Hydrogen sulfide-modified extracellular vesicles from mesenchymal stem cells for treatment of hypoxic-ischemic brain injury

We previously reported that preconditioning of mesenchymal stem cells (MSCs) with hydrogen sulfide (H₂S) improved their therapeutic potential in cerebral ischemia. However, the mechanisms involved with this effect have not been determined. As one approach to address this issue, we focused on a neuroprotective role of modification of MSCs-derived extracellular vesicles (EVs) with H₂S treatment, and further examined the underlying mechanisms during hypoxia-ischemia (HI) injury in neonatal mice. At 24 h following HI insult, neonatal mice received either systemically administered EVs (derived from MSCs) or H₂S-EVs (derived from NaHS-preconditioned MSCs). Both treatments reached the injured region of the ipsilateral hemisphere within 2 h after administration and were incorporated into microglia and neurons. Mice receiving H₂S-EVs exhibited substantially lower amounts of brain tissue loss, decreased levels of pro-inflammatory mediators, and a skewed distribution of CD45^low microglia and CD45^high brain mononuclear phagocytes toward a more anti-inflammatory condition as compared with that in mice receiving only EVs. Moreover, these neuroprotective and anti-inflammatory effects of H₂S-EVs were accompanied with long-term preservation of cognitive and memory functions, in contrast to the functional deficits observed in mice receiving only EVs. This H₂S preconditioning upregulated miR-7b-5p levels in EVs as determined with next-generation sequencing, while knockdown analyses revealed that inducing miR-7b-5p expression and targeting FOS in the ipsilateral cortex were essential for the neuroprotective and anti-inflammatory effects of H₂S-EVs following HI exposure. Taken together, these results demonstrate that miR-7b-5p transferred by H₂S-EVs into the ipsilateral hemisphere further induced miR-7b-5p expression, which promoted CD45^low microglia and CD45^high brain mononuclear phagocytes toward a beneficial phenotype and improved HI-induced cognitive impairments in neonatal mice.