| BackgroundThe syndrome of stagnation of liver qi and spleen deficiency is a common symptom of traditional Chinese medicine(TCM),and its biological basis is not yet completely clear.Modern medical research believes that emotional changes are the etiological basis of liver-stagnation and spleen deficiency-related diseases.The emotional response axis is the nerve center-hypothalamus-brain-stem autonomic nervous system-effect device.When the liver fails to control conveyance and dispersion,the body’s response to emotional changes can lead to dysfunction of the brain’s central nervous system,the excitement of the central cortex,and the corresponding functional and organic changes in the body’s system organs through the transmission of autonomic nerves.During the stage of stagnation of liver-qi stagnation and spleen deficiency,the body often has other changes such as digestive system,nervous system,and gastrointestinal function.Therefore,brain-gut peptide(BGP),a peptide regulating the mood and feeding,has become one of the hot topics in the study of syndromes of liver stagnation and spleen deficiency.BGP is a type of neurotransmitter that is dually distributed in the central nervous system and the gastrointestinal tract.It is the molecular basis of the two-way traffic pathway that connects the cognitive and affective centers with the neuroendocrine,endocrine,and immune systems.It plays an important role in regulating emotions,memory,visceral sensation,secretion,and exercise.BGP is also an important gut hormone involved in food intake and plays a key role in regulating the feeding behavior of the brain’s gut axis.As a typical brain-gut peptide,cholecystokinin(CCK)is secreted by the small intestine type I cells and neurons,and is widely distributed in the gastrointestinal tract(duodenum,jejunum and colon).In the central and peripheral nervous system,it plays an important physiological role in the form of neurotransmitters.CCK not only plays an important role in the regulation of gastrointestinal function,but also in the central nervous system,especially in the emotions and appetite.Xiaoyao Powder is a classic prescription of traditional Chinese medicine for the treatment of liver depression,spleen deficiency and blood deficiency,and has been widely used in the related research of liver depression and spleen deficiency syndrome.Our previous experimental study showed that the rat model of liver epression and spleen deficiency syndrome could be successfully established with the 21 days of chronic restraint stress.The treatment of Xiaoyaosan could play a significant role in the regulation of liver depression and spleen deficiency syndrome,and its mechanism of action may be related to the regulation of BGP.However,whether Xiaoyao Powder can improve liver-qi spleen deficiency syndrome by adjusting the central-periphery network of CCK and CCK1R is not yet clear.ObjectiveBased on the relationship between liver stagnation and spleen deficiency and brain-gut peptides,in this study we duplicates the rat model of liver-qi and spleen-deficiency syndrome with the 21-day chronic restraint stress method.Then,the animal’s general state,behavioral performance,macroscopic representation(food intake,body weight)and microscopic indicators were used to assess the rat model.Using molecular biology techniques,the expression changes of CCK and its receptors in the duodenum and hypothalamus of rats were detected from the gene and protein levels.At the same time,the regulatory effects of Xiaoyao Powder on the measurement of site markers were observed.By the observation of the changes of CCK and CCK1R in the central and peripheral nerves under chronic restraint stress,the purpose of this study is to explore the central-periphery microcosmic mechanism of the onset of liver depression and spleen deficiency syndrome,and to enrich the theoretical basis for the interpretation of the scientific connotation of the syndrome of liver qi stagnation and spleen deficiency,and thus provide a basis for the clinical efficacy evaluation of Xiaoyao Powder.MethodsEighty adult male SD rats were randomly divided into normal group(A group),model group(B group),fluoxetine group(C group)and Xiaoyaosan group(D group)according to the random number table method,20 in each group.In this experiment,we used the method of chronic restraint stress in the study group to replicate the rat model of liver-qi-stagnation and spleen-deficiency syndrome.Rats in group A were free from restraints and interventions every day and were fed freely;B,C,and D rats were bundled daily.On the special restraint frame,its freedom was limited,and the time of restraint was random,3h/d;After the end of the bondage,gavage was performed at the same time.The group C rats were given fluoxetine 2mg/kg body weight,and group D rats were taking Xiaoyao Powder 2.224g/kg body weight,group B rats were given an equal dose of deionized water for 21 days.During the experiment,the changes of the general state,food intake,and body weight of the rats in each group were observed.The behavior of rats was evaluated by using the water-sugar preference test,forced-swimming test,and open field test;The expressions of CCK and CCK1R protein and mRNA in the duodenum and hypothalamus were observed using immunohistochemistry and real-time quantitative PCR,respectivly.ResultsRats exposed to chronic restraint stress were gradually showed symptoms of liver depression and spleen deficiency,such as depression,loss of hair yellow,loss of tiredness,exercise,sleeplessness,stool odor,dryness and dampness,and et al.At the same time,there was a decrease in food intake,weight loss,etc.On the 21st day of modeling,the body weight and food intake of the model group rats were significantly lower than those of the control group,fluoxetine group,and Xiaoyaosan group(P<0.01),indicating that chronic restraint stress affected the appetite and Changes in body weight.In the sugar-water preference test,open-field test and forced-swimming test,the intake of sugar water in the model group was significantly lower(jP<0.01),and the number of standing,passing times,and modification times were significantly reduced(P<0.01).The immobility time was significantly prolonged within 5 minutes(P<0.01),indicating that chronic restraint stress led to behavioral changes in emotional depression in model rats,showing decreased interest,anhedonia,despair and helplessness,and Fluoxetre.All the behavioral indexes of the Ting group and Xiaoyaosan group were significantly improved.RT-qPCR results showed that compared with the normal group,the mRNA expression of CCK and CCK1R in the duodenum and hypothalamus of the model group was significantly increased(P<0.01).Compared with the model group,after drug intervention,the mRNA expression of CCK and CCK1R in the duodenum and hypothalamus was significantly down-regulated in the fluoxetine group and Xiaoyaosan group(P<0.01).The results of immunohistochemistry showed that there were many CCK positive brown particle distributions on LHA and VMH two nuclei.The expression of CCK and its receptor CCK1R in the hypothalamus and duodenum of the model group was significantly higher than that of the normal group.It means that it affects not only emotion but also appetite and gastrointestinal motility.Compared with the model group,the CCK and CCK1R protein expression levels in the duodenum and hypothalamus of fluoxetine group and Xiaoyaosan group were significantly decreased after drug intervention,which were further confirmed the results of RT-qPCR.ConclusionThis study successfully replicated the combination of syndromes of liver stagnation and spleen deficiency syndrome with a rat model by chronic restraint stress.The expression of CCK and its receptor CCK1R in the hypothalamus and duodenum of the model group showed the same tendency in the expression of genes and proteins,and Xiaoyao Powder can down-regulate its expression level through the central-peripheral regulation mechanism of brain-gut peptides,thereby improving this imbalancedstate and reducing gastrointestinal symptoms while regulating emotional changes,which has guiding significance for the treatment of liver stagnation and spleen deficiency syndrome. |
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