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Exploring underlying mechanisms driving the onset of stress-induced insulin resistanceOtto, Delita 03 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Physical and psychological stressors trigger activation of the hypothalamo-pituitary-adrenocortical
(HPA) axis that leads to enhanced secretion of glucocorticoids e.g. cortisol. Moreover, chronic activation
of this pathway may elevate oxidative stress that is linked to the onset of insulin resistance and
cardiovascular diseases (CVD). Our laboratory previously found that oxidative stress increases flux
through metabolic circuits such as the hexosamine biosynthetic pathway (HBP), in effect increasing its
modification of target proteins post-transcriptionally with O-GlcNAc moeities. This in turn may alter
protein function and contribute to the onset of myocardial insulin resistance and impaired contractile
function. Since the underlying mechanisms linking chronic stress to cardiometabolic pathophysiology
are poorly understood, we hypothesised that cortisol elicits myocardial oxidative stress, HBP activation,
and decreased glucose uptake (due to attenuated glucose transport functionality) with detrimental
outcomes, i.e. insulin resistance and apoptosis. To investigate this hypothesis we established an in vitro model using HL-1 cardiomyocytes, with which
we evaluated the degree of O-GlcNAcylation and oxidative stress in response to a range of time-dose
treatments with dexamethasone (synthetic glucocorticoid). Glucose transporter 4 (GLUT4) translocation
to the sarcolemma was also assessed. In agreement with the literature, results suggest that GLUT4
translocation is significantly decreased subsequent to dexamethasone treatment. Although no significant differences were observed with regards to oxidative stress or O-GlcNAcylation, the data show that
dexamethasone increased the latter with a maximal effect after two hours exposure to the 10-6 M dose.
Although our results were not conclusive, the data suggest a potential novel link between dexamethasone
exposure, HBP activation and decreased GLUT4 translocation. Based on our findings we propose
that detrimental effects of chronic stress on the heart may be mediated by increased HBP flux. Given
that glucocorticoid excess and GLUT4 dysregulation have been associated with insulin resistance (and
related metabolic derangements and diseases), these results provide new targets for potential therapeutic
agents. / AFRIKAANSE OPSOMMING: Fisiese sowel as psigologiese stressors veroorsaak die aktivering van die hipotalamiese-hipo seale-bynier
(HHB) pad wat lei tot die verhoogde sekresie van glukokortikoïede soos kortisol. Kroniese aktivering van
hierdie pad kan ook oksidatiewe stres verhoog wat weer tot insulienweerstandigheid en kardiovaskulêre
siektes (KVS) kan lei. Navorsing uit ons laboratorium het voorheen bewys dat oksidatiewe stres 'n toename
in vloei deur metaboliese paaie soos die heksoamine biosintetiese pad (HBP) kan veroorsaak deur
die modi sering van teikenproteïene met O-GlcNAc motiewe. Dit kan weer proteïen funksie verander
en bydra tot die ontstaan van miokardiale insulienweerstandigheid en verswakte kontraktiele funksie.
Die onderliggende meganismes wat kroniese stres aan kardiometaboliese pato siologie verbind word
nog nie goed verstaan nie, daarom is ons hipotese dat kortisol miokardiale oksidatiewe stres veroorsaak,
die HBP pad aktiveer, en glukose opname verminder (deur die funksionele onderdrukking van
glukose transport), wat nadelige uitkomste soos insulienweerstandigheid en apoptose tot gevolg kan hê.
Om hierdie hipotese te ondersoek, is 'n in vitro model van HL-1 kardiomiosiete gebruik waarmee
die graad van O-GlcNAsilering en oksidatiewe stres in reaksie op 'n reeks tyd-konsentrasie behandelings
met deksametasoon (sintetiese glukokortikoïed), bepaal is. Glukose transporter 4 (GLUT4)
translokasie na die sarkolemma is ook geasseseer. In ooreenstemming met die literatuur, is GLUT4
translokasie insiggewend onderdruk tydens deksometasoon behandeling. Alhoewel geen insiggewende
verskille rakende oksidatiewe stres en O-GlcNAsilering gevind is nie, het ons data aangedui dat laasgenoemde
deur deksametasoon vermeerder het na twee ure van blootstelling aan die 10-6 M konsentrasie.
Alhoewel ons resultate geen afdoende bewys lewer nie, stel dit wel voor dat daar 'n potensiële verbintenis
tussen deksametasoon behandeling en 'n afname in GLUT4 translokasie is. Gebasseer op ons
bevindings, stel ons voor dat die nadelige e ekte van kroniese stres op die hart bemiddel kan word
deur 'n toename in vloei deur die HBP. Gegewe dat 'n oormaat glukokortikoïede en GLUT4 wanregulering
geassosieer is met insulien weerstandigheid (en verbandhoudende metaboliese veranderinge en
siektes), verskaf hierdie resultate nuwe teikens vir potensiële terapeutiese ingrepe.
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Increased flux through the hexosamine biosynthetic pathway leads to the induction of acetol-CoA caboxylase gene expression in the heartImbriolo, Jamie 03 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: Gene expression of the cardiac isoform of acetyl-CoA carboxylase (ACCb) is induced
in a glucose-dependent manner. ACCb produces malonyl-CoA, a potent inhibitor of
mitochondrial fatty acid uptake. Previous studies show that increased flux through the
hexosamine biosynthetic pathway (HBP) under hyperglycaemic conditions may
contribute to the development of insulin resistance. In light of this, we hypothesised
that increased HBP flux induces cardiac ACCb gene expression thereby contributing
to the onset of insulin resistance.
We tested our hypothesis by transiently transfecting cardiac-derived rat H9c2
myoblasts with a 1,317 bp human ACCb promoter-luciferase construct (pPIIb-1317)
and an expression construct encoding the rate-limiting step of the HBP i.e. glutamine:
fructose 6-phosphate amidotransferase (GFAT). Overexpression of GFAT increased
ACCb gene promoter activity by 75 ± 23% versus controls (n=6, p<0.001). When cotransfection
experiments were repeated in the presence of varying concentrations of
L-glutamine (0 mM, 4 mM, 8 mM), a substrate for the HBP, ACCb promoter activity
was dose-dependently increased. To further corroborate these findings, we
employed two inhibitors of GFAT, i.e. 40 μM azaserine and 40 μM 6-diazo-5-oxo-Lnorleucine
were administered to transfected cells for a period of 24 hours. Here both
azaserine and 6-diazo-5-oxonorleucine attenuated ACCb gene promoter activity.
In agreement, co-transfections with two dominant negative GFAT constructs also
diminished ACCb gene promoter activity. We next inhibited two enzymes of the HBP
acting downstream of GFAT, i.e. O-GlcNAc transferase and O-GlcNAcase using
alloxan (0.1 mM, 1 mM and 2 mM) and streptozotocin (5 mM and 10 mM), respectively, for a period of 24 hours. Addition of alloxan attenuated ACCb gene
promoter activity by 35.6 ± 1.9% (n=16, p<0.001) and streptozotocin increased
activity by 32 ± 12% (n=12, p<0.001). We also investigated USF1 and USF2 as
transcriptional regulatory candidates for HBP-induced ACCβ promoter regulation.
Our data implicates USF2 as an important transcriptional regulator of HBP-induced
ACCβ promoter regulation.
In summary, this study demonstrates that increased flux through the hexosamine
biosynthetic pathway induces ACCb gene promoter activity. We further propose that
such an induction would reduce cardiac fatty acid oxidation, thereby leading to
intracellular lipid accumulation due to a mismatch between sarcolemmal FA uptake
and mitochondrial FA oxidation in the insulin resistant setting (i.e. hyperlipidaemia). / AFRIKAANSE OPSOMMING: Geen uitdrukking van die kardiale isoform asetiel-KoA karboksilase (ACCb) word in ‘n
glukose afhanklike wyse geïnduseer. ACCb produseer maloniel-KoA, ‘n kragtige
inhibeerder van mitochondriale vetsuuropname. Vorige studies toon aan dat
verhoogde fluks deur die heksosamien biosintestiese weg (HBW) onder
hiperglukemiese toestande bydra tot die ontwikkeling van insulienweerstand. In die
lig hiervan, word daar gehipotetiseer dat verhoogde HBP fluks kardiale ACCb
geenuitdrukking induseer en so bydra tot die ontstaan van insulienweerstand.
Ons hipotese is getoets deur die kardiale afkomstige rot H9c2 mioblaste met ‘n 1.317
bp mens ACCb-lusiferase promotor konstruk (pPII-1317) te transfekteer en ‘n
uitdrukking te konstrueer wat die tempo bepalende stap van HBP i.e. glutamien:
fruktose-6-fosfaat amidotransferase (GFAT) kodeer. Ooruitdrukking van GFAT
verhoog ACCb geenpromotor aktiviteit deur 75 ± 23% teenoor kontrole (n=6,
p<0.001). Die herhaling van ko-transfeksie eksperimente is herhaal in die
teenwoordigheid van variëerbare L-glutamienkonsentrasies (0 mM, 4 mM, 8 mM), ’n
substraat vir die HBP, ACCb promotor aktiwiteit is dosisafhanglik verhoog. Om die
bevindinge verder te staaf, is twee inhibeerders van GFAT, i.e. 40 μM azaserien en
40 μM 6-diazo-5-oxo-L-norleusien aan transfeksie selle toegedien vir ’n tydperk van
24 uur. Beide azaserien en 6-diazo-5-oxo-L-norleusien verlaag ACCb geenpromotor
aktiwiteit.
In ooreenstemming met die bogenoemde het ko-transfeksies met twee dominante
negatiewe GFAT konstrukte ook ACCb geenpromoter aktiwiteit verminder. Die
volgende stap is om twee ensieme van die HBP wat stroomaf van GFAT aktief is, vir ‘n periode van 24 uur te inhibeer i.e. O-GlcNAc transferase en O-GlcNAcase deur
alloxan (0.1 mM, 1 mM en 2 mM) and streptozotosien (5 mM en 10 mM)
onderskeidelik vir ‘n 24 uur periode te gebruik. Toevoeging van alloxan het die ACCb
geenpromotor aktiwiteit by 35.6 ± 1.9% (n=16, p<0.001) verlaag en streptozotosien
aktiwiteit verhoog by 32 ± 12% (n=12, p<0.001). Ons het ook die USF1 en USF2 as
transkripsie regulerings kandidate vir HBP-geïnduseerde ACCβ promotor regulering
ondersoek. Ons data impliseer dat USF2 as ‘n belangrike transkripsie reguleerder
van HBP-geïndiseerde ACCβ promotor regulering is.
Samevattend het hierdie studie demonstreer dat verhoogde fluks deur die
hexosamien biosintetiese weg ACCb geenpromotor aktiwiteit induseer. Ons stel
verder voor dat hierdie induksie die kardiale vetsuuroksidasie verlaag wat daartoe lei
dat intrasellulêre lipied akkumulasie as gevolg van onparing tussen sarkolemma
vetsuuropname en mitochondriale vetsuuroksidasie in ’n insulien weerstandige
situasie (i.e. hiperlipidaemia).
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Antioxidant (Oxiprovin TM) supplementation and muscle recovery from contusion injury - an in vivo studyKruger, Maria Jacoba 12 1900 (has links)
Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Human studies on the response of muscle to contusion injury are limited,
probably due to the large variability in injury severity and the non-specificity of
clinical symptoms reported. To circumvent this problem, several experimental
animal models have been designed to study muscle damage and regeneration
after contusion injuries. However, the majority of techniques currently used to
induce contusion injury are very invasive and therefore not optimal. Furthermore,
published studies regarding clinical treatment of such injuries are limited. The
main aims of this study were therefore: a) to establish and characterise an in vivo
model of non-invasive contusion injury, and b) to assess the effect of pre-injury
chronic administration of the antioxidant supplement Oxiprovin™ - a natural
grape seed extract (GSE) - on skeletal muscle recovery after experimentallyinduced
injury.
Two groups of male Wistar rats were subjected to 14 days of oral administration
of isovolaemic placebo (sterile isotonic saline) or GSE (20 mg/kg/day) prior to
induced contusion. Contusion injury was induced with the mass-drop technique,
and recovery parameters assessed for up to 14 days post-injury. Placebotreated
rats on average exhibited a 56 % higher creatine kinase (CK) activity
when compared to the GSE-treated rats when area under the curve (AUC) was
calculated for 14 days post-injury (p < 0.001). In the placebo group, plasma
oxygen radical absorbance capacity (ORAC) was unchanged over time, but
muscle ORAC was significantly increased by day 7 post-injury (p < 0.001). In the
GSE group, a significant decrease in both plasma (p < 0.01) and muscle ORAC
(p < 0.001) was evident 4 hr after injury, followed by a significant increase by day
3 (p < 0.05 and p < 0.001 respectively). CD34+ satellite cell (SC) numbers (quiescent and activated) peaked earlier in GSE-treated rats when compared to
placebo-treated rats (4 hours vs. day 7 post-injury). Total satellite cell number
(CD56+) also peaked earlier in GSE-treated rats than in placebo-treated rats (4
hours vs. 3 days post-injury), while M-cadherin+ SC numbers (quiescent,
activated or proliferating) in both treatment groups were significantly increased 4
hours post-injury (p < 0.001), but more so in the placebo group. In GSE-treated
rats when compared to placebo-treated rats, newly generated muscle fibres
(displaying central nuclei and MHCf
+) both appeared (day 3 vs. day 7 post-injury)
and peaked in number (day 3 vs. day 7 post-injury; increase from baseline p <
0.001 for both) earlier.
The results of this study demonstrate that we have successfully established an in
vivo model for non-invasive contusion injury in rats. Furthermore, we have shown
that Oxiprovin™: a) increased the ability to scavenge reactive species generated
after injury and b) resulted in the activation of satellite cells and formation of
newly generated muscle fibres at an earlier time point, thus accelerating the
recovery of skeletal muscle after a standardised contusion injury. / AFRIKAANSE OPSOMMING: Eksperimente aangaande die reaksie van spier op kneusings in mense is beperk,
waarskynlik as gevolg van die groot verskeidenheid simptome wat mag voorkom
en die verskille in die ernstigheid van beserings. Ten einde hierdie problem te
oorbrug, is verskeie eksperimentele diermodelle opgestel om kneusings en die
herstel van spier daarna te ondersoek. Die tegnieke wat grootendeels vandag
gebruik word om kneusings te veroorsaak, maak inbraak op die spier deur die
spier te ontbloot voor besering, en is dus nie ideaal nie. Daar is ook nie baie
bewyse aangaande die mees geskikte manier om so ‘n besering klinies te
behandel nie. Die doel van hierdie studie was dus om: a) ‘n in vivo model van
kneusings op te stel en te omskryf, en b) die effek van chroniese toediening van
die antioksidant Oxiprovin™ - ‘n natuurlike druifsaad ekstrak (DSE) – op die
herstel van skeletspier na ‘n kneusing te ondersoek.
Twee groepe manlike Wistar rotte is onderwerp aan mondelikse toediening van
isovolemiese plasebo (steriele isotoniese soutoplossing) of DSE (20 mg/kg/dag)
vir ‘n tydperk van 14 dae voor kneusing. Kneusing is geïnduseer met die “massdrop”
tegniek, en parameters van herstel is ondersoek tot en met 14 dae na
besering. Plasebo-behandelde rotte het gemiddeld 56 % hoër kreatien kinase
(KK) aktiwiteit in vergelyking met DSE-behandelde rotte (p < 0.001), toe die
oppervlak onder die kurwe (OOK) tot en met 14 dae na besering bereken is.
Geen verskil oor tyd is in die plasebo groep opgemerk toe plasma suurstof
radikaal absorpsie kapasiteit (SRAK) bepaal is nie, maar ‘n betekenisvolle
toename in spier SRAK teen dag 7 (p < 0.001) is waargeneem. ‘n Betekenisvolle
afname in beide plasma (p < 0.01) en spier (p < 0.001) SRAK van die DSE is
teen 4 hr waargeneem, gevolg deur ‘n betekenisvolle toename teen dag 3 na besering (p < 0.05 en p < 0.001 onderskeidelik). Die aantal CD34+ satelliet selle
(SS – rustend en geaktiveerd) het beduidend vroeër in die DSE groep gestyg in
vergelyking met die plasebo groep (4 uur vs. 7 dae na besering). Die totale
aantal SS (CD56+) het ook vroeër in die DSE-behandelde rotte as die plasebobehandelde
rotte gestyg (4 uur vs. 3 dae na besering), terwyl die aantal Mcadherin+
SS (rustend, geaktiveerd of prolifererend) betenisvol gestyg het in
beide groepe teen 4 uur (p < 0.001) na besering, maar hoër in die plasebo groep
was. Die aantal nuutgevormde spiervesels (met sentraal geleë nukleï en MHCf
+)
het beide vroeër verskyn en gepiek in die DSE-behandelde rotte in vergelyking
met die plasebo-behandelde rotte (dag 3 vs. dag 7 na besering).
Die resultate van hierdie studie dui aan dat ons instaat was om ‘n in vivo model
van nie-indringende kneusing in rotte op te stel. Verder, het ons ook bewys dat
Oxiprovin™ toediening die vermoë verleen het om: a) reaktiewe spesies wat na
beserings gevorm word, meer doeltreffend te verwyder en b) satelliet selle vroeër
te aktiveer en die vorming van nuwe skeletspiervesels te vervroeg, om sodoende
die herstel van skeletspier na ‘n gestandardiseerde kneusing vinniger te
bewerkstellig.
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Can the Sutherlandia herb or resistance exercise reverse the stress inducing effects of a mild-intermittent stress procedureNeethling, Ian Garth 03 1900 (has links)
Thesis (MSc (Physiological Sciences))--University of Stellenbosch, 2006. / This study aimed to assess the effect of mild psychological stress in male Wistar rats using incremental, intermittent stress on parameters of atrophy, including body mass, soleus and extensor digitorum longus (EDL) muscle mass, and mechanisms possibly contributing to atrophy. Serum corticosterone concentrations, 20s proteasome activity, glutamine synthetase (GS) and tyrosine amino-transferase (TAT) activities were determined. I also assessed whether Sutherlandia (Su) or resistance exercise was able to reverse the effects of stress on any of these parameters.
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Acute simulated hypoxia and ischemia in cultured C2C12 myotubes : decreased phosphatidylinositol 3-kinase (PI3K)/Akt activity and its consequences for cell survivalThomas, Mark Peter 12 1900 (has links)
Thesis (MSc (Physiological Sciences))--Stellenbosch University, 2008. / Cells are equipped with an array of adaptive mechanisms to contest the undesirable effects of
ischemia and the associated hypoxia. Indeed, many studies have suggested that there is an
increase in the PI3K/Akt pathway activation during hypoxia and ischemia. Damaged muscle can
be regenerated by recruiting myogenic satellite cells which undergo differentiation and
ultimately lead to the regeneration of myofibres. The C2C12 murine myogenic cell line is
popular for studying myogenesis in vitro, and has been used in many studies of ischemic
microenvironments. PI3K/Akt pathway activity is increased during C2C12 myogenesis and this
is known to produce an apoptosis resistant phenotype. In this study, we provide evidence that
high basal levels of PI3K activity exist in C2C12 myotubes on day ten post-differentiation.
Ischemia is characterized by depleted oxygen and other vital nutrients, and ischemic cell death is
believed to be associated with an increasingly harsh environment where pH levels decrease and
potassium levels increase. By employing a model that mimics these changes in skeletal muscle
culture, we show that both acute simulated ischemia and acute hypoxia cause decreases in
endogenous levels of the p85 and p110 subunits of PI3K and a consequent reduction in PI3K
activity. Supplementing skeletal muscle cultures with inhibitors of the PI3K pathway provides
evidence that the protective effect of PI3K/Akt is subsequently lost in these conditions. Using
Western blot analysis, a PI3K ELISA assay as well as known inhibitors of the PI3K pathway in
conjunction with the MTT assay we are able to demonstrate that the activation of downstream effectors of PI3K, including Akt, are concurrently decreased during acute simulated ischemia
and acute hypoxia in a manner that is independent of PDK-1 and PTEN and that the decreases in
the PI3K/Akt pathway activity produce a knock-on effect to the downstream signalling of
transcription factors, such as Fox01 and Fox04, in our model. We proceed to provide compelling
evidence that the apoptotic resistance of C2C12s is at least partially lost due to these decreases in
PI3K/Akt pathway activity, by showing increased caspase-3 and PARP cleavage. Then, using
vital staining techniques and a DNA fragmentation assay, we demonstrate increased cell
membrane impairment, cell death and apoptosis after three hours of simulated ischemia and
hypoxia in cultured C2C12 myotubes. In addition to the main findings, we produce evidence of
decreased flux through the mTOR pathway, by showing decreased Akt-dependant
phosphorylation at the level of TSC2 and mTOR during simulated ischemia and hypoxia.
Finally, we present preliminary findings indicating increased levels of HIF1α and REDD-1,
representing a possible oxygen sensing mechanism in our model. Therefore, we show that there
is in fact a rapid decrease in PI3K/Akt activity during severe, acute simulated ischemia and
hypoxia in C2C12 myotubes on day ten post-differentiation, and this causes a concomitant down
regulation in cell survival pathways and increased activity of cell death machinery. Thereafter,
we propose a possible mechanism of action and provide a platform for future studies.
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An investigation into the P13-K/AKT signalling pathway in TNF-a-induced muscle proeolysis in L6 myotubesSishi, Balindiwe J. N. 12 1900 (has links)
Thesis (MSc (Physiological Sciences))--Stellenbosch University, 2008. / Introduction: Skeletal muscle atrophy is a mitigating complication that is characterized by
a reduction in muscle fibre cross-sectional area as well as protein content, reduced force,
elevated fatigability and insulin resistance. It seems to be a highly ordered and regulated
process and signs of this condition are often seen in inflammatory conditions such as cancer,
AIDS, diabetes and chronic heart failure (CHF). It has long been understood that an
imbalance between protein degradation (increase) and protein synthesis (decrease) both
contribute to the overall loss of muscle protein. Although the triggers that cause atrophy are
different, the loss of muscle mass in each case involves a common phenomenon that induces
muscle proteolysis. It is becoming evident that interactions among known proteolytic systems
(ubiquitin-proteosome) are actively involved in muscle proteolysis during atrophy. Factors
such as TNF-α and ROS are elevated in a wide variety of chronic inflammatory diseases in
which skeletal muscle proteolysis presents a lethal threat. There is an increasing body of
evidence that implies TNF-α may play a critical role in skeletal muscle atrophy in a number of
clinical settings but the mechanisms mediating its effects are not completely understood. It is
also now apparent that the transcription factor NF-κB is a key intracellular signal transducer
in muscle catabolic conditions. This study investigated the various proposed signalling
pathways that are modulated by increasing levels of TNF-α in a skeletal muscle cell line, in
order to synthesize our current understanding of the molecular regulation of muscle atrophy.
Materials and Methods: L6 (rat skeletal muscle) cells were cultured under standard
conditions where after reaching ± 60-65% confluency levels, differentiation was induced for a
maximum of 8 days. During the last 2 days, myotubes were incubated with increasing
concentrations of recombinant TNF-α (1, 3, 6 and 10 ng/ml) for a period of 40 minutes, 24
and 48 hours. The effects of TNF-α on proliferation and cell viability were measured by MTT
assay and Trypan Blue exclusion technique. Morphological assessment of cell death was
conducted using the Hoechst 33342 and Propidium Iodide staining method. Detection of
apoptosis was assessed by DNA isolation and fragmentation assay. The HE stain was used for
the measurement of cell size. In order to determine the source and amount of ROS production,
MitoTracker Red CM-H2 X ROS was utilised. Ubiquitin expression was assessed by
immunohistochemistry. PI3-K activity was calculated by using an ELISA assay and the
expression of signalling proteins was analysed by Western Blotting using phospho-specific and total antibodies. Additionally, the antioxidant Oxiprovin was used to investigate the
quantity of ROS production in TNF-α-induced muscle atrophy.
Results and Discussion: Incubation of L6 myotubes with increasing concentrations of
recombinant TNF-α revealed that the lower concentrations of TNF-α used were not toxic to
the cells but data analysis of cell death showed that 10 ng/ml TNF-α induced apoptosis and
necrosis. Long-term treatment with TNF-α resulted in an increase in the upregulation of TNF-
α receptors, specifically TNF-R1. The transcription factors NF-κB and FKHR were rapidly
activated thus resulting in the induction of the ubiquitin-proteosome pathway. Activation of
this pathway produced significant increases in the expression of E3 ubiquitin ligases MuRF-1
and MAFbx. Muscle fibre diameter appeared to have decreased with increasing TNF-α
concentrations in part due to the suppressed activity of the PI3-K/Akt pathway as well as
significant reductions in differentiation markers. Western blot analysis also showed that
certain MAPKs are activated in response to TNF-α. No profound changes were observed with
ROS production. Finally, the use Oxiprovin significantly lowered cell viability and ROS
production. These findings suggest that TNF-α may elicit strong catabolic effects on L6
myotubes in a dose and time dependent manner.
Conclusion: These observations suggest that TNF-α might have beneficial effects in
skeletal muscle in certain circumstances. This beneficial effect however is limited by several
aspects which include the concentration of TNF-α, cell type, time of exposure, culture
conditions, state of the cell (disturbed or normal) and the cells stage of differentiation. The
effect of TNF-α can be positive or negative depending on the concentration and time points
analysed. This action is mediated by various signal transduction pathways that are thought to
cooperate with each other. More understanding of these pathways as well as their subsequent
upstream and downstream constituents is obligatory to clarify the central mechanism/s that
control physiological and pathophysiological effects of TNF-α in skeletal muscle.
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An integrative approach to the effect of interleukin-6 on adaptation to restraint stress in ratsViljoen, Monet 12 1900 (has links)
Thesis (MSc (Physiological Sciences))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Bi-directional communication exists between HPA-axis activation and interleukin-6
(IL-6). However, the relative contribution of centrally versus peripherally secreted IL-
6 remains unclear, especially under psychological stress conditions. We
hypothesised that the HPA response to mild psychological stress is dependent on IL-
6, both centrally and peripherally.
120 male Wistar rats were divided into four groups, depending on whether they
received an anti-IL-6 antibody (Ab) (2μg/ml/kg body weight) or a placebo (sterile
saline) injection and whether or not they were subjected to 1 hour of restraint stress
for 1, 2 or 3 days. Rats were euthanized 24 hours after stress exposure.
Plasma corticosteroid (GC) levels remained significantly increased 24 hours after a
single stress exposure (control placebo (CP) versus stress placebo (SP): p < 0.05).
The undetectable plasma IL-6 levels evident across all groups may be explained by
the short half-life of IL-6. Plasma IL-1β levels decreased when IL-6 was blocked in
unstressed animals (CP versus CAb: p < 0.05), suggesting a role for IL-6 in the
maintenance of IL-1β levels under tonic physiological conditions.
At tissue level, pituitary gland mass increased significantly at time point 2,
independently of stress when blocking IL-6 (CAb: p < 0.05). This suggests that when
normal homeostasis is threatened, immediate adaption or at least compensation
may occur. It was observed that GR, IL-1β, IL-1βR, IL-6, IL-6R and GABAARα1
showed no response to stress alone in the pituitary. It is therefore more likely that
resistance to adaptation exists centrally. IL-1β and IL-1βR (p < 0.05) and
GABAARα1 (p < 0.005) expression increased in the CAb group in the pituitary, again
suggesting a role for IL-6 under control conditions. In terms of the adrenal, blocking IL-6 resulted in decreased glandular mass at time point 1, independent of stress
(CAb and SAb: p < 0.005). The up-regulation in GR expression seen in CAb and
SAb (p < 0.05) may be the effect of a compensatory mechanism to increase IL-6
dependent bioactivity of GCs. The fact that expression of IL-6, IL-6R, IL-1β and IL-
1βR consistently increased in the Ab groups, and mostly in the zona fasciculata and
zona reticularis, suggests that lack of local direct negative cytokine feedback
occurred in response to very low plasma IL-6 levels and that this contributes more
than GCs in the down-regulation of inflammatory cytokine release.
In conclusion, consistent effects of the Ab were apparent in the tissues investigated,
even in control conditions, suggesting that IL-6 plays a role in the maintenance of
basal homeostasis, including its regulation of the response to psychological stress.
We found differential regulation in terms of cytokines and GCs when comparing
peripheral versus central effects of stress and Ab, as well as the levels of cytokines
in the blood compartment, compared to within tissues. / AFRIKAANSE OPSOMMING: Daar bestaan twee-rigting kommunikasie tussen HPA-as aktivering en interleukin-6
(IL-6), allhoewel die relatiewe bydrae van sentraal versus perifeer afgeskeide IL-6
nog onduidelik is, veral gedurende sielkundige strestoestande. Ons hipotese is dat
die HPA reaksie tot sielkundige stres afhanklik van IL-6 is, beide sentraal en in die
periferie.
120 manlike Wistar rotte is in vier groepe verdeel, afhangende van of hulle ‘n anti-IL-
6 teenliggaampie (Ab) (2μg/ml/kg liggaamsgewig) of ‘n plasebo (steriele
soutoplossing) inspuiting gekry het, en of hulle onderworpe was aan 1 uur van
vaskeer-stres vir 1, 2 of 3 dae. Rotte is 24 uur na blootstelling aan stres aan
genadedood onderwerp.
Bloed kortikosteroïed (GC) vlakke het beduidend toegeneem binne 24 uur na ‘n
eenmalige stres blootstelling (kontrole plasebo (CP) versus stres plasebo (SP): p <
0.05). Die onmeetbaar lae vlakke van IL-6 regoor al die groepe, kan verduidelik
word na aanleiding van die kort half-leeftyd van IL-6. Bloed IL-1β vlakke het
afgeneem in kontrole rotte wanneer IL-6 geblok is (CP versus CAb: p < 0.05). Dit kan
beteken dat IL-6 noodsaaklik is vir die onderhoud van IL-1β vlakke gedurende
basale toestande.
Op weefselvlak het die hipofise massa toegeneem by tydpunt 2 toe IL-6 geblok is,
onafhanklik van stres (CAb: p < 0.05). Dit dui aan dat wanneer normale homeostase
bedreig word, daar onmiddelike aanpassing of kompensasie plaasvind. Dit is
opvallend dat GR, IL-1β, IL-1βR, IL-6, IL-6R en GABAARα1 geen respons in terme
van stres alleen in die hipofise getoon het nie. Na aanleiding daarvan is dit meer
waarskynlik dat weerstand tot aanpassing sentraal bestaan. IL-1β and IL-1βR (p <0.05) en GABAARα1 (p < 0.005) uitdrukking in die hipofise het toegeneem in die CAb
groep, wat weereens ‘n rol vir IL-6 onder kontrole toestande uitwys. In terme van die
bynier, het die blok van IL-6 ‘n afname in massa veroorsaak by tydpunt 1, wat weer
onafhanklik van stres was (CAb en SAb: p < 0.005). Die opregulering in die CAb en
SAb groepe (p < 0.05), kan wees as gevolg van ‘n kompensasie meganisme om IL-6
afhanklike GC aktiwiteit te verhoog. Die feit dat die uitdrukking van IL-6, IL-6R, IL-1β
and IL-1βR in die Ab groepe deurlopend verhoog was, en meeste in die zona
fasciculata en zona reticularis, stel voor dat daar ‘n tekort aan plaaslike, direkte
sitokien negatiewe terugvoering was, as gevolg van die merkwaardige lae bloed IL-6
vlakke en dat dit meer bydra as GCs in die afregulering van inflammatoriese sitokien
vrystelling.
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The role of MKP-1 in autophagy, apoptosis and necrosis during ischaemia/reperfusion injury in the heartVermeulen, Michelle 12 1900 (has links)
Thesis MSc (Physiological Sciences))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Ischaemic heart disease is a leading cause of death worldwide and is also
largely contributing to deaths in Africa. Better treatment or even prevention of
ischaemia/reperfusion injury in the heart, necessitates a better understanding
of the molecular pathways and mechanisms of cell death. Three types of cell
death can occur in the diseased myocardium. Type I, better known as
apoptotic cell death, is characterised by cell shrinkage and chromatin
condensation, type II, known as autophagic cell death, is characterised by
intracellular accumulation of double membranes vacuoles and type III,
necrotic cell death, is characterised by cellular swelling and loss of
membrane integrity. Many signaling pathways are activated during
ischaemia/reperfusion injury which include the mitogen activated protein
kinases (MAPKs), such as extracellular signal-regulated protein kinase
(ERK), c-Jun NH2-terminal protein kinase (JNK) and p38 MAPK. These
kinases are dephosphorylated by appropriate phosphatases. MAPK
phosphatase-1 (MKP-1), a dual specificity phosphatase, inactivates the
MAPKs by dephosphorylating specific Thr/Tyr residues. Upregulation of
MKP-1 during ischaemia/reperfusion injury has been shown to be
cardioprotective, however no knowledge regarding a role of MKP-1 in
autophagy exists. Therefore the aim of this study is to investigate the role of
MKP-1 in autophagy, apoptosis and necrosis during simulated
ischaemia/reperfusion injury in the heart.METHOD: H9C2 cells (rat cardiomyocytes) were cultured under standard conditions.
Upon reaching 75-80% confluency, cells were treated for 30 min during
normoxic conditions with dexamethasone, to induce MKP-1 expression, or
sanguinarine, to inhibit MKP-1 induction. Thereafter, they were exposed to 3
hrs simulated ischaemia (induced by an ischaemic buffer and 5% CO2/1%
O2) in the presence of the above mentioned treatments. Cells were then
allowed to reperfuse for 30 min in the presence of dexamethasone or
sanguinarine. Samples were analysed after simulated ischaemia and after
reperfusion. Cell viability was measured by MTT assay. Propidium iodide and
Hoechst staining were used to assess morphological markers of apoptosis
and necrosis. LDH release during reperfusion was assessed as indicator of
necrotic cell death. LysoTracker®Red was used to visualise the autophagic
flux occurring during ischaemia/reperfusion in the cell. Flow cytometry was
used to quantify cells stained with acridine orange as indicator for autophagy.
Autophagic and apoptotic protein markers as well as MAPK and MKP-1
activity were analysed by Western Blotting. RESULTS: Our results indicate a clear relationship between MKP-1 induction,
autophagy and cell survival during simulated ischaemia/reperfusion (SI/R).
MKP-1 inhibition during SI/R resulted in decreased autophagy activity
accompanied by significant apoptotic and necrotic cell death. Increased MKP-1 induction, on the other hand, during SI/R resulted in increased levels
of autophagy activity and subsequent attenuation of apoptotic and necrotic
cell death. p38 MAPK phosphorylation was significantly higher while MKP-1
was inhibited and significantly lower while MKP-1 was induced. This strongly
indicates that upregulation of MKP-1, known to attenuate
ischaemia/reperfusion injury, has an important role in cell survival during
ischaemia/reperfusion injury in the heart, through its involvement in the
regulation of autophagic activity as a stress response against apoptotic or
necrotic cell death. / AFRIKAANSE OPSOMMING: Iskemiese hartsiekte is een van die grootste oorsake van sterftes wêreldwyd
en dra ook beduidend by tot sterftes in Afrika. Om iskemiese hartsiektes te
behandel of selfs te voorkom, is 'n goeie begrip van die molekulêre paaie wat
betrokke is tydens iskemie/herperfusie, noodsaaklik. Drie tipes seldood kom
tydens patologiese toestande in die hart voor. Tipe I, ook bekend as
apoptotiese seldood, word gekenmerk deur selkrimping en kromatien
kondensasie, tipe II, ook bekend as autofagiese seldood word gekenmerk
deur intrasellulêre opeenhoping van dubbelmembraan vakuole en tipe III,
bekend as nekrotiese seldood, word deur sellulêre swelling en verlies van
membraan integriteit gekenmerk. Iskemie/herperfusie lei tot die aktivering
van seintransduksiepaaie wat die MAPKs, soos p38, ERK en JNK insluit.
Hierdie kinases word deur die gepaste fosfatases gedefosforileer. MKP-1, 'n
dubbele spesifieke fosfatase, deaktiveer MAPKs deur hul Thr/Tyr eenhede te
defosforileer. Alhoewel daar al voorheen getoon is dat verhoogte MKP-1 ‘n
beskermende funksie in die hart tydens iskemie/herperfusie het, is daar nog
geen bewyse vir ‘n rol van MKP-1 tydens autofagie nie. Die doel van hierdie
studie is dus om die rol van MKP-1 in autofagie, apoptose en nekrose te
ondersoek tydens gesimuleerde iskemie/herperfusie in die hart. METODE: H9C2 selle (rot ventrikulêre hartselle) is onder standaard toestande
gekweek. Wanneer die selle 75-80% konfluensie bereik het, is dit behandel
met dexamethasone of sanguinarine onder standaard toestande vir 30 min.
Daarna is selle blootgestel aan 3 ure iskemie, in die teenwoordigheid van
dexamethasone of sanguinarine. Selle is dan toegelaat om vir 30 min te
herperfuseer, weer in die teenwoordigheid van dexamethasone of
sanguinarine. Monsters is na iskemie en herperfusie geneem vir analise.
Selvatbaarheid is gekwantifiseer deur ‘n MTT bepaling. Morfologiese
merkers van seldood is bepaal met behulp van propidium iodide en Hoechst
kleuringsmetodes. Laktaatdehidrogenase (LDH) vrystelling tydens
herperfusie is as merker van nekrose gebruik. Autofagie is gevisualiseer
deur gebruik te maak van LysoTracker®Red kleuring tydens iskemie en
herperfusie. Akridienoranje is gebruik om suur kompartemente te kleur.
Vloeisitometrie is as kwantifiseringstegniek vir autofagie gebruik. Western
Blotting is gebruik om uitdrukking van merkerproteïene van autofagie en
apoptose sowel as MAPK en MKP-1 aktiwiteit tydens iskemie/reperfisie te
bepaal. RESULTATE: Ons resultate toon ‘n verband tussen MKP-1 induksie, autofagie en
seloorlewing gedurende gesimuleerde iskemie/herperfusie (SI/R) aan. MKP-
1 inhibisie gedurende SI/R het tot ‘n afname in autofagie gelei tesame met ‘n beduidende toename in apoptotiese en nekrotiese seldood. Verhoogde
MKP-1 induksie gedurende SI/R, daarteenoor, het autofagiese aktiwiteit
verhoog, gepaardgaande met ‘n verlaging in apoptose en nekrose. p38
MAPK fosforilasie was beduidend hoër tydens MKP-1 inhibisie en laer met
MKP-1 induksie. Hierdie resultate toon dat MKP-1 ‘n belangrike rol in
seloorlewing speel tydens iskemie/herperfusiesskade in die hart, deur sy
deelname in die regulering van autofagiese aktiwiteit as ‘n stres reaksie teen
apoptotiese en nekrotiese seldood.
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Cytokine super-families affect adult stem cells : IL-6 and the skeletal muscle nicheSteyn, Paul 03 1900 (has links)
Thesis (MSc (Physiological Sciences))--University of Stellenbosch, 2011. / Includes bibliography. / ENGLISH ABSTRACT: Background: IL-6 belongs to a cytokine super-family known to affect cell proliferation,
although other family members are better characterized. Proliferation promoting
factors (IL-6) compete with differentiation promoting factors (myogenic regulatory
factors: MyoD and myogenin) to affect cell cycle. Cell cycle progression is assessed by
determining the proportion of cells shifting from arrest to chromatin synthesis and
mitosis phases (G0/G1 and S and G2/M respectively).
Methods: This study assessed the effects of IL-6 on cell cycle progression and
proliferation vs. differentiation of C2C12 skeletal myoblasts. Physiological doses (10 or
100 pg/ml) were compared to a high dose (10 ng/ml), with exposure lasting 48 hours
(addition of IL-6 dose to proliferation medium at 0 and 24 hours). Acute signaling
downstream of the IL-6 gp130 receptor was assessed after the first exposure.
Results: Propidium iodide analysis of nuclear material using flow cytometry indicated
shifts in forward scatter. Both Low and Medium doses shifted a greater proportion
(p<0.05) of cells from G0/G1 to S and G2M phases at 24 hours and all doses resulted
in the same shift (p<0.05) at the 48 hour time point. However, the High dose
significantly (p<0.05) increased myogenin expression at the 48 hour time point.
Microscopy indicated that confluence was prevented by low seeding density and did
not influence the result. Cells harvested at 5 minutes post stimulation indicated that
all doses significantly increased STAT3 phosphorylation. 10 minutes post stimulation
the High dose group sustained elevated levels of STAT3 phosphorylation.
Conclusions: Low and medium doses of IL-6 increase proliferation in a muscle
satellite cell line by activating cell division and allowing myoblasts to remain in the
active cell cycle. High doses of IL-6 increase differentiation by mediating upregulation
of myogenic regulatory factors and this is thought to be due to prolonged STAT3
activation. Physiological control of myoblast behaviour by cytokines is evident and
such control would be influenced by the severity of the endogenous cytokine
response to various stimuli. / AFRIKAANSE OPSOMMING: Agtergrond: IL-6 behoort aan n sitokien super-familie bekend vir die affektering van
sel verspreiding, alhoewel ander familie lede beter gekenmerk is. Bevordering van
verspreiding faktore (IL-6) kompeteer met bevordering van differensiasie fatore
(myogenic regulatory factors: MyoD en myogenin) om die sel siklus te affekteer. Sel
siklus progressie word geassesseer deur die bepaling van die proporsie selle wat
verskuif van arrestasie na chromatien sintese en mitose fases (G0/G1 en S en G2/M
onderskeidelik).
Metodes: Hierdie studie het die effekte van IL-6 op die progressie van die sel siklus
geassesseer asook die proliferasie vs. differensiasie van C2C12 skelet spier satelliet
selle. Fisiologiese dosisse (10 en 100 pg/ml) was vergelyk tot n hoog dose (10 ng/ml),
met blywende blootstelling van 48 uur (byvoeging van IL-6 dose tot verspreidings
medium op 0 and 24 uur). Akute sein stroomaf van die IL-6 gp130 reseptor was ook
geassesseer na die eerste blootstelling.
Resultate: Propidium iodide analise van kern materiaal deur vloei sitometrie het
voorwaarts verskuiwing aangedui. Beide Laag and Medium doses het n groter
proporsie (p<0.05) selle verskuif van die G0/G1 tot die S en G2M fases na 24 uur en
alle dosisse het gelei in die selfde verskuiwing (p<0.05) by die 48 huur tyd punt.
Alhoewel die Hoog dose myogenin uitdrukking aansienlik (p<0.05) verhoog het na 48
uur. Mikroskopie het aangedui dat samevloeiing voorkom was deur n lae loting
digtheid en dit het nie resultate geaffekteer nie. Selle wat geoes was 5 minute na
stimulasie het aangedui dat alle dosisse STAT3 fosforilasie laat toeneem het. 10
minute na stimulasie het die Hoog dose groep volgehoue vlakke van STAT3 fosforilasie
besit.
Gevolgtrekkings: Laag en Medium dosisse van IL-6 verhoog verspreiding in n spier
satelliet sel lyn deur die aktivering van sel deling en deur selle toe te laat om in die
aktiewe sel siklus te bly. Hoog dosisse van IL-6 verhoog differensiase deur
bemiddelende opstoot van myogenic regulatory factors en die gedagte is dat dit
bewerkstellig word deur aanhoudende aktivering van STAT3. Fisiologies beheer van
satelliet selle deur sitokiene is duidelik en die beheer sal beinvloed word deur die erns
van die endogene sitokien reaksie op verskillende stimuli.
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Hypothalamic-pituitary-adrenal-axis vs. the sympatho-adrenal medullary system in the acute response to psychological stressJanse van Vuuren, Marthinus 03 1900 (has links)
Thesis (MSc (Physiological Sciences))--University of Stellenbosch, 2011. / Includes bibliography. / ENGLISH ABSTRACT: The hypothalamic-pituitary-adrenal-(HPA) axis has long been closely associated with
psychological stress-induced activation of the adrenal cortex and subsequent glucocorticoid
production. Another, less known peripheral limb of the psychological stress response, is the
sympatho adrenal medullary pathway.
We hypothesized that the sympatho-adrenal medullary system constitutes the primary response
to acute psychological stress, with the HPA-axis functioning as a secondary response. We tested
our hypothesis by manipulating a model of acute mild psychological stress (restraint) by
blocking IL-6, a valuable constituent of the sympatho-adrenal medullary system.
Serum corticosterone concentration increased in response to stress (7 ± 3 vs. 57 ± 4 ng/ml;
P<0.0001), a response attenuated when IL-6 was blocked (17 ± 7 ng/ml). Stress increased
pituitary mass only when IL-6 was blocked (38 ± 3 vs. 65 ± 6 mg; P <0.001). Stress increased
left adrenal mass only in the presence of IL-6 (34 ± 1 vs. 73 ± 8 mg; P <0.00001). Stress did not
influence the circulating levels of TNF-α, IL-1β or IL-6 significantly. IL-1β and TNF-α
concentrations in the unstressed rats were lower when IL-6 was blocked.
We then manipulated the stress model by administering S. frutescens extract to elucidate both the
central and peripheral effects of acute S. frutescens administration on the psychological stress
response.
Restraint caused decreases in hippocampal GR levels when compared to respective controls. S.
frutescens administration and exposure to restraint synergistically decreased hippocampal
GABAAR levels. In addition, exposure to both stress and S. frutescens led to a noteworthy
increase in pituitary mass (P = 0.078), as well as pituitary ACTH levels (P < 0.01). Similarly,
differences in circulating ACTH levels showed an effect of stress on ACTH secretion only in the
presence S. frutescens (P < 0.05). Adrenal mass was significantly increased in S. frutescenstreated
animals that were also exposed to restraint (P < 0.05). Adrenal levels of ACTH showed a
reciprocal trend to pituitary and circulating ACTH levels. No statistically significant differences
were seen in adrenal IL-6 content. However, marked increases in IL-6 levels were seen at this
level with administration of S. frutescens stress exposure and a cumulative increase seen with
both S. frutescens-treatment and stress exposure.
Hippocampal GABAAR, pituitary mass, pituitary ACTH and circulating ACTH levels showed a
similar trend towards a synergistic effect of S. frutescens and restraint in activation of the
psychological stress response, while adrenal ACTH levels showed an inverse trend.
Hippocampal GR did not show any effect of stress or S. frutescens-treatment.
The results from these two experiments indicate that the sympatho-adrenal medullary system
constitutes the primary response to acute mild psychological stress and that the HPA-axis is only
activated during an exacerbated stress response or when the sympatho-adrenal medullary
contribution is inadequate. Furthermore, the acute administration of S. frutescens possibly led to
a functional shift in GABAergic function, resulting in activation of the stress response. The
anecdotal reports of a “docile” effect of S. frutescens most likely results from activation of the
mesolimbic dopaminergic system by the hippocampus and amygdala. These results have
dramatic consequence in GABA-based anxiety-treatments. / AFRIKAANSE OPSOMMING: Die hipotalamo-pituïtêre-adrenale (HPA)-as is lank bekend as ‘n primêre rolspeler in die respons
op emosionele stres en daaropvolgende glukokortikoïed produksie. ‘n Ander, minder bekende
arm van die sielkundige stres respons is die simpatiese bynier-medulla-sisteem.
Ons hipotese was dat die laasgenoemde simpatiese bynier-medulla-sisteem die primêre respons
tot sielkundige stres behartig terwyl die HPA-as ‘n sekondêre respons bied. Ons het ons hipotese
getoets deur die manupilering van ‘n beproefde stres model waar ons IL-6, ‘n waardevolle
rolspeler in die simpatiese bynier-medulla-sisteem, onderdruk het.
In respons op stress, het serum kortikosteroon konsentrasies toegeneem slegs in die
teenwoordigheid van IL-6 (7 ± 3 vs. 57 ± 4 ng/ml; P<0.0001), maar nie wanneer IL-6 onderdruk
is nie (17 ± 7 ng/ml). Stres het ‘n verhoging in hipofise massa teweeggebring slegs tydens die
onderdrukking van IL-6 (38 ± 3 vs. 65 ± 6 mg; P <0.001). Stres het ook linker-byniermassa
verhoog slegs wanneer voldoende IL-6 beskikbaar was (34 ± 1 vs. 73 ± 8 mg; P <0.00001).
Stres alleen het geen invloed gehad op serum IL-1β, IL-6 of TNF-α nie, maar die onderdrukking
van IL-6 het wel ‘n inhiberende effek op basale IL-1β en TNF-α gehad.
Daarna het ons weer eens die stresmodel manipuleer deur die rotte ‘n S. frutescens ekstrak te gee
in ‘n poging om beide die sentrale en perifere effekte daarvan op die sielkundige stres respons te
evalueer.
Stres alleen het gelei tot ‘n afname in GR terwyl ‘n kombinasie van stres en S. frutescens
administrasie tot ‘n afname in GABAARα1 in die hippokampus gelei het. Hierdie kombinasie
het ook tot ‘n merkwaardige toename in hipofise massa (P = 0.078) sowel as ACTH-inhoud van
die hipofise (P < 0.01) gelei. ‘n Soortgelyke patroon is waargeneem betreffende sirkulerende
ACTH en byniermassa met P < 0.05 vir elk. Bynier ACTH inhoud, aan die ander kant, het ‘n
omgekeerd eweredige verhouding met ACTH in die hipofise en in sirkulasie getoon. Bynier IL-
6 inhoud het geen statisties beduidende verskille getoon nie, maar ‘n merkwaardige verhoging is
weereens gesien met ‘n kombinasie van stres en S. frutescens administrasie.
Die soortgelyke tendens wat waargeneem word in GABAAR in die hippokampus, asook
hipofise- en sirkulerende ACTH vlakke, en dui op ‘n samewerkende rol van stres en S. frutescens
in die aktivering van die sielkundige stres respons. GR in die hippokampus toon geen
veranderinge nie. Die resultate van die twee eksperimente dui op ‘n primêre rol van die
simpatiese bynier-medulla-sisteem in die respons op ‘n akute stressor en dat die HPA-as net
geaktiveer word tydens ‘n ooreiste stres reaksie of indien die simpatiese bynier-medulla-sisteem
onderdruk word. Die waargenome “verdowings”-effek van S. frutescens word moontlik deur
aktivering van die mesolimbiese dopamien pad deur die hippokampus en amigdala bewerkstellig.
Die resultate mag ook lei tot die heroorweging van GABA-gebaseerde angs medikasies.
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