The role of p38 MAPK activation in preconditioning mediated protection against ischaemia/reperfusion injury

Hartley, Shahiem (2002-12)

Thesis (MSc)--Stellenbosch University, 2002.


ENGLISH ABSTRACT: The ultimate consequence of the interruption of blood flow to the myocardium is necrosis. In view of the prevalence of coronary artery disease in the general population, and the deleterious effects of myocardial ischaemia on myocardial tissue, it is important to develop new strategies to protect the myocardium against ischaemia. Necrosis of myocardial tissue has for a long time been considered to be the main component of the damage incurred by myocardial infarction. Recently the importance of the contribution of apoptotic cell death in the context of myocardial ischaemia/reperfusion injury has become apparent. There is a general agreement that early reperfusion is necessary to salvage myocardial tissue from cell death. Preconditioning is the phenomenon whereby brief episodes of ischaemia and reperfusion protect the heart against a subsequent longer period of ischaemia. This endogenous mechanism is the strongest form of protection against myocardial infarction that has yet been described. Apart from ischaemie preconditioning (IPC), protection can also be elicited with pharmacologic agents, such as activation of the beta-adrenergic receptor with isoproterenol. Ischaemie preconditioning protects the myocardium against necrosis, arrhythmias and apoptosis, and increases functional recovery upon reperfusion. Betaadrenergic receptor stimulated preconditioning (PPC) has been shown to improve post-ischaemie functional recovery, but it is not known whether it also protects against myocardial infarction and apoptosis. The signaling pathways involved in preconditioning have been extensively studied. A distinction is usually made between factors that act as triggers, or as mediators of protection. Triggers activate cellular responses before the onset of sustained ischaemia, and its involvement is demonstrated by showing that inhibitors of the trigger bracketing the preconditioning protocol can block its protective effect, or that transient administration with washout before sustained ischaemia can activate a protective effect. A mediator operates during sustained ischaemia, and its involvement is demonstrated by showing that infusion of an inhibitor of its action immediately prior to sustained ischaemia (without washout) can block its protective effect. Another approach to demonstrate a mediator role is to attempt to activate signal transduction pathways during sustained ischaemia. As it is not possible to infuse substances during ischaemia, activators are infused immediately prior to ischaemia without washout of the agent and subsequently its effect on protection is observed. It is clear that the evolutionary conserved stress activated pathways are involved in preconditioning. There are three pathways i.e., the extracellular receptor activated pathways (ERK), c-jun terminal activated kinases (JNK) and p38 mitogen-activated protein kinases (MAPK). The precise role of the p38 MAPK pathway has not been elucidated. Experimental evidence has suggested a role for the activation of p38 MAPK as a trigger, as well as a mediator of the protective effect of preconditioning. There is however also strong evidence that the attenuation of p38 MAPK activation during sustained ischaemia, rather than its activation, is responsible for the protection that is observed. Furthermore, the role of p38 MAPK has only been investigated in relation to its protection against necrosis, but not apoptosis. AIMS: The aim of this study was to: (I) Establish a model of preconditioning in neonatal cardiomyocyte cell culture. The reason was that such a model could potentially enable one to rapidly elucidate the signal transduction pathways in an environment without the influence of non-cardiac cells. (II) Investigate whether IPC and ~PC protect against necrosis and apoptosis. (III) Elucidate the role of the stress-activated kinase, p38 MAPK, in preconditioning. METHODS: 1. Neonatal rat cardiomyocyte cell culture model A viability assay with 3-[4,5- Dimethylthaizol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) was first developed using different concentrations - a concentration of 0.25% was found to be optimal to determine viability. Neonatal cardiomyocyte cell cultures were subjected to sustained simulated "ischaemia" by using either 5 mM KCN plus deoxyglucose (DOG) for 5 min or potassium cyanide (KCN) for 45 min. Some cell cultures were preconditioned with either chemical ischaemia (5 mM KCN for 5 min) or isoproterenol (10-7 M) for 5 min and 60 min reoxygenation before being exposed to sustained simulated ischaemia. 2. Isolated adult rat cardiomyocyte model Isolated cardiac myocytes were exposed to 2 hours of hypoxia, which was induced by pelletting the cells by centrifugation, and covering them with a thin layer of mineral oil. Some groups were preconditioned with either hypoxia for 10 min at 37° C or isoproterenol (10-7 M) for 5 min, followed by reoxygenation for 20 minutes. The trypan blue exclusion method and MTT method developed in the neonatal cardiomyocytes were used to assess viability. 3. Isolated perfused rat heart model 3.1 Infarct size was determined in a model of regional ischaemia by using tetrazolium staining and determining the area of necrosis (exclusion of tetrazolium) as a percentage of area at risk. These hearts were subjected to 35 min global ischaemia and 30 min reperfusion. Some groups were preconditioned by three cycles of 5 min global ischaemia or addition of isoproterenol (10-7 M) for 5 min, followed by 5 min reperfusion before the onset of sustained regional ischaemia. 3.2 p38 MAPK activation and markers of apoptosis: p38 MAPK activation was determined using antibodies against dual phosphorylated p38 MAPK (i.e. activated p38 MAPK). Apoptosis was measured by using antibodies against activated caspase-3, and against a fragment of PARP (PARP cleavage). For these experiments isolated rat hearts were exposed to global ischaemia for 25 min followed by 30 min reperfusion. Some groups were preconditioned with three cycles of 5 min global ischaemia. A global ischaemia model was used in order to have sufficient tissue available for the Western blot determinations. This necessitated a shorter period of sustained ischaemia, as the globally ischaemie heart does not recover sufficiently after a longer period of ischaemia such as is necessary in regional ischaemia experiments. 3.3 The role of p38 MAPK in ischaemie preconditioning was investigated by administration of SB 203580 (1IJM),a selective inhibitor of p38 MAPK, either bracketing the preconditioning (i.e. to determine its role as a trigger) or for 10 min immediately prior to sustained ischaemia (i.e. to determine its role as a mediator). The second approach was to use anisomycin, an activator of p38 MAPK, as a trigger (infusion for 10 min followed by wash out) or as a mediator (10 min immediately prior to sustained ischaemia) in the same model as used for determination of p38 MAPK activity. The infusion of anisomycin for 10 min has been shown to elicit activation of p38 MAPK to a similar extent as has been observed with an ischaemie preconditioning protocol. The endpoints used were infarct size and markers of apoptosis. RESULTS: 1. Neonatal rat cardiomyocyte cell culture model It was not possible to establish a model of preconditioning of neonatal cardiomyocytes that was consistently successful. It was therefore decided to abandon the attempts and to use a different cell model. 2. Isolated adult rat cardiomyocyte model Isolated adult cardiomyocytes were preconditioned successfully, but produced too little material to perform simultaneous determinations of cell viability and Western blots (p38 MAPK activation and markers of apoptosis). It was therefore decided to use the isolated perfused adult rat heart. 3. Isolated perfused adult rat heart model 3.1 Both IPC and PPCprotect against infarction and apoptosis: Using two models of preconditioning i.e., IPC and PPC, the protective effects of preconditioning were demonstrated convincingly against infarction (necrosis). IPC and PPC both caused a significant reduction in infarct size (12.2±1.4 and 15.2±2.6%) versus Non-PC hearts (29.6±2.9%) (p < 0.001). Both forms of preconditioning also protected against apoptosis, by significantly reducing the markers of apoptosis, caspase-3 activation and PARP cleavage. The protection afforded by both forms of preconditioning was accompanied by a marked decrease in activation of p38 MAPK upon reperfusion. The relationship between p38 MAPK and the protection that was elicited by preconditioning was then investigated, namely whether p38 MAPK acted as a trigger, or as a mediator of protection. To investigate the role of p38 MAPK as a mediator or a trigger in preconditioning, use was made of (i) a specific inhibitor of p38 MAPK activation i.e., SB 203580 and (ii) a known activator of p38 MAPK i.e., anisomycin. 3.2 p38 MAPK as a trigger of protection: Administration of SB 203580 during the IPC protocol and washed out before sustained ischaemia did not abolish the protective effect of ischaemie preconditioning, and resulted in a small, but significant increase in caspase-3 activation and PARP cleavage. On the other hand, activation of p38 MAPK with anisomycin for 10 min followed by washout also resulted in a significant reduction in necrosis (infarct size 14.9±2.2 versus 29.6±2.9% in Non-PC hearts) (p < 0.001) and both markers of apoptosis. The latter results suggested that p38 MAPK was a trigger of preconditioning. If this was the case, why didn't SB 203580 abolish the protection of IPC? The most likely explanation was that multiple protective mechanisms were activated during a multi-cycle protocol of ischaemic preconditioning, of which activation of p38 MAPK was only one. Inhibition of p38 MAPK with SB 203580 would therefore not be expected to block the activation of those mechanisms that were independent of p38 MAPK, but were still capable of protecting against necrosis or apoptosis. It is very interesting that a small increase in apoptosis was observed when SB 203580 was used in this situation, as it may indicate that the protection against apoptosis was more dependent on the activation of p38 MAPK than the protection against necrosis, as no effect was seen on infarct size. Another explanation could be that infarct size determination was not sensitive enough to detect such small effects. 3.3 p38 MAPK as a mediator of protection: Inhibition of p38 MAPK activation with SB 203580 administered 10 min before sustained ischaemia caused a significant decrease in infarct size compared to Non-PC hearts (12.6±1.9 vs 29.6±2.9%) (p < 0.001) equivalent to that of hearts preconditioned with ischaemia. This was accompanied by a similar pattern of protection against apoptosis, with significantly reduced activation of caspase-3 activation and PARP cleavage. These results strongly supported a role for the attenuation of p38 MAPK activation as a mediator of preconditioning against ischaemia/reperfusion-mediated necrosis and apoptosis. However, the results of the experiments with anisomycin were at first glance not compatible with such a conclusion. The administration of the activator of p38 MAPK, anisomycin, for 10 min immediately prior to sustained ischaemia resulted in significant protection against necrosis (infarct size 16.6±2.4% vs 29.6±2.9% in Non-PC hearts) (p < 0.01) and reduced caspase-3 activation and PARP cleavage indicating less apoptosis. The reason for these findings were probably that this method of administration of anisomycin did in fact not activate p38 MAPK during sustained ischaemia, but actually served as a trigger to protect against ischaemia - similarly as if it had been infused with washout of the drug. Support for this notion was found in the fact that p38 MAPK activation was decreased upon reperfusion. These results suggested that the logistical problem of not being able to infuse a drug into the myocardium during ischaemia could not be overcome by immediate prior infusion, and that the administration of anisomycin in this way had activated downstream effectors of the p38 MAPK signal transduction pathway. An important contender for such an effector would be heat shock protein 27 (HSP27), which has been shown to play an important role in protection against apoptosis, and stabilisation of actin, and thus the cytoskeleton. Another possibility was that anisomycin had activated the JNK stress activated kinases. The elucidation of a role of this signal transduction pathway would necessitate the use of anisomycin in the presence of an agent such as curcumin, an inhibitor of JNK. Final conclusion: The work in this thesis showed that the stress activated kinase, p38 MAPK, was involved in the protective effect of ischaemie preconditioning. The results suggested a role for the activation of p38 MAPK as a trigger of protection, and the attenuation of p38 MAPK as a mediator of protection, which was observed in the reduction of both necrosis (infarct size) and apoptosis as determined with caspase- 3 activation and PARP cleavage.

AFRIKAANSE OPSOMMING: Die afsluiting van bloedvloei na die miokardium gee aanleiding tot nekrose. In die lig van die voorkoms van koronêre bloedvatsiekte onder die algemene populasie, en die nadelige effekte van miokardiale isgemie op miokardiale weefsel, is dit belangrik om nuwe strategieë te ontwikkel wat die miokardium teen isgemie beskerm. Nekrose van miokardiale weefsel word tradisioneel as die belangrikste komponent van die skade aangerig deur miokardiale infarksie beskou. Die belang van apoptotiese seldood in die konteks van miokardiale isgemie/herperfusie (I/R) het onlangs na vore getree. Dit word algeneem aanvaar dat vroeë vroegtydige herperfusie noodsaaklik is om miokardiale weefsel te beskerm teen seldood. Prekondisionering is 'n verskynsel waartydens kort episodes van IIR die hart teen 'n daaropvolgende langer periode van isgemie beskerm. Hierdie endogene meganisme is die kragtigste vorm van beskerming teen miokardiale infarksie tot dusver beskryf. Afgesien van isgemiese prekondisionering (IPC), kan beskerming ook deur farmakologiese middels, soos byvoorbeeld die aktivering van die beta-adrenerge reseptore met isoproterenol, ontlok word. IPC beskerm die miokardium teen nekrose, arritmieë en apoptose, en verhoog funksionele herstel na herperfusie. Daar is reeds aangetoon dat betaadrenerge prekonsionering (~PC) post-isgemiese funksionele herstel verbeter, maar dit is nog onbekend of beskerming ook teen miokardiale infarksie en apoptose verleen word. Die seintransduksie paaie betrokke tydens prekondisionering is reeds in detail bestudeer. Daar word gewoonlik tussen faktore wat optree as snellers, of as mediators van beskerming, onderskei. Snellers aktiveer sellulêre response voor die aanvang van volgehoue isgemie, en hul betrokkenheid word aangetoon deurdat inhibisie van snellers tydens die prekondisionering protokol, beskerming ophef. Snellers se effekete kan ook ontlok word deur hulle tydelike toe te dien en dan net voor volgehoue isgemie weer uit te was. Mediators oefen hulle effek tydens volgehoue isgemie uit, en hulle betrokkenheid word gedemonstreer deurdat toediening van inhibitors net voor volgehoue isgemie (sonder uitwas) hulle beskermende effekte ophef. Mediators se rol kan ook aangetoon word deur te poog om seintransduksie paaie tydens volgehoue isgemie te aktiveer. Aangesien dit ontmoontlik is om middels tydens isgemie te infuseer, word aktiveerders onmiddelik voor die aanvang van isgemie toegedien sonder om hulle uit te was, sodat hulle effekte op beskerming vervolgens bestudeer kan word. Dit is duidelik dat die evolusionêr-behoue stres geaktiveerde paaie tydens prekondisionering betrokke is. Daar is drie paaie nl. die ekstrasellulêre reseptor geaktiveerde pad (ERK), c-jun terminaal geaktiveerde kinases (JNK) en p38 mitogeen geaktiveerde proteïen kinases (MAPK). Die spesifieke rol van die p38 MAPK pad is nog nie ontrafel nie. Eksperimentele bewyse stel 'n rol vir die aktivering van p38 MAPK as 'n sneller, sowel as 'n mediator van die beskermende effek van prekondisionering, voor. Daar is egter ook sterk bewyse dat 'n afname in p38 MAPK aktivering tydens volgehoue isgemie, eerder as sy aktivering, verantwoordelik is vir die waargenome beskermende effek. Verder is die rol van p38 MAPK slegs in die konteks van beskerming teen nekrose, maar nie teen apoptose nie, bestudeer. DOELWITTE: Die doelwit van hierdie studie was: (I) Die vestiging van 'n prekondisionering model in neonatale kardiomiosiet in selkultuur. Hierdie model sou potensieel 'n spoedige ontrafeling van die seintransduksie paaie sonder die invloed van nie-kardiale selle bewerkstellig. (II Om ondersoek in te stelof IPC en PPCteen nekrose en apoptose beskerm. (III) Die ontrafeling van die rol van die stres geaktiveerde kinase, p38 MAPK, tydens prekondisionering. METODES: 1. Neonatale rot kardiomiosiet weefselkultuur model 'n Lewensvatbaarheids essai is ontwikkel deur van verskillende konsentrasies van 3-[4,5-dimetielthiazol-2-yl]-2,5-difeniel-tetrazolium bromied (MTT) gebruik te maak - 'n konsentrasie van 0.25% was optimaalom lewensvatbaarheid te bepaal. Neonatale kardiomiosiet weefselkulture is onderwerp aan volgehoue gesimuleerde "isgemie" deur gebruik te maak van 5 mM KCN plus deoksiglukose (DOG) vir 5 minute of 45 min KCN. Sommige weefselkulture is geprekondisioneer deur middel van chemiese isgemie (5 mM KCN vir 5 min) of van isoproterenol (10-7 M) vir 5 minute en 60 minute reoksigenasie alvorens dit bloot gestel is aan volgehoue gesimuleerde isgemie. 2. Geïsoleerde volwasse rot kardiomiosiet model Geïsoleerde kardiomiosiete is aan twee uur hipoksie blootgestel deur selle in 'n pellet te sentrifugeer en met 'n dun lagie mineraalolie te bedek. Sommige groepe is geprekondisioneer deur middel van 10 minute hipoksie by 37°C, of toediening van isoproterenol (10-7 M) vir 5 minute gevolg deur 20 minute reoksigenasie. Die tripaanblou uitsluitings metode en MTT metode soos ontwikkel in die neonatale kardiomiosiet model is gebruik om lewensvatbaarheid te bepaal. 3. Geïsoleerde geperfuseerde volwasse rot hart model 3.1 Infarkgrootte is bepaal met 'n model van streeks isgemie deur van tetrazolium kleuring gebruik te maak, waarna die area van nekrose (uitsluiting van tetrazolium) as 'n presentasie van die risiko area bepaal is. Hierdie harte was onderwerp aan 35 minute globale isgemie en 30 minute herperfusie. Sommige groepe is geprekondisioneer met 3 siklusse van 5 minute globale isgemie, of die toevoeging van isoproterenol (10-7 M) vir 5 minute, gevolg deur 5 minute herperfusie voor die aanvang van volgehoue streeks isgemie. 3.2 p38 MAPK aktivering en merkers van apoptose: p38 MAPK aktivering is bepaal deur gebruik te maak van anti-liggame teen tweeledige gefosforileerde p38 MAPK (d.w.s. geaktiveerde p38 MAPK). Apoptose is bepaal deur gebruik te maak van anti-liggame teen geaktiveerde kaspase-3, en teen 'n fragment van PARP (PARP kliewing). Tydens hierdie eksperimente is geïsoleerde rotharte bloot gestel aan 25 minute globale isgemie gevolg deur 30 minute herperfusie. Sommige groepe is geprekondisioneer met drie siklusse van 5 minute globale isgemie. Om voldoende weefsel vir Westerse klad tegnieke te verkry, is gebruik gemaak van 'n globale isgemie model. As gevolg hiervan was 'n kort periode van volgehoue isgemie genoodsaak, aangesien die globale isgemiese hart nie voldoende herstel na 'n langer periode van isgemie nie, soos wat benodig word in streeks isgemiese eksperimente. 3.3 Die rol van p38 MAPK tydens IPC is bepaal deur die toediening van 'n 1IJM konsentrasie van SB 203580, 'n selektiewe inhibitor van p38 MAPK, hetsy tydens prekondisionering (d.w.s. om die rol as 'n sneller te bepaal), óf vir 10 minute direk voor die aanvang van volgehoue isgemie (d.w.s. om dus sy rol as mediator te bepaal). Die tweede benadering was om anisomisien, 'n aktiveerder van p38 MAPK, as sneller (toediening vir 10 minute gevolg deur uitwassing) of as mediator (10 minute direk voor aanvang van volgehoue isgemie) in dieselfde model as in die geval van p38 MAPK aktiviteit bepaling, te gebuik. Die toediening van anisomisien vir 10 minute het aangetoon dat dit p38 MAPK aktivering kan ontlok tot dieselfde maate as die IPC protokol. Die eindpunte was infarkgrootte en merkers van apoptose. RESULTATE: 1. Neonatale rot kardiomiosiet weefselkultuur model Dit was nie moontlik om 'n suksesvolle model met konsekwente resultate vir die prekondisionering van neonatale kardiomiosiete te vestig nie. Daar is dus besluit om af te sien van hierdie pogings en eerder 'n alternatiewe selmodel te gebruik. 2. Geïsoleerde volwasse rot kardiomiosiet model Geïsoleerde volwasse kardiomiosiete is suksesvol geprekondisioneer, maar het te min materiaalopgelewer vir die gelyktydige bepaling van sellewensvatbaarheid, p38 MAPK aktivering en merkers vir apoptose. Daar is dus besluit om die geïsoleerde geperfuseerde volwasse rothart te gebruik. 3. Geïsoleerde geperfuseerde volwasse rothart model 3.1 Beide IPC en PPCbeskerm teen infarksie en apoptose: Deur gebruik te maak van twee prekondisionering modelle d.w.s. IPC en PPC, is die beskermende effekte van prekondisionering teen infraksie (nekrose) oortuigend gedemonstreer. Beide IPC en PPC het In betekenisvolle afname in infarkgrootle veroorsaak (12.2 ± 1.4 en 15.2 ± 2.6% respektiewelik), vs Nie-PC harte (29.6 ± 2.9%)(p < 0.001). Beide vorme van prekondisionering het ook teen apoptose beskerm deur die apoptose merkers, kaspase-3 aktivering en PARP kliewing te verlaag. Die beskerming verkry deur beide vorms van prekondisionering is geassosieer met In merkbare afname in die aktivering van p38 MAPK na herperfusie. Die verband tussen p38 MAPK en die beskerming ontlok deur prekondisionering is gevolglik ondersoek, naamlik of p38 MAPK optree as 'n sneller of as 'n mediator van beskerming. Om die rol van p38 MAPK as 'n mediator of sneller tydens prekondisionering te ondersoek is daar gebruik gemaak van (I) 'n spesifieke inhibitor van p38 MAPK aktivering nl. SB 203580 en (II) 'n bekende aktiveerder van p38 MAPK nl. anisomisien. 3.2 p38 MAPK as 'n sneller vir beskerming: Toediening van SB 203580 tydens die IPC protokol en uitwassing daarvan voor die aanvang van volgehoue isgemie het nie die beskermende effek van IPC opgehef nie, en het gelei tot 'n klein maar betekenisvolle verhoging in kaspase-3 aktivering en PARP kliewing. Andersins het die aktivering van p38 MAPK met anisomisien vir 10 minute gevolg deur In uitwas ook tot In betekenisvolle afname in nekrose (infarkgrootte 14.9 ± 2.2 vs 29.6 ± 2.9% in Nie-PC harte) (p < 0.001) in beide merkers van apoptose gelei. Laasgenoemde resultate dui daarop dat p38 MAPK inderdaad 'n mediator van prekondisionering is. Indien dit die geval is, waarom het SB 203580 nie die beskermende effek van IPC opgehef nie? Die mees waarskynlike verklaring is dat veelvuldige beskermingsmeganismes tydens 'n multi-siklus protokol van IPC geaktiveer word, waarvan p38 MAPK aktivering slegs een is. Dit is dus onwaarskynlik dat die inhibisie van p38 MAPK met SB 203580 die aktivering van daardie meganismes onafhanklik van p38 MAPK sal blokkeer en steeds in staat sal wees tot beskerming teen nekrose en apoptose. Dit is interessant dat In klein verhoging in apoptose waargeneem is toe SB 203580 gebruik is onder hierdie toestande, aangesien dit daarop kan dui dat die beskerming teen apoptose meer afhanklik was van die aktivering van p38 MAPK as die beskerming teen nekrose, siende dat geen effek op infarkgrootte waargeneem is nie. 'n Verdere verklaring kan wees dat die bepaling van infarkgrootte nie sensitief genoeg is om sulke klein effekte waar te neem nie. 3.3 p38 MAPK as 'n mediator vir beskerming: Inhibisie van p38 MAPK aktivering deur SB 203580 toediening 10 minute voor volgehoue isgemie het 'n betekenisvolle verlaging in infarkgrootte in vergelyking met Nie-PC harte veroorsaak (12.6 ± 1.9 vs 29.6 ± 2.9%) (p < 0.001) soortgelyk aan dié van harte geprekondisioneer met isgemie. Dit is geassosieer met In soortgelyke patroon van beskerming teen apoptose, met betekenisvolle verlaagde kaspase-3 aktivering en PARP kliewing. Hierdie resultate ondersteun die rol van die afname van p38 MAPK aktivering as 'n mediator van prekondisionering teen I/R-gemedieerde nekrose en apoptose. Die resultate van die anisomisien eksperimente was met die eerste oogopslag nie in oorstemming met hierdie gevolgtrekking nie. Die toedienning van die p38 MAPK aktiveerder, anisomisien, vir 10 minute voor volgehoue isgemie het tot 'n betekenisvolle beskerming teen nekrose aanleiding gegee (infarkgrootte 16.6 ± 2.4 vs 29.6 ± 2.9% in Nie-PC harte) (p < 0.01) en verlaagde kaspase-3 aktivering en PARP kliewing wat dui op verlaagde apoptose. Die rede vir hierdie bevindings is moontlik dat die metode van anisomisien toediening nie p38 MAPK geaktiveer het tydens volgehoue isgemie nie, maar eintlik gedien het as 'n sneller vir beskerming teen isgemie - amper asof dit toegedien sou word sonder om uitgewas te word. Ondersteuning vir hierdie aanname word gevind in die feit dat p38 MAPK aktivering verlaag is na herperfusie. Hierdie resultate stel voor dat die logistiese probleem dat In middel nie tydens isgemie toegedien kan word nie, nie oorkom kan word deur onmiddelike voortydige infusie nie, en dat die toediening van anisomisien op hierdie manier gelei het tot die aktivering van stroom-af effektors van die p38 MAPK seintransduksie pad. 'n Belangrike kandidaat vir so 'n effektor is "heat shock protein 27" (HSP27), wat reeds aangetoon is om 'n belangrike rol in die beskerming teen apoptose en destabilisering, en dus die sitoskelet, te speel. 'n Ander moontlikheid is dat anisomisien die JNK stres geaktiveerde kinases geaktiveer het. Die ontrafeling van die rol van hierdie seintransduksie pad noodsaak die gebruik van anisomisien in die teenwoordigheid van 'n agent soos curcumin, 'n JNK inhibitor. Finale gevolgtrekking: Die werk soos vervat in hierdie tesis toon aan dat die stres geaktiveerde kinase, p38 MAPK, betrokke is in die beskermings effek van isgemiese prekondisionering. Die resultate dui op 'n rol vir die aktivering van p38 MAPK as 'n sneller vir beskerming, en die afname in p38 MAPK as 'n mediator vir beskerming, soos waargeneem in die vermindering van veranderlikes van beide nekrose (infarkgrootte) en apoptose soos bepaal deur kaspase-3 aktivering en PARP kliewing.

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