Sleep is a fundamental physiological process essential for maintaining nervous system function and immune homeostasis, with sleep homeostasis regulation involving a bidirectional interplay with the neuroimmune network [1, 2]. Clinical and animal studies suggest that disrupting sleep homeostasis can activate the immune system, exacerbate the increase in peripheral proinflammatory cytokines, and stimulate microglial activation within the central nervous system (CNS), thereby increasing the risk of infectious, autoimmune, and neurodegenerative diseases [3, 4]. Microglia, the primary immune cells in the brain, regulate wake-sleep cycles by modulating neuronal activity in specific brain regions [5, 6]. For instance, the activation of inhibitory G-protein signaling within microglia has been shown to promote sleep. Conversely, sleep states can regulate the morphology of microglia. Notably, animal studies have associated enhanced neuronal activity during wakefulness with elevated microglial branch extension and dynamic synaptic interactions, whereas microglia exhibit a more quiescent morphology during sleep. These findings closely associate microglial morphology with synaptic plasticity during the sleep-wake cycle [7, 8, 9]. Microglia undergo a morphological shift toward a proinflammatory phenotype, characterized by reduced branching and increased phagocytic activity, during sleep deprivation (SD). This shift, along with inflammatory infiltration in the meninges and brain parenchyma, reveals that sleep states influence immune responses by regulating microglial morphology [10]. Therefore, exploring approaches to maintain sleep homeostasis through immune system modulation provides a theoretical foundation for developing “anti-inflammatory–sleep-promoting” intervention strategies.

Quercetin, a natural flavonoid abundantly present in Chinese herbal medicines, is well known as its anti-inflammatory, antioxidant, and neuroprotective properties [11, 12]. Recent study has shown that quercetin nanoparticles can modulate the gut-brain axis, attenuate the translocation of proinflammatory cytokines into the brain, and improve sleep quality [13]. Additionally, quercetin has been reported to alleviate SD-induced behavioral alterations by inhibiting the inflammatory pathway in the hippocampus and restoring brain-derived neurotrophic factor-mediated neuroplasticity in SD models [14]. The anti-neuroinflammatory effects of quercetin mainly include suppression of the nuclear factor-$\kappa$B and mitogen-activated protein kinase signaling pathways, reduction in proinflammatory factor levels (including interleukin [IL]-1$\beta$, tumor necrosis factor [TNF]-$\alpha$, and nitric oxide [NO]), and upregulation of anti-inflammatory cytokines such as IL-10 [15]. Furthermore, quercetin has been shown to alleviate SD-induced hippocampal neuronal pyroptosis by inhibiting the activation of the nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3) inflammasome [16], thereby enhancing cognitive function through microglial regulation in the hippocampus [17]. Notably, quercetin regulates microglial activation to improve memory impairment, highlighting its significant neuroprotective role [18, 19]. Hence, the inhibitory effect of quercetin on microglia may serve as a central mechanism linking immune regulation and sleep improvement [20].

This study aimed to employ polysomnography and SD paradigms to systematically investigate quercetin-mediated reshaping of sleep architecture in mice under both basal and homeostatic imbalance conditions. Additionally, the effects of quercetin on brain region-specific neuronal activity and microglial dynamics were investigated to elucidate the “natural compound–neuroimmune–sleep homeostasis” regulatory axis and provide mechanistic insights for interventions in sleep disorders.

This study aimed to systematically elucidate the regulatory effects of quercetin in sleep homeostasis and its underlying neuroimmunological mechanisms. Notably, quercetin exerts an NREM sleep-promoting effect under basal conditions, and after ASD, it modulated sleep architecture, thereby suppressing excessive neuronal activation in specific brain regions and alleviating microglial inflammatory responses. Altogether, these findings provide novel insights into the mechanisms underlying the natural compound-mediated regulation of sleep and neuroimmunity.

Due to its limited blood–brain barrier (BBB)-penetrating ability, quercetin requires higher doses in studies on CNS mechanisms in mice compared to those in peripheral anti-inflammatory/antioxidant experiments. According to reports, doses of 10–200 mg/kg are considered effective for quercetin-mediated regulation of sleep-wake cycles in rodents [21, 22]. Recent study on regulating CNS-mediated sleep regulation has consistently used a dose of 200 mg/kg, which effectively targets brain neurotransmitter systems to modulate sleep [23]. Regarding safety, a toxicity study on the hydroethanolic extract of Dolichandra unguis-cati L. leaves showed that a single, high-dose oral administration did not cause mortality or abnormal behaviors in male and female rats over 14 d. Furthermore, the rats exhibited normal weight gain, with no pathological abnormalities in core indicators of liver and kidney function [24]. Based on these findings, the 200 mg/kg dose setting (upper limit) was included in this study, which also exhibits basic safety.

A Study investigating quercetin’s role in treating sleep deprivation-related memory impairment has shown that acute REM SD can activate inflammatory signaling pathways in the hippocampus [18]. A study by Wu et al. [13] reported that orally administered quercetin nanoparticles could regulate sleep duration under high-altitude conditions, providing critical evidence for developing drug treatments for altitude-related sleep disorders. The present study induced SD by disrupting sleep via the rotarod method and employed the i.p. method to administer quercetin, thereby expanding its application in modulating normal sleep rhythms and addressing both ASD and CSD. Additionally, this study investigated cellular activity and microglial activation in key brain regions involved in sleep regulation.

Despite accumulating evidence indicating the therapeutic potential of quercetin in sleep research, various challenges persist in its clinical translation. For instance, free quercetin can be enzymatically hydrolyzed in the gastrointestinal tract or be challenging to absorb owing to low solubility [25]. Nevertheless, recent advancements in nanoformulation technology have provided promising solutions to these issues. For example, quercetin-maize peptide nanoliposomes, synthesized using multifrequency pulsed ultrasonic methods, exhibited significantly enhanced quercetin-encapsulation efficiency, gastrointestinal stability, and transmembrane transport efficiency (they could penetrate the tight junctions of small intestinal epithelial cells) [25], thereby enhancing their potential for CNS targeting. Regarding the clinical translation of neuropsychiatric disease treatment, quercetin-loaded chitosan nanoparticles (Qu-Ch NPs) have demonstrated notable advantages, including a low dose and high safety [26]. Low-dose Qu-Ch NPs exert strong neuroprotective effects by modulating antioxidant and anti-inflammatory pathways.

Advances in nose-to-brain delivery technology have further established an effective route for quercetin entry into the CNS. Notably, traditional oral and intravenous methods achieved extremely low quercetin concentrations in the brain due to the restrictive BBB. In contrast, intranasally delivered quercetin nanoparticles exhibited direct entry into the brain via the olfactory nerve pathway [27]. Notably, mitochondria-targeted quercetin nanoformulations have been shown to combat Alzheimer’s disease-related ferroptosis through a triple mechanism of “iron chelation-pathway regulation-lipid protection” [28], offering strong experimental evidence for their potential in treating neurodegenerative diseases. However, regardless of their potential for clinical translation, further research is necessary on quercetin nanoformulations to clarify the correlation between “dose-central concentration-pharmacodynamic effect” and comprehensive long-term safety. Overall, optimizing quercetin nanoformulations and delivery systems can effectively overcome the major limitation of low bioavailability, highlighting their substantial potential for clinical application in treating neuropsychiatric diseases.

The power of delta waves (0.5–4 Hz) during NREM sleep is a classical indicator of sleep pressure dynamics. Additionally, sleep latency directly reflects the intensity of sleep drive and represents an intuitive metric of sleep pressure. Reportedly, the interaction between “homeostatic pressure (Process S)” and “circadian rhythm (Process C)” governs the sleep-wake balance [29]. Notably, Process S, also referred to as the “traditional homeostatic pathway” of sleep, has the following characteristic core feature: the longer the duration of wakefulness, the greater the accumulation of sleep pressure. Accumulated sleep pressure results in a compensatory increase in delta wave power during sleep, ultimately facilitating pressure dissipation [30, 31]. During wakefulness, neuronal activity-associated metabolite accumulation, coupled with elevated proinflammatory cytokine levels, enhances neuronal excitability and synergistically promotes the buildup of sleep pressure [32]. Together, they act on specific brain regions, including the basal forebrain and hypothalamus, to inhibit wake-promoting pathways, activate sleep-promoting pathways, and induce sleep [33]. Following sleep onset, slow-wave sleep (SWS) and delta wave activity direct the traditional homeostatic pathway into a pressure-dissipation phase, as this pathway activation relies on various factors beyond delta wave power alone, including adenosine concentration, sleep latency, and SWS proportion. It is important to emphasize that the traditional sleep homeostatic pathway does not regulate sleep independently.

Reportedly, sleep initiation involves two parallel pathways: the “homeostasis-dependent” and the “homeostasis-independent” pathways [34]. The traditional pathway relies on the accumulation of homeostatic factors, such as adenosine. In contrast, the homeostasis-independent pathway initiates sleep by directly regulating sleep-wake switching neurons, for example, through gamma-aminobutyric acid (GABA), which mediates receptor-driven inhibition of wake-promoting pathways [35]. Quercetin has been shown to act on the CNS primarily by inhibiting neuroinflammation and regulating oxidative stress levels [21, 36].

In the present study, quercetin treatment reduced sleep latency under basal conditions; however, it exhibited no significant effect on post-SD sleep latency. Additionally, quercetin did not significantly affect delta wave activity during sleep under both baseline conditions and post-SD recovery phase. Integrating the aforementioned literature findings with the present results, we hypothesize that quercetin regulates sleep by modulating cerebral neuronal excitability and microglial activity, rather than interfering with sleep pressure accumulation reflected by delta waves. This finding is consistent with a previously reported study [21]. Additionally, this process is likely associated with the cortical-limbic system and brainstem multilevel neural circuits that maintain sleep-wake homeostasis. To clarify this association, the roles of key sleep-regulatory brain regions were delineated in this study.

Notably, sleep-wake homeostasis regulation depends on coordination among multilevel neural circuits spanning the cortex, limbic system, and brainstem. Insomnia is often associated with hyperactivity or disrupted network connectivity in different important brain regions. For instance, the mPFC has been shown to drive wakefulness by enhancing thalamocortical synchronization [37]. Similarly, the NAc regulates sleep-wake cycles primarily via dopaminergic and glutamatergic signaling, with hyperactivity of dopaminergic neurons potentially prolonging wakefulness in chronic stress or insomnia conditions [38]. Furthermore, the BNST and BLA integrate limbic inputs to modulate the sleep-wake balance. The BNST affects this balance by regulating GABAergic signaling. For example, pharmacological or optogenetic inhibition of GABAergic neurons in the BNST reportedly improved sleep continuity and reduced stress-induced wakefulness [39]. The BLA processes stress and emotional memory, structural and functional alterations and chronic insomnia [40, 41]. Notably, acute stress can trigger the PVT, a relay station connecting the cortex, limbic system, and brainstem, to activate glutamatergic projections to the central amygdala, thereby promoting hyperarousal [42]. Additionally, the hippocampus and ventrolateral PAG (vlPAG) further contribute to sleep-wake regulation via homeostatic and neuroprotective mechanisms. For instance, the hippocampus can suppress excessive stress responses via the hypothalamic-pituitary-adrenal (HPA) axis, and its dysfunction has been associated with anxiety and depression comorbid with insomnia [43]. In contrast, neural pathways in the vlPAG are critically implicated in stress-induced recovery sleep. For example, neurotensinergic neurons in the vlPAG have been shown to promote NREM sleep via GABAergic projections to arousal-promoting nuclei, directly linking brainstem sleep circuits and stress [44]. Altogether, these findings suggest that dysfunction across multiple brain regions can induce insomnia. Further studies on dynamic interactions between these neural circuits are required to fully elucidate the mechanisms underlying quercetin-induced precise therapeutic effects on insomnia.

The results of this study showed that quercetin facilitated a compensatory increase in NREM sleep during the early recovery phase (1–6 h) post-ASD, whereas in the late recovery phase (19–24 h), it promoted normalization of the sleep-wake cycle. Notably, the compensatory increase in NREM sleep was primarily concentrated in the early recovery phase. Importantly, NREM sleep contributes to synaptic pruning, metabolic waste clearance, and overall bodily recovery [45]. These results suggest that quercetin-mediated regulation of NREM sleep during the early recovery phase aligns with the physiological demands of the body and provides important support for restoring physiological homeostasis.

SD-induced neuroinflammation in the brain has been characterized by the upregulation of inflammatory factors, such as IL-6, IL-1$\beta$, TNF-$\alpha$, cyclooxygenase-2, inducible NO synthase, sirtuin 6, and high mobility group box 1, in the mPFC of mice [46]. A recent study showed that SD causes region-specific changes in microglial density and morphology in brain regions such as the NAc, hippocampus, hypothalamus, mPFC, and thalamus in young mice. Among these, the highest density of Iba1-positive cells was found in the NAc, whereas hippocampus and mPFC exhibited the greatest total length and average number of microglia [5]. The increased microglial expression in the mPFC and NAc observed in these studies is consistent with the findings of the present study, underscoring the critical role of the mPFC and NAc in SD-induced neuroinflammation.

A key aspect warranting detailed discussion is that quercetin exerts sleep-regulating effects by attenuating neuroinflammation within specific brain regions. Recent studies report that quercetin exerts anti-inflammatory effects in neurodegenerative diseases by modulating inflammatory cytokine levels in the brain [47, 48]. Furthermore, quercetin can cross the BBB and is widely distributed across the cerebral cortex and limbic system. A previous study confirmed that quercetin mitigated sodium fluoride-induced structural and functional damage to the rat mPFC by enhancing antioxidant capacity and inhibiting apoptosis, offering a potential strategy for protecting the CNS from neurotoxicity [49]. However, direct evidence for the anti-inflammatory effect of quercetin within the NAc remains limited. Therefore, the observed reduction of microglia in the NAc in the present study may be attributed to quercetin-mediated suppression of inflammation in the mPFC, which then reduced the transmission of pro-inflammatory signals to the NAc, thereby alleviating local neuroinflammation.

c-Fos, a key marker of neuronal activation, plays a critical role in assessing neural activity in sleep-regulatory brain regions. This study showed that quercetin inhibited c-Fos expression in the mPFC, PVT, and BLA, while increasing its expression in the BNST, compared with that in the control group. This pattern needs to be further explored to elucidate underlying mechanisms. As a key component of the extended amygdala, the BNST is closely linked with high-arousal behavioral states. It is a highly heterogeneous region, exhibiting extensive diversity in cell types, with notable variations in spatial organization, molecular expression, synaptic inputs, axonal outputs, and functional regulation [50]. Various subtypes of BNST neurons project to brain regions essential for sleep-wake regulation, including the lateral hypothalamus (LH), ventral tegmental area, and arcuate nucleus [50]. Notably, optogenetic activation of BNST GABAergic neurons in mice has been shown to promote rapid transitions from NREM sleep to wakefulness; these neurons are also highly active during wakefulness and REM sleep [51]. Studies have confirmed bidirectional functional connectivity between the LH and BNST [52, 53]. Additionally, excitatory BNST projections have been reported to activate REM-active neurons in the sublaterodorsal nucleus of the brainstem, contributing to REM sleep regulation [54]. Notably, quercetin treatment further increased c-Fos expression in the BNST in this study, suggesting that its effects may involve the neurochemical and cellular heterogeneity of the BNST. Future studies need to focus on integrating EEG-EMG and fiber photometry recordings, along with two-photon imaging, to monitor quercetin-induced sleep regulation in the cell-type-specific BNST neurons.

In contrast to its pronounced efficacy in ASD models, quercetin exhibited limited effectiveness in restoring post-CSD sleep homeostasis, showing only a reduction in REM sleep during the early recovery phase. ASD mainly impairs attention and working memory, along with inducing transient oxidative stress in the hippocampus and prefrontal cortex [55]. Mechanistically, ASD transiently activates the HPA axis and triggers microglial activation, leading to localized inflammation without systemic spread. In contrast, CSD causes “multi-target, irreversible” damage. Prolonged overactivation of the HPA axis results in a “depleted state”, accompanied by dysregulation of multiple neurotransmitter systems [4]. Reportedly, a study on rodents has shown that CSD disrupts BBB integrity, exacerbates microglial activation, and elevates proinflammatory cytokine levels (such as TNF-$\alpha$ and IL-1$\beta$) in the brain compared with the effects of ASD [56]. In the present study, quercetin displayed a tendency to enhance the restoration of post-ASD sleep homeostasis but exerted only a weak effect in maintaining sleep homeostasis during CSD. This discrepancy may be attributable to the single-dose administration design used in this study. To overcome this limitation and facilitate clinical translation, future experiments should focus on identifying suitable delivery methods and optimizing quercetin dosage regimens to effectively regulate sleep homeostasis in various sleep disorder models.

The findings of this study reveal the potential of quercetin in promoting NREM sleep under basal conditions and post-ASD sleep homeostasis restoration, without influencing sleep pressure. The underlying mechanisms may involve inhibiting neuroinflammation and excessive neuronal activation in specific sleep-regulatory brain regions. These findings provide novel evidence supporting the role of natural compounds in regulating sleep and neuroimmune interactions, along with offering quercetin as a promising candidate for treating acute sleep disorders. Future research needs to aim to refine quercetin administration protocols, elucidate region-specific molecular mechanisms, and develop formulations with improved bioavailability, thereby laying a solid foundation for the clinical treatment of sleep disorders.

The data during the current study are available from the corresponding author on reasonable request.

RH and BW designed the research study. BW, ZH, and PF performed animal surgeries, sleep recordings, and immunohistochemistry. ZL, BN, and JZ analyzed EEG data and conducted statistical analyses. RH and BW wrote the initial draft. JZ offered experimental guidance. All authors contributed to editorial changes in the manuscript. All authors revised and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

All animal experiments were approved by the Institutional Animal Care and Use Committee of Xinxiang Medical University (IACUC Protocol No. XYLL-20240521), and we comply with all relevant ethical regulations. Every effort was made to minimize the number of animals used and reduce their suffering throughout the study. This study was conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals (8th edition, National Institutes of Health, USA). All protocols were designed to adhere to the 3Rs principles (Replacement, Reduction, Refinement) of animal research and were periodically reviewed by the IACUC to ensure ongoing compliance with ethical standards.

We sincerely thank Professor Hui Wang for providing the valuable experimental platform and offering rigorous guidance on the experiments. We thank Dr. Yunwei Lou (Xinxiang Medical University) and Dr. Zhitao Gao (Xinxiang Medical University) for their technical guidance and constructive comments. Additionally, the authors appreciate the contributions of all laboratory members for their help with animal care and sample preparation during the study.

This work was financially supported by Key Scientific and Technological Projects of Henan Province (Grant No. 242102310372), and Key Scientific Research Projects of Higher Education Institutions in Henan Province (Grant No. No.23B320001), and China National University Student Innovation & Entrepreneurship Development Program of Xinxiang Medical University (Grant No. 202410472007), and the Natural Science Foundation of Henan Province (Grant No. 202300410314), and Higher Education Discipline Innovation Project (111 Project, No. D20036).

The authors declare no conflict of interest.