| Objective:1. Chronic emotional stress (body weight, attacking behavior, corticosterone concentrations in serum) and sleep pattern (sleep, rest-activity rhythms, sleep-wake rhythm) were used as indicators to establish model of rats with chronic emotional stress-sleep disorders.2. In order to study the antagonistic effect and mechanism of the Sinisan active ingredient on chronic emotional stress-sleep disorder model in rats.Methods:1. Part one:The establishment of the model of chronic emotional stress induced by empty bottle stimulation.18 male SD rats were split into:group ES, group PS, group C. Establish an empty bottle stimulation model for 14 days. The rat’s body weight, attacking behavior, serum CORT level were measured.2. Part two:The effect of empty bottle stimulation on sleep pattern in rats.36 male SD rats were split into:group ES, group PS, group C. Establish an empty bottle stimulation model for 14 days. Sleep high-throughput technology (CLAMS) were used to study the rats’ sleep pattern. The rat’s sleep time, sleep-wake rhythm, rest-activity rhythm were measured.3. Part three:Mechanism of sleep disorder in rats induced by chronic emotional stress.To undertake the second part of the experiment, after the end of the sleep monitoring the rats were killed by cutting down the head to remove the brain. Half rats were prepared whole brain, the other rats were prepared hypothalamus and cortex to determine sleep related cytokines including IL-1β, TNF-α, IL-4, IL-10 level’s changes after chronic emotional stress through enzyme-linked immunosorbent assay (ELISA) technology.4. Part four:Intervention effect and mechanism of Sinisan active ingredients on the sleep disorder induced by chronic emotional stress in rats.24 male SD rats were split into:group ES, group C. group DZP (Diazepam 1mg/kg), group SNS (Sinisan active ingredients 450mg/kg). Establish an empty bottle stimulation model for 14 days. At the beginning of the week 2 of stimulation, the medication groups were treated with corresponding medicine. Group ES and group C were treated with saline. The rat’s body weight, attacking behavior, serum corticosterone content were measured to study on the intervention effect of the sinisan active ingredients on the emotional stress.48 male SD rats were split into:group ES, group C. group DZP (Diazepam lmg/kg), group SNS (Sinisan active ingredients 450mg/kg). Establish an empty bottle stimulation model for 14 days. At the beginning of the week 2 of stimulation, the medication groups were treated with corresponding medicine. Group ES and group C were treated with saline. The rat’s sleep time, sleep-wake rhythm, rest-activity rhythm were measured to study on the intervention effect of the sinisan active ingredients on the emotional stress model rat’s sleep pattern. After the end of the sleep monitoring the rats were killed by cutting down the head to remove the brain. Half rats were prepared whole brain, the other rats were prepared hypothalamus and cortex to determine sleep related cytokines including IL-1β, TNF-α, IL-4, IL-10 level’s changes to study on the mechanism of the sinisan active ingredients improve the sleep disorders rats caused by chronic emotional stress.Results:1. Part one:The establishment of the model of chronic emotional stress induced by empty bottle stimulation.The experimental results showed that at the end of the adaptation period, on body weight has no difference among group ES, group PS, group C (P>0.05). After the empty bottle stimulation, group ES and group PS rat’s weight decreased (P<0.01, P<0.01); Group C and group PS rat’s attack behavior did not appear during the stimulation; Empty bottle stimulation group’s attacks behavior appears lots during the stimulation (P<0.01); Group ES rat’s serum corticosterone concentration significant increased (P<0.01).2. Part two:The effect of empty bottle stimulation on sleep pattern in rats.The experimental results showed that group ES and group PS rat’s total sleep time and night sleep time significant reduced, (P<0.05, P<0.05); In the rest-activity rhythm:Each group rat’s rest-activity rhythm showed circadian rhythm changing. Turn off lights (time 19:00) and drinking water (9:00 am and 21:00 pm) caused obvious activity peak. After the water supply, group ES and group PS has more activities; In the sleep-wake rhythm:Each group rat’s sleep-wake rhythm showed circadian rhythm changing. After drinking water at 9 in the morning, group ES rats has significant sleep fragmentation; After drinking water at 21 in the night, group ES rats has more activity, less sleep, and recovery of sleep fore a long time.3. Part three:Mechanism of sleep disorder in rats induced by chronic emotional stress.The experimental results showed that:The TNF-a level in the whole brain and hypothalamus decreased in both group ES and group PS (P<0.01, P<0.01). The IL-4 level in the hypothalamus and cortex increased in group ES (P<0.05, P<0.05).4. Part four:Intervention effect and mechanism of sinisan active ingredients on the sleep disorder induced by chronic emotional stress in rats.The experimental results showed that:In terms of antagonistic emotional stress:Group DZP and group SNS rat’s weight has the trend of increasing (P>0.05, P>0.05). Compared with group ES, sinisan active ingredients group’s attack behavior decreased (P<0.05), serum corticosterone level decreased (P<0.01). There were no different on the serum CORT level between group DZP and group ES.In the effect of sleep pattern:Group DZP and group SNS rat’s total sleep time increased(P<0.01, P<0.05), night sleep time increased at the same time(P<0.05, P<0.05); In the rest-activity rhythm:The group DZP and group SNS rat’s activity is lower than the empty bottle stimulation group, especially in the night and after water supply; In the sleep-wake rhythm:After drinking water, group DZP and group SNS rats ues a long time to recovery of sleep, sleep fragmentation is not evident and there is a trend of integration.In the mechanism:The TNF-a level in the whole brain increased in both group DZP and group SNS (P<0.05, P<0.05); TNF-α level increased in the hypothalamus of group SNS (P<0.05);IL-4 level decreased in the cortex in both positive drug group and sinisan active ingredients group (P<0.05, P<0.05).Conclusion:1.The animal model of chronic emotional stress-sleep disorders in rats was successfully reproduced by the empty bottle stimulation. After the model established, rats body weight decreased, attacking behavior increased, serum corticosterone concentration increased. Sleep disorder in rats is mainly manifested as the decrease of the whole day’s sleep time, the activity of the night increased, and the sleep fragmentation increased.2. Sleep disorder induced by chronic emotional stress caused by empty bottle stimulation. The levels of TNF-α in the whole brain and hypothalamus of rats were decreased, and the levels of IL-4 in the hypothalamus and cortex were increased.3. Sinisan active ingredient has the function of resisting the chronic emotional stress induced by the empty bottle stimulation, can make the rat’s attacking behavior decreased, serum corticosterone concentration decreased, it also can improve the sleep disorder caused by chronic emotional stress at the same time. Mainly for increased chronic emotional stress rat whole day’s sleep time, reduce the activity of the night and sleep fragmentation has integration trend.4. Sinisan active ingredient can increase the level of TNF-α in the whole brain and hypothalamus of the rats with chronic emotional stress, and reduce the IL-4 level in the cortex to improve the sleep disorder induced by chronic emotional stress. |
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