Walnut Kernel Oil and Defatted Extracts Enhance Mesenchymal Stem Cell Stemness and Delay Senescence

Abstract

Decreased stemness and increased cellular senescence impair the ability of mesenchymal stem cells (MSCs) to renew themselves, change into different cell types, and contribute to regenerative medicine. There is an urgent need to discover new compounds that can boost MSCs' stemness and delay senescence. Therefore, this study aimed to investigate the impact of walnut kernel oil (WKO) and defatted (WKD) extracts on bone marrow (BM)-MSC stemness and senescence. Premature senescence and inflammation were induced in BM-MSCs using H₂O₂ and LPS, respectively. Phytochemical constituents of WKO and WKD extracts were detected by HPLC. The stemness (proliferation and migration), senescence-related markers (p53, p21, SIRT1, and AMPK), oxidative stress/antioxidant markers, inflammatory cytokines, and cell cycle of BM-MSCs were measured by MTT assay, qPCR, ELISA, and flow cytometry. WKO and WKD extracts improved rat BM-MSC stemness, as evidenced by (1) increased cell viability, (2) decreased apoptosis (low levels of <i>Bax</i> and caspase3 and high levels of <i>Bcl2</i>), (3) upregulated <i>MMP9</i> and downregulated <i>TIMP1</i> expression, and (4) cell cycle arrest in the G0/G1 phase and declined cell number in the S and G2/M phases. Additionally, WKO and WKD extracts reduced rat BM-MSC senescence, as indicated by (1) decreased <i>p53</i> and <i>p21</i> expression, (2) upregulated expression and levels of SIRT1 and AMPK, (3) reduced levels of ROS and improved antioxidant activity (higher activity of CAT, SOD, and GPx and upregulated expression of <i>NrF2</i> and <i>HO-1</i>), and (4) declined levels of TNFα, IL1β, and NF-κB. When compared to the WKO extract, the WKD extract had a greater impact on the induction of stemness and reduction of senescence of BM-MSCs due to its stronger antioxidant activity, which could be attributed to its higher levels of flavonoids and phenolic compounds, as detected by HPLC analysis. WKO and WKD extracts enhance rat BM-MSC stemness and protect them from senescence, suggesting their potential use as enhancers to increase MSCs' therapeutic efficacy.