The Function And Mechanism Of Bradykinin Receptors In Neuronal Injury From Hypoxia/reoxegenation

Bradykinin(BK) is a biological active peptide derived from the kalligen under the effect of kallikrein.BK acts on its receptor and exerts various biological functions such as vessel dilatation,hypotention and inhibit the proliferation of the smooth muscle cells.The bradykinin receptors distribute widely in animal bodies and almost exist in all kinds of the tissues.The receptor has 2 subtypes,one is B1 receptor(B1R),the other is B2 receptor(B2R).in physiological conditions,B2R is the dominant bk receptor whereas the B1R is underexpressed.However,in stress(such as ischemia,inflammation and injury),the expression of B1R increase obviously.There are reports that the functions of B1R and B2R are different in peripheral ischemic insults,B1R promotes the injury whereas B2R alleviate the injury.In this study,we used cultured neurons and astrocytes,built the hypoxia/reoxygenation(H/R) model to investigate the expression of bradykinin receptors and their changes in H/R.We also use specific agonist and antagonist of B IR and B2R to observe the function of these 2 receptors in neuronal H/R,and furthermore,to explore the mechanism behind it.Partâ… The expression in cultured neurons and their changes after H/RObjective:to investigate the expression of bradykinin B1R and B2R in cultured neurons in normal condition and after H/R is the premise and foundation to study the functions of these 2 receptors in H/R in neuronal cells.in this study,we used the hypoxia/reoxygenation model to investigate the expression of bradykinin B1R and B2R in cultured neurons both in normal condition and after H/R.Methods:the immunofluoence double label method was applied to observe the distribution of bradykinin receptors in neurons,used RT-PCR to examine the mRNA expression changes of B1R and B2R in different H/R time point and used western blot to investigate the changes of B1R and B2R protein levels.the time point choosed is before-hypoxia,hypoxia 90 min,reoxgenationl h,reoxygenation 4 h,reoxygenation 12 h and reoxygenation 24h.Results:1.The cell body of neuronal cell was swelling after 90min hypoxia and 4h reoxygenation insult,MAP-2 immunofluorecence revealed some neuraxon appeared as string-of-beads or broken,MTT assay suggested the neuronal survival rate deceased to 50%compared to normal condition.2.there are bradykinin B1R and B2R expression in cultured neurons,the distribution of these 2 receptors was different.B1R distributed largely on the cell membrane and the B2R most distributed on the neuclei membrane.3.In normal condition,the expression of B 1R mRNA and protein level is very low.however,it could be induced obviously after H/R.The B1R mRNA and protein levels reached its peak at the time point of 1 h after reoxygenation and decreased rapidly.On another hand,there are expressions of B2R mRNA and protein in normal condition.It could be induced by H/R too.The B2R mRNA expression reached its peak at the time point of 1 h after reoxygenation,and the B2R protein expression reached its peak on the time point of 1-4 h after H/R and maintain high expression till 24h of reoxygenation.Conclusion:1.The H/R model is effective and the time point of hypoxia 90 min reoxygenation 4 h can be the time point to following experiments.2.There are bradykinin B1R and B2R expression in cultured neurons,which has different manner in cell distribution and changes after H/R.this may be the foundation of these 2 receptors exert their physiological functions.Partâ…¡Study of the function of bradykinin receptors in neuronal H/RObjective:To study the function of bradykinin B1R and B2R in H/R injury in curtured neurons.Methods:The specific agonist and antagonist of B1R and B2R were used to observe the neuronal survival rate by MTT,LDH release assay was used to investigate the cytotoxicity and the TUNEL assay was applied to tested the apoptosis ratio of neuronsResults:1.The neuronal survival rate decreased to 50%of that before hypoxia after 90 min hypoxia 4h reoxygenation,with obvious increase of LDH release and apoptosis ratio.2.The specific B1R agonist des-Arg9-BK inhibited the survival of neurona after H/R,increased the apoptosis ratio and the cytotoxicity.This detrimental effect of B1R could be partially inhibited by the specific B1R antagonist Lys-(des-Arg9-Leu8)-BK.3.Bradykinin B2R agonist BK activated B2R,increased the neuronal survival rate after H/R insult,reduced apoptosis of neurons,alleviated cytotoxycity.This protective effect was inhibited by its specific antagonist HOE140.Conclusion:1.H/R decreased the neuronal survival rate and increased the LDH release,induced neuronal apoptosis.2.Bradykinin B1R and B2R exert different functions in neuronal H/R insults,in which B 1R exerts a detrimental effect whereas B2R a protective effect.Partâ…¢The function of bradykinin receptors in cultured astrocytesObjective:1.To observe the expression of bradykinin B1R and B2R in cultured astrocytes in normal condition and after H/R.2.Investigate the function of B1R and B2R in astrocytes after H/R.Methods:The expression of bradykinin 2 receptors on cultured astrocytes was examined by immunofluorescence double labeled methods,observe the change of bradykinin B1R and B2R mRNA and protein levels in cultured astrocytes after H/R.The B1R and B2R specific agonist and antagonist were used to examine the effect of bradykinin 2 receptors on the secreation of cytokine in astrocytes after H/R.Results:1.There are bradykinin B1R and B2R expressions in cultured astrocytes, the B1R distributed largely on the cell membrane and the B2R most distributed on the neuclei membrane,which is similar with the neurons.In normal condition,there was expression of B1R mRNA and protein level in astrocytes but the ratio is low.However,it could be induced obviously after H/R.The B1R mRNA reached its peak at the time point of 3h after reoxygenation,and protein reached its peak at the time point of 6h after reoxygenation and maintained a high expression till 24h after reoxgenation.On another hand,there are expressions of B2R mRNA and protein in normal condition,it could be induced by H/R too.The B2R mRNA and protein reached its peak both at the time point of 3h after hypoxia,and maintain high expression till 24h of reoxygenation.3.H/R induced the secretion of VEGF and GDNF of astrocytes after H/R.4.Bradykinin B1R specific agonist des-Arg9-BK inhibited the secretion of VEGF and GDNF,which could be eliminated by its specific antagonist Lys-(des-Arg9-LeuS)-BK.on the contrary,bradykinin B2R agonist BK increased the secretion of VEGF and GDNF after H/R,which could be inhibited by HOE140,the specific antagonist of B2R.Conclusion:1.There are B1R and B2R expression in cultured astrocytes which can be induced by H/R.2.The secretion of VEGF and GDNF in astrocytes is increased after H/R insult.activate bradykinin B 1R inhibits this effect whereas activation of B2R promote it.Partâ…£Mechanism of bradykinin receptors effect in neuronal H/RObjective:To study the signal transduction mechanism of bradykinin B1R and B2R by using cultured neuronal H/R model.Methods:Constructed the neuronal H/R model,examined the activation of ERK1/2,JNK and p38 pathway induced by H/R by using of ELISA assay kits.Examined the function of specific agonist and antagonist of B1R and B2R in the expression of phosphor-ERK 1/2,JNK and p38 pathway.Used the specific antagonist of ERK 1/2 pathway(PD98059),JNK pathway(SP600125) and p38 pathway(SB203580) to block theses 3 pathways and examined the changes of the bradykinin B1R and B2R function in neuronal H/R.Cell survival rate was examined by MTT,cytotoxicity was examined by LDH release.We also observed the effect of the bradykinin 2 receptors on caspase-3 activation in neuronal H/R by absorption spectrometry method.Results:1.The ERK1/2,JNK and p38 pathway were activated by H/R.The expression of phosphor protein levels changed with the time of reoxgenation.the phospho-ERK 1/2 protein reached its peak at the time of 1 h after reoxygenation,the phospho-JNK protein,1-2 h and the phosphor-p38 protein,30 min.2.The B1R agonist increased the expression of phospho-ERK1/2,JNK and p38 proteins which could be partially inhibited by the specific B 1R antagonist Lys-(des-Arg9-Leu8)-BK.The B2R agonist BK increased the expression of phospho-ERK 1/2 protein but decreased the expression of phospho-JNK and p38 proteins,this effect could be inhibited partially by HOE 140.3.The specific antagonist of JNK pathway,SP600125,could inhibit the cytotoxicity induced by activation of B1R and increase the cell survival rate.The antagonist of ERK1/2 pathway PD98059 could inhibit the protective effect of B2R,increased the cytotoxity and decreased the cell survival rate.4.The caspase-3 was activated after H/R,which enhanced by activation of B1R and alleviated by agonist of B2R.Conclusion:Bradykinin B 1R and B2R may exert their function through different pathways,the B1R can activate the JNK and caspase-3 pathway to promote the neuronal injury and B2R can activate the ERK1/2 pathway,inhibit activation of caspase-3 to exert a protective effect in neuronal H/R.