Browsing by Author "Rawstorne, Jordyn"
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- ItemThe effects of melatonin supplementation on vascular tissue during first line ART: an in vivo, ex vivo and in vitro study(Stellenbosch : Stellenbosch University, 2018-03) Rawstorne, Jordyn; Webster, Ingrid; Strijdom, Hans; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical Physiology.Introduction: Although Antiretroviral therapy (ART) has dramatically reduced HIVassociated morbidity and mortality, non-HIV-related conditions and comorbidities continue to rise in this population. Cardiovascular disease (CVD) has been reported to be the leading cause of death in the HIV-positive population receiving ART. ART is thought to impair vascular endothelial function through increased reactive oxygen species (ROS) and reactive nitrogen species (RNS) production. In this study, we aim to assess the effects of melatonin - a potent antioxidant -supplementation during ART on specific intracellular products of rat Aortic Endothelial Cells (AECs) as well as on the vascular reactivity of rat aortas. Methods: Cells were serum starved and treated with three different melatonin (1nM; 1uM; 10uM) or ART (Low: EFV: 5μM; FTC: 5μM; TDF: 80nM; Mid: EFV: 8μM; FTC: 7.5μM; TDF: 400nM; High: EFV: 12μM; FTC: 10μM; TDF: 1μM) concntrations for up to 24 hours. Nitric oxide (NO), RNS and necrosis were measured with a platereader (control expressed as 100%). The concentration that resulted in the greatest differences, compared to untreated cells, was selected for co-treatment studies and protein investigations, where the same parameters were measured. The effects of acute melatonin and ART administration on vascular reactivity was measured by aortic ring isometric tension studies in aortas extracted from control male Wistar rats. The endothelium-dependent and independent vascular reactivity was measured by isometric tension studies on aortas harvested from male Wistar rats that were treated for 8 weeks with melatonin (10mg/kg/day) and/or ART (EFV: 51.6 mg/kg; FTC: 17.4 mg/kg; TDF: 25.8 mg/kg). Signalling proteins involved in these changes were measured by western blot analyses. Results (Mean±SEM): Dose Response Studies: 1nM melatonin decreased necrosis [92.56±3.11%;p=0.0004] compared to untreated controls [100.00±1.04%; p=0.0004]. Highconcentration ART lead to increased NO production [112.70±2.17%; Control:100.00±2.22%;p=0.0015], RNS production [108.80±2.20%; Control:100±0.79%;p=0.0152] and necrosis [107.30±1.34%; Control: 100.00±0.86%;p=0.0251], compared to untreated controls. Main Studies: 1nM melatonin decreased necrosis [92.43±3.75%;p=0.0002], while Highconcentration ART increased necrosis [121.30±9.11%;p=0.0002] compared to untreated cells [100.00±1.07%;p=0.0002]. When combined, 1nM melatonin + High-concentration ART decreased necrosis [94.17±5.08%;p=0.0002] compared to ART alone. Western blot analyses (arbitarty units) showed that ART increased nitrotyrosine levels [2.31±0.34; Control:1.00±0.18;p=0.0486], but decreased p22 PHOX [0.20±0.043; Control:1.00±0.15;p<0.0001], and cleaved caspase-3 [0.25±0.038; Control:1.00±0.18;p=0.0005], expression. In actute aortic ring experiments, ART exposure elicited a burst of contraction during the treatment period, followed by a significant attenuation in accumulative contraction compared to all other groups. In endothelium-dependent and independent contraction studies on aortas from treated rats, all groups showed a pro-contractile response compared to the control. Western blot analyses showed that ART decreased cleaved caspase-3 [0.27±0.08;p=0.0055] expression. Conclusion: Decreased necrosis in AECs treated with combined melatonin and ART, compared to AECs treated with ART alone shows the protective effect of melatonin. Further specific protein investigations are needed to elucidate this mechanism. Western blots showed that ART induced anti-apoptotic effects and increased RNS production, but not NADPH-oxidase activity. The initial contractile burst following acute ART exposure may precondiition the aortas, resulting in the decreased accumulatve contractile capacity. Chronic ART treatment studies showed that ART treatment does not seem to affect vasorelxation. Blot data reconfirmed that ART is also anti-apoptoic in vivo.