Sodium selenite preserves rBM-MSCs' stemness, differentiation potential, and immunophenotype and protects them against oxidative stress via activation of the Nrf2 signaling pathway

Abstract

Background: The physiological level of reactive oxygen species (ROS) is necessary for many cellular functions. However, during the in-vitro manipulations, cells face a high level of ROS, leading to reduced cell quality. Preventing this abnormal ROS level is a challenging task. Hence, here we evaluated the effect of sodium selenite supplementation on the antioxidant potential, stemness capacity, and differentiation of rat-derived Bone Marrow MSCs (rBM-MSCs) and planned to check our hypothesis on the molecular pathways and networks linked to sodium selenite's antioxidant properties.

Methods: MTT assay was used to assess the rBM-MSCs cells' viability following sodium selenite supplementation (concentrations of: 0.001, 0.01, 0.1, 1, 10 µM). The expression level of OCT-4, NANOG, and SIRT1 was explored using qPCR. The adipocyte differentiation capacity of MSCs was checked after Sodium Selenite treatment. The DCFH-DA assay was used to determine intracellular ROS levels. Sodium selenite-related expression of HIF-1α, GPX, SOD, TrxR, p-AKT, Nrf2, and p38 markers was determined using western blot. Significant findings were investigated by the String tool to picture the probable molecular network.

Results: Media supplemented with 0.1 µM sodium selenite helped to preserve rBM-MSCs multipotency and keep their surface markers presentation; this also reduced the ROS level and improved the rBM-MSCs' antioxidant and stemness capacity. We observed enhanced viability and reduced senescence for rBM-MSCs. Moreover, sodium selenite helped in rBM-MSCs cytoprotection by regulating the expression of HIF-1 of AKT, Nrf2, SOD, GPX, and TrxR markers.

Conclusions: We showed that sodium selenite could help protect MSCs during in-vitro manipulations, probably via the Nrf2 pathway.

Keywords: Mesenchymal stem cells; Nrf pathway; Reactive oxygen species (ROS); Selenium selenite; Stemness.

Fig. 1

Isolation, cultivation and characterization of rBM-MSC. A The rBM-MSCs morphology was spindle shape, and they established adherent culture within one week. B Most (97.63%) of rBM-MSCs showed positive surface expression for CD90 as a surface marker in MSCs. Also, 99.35% of them were positively stained for CD73. An ignorable percentage (less than 1%) of the rBM-MSCs showed expression of CD34 and CD45, which are markers of hematopoietic lineages. Ca Confirmation of adipogenesis potential was done by the presence of detection of intracellular lipid droplets. Cb Confirmation of osteogenesis potential was done by the presence of mineralization particles stained red. All experiments were done in triplicate

Fig. 2

Effects of Sodium selenite on the viability of rBM-MSCs, determined by MTT assay after 24 (A) and 72 (B), hours incubation with different concentrations (0.001, 0.01, 0.1, 1, 10 µM) concentration in comparison to the control group. In the rBM-MSCs optimum OD was obtained at 0.1 µM of Sodium selenite, which showed the best viability and proliferation.*p < 0.05. Number of samples = 3, Data: Mean ± SD

Fig. 3

Relative gene expression of Oct-4, NANOG, and SIRT1 at 0.1 µM Sodium Selenite and control groups. The Oct-4, NANOG, and SIRT1 were significantly upregulated. The error bars show mean ± SD.***(p-Value ≤ 0.001), **(p-Value ≤ 0.01). Number of samples = 3, Data: Mean ± SD

Fig. 4

ROS generation and viability assessment by flow cytometry analysis. ROS generation in rBM-MSCs (A) cultured with Sodium Selenite (0.1 µM) (B). The ROS levels were determined by DCFH staining and flow cytometry. The viability of cells before and after Sodium selenite treatment was studied (C), which showed higher live cell populations after treatment. The ROS levels reduced after sodium selenite treatment (D). Number of samples = 3, Data: Mean ± SD

Fig. 5

Morphological observation and adipogenic differentiation of control compared to Sodium selenite treated rBM-MSCs. Control rBM-MSCs (a) rBM-MSCs treated with sodium selenite exhibited spindle-shaped and elongated without significant morphological changes (b). rBM-MSCs subjected to adipogenesis (c), rBM-MSCs treated with sodium selenite showed increased lipid droplets after oil red o staining (d). The percentage of oil droplets were quantified and reported (e). Number of samples = 3, Data: Mean ± SD

Fig. 6

FACS characterization of markers after Sodium Selenite treatment of the rBM-MSCs. Based on the FACS analysis of the rBM-MSCs, the number of cells for CD44 and CD105 markers increased by about 4.6% and 37%, respectively, after sodium selenite treatment. FACS characterization of negative markers for control (A,C, E,G) and Sodium Selenite treatment (B,D, F, H) of the rBM-MSCs. Based on the FACS analysis of the rBM-MSCs, the number of cells for CD31 and CD45 markers was comparable. Number of samples = 3, Data: Mean ± SD

Fig. 7

Western blot comparison of signaling molecules in response to Sodium Selenite (0.1 µM) in the rBM-MSCs. Expression of Nrf-2 and Selenium dependant antioxidant enzymes, such as SOD, GPX, and TrxR, increased compared with the control group. The level of AKT, P, increased after 72 h treatment with 0.1 µM sodium selenite. Data are represented as mean ± SD* p < 0.05, ** p < 0.01 significant level of treatment (T) vs. control (C) group. C1,2, and 3 are the three replicates of the control groups and T1,2, and 3 are three replicates of selenium (0.1 µM)treatment group. Full length blots are presented in the supplementary data. Number of samples = 3, Data: Mean ± SD

Fig. 8

Schematic illustration of Nrf2 signaling pathway in oxidative stress (A) and protein network STRING (B). Nrf2-related signaling pathway plays a key role in directly regulating oxidative stress signaling pathway by overexpression of antioxidant enzymes (TrxR, SOD, GPX). Nrf2 is regulated by Keap1 in constitutive/oxidative conditions. Proteins coded light green were investigated in the present study, and those coded in dark green are bioinformatic predictions of the next layers in the network. The light green proteins was selected based theit ipmrtance in the siganling pathways, shown by the previously published reports (summerized in Table 3). The importance of these proteins were also confirmed by our by our bioinformatic analysis and pathway investigation by the String tool. A There are meaningful interactions among proteins (Nrf2, SOD, TrxR, GPX, SIRT1, AKT, P38, HIF1) based on the network and pathway analysis by STRING online tool. The different color lines show various interaction types among proteins on the system level. Dotted lines indicate inter-cluster and straight lines show cluster association (B)

Fig. 9

Supplementation of rBM-MSCs culture with sodium selenite caused Nrf2 overexpression, reduced the ROS level, improved cytoprotection by regulating the expression of HIF-1 of AKT, SOD, GPX, and TrxR markers and enhanced the expression of stemness related OCT-4, Sox2, Nanog. Number of samples = 3, Data: Mean ± SD