SHLP2 restores pre-osteoblastic cells against oxidative stress-induced inflammaging

Abstract

This study aimed to evaluate the cytoprotective effects of the mitochondrial-derived peptide, Small Humanin-Like Peptide-2 (SHLP2), on pre-osteoblastic cells exposed to sub-toxic oxidative stress, with the aim of preserving bone homeostasis under conditions of inflammaging. Pre-osteoblastic MC3T3-E1 cells were cultured under sub-toxic oxidative stress induced by 600 mu M H2O2. The study evaluated the effects of SHLP2 (at 10 mu M concentration) through assays for mitochondrial activity, reactive oxygen species (ROS) generation, apoptosis markers, and osteogenic differentiation. Quantitative polymerase chain reaction, alkaline phosphatase (ALP) staining, and Alizarin Red S biomineralization assays were performed to assess gene expression, osteogenic activity, and biomineralization. Oxidatively stressed but untreated cells served as the positive control (PC), while oxidative stress-free cells were used as the mock control. Statistical analyses were performed using one-way ANOVA and t-tests. SHLP2 treatment significantly (p < 0.001) improved cell viability and reduced ROS activity in oxidatively stressed cells. A significant (p < 0.001) decrease in apoptotic markers, including p53 and BAX, and an increase in anti-apoptotic BCL-2 levels, were observed. Additionally, SHLP2 treatment upregulated key osteogenic markers, including RUNX2, OSX, and ALP, compared to PC. When compared to the mock group, SHLP2 restored ALP activity to 95.6% by day 14. By day 21, the biomineralization assay demonstrated 94.92% activity following SHLP2 treatment. SHLP2 treatment effectively mitigates oxidative stress in pre-osteoblastic cells, providing apoptosis protection and preserving osteogenic activity. These findings underscore the potential of SHLP2 as an adjuvant therapeutic agent for enhancing the tissue microenvironment in conditions such as periodontitis and inflammaging.