Bushen Mingmu Wan attenuates excitotoxic retinal injury and ferroptosis-associated pathology with involvement of CYP1B1-related PI3K/AKT signaling.

OBJECTIVE: This study investigated whether Bushen Mingmu Wan (BSMMW) attenuates excitotoxic retinal injury and explored the involvement of CYP1B1-related PI3K/AKT signaling in ferroptosis-associated pathological changes. METHODS: The chemical profile of the tested BSMMW batch was characterized by UPLC-Q-TOF-MS. Candidate glaucoma-related targets were screened using network-based analysis and molecular docking. The predicted biological response was then examined in orally treated rats with NMDA-induced retinal injury and in glutamate-injured R28 cells directly exposed to BSMMW. Retinal structure, Brn3a-positive RGC density, R28 cell viability, and cell death-related changes were evaluated by H&E/PAS staining, retinal flat-mount Brn3a counting, Brn3a immunofluorescence, TUNEL staining, Hoechst/PI staining, CCK-8 assay, and LDH release assay. Ferroptosis-associated alterations were assessed using electron microscopy, Prussian blue staining, GPX4 immunostaining, and GPX4/FTH1 detection by RT-qPCR and western blotting. CYP1B1 gain- and loss-of-function experiments, together with PI3K inhibition, were used to examine the involvement of CYP1B1-related PI3K/AKT signaling. RESULTS: UPLC-Q-TOF-MS provided a preliminary chemical profile of the tested BSMMW batch. Target screening and docking prioritized CYP1B1 as a candidate BSMMW-associated target for experimental investigation. In NMDA-injured rats, oral BSMMW administration reduced retinal structural damage, preserved Brn3a-positive RGC density, decreased TUNEL-positive cells, and improved ferroptosis-associated retinal abnormalities, including mitochondrial damage, iron deposition, and loss of GPX4 and FTH1 expression. In R28 cells directly exposed to BSMMW, cell viability and Brn3a expression were increased, whereas PI staining intensity and LDH release were reduced, accompanied by recovery of GPX4 and FTH1 expression. BSMMW restored CYP1B1 expression and increased the p-PI3K/PI3K and p-AKT/AKT ratios. CYP1B1 knockdown weakened the BSMMW-associated increase in PI3K/AKT phosphorylation and protective response, whereas PI3K inhibition attenuated the BSMMW-associated recovery of cell viability and ferroptosis-related proteins. CONCLUSION: BSMMW attenuated NMDA-induced retinal injury and glutamate-induced R28 cell injury, accompanied by reduced ferroptosis-associated pathological changes and restoration of CYP1B1-related PI3K/AKT signaling. CYP1B1 knockdown or PI3K inhibition weakened the BSMMW-associated protective response, supporting the involvement of CYP1B1-related PI3K/AKT signaling in this response.