A Study On The Effects Of Tissue Kallikrein On Ischemic/Hypoxic Neuronal Injury In Vitro And Its Involving Mechanisms

Tissue kallikrein (TK),an important component of the kallikrein/kinin system(KKS),is a serine proteinase capable of cleaving low molecular weight kininogen torelease vasoactive kinins.Kinins in turn enable to tigger a series of biological effectsmainly by binding to bradykinin B2 (B2R) and B 1 receptor (B 1R).All components ofthe KKS are expressed and widely distributed throughout many mammalian tissuesand are up-regulated by ischemic stroke.Although in recent,accumulating evidencesuggests that humane TK gene transfer or exogenous TK administration,mainly viaB2R,exerts a beneficial effect on cerebral ischemia/reperfusion (I/R) injury,however,little is known about its definitely neuroprotective mechanisms.Among the diversemechanisms involved in the pathophysiology of post-ischemic injuries,it has beingincreasingly accepted that intracellular Ca²⁺ overload is a crucial factor ofischemic/hypoxic brain damage.There are two main pathways underlyingintracellular Ca²⁺ overload during brain ischemia/ hypoxia:One is theglutamate-dependent pathway mediated by ionotropic glutamate receptors includingNMDA and AMPA receptors,and another is the glutamate-independent pathwaymediated by a class of plasma-membrane proteins including acid sensing ion channels(ASICs).In order to explore the mechanism involved in the neuroprotection of TKagainst cerebral ischemia/hypoxia,in the present study we designed to investigate atlength the potential effects of exogenous human TK on glutamate-inducedneurotoxicity and ASICs-mediated neurotoxicity in vitro and its underlyingmechanisms.Partâ… Effects of tissue kallikrein on glutamate-inducedneurotoxicity and its underlying mechanisms1.Effects of tissue kallikrein on glutamate-induced neurotoxicityObjective:To investigate the potential role of TK in the high concentration ofglutamate-induced neurotoxicity on cultured cortical neurons. Methods:In the present study,we established the glutamate (Glu) neurotoxicitymodel by the exposure of cultured cortical neurons to modified Mg²⁺-free Earle'sbalanced salt solution (EBSS) containing 100μM Glu and 10μM glycine for 1 hfollowed by 0.5-24 h of restoration or reperfusion.Cell injury was determined bymeasuring the amount of LDH released into the medium during 1-24 h of restorationafter 1 h Glu exposure.Cell viability was assessed with a WST-8 assay using a CellCounting Kit-8 (CCK-8) after the Glu exposure and 24 h restoration.To compare theeffects of different agents on Glu-induced neuronal damage,LDH release and cellviable at 24 h restoration was measured after pretreatment of cells with TK (1.0nM-1.0μM),B2R agonist or antagonist for 24 h,or glutamate receptor (GluR)inhibitors 30 min before and during the Glu insult.The immunofluoresence method tolabel a neuron marker MAP-2 was applied to observe the morphological changes ofneurons exposed to 1 h Glu and 3 h restoration with or without TK and GluRinhibitors pretreatment.Apoptosis were assessed by TUNEL staining DNA fragmentand Hoechst neuclear staining.Results:1.Cultured neurons exposed to 1 h of Glu (100μM) combined withglycine (10μM),displayed a time-dependent increase in LDH release during thesubsequent 24 h of reperfusion.2.GluR antagonists induced a reduction in LDHrelease and an increase in neuronal survival.Pretreatment with TK (1.0 nM-l.0μM)elicited suppression of LDH release and enhancement of cell viability in aconcentration-dependent manner.Furthermore,the effects of TK (100 nM)adminstration was abolished by a specific B2R inhibitor HOE140 but simulated by aspecific B2R agonist BK.3.After 3 h of reperfusion following the Glu exposure,cortical neurons showed obvious morphological changes compared to the untreatednormal cells such as prominent somatic swelling,dendritic beading and disappearanceand a loss of cellular MAP-2 immunoreactivity.However,cells pretreated with TK orGluR antagonists had less such changes.Conclusion:A brief exposure to a high concentration of Glu followed byrestoration (reperfusion) induces obvious neuronal damage.TK administration,viaB2R,has comparable protective effects of GluR blockade on Glu-induced neuronalinjury. 2.Effects of tissue kallikrein on glutamate-induced oxidative/nitrosative stressObjective:To observe the pontential role of TK in Glu-induced oxidative andnitrosative stress.Methods:Cells on DIV 8-10 were randomly assigned to various pretreatmentconditions including normal control,untreated model control,TK,B2R agonist orB2R antagonist pretreatment 24 h,and GluR inhibitors pretreatment 30 min beforeand during the 100μM Glu exposure.1.Intracellular reactive oxygen species (ROS)were labeled by using fluorescent probe 2',7'-dichlorofluorescin diacetate(DCFH-DA).DCF fluorescent photomicrographs of cortical neurons on glass-bottomdishes were obtained with a fluorescent microscope and DCF fluroscent intensity ofcells on 96-well plates was read on a fluorescent microplate reader at an excitation of488 nm and an emission of 525 nm.2.NO was monitored by the NO-sensitive dye4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM/DA),and the cellularDAF-FM fluorescence intensity was obtained by using a fluorescence microplatereader with excitation at 495 nm and emission at 519 nm.3.Under the condition ofplentiful L-Argine,intracellular NO production can represent the total NOS activity.The nNOS activity was represented by the difference between NO production in thepresence and absence of calcium.4.Mitochondrial membrane potential (MMP) wasmonitored with the fluorescent cationic dye Rhodamine 123 (Rh 123) and the cellularfluorescence intensity of Rh 123 was quantified using a fluorescence microplatereader with excitation at 488 nm and emission at 525 nm.5.Intracellular free Ca²⁺level was detected by using Ca²⁺-sensitive fluorescent probe Fluo-3/AM,and Fluo-3fluorescence intensity was obtained by using a fluorescence microplate reader withexcitation at 488 nm and emission at 525 nm.Intracellular ROS and NO production,MMP and free Ca²⁺ level was expresses the fluorescent intensity of treated groupsfold relative to the normal control after background fluorescence obtained frommedium alone was subtracted.The nNOS activity was expressed a ratio of thedifference between fluorescence DAF-AM intensity in the presence and absence ofCa²⁺,over that of the fluorescence intensity in the presence of Ca²⁺.Each group hadeight samples,and each experiment was repeated at least 3 times.Resutls:1.When compared to the normal control,cells exposed continuously toGlu for 0.5- 6 h had higher ROS and NO production,the increased Ca²⁺ but thereduced MMP level at the same time points.2.Pretreatment with TK,B2R agonist or GluR inhibitors 24 h belfore and during the Glu exposure significantly reducedgeneration of intracellular ROS and NO and the Ca²⁺ level,but elevated the MMPlevel at the same time points of Glu exposure,particularly at the time point of 2 h.3.Addition of B2R antagonist before TK pretreatment could partially block the TKeffects.4.According to the results from the assay of nNOS activity at 2 h of Gluexposure,when compared to cultures under normal circumstances,the Glu exposureelicited a significant increase in nNOS activity.TK or GluR inhibitors pretreatmentmarkedly reduced the elevated nNOS activity.Blockade of B2R prior to TKpretreatment could abrogate the suppressive effects of TK on nNOS activity.Conclusion:TK is capable to prevent Glu-induced oxidative/nitrosative stress,viaactivation of B2R,by inhibiting intracellular ROS and NO production,nNOS activityand Ca²⁺ overload,and preventing mitochondrial depolarization to maintain the MMPlevel.3.The protective mechanisms of tissue kailikrein against glutamate-inducedneurotoxicityObjective:To elucidate the underlying signaling mechanisms of TK againstGlu-induced neurotoxicity.Methods:Immunocytochemical staining was performed at 1 h of restorationafter 1 h of Glu exposure to examine the expression of phospho-ERKl/2 (p-ERK1/2).At the end of Glu exposure immediately and at 1 h of reperfusion after the exposure,Western blot was performed to analyze the expression of phospho-Akt (p-Akt),phospho-JNK (p-JNK),phospho-P38 (p-P38),p-ERK1/2 and phospho-IKKα/β(p-IKKα/β).Results:1.Immunocytochemical staining showed that cortical neurons exposedto Glu alone showed stronger red fluorescence in their cytoplasm than those undernormal conditions,while neurons pretreated with TK prior to Glu insult exhibitedeven stronger fluorescence than those challenged by Glu alone.2.Western blotanalysis showed that one hour exposure to Glu immediately increased the level of p44-ERK1 by 153.7-fold and the p42-ERK2 level by 82.33-fold relative to normalcontrol levels.At 1 h of reperfusion,the increased p-ERK1/2 level that wassignificantly declined to 6.79- and 20.39-fold that of the normal control.Theexpression of p-ERK1/2 was almost totally blocked by GluR inhibitors or theERK-protein-kinase inhibitor PD98059.When compared to Glu exposure alone,a 24h pretreatment with TK before Glu insult induced a much greater increase in the levelof p44-ERK1,to 566-fold relative to the normal control.At 1 h of reperfusion,TKpretreatment elevated the p-ERK1 level to 51.05-fold,but not affect the expression ofp-ERK2.The B2R agonist BK pretreatment induced another 12.05-fold increase inthe level of p-ERK1 at 1 h of reperfusion after Glu exposure.The B2R antagonistHOE140 had a dramatic effect in preventing both the activation of ERK1 and 2induced by TK pretreatment.3.The expression of p-Akt was inhibited by Glu insultalone,and gradually increased during the subsequent reperfusion.Although TK andBK slightly increased the expression of p-Akt in neurons exposed to Glu,however theB2R blockade attenuated significantly the expression of p-Akt 1 h after Glu exposure.4.Neurons in normal conditions seldom expressed p-P38,but those insulted by Gluhad a significantly enhanced level of p-P38 expression,which decreased during thesubsequent reperfusion.Neither TK nor BK pretreatment reduced obviously theincreased expression of p-P38 induced by the Glu exposure.5.The exposure ofneurons to Glu produced an increase in the level of p-IKKα/β,TK pretreatmentenhanced the level of p-IKKα/βexpression in the normal cultured cells and thosechallenged by Glu.A specific ERK inhibitor PD98059 could reduce the level ofp-IKKα/βexpression 1 h after Glu exposure.6.The expression of p-JNK increasedsignificantly at the end of Glu exposure immediately and decreased to the basal levelat 1 h after Glu exposure.Either TK or B2R agonist pretreatment reduced theincreased level of p-JNK bythe exposure of Glu.The B2R inhibitor HOE140 couldabrogate the inhibition ofp-JNK expression induced by TK application.Conclusion:The exposure of cultured neurons to a high concentration of Glubriefly activates the three MAPK signal pathways,particularly ERK1/2,and NF-κBpathway but suppresses activation of PI3K/Akt pathway.TK plays a protective role inGlu-induced neurotoxicity via activation of B2R,correlating with differentiallyinfluencing activation of these signal pathways,strongly promotiong activation of theERK1/2,particularly by activation of the ERK1,and NF-κB pathways,slightlyenhancing activation of the PI3K/Akt pathway,moderately inhibiting the JNK activity, but does not influence the P38 activity.Partâ…¡Effects of TK on acid sensing ion channels-mediatedneurotoxicity and its involving mechanisms1.Effects of acid sensing ion channels activation on cultured neuronsObjective:To investigate the effects of acid-sensing-ion channels (ASICs)activation,especially activation of ASIC1a on cultured neurons under various acidpathological conditions.Methods:In the presence of blockers of major Ca²⁺ entry pathways includingiontrophic GluRs and voltage-gated Ca²⁺ and Na⁺ channels,we used primary culturesof cortical neurons to establish several acidosis (pH6.0) insult models includingacidosis/reperfusion (Ac),continuous acidosis (C-Ac),oxygen and glucosedeprivation (OGD) combined with acidosis (OGD-Ac) followed by reperfusion.Cytotoxicity was determined by assaying the amount of LDH released into themedium.Cell viability was measured with an assay kit of CCK-8.Theimmunofluoresent double label method was applied to observe the distribution ofASIC1a and the morphological changes of neurons exposed to 90 min of OGD-Acand 4 h ofreperfusion.Results:1.Cytotoxicity gradually increased and cell viability progressivelyreduced during reperfusion following 2 h acid incubation (pH6.0).At 24 h ofreperfusion,the relative LDH release attained 45.6% and cell survival decreased to58.5%,when compared to the normal control.2.Continuous acid incubation (pH6.0)induced a more markedly increase in LDH release and decrease in cell viability thanthe Ac model.At the end of 24 h of C-Ac treatment,only about 10% neuronsremained alive.3.Neurons exposed to OGD-Ac 90 min followed by reperfusionexhibited more remarked injury,had a greater increase in LDH release and decreasein cell viability than those challenged by OGD 90 min with reperfusion.3.Blockadeof ASIC1a with a specific inhibitor PcTX1 or nonspecific inhibitor Amiloridealleviated neuronal damage induced by C-Ac and OGD-Ac.The immunofluorescent staining showed:at 4 h of reperfusion following OGD-Ac 90 min,cortical neuronsshowed obvious morphological changes relative to normal condition-treated cells,such as prominent somatic swelling,neurite disruption and loss,nuclear pyknosis(condensation) and a loss of cellular MAP-2 immunoreactivity.ASICla normallydistributed on cytoplaysm membrane and axons,but mainly on cytoplyasm membraneafter the OGD-Ac insult.Conclusion:In the presence of a cocktail of ionotropic Glu andvoltage-dependent Ca²⁺ and Na⁺ channels blockers,the Ac,C-Ac and OGD-Acmodels are effective to induce cultured neurons injury.Under various acidpathological conditions,ASICs,especially ASICla activation,inducesGlu-independent neuronal injury wherease ASICla blockage elicits a markedlyneuroprotective effect.2.Effects of TK on acidosis-induced neurotoxicity in vitroObjective:To observe the effects of TK on ASICs-mediated neurotoxicity,weintestigated the potential role of TK administration in acidosis- and OGD-combinedwith acidosis-induced neuronal injury.Methods:In the presence of a cocktail of glutamate and voltage-dependent Ca²⁺and Na⁺ channels blockers,neurons were treated with TK,B2R agonist and antagonistfor 24 h,or PcTX1 for 30 min before exposed to Ac,C-Ac,OGD alone or OGDcombined with acidosis,Cytotoxicity was determined by assaying LDH release.Neuronal viability was measured by CCK-8.Death cells were observed by PI/Hoechststaining.Apoptotic cells were analyzed by TUNEL staining at 24 h reperfusion afterOGD-Ac 90 min.Immunocytochemical staining was applied to observe thedistribution of ASICla and the effects of TK pretreatment on the morphological ofneurons exposed to OGD-Ac 90 min followed by 4 h reperfusion and C-Ac for 3 h.Results:1.Results from measurement of LDH release and cell viability showed:Primary cortical neurons were treated with various concentration of TK (0.01-1.0μM)for 24 h prior to C-Ac,2 h of acidosis with reperfusion or 90 min of OGD-Acfollowed by reperfusion for 1-24 h.TK elicited a concentration-dependent increase in cell viability and decrease in LDH release.2.PI/Hoechst staining revealed that TKpretreatement reduced the increased ratio of PI positive cells in various acidosismodels.3.PcTX1 and B2R agonist had comparable effects of TK wherease B2Rinhibitor HOE140 had an inverse effect.3.The immunofluoresent stainingdemonstrated that the addition of 100 nM TK 24 h before and during OGD-Ac or acidincubation remarkably inhibited the morphological changes of neurons exposed to 4 hreperfusion following OGD-Ac or 3 h of acidosis,and did not affect the expression ofASIC1a.5.TUNEL staining showed that at 24 h reperfusion after OGD-Ac 90 minTK and PcTX1 pretreated groups had lowered ratio of apoptotic cells than that ofuntreated OGD-Ac.Conclusion:These findings reveal that TK may have antioxidant properties.TKadministration produces a protective effect on acidisos- or OGD-Ac-induced neuronaldamage,similar to that seen with ASICs blockade,by alleviating neuronal injury,reducing cell death and apoptosis and promoting cell survival.3.Effects of TK on the expression of ASICs in cultured neuronsundergoing oxygen and glucose deprivationObjective:To investigate the effects of TK on the expression of ASICs mRNAand proteins.Methods:RT-PCR was used to examine the mRNA expression changes ofASIC1a,ASIC2a and ASIC2b in normal cultured neurons and those exposed to OGD90 min followed by reoxygenation l h,2 h,3 h,6 h and 12 h.Western blot was usedto investigate the changes of ASICla,ASIC2a and ASIC2b protein expression innormal control neurons and those challenged by 90 min of OGD with reoxygenation 1h,2 h,3 h,6 h,12 h and 24 h.And we further examine the effects ofTK pretreatmentfor 24 h on the expression ofASICla,ASIC2a and ASIC2b mRNA and proteins.Results:1.The expression of ASICla,ASIC2a and ASIC2b mRNA in culturedneurons was plentiful but had an obvious decline during 1-12 h of reoxygenationfollowing OGD 90 min.The expression ofASICla mRNA level had a slow decreaseduring the first 6 h of reoxygenation,but such a decrease became accelerated after that and reached its minimum at 12 h.The expression of ASIC2b mRNA level graduallyreduced during 1-12 h of reoxygenation.However,the ASIC2a mRNA expressionincreased slightly at the early stage of reoxygenation,reached it peak at 2 hreoxygenation and decreased after that.2.TK pretreatment significantly inhibited allthe three subunits of ASICs mRNA expression.3.Western blot results showed:ASIC la protein expression changed largely during the course of 1-24 h reoxygenation:during the first 6 h ofreoxyenation,its expression had no obvious change,during 6-12h of reoxyenation displayed a remarkable decrease and reached the minimal level at12 h reoxyenation;however,after that time point ASICla protein expressionexhibited a significant increase.TK treatment could reduce the expression ASIClaprotein level during 1-6 h and 12-24 h of reoxygenation.At the early stage ofreoxygenation,the level of ASIC2a protein expression had a significant increase,especially at the time point of 2 h,then experienced a slow increase from 3 h to 12 h.TK pretreatment did not reduce but enhanced slightly the ASIC2a protein expression.The ASIC2b protein expression exhibited a progressive reduction during the wholecourse of reoxygenation.TK treatment had no effect on the change of ASIC2b proteinexpression.Conclusion:Although during the course of reoxygenation following OGD 90min,ASICla,ASIC2a and ASIC2b expression experiences a reduction in the level ofmRNA,however their protein expression changes differently.TK has an inhibitoryeffect on the level of ASICla,ASIC2a and ASIC2b mRNA expression,butdifferential effects on their protein expression:significantly inhibits ASIClaexpression,slightly enhances the ASIC2a expression level but does not influenceASIC2b expression.TK application elicits a neuroprotective effect on acidosis or/andOGD-induced damage,in part by down-regulation of ASIC1a and up-regulation ofASIC2a protein expression.4.The intracellular signaling mechanisms of tissue kallikrein alleviatingneurotoxicity induced by the combination of OGD and acidosisObjective:To elucidate the underlying signaling mechanisms of TK alleviating neurotoxicity induced by the combination of OGD and acidosis.Methods:lmmunocytochemical staining was performed at 1 h of reperfusionfollowing the combination of OGD and acidosis for 90 min,to check the expressionof p-Akt,p-ERK1/2,p-JNK and p-P38.Western blot was used to quantitativelyanalyze the expression of p-Akt,p-ERK,p-JNK and p-P38 proteins at the end of 90min of OGD-acidosis insult and 1 h reperfusion after the insult.During theOGD-acidosis insult,a cocktail of ionotropic glutamate and voltage-dependent Ca²⁺and Na⁺ channels blockers was present to abolish activation of ionotropic glutamateand Ca²⁺ and Na⁺ channels.We further observed the effect of the ERK1/2 blockade onTK-promoting survival by assaying cell viability at 4 h of reperfusion following 90min of OGD-Ac.Results:1.Immunocytochemical staining revealed that neurons exposed toOGD-Ac insult with 1 h reperfusion exhibited stronger red fluorescence of p-Akt,p-ERK,p-JNK and p-P38 proteins than those in normal conditions.2.Western blotresults demonstrated that the p-Akt protein expression was inhibited by OGD-Acinsult alone,and significantly increased during subsequent reperfusion.TKpretreatment had no significant effect on the level of p-Akt expression.3.Culturedneurons in normal conditions could express p-ERK1/2,p-JNK and p-P38 proteins.At1 h of reperfusion following the 90 min of OGD-Ac,cells had significant increasedlevels ofp-ERKl/2 and p-JNK protein expression but no obvious change in the p-P38expression.4.TK treatment had differential effects on activation of MAPK signalpathways:At the end of OGD-Ac 90 min and subsequent reperfusion 1 h,cellspretreated with TK had a marked rise in the expression of p-ERK1/2,moderatereduction in that of p-JNK,however no significant effect on the p-P38 expression.PcTX1 pretreatment had effects consistent with those of TK adminstration.5.Treatemet with an inhibitor ERK1/2 PD98059 for 1 h,prior to TK pretreatment,significantly inhibited the survival-promoting effect of TK on cells exposed toOGD-Ac 90 min with reperfusion 4 h.Conclusion:The exposure of cultured neurons to OGD-Ac 90 min brieflyactivates the JNK and P38 but suppresses the PI3K/Akt pathways;however,1 h ofsubsequent reperfusion following the OGD-Ac induced activation of the PI3K/AktERK1/2 and JNK pathways.TK may play a protective role in OGD-Ac-inducedneurotoxicity on primary cultures of cortical neurons via potently promotingactivation of the ERK1/2,moderately inhibiting activation of the JNK pathway while the PI3K/Akt and the P38 signal pathways are not required for TK-mediatedneuroprotection.Conclusion1.TK,acting mainly through B2R,plays a protective role in glutamate-inducedneurotoxicity in cultured cortical neurons,by conferring resistance tooxidative/nitrosative stress,suppressing neuronal apoptosis and promotingneuronal survival through activation of the ERK1/2,in particular ERK1,theIKKα/β-NF-κB and the PI3K/Akt signal pathways,at the same time inhibition ofthe JNK signal pathway.2.TK adminstration,like ASICla blockade,protects cortical neurons againstdamage induced by ischemia-acidosis/reperfusion in vitro via allevation ofneuronal injury and apoptosis and enhancement of neuronal viability.And theneuroprotective mechansims of TK against neuronal injury produced by acidosisand/or hypoxia mainly involve down-regulation of the ASICla expression andup-regulation of the ASIC2a expression,probably through activation of theERK1/2 signal pathway and inhibition of the JNK signal pathway.3.In summary,based on our observations,we propose that TK,as a well-establishedneuroprotective agent,to a certain extent,may directly or indirectly interfere withglutamate-dependent and ASICs-mediated glutamate-independent Ca²⁺ neurotoxicmechanisms to ameliorate ischemic/hypoxic brain damage.