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Doctoral Degrees (Medical Biochemistry)

Permanent URI for this collectionhttps://hdl.handle.net/10413/7038

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Browsing Doctoral Degrees (Medical Biochemistry) by Author "Ghazi, Terisha."

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    A Biochemical assessment of the potential of Spirulina Platensis to Ameliorate the adverse effects of highly active Antiretroviral therapy In Vitro.
    (2022) Sibiya, Thabani.; Chuturgoon, Anil Amichund.; Ghazi, Terisha.
    The human immunodeficiency virus (HIV) has been one of the prevalent causes of diseases on a global scale over four decades of its emergence. It is estimated that about 37.7 million people are infected with HIV globally, and 8.2 million persons are in South Africa. The highly active antiretroviral therapy (HAART) involves combining various types of therapies that are dependent on the infected person’s viral load. HAART helps to regulate the viral load and prevents its associated symptoms from progressing into acquired immune deficiency syndrome (AIDS). Despite its success in prolonging HIV-infected patients' lifespan, the long-term use of HAART promotes metabolic syndrome (MetS) through an inflammatory pathway, excess production of reactive oxygen species (ROS), and mitochondrial dysfunction. Interestingly, Spirulina platensis (SP), a blue-green microalga commonly used as a traditional food by Mexican and African people, has been demonstrated to mitigate MetS by regulating oxidative stress and inflammation. This study examined the protective role of SP against HAART-induced oxidative stress and inflammation in human hepatoma (HepG2) liver cells. The first published manuscript (appendix A) is a literature review on the potential of SP to ameliorate adverse effects of HAART: An update focusing on highlighting the potential positive synergistic effects of SP and HAART. This review provides introductory background of spirulina and its protective attributes. Thereafter, a study in an in vitro model was carried out by measuring oxidative stress, antioxidant, and inflammation markers. The HepG2 cell line was used as an in vitro model. Changes were investigated in cellular redox status, inflammation, and antioxidant response. The data analysis followed prolonged [96 hours (hrs)] exposure to HAART and acute (24 hrs) exposure to SP. HAART (Lamivudine (3TC): 1.51 μg/ml, tenofovir disoproxil fumarate (TDF): 0.3 μg/ml and Emtricitabine (FTC): 1.8 μg/ml) in HepG2 cells was investigated for 96 hrs and thereafter, treated with 1.5 μg/ml SP for 24 hrs. The HepG2 cells that served as control contained complete culture medium (CCM) only. 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide (MTT) assay was used to determine cell viability following SP treatment. Cellular redox status was assessed using the quantification of intracellular reactive oxygen species (ROS), lipid peroxidation, and lactate dehydrogenase (LDH) assay. The fluorometric, JC-1 assay was used to determine mitochondrial polarisation. Protein expression was determined using western blots. Quantitative Polymerase Chain Reaction (qPCR) was also employed for micro-RNA and gene expressions. The findings from these investigations led to further analyses as depicted and described in our second, third, and fourth manuscripts. In the second published manuscript (chapter three), antioxidant markers and Nuclear erythroid 2 related factor 2 (NRF-2), a key regulator of antioxidants, was investigated. The results show that SP exposure induces an antioxidant response. The results further reveal that prolonged exposure with HAART followed by SP treatment induced an antioxidant response through upregulating NRF-2 (p < 0.0001), CAT (p < 0.0001), and NQO-1 (p < 0.0001) mRNA expression. Furthermore, NRF-2 (p = 0.0085) and pNRF-2 (p < 0.0001) protein expression was upregulated in the HepG2 cells postexposure to HAART-SP. In the third manuscript (chapter four), microRNAs and genes involved in inflammatory response were analysed. SP prevented the inhibition of microRNAs involved in the regulation of inflammation. MiR- 146a (p < 0.0001) and miR-155 (p < 0.0001) levels increased in SP treated cells. However, only miR- 146a (p < 0.0001) in HAART-SP indicated an increase, while miR-155 (p < 0.0001) in HAART-SP treatment indicated a significant decrease expression. SP may mitigate the inhibition of selected miRNAs that regulate inflammation in HAART treated HepG2 cells. Further, analysis revealed that Cox-1 mRNA expression was significantly increased in HAART-SP treated cells (p < 0.0001). Moreover, HepG2 cells exposed to HAART-SP treatment showed a significant decreased Cox-2 (p < 0.0001) expression, therefore, SP potentially controls inflammation by regulating microRNA and gene expressions. Moreover, the positive synergistic effect is indicated by normalised intracellular ROS levels (p < 0.0001) in HAART-SP treated cells. In the fourth manuscript (chapter five), it was shown how SP mitigates inflammation induced by HAART in HepG2 liver cells. SP inhibits the inflammatory pathway, by significantly decreasing iNOS (p < 0.0001), IκB-α (p < 0.0001), NF-κB (p < 0.0001), IL-1β (p = 0.0002) and TNF-α (p = 0.0074) mRNA levels. The HAART-SP post treatments reduced inflammation as evidenced by decreased mRNA levels of NF-κB (p < 0.0001), IL-1β (p < 0.0001), IL-12 (p < 0.0001), TNF-α (p < 0.0001). Furthermore, NF-κB (p < 0.0001) protein expression was downregulated. Thus, SP has the potential to inhibit inflammation induced by HAART (3TC, TDF and FTC) in HepG2 cells. Finally, the overall results show that SP mitigates HAART-adverse drug toxicity in HepG2 cells, by activating the antioxidant response in HepG2 cells.
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    Current Antiretroviral Drugs- An investigation of metabolic syndrome promotion in HepG2 cells.
    (2022) Mohan, Jivanka.; Chuturgoon, Anil Amichund.; Ghazi, Terisha.
    Metabolic Syndrome (MetS) affects more than 20% of adults globally. Furthermore, the prevalence of MetS in HIV-infected patients on chronic antiretroviral (ARV) therapy continues to rise rapidly. This is alarming as a significant portion of people are HIV-infected worldwide, with the highest incidence experienced in Sub-Saharan Africa. An estimated 21% of people receiving ARV treatment display insulin resistance associated with mitochondrial dysfunction and inflammation. The current study aimed to determine the disruptions of metabolic processes associated with ARV use (Tenofovir disoproxil fumarate (TDF), Lamivudine (3TC) and Dolutegravir (DTG)) following a 120-h exposure period in HepG2 liver cells. Thereafter mitochondrial stress, inflammasome activation and insulin resistance promotion were assessed. Following HepG2 cellular ARV exposure, it was found that mitochondrial stress proteins SIRT3 and UCP2 expressions were significantly suppressed. Due to these aberrations, endogenous cellular attempts to activate the antioxidant responses (pNrf2, SOD2, CAT) and mitochondrial maintenance systems (PINK1 and p62) in selected singular and combinational ARV treatments seemed insufficient. This resulted in lipid oxidative damage and reduced ATP production. These results indicate that ARVs induce mitochondrial dysfunction in liver cells. Furthermore, it was deduced that combinational ARV exposure promoted inflammasome activation at a genomic level. This was seen in increased expression of NLRP3 mRNA expression and caspase-1 activity with coinciding elevation in IL-1β in mRNA expression. Additionally, JNK expression was upregulated, with correlating increases in p-IRS1 protein expression and decreased IRS1 mRNA expression being observed. Consequently, both PI3K and AKT mRNA expression was suppressed, whilst miR-128a expression was significantly upregulated. It can be deduced that the combinational use of ARVs induced mitochondrial dysfunction and subsequently prompted inflammasome activation. This led to dysregulation of the IRS1/PI3K/AKT insulin signalling pathway and the initiation/promotion of insulin resistance. This is further supported through miRNA activation, suggesting possibilities for future studies on in vivo ARV use and related epigenetic changes.
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    An investigation into the molecular and Epigenetic alterations associated with Fumonisin B1-induced toxicity in human liver (HEPG2) cells.
    (2020) Arumugam, Thilona.; Chuturgoon, Anil Amichund.; Ghazi, Terisha.
    The contamination of agricultural commodities with Fusarium mycotoxins is a global issue in food safety, with fumonisin B1 (FB1) being the most prevalent contaminant. FB1 is not only phytotoxic, but it induces a wide range of toxic effects in animals and humans and is associated with carcinogenesis in animals and humans. Intense research has uncovered several mechanisms by which FB1 induces toxicity. Recent evidence suggests that epigenetic mechanisms may also contribute to the toxic effects of FB1. Epigenetic modifications including DNA methylation, histone methylation, N-6- methyladenosine (m6A) RNA methylation, and non-coding RNAs such as microRNAs (miRNA) and long non-coding RNA (lncRNA) are central mediators of cellular function and cellular stress responses and disruption may be pertinent in FB1-induced toxicities. This study aimed to determine the epigenetic mechanisms of FB1-induced hepatotoxicity by specifically investigating changes in DNA methylation, histone 3 lysine 4 trimethylation (H3K4me3), m6A RNA modification, and noncoding RNA in human hepatoma (HepG2) cells. The effect of these FB1-induced epigenetic modifications on stress responses was further investigated. FB1 impairs DNA repair processes via epigenetic mechanism. FB1 reduced the expression of histone demethylase, KDM5B, which subsequently increased the total H3K4me3 and the enrichment of H3K4me3 at the PTEN promoter region; this led to an increase in PTEN transcript levels. However, miR-30c inhibited PTEN translation. Thus, PI3K/AKT signaling was activated, inhibiting CHK1 activity via phosphorylation of its serine 280 residue. This hampered the repair of oxidative DNA damage that occurred as a result of FB1 exposure. Exposure to FB1 not only induced oxidative DNA damage but elevated levels of intracellular ROS triggering cell injury. In response to oxidative injury, cells induce Keap1/Nrf2 signaling which is regulated by epigenetic mechanisms. FB1 elevated global m6A RNA levels which were accompanied by an increase in m6A “writers”: METTL3 and METTL14, and “readers”: YTHDF1, YTHDF2, YTHDF3 and YTHDC2 and a decrease in m6A “erasers”: ALKBH5 and FTO. Hypermethylation occurred at the Keap1 promoter, resulting in a reduction of Keap1 transcripts. The hypomethylation of Nrf2 promoters and decrease in miR-27b expression led to an increase in Nrf2 mRNA expression. m6A-Keap1 and m6A-Nrf2 levels were both elevated; however, protein expression of Keap1 was reduced whereas Nrf2 was increased. Collectively, these epigenetic modifications (promoter methylation, miRNA-27b and m6A RNA) activated antioxidant signaling by reducing Keap1 expression and increasing Nrf2 expression. If cells are unable to cope with stress, p53-mediated apoptosis is activated. Crosstalk between the lncRNA, HOXA11-AS, miR-124 and DNA methylation can influence p53 expression and apoptosis. FB1 upregulated HOXA11-AS leading to the subsequent decrease in miR-124 and increase in SP1 and DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B). This promoted global DNA methylation and hypermethylation of p53 promoters, thereby reducing p53 expression and caspase activity. Taken together, the data suggests that FB1 inhibits p53-dependent apoptosis via HOXA11- AS/miR-124/DNMT axis. Collectively, this study provides novel insights into additional mechanisms of FB1-induced toxicities by epigenetically modulating stress response mechanisms.