Hehuan Anshen decoction inhibits ferroptosis to ameliorate p-Chlorophenylalanine-induced insomnia by activating GPX4 pathway

• Hehuan Anshen decoction (HHASD) alleviates p-Chlorophenylalanine-induced insomnia in mice. • HHASD inhibits ferroptosis of hypothalamus in insomniac mice. • HHASD improves ferroptosis by GPX4 pathway activation. Insomnia is a chronic disease affecting a third of the global adult population. 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). The active constituents of HHASD were analysed by means of UPLC HRMS. PCPA mice were administered HHASD orally for 7 days. The anti-insomnia phenotype of HHASD were assessed by 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 biochemistry, genetics and morphology. Additionally, ferroptosis inhibitor RSL3 was used to observe the underlying mechanism of the effects of HHASD. HHASD could effectively improve the insomnia behaviors induced by PCPA, resulting in increased movement trajectories, 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 PCPA mice. Mechanistic analysis revealed that HHASD could reverse the hypothalamic ferroptosis phenotype of insomnia mice by restoring the lowered levels of glutathione (GSH), 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. HHASD ameliorates insomnia-related behaviors and protects against neuronal damage by suppressing ferroptosis by regulation GPX4 signaling. This study is not only a valuable attempt to explore the potential mechanism of Traditional Chinese formula but also will provide a novel targeted therapy for the treatment strategy of insomnia.