| Stress refers to the non-specific systemic reactions that occur when the body is stimu-lated by various internal and external environmental factors.The factors that can cause stress are called stressors.According to their sources,they can be divided into three categories:ex-ternal environmental factors,internal factors of the body,psychological and social environ-mental factors.According to the bodyy's response to stress,people divide stress response into benign stress and inferior stress,so stress has advantages and disadvantages for human health.Reasonable stress contributes to the improvement of the body's ability to adapt,while exces-sive stress can invalidate the body's adaptation mechanism and lead to dysfunction of the body.The most direct manifestation of stress is mental stress,a large number of studies have shown that mental stress can affect the entire body to make corresponding mental and physi-ological reactions,which can lead to insomnia,depression,functional gastrointestinal diseas-es,hypertension,chronic fatigue syndrome,post-traumatic stress disorder and other diseases.Modern studies have shown that intracerebral neurotransmitters plays an important role in the development of stress,and the gut flora colonizing the digestive tract can be achieved through the intestinal flora-intestinal-brain axis.At present,the treatment under stress is mainly based on psychological intervention and drug therapy,However,there are many problems to be solved,such as obvious side effects,lagging efficacy and so on.Traditional Chinese medicine has certain advantages in alleviating the body discomfort caused by stress.This study is based on the understanding of pathogene-sis of inferior stress in TCM theory,and on the basis of clinical observation,a new compound Tiaoqi Anshen decoction has been formed.The pharmacological mechanism of intervention of stress is not completely clear.In this study,acute stress model was established by single prolonged stress(SPS),chronic stress model was prepared by chronic restraint stress(CRS).The effects of Tiaoqi Anshen decoction on behavior,intestinal flora and neurotransmitters in the brain of stress model rats were observed in order to reveal its pharmacodynamic targets and to further study its pharmacodynamics.Lay the foundation for the mechanism and future clinical development.Objective:To observe the effect of Tiaoqi Anshen decoction on behavior of acute and chronic stress model rats,and to verify the effect of Tiaoqi Anshen decoction on mental and behavioral ab-normalities caused by stress.Furthermore,the changes of intestinal flora and neurotransmit-ters in the brain of stress rats were detected to elucidate the intrinsic mechanism of Tiaoqi Anshen decoction in the intervention of stress,and to provide evidence for the role of flo-ra-intestine-brain axis in the stress process.Methods:1.Effects of Tiaoqi Anshen decoction on the behavior of acute and chronic stress model rats.The experiment was divided into two parts:acute and chronic Stress.Each part of SD rats was randomly divided into five groups:control group,model group,positive drug group(Paroxetine),tangerine peel and aurantii fructus group and Tiaoqi Anshen decoction group.After adaptive feeding,acute stress model was duplicated by single prolonged stress method,chronic stress model was duplicated by chronic restraint stress method.After 7 days of intra-gastric administration,behavior changes of rats were examined by open field test and freez-ing time,and the pharmacodynamic effect of Tiaoqi Anshen decoction was observed.2.The effect of Tiaoqi Anshen decoction on the intracerebral neurotransmitters and their receptors in acute and chronic stress model rats:experimental grouping and treatment as above,DA,NA,5-HT,GABA in the cortex of each group were determined by ELISA.The content of DRD2,ADRB1 and GABARA2 were measured by RT-PCR.To observe the effect of Tiaoqi Anshen decoction on neurotransmitters in the brain of acute and chronic stress rats.3.The effects of Tiaoqi Anshen decoction on gut flora of acute stress rats:To establish the model of acute stress rats.The experimental groups and treatments were the same.The distribution of gut flora in rats of each group was detected by 16srRNA.The effects of Tiaoqi Anshen decoction on gut flora of rats under acute stress were revealed by Alpha and Beta di-versity analysis.4.Ultra-high performance liquid chromatographic analysis of Tiaoqi Anshen decoction:The composition difference between Tiaoqi Anshen decoction and granules was analyzed by ultra-high performance liquid chromatography,and the repeatability and preliminarily evalu-ate stability of Tiaoqi Anshen decoction.5.Acute toxicity effects of Tiaoqi Anshen decoction on rats:SD rats were randomly di-vided into 10 groups according to male and female,and a single oral fixed dose method was adopted after adaptive feeding.The dosage of 0,5,50,500 and 2000 g/person/day was con-verted into the dosage of SD rats.After two weeks of administration,recorde the time of emergence and disappearance of various toxic reactions,and calculate the weight change and mortality of experimental animals.Results:1.Tiaoqi Anshen decoction can improve the behavioral of acute and chronic stress mod-el rats:In the acute stress experiment,compared with the control group,the number of pene-trating,central dwell time,erecting times and the freezing time in the model group were sig-nificantly reduced(p<0.01),suggesting that the single prolonged stress method can induce anxiety,tension and fear in rats.Compared with the model group,erecting times in the tange-rine peel and aurantii fructus group increased significantly(p<0.01),while the number of penetrating,erecting times and freezing time in the Tiaoqi Anshen decoction group increased significantly(p<0.05,p<0.01),suggesting that the Tiaoqi Anshen decoction and its core drugs can alleviate the anxiety and tension of rats.In the chronic stress experiment,compared with the control group,central dwell time,the number of penetrating,erecting times and stiffness time in the model group were significantly reduced(p<0.05,p<0.01),suggesting that chronic restraint stress can induce tension,anxiety and fear in rats.Compared with the model group,erecting times and central dwell time in the treatment group were significantly increased(p<0.05,p<0.01).The number of penetrating,central dwell time and erecting times in the tangerine peel and aurantii fructus group were significantly increased(p<0.05,p<0.01),suggesting that Tiaoqi Anshen decoction and its core drugs have a certain role in regulating chronic stress state.2.Tiaoqi Anshen decoction can regulate the abnormalities of intracerebral neurotrans-mitters and their receptors of rats with acute and chronic stress:In the acute stress experiment,compared with the control group,5-HT,DRD2,GABRA and ADRB1 were significantly decreased in model group,NA,DA and GABA increased significantly(p<0.05,p<0.01).These results suggest that intracerebral neurotransmitters and their receptors are disturbed during acute stress.Compared with the model group,the 5-HT,DRD2,GABRA and ADRB1 of rats in the tangerine peel and aurantii fructus group increased significantly,while NA and DA decreased significantly(p<0.05,p<0.01).In the Tiaoqi Anshen decoction group,5-HT,DRD2,GABRA and ADRB1 increased significantly,DA,NA and GABA decreased significantly(p<0.05,p<0.01),suggesting that Tiaoqi Anshen decoction can effectively improve the disorder of intracerebral neurotransmitters and their receptors of acute stress rats.In the chronic stress experiment,compared with the control group,NA,DA,DRD2,GABRA and ADRB1 in the model group were significantly de-creased,while 5-HT and GABA were significantly increased(p<0.05),suggesting that the disorder of intracerebral neurotransmitters and their receptors were involved in the patholog-ical process of chronic stress.Compared with the model group,5-HT,DRD2,GABRA and ADRB1 in the tangerine peel and aurantii fructus group increased significantly(p<0.05,p<0.01).While the contents of NA,DA,5-HT,DRD2,GABRA and ADRB1 in the Tiaoqi Anshen decoction group increased significantly(p<0.05,p<0.01),and GABA decreased significantly(p<0.01),suggesting that Tiaoqi Anshen decoction can regulate the disorder of intracerebral neurotransmitters and their receptors.3.The tangerine peel and aurantii fructus can change the distribution of gut flora in rats with acute stress:Compared with the model group,the Beta diversity index of the tangerine peel and au-rantii fructus group decreased(p<0.05),suggesting that the tangerine peel and aurantii fruc-tus can regulate the distribution of gut flora in rats under stress,while Tiaoqi Anshen decoc-tion has no effect on the distribution of gut flora in rats under stress.4.The ingredients of Tiaoqi Anshen decoction are relatively stable.The fingerprints showed that there was little difference between the active ingredients of granules and decoc-tion,but the content was relatively small.The stability of decoction was relatively good.5.No acute toxic effect of Tiaoqi Anshen decoction on rats was observedThere was no death in the rats in each group during the observation period,so the half-lethal dose was not reached at 2000g/person/day.There was no difference in body weight between the female and male dose groups compared with the normal group(p>0.05).There was no abnormality in naked eye examination after execution in each group.Conclusion:1.Tiaoqi Anshen decoction and its core drugs can effectively alleviate anxiety,tension,fear and other behavioral changes of stress rats,showing a good effect of improving stress state.2.Tiaoqi Anshen decoction can regulate the disorder of intracerebral neurotransmitters and their receptors of stress rats,suggesting that the improvement of stress state may be re-lated to the regulation of intracerebral neurotransmitters and their receptors.3.The tangerine peel and aurantii fructus can regulate gut flora in rats with acute stress,which may be one of the internal ways to regulate central nervous system function and im-prove behavioral after stress.It also provides modern biological basis for the treatment think-ing of stress in traditional Chinese medicine "taking spleen and stomach as the center and regulating qi of five Zang-organs". |
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