Cerebral ischemia-reperfusion injury (CIRI), a subsequent injury caused by thrombolytic reperfusion post ischemic stroke (IS). Naotaifang (NTF) formula, a novel traditional Chinese medicine (TCM) remedy against IS, was shown to exert beneficial effects in inhibiting inflammation and inhibiting lipid peroxide synthesis in our previous research. This study aimed to further explore the role of NTF in attenuating oxygen-glucose deprivation//reoxygenation (OGD/R)-induced inflammation and ferroptosis by regulating microglial M1/M2 polarization through the bone morphogenetic protein 6（BMP6）/SMADs signaling pathway. BV2 microglia were used to establish an OGD/R model. The effects of NTF on inflammation and ferroptosis in OGD/R-injured BV2 cells were separately detected by immunofluorescence assay, fluorescent probe, DCFH-DA flow cytometry, enzyme-linked immunosorbent assay, and western-blot. The present results revealed that the M1 phenotype of microglia promoted the secretion of pro-inflammatory cytokines and aggravated ferroptosis and brain damage following OGD/R. However, an inhibitor of BMP6, LND-193189, reversed the aforementioned effects. Similarly, NTF promoted the shift of microglia from M1 to M2. Besides, NTF treatment effectively inhibited the expression of hepcidin, BMP6, SMADs and promoted the expression of ferroportin (FPN, SLC40A1) and γ-L-glutamyl-L-cysteinylglycine (glutathione or GSH) peroxidase 4 (GPX4). Microglial M1/M2 polarization plays a pivotal role in inflammation and ferroptosis during OGD/R. The BMP6/SMADs signaling pathway is a potential therapeutical target of inflammation and ferroptosis induced by the transformation of microglia. Moreover, NTF could alleviate inflammation and ferroptosis through the BMP6/SMADs signaling pathway in OGD/R-injured microglia.

Introduction: This study aimed to investigate whether activating the Nrf2/TFAM pathway boosts mitochondrial biogenesis, reduces ferroptosis in ischemic stroke (IS), and evaluates Naotaifang (NTF) formula's therapeutic potential. Methods: Ferroptosis and mitochondrial biogenesis indicators were measured at various time points following MCAO. Various methods, including transmission electron microscopy, immunofluorescence assay, enzyme-linked immunosorbent assay, Western blotting assays, and real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR), were employed to evaluate the impact of NTF on mitochondrial biogenesis and ferroptosis in vivo and in vitro. Results: IS significantly inhibits mitochondrial biogenesis and increases neuronal ferroptosis, with brain damage worsening over time. MCAO groups showed reduced expression of Nrf2, TFAM, ATP, CISD2, FPN, GPX4, SOD, and HO-1, alongside elevated Fe²⁺, ROS, and LPO (P < 0.05) compared to the control group. Both sulforaphane and NTF treatment reversed these effects; NTF treatment effectively increased the expression of Nrf2, TFAM, FtMt, CISD1, CISD2, FPN, and GPX4 while inhibiting the levels of Fe2+, ROS, and LPO. (P < 0.05). Discussion: This finding clarifies mitochondrial biogenesis's crucial role, proposes a new "pathway + molecule" strategy for IS treatment, and supports NTF's clinical potential, though larger animal models and long-term safety studies are needed. Conclusion: In the context of IS, reduced mitochondrial biogenesis plays an important role in ferroptosis. Targeting the Nrf2/TFAM signaling pathway may improve mitochondrial biogenesis in IS. Furthermore, NTF can mitigate ferroptosis by promoting mitochondrial biogenesis through the Nrf2/TFAM signaling pathway