Mg supplementation protects against ritonavir-mediated endothelial oxidative stress and hepatic eNOS downregulation

Abstract

Ritonavir (RTV), a prototypical protease inhibitor currently used as a key component for anti-HIV therapy, is known for its endothelial and hepatic toxicity. The effects of RTV and Mg-supplementation on cultured bovine endothelial cells (EC) and rat hepatic endothelial nitric oxide synthase (eNOS) status were investigated. RTV dose-dependently (5–30µM) decreased EC viability after 48hrs; high Mg (2 mM) significantly attenuated the lost viability. ECs incubated with 15 µM RTV for 6 to 24 hrs. resulted in 2–4-fold elevation of oxidized glutathione and a 25% loss of total glutathione. At 24 hrs., EC superoxide production due to RTV was detected by dihydroethidium staining, and increased 41% when quantified by flow cytometry; both altered glutathione status and superoxide levels were substantially reversed by 2 mM Mg. RTV reduced eNOS mRNA (−25% at 24 hrs.), and led to decreased eNOS dimer/monomer ratios; nitric oxide (NO)-derived products decreased 40%; both changes were attenuated by Mg-supplementation. In male LewXBrown-Norway rats, RTV administration (75 mg/kg/day, 5 weeks) resulted in an 85% increase in plasma 8-isoprostane, a 30% decrease of hepatic eNOS mRNA; concomitantly, eNOS protein decreased 72%, whereas plasma nitrite level was reduced 49%. Dietary Mg-supplementation (6-fold higher than control) prevented the eNOS mRNA decrease along with lowering 8-isoprostane, and restored the eNOS protein and plasma nitrite levels comparable to controls. Conclusion Mg attenuates RTV-mediated EC oxidative eNOS dysfunction, and down-regulation of hepatic eNOS expression; we suggest that Mg can serve as a beneficial adjunct therapeutic against RTV-mediated eNOS toxicity.

Mg-supplementation attenuated lipogenic and oxidative/nitrosative gene expression caused by Combination Antiretroviral Therapy (cART) in HIV-1-transgenic rats

“…The expression of selected hepatic lipogenic gene plus several selected genes related to oxidative/nitrosative stress and inflammatory activity were determined by a real-time quantitative PCR procedure similar to that described [18,19]. Briefly, 30 mg of liver samples from each rat were homogenized extracted for total RNA by TRIzol reagent (Invitrogen) and further purified using RNeasy mini spin columns (Qiagen, Valencia, CA), according to the manufacturer’s procedures.…”

“…Plasma cholesterol levels were determined by a Cholesterol Fluorometric Assay kit (Cayman Chemical), and total cholesterol content for each sample was estimated on the basis of relative fluorescence (excitation 550 nm, emission 590) obtained from standards. Plasma 8-isoprostane levels at 18 weeks were determined by a Cayman Enzyme Immunoassay Kit as described [8, 17–19]. RBC reduced (GSH) and oxidized (GSSG) glutathione levels were assessed enzymatically by the DTNB-GSSG reductase method [17–19].…”

“…Mg-supplementation at high doses has been reported to provide clinical beneficial effects for various cardiovascular disorders such as hypertension, atherosclerosis and CAD [13–16]. By using normal control rats, we also reported the protective effects of Mg-supplementation against AZT and RTV-induced oxidative, endothelial and cardiac toxicity [17–19]. It is unclear whether these antioxidant and anti-calcium properties of Mg influenced cART-induced metabolic and related side effects in HIV-1 expressed Tg animals; more importantly, we examined whether any of the Mg protective effects were related to transcriptome modification.…”

Human Immunodeficiency Virus–Associated Pulmonary Arterial Hypertension

“…104 These deleterious effects are inhibited by nonspecific antioxidants (eg, dihydroxybenzyl alcohol) 105 or magnesium supplementation. 106 Conversely, some antiretroviral agents have been shown to inhibit pulmonary artery smooth muscle cell proliferation in vivo in the monocrotaline-and hypoxia-induced models of experimental pulmonary hypertension, most likely via the inhibition of Akt phosphorylation. 107 Taken together, these findings suggest that there are dual (opposing) effects of antiretroviral therapy on pulmonary arterial remodeling, and each drug may differentially affect pulmonary hypertension in patients.…”

Effect of magnesium on vascular reactivity in NOS inhibition-induced hypertension

“…Thus, the reducing effect of high magnesium on the constriction responses of mesenteric arteries as was demonstrated in this study, might be explained by the mechanism(s) proposed above. On the other hand, magnesium is known to have antiperoxidative properties and is suggested to preserve glutathione status [33]. Thus, the antiradical effects of magnesium might also contribute to improvement of vascular response.…”