| ObjectiveIt is needed to combine the chronically psychological stress with qing zhi disease. According to the TCM emotional which will most easily to cause of liver disease may lead to many internal injuries. The change of the disease-location and the changes of syndromes of the disease which were caused by different chronically psychological stress time will be observed and compared with each other. As well as to observe the change of autophagy activity in the cells of the hippocampus and hepatocytes, and so on. In order to study the best time for the animal experiments in the next step for the anti stress damage animal of TCM. As well as to explain the visceral manifestation of the liver controlling dispersing from the perspective of autophagy theory, which will provide experimental basisMethodsThe open-field test has shown that different rats may have different movement distance. There was no difference in activity distance before the experiment. It was based on the activity ways, the rats were divided into five groups,such as blank control group, model 10 days group, model making 20 days group,30 days group and 40 days group modeling. The six chronically multi-phase stress methods contain fasting, water deprivation, ice swimming, clip tail, electric shock and bondage were used to rats randomly arrange in the process of experiment. The open-field test was used to judge the establish time of the chronically psychological stress model. The euzymelinked immunosorbent assay was used to analyze rat serum motilin, gastrin. On the one hand, the chronically psychological stress had effect on neurons autophagosome in the rat hippocampus CA1 zone. On the other hand, the chronically psychological stress influenced the rat hippocampus neurons, hepatic cell in deliver oneself of autophagy related protein.Results1、The chronically psychological stress play a role on rat spatial memory, learning ability and anxiety emotion.There was no difference in total distance before the building model of different rats groups in open-field test (P>0.05). Compared with blank control group, the model 10 days group had shorter distance in five minutes movement, and had obvious diversity(P>0.05). Nevertheless, It was clear discrepancy to model making 20 days group compare with blank control group(P <0.05). Yet there was obvious difference in 30 days group and 40 days group modeling to compare with blank control group(P<0.01). In three minutes movement, compared with blank control group, there is a clear difference in the model 10 days group(P<0.05). But it is a big diversity among in model 20 days group,30 days group and 40 days group model ing (P<0.01).2、The chronically psychological stress affect rats gastrointinal hormonesWhen the building model have finished, it was to get the rats apex of blood. In order to determine the internal secretion such as motilin and GAS. Compared with the blank control group, the motilin in serum was decreased in the model 10 days (P<0.05). Yet there was an obvious difference among in 20 days group,30 days group and 40 days group modeling(P<0.01). The gastric and dynamic element in serum declined as well, there was an obvious difference compared with the blank control group. The model 10 days and 20 days group have no difference compared with the blank control group (P>0.05), but there was a big difference in the blank control group to compare with 30 days group and 40 days group model ing (P<0.05). The most important was that with the modeling time going on, the gastrointinal hormones declined step by step.3、The chronically psychological stress affect the cell autophagy related indexes in rats.3.1 The chronically psychological stress affect on the cell autophagy related protein TSC2 in rats.When the model experiment was finished, hepatic tissues and hippocampus were needed to detect in immunohistochemistry inverted microscope. The hepatic tissues and hippocampus were stained with immunohistochemistry. Cytoplasm will be like yellow while the nucleus were like blue which were stained with immunohistochemistry. In the blank control group, cell hippocampus dedicated that the yellow immunohistochemistry was very little filled with cell nucleus. Compared with the blank control group, there were no big difference among the model 10 days group (P>0.05). Yet, there was the obvious difference between the blank control group and model 20 days group (P<0.05). The biggest difference was appeared in the blank control group compared with model 30 days group and 40 days group modeling (F<0.01). The same condition was also observed in the hepatic tissues, which also had dimly yellow immunohistochemistry, hepatic cords were clear and the structure of cell was distinct. With the modeling time was longer, the hepatic tissues were more and more yellow. The 40 days group had most yellow immunohistochemistry. The hepatic cords were less clear and the structure of cell was blurry. Compared with the blank control group, there was no difference among the model 10 days group (P>0.05). The biggest difference showed in the blank control group and 20 days group modeling and the model 30 days group and the model 40 days group (P<0.01).3.2 The chronically psychological stress affect on the cell autophagy related protein ULK1 in rats.It was mostly likely the condition of TSC2. In the blank control group, the cell hippocampus showed that the yellow immunohistochemistry was very dark and filled with cell nucleus. Compared with the blank control group, there were no big difference among the model 10 days group CP>0.05). Yet, there was the obvious difference between the blank control group and model 20 days group (P<0.05). The biggest difference was appeared in the blank control group compared with 30 days group modeling and the model 40 days group (P<0.01). The hepatic tissues also had darkly yellow immunohistochemistry, hepatic cords were precise and the structure of cell was clear. With the modeling time going on, the hepatic tissues were more yellow. The 40 days group was most yellow immunohistochemistry. The hepatic cords were less precise and the structure of cell was blurry. Compared with the blank control group, there were no big difference in the model 10 days group(P>0.05). There was the obvious difference between the blank control group and model 20 days group (P<0.05). The biggest difference was appeared in the blank control group compared with the 30 days group modeling and 40 days group modeling (P<0.01).4、The chronically psychological stress affect on neurons autophagosome in the rat hippocampus CA1 zone.While the making rats model finished, transmission electron microscopy was used to observe the hippocampus zone. In the blank control group, the structure in hippocampus cell was distinct. It can see that there had the complete hippocampus nerve cell wall. In the other hand, the cell was filled with cytoplasm, the cell structure was clear and no swelling and necrosis. In another hand, it could see a little autophagosomes. In the model 10 days group, the structure of hippocampus cell was almost like the blank control group. The structure of model 10 days group, hippocampus nerve cell had more broken than 10 days group modeling. In the 30 days group, there were more destroyed than model 20 days group. Yet the 40 days group had the most broken structure in hippocampus nerve cell and have the maximum autophagosomes. Compared with the blank control group, there was no big difference among the model 10 days group and 20 days group modeling (P>0.05). Yet, there was the obvious difference between the blank control group and model 20 days group (P<0.05). The significant difference was appeared in the 30 days group and 40 days modeling when compared with blank control group (P<0.01).ConelusionThe open-field test is very useful to test the chronically psychological stress. As well the chronically psychological stress impacted learning and memory, making out the rats depressed. In the open-field test, the chronically psychological stress time was longer, the rats emotion was more depressed and movement distance was shorter. To based on the open-field test, we can conclusion that the model 30 days is the best time to chronically psychological stress.The chronically psychological stress may affect the gastrointestinal hormone. When the different time of chronically psychological stress was used, the gastrointestinal hormone was affected obvious difference. With the modeling time going on, the gastrointestinal hormone would decrease. Chronic psychological stress affects the liver function, leading to the dysfunction of liver, "Liver disease will transfer to spleen". Therefore, the chronic psychological stress with the extension of time, the symptoms may change and this change will from liver qi stagnation to stagnation of liver and deficiency of spleen.The chronically psychological stress would affect the excessive activation of hippocampus nerve cell in the rat hippocampus CA1 zone and destroy the structure of cell. When it was different in modeling time, the hippocampus nerve cell had not the same representation. To extend the modeling time, the levels of hippocampus autophagy would increase and the cell damage had also increased.The chronically psychological stress could affect the hepatic tissues,leading to hepatic cell was destructive. It was very distinct in modeling time with different expression. The modeling time was longer, the more serious the degree of autophagy and the cell damage was more serious. |
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