Sleep is one of the most essential physiological phenomena for maintaining human health (Imeri and Opp, 2009), and sleep disorders can adversely affect both psychological and physical health (Baranwal et al., 2023). Insomnia, one of the most common sleep disorders, is characterized by persistent difficulty in initiating or maintaining sleep, often accompanied by psychiatric or physical conditions such as impaired attention, anxiety, or stress (Winkelman, 2015). A variety of medications are utilized to treat insomnia, including benzodiazepines, antidepressants, and antihistamines; nevertheless, concerns exist regarding potential side effects such as tolerance with prolonged use, withdrawal symptoms, and addiction (Saddichha, 2010). To mitigate these side effects, interest in alternative medicines such as medicinal plants and dietary supplements for insomnia has grown (Lee et al., 2023; Um et al., 2023).

The sleep-wake states are regulated through the interaction of various brain regions and are controlled by reciprocal competition between wake-active and sleep-active neurons through a variety of neuromodulators and neurotransmitters secreted by distinct neuronal populations (Oh et al., 2019). Wakefulness-related regions include the basal forebrain (BF), tuberomammillary nucleus (TMN), lateral hypothalamus (LH), and dorsal raphe nucleus, each of which secretes wake-active neurotransmitters such as acetylcholine, histamine, orexin, and serotonin (Eban-Rothschild et al., 2018). Sleep-related regions include the ventrolateral preoptic nucleus (VLPO) and pineal gland, each of whose neurons secrets sleep-active neurotransmitters such as GABA and melatonin (Scammell et al., 2017). Strategies aimed at activating sleep-promoting neurons or inhibiting wake-promoting neurons may provide a novel approach to the development of novel hypnotics.

Adenosine is also a crucial neuromodulator in the regulation of the sleep-wake cycle, acting through its interaction with adenosine receptors (Huang et al., 2024). In the central nervous system, four subtypes of adenosine receptors (A₁, A_2A, A_2B, and A₃) are expressed (Liu et al., 2019). Among these receptors, the adenosine A₁ receptor (A₁R) and adenosine A_2A receptor (A_2AR) are particularly implicated in the regulation of sleep (Jamwal et al., 2019). A₁R is proposed to indirectly promote sleep by disinhibiting sleep-active neurons, whereas A_2AR is reported to directly promote sleep by stimulating these neurons (Garrido-Suárez et al., 2022). Both A₁R and A_2AR exhibit significant potential as biomarkers or therapeutic targets for the treatment of insomnia; however, therapeutics for insomnia have been developed targeting A₁R.

β-lapachone (β-Lap), 3,4-dihydro-2,2-dimethyl-2H-naphtho[1,2-b] pyran-5,6-dione, is a natural naphthoquinone compound isolated from the lapacho tree (Tabebuia avellanedae) (Gomes et al., 2021). Recent studies have shown that several naphthoquinones, such as dinaphtodiospyrol (A-F), dinaphthodiospyrol G, and dinaphthodiospyrol H, have sedative or hypnotic effects (Rauf et al., 2020; Al-Awthan et al., 2021; Bawazeer and Rauf, 2021). However, there is no information regarding the hypnotic effect of β-Lap, despite its demonstrated pharmacological activities, including anti-cancer, anti-inflammatory, and neuroprotective effects (Lee et al., 2015; Silvers et al., 2017; Park et al., 2019). In this study, we investigated whether β-Lap exerts hypnotic effects using a pentobarbital-induced sleep model, and if so, elucidated the underlying mechanisms involved in its hypnotic action.

Animals

All experimental animals were acquired from Orient Bio Inc. (Seongnam, Korea). Male ICR mice (8 weeks old, 30-35 g) were used for pentobarbital-induced sleep model and western blot analyses. Male C57BL/6N mice (8 weeks old, 20-25 g) were used for immunohistochemistry and immunofluorescence. The animals were maintained under stringent conditions: constant humidity (30 ± 10%) and temperature (23 ± 1°C) under an automatically controlled 12 h light/dark cycle with lights on at 9:00 and lights off at 21:00. The animals were provided with ad libitum access to food and water and were allowed to acclimate for at least 7 days before experiments. All effort were made to minimize animal suffering, and only the number of animals necessary to produce reliable scientific data was used. All animal-related experiments were conducted in compliance with guidelines from Committee on Animal Research at Ajou University (Permission No. 2022-0036).

Materials

Pentobarbital sodium was obtained from Hanlim Pharm. Co., Ltd. (Seoul, Korea). Diazepam (DZP), used as a positive control, was acquired from Samjin Pharm. Co., Ltd. (Seoul, Korea). β-Lap and flumazenil (FLU) were acquired from Sigma-Aldrich (St. Louis, USA). 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), 2-(Furan-2-yl)-7-phenethyl-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine (SCH), 8-hydroxy-2-(di-n-propyl-amino) tetralin hydrobromide (DPAT), and 2-Pyridineethanamine dihydrochloride (PEA) were acquired from Tocris Bioscience (Avonmouth, UK). Luzindole (LUZ) was purchased from TCI Chemicals (Tokyo, Japan). YNT-185 dihydrochloride (YNT) was acquired from MedChemExpress (NJ, USA). 1,4-Diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene (U0126) and anti-c-Fos rabbit antibody were acquired from Cell Signaling Technology (Danvers, MA, USA). Anti-choline acetyltransferase (ChAT) goat antibody, anti-orexinA (OrxA) mouse antibody, and anti-GAD67 mouse antibody were obtained from Millipore (Burlington, MA, USA). All psychoactive substances were used with permission from the Ministry of Food and Drug Safety (Approval No. 236).

Pentobarbital-induced sleep model

Pentobarbital-induced sleep test was performed at 13:00 PM. All mice were subjected to a 24 h fasting period prior to the experiment. DZP and Pentobarbital sodium were dissolved in 0.9% physiological saline, and β-Lap was suspended in 70% polyethylene glycol (70% PEG). Briefly, β-Lap (1 mg/kg; per oral (p.o.)) was administered to the mice, and 30 min later, pentobarbital (45 mg/kg; intraperitoneal (i.p.)) was injected. FLU, DPCPX, SCH, DPAT, PEA, LUZ, and YNT were dissolved in 1% dimethyl sulfoxide (DMSO) and administered orally, followed by β-Lap 15 min later.

Additionally, we evaluated whether the hypnotic effects of β-Lap were affected by U0126, a mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibitor, and SN-50, a nuclear factor-kappa B (NF-κB) inhibitor. U0126 (10 mg/kg) or SN-50 (1 mg/kg) was dissolved in 1% DMSO and injected intraperitoneally to mice 30 min before administration of β-Lap. 70% PEG or β-Lap was administered orally, followed by an injection of pentobarbital 30 min later. Following the pentobarbital injection, the mice were immediately placed in individual cages for assessment. Sleep onset latency was defined as the interval from pentobarbital injection to the loss of the righting reflex, while total sleep time was defined as the interval from the loss to the recovery of the righting reflex.

c-Fos Immunohistochemistry

Immunohistochemistry (IHC) was conducted to assess the effect of β-Lap on c-Fos positive cell expression in wakefulness-related regions (the BF and LH) and the sleep-related region (the VLPO). At 07:45, DPCPX was administered, followed by the oral administration of 70% PEG or β-Lap at 08:00. 1 h later, the mice were anesthetized with a mixture of ketamine (2 mg/kg, i.p.) and xylazine (0.4 mg/kg, i.p.). The animals were then perfused with saline via the heart, and their brains were subsequently extracted. The brain was post-fixed in 4% paraformaldehyde (PFA) for 24 h and subsequently stored in 30% sucrose for 48 h at 4°C. To produce frozen tissue section blocks, brains were frozen in Tissue-Tek O.C.T compound (Torrance, CA, USA) at –80°C for at least 48 h. After that, the brain was cryosectioned into 20 µm thickness coronal sections using a Leica SM2400 microtome (Leica Biosystems, Buffalo Grove, IL, USA) at −20°C. All sections were washed and stored at –20°C until immunostaining.

Antigen retrieval of all sections was conducted using 10 mM sodium citrate buffer (pH 6.0). Sections were incubated in 3% hydrogen peroxide (H₂O₂) for 5 min to quench endogenous peroxidase activity, then rinsed with phosphate-buffered saline (PBS, pH 7.4). As a blocking solution, 5% bovine serum albumin (BSA) dissolved in PBS containing 0.1% Triton X-100 was used and incubated for 60 min. The sections were then incubated with a rabbit anti-c-Fos primary antibody (1:400) diluted in blocking solution for 48 h at 4°C, followed by thoroughly washing with PBS. The sections were subsequently incubated with a biotinylated goat anti-rabbit secondary antibody (1:500) in PBS for 60 min. After washing three times with PBS for 5 min, the sections were reacted using the Dako Liquid 3,3’-diaminobenzidine (DAB)+ Substrate Chromogen System kit (Agilent, Carpinteria, CA, USA) for 6 min to visualize a brown reaction product in the cell nuclei. The sections were rinsed twice with distilled water for 10 min each, and the expression of c-Fos positive cells was confirmed through dense brown nuclear staining using a light microscope (Korea Lab Tech, Seongnam, Korea). Brain locations were identified with reference to mouse brain atlases. A blinded technician counted and averaged three sections through the middle of each structure per animal.

c-Fos Immunofluorescence

Immunofluorescence was further conducted to evaluate the effect of β-Lap on the expression of c-Fos positive neurons in the BF, LH, and VLPO. Sections were incubated in a blocking solution for 60 min and then rinsed with PBS. Subsequently, the sections were incubated at 4°C for 48 h with rabbit anti-c-Fos antibody (1:400) diluted in blocking solution, along with goat anti-choline acetyltransferase antibody (1:500), mouse anti-orexin A antibody (1:500), or mouse anti-GAD67 antibody (1:500). After washing again, the sections were incubated rabbit anti-goat alexa488 (1:500) or goat anti-mouse alexa488 (1:500) with goat anti-rabbit alexa568 (1:500) (Invitrogen, Carlsbad, CA, USA) in PBS for 60 min. Then, the sections were incubated with DAPI (1:500) (Molecular Probes, Eugene, OR, USA) diluted in PBS for 10 min. After a final wash with PBS, coverslips were attached using Vectashield antifade mounting medium (Vector Labs, Peterborough, UK) and the cross section was observed using a Nikon A₁ confocal microscope (Nikon, NY, USA).

Western blot analysis

DPCPX was administered at 07:45, followed by oral administration of 70% PEG or β-Lap at 08:00. 1 h later, the mouse brain was removed, and the cortex was separated. Nuclear and cytoplasmic proteins were extracted from the brain tissue using a Nuclear and cytoplasmic extraction kit (Thermo Scientific, Rockford, IL, USA). The dissected brain tissue samples were homogenized in RIPA buffer and incubated on ice for 1 h. The supernatant was then separated by centrifugation at 14,000 rpm for 15 min at 4°C and subsequently stored at –70°C. Protein quantification was performed using a BCA assay, 20 μg of protein was subjected to electrophoresis on a 10% SDS-polyacrylamide gel. The separated proteins were transferred to PVDF blotting membranes and blocked with 10% skim milk for 3 h. The membranes were then rinsed four times for 10 min each with Tris-buffered saline containing 0.1% Tween-20 (TBS-T) and incubated overnight at 4°C with primary antibodies against p-ERK (1:1000), ERK (1:1000), p65 (1:500), Lamin B1 (1:500), GAPDH (1:5000), and β-actin (1:1000). Following three times with TBS-T for 10 min, the membranes were incubated with a specific HRP-conjugated secondary antibodies for 1 h at room temperature. Then, rinsing four times with TBS-T for 10 min, the membrane was reacted with ECL detection reagent for 1 min and bands were visualized using a Cytiva Amersham ImageQuant 800 device (GE Healthcare, Uppsala, Sweden).

Quantitative Real-Time PCR

β-Lap was administered 1 h before the mouse brain was removed and the cortex was separated. The collected tissues were stored at –80°C until RNA extraction. Total RNA was isolated from the cortex using TRIzol reagent (Invitrogen) following the manufacturer’s instructions. The concentration of the purified RNA was measured using a NanoDROP spectrometer (Thermo Fisher Scientific, MA, USA). Subsequently, 2 μg of total RNA was synthesized into cDNA using the amfiRivert cDNA Synthesis Platinum Master Mix (GenDEPOT, TX, USA). Quantitative real-time PCR (qRT-PCR) was then conducted using the mfiRivert qGreen Q-PCR Master Mix (GenDEPOT). The cycling parameters for qRT-PCR were initial denaturation step of 3 min at 95°C, a denaturation step of 5 s at 95°C, an annealing step of 15 s at 60°C, and an extension step of 10 s at 72°C followed by 40 amplification cycles. The target genes were as follows: mouse A₁R forward: 5’-AGAACCACCTCCACCCTTCT-3’, reverse: 5’-TACTCTGGGTGGTGGTCACA-3’, and mouse β-actin forward: 5’-CCCAGATCATGTTTGAGACCT-3’, reverse: 5’-ATGTCACGCACGATTTCCC-3’. Gene expression levels were normalized to the mRNA level of β-actin.

Statistical analysis

All values are represented as mean ± standard error of the mean (SEM). Statistical analysis was analyzed with the GraphPad Prism 8.0.2 software (GraphPad Software, San Diego, CA, USA). Data were evaluated using One-way AVOVA, and the significance of differences between two-group data was assessed using Tukey’s post-hoc test. For all tests, a difference of p<0.05 was considered statistically significant.

β-Lap exerts hypnotic effects in pentobarbital-induced sleep model in mice

To examine hypnotic effect of β-Lap, we evaluated the effects of various doses of β-Lap (0.1, 0.3, or 3 mg/kg) in a pentobarbital-induced sleep model. β-Lap (0.3 and 1 mg/kg) showed decreased sleep onset latency (163.3 ± 6.4 and 140.6 ± 8.3 s, respectively) (Fig. 1A) and increased total sleep time (89.6 ± 2.9 and 104.6 ± 2.6 min, respectively) (Fig. 1B). As expected, DZP (1 mg/kg), the positive control, showed decreased sleep onset latency (152.0 ± 7.0 s) and increased total sleep time (117.1 ± 6.7 min) compared to the control group. Notably, β-Lap (1 mg/kg) has a hypnotic effect similar to that of the positive control on pentobarbital-induced sleep test in mice. Therefore, we used the 1 mg/kg dose of β-Lap, which was the most hypnotically effective, in all subsequent in vivo experiments.

Figure 1. Effect of β-Lap on pentobarbital-induced sleep in mice. The changes in (A) sleep onset latency and (B) total sleep time. β-Lap (0.1, 0.3, and 1 mg/kg, p.o.) or DZP (1 mg/kg, p.o.) was administered to mice 30 min before pentobarbital injection (45 mg/kg, i.p.). Data are expressed as mean ± SEM (n=6-10). *p<0.05 vs CTL; **p<0.01 vs CTL. CTL, Control; β-Lap, β-lapachone; DZP, Diazepam; p.o., Per oral; i.p., Intraperitoneal.

A₁R antagonists reverse the hypnotic effect of β-Lap

We investigated whether the hypnotic effect of β-Lap is affected by various receptor agonist and antagonists against neurotransmitters and neuromodulators related to sleep-wake regulation. β-Lap decreased sleep onset latency (149.9 ± 1.5 s) (Fig. 2A) and increased total sleep time (105.5 ± 2.6 min) (Fig. 2B). These effects were only reversed by the A₁R antagonist (DPCPX) (174.3 ± 4.4 s and 64.5 ± 3.8 min, respectively) (Fig. 2). On the other hand, it was not affected by other agonists/antagonists such as adenosine A_2A receptor antagonist (SCH), GABA_AR-BDZ antagonist (FLU), orexin 2 receptor agonist (YNT), serotonin 5-HT1A receptor agonist (DPAT), melatonin 1/2 receptor antagonist (LUZ), and histamine1 receptor agonist (PEA). These results suggest that the hypnotic effect of β-Lap involves A₁R.

Figure 2. Effects of A₁R antagonists on the hypnotic effect of β-Lap in pentobarbital-induced sleep in mice. The changes in (A) sleep onset latency and (B) total sleep time. β-Lap (1 mg/kg, p.o.) was administered to mice 30 min before pentobarbital injection (45 mg/kg, i.p.). DPCPX (5 mg/kg, p.o.), SCH (5 mg/kg, p.o.), FLU (5 mg/kg, p.o.), YNT (40 mg/kg, i.p.), DPAT (0.5 mg/kg, i.p.), LUZ (30 mg/kg, i.p.), or PEA (150 mg/kg, i.p.) was administered to mice 15 min before administration of β-Lap. Data are expressed as mean ± SEM (n=7-10). *p<0.05 vs CTL; **p<0.01 vs CTL; ^#p<0.05 vs VEH; ^##p<0.01 vs VEH. CTL, control; VEH, vehicle; β-Lap, β-lapachone; DPCPX, 8-cyc lopentyl-1,3-dipropylxanthine; SCH, 2-(furan-2-yl)-7-phenethyl-7H-pyrazolo[4,3-e][1,2,4] triazolo[1,5-c]pyrimidin-5-amine; FLU, flumazenil; YNT, YNT-185 dihydrochloride; DPAT, 8-hydroxy-2-(di-n-propyl-amino) tetralin; LUZ, luzindole; PEA, 2-pyridineethan amine dihydrochloride; p.o., per oral; i.p., Intraperitoneal.

β-Lap modulates the neuronal activity of sleep-wake regulatory regions in mouse brain

We performed IHC experiments to investigate whether the hypnotic effect of β-Lap is related to the activity of sleep-wake regulatory neurons in the mouse brain. Neuronal activity was assessed by measuring the number of c-Fos positive neurons, a well-known marker of neuronal activity. To assess the effect of β-Lap (1 mg/kg) on wake-promoting neurons, we examined the number of c-Fos immunoreactive neurons in the BF and LH of mice. β-Lap reduced c-Fos expression in BF cholinergic neurons (31.3 ± 0.9 cells) (Fig. 3). Additionally, β-Lap decreased c-Fos expression in LH orexinergic neurons (27.0 ± 2.0 cells) (Fig. 4). These effects were reversed by DPCPX (55.2 ± 5.8 and 54.4 ± 3.4 cells, respectively). To evaluate the effect of β-Lap on sleep-promoting neurons, we examined the number of c-Fos immunoreactive neurons in the VLPO. β-Lap significantly increased c-Fos expression in VLPO GABAergic neurons (53.4 ± 2.6 cells) (Fig. 5). However, this effect was reversed by DPCPX (31.4 ± 2.1 cells). These results suggest that β-Lap may contribute to the hypnotic effect by inhibiting wake-active neurons, BF cholinergic neurons and LH orexinergic neurons, and activating sleep-active neurons, VLPO GABAergic neurons, associated with the A₁R.

Figure 3. Effect of β-Lap on cholinergic neuronal activity in BF. β-Lap (1 mg/kg, p.o.) was administered 1 h before brain extraction. β-Lap was administered 15 min after administration of DPCPX (5 mg/kg, p.o.). (A) Photomicrographs of c-Fos expression (black arrows) at low magnification and high magnification representative of BF. (B) Quantitative analysis for c-Fos-positive cells. (C) Triple immunofluorescence image showing choline acetyltransferase (ChAT; green), c-Fos (red), and DAPI (blue), with white arrows showing c-Fos merging with the DAPI in BF. Data are expressed as means ± SEM (n=7-10). **p<0.01 vs CTL; ^##p<0.01 vs VEH. CTL, control; VEH, Vehicle; β-Lap, β-lapachone; DPCPX, 8-cyc lopentyl-1,3-dipropylxanthine; p.o., per oral.

Figure 4. Effect of β-Lap on orexinergic neuronal activity in LH. β-Lap (1 mg/kg, p.o.) was administered 1 h before brain extraction. β-Lap was administered 15 min after administration of DPCPX (5 mg/kg, p.o.). (A) Photomicrographs of c-Fos expression (black arrows) at low magnification and high magnification representative of LH. (B) Quantitative analysis for c-Fos-positive cells. (C) Triple immunofluorescence image showing orexin-A (OrxA; green), c-Fos (red), and DAPI (blue), with white arrows showing c-Fos merging with the DAPI in LH. Data are expressed as means ± SEM (n=7-10). **p<0.01 vs CTL; ^##p<0.01 vs VEH. CTL, control; VEH, Vehicle; β-Lap, β-lapachone; DPCPX, 8-cyc lopentyl-1,3-dipropylxanthine; p.o., Per oral.

Figure 5. Role of ERK1/2 in the hypnotic effect of β-Lap. Effect of MEK/ERK inhibitor on the changes in (A) sleep onset latency and (B) total sleep time. U0126 (10 mg/kg, i.p.) was injected to mice 30 min before administration of β-Lap (1 mg/kg, p.o.). Pentobarbital (45 mg/kg, i.p.) was injected 30 min after administration of β-Lap. Data are expressed as mean ± SEM (n=4-5). The changes in (C) p-ERK expression in the cortex of ICR mice. Western blot analysis of expression level of p-ERK was normalized against unphosphorylated form. β-Lap was administered, and 1 h later, the mice were sacrificed. DPCPX (5 mg/kg, p.o.) was administered 15 min before administration of β-Lap. Data are expressed as mean ± SEM (n=4). **p<0.01 vs CTL; ^#p<0.05 vs VEH; ^##p<0.01 vs VEH. CTL, control; VEH, Vehicle; β-Lap, β-lapachone; DPCPX, 8-cyc lopentyl-1,3-dipropylxanthine; U0126, 1,4-Diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene; p.o., Per oral; i.p., Intraperitoneal.

The hypnotic effect of β-Lap is related to the ERK1/2 signaling molecule

To identify the signaling molecules involved in the hypnotic effect of β-Lap, we first investigated whether they were affected by a MEK/ERK inhibitor. β-Lap decreased sleep onset latency (151.4 ± 3.7 s) (Fig. 6A) and increased total sleep time (100.0 ± 5.9 min) (Fig. 6B). These effects were reversed by U0126 (10 mg/kg), an MEK/ERK inhibitor (169.0 ± 4.5 s and 70.6 ± 1.8 min, respectively). Additionally, we used western blot analysis to investigate the phosphorylation level of ERK1/2. Compared with the control group, β-Lap (1 mg/kg) increased the phosphorylation of ERK1/2 (173.6 ± 17.8%) (Fig. 6C). However, this effect was reversed by DPCPX (120.8 ± 6.2%). Therefore, these results suggest that the hypnotic effect of β-Lap is related to the ERK1/2 molecule, a downstream mechanism of A₁R.

Figure 6. Effect of β-Lap on GABAergic neuronal activity in VLPO. β-Lap (1 mg/kg, p.o.) was administered 1 h before brain extraction. β-Lap was administered 15 min after administration of DPCPX (5 mg/kg, p.o.). (A) Photomicrographs of c-Fos expression (black arrows) at low magnification and high magnification representative of VLPO. (B) Quantitative analysis for c-Fos-positive cells. (C) Triple immunofluorescence image showing GAD67 (GAD; green), c-Fos (red), and DAPI (blue), with white arrows showing c-Fos merging with the DAPI in VLPO. Data are expressed as means ± SEM (n=7-10). **p<0.01 vs CTL; ^##p<0.01 vs VEH. CTL, control; VEH, Vehicle; β-Lap, β-lapachone; DPCPX, 8-cyc lopentyl-1,3-dipropylxanthine; p.o., Per oral.

The increase in mRNA levels of A₁R by β-Lap is related to NF-κB translocation

To determine the effect of β-Lap on levels of A₁R mRNA and NF-κB p65 translocation, we conducted qRT-PCR and western blot analysis. β-Lap (1 mg/kg) increased A₁R mRNA levels by 2.4-fold, but this effect was inhibited by DPCPX (Fig. 7A). β-Lap increased the translocation of NF-κB p65 from the cytoplasm to the nucleus (139.8 ± 12.7%) (Fig. 7B, 7C). However, this effect was reversed by DPCPX (70.9 ± 10.4%). Additionally, we investigated whether the hypnotic effect of β-Lap was affected by an NF-κB inhibitor. β-Lap decreased sleep onset latency (149.6 ± 1.9 s) (Fig. 7D) and increased total sleep time (91.4 ± 6.7 min) (Fig. 7E). These effects were reversed by SN-50 (1 mg/kg), an NF-κB inhibitor (195.8 ± 6.2 s and 68.6 ± 2.1 min, respectively). Therefore, these results suggest that β-Lap increases A₁R mRNA levels by increasing NF-κB p65 translocation from the cytoplasm to the nucleus, thereby contributing to its hypnotic effect.

Figure 7. Effect of β-Lap on levels of A₁R mRNA and NF-kB p65 protein. The changes in (A) A₁R mRNA levels in the cortex of ICR mice. Gene expression was normalized to the mRNA levels of β-actin. β-Lap (1 mg/kg, p.o.) was administered, and 1 h later, the mice were sacrificed. DPCPX (5 mg/kg, p.o.) was administered 15 min before administration of β-Lap. The changes in (B) p65 expression in the nucleus extracts. Western blot analysis of the expression level of p65 in the nucleus was normalized to Lamin B1. The changes in (C) p65 expression in the cytoplasm extracts. Western blot analysis of the expression level of p65 in the cytoplasm was normalized to β-actin. Data are expressed as mean ± SEM (n=3). Effect of NF-κB inhibitor on the changes in (D) sleep onset latency and (E) total sleep time. SN-50 (1 mg/kg, i.p.) was injected to mice 30 min before administration of β-Lap (1 mg/kg, p.o.). Pentobarbital (45 mg/kg, i.p.) was injected 30 min after administration of β-Lap. Data are expressed as mean ± SEM (n=5). *p<0.05 vs CTL; **p<0.01 vs CTL; ^#p<0.05 vs VEH; ^##p<0.01 vs VEH. CTL, control; VEH, Vehicle; β-Lap, β-lapachone; DPCPX, 8-cyc lopentyl-1,3-dipropylxanthine; p.o., Per oral; i.p., Intraperitoneal.

To our knowledge, this study is the first to demonstrate the hypnotic effects of β-Lap, showing that β-Lap significantly decreases sleep onset latency and increases total sleep time in a pentobarbital-induced sleep model in mice. These effects of β-Lap were remarkably inhibited by not only DPCPX but also U0126, suggesting the possible involvement of A₁R and ERK1/2 in the hypnotic effect of β-Lap. Immunostaining results showed that β-Lap alone, without pentobarbital injection, decreased neuronal activity in wakefulness-active brain regions and increased neuronal activity in sleep-active brain regions, suggesting that β-Lap itself may have hypnotic effects. Western blot analysis revealed that β-Lap increased ERK1/2 phosphorylation and NF-κB translocation from the cytoplasm to the nucleus, both of which were inhibited by DPCPX. Furthermore, our results indicated that β-Lap can upregulate A₁R mRNA expression. Taken together, these findings suggest that β-Lap plays a significant role in mediating hypnotic effects through a mechanism involving A₁R and ERK1/2.

The balance between wakefulness-active neurons, such as the BF and LH, and sleep-active neurons, such as the VLPO, is known to be critical for the regulation of the sleep-wake cycle (Oh et al., 2019). As the state of wakefulness is prolonged, adenosine levels in the BF increase, inhibiting the activity of cholinergic BF neurons and thereby facilitating the transition to sleep (Strecker et al., 2000). In fact, animal studies have shown that the A₁R agonist, N6-cyclohexyladenosine, reduces the discharge activity of BF wake-active neurons, leading to sleep (Thakkar et al., 2003). Adenosine can also promote sleep by binding to A₁Rs on orexin neurons in the LH, thereby inhibiting their activity (Thakkar et al., 2002, 2008). Additionally, perfusion of A₁R agonist into the perifornical-LH area has been shown to suppress arousal and increase non-rapid eye movements (NREM) sleep (Alam et al., 2009). A₁R are known to inhibit adenylyl cyclase via Gi-coupled inhibitory signaling. Therefore, the sleep-promoting activity of adenosine in BF and LH is thought to occur through inhibitory signaling of A₁R against wakefulness-active cholinergic and orexinergic neurons, respectively. An additional mechanism by which adenosine promotes sleep through A₁R may be by disinhibiting sleep-active neurons in the VLPO, thereby promoting sleep (Chamberlin et al., 2003; Morairty et al., 2004). Neuronal activity in the VLPO can be suppressed by several wakefulness-promoting neurons during arousal (Wang et al., 2018). During sleep, these inhibitory signals are diminished, resulting in the activation of the VLPO, and consequently sleep-active neurons in the VLPO can propagate GABAergic signals throughout the wakefulness-active system, thereby inhibiting arousal (Saper and Fuller, 2017). Studies have shown that A₁R transmits Gi-coupled inhibitory signals to specific wakefulness-active neurons that act to inhibit the VLPO (Morairty et al., 2004). In this context, sleep promotion by A₁R may be mediated through disinhibition of sleep-active neurons in the VLPO. In the present study, to investigate which brain regions are involved in the hypnotic effects of β-Lap, we measured the neuronal activity of the BF, LH, and VLPO in mouse brain by employing IHC techniques with specific markers for the neurons in each region. Our results have shown that β-Lap reduces neuronal activity in the BF and LH, wakefulness-promoting regions, and increased neuronal activity in the VLPO, sleep-promoting region, in mice; all these effects were abolished by DPCPX. These results, along with the finding that the hypnotic effects of β-Lap in the pentobarbital sleep model are inhibited by DPCPX, suggest that the hypnotic effects of β-Lap may be associated with the A₁R.

A₁Rs are known to activate several signaling molecules, such as protein kinase C and phospholipase C (PLC) (Spanoghe et al., 2020). Signal transduction mediated by A₁R is largely driven by coupling to heterotrimeric G-protein complexes, with consist of a Gi-subunit and a Gβγ heterodimeric complex (Trinh, 2021). Activation of A₁R by adenosine can stimulate ERK1/2 activity, a major protein kinase in the brain, through βγ-subunits-dependent mechanisms (Trinh et al., 2022). A₁Rs are expressed at high levels in several brain regions including the cortex (Sachdeva and Gupta, 2013). The cortex also plays a central role in sleep-wake regulation, through processing integrated signals projected from various sleep-wake regulatory neurons, ultimately influencing overall wakefulness and sleep through the release of neurotransmitters and neuromodulators, such as acetylcholine, orexin and adenosine (Horner and Peever, 2017). In the cortex, the signaling pathway for A₁Rs is well understood: ERK phosphorylation, a marker for ERK activation, increases as a downstream signal for A₁Rs and mediates sleep promotion by regulating sleep-wake-related genes such as Arc and Homer1a (Mikhail et al., 2017). Given that the cortex expresses high levels of A₁Rs and processes downstream signals for A₁R in sleep-wake regulation, we investigated the mRNA levels of A₁R and ERK activation in the cortex to better understand the underlying mechanisms involved in β-Lap-induced hypnotic effect. Our results showed that the hypnotic effects of β-Lap were remarkably inhibited by U0126, suggesting the possible involvement of ERK1/2 in the effect of β-Lap. In addition, this study also showed that β-Lap increased ERK1/2 phosphorylation, and this activation of ERK1/2 by β-Lap was inhibited by DPCPX. From these results, it is suggested that the hypnotic effects of β-Lap are associated with increased ERK1/2 phosphorylation as a downstream mechanism of A₁R.

Stimulation of A₁Rs has been shown to increase phosphorylation of the inhibitory protein IκB and nuclear translocation of NF-κB dimers via the activation of the PLC pathway, thereby upregulating A₁R levels (Brown et al., 2012). The expression of A₁Rs has been shown to be reduced in the cortex of mice with a deletion in the p50 subunit of NF-κB (Sheth et al., 2014), suggesting that NF-κB activation controls the expression of A₁Rs. Sleep deprivation has been shown to increase A₁R density in the cerebral cortex of rats, suggesting that A₁Rs are involved in sleep deprivation and sleep regulation (Elmenhorst et al., 2009). In humans, positron emission tomography studies have shown that sleep deprivation upregulates A₁R in the cortex, which is associated with the homeostatic regulation of sleep (Elmenhorst et al., 2007). Microinjection of DPCPX, an A₁R antagonist, into the cortex of mice has been found to increase wakefulness and decrease NREM sleep (Van Dort et al., 2009). In this context, it is suggested that increased expression of A₁R in the cortex may play a role in promoting sleep. Consistent with this concept, our results showed that β-Lap increased NF-κB p65 translocation from the cytoplasm to the nucleus and increased mRNA levels of A₁R, and these effects of β-Lap were significantly inhibited by DPCPX. Taken together, our results in this study suggest that β-Lap can exert a significant hypnotic effect, and A₁R and ERK1/2 signaling may be involved in the underlying mechanisms.

Although data on the permeability of β-Lap to the blood-brain barrier (BBB) are missing so far, this compound is a small molecule with a molecular weight of 242.29 g/mol (Bermejo et al., 2017) and is hydrophobic (Li et al., 2023), allowing us to predict its potential to pass through the BBB. Furthermore, our immunostaining results showed that oral administration of β-Lap significantly altered neuronal activity in the brain parenchymal region, which indirectly suggests that β-Lap may be capable of crossing the BBB. Nonetheless, further studies are necessary to definitively determine whether β-Lap can directly cross the BBB.

This research was supported by the GRRC program of Gyeonggi province (GRRCAjou2023-B01).

The authors declare that there are no conflicts of interest.