Antioxidant and Neuroregenerative Properties of hDPSC Secretome Identified by LC-MS/MS Proteomics in Ischemic Stroke Models

Abstract

Ischemic stroke causes extensive neuronal damage due to oxidative stress, mitochondrial dysfunction, and apoptotic cell death. The secretome derived from human dental pulp stem cells (hDPSCs) has emerged as a promising paracrine therapeutic candidate, offering antioxidant and neuroregenerative potential. In this study, we performed a comprehensive proteomic analysis of the hDPSC secretome using LC-MS/MS to elucidate its mechanism of action, with a focus on its effects across multiple models of ischemia-induced neural injury, including HT22 hippocampal neurons, BV-2 microglial cells, and mouse brain cortex and hippocampus tissues. Proteins identified from the hDPSC secretome were compared with those present in serum-free DMEM to isolate high-confidence factors, resulting in the selection of 346 proteins detected consistently in at least two biological replicates. Gene Ontology (GO) enrichment analysis revealed that these proteins were strongly associated with extracellular vesicles, extracellular matrix structural components, and antioxidant activity. Molecular function and pathway enrichment pointed to regulation of oxidative stress, apoptosis, and neurogenesis, with significant representation in processes such as “wound healing,” “response to reactive oxygen species,” and “negative regulation of apoptotic process.” In HT22 hippocampal neurons subjected to hypoxic stress, proteomic analysis of the hDPSC secretome revealed DEPs associated with mitochondrial homeostasis. Notably, components related to electron transport and mitochondrial permeability transition were reduced, suggesting mitigation of mitochondrial ROS generation. In parallel, proteins linked to mitophagy regulation were also identified, indicating a potential role of the secretome in modulating mitochondrial turnover. These observations support the role of the hDPSC secretome in reducing oxidative stress and suppressing mitophagy-associated apoptotic pathways under hypoxic conditions. In BV-2 microglial cells exposed to hypoxic conditions, proteomic analysis indicated that the hDPSC secretome modulated not only components of the TNF signaling pathway, a key mediator of neuroinflammatory responses, but also pathways related to the cell cycle and oxidative phosphorylation. These findings suggest that the secretome attenuates pro-inflammatory activation and supports a shift toward an anti-inflammatory, M2-like microglial phenotype. In ischemia-affected cortex and hippocampus tissues, proteomic signatures of the hDPSC secretome included factors involved in extracellular matrix remodeling and synaptic maintenance. In the hippocampus specifically, a reduction in ROS levels was observed, potentially mediated by antioxidant enzymes and the regulation of mitophagy-related pathways. These findings point to a coordinated mechanism through which the secretome supports mitochondrial stability and reduces oxidative damage in ischemic brain regions. In summary, LC-MS/MS-based proteomic profiling of the hDPSC secretome reveals a diverse array of neuroprotective proteins that collectively regulate oxidative stress, mitochondrial dynamics, and inflammatory signaling across multiple ischemia-relevant models. These findings highlight the therapeutic potential of the hDPSC secretome as a cell-free strategy for treating ischemic stroke and provide a molecular framework for its neuroprotective effects.