Liver isoform of phosphofructokinase (PFKL)-Mediated Hypoxic Preconditioned Bone Marrow-Derived Mesenchymal Stem Cells Attenuate Cardiac Arrest-Induced Pyroptosis in Rat Cortical Neurons by Protecting Mitochondrial Function from Oxidative Damage

Abstract

Abstract Introduction: Cardiac arrest (CA) often leads to severe neurological dysfunction due to inflammation, mitochondrial dysfunction, and post-cardiopulmonary resuscitation (CPR) neurological damage. Bone marrow-derived mesenchymal stem cells (BMSCs) show promise for neurological diseases, but optimizing their therapeutic potential and neuroregulation post-CA remains unclear. Methods: We established an in vitro co-culture model with BMSCs and post-oxygen-glucose deprivation (OGD) primary neurons, confirming that hypoxic preconditioning enhances BMSCs' resistance to neuronal pyroptosis. We induced an 8-minute CA model through asphyxia induction and assessed hypoxic preconditioned bone marrow-derived mesenchymal stem cells (HP-BMSCs) on post-resuscitation neuronal mitochondrial oxidative stress and pyroptosis using neurological deficit scores (NDS), brain tissue oxidative stress markers, apoptosis-related proteins, mitochondrial area, and damage markers. Mechanistic studies knocked down PFKL expression in HP-BMSCs via si-RNA, verifying potential mechanisms in animals and cells. Results: Hypoxic preconditioning boosted BMSCs' neuroprotective effect against neuronal pyroptosis, possibly through MAPK and NF-κB pathway inhibition. Consequently, we pursued HP-BMSCs as a neuroprotection strategy, with RNA sequencing suggesting liver isoform of phosphofructokinase (PFKL) as a regulatory molecule. HP-BMSCs significantly reduced neuronal pyroptosis, oxidative stress, and mitochondrial damage induced by CA. This manifested as improved oxidative stress markers, decreased apoptosis-related protein levels, enhanced cell membrane and mitochondrial structures, and reduced mitochondrial damage markers. Transfection of PFKL-targeted si-RNA into HP-BMSCs weakened their protective effects. We also established an in vitro co-culture model to confirm HP-BMSCs' role in improving neuronal energy metabolism following OGD. HP-BMSCs lowered apoptosis-related protein levels and mitochondrial damage markers in primary neurons. Intracellular and mitochondrial reactive oxygen species (ROS) levels dropped, as detected by DCFH-DA and MitoSOX probes. Notably, knocking down PFKL expression in HP-BMSCs reversed these protective effects. Conclusion: In conclusion, HP-BMSCs offer a promising therapeutic approach for brain injury post-CA by reducing cell pyroptosis mediated by mitochondrial ROS, potentially linked to elevated PFKL expression following hypoxic preconditioning.