The Mechanism Investigation Of HMGB1/RAGE Pro-inflammatory Axis In Vascular Endothelial Cells Injury Induced By Limb Ischemia Reperfusion

Chapter ? Variation of HMGB1/RAGE axis in limb ischemia reperfusion injury1 Research background and objectiveAcute limb ischemia reperfusion injury(ALI)is one of common sever emergency in surgery department and could be caused by different reasons induced suddenly lumen occluded.Although the treatment technologies have been obviously improved,the rates of complication,mortality,and limb loss after acute limb surgery remains higher in the current.Restoring blood flow is a requirement condition to security the normal function of ischemia limb,but restoring blood flow could induce a series of stress responses and will results in reinjury for ischemia tissue,which called ischemia reperfusion injury(I/R).IR injury could induce different levels of injury,which could develop from a mild impairment to a systematical inflammation reaction and result in several organ injuries.ALI is a complicated inflammatory response process companied with different degrees of oxidative stress responses.The common inflammatory cytokines are including Interleukins(ILs),intercellular adhesion molecules(ICAM),Granulocyte colony-stimulating factor(G-CSF),Tumor necrosis factor(TNF-?),Monocyte chemoattractant protein 1(MCP1),and so on.During this process,inflammation is inevitable occurs in vascular tissue with increase expression of aforementioned cytokines,and induce dysfunction of vascular constrictor,increase of vascular permeability,accumulation of white blood cell,and formation of microthrombus,enhancing IR injury.Thus,it is of importance to alleviate the inflammatory response in vascular of limb to protect vascular structure and function,relieving limb IR injury.High mobility group box protein 1(HMGB1)is a pro-inflammatory cytokine which could transfer from intracellular condition to extracellular modulating the pathogenesis of IR injury.Previous studies of our group have shown that HMGB1 plays an important pro-inflammatory role in the induction and maintenance of inflammatory response in acute limb ischemia-reperfusion,but it is still unclear which receptors it binds to mediate signal transmission?HMGB1 mainly has two receptors,the receptor for advanced glycation end products(RAGE)and the toll-like receptor 2/4(TLR 2/4).The HMGB1/RAGE signal axis activates the RAGE-dependent signal transduction mechanism,such as the MAPK pathway,to activate nuclear factor kappa B(NF-?B)to mediate inflammatory response,which plays an important role in brain,liver,kidney and other tissues during ischemia-reperfusion injury.As a RAGE specific blocker,PFS-ZM1 can block the binding of RAGE to HMGB1 and the signaling mediated by the combination of the two.In this study,PFS-ZM1 was used to pretreat lower limb ischemia-reperfusion rats,to explore the changes of HMGB1/RAGE signal axis in acute lower limb ischemia-reperfusion injury in rats,and to provide new data support for the molecular mechanism of HMGB1-dependent inflammatory signals in acute lower limb ischemia-reperfusion injury2 MethodsA total of 24 male SD rats were randomly divided into three groups:Sham,IR,and IR+FPS-ZM1.After the IR model was constructed,the tissue samples were harvested and the following experiments were conducted:H&E staining and EVG staining were performed to detect the pathology changes,qRT-PCR was used to detect the mRNA expression of HMGB1 and RAGE,and western blot was used to examine the expression of HMGB1,RAGE,P38,p-P38,ERK1/2,pERKl/2,P65,p-P65,IKBa,p-IKBa,NLIRP3,P65 nucleus location.In addition,the blood samples of rat were also collected and used to determine the expression HMGB1,IL-6,and TNF-?using the ELISA method.3 Results1)H&E and EVG stainings were used to explore the pathology changes in different group of rats.The results showed that IR could significantly promote the secretion of inflammatory cytokines and the injury degradation of collagen fibers and elastic fibers,as well as inhibit the survival of endothelial cells.Blocker FPS-ZM1 pre-treatment could significantly reverse the injury of IR on the femoral artery of rats.2)IR injury could significantly upregulate the expression of HMGB1,TNF-?,and IL-6 in the serum of rats;however,FPS-ZM1 pre-treatment could only inhibit the expression of TNF-? and IL-6 induced by IR injury.3)IR injury could significantly increase the expression of HMGB1 and RAGE in femoral artery of rats,and FPS-ZM1 pre-treatment had no significantly effect on the expression of HMGB1 and RAGE in femoral artery of rats after IR.4)IR injury could significantly increase the expression of p-P38,p-ERK 1/2,p-P65,p-IKBa,NLRP3,and P65 nucleus location,but FPS-ZM1 pre-treatment could significantly reverse the effect of IR injury on the expression and activation of effector cytokines in p38/NF-?B signaling pathway4 ConclusionAfter ischemia-reperfusion,the expression of HMGB1 and RAGE in vascular tissue and blood increased significantly,the inflammatory response was up-regulated,and the vascular injury was aggravated.Blocking the binding of RAGE and HMGB1 by FPS-ZM1 can significantly inhibit the inflammatory response level and MAPK signaling pathway activity after ischemia/reperfusion of lower extremity in rats,inhibit the activation of NF-?B,and destroy the positive feedback mechanism of the inflammatory response signaling pathway,thus reducing tissue damage,but has no significant effect on the expression of HMGB1/RAGE.Chapter 2 HMGB1 interacted with RAGE to mediates the inflammatory response in the injury of vascular endothelial cells induced by ischemia reperfusion1 Research background and objectiveAcute ischemia reperfusion injury is a common symptom in clinic,which could not only impair the construction and function of local ischemia tissue,but also could influence the function of remote organ,even sever acute ischemia reperfusion could threaten the security of patients.Vascular endothelial cells(VECs)is a mechanical barrier between vascular smooth muscle and blood,and it has play critical roles in the message delivery,smooth muscle contraction,vascular permeability,and blood coagulation.Previous studies reported that VEC function injury is a trigger point in the process of IR,which could lead to a reduction of NO secretion and induce chemoattractant to recruit neutrophile granulocyte,adhesion molecules to the injury location of vascular inducing a further injury on vascular.Thus,it is important to explore the mechanism changes of VEC during the IR injury,which might be of significance in IR clinical prevention and treatment.To explore the the mechanism of HMGB1/RAGE pro-inflammatory in VECs injury induce by IR,HUVEC cells were introduced in this study,and a hypoxia/re-oxygen(HR)model was constructed to mimic IR injury in vivo.Then,the effect of FPS-ZM1 on HMGB1/RAGE pro-inflammatory axis mediated inflammatory response and cell injury induced by HR was determined.2 MethodsHR model in HUVEC were used to mimic the IR injury in in vivo.Then,different concentration of FPS-ZM1 were used to treatment HR induced HUVECs and cell survival was determined to select a suitable FPS-ZM1 treatment concentration.Following this,the cells were divided into three groups:Control,HR,HR+FPS-ZM1,and the following experiments were performed:qRT-PCR assay was used to determine the mRNA expression of HMGB1 and RAGE,Co-IP assay was used to determine the interaction between HMGB1 and RAGE,Hoechst assay was used to detect the injury levels of cells,flow cytometry was used to detect the apoptosis of cells,immunofluorescence(IF)was used to detect the expression and location of P65 and NLIR3,western blot was used to detect the expression of HMGB1,RAGE,P38,p-P38,ERK1/2,pERK1/2,P65,p-P65,IKBa,p-IKBa,and NLIRP3,and the nucleus location of P65 in cells,and ELISA assay was used to determine the expression of HMGB1,IL-6,and TNF-ain cell medium.3 Results1)CCK-8 was used to determine cell survival of HUVECs after treating with gradient concentration of FPS-ZM1 and found that 1.0 nM FPS-ZM1 could significantly inhibit the survival of HUVEC after HR.Thus,0.5 nM FPS-ZM1 was selected and used in the following experiments.2)CCK-8 assay showed that HR treatment could significantly inhibit the proliferation of HUVEC,but FPS-ZM1 pre-treatment could partially reverse the effect of HR on the proliferation of HUVEC.3)Flow cytometry and Hoechst assay showed that HR could significantly increase the apoptosis and injury of HUVEC,but FPS-ZM1 pre-treatment could partially reverse the effect of HR on the apoptosis and injury of HUVEC.4)ELISA assay results showed that HR treatment could significantly upregulate the expression of HMGB1,TNF-?,and IL-6 in cell medium,while pre-treatment could suppress the upregulation of TNF-aand IL-6 but had no effect on HMGB1 expression after HR.5)Co-IP assay showed that HMGB1 could directly interacted with RAGE,and FPS-ZM1 pre-treatment could significantly suppress the interaction between HMGB1 and RAGE.6)IF assay results presented that HR could significantly increase the nucleus location of P65 and expression of NLRP3,while FPS-ZM1 pre-treatment could suppress these changes.7)Western blot results showed that HR treatment could obviously increase the expression of p-P38,p-ERK1/2,p-P65,p-IKBa?and NLRP3,as well as the nucleus location of P65,while FPS-ZM1 pre-treatment could suppress these changes4 ConclusionHR treatment could significantly increase the expression of HMGB1,RAGE,and inflammatory cytokines,as well as the activation of inflammatory associated signaling pathway to induce cell apoptosis.FPS-ZM1 pre-treatment could significantly block the interaction between HMGB1 and RAGE as well as their mediated inflammatory signaling to alleviate the injury and apoptosis of HUVEC induced by IR.Chapter 3 HMGB1/RAGE pro-inflammatory axis mediated autophagy of HUVEC induced by HR1 Research background and objectiveAutophagy is a stress response of cells to changes of intracellular and extracellular environment.At normal condition,autophagy keep at a basic level and play critical role in cell homeostasis.However,immoderate autophagy might impair the construction and function of cells and induced autophagic cell death.It is reported that autophagy level is significantly increased in the process of ischemia reperfusion,indicating that autophagy might play critical role in the IR injury.To explore the mechanism of HMGB1/RAGE pro-inflammatory axis in the autophagy induced by IR injury,autophagy inhibitor 3-MA,autophagy inducer rapamycin(RAPA),and FPS-ZM1 were used to treat HR induced HUVEC cells and the expression of HMGB1,RAGE,inflammatory cytokine,and autophagy levels of HUVECs were determined in this study.2 MethodsHR treatment in HUVEC were used to mimic IR injury in vivo,Autophagy inhibitor 3-MA,autophagy inducer RAPA,and FPS-ZM1 were used to treat HR induced HUVEC cells and divided cells into five groups:control,IR,IR+3-MA,IR+FPS-ZM1,and IR+FPS-ZM1+RAPA.Then,the following investigations were performed:qRT-PCR assay was used to determine the mRNA expression of HMGB1 and RAGE,western blot was used to determine the expression of HMGB1,RAGE and autophagy markers:LC3 ?/?,Beclin 1,NLRP3,TNF-?,IL-6,and P62.Transmission electron microscope(TEM)was used to analyze the formation of autophagosome,IF was used to determine the expression of LC3B,and ELISA was used to determine the expression levels of IL-6 and TNF-ain cell medium.3 Results1)ELISA assay showed that IR HR injury could significantly increase the expression of TNF-? and IL-6 I in medium of HUVEC,but 3-MA or FPS-ZM1 treatment could partially inhibit the expression of TNF-? and IL-6 I in medium of HUVEC induced by HR.However,FPS-ZM combined RAPA treatment could further enhance the expression of TNF-? and IL-6 I in medium of HUVEC after HR2)qRT-PCR and western blot results showed that HR treatment could significantly increase the expression of RAGE and HMGB1 in HUVECs.In addition,3-MA,FPS-ZM 1,or FPS-ZM1+RAPA had no significantly influence on the expression of RAGE and HMGB1 in HUVECs after HR?3)HR treatment could markedly increase the expression levels of LC3 ?,Beclin ?,NLRP3,IL-6,and TNF-abut decrease the expression of P62.However,3-MA or FPS-ZM1 treatment could partially reverse these changes,while FPS-ZM1 combined RAPA treatment could further enhance the autophagy effect of HR on HUVECs.4)HR injury could markedly increase the autophagosome in HUVECs,but 3-MA or FPS-ZM 1 treatment could partially inhibit the formation of autophagosome in HUVEC after HR.However,FPS-ZM combined RAPA treatment could further enhance the formation of autophagosome in HUVECs after HR.5)IF results showed that HR injury could significantly increase the expression of LC3B,but 3-MA or FPS-ZM1 treatment could partially inhibit the expression of LC3B induced by HR.However,FPS-ZM combined RAPA treatment could further enhance the expression of LC3B in HUVECs after HR.4 ConclusionHR injury could significeantly increases the expression of HMGB1 and RAGE,the inflammatory signaling,and autophagy activation of HUVEC,inhibition of the HMGB1/RAGE pro-inflammatory signal axis could significantly inhibit the cellular inflammatory response and autophagy activities caused by ischemia-reperfusion,suggesting that the HMGB1/RAGE pro-inflammatory signal axis promoted ischemia-reperfusion induced autophagy by mediating cellular inflammatory response.