Orthosiphon stamineus Proteins Alleviate Hydrogen Peroxide Stress in SH-SY5Y Cells

The neuroprotective potential of Orthosiphon stamineus leaf proteins (OSLPs) has never been evaluated in SH-SY5Y cells challenged by hydrogen peroxide (H₂O₂). This work thus aims to elucidate OSLP neuroprotective potential in alleviating H₂O₂ stress. OSLPs at varying concentrations were evaluated for cytotoxicity (24 and 48 h) and neuroprotective potential in H₂O₂-induced SH-SY5Y cells (24 h). The protective mechanism of H₂O₂-induced SH-SY5Y cells was also explored via mass-spectrometry-based label-free quantitative proteomics (LFQ) and bioinformatics. OSLPs (25, 50, 125, 250, 500, and 1000 µg/mL; 24 and 48 h) were found to be safe. Pre-treatments with OSLP doses (250, 500, and 1000 µg/mL, 24 h) significantly increased the survival of SH-SY5Y cells in a concentration-dependent manner and improved cell architecture-pyramidal-shaped cells, reduced clumping and shrinkage, with apparent neurite formations. OSLP pre-treatment (1000 µg/mL, 24 h) lowered the expressions of two major heat shock proteins, HSPA8 (heat shock protein family A (Hsp70) member 8) and HSP90AA1 (heat shock protein 90), which promote cellular stress signaling under stress conditions. OSLP is, therefore, suggested to be anti-inflammatory by modulating the "signaling of interleukin-4 and interleukin-13" pathway as the predominant mechanism in addition to regulating the "attenuation phase" and "HSP90 chaperone cycle for steroid hormone receptors" pathways to counteract heat shock protein (HSP)-induced damage under stress conditions.