Chuturgoon, Anil Amichund.Ghazi, Terisha.Sibiya, Thabani.2023-09-142023-09-1420222022https://researchspace.ukzn.ac.za/handle/10413/22253Doctoral Degree. University of KwaZulu-Natal, Durban.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.enHighly Active Antiretroviral Therapy (HAART).Spirulina Platensis (SP).Inflammation.Oxidative stress.Metabolic Syndrome (MetS).Thesis