| ObjectiveThe study aims to investigate the expression of Apelin and APJ receptor in hypothalamus of model mice with stagnation of liver qi and spleen deficiency and the regulatory role of Xiaoyao san. Studying on the action mechanism of APJ/Apelin system and exploring the relationship between APJ/Apelin system and stress can provide new ideas for the study of syndrome of stagnation of liver qi and spleen deficiency. Apelin and APJ receptor involve in the physiological activities of the nervous, endocrine, immune, digestive and circulatory systems, also in a variety of biological function of cardiovascular, central nervous system, fluid balance, gastrointestinal function, feeding and drinking water has a regulatory role, the as neuropeptide in hypothalamic paraventricular nucleus and the supraoptic nucleus expression is abundant in the physiological activities of the hypothalamus. This study makes the observation of the expression of hypothalamic Apelin and APJ receptor changes in stagnation of liver qi and spleen deficiency model mouse and the regulation of Xiaoyao san, duplicate the model of C57BL/6J mice with stagnation of liver qi and spleen, and animal model is evaluated from the aspects of animal behavior, macroscopic representation, objective index to understand and control the treatment effect of Xiaoyao san group and control drug group. The expression of APJ/Apelin as the breakthrough point, by using immunohistochemistry and cell morphology, molecular biology technology from gene expression level to detect the effect of Xiaoyao san on the expression of APJ/Apelin in hypothalamus of mice to study the relationship between syndrome of stagnation of liver qi and spleen deficiency and the expression of APJ/Apelin in hypothalamus and the effect of Xiaoyao san on the expression of APJ/Apelin.MethodOn the basis of preliminary experiment of research group,80 adult C57BL/6J mice are chosen at 10 to 12 weeks of age, and divide into 4 groups with 20 rats in each group, respectively:normal control group, model group, Xiaoyao san group, fluoxetine group, adaptive feeding for 7 days. Then using bound+fatigue stimulation +solitary ways to make stagnation of liver qi and spleen deficiency mice 21 days model. Each stimulation method is used 2-3 times, and the model last for 21 days.Using the Holland Noldus company behavior equipment (Etho Vision 3 behavioral analysis system) to observe stress, depression and other related manifestations of model mice, and the behavior mainly includes:macroscopic representation, open field test and sucrose preference test, etc. Observe the effect of Xiaoyao san on morphology of hypothalamus in model mice and the expression of Apelin and its receptor APJ by immunohistochemical method. Hypothalamus is taken on the ice, protein expression of Apelin and its receptor APJ is detected by blot Western and the expression of Apelin and its receptor APJ mRNA is detected by real-time fluorescence quantitative PCR. ResultsStagnation of liver qi and spleen deficiency mice model is established successfully. Real-time fluorescence quantitative PCR is used to detect mouse hypothalamic Apelin/APJ mRNA levels. The results showed that among model group, xiaoyao san group and fluoxetine group, there was no significant difference in Apelin mRNA relative expression, and they are significantly lower than normal group mice hypothalamic Apelin mRNA relative expression level (P<0.01). The expression of APJ receptor mRNA in hypothalamus of model group was significantly lower than that in normal group, xiaoyao san group and fluoxetine group (P<0.01).Western blot method is used to detect the mouse hypothalamic Apelin and APJ receptor protein expression. The results showed that in model group and xiaoyao san group, expressions of mouse hypothalamic Apelin were significantly lower than that of normal group (P<0.01), model group mouse hypothalamus APJ receptor content is lower than normal group and xiaoyao san group (P<0.05), also fluoxetine group mouse hypothalamic Apelin and APJ receptor are lower than the normal group (P＜0.05).Each mouse hypothalamic Apelin and APJ receptor protein expression in supraoptic nucleus and paraventricular nucleus are observed by immunohistochemical method. The results show that in the supraoptic nucleus, the Apelin positive cell number in in normal group, xiaoyao san group and fluoxetine group is significantly higher than that in model group (P<0.01); in the hypothalamic paraventricular nucleus. Apelin positive cell number in xiaoyao san group is more than that in normal group, model group and fluoxetine group, and it has significant difference (P<0.01). In the supraoptic nucleus. Apelin average optical density value in normal group, xiaoyao san group and fluoxetine group is greater than that of model group and it is significantly differences (P<0.05). At the same time, Apelin the average optical density value in xiaoyao san group and fluoxetine group is greater than that in normal group (P<0.05, P<0.01); in the hypothalamic paraventricular nucleus, Apelin average optical density value in xiaoyao san group is significantly higher than that of the normal group, model group and fluoxetine group (P<0.01). In the supraoptic nucleus, the APJ receptor positive cell number in xiaoyao san group is significantly lower than that in normal group and model group (P<0.05), and APJ receptor positive cell number in in the fluoxetine group is significantly less than that of the other three groups (P<0.01); in the hypothalamic paraventricular nucleus, the APJ receptor positive expression cell in in model group is more than that in normal group, xiaoyao san group and fluoxetine group, and the difference is statistically significant (P<0.05). In the supraoptic nucleus, APJ receptor average optical density value in xiaoyao san group and fluoxetine group are lower than that in normal group and model group (P<0.05), and between the normal group and model group, there are no significant difference; in the hypothalamic paraventricular nucleus, APJ receptor average optical density value in model group is greater than that of normal group (P<0.05), also it is significantly higher than that of xiaoyao san group and fluoxetine group (P<0.01), and APJ receptor average optical density value in fluoxetine group is smaller than that of normal group (P<0.05). ConclusionsThe corresponding changes are taking place in the content of stagnation of liver qi and spleen deficiency mice model hypothalamic Apelin and APJ receptor. This change are the upregulation of the expression of Apelin expression and downregulation of APJ receptor, the Apelin/APJ system involves in neuroendocrine activity way and Xiaoyao san has a regulation effect on this change. Series of changes of Apelin/APJ system in the model of syndrome of stagnation of liver qi and spleen deficiency in a and the specific effects of Xiaoyao san are still needed more research to explore. |
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