| Objective:Chronic emotional stress model induced by empty bottle stimulation in rat was set up in an attempt to explore its neurobiological mechanism and the interfering effect of Sinisan active ingredients on it.Methods:1. Replication of chronic emotional stress model in rat using empty bottle successive stimulation for thirty-five days. Chronic emotional stress model of Sprague Dawley rats was established by empty bottle stimulation for 35 days being judged by the main parameters such as body weight, behavioristics (aggressive, exploratory and grooming behavior), and serum corticosterone content.2. Study of the neurobiological mechanism of chronic emotional stress in rat induced by empty bottles stimulation for twenty-one days. Sprague Dawley rats were continuously stimulated by bottles stimulation for 21 days to establish the model of chronic emotional stress. The organizational changing of hippocampal neurons were observed by histopathological technique. The protein expression of central corticosterone receptor (GR), glutamate receptors (NMDAR 2B), brain-derived neurotrophic factor (BDNF) in hippocampus were assessed by immunohistochemistry technique. Furthermore, in amygdale region, the CB1 (cannabinoid type 1), CRF (corticotropin releasing factor) and BDNF protein expression were observed. In addition, FAAH (fatty acid amid hydrolase) and CB1 receptor gene expression in amygdala were determined by quantitative PCR.3. Study on the antagonistic effect and the neurobiological mechanism of Sinisan active ingredients on chronic emotional stress rat induced by empty bottles stimulation for twenty-one days. Sprague Dawley rats were induced by empty bottles stimulation for twenty-one days combined with Sinisan-treatment. The changing of body weight, behavioristics, serum corticosterone and central hippocampus neuronal tissue in rats were employed as the mainly indexes in order to observe the effect of Sinisan active ingredients on chronic emotional stress in rat. Moreover, protein expressions of GR, NMDAR 2B and BDNF in hippocampus and the amygdala CB1, CRF, BDNF FAAH, CB1 receptor gene expression in chronic emotional stress model of rat was selected as the main indicators with aim of exploring the interfering neurobiological mechanism of Sinisan active ingredients.Results:1. Establishment of chronic emotional stress model by empty bottle stimulation for thirty-five days in rat.Our results showed that the rat body weight, behavioristics (aggressive, exploratory and grooming behaviors) and changing of serum corticosterone were significant difference between stimulation group and control group. The body weight in stimulated rats was significantly lower than control group (p<0.05) from the second week to the fourth Week. Besides, with respect to behavioristics, stimulation group rats brought out more aggressive than control group from first week (p<0.01) and enhancement of aggressive behavior was as time-manner from the second to the fifth week with a windless tendency. Meanwhile, the aggressive behaviors were not found in control group and physiological group. Additionally, exploratory behavior in stimulatioin group was significantly increased when compared with control group especially at the 2,3 and 4 week (P<0.01). Additionally, grooming behavior in stimulation group were significant lower than the control group (p<0.01) at the 2 and 3 week (P<0.05). Moreover, the serum corticosterone level in stimulation group were significantly increased at 1,2,3,4 and 5 week when compared with control group and physiological group (P<0.01).2. The neurobiological mechanism of chronic emotional stress induced by empty bottles stimulation for twenty-one days.The serum corticosterone levels of physiological group and stimulation group were significantly enhanced with a significant difference (P<0.01) relative to control group. Simultaneously, compared with physiological group, the serum corticosterone level in stimulation group was significantly boosted (P<0.01). As regards histopathological variation in the hippocampus, the obvious change of hippocampal tissue emerging in stimulation group included relaxation of neurons arrangement, fractional lack of neuron in difference region, cell swelling, partial degeneration and dissociation compared with the control group. The same pathological phenomenon described at the above was observed in stress group with a slight degree. Compared with the control group, the expression of hippocampal GR and BDNF positive neurons and the expression of NMDAR 2B-positive neurons in stimulation group were significantly decreased and increased (P<0.01), respectively. Amygdala CB1 positive neurons was significantly reduced and amygdala BDNF positive neurons was significantly increased (P<0.05) contrasted to control group. Additionally, CRF positive neurons in amygdala was significantly increased (P<0.01) than control group. To compare between control group and stimulation group, mRNA content of CB1 gene and mRNA content of FAAH gene in stimulation group was significantly reduced and increased, respectively, with a significant differences (P<0.01).3. The antagonistic effect and neurobiological mechanism of Sinisan active ingredients on chronic emotional stress model induced by empty bottles stimulation for twenty-one days. About the antagonistic effect of Sinisan active ingredients, the body weight of treatment group was significantly increased (P<0.01) when compared with stimulation group. In behavioral aspect, to compare with stimulation group, the treatment rats showed that the aggressive and exploratory behaviors were significantly reduced (P<0.05), while grooming behavior was significantly increased with no significant difference between treatment group and normal group (P>0.05). Serum corticosterone was extremely decreased (P<0.01) in treatment group in comparation with stimulation group and was no significant difference relative to normal group (P> 0.05). To compare the histological changes of hippocampus in stimulation group, the treatment group manifested a significantly improvement in the histopathological hippocampus changes induced by emotional stress, accompanied with tightness of intercellular arrangement, clear framework, occasional cell swelling and rare losing of neuron. Concerning the mechanism of Sinisan active ingredients, there was a significant difference (P<0.01) between stimulation group and treatment group, showing the increasing of GR and BDNF positive neurons expression and the reduction of NMDAR 2B positive neuron expression in treatment group. Moreover, compared with stimulating group, results from treatment group implied that CB1 expression significantly increased, while BDNF expression was markedly decreased significance (P<0.05), and CRF positive neurons also dramaticlly reduced expression (P<0.01) in amygdale area. In addition, CB1 mRNA expression and the amount of FAAH mRNA expression was increased (P<0.01) and decreased (P<0.01), respectively, in treatment group compared with model group.Conclusion:1. The body weight, behavior and serum corticosterone in rat were consecutive changed in the process of chronic emotional stress induced by empty bottle stimulation for thirty-five days, indicating that animal were under the state of chronic emotional stress, which proved that this animal model was reliability and stability.2. The neurobiological mechanism of chronic emotional stress model induced by empty bottles stimulation for twenty-one days in rat was the sustained HPA (hypothalamic-pituitary-adrenocortical) axis hyperactivity, leading to corticosterone toxemia, reduction of GR and BDNF expression in the hippocampus, increasement of NAMD receptor expression, reduction of amygdala CB1 and CB1mRNA expression, and augment of CRF, BDNF and FAAH mRNA expression. In a word, pathological physiological and behavioral changes was produced by stimulate chronic emotional stress provoked by empty bottles in rat.3. Active ingredients of Sinisan could effectively ameliorate receptors, genes and neuronal factors related to chronic emotional stress, keep the normal hippocampus and amygdala physiological functions, and antagonize pathophysiological and behavioral change in rat suffering from chronic emotional stress. |
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