NPB-1575 attenuates neuroinflammation and resists ferroptosis in rat ischemic stroke via IRS2 signaling pathway

Ischemic stroke is one of the most common forms of stroke. There are no effective pharmacological agents to promote recovery yet. Dendrobium is a valuable herb in traditional Chinese medicine, which has shown antiviral, anti-inflammatory, antioxidant and immunomodulatory activities. We previously used dendrobium to synthesize a novel bibenzyl compound NPB-1575 with the ability to scavenge free radicals in vitro. Increasing evidence shows that IRS2 has biological functions other than participating in the insulin signaling pathway. In this study we utilized NPB-1575 to explore the role of IRS2 in neuroinflammation and ferroptosis during cerebral ischemia. Rats were subjected to permanent middle cerebral artery occlusion (pMCAO) surgery. NPB-1575 (25 mg/kg) was orally administered to the rats 5 min to 4 h after the surgery. We showed that NPB-1575 administration significantly reduced the infarct volume and improved neurological outcome at different stages of ischemic stroke in pMCAO rats. In the brain tissue of pMCAO rats and LPS-stimulated BV2 cells, we demonstrated that NPB-1575 exerted the anti-inflammatory effect on microglia through upregulating Nrf2 and inhibiting FOXO1 via IRS2. NPB-1575 might effectively interact with IRS2 to enhance the stability of IRS2 protein. Knockdown of IRS2 in BV2 cells reversed the protective effect of NPB-1575 against cerebral ischemic injury, manifested by upregulated NF-κB and inactivated ferroptosis defense system. In conclusion, we demonstrate that IRS2 might be a novel target in regulation of neuroinflammation and played an indispensable role in anti-ischemic injury of the brain. NPB-1575 mitigates neuroinflammation and resists ferroptosis through the IRS2/Nrf2/NF-κB axis, demonstrating its potential therapeutic effects on ischemic stroke. Cerebral ischemic injury leads to the translocation of NF-κB into the nucleus of microglia, which initiates the transcription of inflammatory genes and further releases inflammatory factors. The release of NO, IL-6, TNFα further leads to ferroptosis of neurons, which is manifested as iron overload and weakened resistance of the Xct system. NPB-1575 up-regulates IRS2, which further up-regulates pAKT/AKT and Nrf2 signaling way, down-regulates FOXO1 and NF-κB. Then the reduced microglial inflammatory response reduced neuronal ferroptosis.