Doctoral Degrees (Physiology)

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Alzheimer’s disease dementia risk in post-traumatic stress disorder: identification of common underlying mechanisms using rat models.
(2021) Faborode, Oluwaseun Samuel.; Mabandla, Musa Vuyisile.
Post-traumatic stress disorder (PTSD) is a neuropsychiatric disorder that can develop from exposure to a trauma. Studies have shown that people who have PTSD are susceptible to developing dementia, mostly Alzheimer’s disease (AD), suggesting common underlying risk factors in the comorbidity. Although several molecular pathways have been implicated in AD and PTSD, including oxidative stress, cellular apoptosis, synaptic dysfunction and stress dysregulation, the underlying neurobiological mechanisms linking AD and PTSD are less understood. This study, therefore, investigated the effect of trauma-like exposure in an amyloid-beta (Aβ) rat model of AD. Seventy-two adult male Sprague-Dawley rats were used throughout the study. The animals were randomly divided into four groups where they received either footshocks or Aβ(1-42) injection or were exposed to footshocks and Aβ(1-42) injection or remained naive. Following inductions, the animals were tested for cognitive, locomotor and anxiety-like behaviours. Thereafter, brain samples were collected for further neurochemical analyses. Our results show that footshocks increased anxiety-like behaviour and impaired fear memory extinction in Aβ(1-42) lesioned rats. A combination of footshocks and Aβ(1-42) also reduced the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), NAD (P) H: quinone oxidoreductase 1 (NQO1), heme oxygenase-1 (HO-1), and increased the expression of Kelch-like ECH-associated protein 1 (Keap1) in the amygdala and hippocampus. Prior exposure to footshocks before Aβ(1-42) lesion caused a decrease in the number of crossing in the target quadrant of the Morris water maze test and reduced percentage alternation in the Y-maze test, indicating memory deficits. There was an interactive effect of footshocks and Aβ(1-42) lesion on the downregulation of BIN1 and the upregulation of NR2B in the hippocampus. There was also an interactive effect of footshocks and Aβ(1-42) lesion on the upregulation of FKBP5 in the hippocampus, amygdala, and PFC. Our finding suggests that footshock stress can exacerbate AD-like pathology via dysregulated redox balance, BIN1 downregulation, FKBP5 and NR2B upregulation, and increased apoptosis in the brain of Aβ(1-42)-lesioned rats. These molecular changes were associated with increased anxiety, impaired fear extinction and memory deficits. These findings, therefore, suggest common molecular mechanisms in PTSD and AD. Isifo sengcindezi eba semva kwesehlakalo esibi i-post-traumatic stress disorder (i-PTSD) yisifo sokusebenzelana kwezinzwa nensebenzamqondo esidala wukwehlelwa yisehlakalo esibi. Ucwaningo selukhombise ukuthi abantu abane-PTSD basengcupheni yokuba nesifo sokukhohlwa, ikakhulukazi i- Alzheimer’s (i-AD), okuchaza ukuthi izimo eziyingcuphe enkulu ekubeni nezifo eziyizimbelambela. Nakuba izindlelamigudu eziningi zamamolekhyuli zisoleka kwi-AD ne-PTSD, okufaka nengcindezi ye-oxidative, i-cellular apoptosis, i-synaptic dysfunction kanye nokungalawuleki kwengcindezi, izindlelamigudu zempiliswanozinzwa nomzimba ezixhumanisa i-AD ne-PTSD akuqondwa ngokuphelele. Ngakho-ke lolu cwaningo, luphenye umthelela wokubekeka endaweni ecisho ibe yisehlakalo esinzima kwimodeli yamagundane i-amyloid-beta (Aβ) ye-rat model of AD. Amagundane amadala esilisa angamashumi ayisikhombisa nambili asetshenziswa kulolu cwaningo. Izilwane zehlukaniswa ngokungahleliwe zaba amaqembu amane lapho zanikwa khona amafootshocks noma umjovo we-Aβ(1-42) noma ayengathola i-footshocks nomjovo we-Aβ(1-42) noma ahlale engazi. Ukulandela ukwethulwa, izilwane zahlolelwa ukuziphatha kwazo ngokomcabango, ukuhamba nokuba nexhala. Emva kwalokho, amasampula obuchopho aqoqwa ukuze aphinde acutshungulelwe amanyurokhemikhali. Imiphumela yethu iveze ukuthi ama-footshocks enyuse ukuziphatha sakuba nexhala kanye nokuqeda ukukhumbula ukwesaba okugwegwile kuma-Aβ(1-42) onikwe amalesioned rats. Ingxubevange yefootshocks ne-Aβ(1-42) iphinde yehlisa ukuvela kwe-nuclear factor erythroid 2-related factor 2 (Nrf2), i-NAD (P) H: i-quinone oxidoreductase 1 (NQO1), i-heme oxygenase-1 (HO-1), kanye nokuvela okusezingeni eliphezulu kwe-Kelch-like ECH-associated protein 1 (Keap1) kwi-amygdala ne-hippocampus. Ukusondelana nama-footshocks kwangaphambi kwe-Aβ(1-42) kwdala ukwehla esibalweni sokuhlanganayo kwi-target quadrant yokuhlolwa kwe-Morris water maze test nenguqunguquko yephesenti kwi-Y-maze test, okuveza ukushoda kokukhumbula. Kwaba nomthelela onokungenelana wama-footshocks kanye ne-Aβ(1-42) lesion ekulawulenikwehlisa i-BIN1 nasekulawulenikukhuphula i-NR2B kwi-hippocampus. Kwaphinde kwaba nomthelela ongenelanayo we-footshocks ne- Aβ(1-42) lesion ekulawulenikukhuphula i- FKBP5 kwi-hippocampus, i-amygdala, ne- PFC. Esikutholile kuphakamise ukuthi ingcindezi ye-footshock ingabhebhezela isakhiwosifo esifuze i-AD nge-dysregulated redox balance, i-BIN1 downregulation, i-FKBP5 ne-NR2B upreguation, i-apoptosis ephezulu ebuchosheni be-Aβ(1-42)-ekuma-lesioned rats. Lezi zinguquko kumamolekhyuli zazihlotshaniswe nokwenyuka kwexhala, inqedakwesaba egwegwile nokulahlekelwa wukukhumbula. Ngakho-ke lokhu okutholakele, kuphakamisa izindlela ezejwayelekile zomumo wamamolekhyuli kwi-PTSD ne-AD.
Evaluating plasmodium berghei infection influence and asiaticacid administration efficacy in sprague dawley male rats: effects on parasitamia, glucose homeostasis and renal electrolyte handling.
(2016) Mavondo, Greanious Alfred.; Mabandla, Musa Vuyisele.
Abstract Introduction: Five human-infecting Plasmodium species orchestrate varied pathophysiology culminating in malaria. Despite being treatable, malaria has high mortality and morbidity in pregnant women and children under five years. Current treatment is hampered by drug resistance, toxicity and failure to address malaria induced pathology directly. Asiatic acid has antioxidant, pro-oxidant, antihyperglycaemiac renoprotective qualities which may be anti-disease properties in malaria. Very little is currently known or reported about these asiatic acid anti-disease perspectives in malaria and therefore require further investigations. The aim of this study was to investigate the influence of asiatic acid administration and malaria infection on parasitaemia suppression, glucose homeostasis, renal function and electrolyte handling in Plasmodium berghei-infected Sprague Dawley male rats. Methods: Three sub-chronic studies and one acute study were conducted. The sub-chronic protocol involved per-oral pre- and post-infection administration of asiatic acid (5, 10, 20 mg/kg) and post-infection transdermal drug delivery system application of asiatic acid (5, 10, 20 mg/kgamidated hydrogel matrix pectin patch). Acute studies included post-infection oral glucose tolerance response to asiatic acid. Influence of asiatic acid on %parasitaemia changes, physicochemical changes, immunological effects of malaria, haematological results of malaria, antioxidant capacity, glucose homeostasis, renal function and renal electrolyte handling were investigated. Results: Asiatic acid suppressed parasitaemia to varying extents with asiatic acid 10mg/kg and 5mg/kg emerging as the most efficacious amongst the three doses by per oral administration and transdermal delivery drug delivery system, respectively. Malaria suppression occurred in both pre-infection and post-infection administration of asiatic acid. Asiatic acid preserved physicochemical parameters, ameliorated haematological effects of malaria, influenced immunological effects of malaria, modulated glucose homeostasis in malaria, protected renal function and electrolyte handling in malaria. Asiatic acid improved glucose tolerance response in acute malarial states. Antioxidant status was also improved in malaria. Conclusions: Asiatic acid displayed chemoprophylactic and chemotherapeutic effects in malaria. Asiatic acid has both glucose homeostatic and renoprotective properties in malaria. The antioxidant characteristics of the amphiphilic asiatic acid seem to exert anti-disease and antiparasitic effects in malaria. Asiatic acid may be used as an antimalarial compound with ability to ameliorate malaria associated pathophysiology.
Developmental methylmercury toxicity in a 6- hydroxydopamine Parkinsonian rat model: evaluating Searsia chirindensis as a potential neuroprotectant.
(2017) Moosa, Zulfiah Mohamed.; Mabandla, Musa Vuyisile.
Methylmercury (MeHg) pollution in South Africa has escalated due to increased demand from industrial sources such as coal-fired power stations. This had led to a growing interest in the effects of this metal toxin on human health. Prenatal MeHg exposure has been suggested to be a silent neurotoxicant, which may display its effects when triggered by a further neurotoxic insult. MeHg exposure during the perinatal period leads to neurodevelopmental deficits resulting in motor and cognitive dysfunction. This suggests that developmental MeHg exposure may predispose to the development of neurodegenerative diseases such as Parkinson’s disease (PD). In this study, we investigate the effects of prenatal MeHg exposure at adolescence and furthermore when subjected to an additional neurotoxic insult in a parkinsonian rat model. Behavioural tests were conducted to assess motor deficits with neurochemical assessment of trace element levels, total antioxidant capacity, dopamine and cytokine concentrations as well as gene expression profiling. We also investigated a novel plant extract Searsia chirindensis (SC) as a potential neuroprotectant by alleviating neurotoxicity. Overall the results of our study show that prenatal MeHg exposure disrupts trace element homeostasis at adolescence asymptomatically however, these imbalances are exaggerated following a further neurotoxic insult leading to motor deficits. Treatment with SC reduced motor deficits in MeHg-exposed offspring as reflected by higher dopamine levels. Contrastingly, treatment in the absence of MeHg exacerbated motor deficits with higher copper levels and upregulation of antioxidant genes fth1 and nqo1 in response to the neurotoxic effect. Therefore the overall total antioxidant capacity was not affected by SC. We also investigated the effect of SC on normal body parameters to assess for toxicity. Our findings showed that SC did not affect either liver or renal function and therefore does not affect the homeostasis of other body systems. Therefore conclusively our study showed that developmental MeHg exposure results in altered trace element homeostasis which may predispose to the development of neurodegenerative diseases such as Parkinson’s. We also showed that SC stem-bark extract reduced motor deficits caused by 6-hydroxydopamine in MeHg-exposed offspring but exacerbated neurotoxicity in its absence. SC also did not have any adverse effect on the homeostasis of other body systems. Overall, this suggests that SC has potential as a neuroprotectant however further studies must be conducted to fully elucidate the mechanisms involved in its effect.
Characterising epigenetic alterations following cocaine consumption.
(2017) Ajonijebu, Duyilemi Chris.; Mabandla, Musa Vuyisile.
Complementary data from clinical and animal research have converged on the hypothesis that persistent use of psychostimulant drugs such as cocaine may not only involve pathological alterations in neural processes that subserve reward–related learning, but also complex interactions between genes and the environment through epigenetic modifications. Fosb and Crem (cAMP response element modulator) are among the central trans-factors suspected to mediate these long–term neurobiological changes due to their potential roles in drug reward. However, the critical question that concerns inheritance of epigenetic marks associated with parental cocaine experience in social settings, offspring vulnerability and modifying maternal–foetal environment by fostering, remains poorly understood. The present study therefore aimed to investigate possible associations between cocaine–induced behavioural changes in social contexts and DNA methylation patterns of inducible transcription factors in the prefrontal cortex (PFC) and hippocampus (HPC) of the exposed parent mice. We further examined whether the induced epigenetic changes were inheritable and then determined the impact of early postnatal (PN) fostering on associated neurobiological changes in the offspring of drug-exposed parents. In doing so, we were able to investigate the interaction between epigenetic and environmental factors in relation to drug consumption. Behavioural response of C57BL/6 mice to cocaine treatments were examined using conditioned place preference (CPP) and IntelliCage (IC) phenotyping techniques. In the CPP experiment, male mice received 6 cocaine injections (10mg/kg, i.p.) on alternate days followed by 6 days of extinction learning. A subthreshold dose of cocaine (5mg/kg, i.p) was later injected to reinstate CPP behaviour. In the IC, female mice were group–housed and initially had free access to drugs (300mg/L cocaine and 12% v/v ethanol in their drinking bottles) and water for 30 days to investigate consumption preference. Subsequently, withdrawal effect and alternate nose-poke learning tasks (ANT) were examined in the following 28 days with concurrent access to cocaine and water. In both experiments, locomotor activity and novelty exploration/recognition memory of mice were examined post cocaine treatments. DNA methylation status of Crem and Fosb gene promoters, within the HPC and PFC, were also assessed using quantitative real–time polymerase chain reaction. Thereafter, cocaine exposed or unexposed male and female mice were matched for mating to produce offspring with lineal phenotypes. At birth, some of the offspring were cross-fostered to further examine the impact of PN fostering on drug-induced epigenetic changes. Locomotion, memory competence and DNA methylation status were also evaluated in the offspring similar to the parent mice. In male mice, cocaine treatment resulted in significant changes in CPP during conditioning. After extinction learning, the subthreshold dose of cocaine did not reinstate the conditioned behaviour. The treatment increased locomotor activity in the open field but decreased novelty exploration in the object recognition task. These changes were characterized by significant hypomethylation in Crem and Fosb gene promoters only in the PFC. During the first 30 days of free access to cocaine, ethanol or water in the IC, the female mice spent significantly more time licking and consuming cocaine than ethanol, whereas consumption of either drugs was significantly less than water. Overall, the mice exhibited motivational deficits as manifested by their inability to learn the ANT. Our data also showed that prolonged access to cocaine in the IC decreased locomotor activity while recognition memory remained intact in the cocaine–experienced mice compared to their controls. These changes were accompanied by hypomethylation or hypermethylation in the promoters of Fosb and Crem genes in the PFC and HPC of the cocaine–experienced mice, respectively. In the offspring, memory performance and locomotor activity were not affected by parental cocaine exposure, except that recognition memory was impaired by early PN fostering in offspring lineally inclined to either paternal and/or combined parental cocaine experience. Crem was hypomethylated only in the PFC of offspring of cocaine–exposed parent mice, while fostering the offspring reversed the expression. Significant change in Fosb methylation was only observed in the HPC of fostered offspring. Together, these findings suggest differential responses of the substrate brain regions to the converging environmental stimuli and dynamic regulation of induced neurobiological changes via DNA methylation. Overall, the data also provide some evidence that cocaine–induced epigenetic marks can be inherited by the non-drug exposed offspring while early PN fostering may enhance molecular switching that may render the individual vulnerable to drug consumption. Key words: Cocaine; social environment; IntelliCage, conditioned place preference; Crem; Fosb; DNA methylation; hippocampus; prefrontal cortex; epigenetic inheritance; postnatal fostering.
The effect of prenatal Mycobacterium tuberculosis infection on offspring neurodevelopment and autistic-like behaviours in a valproic acid mouse model of autism.
(2021) Manjeese, Wadzanai.; Mpofana, Thabisile.; Mvubu, Nontobeko Eunice.; Steyn, Adrie J.C.
Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by restricted repetitive patterns, communication challenges and lack of social skills. ASD has no distinct biomarkers, with symptoms overlapping with related developmental disorders like Schizophrenia. Maternal immune activation (MIA) is when the maternal immune system is invaded by a pathogen causing an immune response that interferes with the normal fetal brain development process. Mycobacterium tuberculosis (Mtb) infections are common during pregnancy and are known to affect fetal health, often causing spontaneous abortions and low birth weights. Valproic acid (VPA) is an anticonvulsant and mood stabilizer associated with ASD when administered during pregnancy. Gestational VPA exposure of mice on Embryonic day 12.5 (E12.5) induces ASD-traits in offspring, as such, this study employed VPA as a positive control. This study investigated the effects of prenatal exposure to Mycobacterium tuberculosis (Mtb) (singularly and in combination with VPA) on developmental delays and offspring behaviour. Pregnant mice were divided into saline, VPA, Mtb, and VPA+Mtb; treatments were administered on E12.5. Developmental milestones were measured between post-natal day 7 (PND 7) and 28. Offspring were subjected to neurobehavioural studies to test for social interaction and repetitive behaviours on PND 35. Ionised calcium binding molecule 1 (IBA-1) and Glial Fibrillary acid protein (GFAP) expression in the prefrontal cortex (PFC) and cerebellum regions were analysed using immunohistochemistry (IHC). The effect on the BBB’s function was determined using Evans blue dye-albumin extravasation method on PND 35. Additionally, cerebellar tissues were homogenized and processed for molecular analyses of NRXN1, NRXN2, NLGN1, NLGN2 and SHANK3 expression. Changes in expression patterns of NRXNs and NLGNs causes an imbalance in the excitation and inhibition of neurons, a feature associated with ASD. The Mtb treated group had significantly low litter count and high fetal resorption compared to saline treated group. Neuroinflammation was evident in the Mtb offspring at PND 35 as shown by a significant increase in GFAP and IBA-1 expressing astrocytes and microglia in the PFC and cerebellum compared to saline group. The BBB’s integrity was compromised as shown by the increased permeability to EB-dye in the PFC and cerebellum of Mtb, VPA and VPA+Mtb offspring. The Mtb offspring also displayed systemic inflammation and altered ASD-linked behaviours. NRXN1 and NLGN1 were overexpressed in the cerebellum of Mtb-induced MIA offspring compared to saline offspring. Dual exposure to VPA and Mtb restored NRXN1 expression levels, reduced astrocyte and microglia injury in the PFC, rescued social behaviours and restored normal eye-opening patterns in offspring. The study demonstrates impaired fetal development which persists into the post-natal period. The impaired development was accompanied by neuroanatomical changes and behavioural patterns consistent with ASD pathophysiology. These findings might be attributed to Mtb-induced maternal system inflammation in pregnancy that induces fetal inflammation via the placenta and BBB of a developing fetus causing insult in the brain. Immune dysregulation and synaptic defects are hallmarks of ASD. We therefore conclude that prenatal Mtb infection predisposes offspring to a higher risk of neurodevelopmental challenges later in life and dual exposure to VPA and Mtb rescues some of these challenges.
Evaluation of the potential benefits of L-ergothioneine on selected complications associated with type-2 diabetes in a rat model.
(2021) Dare, Ayobami.; Nadar, Anand.; Channa, Mahendra Lala.
Several pathogenic factors promote type-2 diabetic complications in patients, including cardiomyopathy, nephropathy, and non-alcoholic fatty liver disease (NAFLD). Specific nutraceuticals from food may act as a medicinal adjuvant in managing diabetic complications. L-ergothioneine (L-egt), a bioactive compound obtained from medicinal mushrooms, beans and some meat products, has been shown to reduce lipid accumulation, provide cytoprotection in tissue injury and enhances therapeutic efficacy when used as adjuvant. This study investigated the effect of L-ergothioneine with or without metformin on pathogenic metabolic pathways and biomarkers associated with selected diabetic complications in a type-2 diabetic rat model. Ninety (90) adult male Sprague-Dawley (175±20)g rats were divided into three study groups [study 1 (36), study 2 (30) and study 3 (24)]. A 10% fructose solution was provided ad libitum to adult male Sprague- Dawley (175±20)g rats for 14 days followed by a single intraperitoneal injection of low dose streptozotocin (STZ 40mg/kg bwt, i.p) to induce type-2 diabetes after which the animals were randomly divided into six, five, and four groups (n=6) in studies 1 (liver), 2 (kidney), and 3 (heart), respectively. The control groups were administered 1ml/100g distilled water, while L-egt (35mg/kg bwt), metformin (500mg/kg bwt), and losartan (20mg/kg bwt) were administered in the other groups. At the end of each study, animals were euthanized via decapitation, blood samples were collected, while the heart, kidney, and liver tissue were excised and used for biochemical, RT-qPCR, ELISA, western blotting, and histopathological analysis. An in-silico study was done to evaluate the molecular antioxidant mechanism of L-egt. Administration of L-egt, with or without metformin, to diabetic animals positively altered selected biomarkers of hepatic, renal, and cardiac dysfunction and prevented structural damage in these tissues. This treatment regimen mitigated oxidative stress, inflammation, and fibrosis by downregulating (p<0.05) SREBP1c, FAS, NF-kB, fibronectin, TGFβ1, and Keap1 expression and upregulating (p<0.05) Nrf2, Sirt1, NQO1, and HO1 expression compared with the diabetic control animals. Interestingly, co-administration of L-egt and metformin improved glucose homeostasis and reduced HOMA-IR. The in-silico study showed that L-egt binds to the active site of Nrf2 and may serve as a ligand to activate this potent antioxidant molecule. The overall result from this study showed the potential benefits of L-ergothioneine in the management of selected complications associated with type-2 diabetes. This bioactive compound may be an effective adjuvant to attenuate hypertriglyceridemia, oxidative stress, and inflammation, thereby protecting vital organs associated with diabetic complications against injury and improving glycemic control when coadministered with metformin to delay the onset of diabetic complications.
Role of neuroinflammation in an amyloid-beta model of Alzheimer’s disease and the identification of possible biomarker.
(2020) Shallie, Oluwadamilola Faith.; Mabandla, Musa Vuyisile.
Introduction. Alzheimer's disease (AD) is the most common form of neurodegenerative disorder that results in dementia. It currently affects 75 million people worldwide and is predicted to affect as many as 135 million people by 2050. Despite considerable research, current medication provides only modest relief to symptoms and does not cure the underlying disease. The delay in identifying a definitive cure is probably due to the scant knowledge of the cellular and molecular mechanisms implicated in its pathogenesis. However, the role of neuroinflammation has been acknowledged. Neuroinflammation is generally due to sustained activation of the brain's resident immune cells, including microglia and astrocytes. Although the importance of amyloid-beta (Aβ) in the aetiology of AD has recently come into question, there is still consensus that Aβ is closely related to AD. Therefore, a reappraisal of the amyloid hypothesis focusing on the role of neuroinflammation will open new avenues for identifying novel targets for AD treatment. In this study, we sought to assess neuroinflammation and identify possible biomarkers in an Aβ(1-42) rat model of AD over a progressive period of time. Materials and Methods. Male Sprague-Dawley rats were used in all experiments. The animals were randomly divided into a vehicle group of rats that were infused with phosphate-buffered saline (PBS) and an Aβ(1–42) group that was lesioned with the Aβ(1–42) peptide. Each group was further sub-divided into four groups (Day 3 group: animals euthanised 3 days after infusion; Day 7 group: animals euthanised 7 days after infusion; Day 10 group: animals euthanised 10 days after infusion, and Day 14 group: animals euthanised 14 days after infusion). Animals were subjected to neurobehavioral tests pre and postinfusion.The Morris water maze test was used to assess spatial learning and memory and the fear conditioning test was used to assess associative fear learning and memory. After euthanisation, whole blood sample acquired aseptically from both the vehicle and Aβ(1–42) lesioned group of rats was collected into ethylenediaminetetraacetic acid (EDTA) coated tubes for cytokine, oxidative stress markers and microRNA assays using multiplex immunoassay, spectrophotometric and real-time polymerase chain reaction analysis respectively. The excised whole brain was post-fixed in 10% neutral buffered formalin (NBF) for immunofluorescence and immunohistochemical analysis. Other brain tissue was placed in frozen 0.9% saline slush before the hippocampus was carefully dissected out and placed in a bio-freezer at -80 ºC post dissection. The tissue was later used for messenger RNA analysis using the real-time polymerase chain reaction technique. Results. We observed impaired spatial and reduced contextual fear memory, which was exacerbated as the postlesion days increased. Our results also showed increased expression of ionized calcium-binding adaptor molecule 1 (IBA-1), glial fibrillary acidic protein (GFAP), and beta-site amyloid precursor protein cleaving enzyme1(BACE1) antibodies and upregulated mRNA expression levels of cluster of differentiation 33 (CD33) and triggering receptor expressed on myeloid cells 2 (TREM2) genes in the hippocampus, as well as downregulated expression of miRNA107 in the plasma. In addition, our results showed a positive relationship between the activated glial cell markers and lipid peroxidation. Furthermore, elevated plasma concentration of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) with a concomitantly lowered levels of the anti-inflammatory cytokine (IL-10) in the Aβ(1–42) lesioned rats was observed when compared to the vehicle groups. A negative correlation between the decline in spatial memory and plasma levels of the pro-inflammatory cytokines and a positive correlation between the decline in spatial memory and plasma concentration of the anti-inflammatory cytokine was observed. Conclusion. Our findings implicate cellular and molecular mechanisms, as shown by prolonged and progressive activation of the glial cells, resulting in a bidirectional interplay between neuroinflammation and oxidative stress. These interconnections result in the concomitant release of brain cytokines as a secondary response to the hallmarks of AD, which impacts both neural circuit activity and expression of microglial genes regulating neuroinflammation, indicating dynamic crosstalk between the immune and nervous systems. These interactions facilitate the understanding of AD's pathogenesis and provide the basis for an integrative approach to validate the role of neuroinflammation in memory processes and, importantly, identify a potential biomarker for the early diagnosis of Alzheimer's diseases. As a contribution to knowledge, this study unveils the connection between memory decline and plasma cytokine concentration, as well as the relationship between genes regulating neuroinflammation in AD. Therefore, it is incontrovertible that neuroinflammation holds a pivotal role in AD pathology.
Impact of quercetin-3-0-rutinoside on biochemical and reproductive profile of rats prenatally exposed to high fat diet.
(2020) Adeyemi, Toluwalope Esther.; Nadar, Anand.; Channa, Mahendra Lala.
The increasing prevalence of infertility and obesity over the last few decades have become a major public health challenge among individuals within the reproductive age. Consumption of a high-fat diet (HFD) is a harbinger for many metabolic alterations and diseases including infertility and subfertility. Studies have shown that the reproductive health of an individual can be programmed prior birth since exposure to certain environmental factors especially during intrauterine life play significant roles in transcriptional and epigenetic alterations in pivotal genes. However, understanding the molecular mechanisms linking oxidative stress caused by adverse environmental conditions to intrauterine alterations at critical periods of development might help in the clinical management of diet-induced infertility problems. This study therefore aimed at investigating the impact of maternal HFD consumption on sex-linked differences in the reproductive hormone profiles of diet unexposed offspring and examined the therapeutic potential of 150 mg/kg Quercetin-3-O-rutinoside (QR) against the HFD-induced biological changes. Adult female Sprague Dawley rats were randomly divided into two groups and fed either 45% HFD or normal diet (ND) for eight weeks before mating with male rats fed ND. Thereafter, the pregnant rats were divided into four dietary treatment groups: ND, HFD, ND+QR, and HFD+QR. At gestation day 19 (GD19), n=7 animals per group were sacrificed. Blood and tissue samples were collected and stored at -800C for biochemical and molecular analyses. The remaining dams were allowed to litter naturally and sacrificed. The pups were also sacrificed at postnatal day (PND) 21, 28 and 35. Blood and tissue samples were collected and stored for subsequent analyses. Using standard laboratory procedures, we measured oxidative changes in the liver, placenta and brain tissues by assessing levels of malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD), catalase and nitric oxide (NO). Concentrations of hypothalamic gonadotropin releasing hormone (GnRH), serum luteinizing hormone (LH), testicular testosterone, and brain tumour necrosis factor (TNF-α) and glucagon-like peptide 1 (GLP-1) were assessed via enzyme linked immunosorbent assay (ELISA) technique. HFD-induced transcriptional changes in chemerin, chemokine-like receptor (CMKLR 1), TNF-α, GLP-1, interleukin-1 (IL-1β) and nuclear factor kappa B (NFκB) in the hypothalamic and testicular tissues were assessed by reverse transcriptase polymerase chain reaction (RT-PCR). After eight weeks of maternal HFD consumption, lipogram test indicated decreased plasma total cholesterol (TC) level, hypertriglyceridemia and increased low-density lipoprotein (LDL) levels. Our findings also showed that offspring of HFD-fed dams had delayed fur appearance and lower body weight compared to those from the control (ND) dams. These morphological changes were accompanied by elevated MDA levels in placenta, liver and brain tissues of HFD-fed dams and their diet-naïve offspring. Furthermore, there was evidence of hepatic nitrosative stress, time-dependent and sex-linked differences in hepatic SOD and brain GSH levels in the offspring. Also, hypothalamic GnRH and serum LH levels were significantly reduced at PND 28 and 35 in the offspring. Moreover, testicular testosterone was decreased at PND 35 in offspring of HFD-fed dams. Upregulation of chemerin, TNF-α, IL-1β mRNA transcripts in the hypothalamic-gonadal axis of male offspring indicates possible HFD-induced tissue inflammation and consequences for dysregulated steroidogenic and/or reproductive functions. Elevated brain GLP-1 may be linked to activated bioenergetic and homeostatic responses to HFD-induced oxidative stress. Overall, maternal HFD exposure led to induced oxidative stress, low-grade tissue inflammation and decreased levels of gonadotropins and androgens in their diet naïve offspring, whereas QR has little or no significant effects on these parameters.
Investigating immune metabolism and exhaustion in Type 2 Diabetes.
(2020) Nyambuya, Tawanda Maurice.; Nkambule, Bongani Brian.; Dludla, Phiwayinkosi Vusi.
Introduction In the era of rapid modernisation and urbanisation, the global incidence of non-communicable diseases such as type 2 diabetes (T2D) has significantly increased. This has been attributed to increased sedentary lifestyles and the adaptation of unhealthy diets, particularly in low-to-middle-income countries like South Africa. These changes promote the development of obesity, which is one of the major risk factors for T2D. Obesity, further plays a pivotal role in the pathoimmunological changes that are associated with outwards effects of poor glucose control and insulin resistance. Chronic inflammation and increased immune activation are a hallmark of T2D, and both these processes are partially mediated by T-cells. Interestingly, these pathological consequences are identified as early as in pre-diabetes before the onset of overt T2D. Upon activation, T-cells release cytokines that induce the activation of other immune cells and polarises T-cells towards the pro-inflammatory subset. This consequently leads to a pro-inflammatory milieu that alters T-cell function and predisposes individuals with pre-diabetes or patients with T2D to developing cardiovascular disease (CVD). Although few studies have implicated activated T-cells in mediating inflammation and altering myocardial function in poor glucose control, the underlying mechanisms and sequence of events remains scarce. Therefore, this study made use of a short-term high-fat diet (HFD)-induced mouse model of pre-diabetes to investigate inflammation and immune responses mediated by T-cells. Furthermore, it assessed and compared the modulatory effects of low-dose aspirin (LDA), metformin and fluvastatin (statin), well-acknowledged anti-inflammatory, anti-hyperglycaemic and cholesterol lowering drugs, respectively, on inflammation, T-cell activation, and cardiovascular risk. Methods This study involved the use of a diet-induced pre-diabetes and inflammation mouse model of glucose intolerance. Briefly, in phase one of the experiment, a total of 27 six-week-old male C57BL/6 mice were randomised into either a high-fat diet (HFD) (n=21) or low-fat diet (LFD) (n=6) groups for a total of 8 weeks. Phase two of the experiment subsequently initiated at week 9 whereby HFD-fed mice were randomised into a short-term treatment with either metformin, LDA or in combination with metformin (LDA+Met) or statin over an additional 6-week period (n=6/7group). Changes in body weights were monitored on a weekly basis. Glucose profiles, cholesterol levels, complete blood counts, T-cell associated cytokines, and the expression on T-cell markers were measured at the end of phase one (week 8) and phase two (week 14) of the experiments. The Kolmogorov-Smirnov test with Dallal-Wilkinson- Lillie was performed for normality testing. For parametric data, the mean differences between the LFDand HFD-fed groups were assessed using unpaired student t-test and were reported as mean ± standard error. The Man Whitney U test was used for non-parametric data and reported as the median and interquartile range [IQR]. Comparisons across the diet and treatments groups were assessed using a Two-way analysis of variance (ANOVA). A posthoc Tukey's multiple comparisons test was performed if the F-value reached statistical significance (p<0.05). The Kruskal-Wallis test, followed by a Dunn's posthoc test, was used for non-parametric data. A p-value of < 0.05 was considered statistically significant. The GraphPad Prism version 6 software (GraphPad Software Inc, CA, USA) was used for all statistical analysis. Results The HFD-fed group had significantly increased weight gain (29.17%) in comparison to the LFD-fed group (21.74%) after the 8-week period. Notably, HFD-feeding (HFF) was associated with impaired metabolic function marked by poor glucose control and a state of hypercholesterolemia. In particular, the HFD-fed group had increased fasting glucose (p<0.0001) and 2-hour postprandial area under curve (p=0.0029) when challenged with an oral glucose tolerance test in comparison to the LFD group. In addition, total cholesterol (Tc) (p=0.0039) and low-density lipoprotein (LDL)-c (p=0.0447) levels were higher in the HFD-group than LFD-group, whilst high-density lipoprotein (HDL)-c levels were comparable between the groups (p=0.1749). HFF was associated with enhanced levels of inflammation and generalised immune activation, marked by increased white cell count (WCC) (p=0.008) and elevated levels of interleukin (IL)-6 (p<0.0001), IL-2, tumour necrosis factor (TNF)-α (p=0.0312) and IL-17A (p<0.0001). Most importantly, HFF upregulated Fas (CD95) and downregulated CD69 (p=0.0009) expression on T-cells without altering the levels of programmed-cell death 1 (PD-1) (p=0.6408). The elevated levels of Fas were directly associated with body weight gain (r=0.93, p=0.0333). Short-term treatment with LDA+Met lowered insulin levels (p=0.0475) and fasting blood glucose (p<0.0001) when compared the untreated HFD-fed group. Although treatment with LDA monotherapy did not affect any cholesterol levels, metformin monotherapy and statin significantly lowered Tc and LDL-c when compared to the untreated HFD-fed group (p<0.05). Treatment with LDA+Met lowered WCC (p=0.0095), lymphocyte count (p=0.0264), IL-6 (p=0.0002), TNF-α (p=0.0465), IL-2 (p=0.0001) and IL-17A (p<0.0001), when compared to the untreated HFD-fed group. Lastly, LDA+Met (p=0.0010) but not LDA (p=0.147), upregulated the expression of CD69 on T-cells whilst both treatment groups had no impact on PD-1 levels. Treatment with fluvastatin had no effect on the levels of inflammation (p>0.05). Conclusion This study showed that T-cell dysfunction is congruent with a state of inflammation, hypercholesterolaemia and poor glucose control in the early stages of obesity. Notably, the altered Tcell function is partially mediated by the aberrant expression of Fas and CD69. The combinational treatment of LDA with metformin was more effective than the use of LDA only in improving glucose control, ameliorate inflammation, and moderate T-cell functions. These findings outline the pathological link between the development of inflammation, immune activation and altered lipid metabolism in a pre-diabetic state. More importantly, it highlights the cardiovascular risk properties of statins and enhanced anti-inflammatory efficacy of LDA when combined with metformin in poor glucose control.Therefore, alleviating inflammation and lowering glucose levels during the early development of T2D may be an effective strategy to attenuate T-cell remodeling in diet-driven metabolic disturbances.
An investigation into the neurochemical and behavioural patterns of C57Bl6 mice exposed to "Sugars" and its constituents.
(2020) Chetty, Yvette Yolanda.; Nadar, Anand.; Naidoo, Panjasaram.
“Sugars” is an illicit drug cocktail that is a low-grade mixture of heroin and other opioids. The composition of this cocktail is highly varied as other ingredients are added during its manufacturing process to add bulk to the mixture and possibly increase profits of the suppliers. This highly addictive cocktail requires only a single use to initiate dependence and if not used timeously thereafter, severe withdrawal symptoms occur as soon as four hours after the last use. Due to the highly variable composition of this drug cocktail, it has been difficult to create a rehabilitation program with a low relapse rate as the physiological mechanisms of action of this cocktail have not been previously investigated. This study therefore aimed to investigate the physiological effects of “Sugars” and its ingredients in an animal model. This would provide novel findings on the pharmacological actions of the components of “Sugars” in the body as well as the physiological changes that may result during administration and withdrawal of the drug. This thesis is comprised of four manuscripts viz. one review paper that discusses the psychosocial issues of “Sugars” from an ethnographic standpoint and three experimental papers that focus on neurophysiology, behaviour, and immunology. The first experimental paper focuses on dopamine concentrations which were analysed using an ELISA assay and the sucrose preference test which can be used to assess the anhedonic behaviour in an animal model. The second paper focuses on the changes in memory function which was assessed using the Morris water maze and hippocampal mass and the third paper discusses changes in circulating immune cells following the analysis of blood samples with a heamotology analyser . The major findings emanating from this study were that administration of “Sugars” resulted in substantial changes in the dopaminergic system, cognitive abilities and haematological parameters involved in immunity; however, it was also observed that these changes were potentially reversed following a withdrawal period of 10 days in a mouse model. The extent of the effects observed may have also been influenced by the ratio of the ingredients in the cocktail. These novel findings can therefore assist in the formation of a targeted rehabilitation program that factors in the changes in the various physiological systems as discussed in this thesis. Key words: “Sugars”, illicit drug cocktail, dopamine, heroin, hippocampus
The effect of isolated and nanoencapsulated flavonoids from Eriocephalus africanus on apoptotic factors and microRNA expression in cancer.
(2020) Magura, Judie.; Mackraj, Irene.; Moodley, Roshila.
Cancer continues to be a major health burden worldwide, with millions of new cases being diagnosed each year. Among women breast cancer remains a leading cause of cancer-related morbidity and mortality globally, despite the significant advances in detection and individualised treatments. The ideal non-surgical approach for the treatment of breast cancer would be anticancer therapeutics that are delivered directly to the tumour site for complete elimination of cancerous cells without being toxic to surrounding healthy cells. However, current chemotherapeutics encounter numerous challenges due to adverse side effects and progressive drug resistance albeit effective. In light of this, identifying new effective therapies with minimal toxic and chemosensitizing effects as well as target specificity is crucial in combating cancer. Emerging evidence has supported the use of plant-derived chemicals as novel alternative treatment options, owing to their minimal side effects and toxicity. Plant-derived polyphenols have gained considerable research interest due to their ability to inhibit proliferation, initiate apoptosis and arrest the cell cycle of cancerous cells by modulating related pathways. Furthermore, incorporation of active plant-derived polyphenols into novel technologies such as nanosystems, offers more optimal therapies through improved bioavailability and target specificity. In this regard, this study demonstrates, for the first time, the potential of phytochemicals isolated from the methanolic extract of the medicinal plant, Eriocephalus africanus, as an alternative therapeutic strategy in breast cancer treatment using ER-positive human adenocarcinoma (MCF-7) cell lines. Spectroscopic techniques including nuclear magnetic resonance spectroscopy (NMR), infrared spectroscopy (IR) and mass spectrometry (MS) were used to identify the isolated compounds as hesperidin (flavanone), luteolin (flavone) and apigenin (flavone). Preliminary anticancer screening using the 3-(4,5dimethylthiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT) assay revealed hesperidin and luteolin to be potent against MCF-7. Dysregulated cellular apoptotic death is a hallmark of cancer and chemotherapy resistance; thus, the development of anticancer drugs targeting apoptosis is a widely used, effective anticancer treatment strategy. In this study, the efficacy of hesperidin and luteolin in targeting the apoptotic pathway was evaluated. Treatment of breast cancer cells with hesperidin and luteolin resulted in the downregulated expression of key anti-apoptotic Bcl-2; upregulated expression of pro-apoptotic Bax and caspases -8, -9 and -3. In addition, hesperidin and luteolin demonstrated the ability to effect epigenetic control through altering the expression of apoptotic microRNAs (-16, -21 and -34a). Moreover, treatment with hesperidin and luteolin resulted in significant accumulation of MCF-7 apoptotic cells into the G0/G1 and sub-G1 cell cycle phases, respectively. Encapsulation of hesperidin into nanoemulsions improved the cytotoxic and apoptotic effects in MCF-7 without being cytotoxic to non-cancerous human cell lines (HEK 293), halted the progression of the MCF-7 cells in the G2/M phase, and exhibited potential therapeutic activity through inhibiting the expression of oncomirs miR-21 and -155 overexpressed in breast cancer. Encapsulation of luteolin into solid nanoparticles generated from cleaved stearylamine exhibited non-selective cytotoxicity and decreased cell viability (< 10%) in both MCF-7 and HEK 293 cells, thus no further investigations were conducted using luteolin-loaded solid nanoparticles. Collectively, findings from this study provide new evidence on the effects of flavonoids isolated from E. africanus on apoptotic and epigenetic control in breast cancer, increasing our knowledge of the molecular basis of their anticancer activity.
Evaluation of the therapeutic properties of a Ruthenium(ll) uracil-derived diimine complex on selected complications associated with diet-induced pre-diabetes.
(2019) Mabuza, Lindokuhle Patience.; Khathi, Andile.; Ngubane, Phikelelani Siphosethu.
Pre-diabetes is a chronic metabolic condition where blood glucose levels are above the upper threshold considered normal but below the threshold for a diagnosis of diabetes. Pre-diabetes predisposes individuals to a high probability of future progression to overt T2DM. Pre-diabetic patients are at increased risk of developing other pathologies such as NAFLD, diabetic nephropathy (DN) and immune dysregulation complications. As a chronic disease, the long-term implications of diabetes contribute to poor quality of life and significantly increase costs associated with healthcare. Pre-diabetes may however be reversible, through the implementation of lifestyle modification programmes based around dietary modification and increased physical activity. Where lifestyle modifications are ineffective, both pharmacotherapy and lifestyle modification are recommended. However, there is reported poor patient compliance in terms of dietary intervention as patients tend to heavily rely on the pharmacological treatment, thus reducing the efficacy of the drug and increasing the possibility to develop T2DM. Hence, there is a need for novel drugs that will remain therapeutic even in the absence of dietary modification. In our laboratory, we have synthesized a novel ruthenium(II) uracil-derived diimine complex that has been shown to improve insulin sensitivity and restore glucose homeostasis in diet-induced prediabetes. In this study, we further sought to evaluate the effects of this compound on selected complications associated with diet-induced pre-diabetes. Estimating whether ruthenium(II) uracil-derived diimine complex in both the presence and/or absence of dietary intervention will show to be effective in the management of prediabetic-related complications. A high fat high carbohydrate (HFHC) diet was used to induce pre-diabetes for 20 weeks. After the induction, pre-diabetic rats were randomly allocated to the following treatment groups: non-prediabetes (NPD); pre-diabetic (PD); metformin plus HFHC; metformin plus normal diet (ND); Ruthenium plus HFHC and ruthenium plus ND. The animals were treated with subcutaneous injection of ruthenium complex (15 mg/ kg) and oral dose of metformin (500 mg/ kg). The rats were treated once a day every third day at 09:00 am for 12 weeks. Every 4 weeks, parameters such as body weight, food intake, fasting blood glucose, fluid intake and urinary output were monitored for 12 weeks treatment period. In study 1, the administration of ruthenium(II) complex resulted in the restoration of liver and body weights in the pre-diabetic treated rats when compared to the PD group. This treatment also reduced liver damage enzyme biomarkers and plasma total bilirubin levels in the pre-diabetic treated rats when compared to the PD group whilst administration of ruthenium(II) with dietary intervention reduced plasma sterol regulatory element binding protein 1c (SREBP-1c) concentration in the pre-diabetic treated rats when compared to the PD group. These findings were further supported by the histological analysis of the liver, showing reduced hepatic lipid droplet accumulation, hepatocyte ballooning and locular disarray in the ruthenium(II)-treated rats when compared to the PD group as seen in chapter 2 of the study. In study 2, the administration of ruthenium(II) complex resulted in reduced blood glucose, aldosterone, fluid intake and urinary output in the pre-diabetic treated rats when compared to the PD group which positively correlated with a restoration in plasma and urinary electrolytes along with plasma antioxidants concentrations in the ruthenium(II)-treated rats. Furthermore, there was a decrease in kidney injury molecular-1 (KIM-1) concentration, albumin excretion rate (AER) albumin creatinine ratio (ACR) and mRNA expression of podocin in urine in the ruthenium(II)-treaded rats. These observetions were further demonstrated by the histological analysis of the kidney, displaying improved histology of renal glomerulus in ruthenium-treated rats when compared to the PD group as seen in chapter 3 of the study. In study 3, treatment with ruthenium(II) complex resulted in reduction of platelet activation markers mean platelet volume (MPV) and CD40 Ligand (CD40 L) concentrations, which positively correlated with decreased plasma triglycerides (TG) and very low-density lipoproteins (VLDL) levels in the pre-diabetic treated rats when compared to the PD group. Whilst administration of ruthenium(II) with dietary intervention reduced plasma fibrinogen concentration in the pre-diabetic treated rats when compared to the PD group. These was further evidenced by normalization of immune cell counts in the ruthenium(II)-treated rats. Furthermore, there was a decrease in pro-inflammatory cytokines, tumor necrosis factor α (TNF-α) concentration in the ruthenium(II)-treated rats and decreased interleukin-1β (IL-1β) concentration in the ruthenium(II) with dietary interventiontreated rats when compared to the PD group as seen in chapter 4 of the study. Taken together, the results observed suggest that ruthenium(II) complex exhibited hepato and renoprotective effects while ameliorating immune dysregulation underlying pre-diabetes in diet-induced pre-diabetic rats. However, further studies are still required to find out the exact mechanism behind potential effect of this metal-based compound.
Evaluating immune activation and cellular determinants of thrombosis in pre-diabetes.
(2018) Mkandla, Zibusiso.; Nkambule, Bongani Brian.; Dludla, Philani.
Introduction Platelet dysregulation in pre-diabetes plays a major role in the progression of prothrombotic and pro-inflammatory conditions. Cardiovascular disease (CVD), which may occur at the pre-diabetic stage, are a common cause of morbidity in diabetic individuals. Despite treatment, people living with type 2 diabetes are at an increased risk of developing CVD, which is attributed to increased platelet activation and platelet mediated cellular cross-talks. The aim of the study was to investigate platelet activation and function in prediabetes and to further evaluate the effects of oral glucose lowering and anti-inflammatory therapy on platelet function a pre-diabetic state. Methodology Male mice were fed experimental diets, control low-fat diet (D12450J 10 kcal% fat) and a high-fat diet (HFD) (D12492, 60 kcal% fat) (Research Diets, NJ, USA). Platelet activation was determined by measuring the formation of spontaneous platelet-monocyte aggregate (PMA). The high-fat diet (HFD) fed mice were then randomized into 3 treatment groups; metformin (150mg/kg) and low-dose aspirin (3mg/kg) dual therapy; low-dose aspirin (3mg/kg); and clopidogrel (0.25mg/kg). The drugs were administered orally, once a day, every day for 3-weeks. We determined the pre-diabetic status of the mice by measuring their oral glucose tolerance and insulin levels. We further measured the haematological parameters. Platelet function and reactivity were determined by stimulating them with endogenous agonists, adenosine diphosphate (ADP), collagen and arachidonic acid, before and after treatment. Results Overall, there were no significant differences in the baseline characteristics such as body weights and insulin levels. However, after three weeks on the experimental diets, the high-fat diet (HFD) fed group exhibited delayed glucose clearance (p=0.0362). Baseline levels of platelet-monocyte aggregates were increased in the HFD group, p=0.0156. Post-stimulation with 4μM ADP and 20μM ADP, the HFD group expressed elevated levels of activated platelets, p<0.05. Metformin and low-dose aspirin-treatment inhibited reversible platelet aggregation, p=0.0220. While, residual platelet activation was observed at a concentration of 20μM ADP, p=0.0535. In the low-dose aspirin group, there were no significant variations in platelet reactivity following stimulation with ADP, p>0.05. Clopidogrel treatment inhibited the platelet response to 20μM ADP (p=0.0313). Conclusion Despite anti-platelet treatment in pre-diabetes, platelets exhibit a varied response to endogenous agonists. Therapeutic targeting of these pathways may reduce the risk of thrombotic complications in pre-diabetes. We further highlight the potential synergistic benefit of using dual oral glucose lowering and antiplatelet treatment to minimize the high on-treatment platelet responses observed in pre-diabetes and T2DM.
Investigating the effects of bredemolic acid on selected markers of some prediabetes-associated dysfunctions in diet-induced prediabetic rats.
(2019) Akinnuga, Akinjide Moses.; Khathi, Andile.; Sibiya, Ntethelelo Hopewell.; Ngubane, Phikelelani Siphosethu.
Prediabetes is an abnormal glycaemic state between normoglycaemia and chronic hyperglycaemia which is currently prevalent in developing and developed countries due to increased consumption of high caloric diet coupled with sedentary lifestyle. Prediabetes is associated with abnormal glucose metabolism. Additionally, the risk of developing prediabetes-associated complications such as non-alcoholic fatty liver disease (NAFLD), cardiovascular and renal diseases is not only present in overt diabetes mellitus but also in prediabetes. Management of prediabetes involves the combination of dietary and pharmacological interventions, however there is reported low compliance among patients as they tend to become overly dependent on the pharmacological interventions. Consequently, the pharmacological intervention efficacy is reduced as patients still progress to having overt diabetes. Therefore, managing prediabetes with anti-diabetic agents that will remain effective even in the absence of dietary intervention is considered necessary. Triterpenes have been found to have potential as anti-diabetic agents. Bredemolic acid (BA), a pentacyclic triterpene, has been reported to have increased biological activity relative to some other triterpenes. In this study, we sought to investigate the effects of BA on selected markers of some prediabetes-associated dysfunctions such as abnormal glucose homeostasis, hepatic, cardiovascular and renal dysfunctions in a prediabetic rat model in both the presence and absence of dietary intervention. Materials and Methods Thirty six (36) Sprague Dawley male rats that weighed 150 – 180g were divided into two groups: the non-prediabetic (n=6) and the prediabetic groups (n=30) which were fed a normal diet (ND) and high fat high carbohydrate (HFHC) diet respectively for 20 weeks to induce prediabetes. At 20th week, prediabetes was confirmed by assessment of fasting blood glucose (FBG) and oral glucose tolerance test (OGTT). The prediabetic rats were further sub-divided into five groups (n=6) and treated with either BA (80 mg/kg) or metformin (MET, 500 mg/kg) in the presence and absence of diet intervention for 12 weeks. Every 4 weeks of treatment, all the animals were placed in metabolic cages to determine caloric and fluid intake as well as urine output. Also, the body mass index (BMI), waist circumference (WC), blood pressure and heart rate were measured at every 4 weeks of treatment. After the 12 weeks of treatment, the animals were sacrificed, blood samples were collected into EDTA sample bottles and centrifuged to obtain plasma. Also, the skeletal muscle, liver, heart and kidney were collected, weighed, snapped frozen with liquid nitrogen and stored at -80°C before the biochemical analysis of selected markers of glucose homeostasis, hepatic, cardiovascular and renal functions. Results In the first study, the untreated diet-induced prediabetic rats had a significantly increased body weight, increased caloric intake, elevated glycated haemoglobin, increased ghrelin plasma concentration, decreased muscle glycogen concentration, insulin resistance and hyperinsulinaemia compared to the non-prediabetic rats. However, BA treatment with or without diet intervention ameliorated the body weight, caloric intake, glycated haemoglobin, muscle glycogen, glucose tolerance, plasma insulin and increased the expression of glucose transporter 4 (GLUT 4) in the skeletal muscle by comparison to the untreated prediabetic rats. Prediabetic induction in the second study resulted into elevated plasma concentration of liver enzymes, increased liver glycogen and triglyceride concentrations, increased oxidative stress in the liver and decreased sterol regulatory element binding protein (SREBP1c) by comparison to the non-prediabetic animals. Conversely, administration of BA with or without dietary intervention ameliorated liver functions by decreased oxidative stress, decreased liver enzymes, decreased liver glycogen and triglyceride as well as increased hepatic SREBP1c concentration in comparison to the untreated prediabetic animals. The results in the third study showed that the untreated prediabetic rats had a significantly increased body mass index (BMI), waist circumference (WC), blood pressure, heart rate, lipid profile, oxidative stress and inflammatory markers with significantly decreased endothelial nitric oxide synthase (eNOS) by comparison to the non-prediabetic control rats. On the other hand, the administration of BA with or without diet intervention improved cardiovascular functions by a decrease in BMI, WC, total cholesterol concentration, triglyceride concentration, blood pressure, heart rate, oxidative stress and inflammation with significant increase in eNOS plasma concentration in comparison to the untreated prediabetic rats. In the fourth study, the untreated prediabetic rats had a significantly increased fluid intake, urine output, sodium retention, potassium loss, aldosterone concentration, albuminuria, proteinuria, kidney injury molecule (KIM-1) and urinary podocin mRNA expression in comparison to non-prediabetic control and BA treated rats with or without diet intervention. Also, the untreated prediabetic rats presented increased albumin, total protein, urea, uric acid, creatinine and oxidative stress markers concentrations with a significant decrease in glomerular filtration rate (GFR). However, administration of BA with or without diet intervention attenuated oxidative stress, decreased urinary podocin mRNA expression and the aforementioned renal dysfunctions parameters. Conclusion This study showed that long term consumption of high caloric diet-induced prediabetes and resulted in abnormal glucose homeostasis, hepatic, cardiovascular and renal dysfunctions. Also, the results of this study showed that these dysfunctions are not only present during overt type 2 diabetes mellitus but already present at the prediabetic stage due to insulin resistance or hyperinsulinaemia that triggered oxidative stress in the physiological systems that we examined in this study. However, due to amelioration of insulin resistance via improved insulin sensitivity and earlier reported antioxidant activities that are common to all pentacyclic triterpenes, administration of BA significantly ameliorated the prediabetes-associated dysfunctions (abnormal glucose homeostasis, hepatic, cardiovascular and renal dysfunctions) with or without diet intervention in the prediabetic stage.
Investigating the biomarker potential of exosomes in preeclampsia.
(2019) Pillay, Preenan.; Mackraj, Irene.
Preeclampsia is a hypertensive disorder of pregnancy and one of the leading causes of maternal and perinatal morbidity, affecting up to 7 -10% of pregnancies globally which results in 50 000-76 000 maternal deaths per year worldwide. Preeclampsia remains an enigmatic phenomenon due to its unknown etiology. This is attributed to its multifactorial nature, which complicates clinical diagnosis and management, hence the need for a definitive biomarker. Several candidate biomarkers of preeclampsia have been identified but none have the potential to diagnose preeclampsia in early pregnancy. Exosomes have recently emerged as promising biomarkers of the disease and the present study, therefore, focuses on determining the context of the use of exosomes as biomarkers of preeclampsia. In our approach, we developed a theoretical framework for the evaluation of exosomes as potential biomarkers of preeclampsia in terms of the specified criteria defined by the Food & Drug Administration (US) (FDA) Biomarker Workgroup. This subsequently led to the evaluation of the context of use of exosomes as biomarkers of preeclampsia by determining the role of exosomal microRNA in the pathophysiology of preeclampsia using direct digital detection, computational algorithms and biological databases. We identified distinct exosomal miRNAs signatures involved in the aberrant pathophysiology of preeclampsia which serve as promising biomarkers. Furthermore, in terms of the FDA criteria, it is important to incorporate the intent-to-diagnose preeclampsia in combination with comorbidities associated with the increased susceptibility/risk of preeclampsia. It is clinically evident that HIV-positive pregnant women on (Highly Active Antiretroviral Therapy) HAART are at a greater risk of developing preeclampsia due to an unknown immune-related pathology caused by HAART. We, therefore, determined the potential diagnostic application of exosomal cytokines in preeclamptic and HIV-positive preeclamptic women on HAART. In doing so we have identified altered exosomal cytokine levels in both preeclampsia and HIV-positive pregnant women, which suggests an aberrant mechanism of exosomal cytokine encapsulation, which modulates immune responses in both disease states. Importantly, we show that exosomal Tumor necrosis factor alpha (TNF-α) may have clinical validity in diagnosing preeclampsia and preeclampsia in HIV-infected pregnant women. Abstract (isiZulu) I-preeclampsia iyinkinga ephezulu yokukhulelwa kanye neyodwa yezimbangela ezibangela ukugula komama nokubeletha, okuphazamisa ama-7 -10% wezokukhulelwa emhlabeni wonke okuholela ekufeni kwabantu abangu-50 000-76 000 emhlabeni wonke. I-preeclampsia iyisici esiyinkimbinkimbi ngenxa ye-etiology engaziwayo. Lokhu kubangelwa uhlobo olunezinhlobonhlobo zezinto ezihlukahlukene, okubandakanya ukuxilongwa nokuphathwa kwemitholampilo, ngakho-ke isidingo se-biomarker ecacile. Kunezimboni eziningana zepreeclampsia ezikhethwe yi-candidate kodwa azikho ongakwazi ukuhlolisisa i-preeclampsia ekukhulelwe kokuqala. Ama-Exosome asanda kuvela njengezicikumezi ezithembisayo zesifo futhi isifundo samanje, sigxile ekunqumeni umongo wokusetshenziswa kwama-exosomes njengama-biomarkers we-preeclampsia. Endleleni yethu, sathuthukisa uhlaka lwezinhlelo zokuhlola ama-exosomes njengama-biomarkers angaba yi-preeclampsia ngokwemigomo ebekiwe echazwe yi-Food & Drug Administration (US) (FDA) Biomarker Workgroup. Lokhu kwaholela ekuhlolweni kokusetshenziswa kwama-exosomes njengama-biomarkers of preeclampsia ngokunquma indima ye-exosomal microRNA ekuziphatheni kwe-preeclampsia esebenzisa ngokuqondile ukutholakala kwedatha, ukuhlelwa kwezinto zokusebenzisa izibalo kanye nolwazi lwezinto eziphilayo. Sithole amasignesha ama-miRNA ahlukile asebenzayo kuperosis of preeclampsia esebenza njengama-biomarkers athembisayo. Ngaphezu kwalokho, ngokwemigomo ye-FDA kubalulekile ukufaka i-preeclampsia ye-inten-to-diagnostic ngokubambisana nama-comorbidities ehambisana nokwehluleka / ukulimala kwepreeclampsia. Kuyabonakala emitholampilo ukuthi abesifazane abane-HIV abakhulelwe (i-Highly Active Antiretroviral Therapy) i-HAART ingengozini enkulu yokuthuthukisa ipreeclampsia ngenxa yokugula okungaziwa komzimba okubangelwa i-HAART. Ngakho-ke, sinquma ukuthi kungenzeka yini ukuhlolwa kwesifo se-cytokines exosomal ku-preeclamptic nakwabesifazane abane-HIV ngaphambili kwe-HAART. Ngokwenza kanjalo sesiye sabona amazinga e-cytokine ashintshiwe eguquguqukayo kokubili preeclampsia nabesifazane abakhulelwe abane-HIV, okusikisela indlela engavamile yokukhipha i-exosomal cytokine encapsulation, eyenza ukuthi izimpendulo zamasosha omzimba zisetshenziswe. Okubaluleke kakhulu, sibonisa ukuthi i-tumor necrosis factor alpha (TNF-α) ingaba nokusebenza komtholampilo ekuhloleni i-preeclampsia kanye ne-preeclampsia kwabesifazane abakhulelwe abane-HIV.
The cytotoxic effects, anti-iflammatory, antioxidant, antibacterial, and antidiabetic properties of eight selected South African plants for medicinal purposes.
(2020) Nkala, Bongani Alphouse.; Mbongwa, Hlengiwe Prosperity.; Qwebani-Ogunleye, Tozama.
People from the Southern African region have been using the fauna and flora of the region in their homes for millennia to treat all sorts of ailments and complaints with great success. This knowledge transfer was done through ’apprenticeships’ and oral communication. Certain communities consider medicinal plants to be safer than drugs and that they can treat more than one ailment. This study investigated cytotoxic effect, antimicrobial, anti-inflammatory, antioxidant, and antidiabetic properties of eight selected South African plants for medicinal purposes. Plant species were collected from the Walter Sisulu National Botanical-Gardens and were extracted with 90% methanol (1 g/10 ml) and concentrate to 10 mg/ml. Antimicrobial activities were determined by the microplate dilution method to establish the ability of the plant extracts to inhibit or kill pathogenic organisms with minimal inhibitory concentrations and minimum bactericidal concentration. Cytotoxicity effects were determined with Alamar blue and crystal violet cell viability assays, against C2C12 and RAW 264.7 cells. Anti-inflammatory effects were identified with stimulated lipopolysaccharide RAW 264.7 cells, and nitric oxide inhibition was measured with Griess reagent assay. The estimation of preliminary phytochemical, antioxidant (DPPH and ABTS radical scavenging), and alpha-amylase inhibition were determined with standard methods. The plant extracts inhibition and bactericidal effects were observed against all bacteria, namely: Lippia javanica (0.25±0.00 to 1.13±0.29 mg/ml); Ziziphus mucronata leaf (0.44±0.00 to 1.00±0.00 mg/ml); Erythrina lysistemon (0.44±0.00 to 1.08±0.00 mg/ml) and Schkuhria pinnata (0.5±0.00 to 1.34±0.00 mg/ml). All plant extracts exhibited flavonoids, phenols, terpenoids, and coumarins. The antioxidant inhibition was observed above 80% for Schkuhria pinnata, Lippia javanica, Clerodendrum myricoides, and Erythrina lysistemon. Also, these plant species exhibited an alpha-amylase inhibitory effect of 80%. The IC50 values were > 1000 μg/ml. All plant extracts demonstrated some degree of an antiinflammatory effect. However, Clerondendrum myricoides (35% - 89%), Lippia javanica (26% - 77%), Erythrina lysistemon (23% - 76%), Schkuhria pinnata (27% - 65%), and Vernonia oligocephala (16% - 58%) with IC50 value >1000 μg/ml, exhibited a marked antiinflammatory effect. Therefore, the presence of phenolic, flavonoids, anti-inflammatory, antioxidants, and α-amylase properties are potential solutions towards the management of diabetes and other chronic inflammatory diseases. Keywords: medicinal plants, antimicrobial, cytotoxicity, anti-inflammatory, antidiabetic, antioxidant.
An evaluation of the chemical composition and the in vitro and in vivo antihypertensive activity of extracts of Tulbaghia acutiloba Harv. in an L-NAME induced hypertensive model.
(2019) Arhin, Isaiah.; Mackraj, Irene.
The increasing prevalence of hypertension over the years has been identified as a major contributor to high morbidity and mortality, globally and locally, in Africa, posing a serious global health threat. The demand for novel therapeutic strategies has become increasingly important, given that conventional options may be inaccessible and costly, and are often associated with side effects, hampering patient compliance. The scientific validation of alternative strategies, such as phytotherapy has, therefore, become a major focus in the treatment and management of hypertension, as it is perceived as being cheap, accessible, and possessing minimal side effects. Hence the investigation of medicinal plants, within the field of novel drug discovery, is of interest as plants possess various phytochemicals displaying biological activities, which may be beneficial in hypertension, and it’s associated complications. Since there is no existing scientific data available to validate its medicinal usage, this study, therefore, evaluated the in vitro and in vivo antihypertensive effects of Tulbaghia acutiloba. The hydro-methanolic extracts of the plant parts (i.e. leaves, flowers, rhizomes and roots) were initially evaluated in vitro for their phytochemistry, antioxidant potential, angiotensin-converting enzyme (ACEI) inhibition activities, and heavy metal content. The phytochemical investigation of the various parts of the plants showed the presence of phenols, amino acids and alkaloids in all parts, with the leaves exhibiting a higher total phenolic content, in comparison to the other parts. Further analysis, using gas chromatography–mass spectrometry (GC-MS), revealed the presence of bioactive compounds, such as α-linolenic acid, which was found only in the leaves. Other compounds such as oleic acid and palmitic acid were found in all the parts of the plants. All parts of the plant showed antioxidant activity in vitro. Heavy metal toxicity analysis revealed the safety profile for all parts of the plants. All parts also showed a potential ACE inhibitory effect of greater than 50%, with the leaves showing the most significant effects, comparable to the conventional drug, Ramipril. We further investigated the effect of the hydro-methanolic leaf extract on oxidative stress, endothelial function, cardiovascular, renal and haematological parameters, associated with hypertension, in an L-NAME induced hypertensive rat model. The administration of the hydro-methanolic leaf extract of Tulbaghia acutiloba at different concentrations of 40, 60 and 80mg/kg b.w., reduced systolic, diastolic and mean arterial pressure in the model, with a pronounced effect at the dosage of 80mg/kg b.w. Additionally, the leaves of T. acutiloba significantly enhanced bradykinin receptor levels (B1 and B2), nitric oxide (NO) availability, promoted antioxidant activities and significantly reduced ACE activity in serum and cardiac tissues in hypertensive rats. Cardioprotection was significantly enhanced at 80mg/kg b.w. of T.acutiloba, as depicted by the cardiac function and morphology, and cardiac gene expression in experimental rats. There was no evidence of toxicity as depicted in the liver enzymatic activity after the administration of T.acutiloba in the hypertensive rats. Administration of T. acutiloba improved renal function as evidenced by the increased creatinine clearance (Ccr), improved fractional excretion of sodium and decreased urine protein-creatinine ratio (UPr/UCr). Additionally, decreased levels of leucocyte infiltration, decrease in both, neutrophil to lymphocyte ratio (NLR) and lymphocyte to monocyte ratio (LMR), was found after administration of T.acutiloba, with a maximal effect occurring at a dose of 80mg/kg b.w. Together, these findings provide scientific validation for T.acutiloba as a medicinal plant that has cardioprotective and antihypertensive properties, and is able to improve renal function and haematological parameters in an L-NAME induced hypertensive rat model. Overall, the data also provides substantive evidence for the possible usage of T.acutiloba as an alternate antihypertensive agent, in resource limited areas where conventional drugs are inaccessible. Key Words: Tulbaghia acutiloba, phytotherapy, cardioprotection, L-NAME, renal function, haematology.
Fungal endophytes: isolation, identification and assessment of bioactive potential of their natural products.
(2017) Sibanda, Edson Panganayi.; Mduluza, Takafira.; Mabandla, Musa Vuyisile.
Fungal endophytes produce a broad variety of bioactive compounds with potential to address some of the unmet human needs. Medicinal plants have an important role to play in the search for new strains of endophytes fungi, as it is possible that their beneficial characteristics are as a result of the metabolites produced by their endophytic community. However, inspite of this potential as repositories of bioactive compounds, the fungal endophytes of African medicinal plants remain largely underexplored. This thesis reports on studies that were conducted to bioprospect for endophytic fungi with antioxidant and antimicrobial activity hosted by the plants Warburgia salutaris, Annona senegalensis, Kigelia africana and Vitex payos used in Zimbabwean traditional medicine. The surface sterilization technique was used to isolate the endophytic fungi that were identified by ribosomal DNA sequencing of the nuclear ribosomal internal transcribed spacer region. Crude extracts obtained from the fermentation of the isolated endophytic fungi were screened for antimicrobial activity using the agar diffusion method and evaluated for total antioxidant activity using a commercial kit that used the single electron transfer mechanism. Fourier-transform infrared spectroscopy (FT-IR) and Gas Chromatography – Mass Spectrometry (GC-MS) were used to provide a snapshot of the metabolites present in the endophyte fungi extracts. A total of 33 endophytic fungi were isolated from the medicinal plants and the fungal endophyte colonisation rates varied by plant species and plant tissue. The isolated fungi across the different plant species and tissue types were found to be dominated by members of the phylum Ascomycota. The endophytic fungi Penicillium chloroleucon was isolated from all the plant species except for Cladosporium uredinicola and Myrothecium gramineum (both isolated from Kigelia africana) which had an inhibitory effect against Escherichia coli (ATCC1056). Whilst Epicoccum sorghinum isolated from Annona senegalensis exhibited the most potent antioxidant activity, a significant number of the screened endophytic fungi from the different plant species were also found to have some antioxidant activity. The total phenolic content was found to have a positive correlational relationship with total antioxidant activity of the screened endophytic fungi crude extracts. The endophytic fungi were shown to produce a diverse range of metabolites including phenolic and polyphenolic compounds through FT-IR and GC-MS analysis. The isolate Cladosporium uredinicola has potential as a source of antimicrobial compounds whilst the isolate Epicoccum sorghinum has potential as a source of natural antioxidant. Antioxidant activity is a common phenomenon in the studied endophytic fungi and the fungal endophytes of the medicinal plants of Zimbabwe have potential as sources of bioactive compounds. Keywords: Bioprospecting, antioxidant, antimicrobial, endophytic fungi, medicinal plants, Zimbabwe.
Insulin resistance and salt sensitivity in the rat model of hypertension
(2002) Channa, Mahendra Lala.; Somova, Liliana I.
Abstract available in PDF file.
An EM investigation of skeletal muscle regeneration in cultured explants.
(1995) Naidoo, Poovalingam Ramsamy.; Gathiram, Premjith.
Abstract available in PDF file.

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