Nrf2 overexpression reprograms neural stem cell fate: promoting neuronal differentiation and functional recovery post-ischemic stroke via suppression of the ROS/NF-κB axis

Neural stem cells (NSCs) have been experimentally used in multiple models and patients, offering great potential for the treatment of neurological disorders such as ischaemic stroke. However, the proliferative and differentiative limitations of NSCs transplants must be overcome to further exploit the clinical potential of NSCs-based therapies. This study aimed to elucidate the regulatory role of Nrf2 in NSCs proliferation and neuronal differentiation in vitro, to evaluate the therapeutic potential of transplanting Nrf2-overexpressed NSCs for functional recovery in the MCAO mouse model, and to further explore the synergistic benefits of combining this cell therapy with the pharmacological Nrf2 agonist PTE. The study design encompassed in vitro NSC cultures and a mouse MCAO model, involving Nrf2 modulation, transplantation of engineered NSCs, pharmacological activation with PTE, and comprehensive assessment of cellular, molecular, and functional outcomes. Specifically, Nrf2 expression was manipulated using AAV-9. Nrf2-overexpressed NSCs were generated via AAV-9 transduction and stereotaxically transplanted into the lateral ventricle of mice with MCAO, with a subset of these animals additionally receiving the Nrf2 agonist PTE. Nestin, Dcx, BrdU, NeuN, and GFAP expression were evaluated using immunofluorescence; GFAP-positive and NeuN-positive cell proportions were quantified by flow cytometry. Oxidative stress was monitored using DCF fluorescence and MDA ELISA kits. Inflammation was measured with IL-6 and TNF-α ELISA kits. The expression levels of Nrf2, HO-1, NQO1, p65, and NLRP3 were evaluated using western blotting. Neurological deficits were assessed using Clark scores, rotarod test, Morris water maze, grip strength test, cerebral water content measurement, and TTC staining for infarction volume. Overexpression or knockdown of Nrf2 in vitro (in NSCs) and in vivo (in mouse hippocampus) correspondingly regulated the expression of its downstream effectors, HO-1 and NQO1. In vitro, Nrf2 regulated the proliferation and neuronal differentiation of NSCs. In the MCAO mouse model, Nrf2 ameliorated cerebral oxidative stress and inflammation, and improved functional recovery, as evidenced by improved neurological scores, enhanced performance in behavioural tests, reduced infarction volume, and attenuated cerebral oedema. Transplantation of Nrf2-overexpressed NSCs promoted the proliferation and neuronal differentiation of the grafts, enhanced their neuroprotective effects, and led to superior recovery compared to standard NSCs transplantation. Furthermore, administration of the Nrf2 agonist PTE synergized with Nrf2-overexpressed NSCs transplantation, resulting in augmented anti-inflammatory, antioxidant, and functional benefits. This study demonstrates that Nrf2 is a key regulator of NSCs proliferation and neuronal fate, both intrinsically and following transplantation. Furthermore, the combination of Nrf2-overexpressed NSCs transplantation and PTE administration synergistically amplified therapeutic outcomes, offering a promising combinatorial strategy to enhance neurorepair after ischemic stroke.