| Objective This experiment was designed to observe the affect of Xiao Yao San on treating liver depression and spleen deficiency syndrome anxiety by recording the changes of P38 mitogen-activated protein kinase(P38MAPK), phosphorylated P38MAPK, Caspase-3, and discuss anti-anxiety effect and its mechanism of Xiaoyaosan decoction.Method1.28 male SD rats were randomly divided into 4 groups:normal group, model group, Xiao yaosan group and fluoxetine group. We didn’t do any processing in normal group, but model, Xiao Yao San and fluoxetine groups by continuous 14days immobilization stress induced liver depression and spleen deficiency syndrome anxiety, before every morning bondage administered Xiao Yao San (0.61g/100g·d) to the Xiaoyaosan group, fluoxetine (0.2mg/ 100g·d) to the fluoxetine group and administered a deionized water to the normal and model groups.2. We observed macro characterization, weight change, the consumption of sugar, elevated plus maze and novelty suppressed feeding test.3. We used ELISA to measure IL-1β, IL-6, TNF-α Levels in Blood Serum of Rats.4. We used immunohistochemistry, Western blotting, and RT-PCR to measure recording the changes of P38MAPK, phosphorylated P38MAPK, Caspase-3 in Hypothalamus.Results1. Establishment of rat model of syndrome anxiety of liver-depression and spleen-defic lencyAfter 14days, model group rats appeared slow weight gain, the spirit of apathetic or irritable, massy and lackluster hair, like to cluster or dormant corner, and loose stool, etc.5minutes closed arm entries frequency and stay time, total movement distance, central area entries frequency and stay time were all decreased in elevated plus mazetest. Although no statistical significance of sμgar consumption, but the model group was lower than the other three groups, there is a certain trend. Xiaoyaosan group and fluoxetine group of chronic immobilization stress rats weight, elevated plus maze test, novelty suppressed feeding tests, and sugar consumption experiment indicators significantly improved.2. The results of detection of serum Elisa methodIn model group, the levels of IL-1β, IL-6 and TNF-a in serum were higher than normal group, fluoxetine group and Xiaoyaosan group (P<0.05 or P<0.01). Compared with the normal group, there was no statistical difference in the xiaoyaosan group and fluoxetine group (P>0.05), which showed that Xiaoyaosan decoction and fluoxetine decreased the inflammatory factors and had a certain effect for anxiety.3. Immunohistochemical resultsImmunohistochemical observation of P38, P-P38 and Caspase-3 in hypothalamus of rats in each group were analyzed, three indexes of the model group IOD than the rest of the three groups were significantly higher. Compared with the normal group, model group rats three indexes of integral light density than that of the normal group was higher, with statistical significance (P<0.05 or P<0.01). Normal group and Xiaoyaosan group, fluoxetine group does not exist significant difference. Between the drug groups no significant difference (P>0.05). The positive area of model group than the other 3 groups rats obviously increased, and exists statistically significant difference (P<0.01).4. Western-blotting resultsWe observed expression levels of P38MAPK, phosphorylated P38MAPK and Caspase-3 protein in the hypothalamus of rats with chronic immobilization by experiment. The results found that 14days of chronic stress leads that anxiety model rats P38, P-P38, caspase-3 content in hypothalamus is rising, and compared with the normal group had statistical difference (P<0.05, P<0.01), but P38 had no statistical difference, which reason remains to be further studied. Compared with the model group, the protein expression levels of Xiaoyaosan group and fluoxetine group were significantly lower than the model group.5. RT-PCR resultsThe changes of P38MAPK and Caspase-3 mRNA levels in rats were observed by experiment. After 14 days of chronic immobilization stress, the experimental results showe d that the P38 mRNA expression in the hypothalamus of the rats, the normal group and the model group is different (P<0.05). Although there was no statistical significance between the model group and the two drug groups, but model group was higher than the other three groups, there was a certain trend. We observed Caspase-3 mRNA expression, the level expression of model group mRNA was significantly higher than the normal group, there is statistical significance (P<0.01).Conclusion1.The 14days chronic immobilization stress method can be prepared by liver stagnation and spleen deficiency animal models of anxiety. Throμgh animal macroscopic representations, behavioral changes, sugar consumption, Xiaoyaosan decoction intervention to measure the syndrome, suggesting that 14days of chronic immobilization stress method can successfully replicate liver stagnation and spleen deficiency rat models of anxiety.2.The levels of serum inflammatory factors IL-1β, IL-6, TNF-α were increased in chronic immobilization stress anxiety rats, and Xiaoyaosan decoction can reduce the content of serum inflammatory factors.3.Anxiety induced by chronic immobilization stress was associated with increased expression of P38, phosphorylated P38 and Caspase-3 in hypothalamus. The expression of P38, P-P38 and Caspase-3 in hypothalamus was down regulated by Xiaoyaosan decoction and fluoxetine. Suggesting that the activation of signaling pathway can effectively inhibit the activation of the P38MAPK signaling pathway to achieve the anti-anxiety effect. To provide experimental evidence for the rationality and treatment of anxiety disorder. |
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