Spelling suggestions: "subject:"etheses -- physiology"" "subject:"etheses -- hophysiology""
61 |
Anthracycline-induced cardiotoxicity : the role of proteolytic pathwaysSishi, Balindiwe J. N. (Balindiwe Jennifer Nonkosazana) 03 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Introduction: The anthracyclines (ACs), daunorubicin (DNR) and doxorubicin (DXR)
are two of the most effective drugs known for the treatment of systemic neoplasms
and solid tumours. However, their clinical use is often hampered by their dosedependent
cumulative cardiotoxicity, which leads to irreversible and fatal druginduced
congestive heart failure. The mechanism by which ACs induces heart
damage is not fully understood. Recent reports have indicated that DXR activates
autophagy and ubiquitin proteasome-mediated degradation of specific transcription
factors, however, no reports exists on the effect of ACs on the E3 ubiquitin ligases,
MuRF-1 and MAFbx. The aim of the first part of the study was therefore to
investigate the effect of DNR treatment on the protein and organelle degradation
systems in the heart and to elucidate the signalling mechanisms involved.
Although this model was ideal in allowing the investigation of the signalling pathways
which are affected by DNR, it did not allow for further exploration or manipulation of
signalling pathways that may be of potential benefit in this context. The in vitro model
was therefore used to validate the hypothesis that increased autophagy alleviates
AC-induced cardiotoxicity and delays the onset of cardiomyocyte death. The aims for
the second part of the study were (i) to characterize the effect of DXR in H9C2 cells,
(ii) to determine whether the induction/inhibition of autophagy in combination with
DXR alleviates cytotoxicity and (iii) to investigate the influence of
increased/decreased autophagy in combination with DXR on reactive oxygen
species (ROS) production, mitochondrial function, endoplasmic reticulum (ER) stress
and the ubiquitin proteasome pathway. In the final part of this study, an in vivo model
was used to assess the potential benefit of autophagy in a novel GFP-LC-3 tumour
bearing mouse model of acute DXR-induced cardiotoxicity. Material and Methods: Adult rats were divided into two groups where one group
received six intraperitoneal injections of 2 mg/kg DNR on alternate days and the
other group received saline injections as control. Hearts were excised and perfused on a working heart system the day after the last injection and freeze clamped for
biochemical analysis.
H9C2s were cultured and treated with Bafilomycin A1 (10 nM, inhibitor of autophagy)
for 6 hrs, Rapamycin (50 μM, inducer of autophagy) for 24 hrs, DXR (3 μM) for 24
hrs or a combination of these drugs. Following treatment, cells were harvested and
assessed for cell death, proteolytic activity and oxidative stress using western
blotting, fluorescence microscopy and flow cytometry.
In the final phase of the study, twenty-four female mice were injected at 8 weeks with
a mouse breast cancer cell line (EO771) and after observation of tumour growth,
animals were either treated with one injection (i.p.) of Rapamycin (4 mg/kg), two
injections (i.p.) of DXR (10 mg/kg) or a combination of the two drugs. After the
experimental protocol, mice were terminated and their hearts were rapidly excised.
The hearts were divided cross-sectionally and utilized for biochemical and
histological analyses.
Results and Discussion: DNR treatment significantly attenuated myocardial
function and increased apoptosis in the ex vivo heart model. DNR-induced cardiac
cytotoxicity was associated with the upregulation of two E3 ubiquitin ligases, MuRF-1
and MAFbx as well as a significant increase in two markers of autophagy, beclin-1
and LC-3. These changes observed in the heart were also associated with
attenuation of the PI3-kinase/Akt signalling pathway. The augmentation of autophagy with rapamycin before DXR treatment significantly
reduced cell death in the in vitro model. Indeed, rapamycin treatment demonstrated
to be a vital survival mechanism for acute DXR-induced cardiotoxicity as it
decreased cellular ROS production, improved mitochondrial function and prevented
nuclear translocation of DXR. Moreover, these changes in cardiomyocytes were also
associated with a reduction in the ubiquitin-proteasome pathway (UPP). In the final part of this study, a novel tumour bearing GFP-LC3 mouse model was
developed to confirm the results obtained in the in vitro study. It was demonstrated
that acute DXR-induced cardiotoxicity resulted in increased apoptosis, the inhibition
of autophagy and increased proteolysis via the UPP. These findings were associated
with a reduction in body weight and cardiomyocyte cross-sectional area. The
cardiotoxic effects of DXR were substantially reduced when autophagy was induced
with rapamycin. Taken together, our data strongly indicates that it is possible to
attenuate the cardiotoxic effects of doxorubicin in cancer patients by carefully
controlling the levels of autophagy using rapamycin as adjuvant therapy. / AFRIKAANSE OPSOMMING: Inleiding: Die antrasikliene (AC’s), daunorubisien (DNR) en doksorubisien (DKS), is
twee van die mees effektiewe AC wat bekend is vir die behandeling van sistemiese
neoplasmas en soliede tumore. Hulle kliniese gebruik word egter deur dosis
afhanklike kumulatiewe kardiotoksisiteit benadeel, wat tot onomkeerbare en dodelike
kongestiewe hartversaking kan lei. Die meganisme waardeur AC’s hartversaking kan
veroorsaak, word nog nie ten volle verstaan nie. Onlangse navorsing het aangetoon
dat DKS autofagie en die ubikwitienproteosoom-bemiddelde degradasie van
spesifieke transkripsie faktore aktiveer. Daar is egter geen literatuur wat die effek
van AC’s op die E3-ubikwitienligases, MuRF-1 en MAFbx beskryfnie. Die doel van
hierdie eerste afdeling van die studie is om die effek van DNR behandeling op die
proteïen- en organel degradasie sisteme in die hart te ondersoek en om van die
betrokke seinmeganismes te bepaal.
Alhoewel hierdie model ideaal is om sommige seinweë wat deur DNR geaffekteer
word, te ondersoek, kon seinoordragpaaie wat potensieël voordelig in hierdie
konteks is, nie in bg. model gemanipuleer word nie. Die in vitro model is gebruik om
die hipotese dat verhoogde outofagie AC-geïnduseerde kardiotoksisiteit verlaag en
sodoende seldood verminder, te bevestig. Die doel van hierdie afdeling van die
studie was: (i) om die effek van DKS op H9C2 selle te karakteriseer, (ii) om te bepaal
of die induksie/inhibisie van outofagie in kombinasie met DKS kardiotoksisiteit
verbeter (iii) om die invloed van verhoogde/verlaagde outofagie in kombinasie met
DKS op reaktiwe suurstof species (ROS), mitokondriale funksie, endoplasmiese
retikulum (ER) stress en die ubikwitienproteosoompad te ondersoek. In die finale
deel van hierdie studie, is ‘n in vivo model gebruik om die moontlike voordelige effek
van verhoogde outofagie in ‘n GFP-LC-3 tumor-draende muismodel met akute DKSgeïnduseerde
kardiotoksisiteit, ondersoek.
Materiaal en Metodes: Volwasse rotte is in twee groepe verdeel waar een groep
ses intraperitoneale inspuitings van 2 mg/kg DNR op afwissellende dae ontvang het en die andergroep as ‘n kontrole, ‘n soutoplossing gekry het. Die harte is verwyder
en geperfuseer op ‘n werkende hartsisteem een dag na die laaste inspuiting en
gevriesklamp vir biochemiese analises.
H9C2 selle is vir 6 uurgekweek en behandel met Bafilomisien A1 (10 nM, ‘n autofagie
inhibitor), 24 uur met Rapamisien (50 μM, ‘n autofagie induseerder), 24 uur met DKS
(3 μM) of ‘n kombinasie van hierdie middels. Na behandeling is selle ge-oes vir
analises in seldood, proteolitiese aktiwiteit en oksidatiewe stress deur van westelike
kladtegniek, fluoresensie mikroskopie en vloeisitometrie gebruik te maak.
In die finale fase van hierdie studie is vier en twintig, agt weke oue wyfie muise
ingespuit met ‘n muisborskankersellyn (E0771) en is tumorgroei waargeneem; die
diere is of behandel met een rapamisien inspuiting (i.p) (4 mg/kg), of twee DKS
inspuitings (i.p.) (10 mg/kg) of ‘n kombinasie van die twee middels. Na die
eksperimentele protokol, is die muise van kant gemaak en hulle harte vinnig
verwyder. Die harte is in twee verdeel en gebruik vir biochemiese- en histologiese
analises.
Resultate en Bespreking: DNR behandeling het kardiale funksie betekenisvol
verswak en apoptose in die hart verhoog. DNR-geïnduseerde kardiotoksisiteit is
geassosieer met die opregulering van E3-ligases, MuRF-1 en MAFbx en het ook ‘n
betekenisvolle toename in twee outofagie merkers, beclin-1 en LC-3 veroorsaak.
Hierdie veranderinge wat in die hart waargeneem is, is ook geassosieer met ‘n
onderdrukking van die PI3-kinase/Akt seinweg. Die toename in outofagie met rapamisien voor DKS behandeling het seldood in die
vorm van apoptose betekenisvol verlaag. Daarmee saam het verhoogde outofagie ‘n
noodsaaklike oorlewings meganisme vir akute DKS-geïnduseerde kardiotoksisiteit
gedemonstreer. Die rede hiervoor is dat dit ROS produksie verlaag het,
mitokondriale funksie verbeter het en DKS translokasie vanuit die sitoplasma tot binne die nukleus verhoed het. Hierdie veranderinge in kardiomiosiete is ook met ‘n
afname in die ubikwitienproteosoomseinweg (EPS) geassosieer.
In die finale deel van hierdie studie, is ‘n nuwe tumor-draende muismodel ontwikkel
om die resultate wat in die in vitro studie gekry is, te bevestig. Daar is bewys dat
akute DKS-geïnduseerde kardiomiotoksisiteit aanleiding gegee het tot verhoogde
apoptose, outofagie inhibisie en verhoogde proteolise via die EPS. Hierdie
bevindinge is geassosieer met ‘n verlaging in liggaamsgewig en kardiomiosiet
dwarssnit area. Die kardiotoksiese effekte van DKS is insiggewend verminder as
autofagiege ïnduseer is met rapamisien. Om saam te vat: Ons data bevestig dat dit
moontlik is om die kardiotoksiese effekte van DKS in kanker pasiënte te verminder
deur outofagie vlakke te monitor en te kontroleer deur middel van rapamisien
behandeling as bykomende terapie.
|
62 |
The association between genotype and BMI, health and lifestyle indicators as well as weight loss outcomes in overweight/obese Caucasian adultsHarbron, Janetta 03 1900 (has links)
Thesis (PhD (Physiological Sciences))--University of Stellenbosch, 2011. / Includes bibliography. / ENGLISH ABSTRACT: Genetic screening to improve obesity treatment outcomes is available despite the lack of conclusive
evidence, specifically for Caucasian South Africans, in this regard.
The aim of this study was to investigate the association between genotype (seven polymorphisms) and
body mass index (BMI), health and lifestyle indicators in a cross-sectional sample of overweight/obese
Caucasian adults (n=133), as well as the association between genotype and weight loss outcomes following
an intervention (n=88) using a quasi experimental study design (time-series). The intervention consisted of
a 24-week conservative weight loss programme that included dietary, physical activity and behavioural
components.
The primary null hypothesis for the cross-sectional sample, namely that there is no association between
genotype and BMI, has not been rejected. A number of the secondary/exploratory hypotheses were
rejected of which the most plausible associations (based on support by the literature and a physiological
basis for the findng) are: 1) the mutant TT homozygotes of the GNB3 C825T polymorphism may have a
higher risk to develop the metabolic syndrome (MetS) as they had significantly higher fasting triglyceride
and glucose levels, a higher number of traits that met the diagnostic cut-off criteria for MetS and higher
number of these subjects was diagnosed with MetS compared to the wild-type C-allele carriers; and 2)
subjects with mutant alleles of either the FTO rs1421085 or rs17817449 polymorphisms may have poorer
eating behaviours (a higher rigid control, habitual and emotional disinhibition, perceived hunger and
internal locus for hunger) and higher intake of high-fat foods.
The primary null hypothesis for the intervention sample, namely that there is no association between
genotype and weight loss outcome, was not rejected for the FABP2 Ala54Thr, INSIG2 rs7566605, FTO
rs1421085, ADRB3 Trp64Arg and GNB3 C825T polymorphisms. However, it was rejected in some instances
indicating the following associations: 1) The wild-type TT homozygotes of the FTO rs17817449
polymorphism lost significantly more weight during the first two months of the program compared to the
mutant allele carriers (this is a novel finding); 2) The wild-type Arg16Arg homozygotes of the ADRB2
Arg16Gly polymorphism lost significantly more weight during the first month of the program compared to
the mutant allele carriers (this finding is supported by one other intervention study); 3) Subjects with a
mutant C-allele of the INSIG2 rs7566605 polymorphism and a mutant Gly16-allele of the ADRB2 Arg16Gly
polymorphism lost significantly less weight over the six month intervention period (this is a novel genegene
interaction finding). A number of secondary/exploratory hypotheses were rejected, of which the
most plausible finding include that the improvement in emotional disinhibition in the wild-type TT subjects of the FTO rs1421085 polymorphism was associated with a more pronounced decrease in BMI over the six
month weight loss period.
The integration of the results from this study with the literature indicates that there is insufficient evidence
at this stage for genetic screening of the polymorphisms investigated in this study and the provision of
evidence-based personalized recommendations for weight loss in obese individuals. It is recommended
that these associations should be viewed as priority in future research. / AFRIKAANSE OPSOMMING: Genetiese sifting om die resultate van vetsug behandeling te verbeter is beskikbaar ten spyte van ‘n tekort
aan genoegsame bewyse, spesifiek ten opsigte van Kaukasiërs van Suid-Afrika.
Die doel van hierdie studie was om die assosiasie tussen genotipe (sewe polimorfismes) en liggaamsmassa
indeks (LMI), gesondheid en lewenstyl indikatore in ‘n dwarssnit (cross-sectional) steekproef van
oorgewig/vetsugtige Kaukasiër volwassenes (n=133) te ondersoek, asook die assosiasie tussen genotipe en
gewigsverlies uitkomste na afloop van ‘n intervensie (n=88) in ‘n kwasi-eksperimentele studie ontwerp (tydreeks).
Die intervensie het bestaan uit ‘n 24-week konserwatiewe gewigsverlies program met dieet, fisieke
aktiwiteit en gedragskomponente.
Die primêre nul hipotese vir die dwarsnit steekproef, naamlik dat daar geen assosiasie tussen genotipe en
LMI is nie, is nie verwerp nie. ‘n Aantal sekondêre/spekulatiewe hipotesis is verwerp waarvan die mees
geloofwaardige assosiasies (gebasseer op ondersteuning van die literatuur en ‘n fisiologiese basis vir die
bevinding) die volgende insluit: 1) die mutante TT homosigote van die GNB3 C825T polimorfisme het
moontlik ‘n hoër risiko vir die ontwikkeling van die metaboliese sindroom (MetS) aangesien hulle
betekenisvolle hoër vastende trigliseriede en glukose vlakke gehad het, ‘n grooter aantal kenmerke gehad
het wat aan die diagnostiese afsnykriteria vir MetS voldoen en ‘n grooter aantal van hierdie persone was
met MetS gediagnoseer in vergelyking met die wilde-tipe C-alleel draers; en 2) persone met die mutante
allele van die FTO rs1421085 of rs17817449 polimorfismes het moontlik ‘n swakker eetgedrag (‘n hoër
rigiede kontrole, gewoonte en emosionele disinhibisie, waarneembare honger en interne lokus van honger)
en ‘n hoër inname van hoë-vet voedsel.
Die primêre nul hipotese vir die intervensie steekproef, naamlik dat daar geen assosiasie tussen genotipe
en gewigsverlies uitkomste is nie, is nie vir die FABP2 Ala54Thr, INSIG2 rs7566605, FTO rs1421085, ADRB3
Trp64Arg en GNB3 C825T polimorfismes verwerp nie. Dit was egter in sommige gevalle vir die volgende
assosiasies verwerp: 1) Die wilde-tipe TT homosigote van die FTO rs17817449 polimorfisme het
betekenisvol meer gewig in die eerste twee maande van die program verloor in vergelyking met die
mutante alleel draers (dit is ‘n nuwe bevinding); 2) Die wilde-tipe Arg16Arg homosigote van die ADRB2
Arg16Gly polimorfisme het betekenisvol meer gewig gedurende die eerste maand van die program verloor
in vergelyking met die mutante alleel draers (hierdie bevinding word ondersteun deur een ander
intervensie studie); 3) Persone met ‘n mutante C-alleel van die INSIG2 rs7566605 polimorfisme en ‘n
mutante Gly16-allele van die ADRB2 Arg16Gly polimorfisme het minder gewig tydens die ses maande
intervensie periode verloor (dit is ‘n nuwe geen-geen interaksie bevinding). ‘n Aantal sekondêre/
spekulatiewe hipoteses is verwerp, waarvan die mees geloofwaardigste bevinding insluit dat ‘n verbetering in emosionele disinhibisie van die wild-tipe TT persone van die FTO rs1421085 polimorfisme geassosieer
was met ‘n meer prominente daling in LMI oor die ses maande gewigsverlies periode.
Die integrasie van die resultate van hierdie navorsing met die literatuur dui aan dat daar op hierdie stadium
onvoldoende bewyse vir genetiese sifting en die voorsiening van bewys-gebasseerde persoonlike
aanbevelings vir gewigsverlies in vetsugtig individue bestaan vir die polimorfismes wat ondersoek is. Dit
word aanbeveel dat hierdie assosiasies as prioriteit in toekomstige navorsing beskou moet word.
|
63 |
Immune and satellite cells : important role players in muscle recovery after injuryKruger, Maria Jacoba 03 1900 (has links)
Thesis (PhD (Physiological Sciences))--University of Stellenbosch, 2011. / Includes bibliography. / ENGLISH ABSTRACT: Muscle injuries are associated with changes in skeletal muscle as well as the immune
system. All studies investigating possible treatment modalities have found both positive and
negative effects on muscle recovery. Since no universally accepted treatment modality
exists, this thesis aims to determine whether a plant-derived antioxidant, proanthocyanidolic
oligomer (PCO), might prove beneficial as treatment for sports injuries in order for athletes to
return to the sports field quicker. The difference in recovery of muscle following both chronic
(supplementation started 14 days prior to injury and continued thereafter) and acute
supplementation (supplementation started two hours after injury) were also investigated.
Both chronic and acute PCO supplementation in a rat hindlimb contusion injury model
resulted in earlier muscle recovery, verified by an earlier satellite cell response compared to
the placebo group. This effect was most prominent already at the four hour time point
following injury, compared to day seven and three after chronic and acute placebo treatment
respectively. PCO supplementation also resulted in quicker foetal myosin heavy chain
(MHCf) expression compared to placebo treatment. Chronic supplementation specifically
resulted in a blunted circulatory pro-inflammatory cytokine response, whilst allowing for a
significant increase in IL-10, an anti-inflammatory cytokine, on day three (in the PCO group
only). At tissue level, the response of the muscle pro-inflammatory cytokines, TNF- and IL-
6, coincided with the satellite cell response. Macrophage infiltration into the injured muscle
also followed a similar pattern to that seen for the pro-inflammatory cytokines. Macrophages
invaded the injured area quicker when supplemented with PCO chronically, however,
macrophage infiltration could not explain the cytokine response seen with acute
supplementation. Both chronic and acute supplementation with PCO was responsible for a
severely blunted neutrophil response, a novel finding of this particular antioxidant.
The main findings of the in vivo rodent study were that PCO was able to blunt the neutrophil
response, whilst allowing for earlier macrophage infiltration. To establish possible
mechanisms by which PCO might exert these beneficial effects, further analysis included determining macrophage phenotypes and neutrophil numbers in circulation. An in vitro
neutrophil migration assay was also employed to further elucidate PCO’s ability to blunt
neutrophil infiltration into the injured area. For this study, conditioned plasma were harvested
from experimental animals and added together with neutrophils from control rats and
granulocyte colony stimulating factor (G-CSF) to the insert of the migration chamber. A
chemotactic factor, N-formyl methionine-leucine-phenylalanine (fMLP), was added to the
bottom well and neutrophils were allowed to migrate for two hours. Results from this study
indicated that neutrophil migration was attenuated in vitro in the presence of conditioned
plasma from PCO supplemented rats only.
The studies in this thesis on the effect of PCO on parameters of muscle and the immune
system led to the following main conclusions: a) PCO supplementation resulted in earlier
muscle recovery as a result of earlier satellite cell activation and MHCf synthesis; b) PCO
favours an anti-inflammatory cytokine reaction, whilst blunting the pro-inflammatory cytokine
response; and c) PCO blunted the neutrophil response whilst facilitating earlier macrophage
infiltration into the injured area. The specific mechanism of action of PCO to blunt the
neutrophil response specifically, possibly includes the ability to suppress adhesion molecule
expression on the neutrophils themselves. However, this warrants further investigation. / AFRIKAANSE OPSOMMING: Spier beserings word geassosiëer met veranderinge in skeletspier sowel as die
immuunstelsel. Meeste studies wat moontlike behandelingsopsies ondersoek, het beide
positiewe en negatiewe spierherstel gerapporteer. Omrede daar geen universele
behandelingsmoontlikheid bestaan nie, is die doel van hierdie tesis om die effek van ‘n
plantgebaseerde anti-oksidant, pro-antosianiedoliese oligomeer (PSO), as ‘n voordelige
behandelingstrategie vir sportbeserings te toets. Die verskil in spierherstel na beide kroniese
(supplementering wat 14 dae voor besering begin is, en volgehou is daarna) en akute
supplementering (supplementering het twee uur na besering begin), is ook ondersoek.
Beide kroniese en akute PSO supplementering, in ‘n rot agterbeen-kneusbeseringmodel, het
gelei tot vroeë spierherstel. Die bevindinge is geverifiëer deur ‘n vroeë satelietselrespons in
vergelyking met die plasebo groep. Hierdie effek was reeds opvallend vier uur na besering,
in vergelyking met die dag sewe en dag drie tydpunt tydens kroniese en acute plasebo
behandeling onderskeidelik. In vergelyking met die kontrole groep, het PSO
supplementering ook gelei to vininger uitdrukking van miosienswaarketting (MHCf). Kroniese
supplementering het spesifiek gelei to ‘n onderdrukte sirkulatoriese pro-inflammatoriese
sitokien response, terwyl ‘n betekenisvolle toename in IL-10 op dag drie (in die PSO groep
alleenlik) waargeneem is.
Op weefselvlak, het die pro-inflammatoriese sitokiene, IL-6 en TNF- , dieselfde patron
gevolg as die van satelietselle. Makrofaaginfiltrasie binne die beseerde spier het ook ‘n
soorgelyke patroon gevolg. Makrofage het die beseerde area vinniger geïnfiltreer in die
kronies PSO-gesupplementeerde groep, maar kon nie die sitokienrespons, wat waargeneem
is met akute supplementasie, verklaar nie. Beide kroniese en akute PSO supplementering
was verantwoordelik vir ‘n onderdrukte neutrofiel respons, wat ‘n nuwe bevinding is vir
hierdie spesifieke anti-oksidant. Die hoof bevindinge in die in vivo rotstudies, is dat PSO instaat is om die neutrofielrespons te
onderdruk, en sodoende vroeë makrofaaginfiltrasie teweeg te bring. Om meganismes
waarby PSO hierdie voordelige effekte veroorsaak te ondersoek, is verdere analises gedoen
om makrofaagfenotipe en neutrofielgetalle in die sirkulasie te bepaal. ‘n In vitro
neutrofielmigrasie studie is ook aangewend om PSO se vermoë om neutrofielinfiltrasie in die
beseerde area te onderdruk, te ondersoek. Neutrofiele van kontrole rotte, tesame met
gekondisioneerde plasma van eksperimentele diere en granulosiet-kolonie stimulerende
faktor (G-KSF), is toegelaat om vir twee ure in die teenwoordigheid van ‘n chemotaktiese
faktor, N-formiel metionien-leusien-fenielalanien (fMLP) te migreer. Resultate van hierdie
studie het aangetoon dat neutrofielmigrasie, in vitro, alleenlik onderdruk word in die
teenwoordigheid van gekondisioneerde plasma van PSO-gesupplementeerde rotte.
Die studies in hierdie tesis oor die effek van PSO op parameters van spier en die
immuunsisteem, het tot die volgende hoofgevolgtrekkings gelei: a) PSO supplementering
het vroeë spierherstel, as gevolge van vroeë satelietselaktivering en MHCf sintese, teweeg
gebring; b) PSO verkies ‘n anti-inflammatoriese sitokien reaksie, terwyl dit die proinflammatoriese
sitokienrespons onderdruk; en c) PSO onderdruk die neutrofielrespons,
terwyl vroeë makrofaaginfiltrasie in die beseerde area gefasiliteer word. Die spesifieke
meganisme van aksie van PSO, om die neutrofielrespons te onderdruk, kan moontlik die
vermoë van neutrofiele om adhesie molekule uit te druk, insluit. Hierdie aanname moet egter
verder ondersoek word.
|
64 |
Characteristics and adaptation of skeletal muscle to endurance exerciseKohn, Tertius A. 10 1900 (has links)
Thesis (PhD)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Skeletal muscle adapts to stimuli by modifying structural and metabolic protein expression.
Furthermore, a muscle group may vary within itself to accommodate specialisation in regions.
Structural and metabolic characteristics of an individual are regulated partly by genotype, but
contraction duration and intensity may play a greater role in muscle phenotype. The aims of this
dissertation were to investigate: structural and metabolic regionalisation in a muscle group, possible
relationships between training volume and intensity and hybrid fibres, muscle characteristics of
athletes from two different ethnic groups, and muscle adaptation in already well-trained athletes
subjected to high intensity interval training.
Myosin heavy chain (MHC) isoform content and citrate synthase (CS) activities were measured in
the Quadriceps femoris (QF) muscle of 18 female rats. Muscle was divided into superficial, middle
and deep, distal, central and proximal parts. MHC IIb and IIx were more abundant in superficial
regions (P < 0.05) with low CS activities compared to deeper parts. Isoform content varied along the
length of deep regions. This study showed that the QF has regional specialisation. Therefore,
standardisation of sampling site is important.
Hybrid fibre proportions in muscle biopsies of 12 middle distance runners and 12 non-runners were
investigated. MHC IIa/IIx correlated with training volume/week in runners (r = -0.66, P < 0.05) and
MHC IIa/IIx correlated with exercise hours/week in non-runners (r = -0.72, P < 0.01). Average
preferred racing distance (PRDA) correlated better with MHC IIa/IIx in runners (r = -0.85, P <
0.001). MHC IIa/IIx may therefore be more closely related to exercise intensity than previously
thought.
Fibre type characteristics and performance markers were investigated in 13 Xhosa and 13 Caucasian
distance runners, matched for performance, training volume and PRDA. Xhosa runners had less
MHC I and more MHC IIa fibres in muscle biopsies than Caucasian runners (P < 0.05). Xhosa
runners had lower plasma lactate at 80% peak treadmill speed (PTS) (P < 0.05), but higher lactate
dehydrogenase (LDH) (P < 0.01) and phosphofructokinase (P = 0.07) activities in homogenate
muscle samples. LDH activities in MHC I (P = 0.05) and IIa (P < 0.05) fibre pools were higher in
Xhosa runners. Xhosa athletes may thus have a genetic advantage or they may have adapted to
running at a higher intensity.
Six weeks of individually standardised high intensity interval treadmill training (HIIT) were
investigated in 15 well-trained runners. PTS increased after HIIT (P < 0.01), while maximum
oxygen consumption (VO2max) only showed a tendency to have increased as a result of HIIT (P = 0.06). Sub-maximal tests showed lower plasma lactate at 64% PTS (P = 0.06), with lower heart
rates at workloads from 64% to 80% PTS (P < 0.01) after HIIT. No changes were observed for
cross-sectional area, capillary supply and enzyme activities in homogenates muscle samples. LDH
activity showed a trend (P = 0.06) to have increased in MHC IIa pools after HIIT. Higher HIIT
speed was related to decreases in MHC I fibres, but increases in MHC IIa/IIx fibres (r = -0.70 and r
= 0.68, respectively, P < 0.05). Therefore, HIIT may alter muscle fibre composition in well-trained
runners, with a concomitant improvement in performance markers. / AFRIKAANSE OPSOMMING: Skeletspier kan adapteer deur strukturele en metaboliese protein ekspressie te verander as gevolg
van stimulante. ‘n Spiergroep kan ook intern verskil om spesialisering in spierdele toe te laat.
Strukturele en metaboliese karaktereienskappe van ‘n individu word deels gereguleer deur gene,
maar kontraksie tydperk en intensiteit mag ‘n groter rol speel in spierfenotipe. Die doelwitte van
hierdie tesis was om ondersoek in te stel in: strukturele en metaboliese eienskappe in
spiergroepstreke, moontlike verhoudings tussen oefeningsvolume of intensiteit en baster vesels,
spier eienskappe in atlete van twee etniese groepe, en spier adaptasie in goed geoefende atlete
blootgestel aan hoë intensiteit interval oefening.
Miosien swaar ketting (MSK) isovorm inhoud en sitraat sintase (SS) aktiwiteite is gemeet in die
Quadriceps femoris (QF) spier van 18 wyfie rotte. Spiere was opgedeel in oppervlakkig, middel en
diep, asook distaal, sentraal en proksimale dele. MSK IIb en IIx was meer oorvloedig in
oppervlakkige dele (P < 0.05) met lae SS aktiwiteite in vergelyking met dieper dele. Isovorm
inhoud het ook verskil oor die lengte van diep dele. Dus bevat die QF gespesialiseerde streke en is
die area van monsterneming belangrik.
Baster vesel proporsies is ondersoek in spiermonsters van 12 middel afstand hardlopers en 12 niehardlopers.
MSK IIa/IIx van hardlopers het met oefeningsvolume/week gekorreleer (r = -0.66, P <
0.05), asook MSK IIa/IIx van nie-hardlopers met oefeningsure/week (r = -0.72, P < 0.01).
Gemiddelde voorkeur wedloop afstand (VWAG) het beter met MSK IIa/IIx gekorreleer in
hardlopers (r = -0.85, P < 0.001). MSK IIa/IIx mag dus meer verwant wees aan oefeningsintensiteit.
Veseltipe eienskappe en prestasie merkers was ondersoek in 13 Xhosa en 13 Caucasian langafstand
atlete, geëweknie vir prestasie, oefeningsvolume en VMAG. Xhosa hardlopers het minder tipe I en
meer tipe IIA vesels in hul spiermonsters gehad as die Caucasian hardlopers (P < 0.05). Xhosa
hardlopers het laer plasma laktaat by 80% van hul maksimale trapmeul spoed (MTS) (P < 0.05),
maar hoër laktaat dihidrogenase (LDH) (P < 0.01) en fosfofruktokinase (P = 0.07) aktiwiteite in
homogene spiermonsters gehad. LDH aktiwiteite in MSK I (P = 0.05) en IIa (P < 0.05)
veselbondels was hoër in Xhosa hardlopers. Xhosa atlete mag dus ‘n genetiese voorsprong geniet, of
hulle het geadapteer om by hoër intensiteite te hardloop.
Ses weke van geïndividualiseerde gestandardiseerde hoë intensiteit interval trapmeul oefening
(HIIT) was ondersoek in 15 goed geoefende hardlopers. MTS het verhoog na HIIT (P < 0.01), en
maksimale surrstof verbruik (VO2max) het ‘n neiging getoon om te verhoog het na HIIT (P = 0.07).
Submaksimale toetse het laer plasma laktaat by 64% MTS getoon (P = 0.06), met laer harttempos by werkladings 64% tot 80% MTS (P < 0.01). Geen veranderings was gemerk vir deursnit area,
kapillêre toevoer en ensiem aktiwiteite in homogene spiermonsters nie. LDH aktiwiteit het ‘n
neiging getoon om te verhoog het (P = 0.06) in MSK IIa veselbondels na HIIT. Hoër HIIT snelhede
was verwant aan ‘n daling in MSK I vesels, maar ‘n verhoging in MSK IIa/IIx vesels (r = -0.70 en r
= 0.68, respektiwelik, P < 0.05). HIIT mag dus spier veseltipe verander in goed geoefende
hardlopers, met gevolglike verbetering in prestasie merkers.
|
65 |
The modulation of various signal transduction pathways in colorectal carcinoma cells by docosahexaenoic acidDu Toit, Joe-Lin 12 1900 (has links)
Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Introduction: The ability of different polyunsaturated fatty acids (PUFAs),
especially n-3 PUFAs, to prevent the development of cancer has been under
intense investigation the past three decades. Numerous studies have shown
that these fatty acids can kill cancer cells in vitro as well as in vivo whilst normal
cells remain unaffected. Unfortunately, the cellular and molecular mechanisms
responsible for this phenomenon are still poorly understood. This study
investigated the signalling pathways modulated by docosahexaenoic acid
(DHA) in an adenocarcinoma cell line, in order to shed some light on these
unknown mechanisms.
Materials & Methods: NCM460 (normal colon epithelial) and CaCo2 (colon
adenocarcinoma) cells were cultured and treated with low doses of palmitic acid
(PMA), oleic acid (OA), arachidonic acid (AA), and DHA. The effects of these
fatty acids on the proliferation of the cells were measured with the MTT assay.
The composition of membrane phospholipids of CaCo2 cells was determined
after 48h supplementation with different fatty acids by gas chromatography.
Also, CaCo2 cells were treated with DHA (10 μM) only and proteins were
harvested at fixed time points ranging from 2 minutes to 48 hours. The protein
inhibitors wortmannin (PI3 kinase inhibitor), PD 98059 (MEK inhibitor) and SB
203580 (p38 inhibitor) and also RNA interference (RNAi) of the p38 MAPK
protein were used to investigate cross-talk between signalling pathways. ERK,
p38 MAP kinase, Akt, and p53 were then analysed by Western blotting using
phospho-specific and total antibodies. The cleavage of the apoptotic proteins,
caspase-3 and PARP were also analysed.
Results and discussion: MTT assays revealed that none of the fatty acids were
toxic to normal cells. In addition, DHA was shown to be most effective to kill
CaCo2 cells whilst protecting NCM460 cells and a subsequent dose response experiment revealed that lower concentrations are most suitable for this
purpose. DHA was also shown to be readily incorporated into phospholipids,
along with AA. This is associated with increased membrane fluidity, which
could affect the localisation, and downstream effects, of various signalling
proteins within the membrane. Western blot analysis revealed a rapid increase
in activity in most proteins under investigation, especially ERK and Akt
(Ser473). Long-term DHA supplementation suppressed the full activation of
Akt. This down regulation of survival signalling could lead to cell death in
CaCo2 cells. In addition, it was shown that after 48h, DHA induced the
cleavage of caspase-3 and PARP, which is indicative of apoptosis. RNAi
experiments suggested a possible role for p38 MAPK in the phosphorylation of
p53 at Ser15, a site which is associated with DNA damage.
Conclusion: DHA exerts its effects by means of cellular signal transduction
pathways, particularly by suppression of the important survival-related kinase,
Akt. This could have implications for future therapeutic interventions in cancer
patients, as fatty acids are safe to use and do not interfere with the functionality
of normal tissue. / AFRIKAANSE OPSOMMING: Inleiding: Die vermoë van verskillende poli-onversadigde vetsure (POVSe),
veral n-3 POVSe, om die ontstaan van kanker te voorkom, is intens nagevors
die afgelope drie dekades. Menigte studies het aangevoer dat hierdie vetsure
kankerselle in vitro asook in vivo kan doodmaak, terwyl normale selle nie
daardeur beïnvloed word nie. Ongelukkig word die sellulêre and molekulêre
meganismes onderliggend tot hierdie verskynsel nie goed begryp nie. Hierdie
studie het verskeie seintransduksie-paaie wat deur dokosaheksaenoësuur
(DHS) in ‘n adenokarsinoom sellyn gemoduleer word, ondersoek.
Materiale & Metodes: NCM460 (normale kolonepiteel) en CaCo2 (kolon
adenokarsinoom) selle is onderhou in ‘n selkultuur-laboratorium en behandel
met lae dosisse palmitiensuur (PMS), oleïensuur (OS), aragidoonsuur (AS), en
DHS. Die invloed van hierdie vetsure op die proliferasie van die selle is d.m.v.
die MTT toets bepaal. The samestelling van membraan-fosfolipiede van CaCo2
selle is na 48h behandeling met die verskillende vetsure bepaal deur middel
van gaschromatografie. Die CaCo2 selle is ook met DHA (10 μM) alleenlik
behandel en teen vaste tydpunte wat wissel van 2 minute tot 48h, waarna
proteïene geëkstraeer is. Die proteïen-inhibitore wortmannin (PI3 kinase
inhibitor), PD 98059 (MEK inhibitor), en SB 203580 (p38 inhibitor) asook RNAinterferensie
(RNAi) teen die p38 MAPK proteïen is ingespan om oorvleueling
tussen seintransduksie–weë te ondersoek. ERK, p38 MAPK, Akt, en p53 is
geanaliseer deur middel van die Western–klad metode met fosfo–spesifieke en
totale antiliggame. Die kliewing van die apoptotiese proteïene caspase-3 en
PARP is ook bepaal.
Resultate en bespreking: MTT toetse het ontul dat geen vetsure toksies was vir
die normale selle nie. Daar is ook gevind dat DHS die mees effektiewe vetsuur
was om CaCo2 selle te dood, terwyl NCM460 selle beskerm word. Gevolglik het ‘n dosis-respons eksperiment getoon dat laer konsentrasies die beste
geskik is vir hierdie doel. Daar is ook gevind dat DHA maklik in fosfolipiede
geïnkorporeer word, tesame met AS. Dit word geassosieer met verhoogde
membraan-vloeibaarheid, wat die ligging, en ook stroom-af werking, van
verskeie seintransduksie proteïene in die membraan, kan beïnvloed. Westernklad
analises het ‘n vinnige verhoging in die aktiwiteite van die meeste
proteïene onder die soeklig, getoon, veral ERK en Akt (Ser473). Langdurige
DHS behandeling het die maksimale aktiwiteit van Akt onderdruk. Hierdie
afname van oorlewing-gerigte seine kan lei tot seldood in CaCo2 selle. Daar is
boonop geving dat DHS die kliewing van caspase-3 en PARP geïnduseer het
na 48, wat dui op apoptose. Uit die RNAi eksperiment kon daar ook ‘n
moontlike rol vir p38 MAPK in die fosforilering van p53 by Ser15, wat
geassosieer word met DNS-skade, getoon word.
Gevolgtrekking: DHS beoefen sy effekte deur middel van seintransduksie
paaie, veral deur die oorlewing-geassosieerde kinase, Akt, te onderdruk. Dit
kan implikasies hê vir toekomende terapeutiese ingrypings in kankerpasiënte,
aangesien vetsure veilig is om te gebruik en nie skadelik is vir normale weefsel
nie.
|
66 |
ART-related body composition changes in adult women in a semi-rural South African contextDe Bruto, Petro C. 12 1900 (has links)
Assignment (MPhil)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: The aim of this study was to investigate practical methods of monitoring AIDS related
wasting and lipodystrophy in a resource-poor clinical setting with HIV infected women as
the population group of interest. Measurement of body composition changes using
anthropometry is both cost- and time-efficient. Various different skinfolds were taken and
two different equations (the equations of Pollock et al. (1975) and Durnin and Womersley
(1974) for calculating body fat were used to determine the most promising method or
methods of monitoring body composition changes in a clinical setting.
Detailed anthropometric measurements were performed, as well as selected measurements
for haematological parameters and quality of life (QoL) for a group of 8 participants on
antiretroviral medication (ART group) and 6 participants who were not on treatment (TN
group). New variables namely, intra-abdominal indicator (IAI) and a percent of ideal body
mass to percent of ideal arm circumference ratio (%IBW:%IAC) were investigated as
possible indicators of lipodystrophy. Although measurements were taken at various timepoints,
three specific time-points were chosen for data-analysis for the ART group and two
time points for the TN group. These three time-points were, baseline (on the day of
recruitment for TN participants and within one month before the initiation of treatment for
ART participants), short-term (2 to 12 weeks after treatment initiation or the baseline
measurement or for the ART and the TN participants) and long-term (within one and a half
year of treatment initiation for the ART group).
ART and TN participants did not differ for many variables at baseline. The major
differences between ART and TN were in measured and derived variables of the arm,
especially percent of ideal arm circumference (%IAC) and upper arm fat area (UAFA),
which were significantly lower in the ART group.
CD4+ and QoL improved significantly for the ART participants from baseline to long-term.
This was not associated with changes in muscle mass, but rather some fat mass variables.
Participants on antiretroviral medication exhibited changes relating to abdominal obesity.
It was concluded that antiretroviral therapy contributed greatly to the QoL of the participants and it probably aided in the recovery from wasting for at least one participant
in this study. Measures of the arm can be used in a rural clinical setting to effectively
monitor patients with regard to AIDS related wasting. The new variables IAI and
%IBW:%IAC could be helpful in the monitoring of lipodystrophy and should be
investigated in future research. / AFRIKAANSE OPSOMMING: Die doelwit van hierdie studie is om praktiese metodes te ondersoek om VIGS-verwante
uittering en lipodistrofie te meet in ‘n plattelandse kliniese omgewing (waar hulpbronne
dikwels beperk is) met MIV ge-infekteerde vroue as populasiegroep. Die gebruik van
antropometrie om veranderinge in liggaamssamestelling te meet is beide koste- en
tydeffektief. Verskeie velvoumetings is geneem en twee verskillende vergelykings (die
vergelykings van Pollock et al. (1975) en Durnin en Womersley (1974)) is gebruik om
liggaamsvetinhoud te bereken, met die doel om ‘n belowende metode te vind om
veranderinge in liggaamssamestelling te meet in ‘n kliniese omgewing.
Verskeie antropometriese metings is geneem, sowel as uitgesoekte hematologiese en
lewenskwaliteitmetings (QoL) vir ‘n groep van agt deelnemers wat antiretrovirale
medikasie ontvang het (ART groep) en ses deelnemers wat nie hierdie behandeling ontvang
het nie (TN groep). Nuwe veranderlikes (binnebuikindikator (IAI) en die verhouding van
persentasie van ideale liggaamsmassa tot persentasie van ideale armomtrek
(%IBW:%IAC)) is ondersoek as moontlike aanwysers van lipodistrofie. Drie spesifieke
tydpunte vir die ART groep en twee tydpunte vir die TN groep is gekies uit die verskeie
tydpunte waarby metings geneem is, nl. basislyn (gedefinieer as die dag wat TN deelnemers
in die studie opgeneem is en 0 tot 4 weke voor die begin van behandeling vir die ART
deelnemers), korttermyn (2 tot 12 weke nadat behandeling begin is of na die basislyn
meting) en lang-termyn (binne een en ‘n half jaar nadat behandeling begin is vir die ART
groep).
By die basislyn tydpunt het min van die ART en TN deelnemers se gemete veranderlikes
verskil. Die ART en TN groepe het hoofsaaklik verskil ten opsigte van veranderlikes wat
betrekking het op die arm, veral persentasie van ideale armomtrek (%IAC) en bo-arm vetarea
(UAFA). Hierdie twee veranderlikes was beduidend laer in die ART groep as in die
TN groep.
CD4+ seltelling en lewenskwaliteit tellings het beduidend verbeter vir die ART deelnemers
van die basislyn tot die lang-termyn tydpunt. Hierdie veranderinge is nie samehangend met veranderinge in spiermassa nie, maar eerder met sommige vetmassa veranderlikes.
Deelnemers wat antiretrovirale medikasie ontvang het, het veranderinge getoon wat gedui
het op ‘n verhoogde neerlegging van vet in die buikarea. Ten slotte is bevind dat
antiretrovirale medikasie bygedra het tot die verbeterde lewenskwaliteit van die deelnemers
en dat dit waarskynlik ook die omkeer van uittering van ten minste een deelnemer
aangehelp het. Daar is ook bevind dat armverwante metinge gebruik kan word in die
plattelandse kliniese omgewing om pasiënte suksesvol te monitor ten opsigte van VIGSverwante
uittering. Die nuwe veranderlikes, IAI en %IBW:%IAC kan moontlik gebruik
word om lipodistrofie-verwante veranderings te meet en die gebruik van hierdie
veranderlikes behoort ondersoek te word in verdere navorsing.
|
67 |
The effect of the TGF-β isoforms on progenitor cell recruitment and differentiation into cardiac and skeletal muscleSchabort, Elske Jeanne 12 1900 (has links)
Thesis (PhD (Physiology (Human and animal))-- University of Stellenbosch, 2007. / Definition: Stem cells are unspecialised cells with the capacity for long-term self-renewal and
the ability to differentiate into multiple cell-lineages.
The potential for the application of stem cells in clinical settings has had a profound effect on
the future of regenerative medicine. However, to be of greater therapeutic use, selection of
the most appropriate cell type, as well as optimisation of stem cell incorporation into the
damaged tissue is required. In adult skeletal muscle, satellite cells are the primary stem cell
population which mediate postnatal muscle growth. Following injury or in diseased
conditions, these cells are activated and recruited for new muscle formation. In contrast, the
potential of resident adult stem cell incorporation into the myocardium has been challenged
and the response of cardiac tissue, especially to ischaemic injury, is scar formation.
Following muscle damage, various growth factors and cytokines are released in the afflicted
area which influences the recruitment and incorporation of stem cells into the injured tissue.
Transforming Growth Factor-β (TGF-β) is a member of the TGF-β-superfamily of cytokines and
has at least three isoforms, TGF-β1, -β2, and -β3, which play essential roles in the regulation
of cell growth and regeneration following activation and stimulation of receptor-signalling
pathways. By improving the understanding of how TGF-β affects these processes, it is
possible to gain insight into how the intercellular environment can be manipulated to improve
stem cell-mediated repair following muscle injury. Therefore, the main aims of this thesis
were to determine the effect of the three TGF-β isoforms on proliferation, differentiation,
migration and fusion of muscle progenitor cells (skeletal and cardiac) and relate this to
possible improved mechanisms for muscle repair.
The effect of short- and long-term treatment with all three TGF-β isoforms were investigated
on muscle progenitor cell proliferation and differentiation using the C2C12 skeletal muscle
satellite and P19 multipotent embryonal carcinoma cell-lineages as in vitro model systems.
Cells were treated with 5 ng/mℓ TGF-β isoforms unless where stated otherwise. In C2C12
cells, proliferating cell nuclear antigen (PCNA) expression and localisation were analysed, and
together with total nuclear counts, used to assess the effect of TGF-β on myoblast
proliferation (Chapter 5). The myogenic regulatory factors MyoD and myogenin, and structural
protein myosin heavy chain (MHC) were used as protein markers to assess early and terminal
differentiation, respectively. To establish possible mechanisms by which TGF-β isoforms
regulate differentiation, further analysis included determination of MyoD localisation and the
rate of MyoD degradation in C2C12 cells. To assess the effect of TGF-β isoforms on P19 cell differentiation, protein expression levels of
connexin-43 and MHC were analysed, together with the determination of embryoid body
numbers in differentiating P19 cells (Chapter 6). Furthermore, assays were developed to
analyse the effect of TGF-β isoforms on both C2C12 and P19 cell migration (Chapter 7), as
well as fusion of C2C12 cells (Chapter 8).
Whereas all three isoforms of TGF-β significantly increased proliferation of C2C12 cells,
differentiation results, however, indicated that especially following long-term incubation,
TGF-β isoforms delayed both early and terminal differentiation of C2C12 cells into myotubes.
Similarly, myocyte migration and fusion were also negatively regulated following TGF-β
treatment. In the P19 cell-lineage, results demonstrated that isoform-specific treatment with
TGF-β1 could potentially enhance differentiation. Further research is however required in this
area, especially since migration was greatly reduced in these cells.
Taken together, results demonstrated variable effects following TGF-β treatment depending
on the cell type and the duration of TGF-β application. Circulating and/or treatment
concentrations of this growth factor could therefore be manipulated depending on the area of
injury to improve regenerative processes. Alternatively, when selecting appropriate stem or
progenitor cells for therapeutic application, the effect of the immediate environment and
subsequent interaction between the two should be taken into consideration for optimal
beneficial results.
|
68 |
Dietary red palm oil-supplementation offers cardioprotection against Ischaemia/Reperfusion injury : possible cellular mechanisms involvedEsterhuyse, Adriaan Johannes 12 1900 (has links)
Dissertation (PhD)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Activation of the NO-cGMP pathway is associated with myocardial protection
against ischaemia/reperfusion injury. However, high-cholesterol diets alter
function of this pathway and these alterations have been implicated in both
ischaemic/reperfusion injury and the development of ischaemic heart disease.
Little is known about the effects of supplements such as Red Palm Oil (RPO)
on the myocardial NO-cGMP-signalling pathway. RPO consists of saturated,
mono-unsaturated and poly-unsaturated fatty acids and is rich in antioxidants
such as β-carotene and Vitamin E (tocopherols and tocotrienols). The aims of
this study were: 1) to determine whether dietary RPO-supplemention protects
against ischaemia/reperfusion injury in rats fed a standard rat chow (control)
and cholesterol-enriched diets and 2) if so, to investigate possible mechanisms
for this protection.
Male Long-Evans rats were fed a standard rat chow or a standard rat chow
plus cholesterol and/or RPO-supplementation for 6 weeks. Myocardial
functional recovery was measured and hearts were freeze-clamped for
determination of myocardial phospholipid, cAMP/cGMP concentrations, total
myocardial nitric oxide concentrations, lipid hydroperoxide production and
superoxide dismutase- and nitric oxide synthase activity in isolated rat hearts
subjected to 25 minutes of normothermic total global ischaemia. In addition,
the degree of phosphorylation of extracellular signal-regulated kinase (ERK),
p38, c-Jun N-terminal protein kinase (JNK) and protein kinase B (PKB/Akt)
was investigated. Furthermore, the effect of RPO-supplementation on
caspase-3 activation and poly (ADP-ribose) polymerase (PARP)-cleavage in
hearts subjected to ischaemia and reperfusion was also investigated. Our data show that dietary RPO-supplementation protects the hearts of rats on
a standard rat chow (control) and hypercholesterolaemic diet against
ischaemia/reperfusion injury as reflected by improved aortic output recovery.
Increased intracellular cardiomyocyte NO concentrations as observed in
control hearts supplemented with RPO after 120 minutes hypoxia may
contribute to the elevated cGMP concentration and may confer some of the
cardioprotection to the ischaemic/reperfused heart. Although improved
functional recovery with RPO-supplementation of a high-cholesterol diet was
also associated with an increase in intracellular cardiomyocyte NO production
after hypoxia compared to the non-hypoxic conditions, it could not be linked to
increased NO-cGMP signalling. These data are in agreement with other
studies, which showed that high-cholesterol diet impairs NO-cGMP signalling
and confirms our hypothesis that elevated cGMP concentrations may not be
the only mechanism of protection. We have also shown that RPOsupplementation
caused increased phosphorylation of p38 and PKB, reduced
phosphorylation of JNK and attenuation of PARP cleavage, which may
contribute to the protection of the cell against apoptosis.
Based on our results we propose that the myocardial protection offered by
RPO-supplementation of rats on a normal and hypercholesterolaemic diet may
be associated with either its antioxidant characteristics and/or changes in the
fatty acid composition of the myocardium during ischaemia/reperfusion.
Furthermore, we demonstrated for the first time that RPO-supplementation
protects the isolated perfused working rat heart during reperfusion from
ischaemia/reperfusion-induced injury through a MAPK-dependent pathway. / AFRIKAANSE OPSOMMING: Aktivering van die NO-cGMP sein transduksie pad word geassosieer met
miokardiale beskerming teen isgemie/herperfusie skade. Hoë cholesterol diëte
verander egter die funksie van die pad en hierdie veranderings speel ‘n rol in
beide isgemie/herperfusie besering en die ontwikkeling van isgemiese
hartsiekte.
Daar is egter min inligting beskikbaar oor die uitwerking van aanvullings soos
rooi palm olie (RPO) op die miokardiale NO-cGMP sein transduksie pad. RPO
bevat versadigde, mono-onversadigde en poli-onversadigde vetsure en is ryk
aan anti-oksidante nl. β-karotene en vitamien E (tokoferole en tokotriënole).
Die doelwitte van hierdie studie was: 1) om vas te stel of ‘n RPO-aanvulling
beskerming bied teen isgemie/herperfusie besering in rotte wat gevoed is met
‘n standaard rotmengsel (kontrole) en cholesterol-verrykte dieet en 2) indien
wel, om moontlike meganismes van beskerming te ondersoek.
Long-Evans manlike rotte is vir 6 weke gevoer met ‘n standaard rotmengsel of
‘n standaard rotmengsel plus cholesterol en/of RPO-aanvulling. Miokardiale
funksionele herstel is gemeet en harte is gevriesklamp vir die bepaling van
miokardiale fosfolipied, cAMP/cGMP, totale stikstofoksied, lipied
hidroperoksied, superoksied dismutase en stikstofoksied sintase in
geïsoleerde rotharte wat vir 25 minute onderwerp was aan normotermiese
totale globale isgemie. Hiermee saam is die graad van fosforilering van
ekstrasellulêre sein gereguleerde kinase (ERK), p38 mitogeen-geaktiveerde
proteïen kinase (p38 MAPK), c-Jun-N-terminale proteïenkinase (JNK) en proteïen kinase B (PKB/Akt) ondersoek, asook kaspase-3 aktivering en poli
(ADP-ribose) polimerase (PARP) kliewing in harte blootgestel aan isgemie en
herperfusie.
Ons resultate toon dat RPO-aanvulling van rotte op ‘n normale en
hipercholesterolemiese dieet die hart beskerm soos getoon deur verbeterde
herstel van aortiese uitset. Verhoogde intrasellulêre miokardiale NO vlakke in
kontrole harte met ‘n RPO-aanvulling wat blootgestel was aan 120 minute
hipoksie, mag bygedra het tot die verhoogde cGMP vlakke en beskerming van
die hart tydens isgemie en herperfusie. Alhoewel verbeterde funksionele
herstel met RPO-aanvulling van ‘n hoë cholesterol dieet ook geassosieer is
met ‘n toename in intrasellulêre miokardiale NO produksie ná hipoksiese
toestande, kon dit nie verbind word met verhoogde aktivering van die NOcGMP
sein transduksie pad nie. Hierdie resultate stem ooreen met ander
studies wat aangetoon het dat hoë-cholesterol diëte die NO-cGMP seinpad
onderdruk. Hierdie bevinding bevestig ons hipotese dat verhoogde cGMP
vlakke moontlik nie die enigste beskermingsmeganisme is nie. Ons resultate
het ook gewys dat RPO-aanvulling fosforilering van p38 en PKB/Akt verhoog,
fosforilering van JNK verminder en PARP kliewing onderdruk. Dit dui op
beskerming van die sel teen apoptose.
Ons resultate dui aan dat die miokardiale beskerming wat RPO-dieet
aanvulling bied moontlik geassosieer kan word met sy anti-oksidant eienskap
en/of veranderinge in die vetsuur samestelling van die miokardium tydens
isgemie/herperfusie. Ons het ook vir die eerste keer bewys dat RPO-aanvulling die geïsoleerde geperfuseerde werkende rothart gedurende herperfusie
beskerm teen isgemie/herperfusie besering deur die aktivering en/of
deaktivering van die MAPK afhanklike pad.
|
Page generated in 0.0463 seconds