Ziziphi Spinosae Semen (ZSS), as the first choice of traditional Chinese medicine in the treatment of insomnia, has attracted extensive attention. However, the current studies on improving sleep mechanism of ZSS are mainly focused on the role of single component, to further reveal the potential mechanism of ZSS in multi-constituents is necessary. In this study, ZSS extract (ZSSE) was extracted from ZSS via detailed modern extraction, separation and purification technologies. The chemical constituents of ZSSE were analyzed by HPLC-MS. In vivo experiments, rat’s model induced by p-chlorophenylalanine (PCPA) has been established to investigate the potential effect and corresponding mechanism of ZSSE on improving sleep. HE stains experimental results indicated that the drug group showed fascinating advantages over model group in improving sleep. Moreover, the levels of GABA, Glu, 5-HT and DA factors of insomnia rats in brain were also monitored via ELISA method to further study the improving sleep mechanism of ZSSE. The results indicate that sleep could be improved effectively via up-regulation of GABA and 5-HT, and down-regulation of Glu and DA, respectively. In addition, molecular mechanisms of ZSSE in improving sleep were also studied by immunohistochemical analysis. The results indicate sleep could be improved by regulating the expression levels of GABAARα1 and GABAARγ2 receptors in hypothalamus and hippocampus tissue sections. Therefore, this work not only identifies the active ingredients of ZSSE, but also reveals the potential pharmacological mechanism of ZSSE for the improving sleep, which may greatly stimulate the prospective development and application of ZSSE.
Research Article
Neuroprotective effect of Ziziphi Spinosae Semen on p-chlorophenylalanine induced insomnia rats by activating GABA A receptor
https://doi.org/10.21203/rs.3.rs-1685189/v1
This work is licensed under a CC BY 4.0 License
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Ziziphi Spinosae Semen
Insomnia
Para-chlorophenylalanine
GABAARα1
GABAARγ2
Recently, sleep disorders have become a growing health problem. Specially, the global rate of sleep disorders has reached 27% according to the World Health Organization (WHO) statistics. Insomnia as one kind of sleep disorders, has become one of the important factors affecting people's quality of life, and may even lead to traffic accidents and other accidents and endanger personal and public safety, which constitutes a serious burden to individuals and society. Therefore, development of highly efficient drugs in treating insomnia to promote people's physical and mental health is particular important. Clinically, benzodiazepines had been a candidate drug in treating insomnia (McKillop et al. 2021). Generally, total sleep time can be effectively increased by treating with benzodiazepines via shortening the sleep latency period and reducing the number of awakenings during nighttime sleep (Lahteenmaki et al. 2019). However, long-term use of benzodiazepines can lead to drowsiness (Qeadan et al. 2022), memory impairment (Kleinman and Weiss 2022), ataxia, drug addiction and other adverse reactions (Murphy et al. 2016). Chinese medicine has shown unique advantages such as Suanzaoren Decoction in the treatment of insomnia (Dong et al. 2021). Therefore, development of natural and non-toxic Chinese herbal medicine to improve sleep function is highly desired.
ZSS, as the preferred medicine in treating insomnia in traditional Chinese medicine, has a long history over 2000 years in clinical application. ZSS contains saponins, flavonoids, fatty acids, alkaloids, proteins and other chemical constituents (He et al. 2020). Benefit from the diversity of chemical constituents of ZSS, the diversity of biological activity and pharmacological action were usually presented. Modern pharmacological studies have confirmed that ZSS exhibited a series of pharmacological effects, such as neuroprotective effect (Liu et al. 2014), antianxiety effect (Li et al. 2019) and improving sleep (Fang et al. 2010). The previous report had certified that ZSS saponins could enhance the hypnotic effect of pentobarbital sodium (Cao et al. 2010). Jujuboside A and B could improve the sleep of Drosophila melanogaster, because of Jujuboside A mainly affects the autonomous activities of Drosophila at night, and Jujuboside B mainly improves the daytime sleep of Drosophila, respectively (Yang Bo 2013). Spinosin one kind of flavonoid in ZSS, assisted pentobarbital to induce sleep time in mice and shorten sleep latency (Wang et al. 2010). ZSS oil could significantly reduce the number of autonomous activities of mice, to prolong the sleep time and shorten the sleep latency of mice, to further improve the sleep quality of mice (Jia Ying 2018). Total alkaloid gavage of ZSS could significantly prolong the sleep time of mice via reducing the spontaneous activity of mice (Fu Jingwei 2005). In summary, all the above constituents of ZSS have shown good pharmacological effect on improving sleep. However, identification of active ingredients of ZSS and the corresponding improving sleep mechanism study are still unclear, which restrict the rapid development of ZSS medicine.
GABAergic neurons are widely distributed in the central nervous system and form neural networks in the brain GABAergic neurons communicate with a variety of neurons through different receptor subtypes to jointly regulate the sleep wake process (Li et al. 2022). GABA receptors are mainly divided into ionic channel GABAA and GABAC receptors and metabolic GABAB receptors (Wisden et al. 2019). At present, many anti-insomnia drugs used in clinic are designed and developed by using the inhibition of these GABA receptor targets (Belelli and Lambert 2005). Among them, GABAA receptor is the most abundant GABA receptor in human brain, it is widely distributed in the hippocampus, hypothalamus and cerebral cortex (Michels and Moss 2007). It is the most important of the three subtypes, because GABAA receptor acts as the target of many major clinical anti-insomnia drugs, such as diazepam, barbiturates, clonazepam and Midazolam (Brickley and Mody 2012). The clinical efficacy of these drugs is well correlated with their affinity for GABAA receptors (Ghit et al. 2021). Therefore, the development of anti-insomnia drugs targeting on GABAA receptor has become a research hotspot.
Although the efficacy of ZSS has been widely recognized by doctors and patients, the underlying treating mechanism on improving sleep is still unclear. In this study, taking ZSSE（which was obtained from ZSS by pharmacodynamic tracking method）as the research object, to clarify the pharmacodynamics material basis of ZSSE and to understand the underlying mechanisms on improving sleep, HPLC, HPLC–MS were performed to systematically analyze the compound composition of ZSSE. On this basis, according to the effective components and action targets obtained by molecular docking, we established a rat model of insomnia induced by PCPA to evaluate the improving sleep effects of ZSSE and investigate the GABA anti-insomnia pathway for exploring the molecular mechanism. This study may provide a new insight on the protective effects and molecular mechanism of ZSSE against insomnia.
Animals
The effect of ZSS on sleep improvement was studied in SPF Kunming mice (body weight 18-22 g). Male SD rats (6 weeks old，body weight 180-220 g) were used to explore the mechanism of sedative-hypnotic effects of ZSSE on PCPA induced insomnia model. All animals are from Liaoning Chang sheng Biotechnology Co. Ltd. (License No. SCKK (Liao) 2020-0001). All experimental procedures in this work conform to the ethical standards of the Medical Ethics Committee of Changchun University of Chinese Medicine, and every effort is made to reduce the number of animals used and any pain and discomfort experienced by the subjects.
Cell culture
Mouse hippocampal neuron cell line HT22 (American Type Culture Collection, Manassas, VA, USA) line were grown in DMEM supplemented with100 U/mL penicillin, 100 mg/mL streptomycin, and 10% calf serum at 37 °C in a humidified incubator with an atmosphere of 95 % air and 5 % CO2. All the reagents were dissolved in the cell culture medium.
Reagents and Instruments
ZSS is provided by the Pharmacy Department of 1st Associated Hospital of Changchun University of Chinese Medicine. Sodium pentobarbital (Lot: F0749) was purchased from Shandong West Chemical Technology Co. Ltd. Diazepam (national drug standard: H37023039) was purchased from Shandong Xinyi Pharmaceutical Co., Ltd. Para-chlorophenylalanine (PCPA) purchased from Alfa Aesar Company. 5-HT, GABA, DA, GLU Elisa kits, PV-6000 immunohistochemical kits, GABAARα1 and GABAARγ2 antibodies were purchased from Beijing Solebao Technology Co., Ltd.
The instruments used in this experiment include KD- MBII biological tissue embedding machine and Leica RM2245 paraffin slicer (Hubei Kanglong Electronic Technology Co., Ltd.), 10AO automatic Photography device, Optical microscope (Olympus, Japan), JY10001 Electronic scale (Shanghai Precision Scientific Instrument Co., Ltd., China). Agilent 1260 HPLC (Agilent Technologies Inc.). UltiMate 3000RS-chromatograph, Q Exactive high resolution mass spectrometer (Semel Fisher Technology (China) Co., Ltd.).
Screening of ZSS extract for improving sleep activity
The extraction method of ZSS is shown in Figure 1A. The effects of ZSS extracts on sleep latency and sleep time of mice were screened by pentobarbital sodium threshold experiment (The disappearance of righting reflex is an indicator of falling asleep). Identify the best extract to improve sleep (ZSSE). The contents of total saponins, polysaccharides, flavonoids and fatty acids were determined by ultraviolet spectrophotometry (UV1700, HIMADZU).
Preparation of ZSSE
After grinding, ZSS was degreased with petroleum ether to obtain powder. Then reflux extraction with 10 times of 70% ethanol for 3 times, 1 hour each time, collect the combined filtrate and dry. Add 10 times n-butanol to the dried alcohol extract, reflux extraction for 3 times, 1 hour each time, filter, collect the filtrate, recover the solvent under reduced pressure, and then dry. The solid obtained is the effective part of the experiment.
Identification of chemical components in ZSSE by LC-MS
On the Ultimate 3000 RS chromatographic system, the chemical composition of ZSSE sample solution was analyzed and identified by high resolution LC-MS. Mass spectrometry conditions: ion source: electrospray ionization source, scanning mode: positive and negative ion scanning switch, detection method: full mass / dd-MS2, resolution: 70000 (Fullmass), 17500 (dd-MS2), scanning range: 150.0 kV (positive), capillary temperature: 300 ℃, collision gas: high purity argon (purity≥99.999), sheath: nitrogen (purity≥99.999, 40 arb). Chromatographic conditions: RP-C18 column (150 mm×2.1 mm, 5 μm Welch), flow rate: 0.30 mL/min, A phase: 0.1% formic acid aqueous solution, B phase: 0.1% formic acid acetonitrile, needle lotion: methanol, column temperature: 35 ℃, injection volume: 5 μL, solvent gradient as shown in Table1.
Table 1
Gradient elution procedures
|
Time (min)
|
A:0.1% Formic acid aqueous solution (%) (A)(%) |
B:0.1%Formic acid acetonitrile (%) (B)(%) |
|
0 |
98 |
2 |
|
1 |
98 |
2 |
|
5 |
80 |
20 |
|
10 |
50 |
50 |
|
15 |
20 |
80 |
|
20 |
5 |
95 |
|
25 |
5 |
95 |
|
26 |
98 |
2 |
|
30 |
98 |
2 |
Determination of ZSSE chemical composition by HPLC
Fifteen batches of ZSSE samples were analyzed by Agilent-C18 column (250 mm×4.6 mm, 5 μm) on Agilent-1260 HPLC system. The mobile phase system consists of mobile phase A (acetonitrile) and mobile phase B (0.1% phosphate water). The solvent gradient is shown in Table 2. The chromatographic conditions are as follows: the detection wavelength is 250 nm, the column temperature is 25 ℃, the flow rate is 1 mL/min, and the injection volume is 20 μL.
Table 2
Gradient elution schedule
|
Time (min) |
A: Acetonitrile (%) |
B: 0.1% phosphate water (%) |
|
0 |
10 |
90 |
|
30 |
|
84 |
|
50 |
25 |
75 |
|
65 |
40 |
60 |
|
75 |
40 |
60 |
16Docking procedure
Molecular docking is a method of placing the ligand in the binding area of the receptor through computer
simulation and calculating its physical and chemical parameters to predict the binding affinity and conformation of the ligand and receptor (Morris and Corte 2021). GABAA receptor, the target of diazepam and barbiturates widely used in clinic (Sigel 2005), was selected for molecular docking with ZSSE active ingredients. Through the analysis of the docking score, the results are obtained and evaluated. Select corresponding gene target proteins through RSCB PDB database (https://www.rcsb.org/) and download 3D structure PDB format files. Download the MOL2 file of the corresponding active ingredient in the TCMSP database. The target protein and small molecule ligand were processed by AutoDock software. AutoDock-Vina calculates the binding energy. Data visualization in Pymol software.
Effect of ZSSE on the activity of HT22 cells
The effect of ZSSE on the activity of HT22 cells injured by Glu was determined by CCK-8 method. HT22 cells were spread in 96 well plates at the concentration of 5×103 cells/well. After 24 hours of culture, the cells were divided into 7 groups: blank group, Glu group, 25, 50, 100, 200 and 400 μ Mol/L ZSSE administration group. The administration group was given 25, 50, 100, 200 and 400 respectively μ Mol/L ZSSE pretreatment for 3 h, L-Glu was added to the cells of model group and administration group to make the final concentration of 20 mmol/ L, and incubated with the cells for 24 h. Then, 10 µl CCK-8 was added to each well and cultured under the same conditions for 2 hours. Optical density (OD) was measured at 490 nm using bio red 550. The reference wavelength is 620nm. The experiment was repeated three times. The effect of ZSSE on the activity of HT22 cells induced by Glu was calculated.
performed with the following equation:
Cell viability (%) = D1/D0×100%
where D0 is the OD value of the control wells, and D1is the OD value of the sample’s wells.
Preparation of PCPA induced insomnia rat’s model
After the rats were adaptively reared for 7 days, the rats were injected intraperitoneally with 350 mg/kg PCPA once a day for 3 consecutive days. The animals showed a loss of circadian rhythm and were active during the day, suggesting that the insomnia model was successfully made. Take 60 rats successfully modeled and divide them into Model Group (MOD), Diazepam Group (DZP, 1.5 mg/kg), Jujuboside B (JUB, 20 mg/kg), ZSSE 13.50g/kg, 9.01g/kg, 4.50g/kg Group. Another 10 unmodeled rats were selected as blank control group (Blank). All groups were given intragastric administration once a day for 7 days (Figure 6A).
Histopathological Analysis
After the last administration for 4 h, the rats were given deep anesthesia. After anesthesia, normal saline was perfused through the heart until the effluent was colorless, and then 4 % paraformaldehyde was perfused. The brain was quickly taken out and placed on an ice-cold petri dish. The blood was washed with ice saline and fixed in a 10 % neutral formaldehyde solution for 24 h. Then the brain tissue was dissected and immersed in different gradients of ethanol and xylene for dehydration. After dehydration, they were embedded in paraffin and sectioned in 4 μm thick coronal plane of the brain. The histomorphological changes of hypothalamus and hippocampus of insomnia rats were observed by hematoxylin and eosin (HE) staining under optical microscope (×100).
Biochemical Analysis
The experimental rats were deeply anesthetized 4 hours after the last administration, and the hypothalamus and hippocampus were quickly removed. Nine times normal saline was added to the hypothalamus and hippocampus tissue, which was then prepared into homogenate by high-speed disperser. The homogenate was centrifuged at 3500 r/min for 10 min to collect the supernatant. The contents of 5-hydroxytryptamine (5-HT), Glutamic acid (GLU), Dopamine (DA), and Gamma-aminobutyric acid (GABA) in the hypothalamus and hippocampus tissue homogenate were determined by ELISA. The possible mechanisms of the sedative-hypnotic effect of ZSSE were investigated by comparing 5-HT, DA, GLU, and GABA content of the hypothalamus and hippocampus tissues of rats in each group.
Immunohistochemistry Analysis
The sections were deparaffinized and rehydrated in xylene and different graded alcohol. Antigen retrieval was done by heating 60 min. The sections were incubated with 3 % H2O2 deionized water for 10 minutes to block the activity of endogenous peroxidase. After being washed by PBS for 3 times, we used antibody diluent blocking buffer to block the nonspecific binding site for 1h at room temperature. Slices added primary antibody (1:200) and then was incubated at 37 °C for 2 h. Washed by PBS, we added second antibody to slices and incubated at 37 °C for 30 min. Then that washed 3 times with PBS, each time for 5 min. Be dyed with DAB substrate kit according to kit instructions. The stained slices were dehydrated, transparent and sealed routinely. The mean optical density (MOD) of GABAARα1 and GABAARγ2 positive cells in the cerebral cortex was determined under light microscopy.
Statistical method
All experiments were conducted at least three times. The data were reported as mean value±standard deviation. Statistical analyses were conducted using SPSS 21.0 and Origin 9.0. For multiple comparisons, data were subjected to one-way ANOVA (Turkey's post hoc) and paired t-test to determine statistical significant. p < 0.05 was considered statistically significant differences between the groups.
ZSS improves sleep activity screening
As shown in Figure 1A, 13 ZSS extracts were obtained by solvent extraction with different solvents. The yield of ZSSE was 2.17 %. At the same time, pentobarbital sodium threshold test was used to screen the sleep improving activity of these 13 extracts. The results are shown in Figure 1B. The results showed that compared with other extracts, ZSSE significantly prolonged the sleep time of mice and shortened the sleep time of mice, indicating that ZSSE has a good effect on improving sleep. In addition, the contents of total saponins, total flavonoids, total alkaloids and total fatty acids in ZSSE were determined by UV spectrophotometry. The results showed that ZSSE contained a large number of saponin and fatty acids（in Table 3）.
Table 3
ZSSE active ingredient content table
|
Plant |
Total saponin content （mg GRE g-1) |
Total flavonoid content （mg RUE g-1) |
Total alkaloid content （mg ACE g-1) |
Total fatty acid content （mg FTE g-1) |
|
ZSSE |
6.851±0.062 |
2.114±0.041 |
0.985±0.005 |
5.833±0.095 |
Note: Data are represented as means±SD (n = 3). GRE: Ginsenoside Re equivalents; RUE: Rutin equivalents; ACE: Aconitine equivalents; FTE: Fatty acid triglycerides equivalents.
HPLC-MS analysis
The chemical constituents of ZSSE were analyzed by HPLC-MS. The molecular ion peaks [M-H]- and [M+H]+ of each chromatographic peak in the positive and negative ion mode were detected by UPLC-Q-Orbitrap-MS, and the possible molecular weight was determined by the molecular ion peak, fragment ions and their chromatographic peak retention time. As shown in Table 4, 34 compounds in ZSSE were preliminarily identified, including Spinosin, Rutin, Ferulic acid, Quercetin, Jujuboside A, Jujuboside B, etc. Secondary mass spectrogram of main compounds in ZSSE (Figure 2).
Table 4
List of ZSSE compounds identified by UPLC-Q-Orbitrap-MS mass spectrometry
|
NO. |
Component Name |
Formula |
Molecular Weight |
tR/min |
Area |
[M+H]+1 |
[M-H]-1 |
|
1 |
α-D-Mannose 1-phosphate |
C6 H13 O9 P |
260.0293 |
1.79 |
2141838.22 |
-- |
259.02 |
|
2 |
Norharman |
C11 H8 N2 |
168.07 |
6.44 |
793182.18 |
169.08 |
-- |
|
3 |
Spinosin |
C28H32 O15 |
608.54 |
8.61 |
17651166.00 |
-- |
607.30 |
|
4 |
Rutin |
C27H30 O16 |
610.15 |
8.69 |
84420324.00 |
-- |
609.14 |
|
5 |
Hesperidin |
C28H34 O15 |
610.56 |
8.60 |
334840273.00 |
-- |
609.18 |
|
6 |
Nuciferin |
C19H21NO2 |
295.37 |
8.83 |
71234552.03 |
296.1645 |
-- |
|
7 |
Ferulic acid |
C10H10 O4 |
194.19 |
9.20 |
138552103.00 |
-- |
193.05 |
|
8 |
Quercetin |
C15H10 O7 |
302.04 |
11.03 |
43616832.00 |
303.03 |
-- |
|
9 |
Jujuboside A |
C58H94 O26 |
1207.35 |
11.65 |
89441488.00 |
-- |
1205.21 |
|
10 |
Citroflex 2 |
C12 H20O7 |
276.12 |
11.78 |
31068100.02 |
277.13 |
-- |
|
11 |
Isorhamnetin |
C16 H12O7 |
316.06 |
12.07 |
1654689.90 |
317.07 |
-- |
|
12 |
Jujuboside B |
C52H84 O21 |
1045.21 |
12.27 |
130876714.00 |
-- |
1043.54 |
|
13 |
Glycitein |
C16 H12O5 |
284.07 |
13.74 |
2513278.92 |
285.07 |
-- |
|
14 |
Bis(4-ethylbenzylidene) sorbitol |
C24H30O6 |
414.20 |
14.76 |
5266759.95 |
415.21 |
-- |
|
15 |
(+/-)12(13)-DiHOME |
C18H34O4 |
296.2344 |
14.94 |
5208181.23 |
297.24 |
-- |
|
16 |
α-Linolenic acid |
C18H30 O2 |
278.22 |
16.09 |
8284980.70 |
279.23 |
-- |
|
17 |
Palmitoleic acid |
C16H30 O2 |
254.22 |
16.61 |
1307156.50 |
255.23 |
-- |
|
18 |
16-Hydroxyhexadecanoic acid |
C16 H32 O3 |
272.23 |
16.70 |
2382085.21 |
-- |
271.23 |
|
19 |
Dibutyl phthalate |
C16 H22O4 |
278.15 |
17.56 |
156100574.70 |
279.16 |
-- |
|
20 |
Palmitoyl ethanolamide |
C18H37 NO2 |
299.28 |
18.61 |
22198505.87 |
300.29 |
-- |
|
21 |
Pinolenic acid |
C18H30O2 |
278.22 |
18.91 |
22576128.92 |
279.23 |
-- |
|
22 |
1-Linoleoyl glycerol |
C21H38 O4 |
336.27 |
19.03 |
22630965.85 |
337.27 |
-- |
|
23 |
Ursolic acid |
C30 H48 O3 |
456.36 |
19.20 |
182129584.80 |
-- |
455.35 |
|
24 |
Ethyl palmitoleate |
C18 H34 O2 |
282.26 |
19.48 |
2865584.15 |
-- |
283.26 |
|
25 |
Oleamide |
C18H35NO |
281.48 |
19.88 |
81821910.05 |
-- |
265.25 |
|
26 |
Monoolein |
C21H40O4 |
356.2917 |
20.33 |
44907121.30 |
-- |
357.29 |
|
27 |
Ethyl myristate |
C16H32 O2 |
256.24 |
21.08 |
5451411.39 |
-- |
255.23 |
|
28 |
Oleic acid |
C18H34O2 |
282.26 |
21.35 |
203048809.08 |
-- |
281.25 |
|
29 |
Linolenic acid ethyl ester |
C20H34 O2 |
306.26 |
22.15 |
1411190.83 |
307.26 |
-- |
|
30 |
Ricinoleic acid methyl ester |
C19H36O3 |
294.26 |
22.53 |
2310061.40 |
295.26 |
-- |
|
31 |
Stearic acid |
C18H36 O2 |
284.27 |
22.86 |
8075045.05 |
-- |
283.26 |
|
32 |
Erucamide |
C22 H43NO |
337.33 |
24.05 |
2135618.78 |
-- |
338.34 |
|
33 |
Ethyl oleate |
C20H38O2 |
310.29 |
24.91 |
310.29 |
311.29 |
-- |
|
34 |
4-Dodecylbenzenesulfonic acid |
C18 H30 O3 S |
326.19 |
27.51 |
5494426.33 |
-- |
325.18 |
HPLC analysis
HPLC was carried out to investigate ZSSE. As shown in Figure 3A, 15 typical ZSSE chromatograms with good quality control lots were observed. Furthermore, as shown in Figure 3B, nine peaks of ZSSE, including Puerarin (No.5), Ferulic acid (No.8), Spinosin (No.10), Rutin (No.11), Hesperidin (No.12), Nuciferin (No.15), Quercetin (No.18), Jujuboside A (No.19) and Jujuboside B (No.20) were identified in comparison with corresponding reference standards. The relative contents of individual constituents in the nine peaks of ZSSE are shown in Table 5.
Table 5
Identification and determination of the compounds in the ZSSE by HPLC.
|
Number |
Retention time of control substance (min) |
Retention time of ZSSE sample (min) |
Compounds |
Contents(mg/g) |
|
5 |
13.47±0.04 |
13.34±0.04 |
Puerarin |
0.069±0.003 |
|
8 |
22.26±0.12 |
22.37±0.06 |
Ferulic acid |
0.026±0.001 |
|
10 |
30.65±0.09 |
30.64±0.05 |
Spinosin |
0.156±0.005 |
|
11 |
21.21±0.07 |
31.25±0.06 |
Rutin |
0.035±0.008 |
|
12 |
41.52±0.02 |
41.99±0.08 |
Hesperidin |
0.050±0.002 |
|
15 |
48.92±0.20 |
48.82±0.41 |
Nuciferin |
0.011±0.005 |
|
18 |
53.51±0.19 |
53.32±0.05 |
Quercetin |
0.024±0.011 |
|
19 |
65.47±0.09 |
65.70±0.01 |
Jujuboside A |
0.837±0.013 |
|
20 |
68.38±0.11 |
68.65±0.01 |
Jujuboside B |
0.654±0.024 |
Molecular docking
GABAA receptor is the target of diazepam and barbiturates, which are widely used in clinic. These drugs bind to the corresponding sites on the GABAA receptor and enhance the effect of GABA on the receptor by changing the conformation of the receptor. Therefore, there is a great correlation between the clinical efficacy of drugs and their affinity for GABAA receptors. GABAA receptor is a pentameric macromolecule composed of five subunits. At present, there are 16 kinds of GABAA receptor subunits, of which α1 and γ2 GABAA receptor subtypes composed of are the most abundant in the brain, especially in the hippocampus and cerebral cortex, accounting for about 43 % of all GABAA receptors. Therefore, we chose GABAARα1 and GABAARγ2 receptor for molecular docking verification. Select the corresponding gene target protein through the PDB database, download the 3D structure, and directly download the mol2 file of the corresponding active ingredient in the TCMSP database. Target proteins and small molecule ligands were docked by AutoDock software. The molecular docking results are shown in Figure 4.
Molecular docking results showed that 8 of the above 9 chemical components were related to GABAARα1 and GABAARγ2 receptors have good binding ability (Table 6). Jujuboside B exhibited best combination ability with GABAARα1 (-10.5 kJ/mol), and the order from strong to weak was as follows: Jujuboside B> Jujuboside A>Rutin > Hesperidin> Spinosin> Puerarin> Quercetin> Ferulic acid. Jujuboside B exhibited best combination ability with GABAARγ2 (-11.2 kJ/mol), and the order from strong to weak was as follows: Jujuboside B> Jujuboside A> Puerarin> Spinosin> Hesperidin > Rutin >Quercetin> Ferulic acid. All the results indicated that Jujuboside B and Jujuboside A of ZSSE exhibited best binding ability to GABAARα1 and GABAARγ2 receptors, which may play the main pharmacological role in improving sleep.
Table 6
Docking results of important components and target analysis
|
Binding energy (kJ/mol) |
Binding energy (kJ/mol) |
||
|
Compounds |
GABAARα1 |
Compounds |
GABAARγ2 |
|
Jujuboside B |
-10.5 |
Jujuboside B |
-11.2 |
|
Jujuboside A |
-10.1 |
Jujuboside A |
-9.8 |
|
Rutin |
-9.7 |
Puerarin |
-8.8 |
|
Hesperidin |
-9.5 |
Spinosin |
-8.4 |
|
Spinosin |
-8.7 |
Hesperidin |
-8.4 |
|
Puerarin |
-7.8 |
Rutin |
-8.3 |
|
Quercetin |
-7.7 |
Quercetin |
-7.6 |
|
Ferulic acid |
-7.6 |
Ferulic acid |
-6.4 |
Effect of ZSSE on the survival rate of HT22 nerve cells induced by Glu
It can be seen from Figure 5 that after 24 hours of Glu modeling, the survival rate of cells in Glu group decreased compared with that in blank group. After ZSSE administration, the cell survival rate was significantly higher than that in Glu group. The survival rate increased with the concentration of the drug. The results showed that ZSSE could reduce the damage of Glu to HT22 cells.
Histopathological Findings
PCPA, one kind of inhibitor, which can highly selective inhibition of tryptophan hydroxylase in the brain, resulting in insomnia via a significant reduction of 5-HT in the brain (Yang et al. 2021). Therefore, PCPA was usually selected to establish insomnia modeling due to its fast, efficient and stable modeling characteristic. Generally, within 26 to 30 hours of modeling, the circadian rhythm of rats was disappeared, accompanied with almost complete sleep deprivation state (Shen et al. 2022). In this work, rat insomnia model induced by PCPA has been established to study the sedative and hypnotic mechanism of ZSSE. As shown in Figure 6, the HE staining results showed that compared with the normal group, a large number of neuronal degenerations was observed in the hypothalamic cortex of the model group, and the neuron of the thalamic nucleus was atrophied. Furthermore, the hippocampal pyramidal cells appeared disordered and loose, and unclear structure of neurons. The above experimental results show that a rat model of insomnia induced by PCPA has been successfully established. After one week of administration intervention, compared with the model group, most of the nerve cells in the hypothalamus and hippocampus were recovered in the ZSSE administration groups, indicating that ZSSE can improve sleep via improving the damage to the rat hypothalamus and hippocampus caused by PCPA.
Effects of ZSSE on GABA, GLU, 5-HT and DA levels in hypothalamus and hippocampus of rats
As shown in Figure 7, the contents of GABA and 5-HT in hypothalamus and hippocampus in the model group decreased significantly in comparison with blank group, while the contents of GLU and DA increased significantly. Furthermore, as shown in Figure 7, compared with the blank group, the ratio of 5-HT/DA in the model group decreased significantly and the ratio of Glu/GABA increased significantly. All the results indicate that the model group has been established successfully. Notably, compared with the model group, the concentrations of GLU and DA in hypothalamus and hippocampus were decreased when administration of ZSSE, and increased the levels of GABA and 5-HT. Compared with the model group, the ratio of 5-HT/DA in hypothalamus and hippocampus increased significantly, and the ratio of Glu/GABA decreased significantly.
Effect of ZSSE on the expression of GABAARα1 and γ2 in the hypothalamus of rats
As shown in Figure 8, under light microscopy, the cytoplasm of the GABAARα1 and γ2 positive cells in the hypothalamus of the rats is brown-yellow. The normal control group exhibited more GABAARα1and γ2 positive cells with darker cytoplasm. In the model control group, the number of GABAARα1 and γ2 positive cells in the hypothalamus was decreased obviously, and the cytoplasm became lighter. One week after the intervention, the number of positive cells in the ZSSE administration group and the positive control group were increased to different degrees, and the staining became darker. All the results showed that the average optical density of the positive cells in the model control group was significantly lower than that in the normal group. Compared with the model control group, the average optical density of the positive cells in each dose group was significantly increased.
3.8 Effect of ZSSE on GABAARα1 and γ2 expression in hippocampus of rats
As shown in Figure 9, under light microscopy, the cytoplasm of the GABAARα1and γ2 positive cells in the rat hippocampus exhibits brownish yellow. The hippocampus of normal control group exhibited more GABAARα1and γ2 positive cells with darker cytoplasm. In the model control group, the number of GABAARα1and γ2 positive cells in the hippocampus was decreased obviously, and the cytoplasm became lighter. One week after the intervention, the number of positive cells in the ZSSE administration group and the DZP group were increased to different degrees, and the staining became darker. All the results indicated that the average optical density of GABAARα1and γ2 positive cells in the model control group was significantly lower than that in the normal group. Compared with the model control group, the mean optical density of GABAARα1 positive cells in the ZSSE 13.5 g/kg group and the positive control group were increased, and the expression of GABAARγ2 in the ZSSE 13.5 g/kg group was significantly different from that in the model group. No significant difference was found in the various dose groups of ZSSE.
The pathogenesis of insomnia is extremely complex, and many questions remain. At present, sleep is considered to be a delicate and complex neurochemical process, which is participated by multiple centers in the brain that promote sleep and bring awakening (W 2020). These sleep centers are mainly found in the hypothalamus of the brain (Dell’Osso et al. 2022). As an important nerve center, the hypothalamus receives projections from neurons in many sleep wake nuclei (Yamagata et al. 2021). Studies have shown that stimulating the posterior medial hypothalamus, the low arousal center reacts, which can make animals wake up and get out of sleep (Bajaj and Kaur 2022). Destroying the hypothalamus can activate the reticular structure in the brain, and large-scale injury will lead to complete insomnia (Yamagata et al. 2021). At the same time, the hippocampus, located on the inner side of the cerebral hemisphere, is an important brain structure mainly involved in neural activities such as memory and thinking (Forouzanfar et al. 2021). A large number of animal experiments show that long-term repeated sleep deprivation will greatly reduce the activity of hippocampus, block the occurrence of neurons and damage the ability of regeneration, cause a certain atrophy of hippocampal volume, and reduce Rapid Eye Movement (REM) sleep (Tapp et al. 2022). The results of HE experiments showed that after PCPA modeling, compared with the normal group, there were a large number of neuronal degenerations in hypothalamic cortex, atrophy of thalamic nucleus neurons, disorder and looseness of hippocampal pyramidal cells and unclear neuronal structure in PCPA group. After one week of administration intervention, compared with PCPA group, most nerve cells in hypothalamus and hippocampus in ZSSE administration group returned to normal, indicating that ZSSE can improve sleep by improving the damage of PCPA to hypothalamus and hippocampus in rats. At the same time, cell experiments showed that ZSSE had a certain protective effect on HT22 cells injury induced by Glu, indicating that ZSSE had a certain neuroprotective effect.
In the brain, there are many endogenous neurotransmitters involved in sleep mechanisms, including amino acids, monoamines and cholinergic neurotransmitters (Jhawar et al. 2022). GABA and Glu is a pair of neurotransmitters with mutual antagonism in the central nervous system. Excitatory neurotransmitter Glu can produce inhibitory neurotransmitter GABA under the action of glutamate decarboxylase (Chen et al. 2021, Simon et al. 2012). Many studies have shown that the imbalance of excitatory / inhibitory function of Glu / GABA nerve is the key mechanism of insomnia, and the ratio of GABA, Glu and Glu / GABA can be used as an objective index to judge insomnia. When the content of GABA decreases and Glu increase in the brain, it can cause insomnia (Li et al. 2018b). 5-HT and DA are important biochemical indicators in the clinical research of drugs for insomnia which are important members of neurotransmitters, and the interaction between them can effectively regulate and control the rhythm of sleep and wakefulness (Volgin et al. 2013, Fagan et al. 2021). Studies have shown that the content of 5-HT decreased significantly and the content of DA increased significantly, when animals were subjected to periodic sleep deprivation (Sun et al. 2020). After injecting 5-HT into pigeons, the sleeping time of the drug group was significantly improved and study had proved that patient can activate the wake-up mechanism, when the patient administers to from an intravenous injection DA under anesthesia (Dos Santos et al. 2015, Dos Santos et al. 2015). This study found that the contents of GABA and 5-HT in hypothalamus and hippocampus of insomnia rats decreased significantly, while the contents of Glu and DA increased significantly. After administration, compared with the model group, the contents of GABA and 5-HT in hypothalamus and hippocampus of insomnia rats in ZSSE administration groups increased significantly, while the contents of Glu and DA decreased significantly. The sleep efficiency of rats was significantly increased and the symptoms of insomnia were significantly relieved.
GABAA receptor is widely distributed in the nervous system of mammals, so it plays an important role in almost all brain physiological functions, and serves as the target of a variety of clinical neuro drugs such as sedatives, diazepam and barbiturates (Ghit et al. 2021). GABAA is a GABA gated chloride channel (Ghit et al. 2021). It is a heterogeneous oligomeric transmembrane glycoprotein polymerized by five subunits embedded in the double lipid layer of nerve cell membrane (H 1995). Its center is chloride channel (K 1999). The five subunits of the receptor contain different drug binding sites, and the GABAA receptor subtype composed of α1 and γ2 has the largest number in the brain (Lorenz-Guertin et al. 2018). Studies have shown that most of the surfaces of GABAA receptor subunits α1 and γ2 have binding sites with diazepam and benzodiazepines (Belelli et al. 2021). The mechanism of action is that when GABA is combined with GABAA receptor, the chloride ion channel on the cell membrane is opened, the chloride ion enters the cell along the concentration difference, and the intracellular potential increases to produce hyperpolarization, so as to inhibit the excitation of neurons and achieve the effects of sedation, hypnosis and antianxiety (Has and Chebib 2018). In this study, the results showed that ZSSE could not only increase the content of GABA in the brain of insomnia rats, but also significantly increase the expression of GABAARα1 and GABAARγ2 receptor positive cells in the brain of insomnia rats compared with the model group. Therefore, the above results show that ZSSE can improve sleep by increasing the content of GABA in the brain of insomnia rats and enhancing the binding between GABA and its specific receptors GABAARα1 and GABAARγ2, so as to inhibit the excitation of neurons.
Natural medicines or Chinese medicines had shown excellent therapeutic effects due to its multiple targets characters of multiple ingredients. In this study, HPLC-MS and HPLC were used to study the chemical constituents of ZSSE and explore its potential therapeutic mechanism. The active ingredients of ZSSE were analyzed by combined HPLC and LC/MS. A total of 35 compounds were identified. In order to further clarify the material basis of ZSSE efficacy, classical receptor targets of benzodiazepines were selected for molecular docking. The docking results show that, compounds Jujuboside A, Jujuboside B, Spinosin, Hesperidin, Quercetin, Rutin, Puerarin and Ferulic acid have strong binding ability with related pathways, play a role in improving sleep. Furthermore, Jujuboside A and Jujuboside B exhibited the most prominent binding ability, which might be the main substances of ZSSE in the treatment of improving sleep. The previous studies had shown that Jujuboside A is a saponin component with the highest content in ZSS, and it is also one of the effective constituents of ZSS in the treatment of insomnia (Has and Chebib 2018). Jujuboside B can significantly reduce the daytime sleep time of light-induced sleep-deprived drosophila (Wang et al. 2012). The above results show that the pharmacological effect of ZSSE on improving sleep can be attributed to the comprehensive effect of various components of ZSSE, which reflects the therapeutic characteristics of multi-component and multi-target of traditional Chinese medicine.
In this study, HPLC and HPLC-MS were performed to identify the active ingredients of ZSSE for the first time. Moreover, molecular docking technology was used to prove the effective substances of ZSSE for improving sleep are Jujuboside B, Jujuboside A, spinosin and other chemical components. At the same time, combined with molecular biological techniques such as Elisa and immunohistochemistry, the mechanism and pharmacologic basis of ZSSE in improving sleep were discussed. ZSSE can improve sleep by activating the expression of GABA specific receptors GABAARα1 and GABAARγ2 in hypothalamic GABA system pathway. Furthermore, ZSSE exhibited the advantages of multi-ingredients, multi-targets, and multi-pathway treatment in comparison with Diazepam. Specially, ZSSE exhibited a unique prevention and treatment effect without obvious adverse reactions or drug tolerance after long-term use. Therefore, ZSSE is an active substance extracted from natural plants to improve sleep, which can be used as the raw material for the development of drugs to prevent and treat insomnia in the future.
Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.
Conflicts of interest
The authors declare that they have no conflicts of interest.
Author Contributions
Fengqin Xiao carried out the experiments, participated in the study design and wrote the paper. Shuai Shao analyzed the active ingredients of the extract. Hongyin Zhang analyzed the data. Guangfu Li the spectral effect was studied. Songlan Piao completed the pathological section and analysis. Daqing Zhao performed the in vitro activity tests. Mingming Yan guided all experiments and revised the article. Guangzhe Li participated in the experiment design and revised the article.
Acknowledgments
This study was supported by grants from the National Key Research and Development Program: research on key technologies of ginseng industry and development of massive health products (2017YFC1702100); the Science and Technology Development Plan Project of Jilin Province (20210401111YY); the Science and Technology Development Plan Project of Changchun City (21ZGY20). (JJKH20210965KJ); the Science and technology project of Jilin Provincial Department of Education (JJKH20210965KJ).
- Bajaj P, Kaur G (2022) Acute Sleep Deprivation-Induced Anxiety and Disruption of Hypothalamic Cell Survival and Plasticity: A Mechanistic Study of Protection by Butanol Extract of Tinospora cordifolia. Neurochemical Research 47(6):1692-1706 https://doi.org/10.1007/s11064-022-03562-8
- Belelli D, Hales TG, Lambert JJ, Luscher B, Olsen R, Peters JA, Rudolph U, Sieghart W (2021) GABAA receptors in GtoPdb v.2021.3. IUPHAR BPS Guide Pharm CITE 2021(3) https://doi.org/10.2218/gtopdb/F72/2021.3
- Belelli D, Lambert JJ (2005) Neurosteroids: endogenous regulators of the GABA(A) receptor. Nat Rev Neurosci 6(7):565-575 https://doi.org/10.1038/nrn1703
- Brickley SG, Mody I (2012) Extrasynaptic GABA(A) receptors: their function in the CNS and implications for disease. Neuron 73(1):23-34 https://doi.org/10.1016/j.neuron.2011.12.012
- Cao JX, Zhang QY, Cui SY, Cui XY, Zhang J, Zhang YH, Bai YJ, Zhao YY (2010) Hypnotic effect of jujubosides from Semen Ziziphi Spinosae. J Ethnopharmacol 130(1):163-166 https://doi.org/10.1016/j.jep.2010.03.023
- Chen X, Jia X, Zhang Y, Zhao Z, Hao J, Li H, Du J, Zhuang W, Szeto I, Zhou W, Chen Q, Ma Y, Feng H, Chen Y (2021) The combined use of gamma-aminobutyric acid and walnut peptide enhances sleep in mice. Ann Palliat Med 10(10):11074-11082 https://doi.org/10.21037/apm-21-2798
- Dell’Osso L, Massoni L, Battaglini S, Cremone IM, Carmassi C, Carpita B (2022) Biological correlates of altered circadian rhythms, autonomic functions and sleep problems in autism spectrum disorder. Annals of General Psychiatry 21(1) https://doi.org/10.1186/s12991-022-00390-6
- Dong YJ, Jiang NH, Zhan LH, Teng X, Fang X, Lin MQ, Xie ZY, Luo R, Li LZ, Li B, Zhang BB, Lv GY, Chen SH (2021) Soporific effect of modified Suanzaoren Decoction on mice models of insomnia by regulating Orexin-A and HPA axis homeostasis. Biomed Pharmacother 143:112141 https://doi.org/10.1016/j.biopha.2021.112141
- Dos Santos TS, Kruger J, Melleu FF, Herold C, Zilles K, Poli A, Gunturkun O, Marino-Neto J (2015) Distribution of serotonin 5-HT1A-binding sites in the brainstem and the hypothalamus, and their roles in 5-HT-induced sleep and ingestive behaviors in rock pigeons (Columba livia). Behav Brain Res 295:45-63 https://doi.org/10.1016/j.bbr.2015.03.059
- Fagan RR, Kearney PJ, Luethi D, Bolden NC, Sitte HH, Emery P, Melikian HE (2021) Dopaminergic Ric GTPase activity impacts amphetamine sensitivity and sleep quality in a dopamine transporter-dependent manner in Drosophila melanogaster. Mol Psychiatry 26(12):7793-7802 https://doi.org/10.1038/s41380-021-01275-y
- Fang X, Hao JF, Zhou HY, Zhu LX, Wang JH, Song FQ (2010) Pharmacological studies on the sedative-hypnotic effect of Semen Ziziphi spinosae (Suanzaoren) and Radix et Rhizoma Salviae miltiorrhizae (Danshen) extracts and the synergistic effect of their combinations. Phytomedicine 17(1):75-80 https://doi.org/10.1016/j.phymed.2009.07.004
- Forouzanfar F, Gholami J, Foroughnia M, Payvar B, Nemati S, Khodadadegan MA, Saheb M, Hajali V (2021) The beneficial effects of green tea on sleep deprivation-induced cognitive deficits in rats: the involvement of hippocampal antioxidant defense. Heliyon 7(11):e08336 https://doi.org/10.1016/j.heliyon.2021.e08336
- Fu Jingwei QW, Chen Chaohui (2005) Experimental study on sedative and hypnotic effects of total alkaloids from Zizyphus jujuba seed. Journal of Tianjin Medical University
- Ghit A, Assal D, Al-Shami AS, Hussein DEE (2021) GABAA receptors: structure, function, pharmacology, and related disorders. J Genet Eng Biotechnol 19(1):123 https://doi.org/10.1186/s43141-021-00224-0
- H HWL (1998) The diversity of GABAA receptors. Pharmacological and electrophysiological properties of GABAA channel subtypes. Molecular Neurobiology
- H RLERSJFD (1995) From ion currents to genomic analysis: recent advances in GABAA receptor research. Synapse
- Has ATC, Chebib M (2018) GABAA receptors: Various stoichiometrics of subunit arrangement in alpha1beta3 and alpha1beta3epsilon receptors. Curr Pharm Des 24(17):1839-1844 https://doi.org/10.2174/1381612824666180515123921
- He SR, Zhao CB, Zhang JX, Wang J, Wu B, Wu CJ (2020) Botanical and Traditional Uses and Phytochemical, Pharmacological, Pharmacokinetic, and Toxicological Characteristics of Ziziphi Spinosae Semen: A Review. Evid Based Complement Alternat Med 2020:5861821 https://doi.org/10.1155/2020/5861821
- Jhawar S, Gopala Krishna G, Chikkanna U (2022) Understanding the pathophysiology of insomnia (Anidra) with special reference to primary insomnia using neurotransmitter sleep theories: a narrative review. Journal of Indian System of Medicine 10(1) https://doi.org/10.4103/jism.Jism_75_21
- Jia Ying GY, sun Shengjie, Wang Yingli, Feng Qianjin (2018) Sedative and hypnotic effects of supercritical CO2 extraction of volatile oil from raw jujube kernel. Chinese Journal of traditional Chinese Medicine
- K MAKTM (1999) An update on GABAA receptors. Brain Research Reviews
- Kleinman RA, Weiss RD (2022) Benzodiazepine-Involved Overdose Deaths in the USA: 2000-2019. J Gen Intern Med https://doi.org/10.1007/s11606-021-07035-6
- Lahteenmaki R, Neuvonen PJ, Puustinen J, Vahlberg T, Partinen M, Raiha I, Kivela SL (2019) Withdrawal from long-term use of zopiclone, zolpidem and temazepam may improve perceived sleep and quality of life in older adults with primary insomnia. Basic Clin Pharmacol Toxicol 124(3):330-340 https://doi.org/10.1111/bcpt.13144
- Li J, Yu T, Shi F, Zhang Y, Duan Z, Fu B, Zhang Y (2018a) Involvement of Ventral Periaqueductal Gray Dopaminergic Neurons in Propofol Anesthesia. Neurochem Res 43(4):838-847 https://doi.org/10.1007/s11064-018-2486-y
- Li L, Zhang MQ, Sun X, Liu WY, Huang ZL, Wang YQ (2022) Role of Dorsomedial Hypothalamus GABAergic Neurons in Sleep-Wake States in Response to Changes in Ambient Temperature in Mice. Int J Mol Sci 23(3) https://doi.org/10.3390/ijms23031270
- Li LB, Kim YW, Wang YH, Bai L, Zhu XD, Zhao ZL, Lee CW, Jiao Y, Wu T, Cai ZZ, Kim SC, An WG, Yang CH, Cui GC, Zhao RJ (2019) Methanol extract of semen Ziziphi Spinosae attenuates ethanol withdrawal anxiety by improving neuropeptide signaling in the central amygdala. BMC Complement Altern Med 19(1):147 https://doi.org/10.1186/s12906-019-2546-0
- Li N, Liu J, Wang M, Yu Z, Zhu K, Gao J, Wang C, Sun J, Chen J, Li H (2018b) Sedative and hypnotic effects of Schisandrin B through increasing GABA/Glu ratio and upregulating the expression of GABAA in mice and rats. Biomed Pharmacother 103:509-516 https://doi.org/10.1016/j.biopha.2018.04.017
- Liu Z, Zhao X, Liu B, Liu AJ, Li H, Mao X, Wu B, Bi KS, Jia Y (2014) Jujuboside A, a neuroprotective agent from semen Ziziphi Spinosae ameliorates behavioral disorders of the dementia mouse model induced by Abeta 1-42. Eur J Pharmacol 738:206-213 https://doi.org/10.1016/j.ejphar.2014.05.041
- Lorenz-Guertin JM, Bambino MJ, Jacob TC (2018) gamma2 GABAAR Trafficking and the Consequences of Human Genetic Variation. Front Cell Neurosci 12:265 https://doi.org/10.3389/fncel.2018.00265
- McKillop LE, Fisher SP, Milinski L, Krone LB, Vyazovskiy VV (2021) Diazepam effects on local cortical neural activity during sleep in mice. Biochem Pharmacol 191:114515 https://doi.org/10.1016/j.bcp.2021.114515
- Michels G, Moss SJ (2007) GABAA receptors: properties and trafficking. Crit Rev Biochem Mol Biol 42(1):3-14 https://doi.org/10.1080/10409230601146219
- Morris CJ, Corte DD (2021) Using molecular docking and molecular dynamics to investigate protein-ligand interactions. Modern Physics Letters B 35(08) https://doi.org/10.1142/s0217984921300027
- Murphy Y, Wilson E, Goldner EM, Fischer B (2016) Benzodiazepine Use, Misuse, and Harm at the Population Level in Canada: A Comprehensive Narrative Review of Data and Developments Since 1995. Clin Drug Investig 36(7):519-530 https://doi.org/10.1007/s40261-016-0397-8
- Qeadan F, Madden EF, Mensah NA, Tingey B, Herron J, Hernandez-Vallant A, Venner KL, English K, Dixit A (2022) Epidemiological trends in opioid-only and opioid/polysubstance-related death rates among American Indian/Alaska Native populations from 1999 to 2019: a retrospective longitudinal ecological study. BMJ Open 12(5):e053686 https://doi.org/10.1136/bmjopen-2021-053686
- Shen C-Y, Li X-Y, Ma P-Y, Li H-L, Xiao B, Cai W-F, Xing X-F (2022) Nicotinamide mononucleotide (NMN) and NMN-rich product supplementation alleviate p-chlorophenylalanine-induced sleep disorders. Journal of Functional Foods 91 https://doi.org/10.1016/j.jff.2022.105031
- Sigel E (2005) The Benzodiazepine Recognition Site on GABAA Receptors. Medicinal Chemistry Reviews - Online https://doi.org/10.2174/1567203054065673
- Simon C, Hayar A, Garcia-Rill E (2012) Developmental changes in glutamatergic fast synaptic neurotransmission in the dorsal subcoeruleus nucleus. Sleep 35(3):407-417 https://doi.org/10.5665/sleep.1706
- Sun Y, Zhang N, Qu Y, Cao Y, Li J, Yang Y, Yang T, Sun Y (2020) Shuangxia decoction alleviates p-chlorophenylalanine induced insomnia through the modification of serotonergic and immune system. Metab Brain Dis 35(2):315-325 https://doi.org/10.1007/s11011-019-00499-w
- Tapp ZM, Cornelius S, Oberster A, Kumar JE, Atluri R, Witcher KG, Oliver B, Bray C, Velasquez J, Zhao F, Peng J, Sheridan J, Askwith C, Godbout JP, Kokiko-Cochran ON (2022) Sleep fragmentation engages stress-responsive circuitry, enhances inflammation and compromises hippocampal function following traumatic brain injury. Exp Neurol 353:114058 https://doi.org/10.1016/j.expneurol.2022.114058
- Volgin DV, Stettner GM, Kubin L (2013) Circadian dependence of receptors that mediate wake-related excitatory drive to hypoglossal motoneurons. Respir Physiol Neurobiol 188(3):301-307 https://doi.org/10.1016/j.resp.2013.04.024
- W VSEJ (2020) Brain mechanisms of insomnia: new perspectives on causes and consequences. Physiological Reviews https://doi.org/10.1152/physrev.00046.2019
- Wang LE, Cui XY, Cui SY, Cao JX, Zhang J, Zhang YH, Zhang QY, Bai YJ, Zhao YY (2010) Potentiating effect of spinosin, a C-glycoside flavonoid of Semen Ziziphi spinosae, on pentobarbital-induced sleep may be related to postsynaptic 5-HT(1A) receptors. Phytomedicine 17(6):404-409 https://doi.org/10.1016/j.phymed.2010.01.014
- Wang X, Yang B, Sun H, Zhang A (2012) Pattern recognition approaches and computational systems tools for ultra performance liquid chromatography-mass spectrometry-based comprehensive metabolomic profiling and pathways analysis of biological data sets. Anal Chem 84(1):428-439 https://doi.org/10.1021/ac202828r
- Wang XX, Ma GI, Xie JB, Pang GC (2015) Influence of JuA in evoking communication changes between the small intestines and brain tissues of rats and the GABAA and GABAB receptor transcription levels of hippocampal neurons. J Ethnopharmacol 159:215-223 https://doi.org/10.1016/j.jep.2014.11.012
- Wisden W, Yu X, Franks NP (2019) GABA Receptors and the Pharmacology of Sleep. Handb Exp Pharmacol 253:279-304 https://doi.org/10.1007/164_2017_56
- Yamagata T, Kahn MC, Prius-Mengual J, Meijer E, Sabanovic M, Guillaumin MCC, van der Vinne V, Huang YG, McKillop LE, Jagannath A, Peirson SN, Mann EO, Foster RG, Vyazovskiy VV (2021) The hypothalamic link between arousal and sleep homeostasis in mice. Proc Natl Acad Sci U S A 118(51) https://doi.org/10.1073/pnas.2101580118
- Yang Bo ZA, Wang Ping, sun Wenjun, Wu Xiuhong, Yan Guangli, Han Ying, sun Hui, Wang Xijun (2013) Experimental study of Zizyphus jujubosides A and B in the treatment of Insomnia Based on Drosophila model. Information on Traditional Chinese Medici https://doi.org/10.19656/j.cnki.1002-2406.2013.05.020
- Yang Y, Wu Y, Xu P, Guo F, Guo F, Yang B (2021) Nyctinastic herbs decoction improves para-chlorophenylalanine-induced insomnia by regulating the expression level of neurotransmitters. Ann Transl Med 9(20):1524 https://doi.org/10.21037/atm-21-4462
No competing interests reported.