| Background and objectivecerebral ischemia-reperfusion injury(CIRI) is the most common pathological process of cerebral infarction.The mechanisms of nerve cells damage induced by CIRI are complex and diverse,and its essential pathological changes of nerve cells is necrosis and apoptosis due to hypoxia and ischemia.It is reported that a lot of cytokines involved in process of CIRI.nuclear factor-κB(NF-κB) is found that it is associated with neuronal apoptosis and necrosis induced by cerebral ischemic injury,and that its functions relate to cell cycle regulation,apoptosis activation,cytokine transcription,and many other aspects. High mobility group protein box 1(HMGB1) are a class of charge-rich non-histone chromatin protein,involved in many pathophysiological process.A large number of clinical studies and experiments in vivo and in vitro,in recent years,identified extracellular HMGB1 as a late inflammatory factor playing an important role at the pathological process of sepsis.Unexpectedly,recent studies have found that HMGB1 is closely related to ischemia/reperfusion injury(IRI).HMGB1 released to extracellular milieu by the injured and necrotic cells,activates neutrophils, macrophages,endothelial cells,glial cells,etc to induce inflammatory response. Furthermore,a large number of inflammatory mediators such as tumor necrosis factor-α(TNF-α),interleukin-1(IL-1),interleukin-6(IL-6),intercellular adhesion molecule-1(ICAM-1),etc produced by the activated cells,result in necrosis and apoptosis of other tissue cells.However,studies about expression and role of HMGB1 in neurocytes in cerebral ischemia / reperfusion have no reported in our country,and the study about impact of HMGB1 from neurons to neurons is more rare.To explore the role of HMGB1 in the mechanisms that neuronal apoptosis can be caused by ischemia/reperfusion injury(IRI) for searching the further clinical measures to CIRI,in this study,we are going to make models on neurons,which cultured in vitro,by hypoxia/reoxygenation(H/R) injured to simulate IRI and by rHMGB1 acted directly(without H/R),and observe apoptosis,expression of HMGB1 and NF-κB in neurons,and the relationship of the three,Materials and methods1.Experimental groupingCortical neurons cultured for 7 days were randomly divided into 4 groups:A (normal control Group),B(hypoxia/reoxygenation alone H/R),C(treatment with rHMGB1,and without H/R) and D(pretreatment with PTDC + H/R).2.Manufacturation of modelGroup A always was cultured in the 95%O2+5%CO2,37℃incubator after its serum-free medium replaced by sugar earle's medium.Group B was deal with H/R: put into constant temperature of 37℃hypoxia bag that was exhausted air,after the culture medium was replaced by sugar-free earle's medium,slowly filling the 95%N2 +5%CO2(flow rate of 1 L/min)for 30min,then cultured in the 37℃,95%O2+5% CO2 incubator after fully enclosing bag.Group C was treated with 0.2ug/L rHMGB1 alone without H/R,and cultured in incubator.Group D,after pretreated with 1μM PTDC for lh,was deal with H/R as Group B.After 50 minutes,all the 4 groups were re-exchanged for serum-free medium,and cultured under normal conditions for 24h.3.content of observationTo observe the cells morphology under the inverted microscope,to detect cells survival rate by MTT colorimetry,to detect apoptotic cells by TUNEL,and to detect the expression of HMGB1,NF-κB using Immunocytochemical technique on each time point(0h,1h,3h,6h,12h,24h) after reoxygenation or after treatment with rHMGB1 of Each group.Results1.The results of cells morphology under the inverted microscopeIn Group A,normal 7d-neurons were active.Refractive index of the cells were strong,nuclei were big and clear,and the neurites interlaced into networks.Compared with Group A,Group B had poor shapes,rounded,shranked,deformated,or broken bodies,and shortening or broken neurites after H/R,and the cells that had shranked of deformated bodies were apoptotic neurons.With reoxygenation prolonged,damaged and apoptotic cells increased in Group B.Similar to group B,damage and apoptotic cells of Group C were increased.In Group D,after treated with PTDC+H/R,the damage and apoptosis cells were less.2.The results of cells survival rate by MTT colorimetryCompared with Group A,the cells survival rate in Group B was low,and the later the lower.and the differences were significant(P＜0.05) on each time point after reoxygenation,similar to group B,in Group C,the survival rate of cells declined too. Compared with group B or C the survival rate was increased in Group D(P＜0.05).But the survival rate in Group D was still lower than Group A on each time point from 6h to 24h.3.The results of cells apoptosis by TUNELCompared with Group A,apoptotic cells increased obviously in Group B and Group C from 3h-point after reoxygenation or treatment of rHMGB1 finished.And the later,apoptotic cells were the more.The differences were significant(P＜0.05) on each time point(3～24h).Compared with Group B and C,apoptotic cells in Group D decreased markedly on each time point(3～24h,P＜0.05).Compared with Group A, apoptotic cells in Group D were more(3～24h,P＜0.05).4.The results of Immunocytochemical for HMGB1the expression of HMGB1 could be in nuclei and cytochylema of normal neurons and was low in Group A.After H/R,in Group B,the expression of HMGB1 in cytochylema increased from 0～3h,on peak at 12h-point,and maintained the high standard to 24h.And the differences were significant on each time point(0～24h, P＜0.05),compared to group A.In Group C,after treatment with rHMGB1,the expression of HMGB1 in cytochylema raised from 1～3h,on peak at 12h-point,and maintain the high standard to 24h.And compared to group A,the differences were significant(P＜0.05) on each time point(3h,6h,12h,24h).The expression of HMGB1 reduced in Group D due to pretreatment of PTDC.And compared to group B and C, the differences was significant(P＜0.05) after reoxygenation.It was higher than that in group A,but the difference was not significant.5.The results of Immunocytochemical for NF-κBThe expression of NF-κB could be in cytochylema of normal neurons and was very low,and it would be difficult to find expression of NF-κB in nuclei of normal neurons in group A.In group B and C,the expression of NF-κB in nuclei of neurons increased significantly(P＜0.05) on each time point(3h,6h,12h,24h),compared to group A.And there was no significant differences between Group B and Group C.The expression of NF-κB reduced in Group D.And compared to group B and C,the differences was significant on each time point(3～24h,P＜0.05).It was higher than that in group A,but the difference was not significant.6.The results of analysis for the correlation HMGB1,NF-κB,and neuronal apoptosisIn Group B,with reoxygenation time extension,Pearson correlation coefficient of HMGB1 expressed in neuronal cytoplasm and TUNEL positive cells:r= 0.66,P＜0.05;Pearson correlation coefficient of NF-κB expressed in neuronal nucleus and TUNEL positive cells:r= 0.774,P＜0.05;Pearson correlation coefficient of HMGB1 and NF-κB:r=0.737,P＜0.05.Conclusions1.ischemia/reperfusion could induce HMGB1 translocate to cytoplasm of neurons,and NF-κB translocate to nuclei of neurons.2.HMGB1 may act directly on neurons and could induce neuronal damage and apoptosis.3.one of the mechanisms of neuronal damage and apoptosis induced by HMGB1 after ischemia / reperfusion may be associated to NF-κB.4.HMGB1 and NF-κB maybe induce each other's expression to increase. |
