Mechanisms of naozhenning in inhibiting neuronal ferroptosis and alleviating brain recurrent mild traumatic brain injury in a rat model.

Abstract

BACKGROUND: Recurrent mild traumatic brain injury (rmTBI) represent a significant predisposing factor for the development of neurodegenerative diseases, including but not limited to Parkinson's disease and Alzheimer's disease. However, its underlying pathological mechanisms remain unclear, and there are currently no definitive clinical treatments available. Naozhenning, an empirical formula used to treat brain concussions, comprises multiple medicinal components and exhibits a potential for treating rmTBI. Nonetheless, its material basis and efficacy for rmTBI alleviation are still unknown. PURPOSE: This research aims to determine the role of ferroptosis in hippocampal neurons following rmTBI and to elucidate Naozhenning's neuroprotective mechanisms towards rmTBI. METHODS: A mild cerebral concussion (MCC) rat model was established using a free-fall injury method. To evaluate the extent and temporal progression of hippocampal ferroptosis, different impact heights and various post-injury time points (48 h, 7, 14, and 21 days) were tested. The changes associated with ferroptosis were assessed using Prussian blue staining, transmission electron microscopy (TEM), and Western blotting. Upon identification of the peak period of neuronal ferroptosis, Naozhenning was introduced to assess its regulatory effects. Subsequently, a comprehensive "drug-component-target-pathway" network was systematically constructed by integrating high-resolution liquid chromatography-mass spectrometry, network pharmacology, and molecular docking. The core active components and key molecular targets predicted by this network were then experimentally validated through in vitro assays. RESULTS: Hippocampal neuronal ferroptosis in MCC model was positively associated with both injury severity and duration. Naozhenning significantly alleviated neuronal injury and improved learning and spatial memory in rats, exerting its effect primarily through the suppression of hippocampal ferroptosis. Integrated chromatographic and computational analyses revealed that quercetin and lutein as key bioactive constituents in Naozhenning. Molecular docking and cellular assays confirmed that these compounds inhibit ferroptosis by modulating the HMOX1 protein. CONCLUSION: Our findings demonstrate that ferroptosis underlies rmTBI pathogenesis, and that Naozhenning confers neuroprotection by mitigating hippocampal neuronal injury and improving cognitive function in MCC rats, primarily through the inhibition of ferroptosis via the actions of quercetin and luteolin on HMOX1.