Hehuan Anshen Decoction Inhibits Hypothalamic Ferroptosis to Ameliorate p-Chlorophenylalanine-induced Insomnia

Abstract Insomnia is a highly prevalent health condition with a global impact. The hypothalamus, a crucial brain region governing sleep-wake cycles, plays a pivotal role in the manifestation of insomnia. Perturbation in the hypothalamus correlates with the development of insomnia by inducing neuronal demise. Furthermore, emerging evidence acknowledges the involvement of ferroptosis, a new form of programmed cell death, in various neurological disorders. Hehuan Anshen Decoction (HHASD), an innovative formula derived from Traditional Chinese medicine, has demonstrated therapeutic efficacy in treating insomnia, however, the potential pharmacological mechanism underlying its anti-insomnia effects remains incompletely elucidated. This study aimed to explore the underlying mechanism of HHASD treatment in mice with insomnia induced by p-Chlorophenylalanine (PCPA). PCPA-treated insomnia mice were administered HHASD orally for 7 days. The main constituents of HHASD were identified by high-performance liquid chromatography (HPLC). The anti-insomnia effects of HHASD were assessed through behavioral tests, encompassing the open field test and pentobarbital sodium-induced sleep test, alongside the measurement of hypothalamic 5-HT levels. Then, we conducted an in-depth analysis of specific ferroptosis markers, considering morphology, biochemistry, and genetics. The results demonstrated that HHASD could effectively improve the insomnia phenotype induced by PCPA, resulting in decreased sleep latency and prolonged sleep duration. Specifically, HHASD exerted a neuroprotective effect by enhancing the integrity of Nissl bodies in the hypothalamus of the insomnia mouse modeling. Mechanistic analysis revealed that HHASD could reverse the hypothalamic ferroptosis phenotype of insomnia mice by restoring the lowered levels of glutathione (GSH) and superoxide dismutase (SOD), inhibiting iron accumulation and elevated malondialdehyde (MDA), and mitigating mitochondrial cristae damage. Furthermore, HHASD enhanced the expression of SLC7A11 and GPX4 and reduced the ASCL4 in the hypothalamus, while the anti-insomnia effect of HHASD in the PCPA mice was eliminated by the GPX4 inhibitor RLS3. In summary, HHASD ameliorates insomnia-related behaviors and protects against neuronal damage by suppressing hypothalamic ferroptosis.