| Objectives Based on insomnia theory of Huangdi’s Inner Classic of Medicine (Huangdi Neijing) and the summary of different insomnia theory of Traditional Chinese Medicine (TCM) and clinical practice, we propose that deficiency of essence of zang-fuviscera and mind dystrophy are the pathogenesis of chronic insomnia, and the strengthening essence and nourishing original qi and nourishing heart and tranquilizing mind are the principle of therapy. The clinical prescription of strengthening essence and tranquilizing mind formula (Gu Ben An Shen Fang in Chinese)is created based on the systematical summary of experiences of medical usage of different doctors and the clinical practices of our tutor. Through the observation of the influence of decoction and wine type of strengthening essence and tranquilizing mind formula on sleep duration induced by above threshold dose of pentobarbital sodium and the rate of falling asleep of mice induced by below threshold dose of pentobarbital sodium, and the observation of the changes of index of the chronic insomnia deprived model rat, such as breathing rate, heart rate, and exhaustive swimming test, and the monitoring analysis of the index such as sleep promoting test, learning and memorizing ability of rats, and the expression analysis of neurotransmitters and their receptors after the treatment of strengthening essence and tranquilizing mind formula, the mechanism of its action is systematically and objectively evaluated. This topic is of a beneficial exploration of insomnia system, and expands the train of thought for the treatment, prevention and health care of insomnia with the product development of TCM.Methods:After the experimental observation of sleep promoting effect by the strengthening essence and tranquilizing mind formula, the healthy Wistar rats were randomly divided into 5 groups,8 rats per group:blank control group, model group, low dose of strengthening essence and tranquilizing mind formula group, high dose of strengtheningessence and tranquilizing mind formula group, and estazolam group. Compared to every sleep deprived group, a complete fine wire instead of a platform was placed on the bottom of the blank control group, making sure of the similar environment as that of model group. In the strengthening essence and tranquilizing mind formula group regardless of the dose, the experimental rats were orally administrated for 7 days after 4 weeks of successfully establishing sleep deprived model, and the concentration was 2ml.100g-1. In the estazolam group, the drug estazolam was made into powder, and proceeded the gavage with the concentration of 0.05 mg. kg1. d-I. All the animals were feed at 18:00 every night. The materials were taken after one week treatment for all rats. T he concrete index tested were as follows:1. Proceed the comparative monitoring analysis of the general things of basic conditions of sleep, amount of activity, eyes, lips, nails, fur, and the breathing rate, weight, food ration, exhaustive swimming test and so on.2. Check the changes of learning and memorizing ability of rats in every group by Morris water maze.3. Test the content of Glu and GABA in the hypothalamus of rats with LC-MS, and the influence on mRNA of the receptor of GABA.4. Test the content of 5-hydroxytryptaphane (5-HT) with HPLC, and the influence on 5-hydroxyindoleacetic acid with E LISA.Results:1. Compared to blank control group, the estazolam group, and the high dose and low dose of strengthening essence and tranquilizing mind formula group, and the Jing Mian wine group can obviously extend the sleep time of mice caused by the above threshold dose of pentobarbital sodium(P<0.01), and obviously increase the rate of falling asleep of mice induced by below threshold dose of pentobarbital sodium(P<0.01), indicating the significant hypnotic effect for mouse. The effect of the high dose of strengthening essence and tranquilizing mind formula group is stronger than that of lose dose, but have no statistical significance (P> 0.05). T he effect of wine type is stronger than that of decoction, but have no statistical significance (P> 0.05).2. The animals of model group have the symptoms of continuous yawn when occurring, doze off, less activity, listlessness of essence-spirit, pale eyes and lips with some pink, glassy eyes, severe withered unkempt fur, drenched, pale ivory, claw armor, and inability to resist when being grasped. The animals of high dose of strengthening essence and tranquilizing mind formula group have the symptoms listlessness of essence-spirit in the day, occasional yawn, pale eyes and lips, little withered unkempt fur, claw armor, and strong resist when being grasped.In comparison with blank control group, the rats of models have the obviously faster breathing rate and faster heart rate, significantly increased systolic blood pressure, significantly shortened exhaustive swimming time, significant weight loss, significant higher ear temperature, and significantly reduced food intake (P<0.01). Compared to model group, the high dose and low dose of strengthening essence and tranquilizing mind formula group have the symptoms of the obviously less breathing rate, obviously lower heart rate, significantly prolonged exhaustive swimming time, significantly lower ear temperature, significantly increased food intake (P<0.01), decreased systolic blood pressure, and increased weight(P<0.05). Compared to model group, the estazolam group have the symptoms of obviously less breathing rate, significantly prolonged exhaustive swimming time (P< 0.01), decreased systolic blood pressure, increased food intake, and lower ear temperature ((P< 0.05)). T he overall effect of strengtheningessence and tranquilizing mind formula group is better than that of the estazolam group, and presenting the dose effect incremental relationship.3. In the experiments of hidden platform test and probe trains, compared to blank contract group, the model group have the symptoms of obviously longer latency period (P<0.01), significantly reduced proportion of swimming distance in platform quadrant (P<0.01), significantly less times striding over the original platform (P<0.01), significantly increased proportion of swimming distance in the area of the 20%-40% pool wall (P< 0.01). Compared to the model group, the estazolam group and the high dose and low dose of strengthening essence and tranquilizing mind formula group have the symptoms of obviously shorter latency period (P<0.01), significantly increased proportion of swimming distance in platform quadrant (P<0.01), significantly more times striding over the original platform (P<0.01), significantly decreased proportion of swimming distance in the area of the 20%-40% pool wall (P<0.01). The overall effect of strengthening essence and tranquilizing mind formulagroup is better than that of the estazolam group.4. Compared to the blank control group, the content of the amino acids neurotransmitter Glu and GABA significantly increased in the hypothalamus of rats in the model group (P<0.01), and the change of GABA was more obvious and the proportion of GIu/GABA decreased. Compared to the model group, the content of the amino acids neurotransmitter Glu and GABA significantly decreased in the hypothalamus of rats in the estazolam group, and the high dose and low dose of strengthening essence and tranquilizing mind formula group (P<0.01), and the change of Glu was more obvious and the proportion of Glu/GABA decreased, which presented a certain quantity effect relationship (P<0.05). Compared to the blank control group, the expression of GABAA-R mRNA in the hypothalamus of rats in the model group decreased significantly (P<0.01). Compared to the model group, the the expression of GABAA-R mRNA in the hypothalamus of rats in estazolam group and the high dose and low dose of strengthening essence and tranquilizing mind formula group increased significantly (P< 0.01).5. Compared to the blank control group, the content of 5-HT and 5-HIAA in the hypothalamus of insomnia rats in the model group decreased significantly (P<0.01), however, they changed little between the model group and the estazolam group that showed non statistic significance (P>0.05). Compared to both the blank control group and the estazolam group, the content of 5-HT and 5-HIAA in the hypothalamus of rats in the high dose and low dose of strengthening essence and tranquilizing mind formula group increased significantly and showed statistic significance (P<0.01) respectively, and the high dose group changed more obviously compared to the low dose group (P< 0.01).Conclusions:1. Based on insomnia theory of Huangdi’s Inner Classic of Medicine (Huangdi Neijing) and integrated with different insomnia theory of Traditional Chinese Medicine (TCM), the pathogenesis of chronic insomnia is considered as the deficiency of essence of zang-fuviscera and mind dystrophy. According to the conception of holism of TCM, the therapies of strengthening essence and nourishing original qi and nourishing heart and tranquilizing mind are proposed, and the clinical prescription of strengthening essence and tranquilizing mind formula is created based on the systematical summary of experiences of medical usage of different doctors and the clinical practices of our tutor.2. The effect of the clinical prescription of strengthening essence and tranquilizing mind formula is the same as that of estazolam and presents dose effect incremental relationship, but is better for treatment of the symptoms of TCM and has minor side effects on the model rats.3. In the high dose and low dose of strengthening essence and tranquilizing mind’formula’group, the moderate and high dose of Jing Mian wine group, and the estazolam group, it is obvious that the amount of sleeping mice induced by below threshold dose of pentobarbital sodium increases significantly and the sleep duration of mice induced by above threshold dose of pentobarbital sodium prolongs significantly, indicating that the strengthening essence and tranquilizing mind formula has a synergistic action with the drug estazolam, and demonstrating that the strengthening essence and tranquilizing mind formula has an obvious sedative and sleep promoting effect which is dose dependent. Wine and medicine have synergistic effect, however, it is not obvious in this experiment that Jing Mian wine is better than the decoction (P>0.05), but the pharmacodynamic tests set a base for further study the mechanism of strengthening essence and tranquilizing mind formula for the chronic sleep deprivation.4. The strengthening essence and tranquilizing mind formula can restore the decreased content of 5-HT and 5-HIAA in the hypothalamus of the experimental rats to their normal level, and decrease the content of the metabolic ingredients in the hypothalamus-amino acids neurotransmitter Glu and GABA and the ratio of Glu/GABA, and increase the expression of GABAA-R mRNA, which exerts its protective effect on the brain for sedative and sleep promoting and improve the ability of learning and memorizing and regulating the deficiency of essence and mind dystrophy. |
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