Thymoquinone regulates RSL3-induced ferroptosis in the alzheimer mouse hippocampus

Abstract

Introduction: Ferroptosis, a type of iron-dependent regulated cell death driven by lipid peroxidation, has emerged as a critical contributing factor to hippocampal neurodegeneration in Alzheimer's disease (AD). Dysregulated iron homeostasis exacerbates oxidative injury, impairing cognitive function. This study provides experimental evidence that thymoquinone (TQ) exerts neuroprotective effects in an AD-related ferroptosis model by modulating iron, lipid, and glutathione metabolism.

Methods: Thirty female C57BL/6 mice (8-10 weeks, 18-22 g) were randomly allocated into five groups: 1- Control, 2- artificial cerebrospinal fluid (aCSF; 20 ng/L), 3- RAS-selective lethal 3 (RSL3) (100 ng/10 µL in aCSF), 4- RSL3 + TQ (50 mg/kg in 1.0% w/v acacia gum), and 5- TQ alone (50 mg/kg in 1.0% w/v acacia gum). A bilateral hole was drilled according to the Bregma coordinates (0.5 mm back, 1 mm left-right, 2,5 mm deep) and a 10 µl Hamilton syringe was inserted perpendicularly from the skull to the brain. RSL3 (100 ng/10 µl) was infused with 4 µl (Intracerebral-IC). Cognitive and anxiety-like behaviors were assessed using the Morris Water Maze (MWM) and the Open Field Test (OFT). Hippocampal oxidative markers, total antioxidant status (TAS), and total oxidant status (TOS), were determined by spectrophotometric quantification. GPx4, XCT, and Fpn mRNA levels were measured by qRT-PCR. Protein expression and distribution were assessed by Western blotting and immunohistochemistry. Iron accumulation was visualized using Perl's Prussian blue staining method.

Results: RSL3-induced ferroptosis impaired spatial learning and increased anxiety-like behavior, accompanied by elevated hippocampal iron accumulation, oxidative stress, and downregulation of GPx4, XCT, and Fpn. TQ treatment significantly restored TAS levels, reduced TOS, suppressed iron deposition, and reversed ferroptosis-associated molecular changes, upregulating GPx4 and Fpn while attenuating XCT downregulation (P < 0.05). Ferroptosis-induced behavioral deficits were significantly improved by TQ.

Conclusion: These findings identify TQ as a potent modulator of ferroptosis, acting through coordinated regulation of iron export, lipid peroxidation, and glutathione-dependent antioxidant defenses. TQ targeting convergent ferroptosis pathways is a promising therapeutic candidate to alleviate hippocampal neurodegeneration and cognitive decline in AD.

Keywords: Alzheimer’s disease; Anxiety; Ferroptosis; Fpn; GPx4; Iron deposition; Memory; XCT.