Effects Of Fear Stress On The Contents Of Monoamine Neurotransmitters In Hypothalamus Of Rats, ACTH, CORT, IL-2, IL-8 And The Intervention Of Chinese Medicine

Background:With the development of society and increasing pressure of competition,spectrum of diseases have changed heavily.There were more and more diseases caused by society,psychology and behavial factor,so is the diseases caused by excess psychological stress.The burden caused by psychosis and nervous disease took the first place in our conntry,which is about 20%of the total burden.according to World Health Organization's calculation.The burden caused by psychosis and nervous disease in China will rise to 25%.it shows there is about 70%human of our population in subhealthy state,with a percentage of 5 to 10 caused by excess psychological stress,physical and mental diseases,psychologic obstacle have been frequently encountered and common diseases.Neurosis take the first place among mental disorder,which is included by anxiety disorders,obsession,phobia and neurastheria mainly.At present,it is one of the important topics to make the foundation and clinical research on psychological disease and stress.Traditional Chinese medicine(TCM)have rich experience of treatment on psychological diseases.There were emotion treatment and acupuncture and moxibustion therapy in Neijing.later generation doctors developed them into the therapy of entrails pathogenesis,and created a series of Formulas.Sangpiaoxiao Decoction(SPXD)is from Bencaoyanyi,which has a good curative effect for phobia.My topic is based on the theory of Seven emotions.we establish the fear stress model according to classical conditioned reflex,and determine the contents of monoamine neurotransmitters and their metabolites by HPLC-ECD.Meanwhile,we determine the average contents of IL-2,IL-8,CORT,ACTH by radio-immunity assay. To reveal the possible mechanism of fear stress and Chinese medicine,we compare the function of tri-transmitter in limbic system and link with the whole function of neuroendocrine system.Methods:1.Male wistar rats weighing 180-220g are divided into 3 groups at random: normal control group,fear model group,breed lonely group.in each group,there are 8 rats.fear model rats are bred in stainless steel cage(50cm×30cm×20cm)separately. After 10 days,let the model rat separately in Photoelectricity stimulation instrument at 1:30PM,which have 1mA alternating current and a 10W daylight lamp above.After 5min adaptation period,let the rat be shocked and flashed for 1 second interval 1min simultaneously,so is repeated on 12(th)13^thday.There are totally 30 min every day.On 14^thday,there was only flash and no shock,then took a photograph of rat's behavior change and record the freezing time.Three observers scored the rat's behavior after seeing the photograph in the blind way.Then the rats were put in open-field box and received the ethology test.breed lonely group rats were bred for 13 days in the same environment,and photographed on 14^thday,so is repeated with model rats.nomal control rats were bred communally and received the ethology test and open-field experiment.On 14^thday every rat was anesthetized and executed after decapitation. We prepared blood serum and plasma and determined the average contents of ACTH,CORT,IL-2,IL-8. 2.Male wistar rats weight 180-220g are divided into 3 groups at random:normal control group,fear model group,medication administration group.in each group,there are 8 rats.fear model rat was done in the same way(see above).Three days before shock eaperiment,medication administration rat was lavaged 2ml Sangpiaoxiao Decoction(SPXD)once a day.On 14^thday every rat was anesthetized and executed after decapitation.we took out of the hypothalamus,homogenated and centrifugated, then determined the contents of monoamine neurotransmitters and their metabolites by HPLC-ECDStatistical analysis:Evaluated and used as nonspecific controls for the localized site injections.Statistical analysis.The overall treatment effects were examined using one-way ANOVA.For each significant F ratio,Fisher's protected least significant difference(PLSD)test was used to analyze the statistical significance of appropriate multiple comparisons (Kirk,1968).A probability level of P＜0.05 was accepted as statistically significant in all of the studies,and all of the post hoc tests were two-tailed.Results:1.Score of ethology and open-field In fear model rats,score of ethology has discrepancy significantly compared with other groups(F=72.582,P=0.000).score of ethology in fear model rats was higher than other groups.there was no discrepancy significantly between normal control group and breed lonely group(P=0.153),there was discrepancy significantly on level score,vertical score and total score in open-field experiment among three groups(F=23.893,52.371,40.963;P=0.000).score of breed lonely group was lower than that of normal control group and higher than fear model group(P＜0.05).we took the Pearson's correlation analysis for vertical score and level score and found there was positive correlation between both variables(r=0.730,P=0.000).2.Average contents of ACTH and CORT There was discrepancy significantly among three groups(F=10.466,13.804;P＜0.01).Fear sress could increase the level of ACTH and CORT in serum,compared with other two groups(P＜0.05).There was no discrepancy significantly between breed lonely group and normal control group(P＞0.0.5).3.Average contents of IL-2 and IL-8 For the level of IL-2,there was no discrepancy significantly among three groups(F=1.355,P=0.280).Fear tress could increase the level of IL-8,compared with normal control group(P＜0.05).4.Contents of monoamine neurotransmitter in hypothalamus The lower contents of NE,E,DA,5—HT had been observed in hypothalamus of model rats compared with normal control grouop(P＜0.01).5.Contents of monoamine neurotransmitter metabolites For the content of MHPG and HVA,There was discrepancy significantly between fear model group and normal control group(P＜0.01).For the content of MHPG and 5-HIAA,There was discrepancy significantly between fear model group and medication administration group(P＜0.01).For the content of HVA and 5-HIAA,There was discrepancy significantly between normal control group and medication administration group(P＜0.05).6.Specific value between monoamine neurotransmitters and their metabolites The lower value of DA/DOPAC had been observed in fear model rats compared with normal control grouop(P＜0.01).7.Proportionality relationship among neurotransmitters There was no discrepancy significantly among three groups(P＞0.05).Conclusion:1.According to the principle of classical conditioned reflex,we establish the fear stress model according toTakashi's method and avoid the impression of body stimulus as possible.The fear stress model is simple,which has a high stability and good feasibility.2.We socored the ethology and open-field and found fear sress could weaken excitability and adaptability of rat.The appearance is similar with emotional reaction of human being.it demonstrated the model could replicate successfully fear emotion,which is an ideal model of internal injury in TCM.3.Fear sress could activate HPA axis,thereby increase the level of ACTH and CORT.4.Fear sress could increase the level of IL-8 and had no influence significantly on the level of IL-2.5.Fear sress could decrease the content of NE,E,DA,5—HT.6.Fear sress could decrease the content of MHPG,HVA.7.Fear sress could decrease the value of DA/DOPAC.8.SPXD could adjust the content of NE,E,5—HT to normal level,restrain the activation of HPA axis and the intensity of stress reaction.In conclusion,we establish the fear stress model according to the principle of classical conditioned reflex and evaluate the ethology index,which accord with the invasion process of internal injury in TCM.fear sress could activate HPA axis, thereby increase the level of ACTH and CORT.The lower contents of NE,E,DA,5—HT had been observed in hypothalamus of model rats,which indicate those neurotransmitters play a important role in fear sress.fear sress could increase the level of IL-8 and had no influence significantly on the level of IL-2.the impression of fear stress on immune function is wating for a further research.SPXD could adjust contents of NE,E,5—HT and exert the action of nerve central regulation.