Protective Role of Whey Protein Isolate on MPP+-Induced Differentiation of SH-SY5Y Cells by Modulating the Nrf2 Antioxidant Pathway

Abstract

The pathogenesis of Parkinson's disease (PD) consists of the apoptosis of dopaminergic neurons in the substantia nigra pars compacta (SNpc) due to oxidative stress. The present study aimed to evaluate the potential antioxidant activity of whey protein isolate (WPI) in PD models, using neurotoxin-exposed SH-SY5Y cells differentiated into dopaminergic-like neurons. Our research shows that WPI's high glutamic acid, aspartic acid, and leucine contribute to its antioxidant and neuroprotective effects, with glutamic acid crucial for glutathione synthesis. In vitro studies found that WPI, at concentrations of 5-1000 µg/mL, is non-toxic to differentiated SH-SY5Y cells. Notably, the lowest con-centration of WPI (5 µg/mL) significantly decreased intracellular reactive oxygen species (ROS) levels in these cells following a 24 h co-treatment with 1-methyl-4-phenylpyridinium (MPP⁺). The antioxidant effects of WPI were also confirmed by the increased expression of HO1 and GPx antioxidant enzymes, which are Nrf2 pathway target genes and were evaluated by real-time PCR. Furthermore, Nrf2 nuclear translocation in the differentiated SH-SY5Y cells was also increased when the cells were exposed to 5 µg/mL of WPI with MPP⁺. These results together suggest that WPI has antioxidant effects on dopaminergic-like neurons in a Parkinson's disease model.

Keywords: MPP+; Nrf2; Parkinson’s disease; antioxidant; whey protein isolate.

Figure 1

SH-SY5Y neurons were differentiated into dopaminergic-like neurons for use as PD models. (a) The morphology of undifferentiated SH-SY5Y cells and differentiated SH-SY5Y cells after incubation with and without 10 µM RA and 80 nM TPA for 6 days. The red arrow is the branching process connecting each cell after differentiation. (b) Immunofluorescent staining of TH in SH-SY5Y cells. TH is represented by green signals around the blue signal, which is the nucleus (scale bar = 30 μm). The intensity of TH in undifferentiated and differentiated SH-SY5Y cells is shown in the bar graph. (c) The relative expression of DRD2 and SLC18A1 genes in undifferentiated and differentiated SH-SY5Y cells. The data are presented as the mean ± SEM; n = 120 for TH staining, and n = 3 for RT-PCR. ** p ≤ 0.01 and *** p ≤ 0.001 indicate significant differences between groups.

Figure 2

Differentiated SH-SY5Y cell viability after exposure to WPI (a) for 24 h or MPP⁺ (b) at various concentrations. The data are presented as the mean ± S.E.M.; n = 3. Statistical analysis revealed significant differences between the control group and test groups (*** p ≤ 0.001).

Figure 3

WPI decreased the production of intracellular ROS in MPP⁺-treated differentiated SH-SY5Y cells through the accumulation of Nrf2 in the nucleus. (a) The assessment of intracellular relative ROS levels in differentiated SH-SY5Y cells exposed to MPP⁺ and WPI, with Hyd used as a positive control. (b) The examination of Nrf2 nuclear translocation in differentiated SH-SY5Y after co-treatment with WPI and MPP⁺. Nrf2 nuclear translocation was confirmed by immunofluorescence staining using Nrf2 antibody (green) and TOPRO-3 dye (red) (scale bar = 100 µm). Merged images displaying a yellow color (resulting from the merging of green and red) within the nucleus indicate the successful nuclear translocation of Nrf2. (c) The quantification of the nuclear–cytoplasmic signal ratio. Data are presented as mean ± SEM; * p ≤ 0.05 and *** p ≤ 0.001 vs. MPP⁺-treated group, # p ≤ 0.05 and ### p ≤ 0.001 vs. control group.

Figure 4

The expression of antioxidant genes in differentiated SH-SY5Y cells was assessed following co-treatment with MPP⁺ and WPI. (a–f) The mRNA levels of SOD1, GST, GCLC, GPX, HMOX1, and NQO1 were assessed using RT-qPCR. The data are presented as the mean ± SEM; n = 3. ** p ≤ 0.01 indicates significant differences compared to the MPP⁺ group.

Figure 5

The schematic of the proposed mechanism illustrates that WPI, at a concentration of 5 µg/mL, exerts antioxidant properties on differentiated SH-SY5Y cells. WPI may be internalized by differentiated SH-SY5Y cells and exert bioactivity. WPI induces the translocation of Nrf2 into the nucleus, resulting in the enhanced expression of antioxidants such as GPx and HO1. Following this, these antioxidants decrease intracellular ROS, a precursor to cell death. Excessive intracellular ROS is induced by mitochondrial dysfunction caused by MPP⁺, which enters differentiated SH-SY5Y cells through the dopamine transporter (DAT). GPx converts H₂O₂ to water molecules. HO1 changes heme, which is a toxic molecule, to biliverdin, which is subsequently converted to bilirubin, which is an antioxidant molecule that can convert hydroxyl radicals (•OH) to water molecules. The figures were produced through the utilization of the BioRender.com platform.