Establish A Sedative Hypnotics Preclinical Pharmacodynamic Evaluation Method And The Research On The West Safflower Hypnotic Effect And Mechanism Of Active Ingredients

Objective:Insomnia is becoming a serious medical and social problem all over the world. Although traditional chemical synthesized hypnotics are clinically effective for insomnia patients temporarily, there are many side effects, such as drug dependence, tolerance, and rebound insomnia after long-term treatment. Chinese herbs have been clinically shown to be effective for insomnia with little adverse reactions. However, it lacks scientific proof owing to the deficiency of insomnia animal model and the evaluation platform for hypnotic drugs. This study intended to establish an animal model treated with low-dose pentobarbital under novel environment or insomnia model to mimick the "first-night effect" transient insomnia, on the basis of a highly automatic recording-analysis system for sleep-wake profile. Using these models, we attempted to evaluate the effect of active ingredients extracted from saffron, a traditional Chinese herb, and to clarify its mechanism.Methods:(1) Infrared detection of locomotor activity (LMA):self-controlled experiments were carried out in40male C57BL/6J mice randomly divided into5groups:mice with intraperitoneally (i.p.) injection of vehicle at20:00on the first LMA detecting day served as the control group and those with i.p. injection of safranal (90,180or360mg/kg), diazepam (3mg/kg), or flumazenil (1mg/kg) plus safranal (360mg/kg) at the same time on the next day served as the experimental groups. LMA was recorded for48hours to observe the sedative effect of safranal and whether this effect can be antagonized by flumazenil.(2) Time for loss of righting reflex (LORR) after the treatment of pentobarbital sodium:the male Kunming mice were divided into10groups. This experiment was carried out in a sound-proof chamber with constant temperature and humidity in the afternoon (13:00-19:00). The sleep latency was defined as the time from the injection of pentobarbital to the onset of LORR.The duration of LORR was determined as the time from the onset to the recovery of LORR. Thirty minutes after the i.p. injection of vehicle, safranal (90,180or360mg/kg) or diazepam (3mg/kg), pentobarbital (45mg/kg) was given i,p. Flumazenil (1or3mg/kg), was injected20min before the injection of safranal to see whether safranal acted on the benzodiazepine receptors.(3) Evaluation of the hypnotic effect of safranal in mice treated with pentobarbital under novel environment (NV-PENT):the male C57BL/6J mice were allowed7days to recovery from chronic implantation of EEG and electromyogram (EMG) electrodes before the execution of continuous polysomnographic recordings for48h without adaption to the recording chambers. Mice were divided into5groups (5-6each) to study the effect of safranal on sleep:vehicle group, safranal group at a dose of90,180or360mg/kg and diazepam group at3mg/kg in which the above corresponding drugs were intragastrically(i.g.) administered at20:00before the i.p. injection of pentobarbital at21:00.(4) Establishment of the first-night effect-mimicked insomnia (FNEI) mice model to evaluate hypnotic effect of crocin:the manipulation is similar to that in (3) before polysomnographic recording except that these mice were allowed three days to adapt to the chambers. Then continuous recording were started at7:00on the next day, lasting for48h. Data from the first recording day served as the baseline, and on the second recording day mice were transferred at10:00to clean cages or dirty cages in which other mice stayed5days, to assess the characteristics of wakefulness in cage chage-induced insomnia mice. Procedure for evaluation of crocin is the same except for an i.p. injection of vehicle or crocin at200or400mg/kg20min before cage change.(5) Neurochemical experiment:mice, divided as that in (3), were decapitated90min after drug administration, with their brains dissected immediately into the cortex, hippocampus, striatum, medulla, midbrain, thalamus and hypothalamus, weighted and stored at-80℃for subsequent detection. Following homogenate and centrifugation, the supernate were detected by high performance liquid chromatography-electrochemical detector (HPLC-ECD) to monitor the level of neurotransmitters.(6) Immunohistochemical experiment:mice treated as (2)/(4), were deeply anesthetized with chloral hydrate (i.p.500mg/kg) and then perfused with50ml saline followed by50ml10%formalin through the heart. The brains were removed, postfixed for4h in10%formalin, and then immersed in10%,20%and30%sucrose. After these treatments, brains were sectioned on a freezing microtome at30μm followed by post hoc staining for c-Fos to judge the changes of neuronal activity.Results:1. Safranal administered i.p. at doses of90,180and360mg/kg dose-dependently decreased LMA in mice, which lasted for2,4, and7h, respectively. Flumazenil (1mg/kg, i.p.), an antagonist of benzodiazepine receptors, reversed the sedative effect of safranal at a dose of360mg/kg from7to2h. Safranal at a dose of90,180or360mg/kg also dose-dependently prolonged the duration of LORR in mice induced by pentobarbital (45mg/kg, i.p.) by35%,68%and176%, respectively. Flumazenil at3mg/kg neutralized partly the extension of LORR caused by safranal at360mg/kg.2. Safranal administered i.g. at doses of90,180and360mg/kg dose-dependently increased NREM sleep rather than REM sleep, which lasted for1,2and3h respectively. And the amounts of NREM sleep in the first3h after administration of safranal increased by35%,68%and176%. In the control group, the latency to NREM sleep was3.47min, and safranal at180and360mg/kg shortened the latency to NREM sleep by1.61min and1.86min, respectively. Neither Safranal at the three doses nor diazepam at3mg/kg had effect on the latency to REM sleep. Safranal at180and360mg/kg enhanced delta power density and increased slow wave activity (SWA) and transition numbers of sleep in the first3hours, which was opposite to that of the diazepam group. Safranal at180mg/kg but not90and360mg/kg shortened the mean duration of NREM sleep. Neurochemistry data showed that in NV-PENT mice, safranal at360mg/kg increased only the level of3,4-dihydroxy-phenylacetic acid (DOPAC), one of metabolites for dopamine (DA), in all catecholamine and metabolites in the cortex, striatum and midbrain and the level of DOPAC or homovanillic acid (HVA), another metabolites for DA in the hippocampus, midbrain and hypothalamus, raised the ratio of (DOPAC+HVA)/DA in the midbrain and hypothalamus, and decreased the level of norepinephrine (NE), an awaking substance, in the hippocampus, midbrain and hypothalamus. In all seven detected regions, level of5-hydroxytryptamine (5-HT) trended to be decreased, while5-hydroxyindole acetic acid (5-HIAA), one of metabolites of5-HT increased. The ratio of5-HIAA/5-HT was raised in the cortex, striatum, midbrain and hypothalamus, indicating the acceleration of metabolism for5-HT, which may be a hypnotic mechanism for safranal. The immunohistochemical data showed that safranal at a high dose enhanced c-Fos expression in the ventrolateral preoptic nucleus (VLPO) in NV-PENT mice compared to the control, indicating that the promotion of NREM sleep by safranal is mainly based on the activity of neurons in the VLPO.3. The FNEI mice model is to cause insomnia in mice under a certain psychological stress through changing their living environmentto a new dirty or clean cage. The data showed that the amount of wakefulness/sleep was increased/decreased in the first hour after changed to either a dirty or a clean cage, compared to the control, which extended even to3h; although there was no statistical difference in wakefulness of each hour, NREM sleep and REM sleep in the first6hours between the dirty cage group and the clean cage group, the amount of wakefulness in clean cage group was increased in the first6hours, compared to the dirty cage group. These results indicated that change of host cage caused transient insomnia in mice with more facility of clean cage. When compared to the baseline, cage change increased the power density of NREM sleep and decreased the transition numbers of sleep and the fragmentation degree. However, only the power density of NREM sleep was increased as the cage became cleaner.4. The evaluation for hypnotic effect of crocin based on the FNEI mouse model is below:crocin at dose of200mg/kg enhanced the Delta power density of NREM sleep with no effects on the amounts of sleep/wakefulness, latency to sleep and SWA; i.p. injection of crocin at dose of400mg/kg trended to increase NREM sleep and REM sleep and decrease wakefulness, which lasted for3h, although there was no significant difference in the amount of sleep/wakefulness; the latencies to NREM sleep and REM sleep were reduced by34.58%and27.97%, respectively; both the delta power density and SWA were decreased. These results indicated that a low dose of crocin augmented sleep depth but not affect the amount of NREM sleep, while the high dose had the opposite effect. Crocin200mg/kg only increased the brief wakefulness, when400mg/kg decreased the mean duration of wakefulness and NREM sleep, as well as increased the amount of NREM sleep, the transition numbers of wakefulness and the episodes of sleep/wakefulnessin FNEI mice. Immunohistochemical data showed that crocin only decreased the expression of c-Fos in the locus coeruleus (LC) when compared to the control, which implicated the decrease of noradrenergic neuronal activity.Summary:1. Safranal dose-dependently decreased LMA and prolonged the duration of LORR caused by pentobarbital, which could be antagonized by flumazenil.2. Safranal increased the transition numbers between wake and NREM sleep and the amount of NREM sleep, shortened NREM latency, enhanced NREM power density and reduced the mean duration of each sleep episode or no change in NV-PENT mice.3. Safranal decreased the level of NE, increased the level of DOPAC/HVA, metabolites of DA in the hippocampus, midbrain and hypothalamus, the ratio of5-HIAA/5-HT in the cortex, striatum, midbrain and hypothalamus, and the expression of c-Fos in the VLPO.4. The FNEI mice model showed increase in wakefulness, time extention to onset sleep, reduced sleep, difficulty in maintainence of sleep, reduced transition numbers, as well as the enhanced power denisty of NREM sleep. Compared to a dirty cage, when mice were changed into a clean cage, the amount of wakefulness and subsequent power density of NREM sleep increased.5. Crocin increased NREM sleep, shortened sleep latency, decreased delta power density of NREM sleep and the expression of c-Fos in the LC of FNEI mice.