Moringa oleifera Lam Leaf Extract Stimulates NRF2 and Attenuates ARV-Induced Toxicity in Human Liver Cells (HepG2)

Abstract

The World Health Organization (WHO) reported that there are 37 million individuals living with the human immunodeficiency virus (HIV) worldwide, with the majority in South Africa. This chronic disease is managed by the effective use of antiretroviral (ARV) drugs. However, with prolonged use, ARV drug-induced toxicity remains a clinically complex problem. This study investigated the toxicity of ARV drugs on mitochondria and the NRF2 antioxidant pathway and its possible amelioration using Moringa oleifera Lam (MO) leaf extracts. This medicinal plant has a range of functional bioactive compounds. Liver (HepG2) cells were treated with individual ARV drugs: Tenofovir disoproxil fumarate (TDF), Emtricitabine (FTC), and Lamivudine (3TC) for 96 h, followed by MO leaf extracts for 24 h. Intracellular ROS, cytotoxicity, lipid peroxidation, total and reduced glutathione (GSH), ATP, and mitochondrial polarisation were determined. Finally, protein (pNRF2, NRF2, SOD2, CAT, and Sirt3) and mRNA (NRF2, CAT, NQO1 SOD2, Sirt3, and PGC1α) expression were measured using Western blot and qPCR, respectively. TDF, FTC, and 3TC significantly increased intracellular ROS and extracellular levels of both MDA and LDH. ARVs also reduced the GSH and ATP levels and altered the mitochondrial polarization. Further, ARVs reduced the expression of NRF2 SOD2, Sirt3, CAT, NQO1, UCP2 and PGC1α mRNA and consequently pNRF2, NRF2, SOD2, Sirt3 and CAT protein. In contrast, there was a significant reduction in the extracellular MDA and LDH levels post-MO treatment. MO significantly reduced intracellular ROS while significantly increasing GSH, ATP, and mitochondrial membrane polarization. The addition of MO to ARV-treated cells significantly upregulated the expression of NRF2, SOD2, Sirt3, CAT, UCP2, PGC1α, and NQO1 mRNA and pNRF2, NRF2, SOD2, Sirt3 proteins. Thus, MO ameliorates ARV-induced hepatotoxicity by scavenging oxidants by inducing the NRF2 antioxidant pathway. MO shows great therapeutic potential and may be considered a potential supplement to ameliorate ARV drug toxicity.

Keywords: Moringa oleifera; NRF2 signalling; antioxidants; antiretroviral drugs; mitochondrial toxicity; oxidative stress.

Figure 1

Assessment of ROS generation post-ARV and post-ARV MO treatment. TDF, FTC, and 3TC significantly increased the levels of ROS, while ARV MO displayed a significant reduction in ROS in HepG2 cells. * Represents a significant difference between the control and ARV (***, p < 0.0001). α Represents a significant difference between ARV and ARV MO (ααα, p < 0.0001).

Figure 2

Assessment of cellular membrane integrity post-ARV and post-ARV MO treatment. (A) TDF, FTC, and 3TC significantly increased the levels of extracellular MDA, while ARV MO displayed a significant reduction in MDA in HepG2 cells. (B) ARVs significantly increased extracellular LDH, whereas ARV MO caused a significant reduction in LDH. * Represents a significant difference between the control and ARV (*, p < 0.05 and **, p < 0.001). α Represents a significant difference between ARV and ARV MO (α, p < 0.05 and αα, p < 0.001).

Figure 3

Assessment of mitochondrial integrity post-ARV and post-ARV MO treatment. (A) TDF, FTC, and 3TC significantly decreased the levels of intracellular ATP, whereas ARV MO displayed a significant increase in ATP in HepG2 cells. (B) ARVs significantly decreased the ΔΨm. In contrast, ARV MO significantly increased ΔΨm in HepG2 cells. * Represents a significant difference between the control and ARV (*, p < 0.05 and ***, p < 0.0001). α Represents a significant difference between ARV and ARV MO (α, p < 0.05 and ααα, p < 0.0001).

Figure 4

Assessment of GSH concentration post-ARV and post-ARV MO treatment. TDF, FTC, and 3TC significantly decreased the levels of intracellular GSH, while ARV MO displayed a significant increase in GSH in HepG2 cells. * Represents a significant difference between the control and ARV (**, p < 0.001 and ***, p < 0.0001). α Represents a significant difference between ARV and ARV MO (α, p < 0.05, αα, p < 0.001 and ααα, p < 0.0001).

Figure 5

Assessment of pNRF2 and NRF2 expression post-ARV and post-ARV MO treatment. (A) TDF, FTC, and 3TC significantly decreased the levels of pNRF2, TDF and 3TC significantly decreased the expression of NRF2, while 3TC caused a slight increase in NRF2 expression. MO increased pNRF2 and NRF2 in ARVs treatments with a slight decrease at 3TC. (B) The NRF2 mRNA was significantly decreased by TDF, FTC, and 3TC while significantly increased by ARV MO. * Represents a significant difference between the control and ARV (*, p < 0.05, **, p < 0.001 and ***, p < 0.0001). α Represents a significant difference between ARV and ARV MO (α, p < 0.05, αα, p < 0.001 and ααα, p < 0.0001).

Figure 6

Assessment of antioxidants CAT, SOD2, and NQO1 expression post-ARV and post-ARV MO treatment. (A) CAT was significantly decreased by TDF, FTC, and 3TC while significantly increased by MO. SOD2 was significantly decreased by TDF and FTC and increased by 3TC. MO significantly increased SOD2 expression at TDF and FTC; however, there was a slight decrease in MO and 3TC treated cells (B) The CAT, SOD2, and NQO1 mRNA was significantly decreased by TDF, FTC, and 3TC while increased by the addition of MO. * Represents a significant difference between the control and ARV (*, p < 0.05, **, p < 0.001 and ***, p < 0.0001). α Represents a significant difference between ARV and ARV MO (αα, p < 0.001 and ααα, p < 0.0001).

Figure 7

Assessment of mitochondrial protective enzymes expression post-ARV and post-ARV MO (A) Sirt3 expression was significantly decreased by TDF, FTC, and 3TC and significantly increased by ARV MO. (B) The mRNA of Sirt3, UCP2, and PGC1α was significantly decreased by ARVs and increased by ARV MO. * Represents a significant difference between the control and ARV (**, p < 0.001 and ***, p < 0.0001). α Represents a significant difference between ARV and ARV MO (αα, p < 0.001 and ααα, p < 0.0001).

Figure 8

A brief overview of MO’s potential to ameliorate the long-term HAART-induced toxicity in HIV-positive individuals. HAART depolarises the mitochondrial membrane and reduces ATP. HAART increases ROS and reduces GSH and other antioxidants, therefore promoting oxidative stress. The addition of MO attenuates ARV toxicity by increasing the polarisation of the mitochondria and ATP synthesis. MO scavenges ROS and restores GSH levels. MO upregulates the NRF2 antioxidant pathway and further reduces oxidative stress. MO leaf extracts reduce MDA and LDH levels by reducing the peroxidation of lipids. Created in Biorender.com (access date: 14 June 2022).