Protandim Protects Oligodendrocytes against an Oxidative Insult

Abstract

Oligodendrocyte damage and loss are key features of multiple sclerosis (MS) pathology. Oligodendrocytes appear to be particularly vulnerable to reactive oxygen species (ROS) and cytokines, such as tumor necrosis factor-α (TNF), which induce cell death and prevent the differentiation of oligodendrocyte progenitor cells (OPCs). Here, we investigated the efficacy of sulforaphane (SFN), monomethyl fumarate (MMF) and Protandim to induce Nrf2-regulated antioxidant enzyme expression, and protect oligodendrocytes against ROS-induced cell death and ROS-and TNF-mediated inhibition of OPC differentiation. OLN-93 cells and primary rat oligodendrocytes were treated with SFN, MMF or Protandim resulting in significant induction of Nrf2-driven (antioxidant) proteins heme oygenase-1, nicotinamide adenine dinucleotide phosphate (NADPH): quinone oxidoreductase-1 and p62/SQSTM1, as analysed by Western blotting. After incubation with the compounds, oligodendrocytes were exposed to hydrogen peroxide. Protandim most potently promoted oligodendrocyte cell survival as measured by live/death viability assay. Moreover, OPCs were treated with Protandim or vehicle control prior to exposing them to TNF or hydrogen peroxide for five days, which inhibited OPC differentiation. Protandim significantly promoted OPC differentiation under influence of ROS, but not TNF. Protandim, a combination of five herbal ingredients, potently induces antioxidants in oligodendrocytes and is able to protect oligodendrocytes against oxidative stress by preventing ROS-induced cell death and promoting OPC differentiation.

Keywords: Nrf2; antioxidant enzymes; multiple sclerosis; reactive oxygen species.

Figure 1

Nrf2-activators dose-dependently increase antioxidant protein expression in OLN-93 cells. HO-1 (A), NQO-1 (B) and p62 (C) protein expression levels after 24 h treatment in the OLN-93 oligodendrocyte cell line with 5 µM or 10 µM SFN, 45 µM or 90 µM MMF, 30 µg/mL or 60 µg/mL Protandim or their respective DMSO or EtOH vehicle control. Protein levels were assayed by Western blotting. Data are presented as percentage of control and expressed as the mean ± SEM of 3 independent experiments. All statistics reflect one-way ANOVA tests with post hoc Bonferroni correction; * p < 0.05, ** p < 0.01; *** p < 0.001.

Figure 2

Sulforaphane and Protandim promote viability of OLN-93 cells under tert-butyl hydrogen peroxide-induced oxidative insult. OLN-93 cells were treated with 5 µM SFN, 90 µM MMF, 60 µg/mL Protandim or their respective DMSO or EtOH vehicle control for 24 h. After removal of medium, cells were subsequently exposed to either control medium or medium with 200 µM tert-butyl hydrogen peroxide. Cell viability was measured using an Invitrogen live/dead cytotoxicity kit. Data are presented as percentage of control and expressed as the mean ± SEM of 3 independent experiments. Statistics reflect one-way ANOVA test with post hoc Bonferroni correction; ** p < 0.01.

Figure 3

Nrf2-activators dose-dependently increase antioxidant protein expression in mature primary rat OLs. HO-1, NQO-1 and p62 protein expression levels after 24 h treatment in mature primary rat OLs, differentiated for 7 days, with 5 µM SFN, 90 µM MMF,30 µg/mL Protandim or their respective DMSO or EtOH vehicle control. Protein levels were assayed by Western blotting. Data are presented as percentage of control and expressed as the mean ± SEM of 3 independent experiments. Statistics reflect one-way ANOVA test with post hoc Bonferroni correction; * p < 0.05.

Figure 4

Protandim increases total glutathione levels in mature primary rat OLs. Mature primary rat OLs differentiated for 7 days were treated for 24 h with 5 µM SFN, 90 µM MMF,30 µg/mL Protandim or their respective DMSO or EtOH control. Total glutathione levels were assayed using the GSH-Glo kit from Promega. Data are presented as percentage of control and expressed as the mean ± SEM of 3 independent experiments. Statistics reflect one-way ANOVA test with post hoc Bonferroni correction; *** p < 0.001.

Figure 5

Protandim promotes viability of mature primary rat OLs under oxidative insult. Mature primary rat OLs differentiated for 7 days were treated for 24 h with 5 µM SFN, 90 µM MMF,30 µg/mL Protandim or their respective DMSO or EtOH vehicle control. After removal of medium, cells were subsequently exposed to either control medium or medium with 100 µM tert-butyl hydrogen peroxide (tbH₂O₂) or glucose oxidase (1:750,000). Cell viability was measured using an Invitrogen live/dead cytotoxicity kit. Data are presented as percentage of control and expressed as the mean ± SEM of 3 independent experiments. Statistics reflect one-way ANOVA test with post hoc Bonferroni correction; *** p < 0.05.

Figure 6

Nrf2-activators dose-dependently increase HO-1 protein expression in primary rat OPCs. After 2 day de-differentiation of OLs with growth factors bFGF-2 and PDGF-AA, primary rat OPCs were treated with 5 µM SFN, 90 µM MMF,30 µg/mL Protandim or their respective DMSO or EtOH vehicle control for 24 h. Protein levels were assayed by Western blotting. Data are presented as percentage of control and expressed as the mean ± SEM of 3 independent experiments. Statistics reflect one-way ANOVA test with post hoc Bonferroni correction; * p < 0.05.

Figure 7

Protandim promotes differentiation of primary rat OPCs under oxidative stress. After 2 day de-differentiation of OLs with growth factors bFGF-2 and PDGF-AA, primary rat OPCs were incubated with 30 µg/mL Protandim or vehicle control (EtOH) for 24 h. After removal of medium, cells were subsequently exposed to either control medium or medium with 10 µM tert-butyl hydrogen peroxide (tbH₂O₂) for 5 days. MBP and Olig2 expression were assayed by immunocytochemistry. Data are presented as percentage of control and expressed as the mean ± SEM of 3 independent experiments. Statistics reflect student’s t-test, one-tailed; * p < 0.05.

Figure 8

Protandim marginally promotes differentiation of primary rat OPCs in the presence of TNF. After 2 days of de-differentiation, primary rat OPCs were incubated with 30 µg/mL Protandim or vehicle control (EtOH) for 24 h. After removal of medium, cells were subsequently exposed to either control medium or medium with 10 ng/mL TNF for 5 days. MBP and Olig2 expression were assayed by immunocytochemistry. Data are presented as percentage of control and expressed as the mean ± SEM of 3 independent experiments. Statistics reflect student’s t-test, one-tailed; * p < 0.05.