Doctoral Degrees (Physiology)

Permanent URI for this collectionhttps://hdl.handle.net/10413/7045

Browse

Now showing 1 - 20 of 41

Glucocorticosteroid receptor characteristics of peripheral blood mononuclear cells in oral steroid dependent asthma : utilization of an in vitro model of steroid resistant asthma to investigate mechanisms of resistance and functional consequences of altered receptor affinity.
(2007) Irusen, Elvis Malcolm.
Background: Although glucocorticoids are the most effective treatment for asthma, some patients show a poor response. In such patients with steroid resistant asthma, this has been ascribed to altered glucocorticoid receptor (GR) ligand-binding affinity induced by IL-2 combined with IL-4 or IL-13 alone- all of which can also modulate glucocorticoid function in vitro. Objective: We sought to assess the ligand-binding affinity in a distinct group of oral steroid-dependent asthmatic subjects and examine the mechanisms by which IL-2 and IL-4 (or IL-13) modify the ligand-binding affinity of the GR. Methods: Using dexamethasone-binding assays, we examined PBMCs ex vivo from healthy subjects, subjects with controlled asthma, and oral steroiddependent subjects with severe asthma. In addition, IL-2 and IL-4 were used to alter GR affinity in vitro. We used mediators or inhibitors of signal transduction to investigate the mechanisms of resistance. We also determined cytokine production of PBMC's by means of ELISA. Results: GR ligand-binding affinity was significantly reduced in the nucleus but not in the cytoplasm of oral steroid-dependent asthmatic subjects compared with that seen in steroid-sensitive and healthy subjects (dissociation constant, 41.37 ± 17.83 vs. 25.36 ± 2.63 nmol/L vs. 9.40 ± 4.01 nmol/L, respectively [p<.05 for both in comparison to normals] ). This difference in ligand-binding affinity could be mimicked by IL-2 and IL-4 co-treatment and was blocked by the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580. PBMC's rendered resistant in vitro demonstrated lower IL-10 and increased GM-CSF production following LPS or PMA & PHA stimulation compared to cells with normal GR affinity. Resistant cells also showed reduced dexamethasone repression of LPSstimulated IL-10 release. These effects were also reversed by SB203580. Inhibition of the ERK MAPK pathway by PD098059 (10 mol/L), phosphoinositol 3 kinase by wortmannin (5 nmol/L) or treatment with IL-10 (10 ng/mL) failed to modulate the effect of IL-2 and IL-4 on receptor affinity. Ro318220 (10 nmol/L), a specific protein kinase C inhibitor and theophylline, similarly, had no effect on affinity. Conclusion: GR ligand binding affinity is tiered; compared to normal subjects; steroid responsive asthmatics have a mild reduction in ligand binding whereas oral steroid dependent asthmatics have greater reductions. When mononuclear cells are rendered resistant in vitro, cytokine production (low IL-10 and high GM-CSF) favours a pro-inflammatory state. Our data do not support the ERK MAPK, phosphoinositol 3 kinase, protein kinase C pathways in steroid resistance. Treatment with IL-10 and theophylline also failed to modulate the effect of IL-2 and IL-4 on receptor affinity. However, P38 MAPK inhibitors may have potential in reversing glucocorticoid insensitivity and re-establishing the beneficial effects of glucocorticoids in patients with severe asthma.
A study of diabetes mellitus in young Africans and Indians (age of onset under 35) in Natal.
(1982) Mahomed, Abdool Khalek Omar.
No abstract available.
The case for critical thought: an investigation into contemporary determinist knowledge, its social effects, and the alternative offered by a 'mode 2' approach to teaching, learning and research.
(2002) Skinner, Jane.
This thesis is centrally concerned with the current nee-liberal world order and its effects upon society. It is concerned to expose the contradictions and weaknesses within the knowledge systems that underpin our political reality. It considers economics as the determining discourse of neo-liberal politics, analytic biology as its determining discourse of individual persons, and analytic and neo-pragmatist philosophy as its leading systems of thought. In each case it finds a linear rationalism compatible with the determinist materialism of noo-Darwinism, and indeed explicitly invoking Darwin. This seems to vindicate Manuel Castells's fmding of this 'Knowledge Society' as driven by 'an abstract, universal instrumentalism'. The thought systems of this economic liberalism have seen politics subsumed within economics, de-humanising most of the institutions of the earlier Liberal tradition, to the detriment of both freedom and democracy. But it disputes Castells's assumption that this is a necessary reality and finds in neo-liberal education the exception to this dehumanising trend. Revitalised as 'Mode 2' knowledge production, this form of teaching, learning and research is found to be ideally suited to challenge the underpinnings of the very social order which initially produced it. The thesis as a whole is designed to employ Mode 2 methods in order to support this contention. Using this approach it seeks to demonstrate that in place of neo-Darwinism the ideas of the South African natural scientist Eugene Marais, concerning the significance of conscious thought itself within evolution, can provide a more convincing epistemoloy than the behaviourism and materialism of analytic biology. It finds John Maynard Keynes's acceptance of economics as a moral and not a natural science, more logically convincing and more inherently useful for social reconstruction than the current mathematicisation of economic theory. Prevalent philosophical approaches appear to serve only to reinforce the systems of thought already found (and found wanting) in politics, biology and economics. But again these philosophies are shown to be vulnerable to a Mode 2 critique, particularly employing the ontological understanding of the contemporary pragmatist philosopher Joseph Margolis, whose strong version of relativism allows for both bivalent and multivalent truth values more appropriate to understanding the complex realities of ethical and democratic societies.
The role of angiotensin ll and oxidative stress in the spontaneously hypertensive rat.
(2011) Govender, Melvin M.; Nadar, Anand.
Oxidative stress resulting from an imbalance between free radicals and antioxidants is considered to be an important etiological factor in the development and maintenance of hypertension. Angiotensin II (Ang II) has been shown to be an important regulator of blood pressure and acts to elevate blood pressure by its pressor effects. The pressor effects of Ang II are well documented but recent evidence has suggested another possible role of Ang II in elevating blood pressure, whereby it acts via an independent mechanism that is directly linked with oxidative stress. The spontaneously hypertensive rat (SHR) is a widely used model in the investigation of the pathophysiological mechanisms involved in hypertension. This study was therefore undertaken to determine whether Ang II acts as a causative factor via oxidative stress in the development and maintenance of hypertension in the SHR. This was elucidated by evaluating the role of both the endogenous in vivo levels of Ang II as well as an exogenous sub-pressor dose of Ang II, on oxidative stress and its associated parameters. The parameters evaluated included, the antioxidant status of the model on the basis of the levels of the major antioxidant enzymes viz. SOD, GPx and catalase; the free radical generating capacity, by assessing the activity of the membrane bound enzyme NADPH oxidase and the levels of H2O2. The study also evaluated the levels of the endogenous vasodilator nitric oxide (NO), remodelling of the vasculature and the level of tissue oxidative stress in the kidney. The results show that the SHR has an elevated plasma Ang II level and an elevated level of oxidative stress, thus showing that in this model there is an intimate link between oxidative stress and Ang II. The SHR also shows depleted levels of NO and thus a decreased vasodilatory capacity and increased remodelling of the vasculature. The kidney showed an increased level of lipid peroxidation, which was due to the elevated levels of oxidative stress. All of these pathophysiological changes contribute to the elevation in blood pressure in this model. The long term infusion of the sub-pressor dose of Ang II affected the SHR to a greater extent than the Wistar. Although the dose of Ang II elevated the blood pressure in both models, the degree of the pathophysiological changes associated with the elevation in blood pressure was greater in the SHR. The Ang II infusion in both these models demonstrated that in the SHR which is genetically predisposed to hypertension, adjustments are made to the antioxidant system, that result in an elevated level of protection against oxidative stress. These results show that Ang II acts as a causative factor in the pathogenesis of hypertension in the SHR via its well documented pressor effects, as well as via a multitude of independent mechanisms that are linked to oxidative stress. This is substantiated by the significant decrease in NO that is caused by the elevated oxidative stress, as well as the previously described pathophysiological changes. This study has therefore shown that Ang II has an intimate causative link with oxidative stress that results in parallel mechanisms that work concomitantly with each other in hypertension in this model.
Chemical investigation of isihlambezo or traditional pregnancy-related medicines.
(2004) Brookes, Kathleen Bridget.; Dutton, Michael Francis.
This study was undertaken to redress the scant knowledge regarding the chemistry and mode of action of pregnancy-related traditional medicines, or isihlambezo (Zulu), which are used by 60 to 80% of women in South Africa. The three selected plants are among the six most frequently cited species from the approximately 90 used by traditional healers. The purpose of the study was to identify components which could cause uterine contractions, those with nutritional value for the foetus and mother, and those with any toxic effects. Plant root extracts were purified via silica gel column chromatography and bioassays were carried out on the fractions, using isolated rat uterine tissue. Purified compounds were identified via spectral techniques, and some were characterised by comparison to authentic standards using HPLC, and others by matching their GC-MS spectra to library standards. Thirty-eight compounds were identified in total, the majority of these being novel to the species concerned. Those isolated from Combretum kraussii were 1 sitosterol, 2 combretastatin, 3 3',4-tri-O-methylellagic acid, 4 combretastatin B-1, 5 combretastatin A-1, 6 3,3'-di-O-ellagic acid lactone, 7a ellagic acid lactone, 7b ellagic acid, 8 and 9 a mixture of combretastatin B-1 and A-1 glucosides, 10 and 11 partly characterised glucosides of ellagic acid. Those isolated from Gunnera perpensa were 12 3',4-tri-methylellagic acid, 13 ellagic acid lactone, 14 1,1'-biphenyl-4,4'-diacetic acid, 15 p-hydroxybenzaldehyde, 16 Z-methyl lespedezate, 17 and 18 partly characterized higher glucosides of Z-methyllespedezate. Those isolated rom Rhoicissus tridentata were 19 (-)-epigallocatechin, 20 (+)-gallocatechin, 21 procyanidin B3, 22 procyanidin B4, 23 (+)-catechin hydrate, 24 (+)-mollisacacidin, 25 (+)-epicatechin, 26 fisetinidol-(4a-8) catechin, 27 (-)-fisetinidol, 28 fisetinidol-(4b-8)catechin, 29 gallic acid, 30 epicatechin-3-0-gallate, 31 partly characterized hydrogel of glucose, 32 sitosterol, 33 sitosterolin, 34 y-sitosterol, 35 oleanolic acid, 36 lupen-3-one, 37 20-epi-y-taraxastananol and 38 triacontanol. The compounds with the greatest in vitro uteroactivity were predominantly proanthocyanidins or phenolic glucosides. It is proposed that effects of phenolic glucosides could be due to the interaction of the sugar moiety as well as the phenolic moiety with the receptor site in muscle tissue. The corresponding phenolic aglycones isolated were only moderately uterotonic, or unreactive by comparison. Non-polar compounds such as sitosterol and sitosterolin showed minimal enhancement of the uterine response at low concentrations, and inhibition at higher concentrations. Aqueous root extracts of the plants were all found to be non-toxic according to cell-viability tests using monkey vero cells and human fibroblasts. Extracts are therefore considered safe for human consumption, although it is recommended that Rhoicissus tridentata be used with caution because it showed the lowest cell viability of the three species, and uterine hyperstimulation has been attributed to this species, as well as CNS depression and respiratory arrest. Ions which could be nutritionally beneficial in pregnancy, calcium, iron, and phospate, were present in low in aqueous extracts. Levels of calcium and potassium ions were considered to be too low to directly stimulate uterine muscle. Proanthocyanidins, combretastatins, ellagic acid derivatives and phytosterols, with health-promoting properties, were also identified.
The possible implication of selected Fusarium Mycotoxins in the aetiology of brain cancer.
(2004) Palanee, Thesla.; Chuturgoon, Anil Amichund.
The central nervous system is a potential site of action for the Fusarium mycotoxin Fumonisin B1 (FB1), and is exemplified in horses by the disease equine leukoencephalomalacia. Structurally resembling sphingoid bases, FB1 inhibits ceramide synthase, an enzyme involved in sphingolipid metabolism, leading to accumulation of free sphinganine (Sa) and sphingosine (So). This investigation focused on FB1, Sa, So and the Fusarium mycotoxins fusaric acid (FA), moniliformin (MaN), zearalenone (ZEA), deoxynivalenol (DON), and T-2 toxin (T2). Effects of the Fusarium mycotoxins and sphingoid bases on the N2a neuroblastoma cell line were assessed using the methylthiazol tetrazolium (MIT) and ApoGlow™ assays. The MIT assay revealed significant differences between the viability of N28 control cells and the cytotoxic effects of FB1 (p=0.001), So (p=1.1 x10-6 ), Sa (p=1.9x10-6 ), MON (p=0.002), DON (p=0.04) and ZEA (p=0.003) on N28 cells between 5-250µM. The cytotoxic effects of FA did not differ significantly from controls (p=0.1). The ApoGlow™ assay revealed that in N28 cells, FB1 at 8µg.ml-1, FA at 128µg.ml-1, and (FBI+FA) combined induced growth arrest at 2 and 4µg·ml-1. Assessment of the effects of FBI and FA on the Jurkat leukaemic suspension cell line revealed that FB1 induced apoptosis at 1.56,12.5 and 50µ.ml-1, growth arrest at 100, 200 and 800µg.ml-1 and proliferation at 400µg.mg-1. Fusaric acid induced proliferation at 1. 56µg.ml-1, apoptosis at 3.15µg.mrl, growth arrest at 100 and 200µg.mrl, and necrosis at 800µg.ml-1. Combined, (FB1+FA) induced apoptosis at 1.56, 3.15,12.5 and 800µg.ml-1. Flow cytometry and fluorescence microscopy revealed that mycotoxins, Sa and So induced varying levels of apoptosis and necrosis in N28 cells. Acridine orange and ethidium bromide staining facilitated discrimination between viable, apoptotic and necrotic cells. Transition of the mitochondrial transmembrane potential was measured using Rhodamine 123 with propidium iodide, and the dual emission potential sensitive stain JC-1. Changes in mitochondrial membrane potential and plasma membrane integrity were expressed as increases or decreases in fluorescence intensity. An increase in mycotoxin concentration from 50 to 200µM was usually paralleled by a decrease in J-aggregate formation, suggesting a decrease in the ?¦¥m. Staining with Rh 123/PI indicated at specific concentrations whether N28 cells were either late apoptotic or necrotic reflected by the levels of PI uptake. No dose dependant mechanism of cell death was established using either method, as fluctuations were evident. Immunolocalisation of T2, ZEA and FB1 within cellular organelles that exhibited ultrastructural pathology provided correlation between mycotoxin exposure and effects. Multinucleate giant cells and retraction of cellular processes were observed. At the electron microscope (EM) level, FB1 was immunolocalised within microsegregated and peripherally condensed nucleoli, the nucleoplasm, distorted mitochondria and dilated endoplasmic reticulum (ER). The capacity of cells to incorporate mycotoxins and effect cytological changes represents a major factor in the potential for initiation of malignant transformation. Exposure of N2a cells to FB1 for 72 hours increased intracellular free Sa and depleted complex sphingolipids. Using High Performance Liquid chromatography (HPLC), acid hydrolysis revealed reduction in Sa from a level of O.6±0.12µM in control cells, to 02±0.lµM in cells exposed to 50µM and lOOµM FB1. Base hydrolyses revealed increase in free Sa: So ratios from 0.52±0.2 in control cells, to 1.14±0.2 and 1.4±0.3 in cells exposed to 50 and l00µM FB1 respectively. The Sa: So ratio in the complete culture media (CCM) increased from 1. 7±0. 3 for control cells to 2.0±0.2 and 2.50±0.4 for cells exposed to 50 and lOOµM FB1 respectively. Correlation coefficients between Sa: So ratios to FB1 exposure in CCM (R=0.75) and within cells (R=0.85), imply that the free Sa: So ratio within cells appears to be a better biomarker for FB1-induced disruption of sphingolipid metabolism in vitro, than the Sa: So ratio in CCM. Optimisation of HPLC analytical procedures improved recovery of FB I from spiked human sera to 95.8% (n=15) and detection limits to -5ng.ml-1 at a signal to noise ratio of 5:1. Optimisation of methods for recovery of Sa and So from spiked sera, led to recoveries of 77.9% and 85.0%, for So and Sa respectively at levels of spiking with lOng per 500µl of serum. Matched sera Sa:So ratios and FB1 levels in brain cancer and non-cancer subjects in KwaZulu-Natal were determined using these optimised methods. Fumonisin B1 was detected in sera of non-cancer (76.7±62.2nM) and brain cancer subjects (l07.38±116nM). Mean serum Sa:So ratios of 21 non-cancer subjects was 1.7±0.7. There was no correlation (R=0.26) between these variables in non-cancer subjects. The mean serum FB1 level in brain cancer subjects was 107.4±116nM (range 10.5-298nM) (n=50) and the mean Sa:So ratio (n=50) was 1.9±1.7 (range 0.40-8.16). No correlation was found between these variables in the brain cancer subjects either (R = -0.23). Fumonisin B1 was irnmunolocalised in 49 of 76 brain tumour tissue samples analysed using immunohistochemistry (IHC). Thirty-eight of the 76 specimens had matched serum FBI levels and Sa: So ratios, and 23 of these were positive for FB1 presence. Although not significantly different (p=0.ll), the FBI sera levels in the cancer group with FBI within the tumour tissue had higher levels of FB1 in sera than the IHC FB1 negative group. Fumonisin B1 was localised within irregular profiles of nuclei, elongated and swollen mitochondria and ER. Immunolocalisation of FB1 within organelles in the brain showing ultrastructural cellular pathology suggests FBI may be implicated in the aetiology of human brain carcinogenesis.
The synthesis of xanthone derivatives and their enzymatic conversion and inhibition of aflatoxin biosynthesis.
(1996) Gengan, Robert Moonsamy.; Mulholland, Dulcie Aca.; Dutton, Michael Francis.
The biosynthesis of Aflatoxin B1 (AFB1) has been the subject of conflicting speculation and numerous reviews. The currently accepted scheme for the aflatoxin pathway is based on data obtained from feeding studies using isotopically labelled precursors. In these studies the conversion of possible intermediate metabolites to AFBl by mutants of Aspergillus parasiticus illustrated their role as biogenetic precursors. Currently there is now agreement on the identity of most of the intermediate Illetabolites involved in the biosynthesis of AFB1. However, there is a lack of clarity on the details of AFB1 biosynthesis including the conversion of sterigmatocystin (ST) to AFB1 via the metabolite O-methylsterigmatocystin (OMST). There is no clear cut evidence of the metabolic role of OMST, i.e., either it is a compulsory intermediate or a shunt metabolite and hence part of a metabolic grid. In order to investigate this step in AFBl biosynthesis, ST was isolated from surface cultures of A. versicolor (M1101) and purified by silica gel column chromatography and repeated recrystallisation. Sterigmatocystin was characterised by thin layer chromatography (t.1.c.), low resolution mass spectrometry (M.S) and nuclear magnetic resonance spectroscopy (N.M.R). A series of seven derivatives of the free hydroxyl group of ST were synthesised by known chemical reactions, purified by silica gel column chromatography and characterised by high resolution mass spectrometry and proton nuclear magnetic resonance spectroscopy. A high pressure liquid chromatography (HPLC) method was developed using a fluorescence detector. The optimum parameters for the separation of the four major aflatoxins, namely AFBl, AFB2, AFGl and AFG2, using trifluoroacetic acid as the derivatising reagent, were obtained for a reversed phase Prodigy C18 column with a mobile phase of water: acetonitrile: isopropanol: acetic acid (8: 1: 0.5: 0.5, v/v). Feeding studies, using whole cells of A. parasiticus (WhI-11-105), showed that ST and the ST derivatives were converted to AFB1. A time courser study for the conversion of ST and selected ST derivatives to AFB1 indicated a decrease in the rate of conversion in the order: a-propyl sterigmatocystin (OPROST) > a-ethyl sterigmatocystin > a-methylsterigmatocystin > Sterigmatocystin> a-benzoyl sterigmatocystin (OBzST). It was apparent that the "enzyme" responsible for the conversion of the derivatives to AFB1 did not display a high degree of substrate specificity, since it was unable to recognize the difference between the various alkyl groups, either as ether or ester functional groups. An HPLC method was developed using a diode array detector. The optimum parameters for the separation of aflatoxin metabolites and the synthesised derivatives were obtained for a reversed phase Lichrosphere RP-I8 column with a 30 minute gradient elution program with water and acetonitrile as the mobile phase. Crude cell-free extracts were prepared by lyophilisation of the mycelia of A. parasiticus (Whl-11l-105) with phosphate buffer. The temperature and pH for the conversion of ST to AFB1, were found to be optimum at 28°C and 7.2, respectively. The addition of SAM (1.5 mM) and NADPH (1.5 mM) increased the conversion of ST to AFBl from 11.21 % to 27.10 %. A time course study with ST, OMST and OPROST showed that the rate of conversion to AFBl was close to linear for an incubation time of up to 60 minutes. Approximation of the reaction rate indicated a decrease in the order: OMST > ST > OPROST. This indicated that the time course reaction using whole cells was in part a measure of membrane permeability rather than substrate specificity. Molecular exclusion chromatography was used to separate enzymatic protein from primary and secondary metabolites, small biomolecules and indigenous co-factors (MW < 10 000) and the partially purified "enzyme" was concentrated by dialysis against solid sucrose. The "enzyme" was subjected to non-denaturing polyacrylamide gel electrophoresis and was found to be made of sub-units ranging from 58 kDa to over 200 kDa. Enzymatic investigations with ST, as substrate, indicated that OMST is a compulsory intermediate in the biosynthesis of AFBl. Also, enzymatic investigations of selected ST derivatives showed that the partially purified "enzyme" displayed relative specificity for these substrates, viz., OMST, OPROST and OBzST. Three xanthones, namely, 1-hydroxy-,6-dimethylxanthone, I-methoxy-3,6-dimethylxanthone and l-acetyl-3,6-dimethylxanthone were synthesised, purified and characterised spectroscopically. Whole cell studies of A. parasiticus (CMI 91019b) and A. parasiticus (Wh1-11-105) showed that these xanthones inhibited AFBl production to varying extents. Kinetic studies of cell-free extracts revealed that the 1-methoxy-3,6-dimethylxanthone derivative was a non-competitive inhibitor. The Michaelis Menten constant (Km) of approximately 5.60 uM (for OMST) was determined for a cell-free reaction at pH 7.2 and 28 QC. A Clark oxygen electrode was used to carry out oxygen consumption studies in a partially purified "enzyme" preparation. A calibration system was designed and the enzymatic conversion of OMST to AFB1 and NADPH consumption were monitored by HPLC and UV spectroscopy, respectively. From the results of these enzymatic reactions, the following stoichiometric relationship was determined: 2 mole oxygen consumed = 1 mole NADPH consumed = 1 mole AFB1 produced A tentative mechanism is discussed for the conversion of OMST to AFB1 which utilizes a monooxygenase and a dioxygenase.
Endotoxaemia in intestinal dysfunction in experimental animals : intestinal ischaemia and hyperthermia.
(1988) Gathiram, Premjith.; Gaffin, Stephen L.
Endotoxins or lipopolysaccharides (LPS), highly toxic component of the outer membrane of gram-negative bacteria, are normally present in the mammalian gut lumen.In this thesis, I investigated, in laboratory animals, whether these gut-derived endotoxins play a role in pathophysiology resulting from intestinal dysfunctions caused by intestinal ischaemia and heat-stress.In primates, reperfusion of the splanchnic region after a temporary ischaemia was followed by a rapid increase in LPS concentration, first in the hepatic portal plasma and, ten minutes later, in the systemic arterial plasma. Rises in plasma LPS concentrations during or following the temporary intestinal ischaemia was prevented by prophylactic administrations of corticosteroids, anti-LPS IgG antibodies and oral, non-absorpable, antibiotics agents which appear to stabilize cellular membranes, aid the reticuloendothelial system in removal of LPS from the circulation and destroy the intestinal aerobic gramnegative bacteria respectively. In addition, administration of therapeutic anti-LPS antibodies also rapidly reduced the plasma LPS concentrations to baseline during an endotoxaemia. In a control heat-stress model, elevations in plasma LPS concentration commenced at rectal temperatures greater than 41,SoC. Like the intestinal ischaemia model, this occurred first in the hepatic portal plasma, and 10-15 minutes later, in the systemic arterial plasma. Peak plasma LPS levels of about 0,3 ng/ml, measured in heat-stressed primates, have proved in previous studies, to be toxic. A rapid decline in mean arterial pressure was followed by increases in plasma LPS concentrations and heart rates. Reductions in splanchnic blood flow and consequent local ischaemia coupled with thermal injury to the intestinal wall and the liver, may have permitted rises in plasma LPS concentration. Furthermore, as in the ischaemia model, prophylactic administrations of corticosteroids, anti-LPS IgG antibodies, and oral, nonabsorbable antibiotics prevented a rise in plasma LPS concentration. Of importance, prophylaxis with intravenous corticosteroids and 'anti-LPS IgG antibodies increased the survival rates significantly in heat stroke in primates. In addition, monkeys having high titres of "natural" antiLPS IgG antibodies had lower plasma LPS concentrations and survived the induced-heat stroke. It is suggested that other pathophysiologic conditions which compromise the integrity of the gut wall would also lead to the development of an endotoxaemia, and that gutderived endotoxins contribute to the athogenesis of heat stroke and treatments with corticosteroids and anti-LPS IgG antibodies may prove beneficial in other endotoxinrelated disorders.
The value of morphological analysis in duodenal ulcer therapy
(1994) Gregory, Michael Alfred.
This study was designed to examine two premises: that the morphological "severity" of duodenal ulcers (DU) may influence the incidence of drug mediated healing and the morphological "quality" of healing after curative therapy may influence t he duration of remission. Biopsies taken at endoscopy from five healthy volunteers and from 84 patients suffering from DU were examined by light and electron microscopy. The endoscopic and morphological appearance of the mucosa within 8mm of the DU or scar, before and up to 1 year after therapy with either sucralfate, cimetidine, pirenzipine or misoprostol are described. Irrespective of the mode of therapy or whether the biopsies were from normal, juxta-DU or scar mucosa, specimens could be divided into 2 primary morphological classes: gastric metaplastic and non-metaplastic. Based on the degree of metaplastic differentiation and nonmetaplastic degeneration, these classes were further divided into 4 sub-classes. When correlated with the incidence of healing and duration of remission, metaplasia was generally found to be a positive and degenerative nonmetaplasia a negative prognostic criterion. Scores were awarded to primary morphological criteria and weighted to give high total s to favourable (metaplastic) and low totals to non-favourable (degenerative non-metaplastic) prognostic features. The sum of scores expressed as a percentage was termed the morphological index. This proved useful as a means of correlating mucosal morphology with DU healing and duration of remission. It also facilitated comparison of morphology within and between groups of patients before and after each drug regimen. The results showed that the morphological appearance of the ulcerative mucosa influenced healing and remission outcome. Discriminant analysis was applied to the numeric data that described the juxta-DU (group 1) and scar (group 2) morphology of patients treated with cimetidine in 2 studies. Separation between healed and not healed DU was achieved in 92% of group 1 and 100% (remission - more or less than 6 months) of group 2. When applied to the juxta-DU data from patients treated with cimetidine in a third study, the formulae predicted correctly in 88% of cases. In addition to predicting outcome, the formulae were used as standards to accommodate for natural variations in the prognosis of individual DU of patients enrolled for comparative drug studies. These data show that morphological analysis may be usefully employed in duodenal ulcer therapy.
The effect of sildenafil citrate and kraussianone-2 on pre-eclampsia-like manifestations in Sprague-Dawley rats.
(2011) Ramesar, Shamal Vinesh.; Mackraj, Irene.; Moodley, Jagidesa.; Gathiram, Premjith.
Pre-eclampsia, often described as toxaemia of pregnancy, historically represents one of the most widely investigated conditions relating to human reproduction. To date no firm cure has been found and a clear, well defined mechanism has not been ascribed to the pathogenesis of the disease. Researchers seem to focus on single pathways in isolation of others. The disease rather represents a multitude of possible underlying pathologies nvolving genetics, immune dysregulation, vascular maladaptation, and sociobiological factors thus complicating the approach to treatment. However, a central theme is the presence of reduced placental perfusion resulting in a hypoxic and/or ischaemic placenta and the subsequent secretion of various factors that initiate the maternal syndrome. It is within this context that we examine how an intervention such as increasing placental perfusion may represent a promising treatment strategy for this disease. We sought to manipulate the vasodilatory mechanisms of the uterine vasculature using sildenafil citrate and a flavonoid extracted from Eriosema kraussianum (Kr2), in Sprague-Dawley rats that exhibited preeclampsia-like manifestations. Both treatment regimens improved fetal outcomes and reduced blood pressure amplification and proteinuria. They also reduced the plasma concentrations of the two anti-angiogenic factors; sFlt1 and sEng. Only sildenafil citrate improved nitric oxide levels which was expected, suggesting that Kr2 causes vasodilation by some other mechanism. Nevertheless, both compounds improved both pup and placental weights, suggesting that they also improve utero-placental perfusion. These findings that selective uterine vascular dilation improves placental perfusion may be promising in averting possible death to mothers and their babies from pre-eclampsia especially in low resource environments.
Immunohistochemical and ultrastructural evaluation of the pathology and aetiopathogenesis of keloid formation.
(2013) Bux, Shamin.; Madaree, Anil.
Introduction Keloids are formed by the excessive production of scar tissue, which extends beyond the margins of the original injury, often resulting in lesions of grotesque dimensions. Keloids present a major dilemma to surgeons because of the high recurrence rate with recurrent growth often larger than the original keloid. The high recurrence rate and the poor response of keloids to therapy present a great challenge to surgeons. The numerous therapeutic regimens demonstrate that to date there is no single therapy that is absolutely successful. Therefore, it is necessary to comprehensively establish the pathology of keloids and to determine the aetiopathogenesis of the lesion in order to eventually provide unfailing specific effective treatment and to better understand the mechanisms regulating fibrosis in various fibroproliferative diseases. Aim To evaluate the pathology and aetiopathogenesis of keloid formation. Methods The research protocol for the study was approved by the Nelson R Mandela Faculty of Medicine Ethics Committee. Informed consent was obtained before the biopsies were taken. Keloid and non-lesional skin biopsies were obtained from thirty two patients who had multiple lesions in various locations, bringing the total number of keloids and apparently normal skin biopsies processed and examined to fifty eight. The biopsied specimens were processed for paraffin wax embedment and routine haematoxylin and eosin, differential and immunocytochemical staining. Sections were scrupulously examined using the Olympus BH-2 microscope; features pertinent to the study were photographed with the Olympus DP 10 microscope digital camera system. The stored images were studied, using the Camedia graphics processing programme. Results The results of the study showed that keloids comprise many distinct regions categorized as: the zone of hyalinising collagen bundles, fine fibrous areas, areas of inflammation, zone of dense regular connective tissue, nodular fibrous area and area of angiogenesis. Fibroblastic phenotypes present ranged from spindle, fibrohistiocytic, epitheloid, elongated flattened condensed fibroblastic cells to few wavy, fuzzy, polygonal and atrophic cell types. Immunocytochemically these cells were vimentin-positive and actin- and desmin-negative. Few myofibroblastic phenotypes were also identified and these were vimentin- and alpha smooth muscle actin-positive and desmin-negative. The fibroblastic and myofibroblastic phenotypes were in proliferative or degenerative stages and pathological features exhibited were the presence of vesicular, degenerate or calcified nuclei; nuclear and plasma membrane damage; cytoplasmic and nucleoplasmic clearing; atrophy, pyknosis and swelling. Severe, moderate to mild paravascular inflammation was observed around the microvessels of the sub-papillary plexus and within the keloid. There was compression and occlusion of small blood vessels, coagulation necrosis and dissolution of mural cells of small blood vessels and small peripheral nerves. Also present in keloids were oedematous areas, disorganised and hyalinised connective tissue fibres and increased numbers of degranulated and degranulating mast cells. Elastic fibres in keloids were minimal or absent whereas at the border of keloids there was an increase.Discussion Degenerate, occluded and compressed microvessels were a widespread pathological feature in keloids. This resulted in impaired vascular supply to each of the keloid regions which impacted directly on the pathology of keloids where degeneration and necrosis, manifesting the lack of nutrients and oxygen to tissue, were found throughout the keloid. The vascular supply was impaired because of the chronic inflammatory destruction of the microvessels and the elevated stress within keloids. Factors contributing to increased intrinsic stress were: 1) the lack of elastic fibres in keloids which decreased the elastic limit, leading to effects of excessive deformational force which were compression and stiffening of tissue; 2) the high tension skin covering keloid prone areas had low stretch and a low elastic modulus; 3). protruding hard connective tissue such as bony prominences or cartilage into the dermis of keloid prone skin; 4) contractile forces exerted by wound healing fibroblastic cells; and 5) external forces. Compression and occlusion of blood vessels induced ischaemic and reperfusion tissue injury. During the reperfusion phase blood rich in growth factors returned to tissue stimulating tissue growth. Tissue growth was also promoted by elevated internal stress which stimulated increasing levels of gene expression, collagen synthesis and mitotic activity. All these growth promoting effects resulted in keloid formation.
Hypoglycaemic effects of some medicinal plant extracts in streptozotocin-induced diabetic rats.
(2007) Mavuto, Masopera Gondwe.; Musabayane, Cephas Tagumirwa.; Ojewole, John Akanni Oluwole.; Shode, Francis Oluwole.
Abstract available in PDF file.
The role of antioxidants in atherogenesis and salt-sensitive hypertension.
(2003) Nadar, Anand.; Somova, Liliana I.
Abstract available in PDF file.
The effects of insulin and Syzygium aromaticum-derived oleanolic acid containing dermal patches on kidney function and renal expression of glucose transporters in streptozotocin-induced diabetic rats.
(2014) Ngubane, Phikelelani Siphosethu.; Musabayane, Cephas Tagumirwa.
Introduction The tight glycaemic control required to attenuate chronic complications in type 1 diabetes mellitus requires multiple daily injections of bolus insulin which have been reported to be associated with Na+ retention resulting in hyperinsulinaemic oedema and hypertension. Current research on insulin delivery methods include buccal, oral, nasal, and transdermal delivery systems. Transdermal delivery system is of great interest as this offers sustained controlled release of insulin into the systemic circulation. We have previously reported that transdermal application of pectin hydrogel insulin (PI) matrix patches sustain controlled insulin delivery into the bloodstream of STZ-induced diabetic rats to perhaps ameliorate diabetic complications. Since we have previously reported that STZ-induced diabetic rats retain Na+ following hypotonic saline challenge, this study investigated whether insulin-containing dermal patches can avert and improve the impaired renal fluid and electrolyte handling of STZ-induced diabetic rats. We have also shown that oral administration of OA in addition to possessing hypoglycaemic effects, improves kidney function STZ-induced diabetic rats. The study therefore also investigated whether OA-containing dermal patches can improve kidney function STZ-induced diabetic rats. Materials and methods Pectin insulin (PI)-containing dermal patches of various doses (3.99, 9.57, 16.80 μg/kg) and pectin oleanolic acid (P-OA) containing dermal patches of various doses (21, 42, 84 mg/kg) were prepared by dissolving pectin/insulin or pectin/OA in deionized water and solidified with CaCl2. Short-term (5 weeks) effects on renal function of thrice daily treatments with PI and P-OA patches 8 hours apart were assessed in diabetic animals. Rats sham treated with the pectin drug free patch and insulin (175 μg/kg sc) acted as negative and positive controls, respectively. Daily urine volume, urinary glucose, Na+, K+ and creatinine excretion rates were monitored over 5-weeks. Blood was collected 6 h following treatments for insulin determination. Blood and kidney samples were also collected after 5 weeks for hormonal analysis and measurement of selected biochemical parameters. Results Untreated STZ-induced diabetic rats exhibited elevated weekly urinary glucose, K+ outputs and depressed urinary Na+ outputs throughout the 5-week compared to non-diabetic control animals. Application of PI-containing dermal patches significantly increased urinary Na+ output and reduced urine volume and urinary outputs of glucose and K+ in weeks 4 and 5. Plasma AVP concentrations of untreated STZ-induced diabetic rats were significantly low at end of the 5-week experimental period by comparison with control non-diabetic animals while plasma aldosterone levels were significantly elevated. The highest dose of the insulin-containing dermal patch (16.80 μg/kg) significantly (p < 0.05) elevated plasma AVP concentrations while decreasing plasma aldosterone concentrations of STZ-induced rats by comparison to untreated STZ-diabetic rats. GFR of untreated STZ-induced diabetic rats was significantly decreased while plasma creatinine concentrations were significantly elevated by comparison to non-diabetic control animals. PI containing dermal patches increased GFR of STZ-induced diabetic rats with a concomitant reduction of plasma creatinine concentrations by comparison to untreated STZ-induced diabetic rats. Interestingly, P-OA dermal patches also increased GFR of STZ-induced diabetic rats while reducing plasma creatinine concentrations. The effects of both PI and P-OA containing dermal patch compared with subcutaneous insulin. Significant increase of MDA and decreases of SOD and GPx were found in the skin, kidney and heart tissues of STZ-diabetic animals as compared to non-diabetic control animals. PI (16.80 μg/kg) -treated STZ-induced diabetic animals however showed low concentrations of MDA and increased the activities of SOD and GPx in the skin, kidney and heart tissues compared to untreated STZ-induced diabetic animals. P-OA-treated STZ-induced diabetic animals similarly and significantly showed decreased MDA, and increased activity of antioxidant enzymes; SOD and GPx in skin, kidney and heart tissues. H and E kidney stained sections of untreated non-diabetic control, untreated STZ-induced diabetic rats and diabetic animals topically applied with insulin and OA-containing dermal patches were observed under light microscope. However, STZ-induced diabetic rats showed thickened basement membrane of the Bowmans capsule, thickened glomerular basement membrane and hypercellularity of the proximal tubules by comparison to the non-diabetic animals after 5 weeks of the study. Treatment with insulin containing dermal patches and subcutaneous insulin for 5 weeks however attenuated these features when compared with the untreated STZ-diabetic rats. Like PI dermal patches, OA containing dermal patches also ameliorated structural changes of kidney of STZ-induced diabetic rats. The increased urinary glucose concentrations of the untreated STZ-induced diabetic rats were associated with increased expression of GLUT 1 and SGLT 1 to normalcy by comparison to nondiabetic rats. The highest dose of PI containing dermal patch however, like subcutaneous insulin, significantly decreased the expressions of GLUT 1 and SGLT 1 by comparison to STZ-induced diabetic controls. Plasma insulin concentrations of untreated STZ-induced diabetic rats were significantly low in comparison with control non-diabetic animals. Acute (6 h) and short-term (5 weeks) daily application of PI containing dermal patches to STZ induced diabetic rats significantly elevated plasma insulin concentrations by comparison with untreated diabetic animals. However, the plasma insulin concentrations in animals treated with the high insulin doses (9.57, 16.80 μg/kg) were significantly higher than those found in diabetic groups treated with the low insulin dose (3.99 μg/kg). There were no differences in the plasma insulin concentrations in STZ-induced diabetic animals treated with P-OA containing dermal patches by comparison to STZ- diabetic untreated controls both acutely and chronically. To determine whether insulin was transported across skin of STZ-induced diabetic rats following topical application of PI and P-OA containing dermal patches, we also monitored the density of phosphorylated insulin receptor substrates (IRS) in the skin by immunohistochemical staining with specific insulin receptor antibodies. Non-diabetic treated skin sections showed slight immunostaining of insulin receptors in comparison STZ-induced diabetic rats which stained negative. Immunohistochemical staining for phosphorylated IRS in the skin of animals following application of insulin and sc insulin treatment for 5 weeks clearly demonstrated widespread localization of IRS in cell bodies of the dermis, collagen and subcutaneous layer. Interestingly, OA-containing dermal patches also showed widespread localization of IRS in cell bodies of the dermis, collagen and subcutaneous layer. H and E skin sections of untreated non-diabetic control, untreated STZ-induced diabetic rats and diabetic animals topically applied insulin and OA-containing dermal patches showed no significant histological differences in dermis compared to the untreated non diabetic control skin sections. Discussion Previous studies indicate compromised renal function in experimental diabetes and diabetic patients. The results herein however indicate that insulin containing dermal patches increase Na+ excretion probably by decreasing plasma aldosterone and increasing plasma AVP concentrations of STZ-induced diabetic rats. PI containing dermal patches also improve kidney function by increasing GFR with concomitant reduction of plasma creatinine concentrations. Like PI containing dermal patches, P-OA containing dermal patches increased Na+ excretion by decreasing plasma aldosterone and increasing plasma AVP concentrations of STZ-induced diabetic rats. P-OA containing dermal patches also increased GFR and reduced plasma creatinine concentrations of STZ-induced diabetic rats. Conclusion From these results, we conclude that PI and P-OA dermal patches deliver physiological amounts that can improve kidney function in diabetes.
Mechanisms of cardiovascular effects of oleanolic acid and related synthetic oleanane derivatives : an experimental study.
(2014) Madlala, Hlengiwe Pretty.; Musabayane, Cephas Tagumirwa.; Van Heerden, Fanie Retief.; Mubagwa, K.
Introduction Despite the various conventional treatments that are available to treat hypertension, this disease continues to be globally responsible for approximately 9.4 million deaths each year. The high mortality can partly be attributed to side effects of available drugs or to the inaccessibility of current synthetic drugs to communities from poor socioeconomic background because of their relative high cost. This problem has resulted in a growing interest in the use of medicinal plant products because they are considered to be cheap, believed to possess few side effects and are easily accessible to the general population in developing countries. Although traditional herbal remedies are widely used in Africa for the management of various disorders including cardiovascular diseases, very little reliable data is available on their therapeutic and pharmacological effects. In search for plants with therapeutic properties for the treatment of hypertension and complications, our laboratory has scientifically evaluated several plant species. In particular, we have isolated Syzygium spp-derived triterpenes and focused on the therapeutic effects of oleanolic acid (OA) and maslinic acid (MA). In the present study, we investigated the effects of Syzygium aromaticum-derived OA and related synthetic derivatives on arterial pressure and evaluated the underlying mechanisms in Wistar, spontaneously hypertensive rats (SHR) and Dahl salt-sensitive (DSS) animals. Materials and methods OA was extracted from dried flower buds of Syzygium aromaticum using a previously validated protocol in our laboratory. Oleanolic acid methyl ester (Me-OA) and a brominated derivative of OA (Br-OA) were synthesised according to methods described by Fu and Gribble. The structures of extracted OA and synthesised derivatives were confirmed using 1H and 13C nuclear magnetic resonance (NMR) spectroscopy and were comparable to previously reported data. Acute renal clearance studies investigated the influence of OA and derivatives on mean arterial pressure (MAP) and Na+ handling in the proximal tubule of anaesthetised Wistar rats using lithium clearance. Animals were given water with lithium (12 mmol L-1) for 48 hours following which they were anaesthetised and cannulated using a previously validated standard protocol that has been reported from our laboratories. After a 3.5 h equilibration, animals were challenged with hypotonic saline for 4 h of 1 h control, 1.5 h treatment and 1.5 h recovery periods. OA, Me-OA and Br-OA were added to the infusate during the treatment period followed by measurements of arterial pressure, fluid and electrolyte handling. Sub-chronic study experiments were restricted to OA because of the low amounts of synthetic derivatives obtained during the synthetic studies to prepare the derivatives. Various doses of OA (30, 60, 120 mg kg-1, p.o.) were administered to separate groups of male Wistar, SHR and DSS rats twice (8 h apart) every third day for nine weeks. Rats given dimethyl sulphoxide (DMSO)-saline (3 mL kg-1, p.o.) acted as untreated controls. Mean arterial pressure (MAP) was monitored every third consecutive day using non-invasive tail cuff method with photoelectric sensors. Measurements of body weight, food and water intake, Na+, K+, Cl-, urea and creatinine were taken 24 h after dosing. At the end of this 9 week study, the cardiac, renal and hepatic tissues harvested from hypertensive animals were evaluated for oxidative status by measuring malonyldialdehyde (MDA, lipid peroxidation marker) and antioxidant enzymes; superoxide dismutase (SOD) and glutathione peroxidase (GPx). In addition, we evaluated the effects of OA and derivatives on aldosterone and arginine vasopressin (AVP) secretion on plasma samples from both sub-chronic and acute experimental settings. Additional Ex vivo studies to unravel the mechanisms of action of OA and derivatives were carried out in isolated ventricular cardiomyocytes and arteries. Measurements of cell shortening and Ca2+ currents were done in cells isolated from Wistar and DSS animals using edge detection and whole cell patch clamp techniques, respectively. Isometric tension measurements were done in endothelium-intact and denuded aortic rings and mesenteric arteries of Wistar and DSS rats. The influence of indomethacin (INDO), N-nitro-L-arginine methyl ester (L-NAME), glibenclamide (Gli) and 4-aminopyridine (AP) on the effects of OA and derivatives was investigated. Results The purity of the plant-derived OA was approximately 98% and the percentage yield varied from 0.79% to 1.72% of the dry plant material. The percentage yield of the synthetic derivatives, Me-OA and Br-OA was 65% and 30%, respectively, from the starting materials. Our results show that OA decreased MAP in both acute and sub-chronic experimental settings, and that the MAP lowering effect was more marked in hypertensive animals compared to normotensive rats. Similarly, the two OA derivatives, used in acute settings, also decreased MAP. OA increased urinary Na+ excretion rate under acute and sub-chronic conditions. A similar but quantitatively more marked increase was obtained with Br-OA derivative under acute conditions. Untreated hypertensive animals had elevated levels of plasma aldosterone and AVP in comparison to Wistar rats. OA treatment significantly reduced aldosterone secretion in these animals but had no influence on plasma concentrations of AVP. Compared with respective control rats, OA-treated animals exhibited significantly lower MDA levels and increased activity of the antioxidant enzymes, SOD and GPx in hepatic, cardiac and renal tissues. OA and derivatives had a positive inotropic effect on isolated ventricular cardiomyocytes in Wistar rats and had no influence on the contraction of cells from hypertensive animals. OA and derivatives caused relaxation in aortic rings and mesenteric arteries of both Wistar and DSS animals. This effect was partly inhibited by INDO and K+ channel blockers when used independently. Addition of L-NAME did not further inhibit the cyclooxygenase (COX)-resistant relaxation. The combination of INDO, Gli and AP completely abolished OA or derivatives-evoked relaxation in endothelium-intact arteries indicating involvement of both endothelium-dependent and independent mechanisms. Discussion The present study investigated the effects of OA and related synthetic derivatives (Me-OA and Br-OA) on blood pressure and thereafter examined the possible underlying mechanisms. The absolute stereo-structure of S. aromaticum-derived OA and synthetic derivatives elucidated from the spectra using 1H- and 13C-NMR was comparable to previously reported data. Our results confirm the previously reported antihypertensive properties of OA in experimental models of hypertension. However, the present study demonstrates a more marked action in SHR and DSS by comparison with non-hypertensive animals, suggesting a specifically enhanced action in disease conditions. We noticed that urinary Na+ excretion in control, untreated animals tended to spontaneously increase with time during the following 9 weeks post weanling. In both hypertensive models, no such increase with time was obtained, instead, urinary Na+ excretion tended to decrease with time after weanling in the DSS model supporting Na+ retention previously reported in these animals. To further support this theory we found increased aldosterone levels in the plasma of non-treated hypertensive rats. Our data show that OA decreased aldosterone secretion and increased urine Na+ excretion, relatively larger increases were obtained in the hypertensive models. This suggests that treatment with the drugs was accompanied by alleviation of Na+ retention in these animals. This increase in urinary Na+ output is, at least in part, mediated via inhibition of proximal tubular Na+ reabsorptions as indicated by increased Li+ clearance. Indeed, we found a positive correlation between the increase in urinary Na+ excretion rate and the decrease in MAP. However, despite the potent natriuretic effects of these triterpenes mediated by decreased aldosterone levels, the urine flow rate was not changed as supported by unchanged levels of AVP. Our experiments indicated that OA induces vasorelaxation of aortic rings and small mesenteric arteries in both normotensive and hypertensive animals. The maximum relaxation evoked by the derivatives, particularly Br-OA, was significantly larger in the mesenteric vessels although these differences were not observed in the aorta. Glibenclamide and 4-aminopyridine used allowed a complete blockade of the relaxation to OA, Br-OA and Me-OA suggesting that ATP-dependent and voltage-activated K+ channels opening mediate the endothelium-independent relaxation. Our experiments using isolated cardiomyocytes showed that OA and derivatives do not decrease but rather tend to increase myocyte shortening and had no influence on Ca2+ currents in Wistar rats. Increase cardiomyocyte contractility implies an increase in the force of cardiac tissue contraction and increase cardiac output which may be ideal for a drug which causes natriuresis. The results showed that OA-treated DSS animals had normal levels of MDA, SOD and GPx in comparison to untreated hypertensive rats. Therefore, we suggest that OA-evoked decreased reactive oxygen species (ROS) and increased antioxidant enzymes may have enhanced the production of prostanoids, thereby improving vasodilation. Hence, we speculate that antioxidant properties of OA could play a role in hypotensive mechanisms of this triterpene. Conclusion The results of this study introduce the first evidence that OA and its oleanane derivatives induce similar effects. These involve 1) increased urinary Na+ output mediated by inhibition of proximal and distal tubular Na+ reabsorption as indicated by increased Li+ clearance and decreased aldosterone levels, respectively. 2) Modulation of oxidative status in cardiac, renal and hepatic tissues in hypertensive animals. As well as 3) decreased vascular resistance via endothelium-dependent COX/prostanoids pathway and endothelium-independent opening of ATP-dependent and voltage-activated K+ channels. The results of this study are novel and clinically relevant because OA and related triterpene derivatives exert multiple blood pressure lowering mechanisms while increasing the force of cardiac contraction hence balancing the fluid volume in the circulatory system so as to avoid a state of hypotension. Limitations and direction for future studies Due to low amounts obtained for the synthetic derivatives, we were unable to study their sub-chronic effects in conscious animals, therefore this should be explored in future. OA effects on renal function seem to mimic oxytocin-like activities i.e. increase Na+ output without changing the urine flow rate, hence future studies should explore whether this hormone can have synergistic effects with OA and derivatives. This study did not evaluate the effects of these triterpenes on nitric oxide production or expression of eNOS and phosphorylation of K+ channels.
Syzygium aromaticum-derived triterpenes modulate intestinal glucose handling in streptozotocin-induced diabetic rats.
(2014) Khathi, Andile.; Musabayane, Cephas Tagumirwa.
Polyphagia in diabetes mellitus ascribed to elevated plasma ghrelin concentrations is associated with prolonged postprandial hyperglycaemia due to increased activities of intestinal carbohydrate hydrolyzing enzymes and glucose transporters. Postprandial hyperglycaemia is a major risk factor in the development of diabetic complications, and as such, should be managed to prevent chronic vascular complications. Previous studies in our laboratory have shown that Syzygium aromaticum-derived oleanolic acid (OA) and maslinic acid (MA) use various mechanisms to lower blood glucose concentrations in experimental diabetes. The effects of these triterpenes, however, on intestinal glucose handling remain unknown. Accordingly, this study was designed to investigate the effects of these triterpenes on intestinal glucose handling in STZ-induced diabetic rats. Materials and methods OA and MA were extracted from Syzygium aromaticum cloves using a previously validated protocol. Briefly, S. aromaticum-derived OA and MA were sequentially extracted with dichloromethane and ethyl acetate to obtain ethyl acetate solubles which contained mixtures of OA/ursolic acid (UA) and methyl maslinate/methyl corosolate. These solubles were purified by silica gel 60 column chromatography with hexane: ethyl acetate solvent systems to produce OA and MA. The structures of these triterpenes were confirmed by using 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopy and were comparable with those previously reported in literature. The in vitro studies investigated the inhibitory effects of OA and MA against enzymes such as α-amylase, α-glucosidase and sucrase. Additionally, the effects of various concentrations of OA and MA (0.82 - 6.56 mmol/L) on intestinal glucose transport were investigated using the everted intestinal sacs protocol. The in vivo studies investigated the effects of OA and MA on intestinal carbohydrate handling in separate groups of non-diabetic and STZdiabetic male Sprague Dawley rats. These studies were subdivided into oral glucose tolerance (OGT) responses which were carried out over two hours following loading with various carbohydrates as well as sub-chronic studies that were carried out over 5-weeks where the rats were kept on standard rat chow. OGT responses were monitored in separate groups of nondiabetic and STZ-induced diabetic animals the rats treated with OA and MA (80 mg/kg, p.o.). The rats were loaded with monosaccharides, disaccharides and polysaccharides after an 18-hour fast. The sub-chronic studies investigated the effects of the triterpenes on blood glucose concentrations over 5-weeks in groups of non-diabetic and STZ-induced diabetic male Sprague- Dawley rats. In those animals in which the effects of OA/MA were investigated, the rats were administered with OA/MA (80 mg/kg, p.o.) twice daily. Blood glucose, body weights as well as food and water intake were assessed every third day for the duration of the experimental period. At the end of the experimental period, the rats were killed and blood was collected for plasma insulin and ghrelin measurements. Furthermore, mid portions of the small intestine were snap frozen in liquid nitrogen and stored in a BioUltra freezer at -70 °C for Western blot analysis of glucose transporters, carbohydrate hydrolyzing enzymes and ghrelin expression. Additionally, the effects of OA and MA on intestinal oxidative stress were evaluated through malondealhyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx) measurements.Results The in vitro studies revealed that OA and MA possess inhibitory effects on the activity of α- amylase, α-glucosidase and sucrase comparable with those of the standard drug acarbose. In addition, OA and MA significantly (p<0.05) inhibited intestinal glucose transport in the everted intestinal sacs in a dose-independent manner. The OGT studies showed that OA and MA had no significant effects on blood glucose concentrations in non-diabetic rats loaded with the various carbohydrates by comparison with the non-diabetic control. However, the triterpene-treated STZdiabetic rats loaded with the various carbohydrate showed significant (p<0.05) reductions in blood glucose concentrations by comparison with untreated STZ-diabetic rats. OA and MA progressively reduced blood glucose concentration as well as food and water intake over the 5- week study in STZ-induced diabetic rats by comparison with untreated STZ-diabetic rats. Treatment with OA and MA had no effects on plasma insulin concentration in STZ-induced diabetic rats. However, these triterpenes significantly (p<0.05) reduced plasma ghrelin concentrations by comparison with untreated STZ-induced diabetic rats. Furthermore, rats treated with OA and MA showed significant (p<0.05) decreases in ghrelin, SGLT1, GLUT2, α- amylase and α-glucosidase expression in the gastrointestinal tract by comparison with untreated STZ-diabetic rats. This was accompanied by improvements in their intestinal antioxidant status as there were significant (p<0.05) reductions in MDA concentrations with significant (p<0.05) increases in SOD and GPx by comparison with the STZ-diabetic control. Additionally, OA and MA-treated rats showed significant (p<0.05) increases in intestinal glycogen concentrations with concomitant significant (p<0.05) increases in the intestinal expression of glycogen synthase by comparison with untreated STZ-diabetic animals. Discussion The results of the present study indicate that the blood glucose lowering effects of OA and MA in STZ -induced diabetic rats are mediated, in part, via modulating postprandial hyperglycaemia. These findings suggest that this is achieved through the ghrelin-mediated reduction in food intake leading to decreased expression of intestinal carbohydrate hydrolyzing enzymes as well as intestinal glucose transporters. This was followed by significant improvements in the antioxidant status in the rats suggesting that these triterpenes could, by preventing chronic postprandial hyperglycaemia, prevent the onset of the development of diabetic complications. The results of this study are of considerable importance as they suggest another mechanism for the anti-diabetic properties of the triterpenes and further explain the role of the gastrointestinal tract in the management of diabetes mellitus. Conclusion The results of the present study suggest that the S. aromaticum-derived triterpenes possess antidiabetic properties that arise, in part, through the modulation of intestinal glucose handling.
Transdermal delivery of insulin and Syzygium aromaticum-derived oleanolic acid by dermal patches in streptozotocin-induced diabetic rats : effects on some selected metabolic parameters.
(2014) Hadebe, Silindile Innocentia.; Musabayane, Cephas Tagumirwa.
The tight glycaemic control required to attenuate chronic complications in type 1 diabetes mellitus often requires numerous daily injections of bolus insulin. Typically insulin is administered by subcutaneous needle injection, insulin pen and catheters connected to insulin pumps. The routine multiple sc injections of insulin cause discomfort resulting in noncompliance, a major factor negating the quality of life of diabetic patients. Studies suggest that the bolus insulin injections are associated with hyperinsulinaemia, insulin resistance, glucose intolerance, weight gain and accelerated development of cardiovascular complications. These challenges of needle phobia and stress have encouraged investigations of possible administration routes of insulin delivery such as oral, nasal, buccal, pulmonary, rectal, ocular and transdermal systems. The skin has increasingly become a route of the delivery of drugs with a range of compounds generating a great deal of interest in this area of research. Studies in our laboratory are concerned with developing optional insulin delivery routes based on amidated pectin hydrogel matrix gel. Investigations described in this thesis were mainly designed to establish whether pectin insulin (PI)-containing dermal patches of different insulin concentrations sustain controlled release of insulin into the bloodstream of streptozotocin (STZ)-induced diabetic rats with concomitant alleviation of diabetic symptoms in target tissues, most importantly, muscle and liver. This study also focused on the hypoglycaemic effects of oleanolic acid (OA) which has been shown to significantly reduce blood glucose concentrations in both non-diabetic and diabetic rats when administered orally. OA does not dissolve easily in water hence we assessed the hypoglycaemic effects of OA via the transdermal route. Materials and methods Oral glucose test (OGT) responses to application of dermal patches containing different insulin concentrations were evaluated in separate groups of STZ-induced diabetic rats according to the method described previously by Musabayane et al., with slight modifications (Musabayane et al., 2007). Similarly, OGT responses to application of dermal patches containing different OA concentrations were also evaluated. Groups of STZ-induced diabetic rats were fasted overnight (18 h), followed by measuring blood glucose (time 0). The animals were given a glucose load of 0.86 g/kg and then the patches were applied on the shaved skin on the dorsal region of the animals. OGT responses to PI dermal matrix patches (2.47, 3.99, 9.57 and 16.80 μg/kg) prepared by dissolving pectin/insulin in deionised water and solidified with CaCl2 were monitored. Likewise, OGT responses to OA-containing dermal matrix patches (21, 42 and 84 mg/kg) were also monitored. Short-term (5 weeks) metabolic effects were evaluated in separate groups of non-diabetic and STZ-induced diabetic rats housed individually in Makrolon polycarbonate metabolic cages (Tecniplats, Labotec, South Africa) and were allowed water ad libitum and daily given 30 g standard rat chow (Meadow Feeds, Pietermaritzburg, South Africa). These animals were treated thrice daily with dermal matrix patches 8 hours apart. Rats treated with drug-free pectin and insulin (175 μg/kg, sc.) acted as negative and positive controls, respectively. Blood, liver, gastrocnemius muscle, pancreas and skin were collected for measurements of selected biochemical parameters after the experimental period. Plasma insulin concentrations were measured from blood samples collected after 6 hours (acute) and after 5 weeks (chronic) of treatment. Results Neither inflammation nor necrosis was detected in the skin of the rats after five weeks of daily treatment with PI-containing dermal patches. The density of phosphorylated IRS in skin tissues determined by immunohistochemical staining showed widespread localisation of IRS in cell bodies of the dermis, collagen and subcutaneous layer following treatment with PI-containing dermal patches. OGT responses and the area under the glucose curve (AUCglucose 0-360min) of untreated STZ-induced diabetic rats remained significantly elevated in comparison to the nondiabetic control rats. Topical application of PI-containing dermal patches on the skin of STZinduced diabetic rats at various doses showed a statistically significant decrease in blood glucose and AUCglucose 0-360min at the end of the 6h experimental period by comparison to respective control rats. However, there was no dose-dependent effect on the magnitude of blood glucose lowering induced by PI-containing dermal patches. The blood glucose-lowering effects evoked by PI-containing dermal patches were similar to those of the standard drug (sc. insulin). Treatment of STZ-induced diabetic rats with OA-containing dermal patches at various doses induced similar effects on the skin, IRS and blood glucose concentrations. Similar trends were observed chronically. Plasma insulin concentrations of untreated STZ-induced diabetic rats were significantly low compared with control non-diabetic rats. All PI treatments elevated plasma insulin concentrations of diabetic rats after the 6 h period but, the levels induced by low doses (2.47 and 3.99 μg/kg) were smaller than those caused by high doses (9.57 and 16.80 μg/kg). However, these effects on plasma insulin concentrations were comparable to those of sc insulin treated animals. Similarly, 5-week treatment with PI-containing dermal patches elevated plasma insulin concentrations although dose-dependent effects were not observed. Interestingly, the sc treated group remained low within levels that were comparable to those of the untreated STZinduced diabetic group. On the other hand, the plasma insulin concentrations of all OA treated groups remained significantly low at the end of the 6 h and 5-week experimental period in comparison to the non-diabetic control. There was no change in plasma insulin concentrations of STZ-induced diabetic rats following acute and short-term daily treatment with OA-containing dermal patches. Untreated STZ-induced diabetic rats exhibited significant depletion of glycogen and the expressions of glucose transporter-4 (GLUT-4) and glycogen synthase (GS) in liver and muscle tissues at the end of the 5-week study by comparison to non-diabetic rats at the corresponding time periods. Treatment with the PI matrix patch restored the glycogen levels and the expressions of GLUT-4 and GS to levels comparable to those of non-diabetic control animals and sc insulin. Moreover, treatment of STZ-induced diabetic rats with PI-containing dermal patches decreased plasma creatinine concentrations and increased GFR without altering with the plasma urea concentrations. Treatment of STZ-induced diabetic rats with OA-containing dermal patches induced similar effects on glycogen, plasma creatinine, GFR as well as plasma urea concentrations. Discussion Dermal patches delivered relevant amounts of pharmacologically active insulin and OA as evidenced by blood glucose lowering effects in STZ-induced diabetic rats. PI and OA dermal matrix patches will be easy to use and will not require elaborative devices to prevent drug leakage as in solution formulations. The findings are of considerable importance because this would free diabetic patients from daily bolus injections of insulin. Pectin has been used as a carrier of a wide variety of biologically active agents, for sustained release applications and targeting drugs to the colon for either local treatment or systemic action (Krusteva et al., 1990; Musabayane et al., 2000). The non-invasive PI- and OA-containing dermal patches may offer minimally invasive drug delivery in clinical applications to perhaps improve drug bioavailability and patient compliance. Interestingly, comparisons of the effects of PI and OA dermal patches of different insulin and OA concentrations on blood glucose lowering could not be separated statistically. The failure to observe these effects cannot be explained by the present study, but may be attributed to the narrow range of the doses used in the present study. These effects were also not statistically different from those of sc insulin. In summary, the ability of PI and OAcontaining dermal patches to reduce blood glucose with concomitant alleviation of symptoms associated with diabetes could be attributed to the ability of pectin to entrap and release drugs in a sustained and controlled manner. The PI- and OA-containing dermal hydrogel matrix patch would also provide patients with pain-free self-administration of insulin thereby improving compliance. Conclusions The current study has demonstrated that the pectin hydrogel insulin and OA dermal patches have the potential to deliver insulin and OA across the skin and into the blood stream and lower blood glucose concentrations and alleviate some symptoms associated with diabetes. Recommendations The limitations of the study include the absence of lipid profile and liver function assessment. In this regard, it is envisaged to utilize the obese Zucker diabetic rat model in future studies. Furthermore, limitations of this study also include the absence of plasma OA measurements.
Effects of maslimic acid and related triterpene derivatives on kidney function of male Sprague-Dawley rats.
(2014) Mkhwanazi, Blessing Nkazimulo.; Musabayane, Cephas Tagumirwa.; Van Heerden, Fanie Retief.
Reports indicate that hyperglycaemia leads to development of kidney complications which result in sodium retention, decrease in glomerular filtration rate (GFR) and high blood pressure. Various biochemical processes such as polyol pathway, AGEs formation are thought to gives rise to a development and progression of these complications. Clinical trials show that there is currently no commercially available compound that lowers blood glucose concentration while alleviating diabetic nephropathy (DN). Current methods involve the use of ACE blockers which are associated with side effects. Previous reports in our laboratories indicate that triterpene constituents of Syzygium spp. such as oleanolic acid (OA) possess hypoglycaemic and renoprotetive effects in STZ-induced diabetic rats. The important question is whether MA, a related triterpene also possesses the same properties. MA is a hydrophobic triterpene and we therefore synthesised derivatives to improve solubility, bioavailability and efficacy. Accordingly this study was designed to investigate the effects of MA and related triterpene derivatives on renal function of STZ-induced diabetic rats.
Mechanisms of the cardiovascular effects of some medicinal plants : an experimental study.
(2008) Kamadyaapa, Davie Rexon Juwa.; Musabayane, Cephas Tagumirwa.; Ojewole, John Akanni Oluwole.; Shode, Francis Oluwole.
Abstract available in PDF file.
Monitoring the change in health behaviour of learners in selected health promoting schools in KwaZulu-Natal.
(2009) Naidoo, Rowena.; Coopoo, Yoganathan.
Abstract available in PDF file.

Browse

Browsing Doctoral Degrees (Physiology) by Date Accessioned