMGB1+si-HER2 group had no significant difference(P=0.307).6.Effect of HMGB1 on ERK1/2 and P38 phosphorylationWestern blot was used to detect the phosphorylated proteins ERK1/2 and P38.The levels of ERK1/2 and P38 phosphorylation significantly increased after stimulated by HMGB1 with 4 hours compared with control group(P<0.01).Compared with HMGB1 group,the phosphorylation levels of ERK1/2 was significantly inhibited in HMGB1+Erlotinib and HMGB1+PD168393 groups,which was statistically significant(P<0.01).HMGB1+ PD168393 group was more significantly than HMGB1+Erlotinib group.The expression levels of P38 phosphorylation between HMGB1+PD 168393 group and HMGB1 group had statistically significant difference(P<0.01),however the expression levels of P38 phosphorylated between HMGB1+Erlotinib group and HMGB1 group had no statistically significant difference(P<0.01).The expression levels of phosphorylated P38 in HMGB1+PD 1683 93 group were significantly lower compared HMGB1+Erlotinib group(P<0.05).Conclusions1.HMGB1 can induce the release of TNF-a in THP1 cell and BMM cells.2.Both EGFR and HER2 are involved in the release of TNF-a induced by HMGB1.3.The possible mechanism that HMGB1 induced the release of TNF-a is that HMGB1 leads to ERK1/2 and P38 phosphorylation by activating EGFR and HER2.Phosphorylation of ERK1/2 and P38 regulate the transcription and generation of TNF-a.