| Research BackgroundDepression is a common psychological and emotional disorder.Severe patients with depression even lose confidence in life,resulting in suicidal tendencies.It is a kind of chronic mental disease characterized by high morbidity,high recurrence rate,high disability rate and high suicide rate[1].With the increasingly fierce social competition and the acceleration of work rhythm,the number of patients suffered from depression is increasing significantly,which seriously affects people's life,work and physical and mental health.Modern clinical research has found that depression is often accompanied by functional gastrointestinal disorders?FGD?[2], which is very similar to the clinical manifestations of depression with the syndrome of stagnation of liver Qi and spleen deficiency.As one of the common syndromes of depression,syndrome of stagnation of liver Qi and spleen deficiency has been widely concerned by domestic scholars in recent years.According to the theory of traditional Chinese medicine?TCM?,the early stage of depression is often manifested as the syndrome of liver depression and Qi stagnation.The stagnation of liver Qi last for a long time and then block the Qi movement.Subsequently,Qi is rising and falling abnormally,and violating the spleen,leading to the formation of the syndrome of stagnation of liver Qi and spleen deficiency.It is often manifested as depression,abdominal distention,poor appetite,diarrhea and other gastrointestinal symptoms.In the world,the clinical research of antidepressants in the treatment of gastrointestinal dysfunction is relatively extensive[3].However,it is easy to produce drug resistance and side effects.TCM has accumulated rich theoretical knowledge and clinical experience in the treatment of depression.In recent years,there have been extensive and effective researches on the etiology and pathogenesis,diagnosis and syndrome differentiation, treatment principles,prescriptions and basic experiments of depression in China.Therefore,under the guidance of the theory of TCM,it is of great significance to use modern scientific methods to study the antidepressant effect and mechanism of TCM and to find and develop safe and reliable antidepressant drugs.Xiaoyao Powder,which originated from Taiping Huimin Heji Jufang,is a classic prescription for relieving depression in TCM.It has the functions of dispersing stagnated liver Qi for relieving Qi stagnation,invigorating spleen and harmonizing yingfen.It has been proved that Xiaoyao Powder has a good effect on depression and its complicated intestinal symptoms[4].Compared with Western medicine antidepressants,it has the advantages of long-lasting antidepressant effects and low side effects,but its specific mechanism is not clear.Therefore,from the aspect of gastrointestinal function,it is of great significance to understand the relationship between depression and intestinal dysfunction,to better understand the pathogenesis of depression,and to provide a new scientific basis for the treatment of depression with FGD by Xiaoyao Powder.Research ObjectiveBased on the previous work of the research group,this study studied the changes of colonic permeability in rats with syndrome of stagnation of liver Qi and spleen deficiency established by chronic unpredictable mild stress for 6 weeks,and explored the mechanism of intestinal permeability changes in depression with the syndrome of stagnation of liver Qi and spleen deficiency and the regulatory effect of Xiaoyao Powder,to lay a foundation for the interpretation of the biological connotation of the syndrome of stagnation of liver Qi and spleen deficienc,and provide a new theory for the antidepressant mechanism of Xiaoyao Powder.Research Methods1.Sixty SPF male SD rats,6-7 weeks old,were randomly divided into normal group,model group,Xiaoyao Powder group and fluoxetine group according to their initial body weight,15 rats in each group,5 rats in a cage.2.After 7 days of adaptive feeding,there was no stimulation except for free drinking and eating in the normal group.In the other three groups,the rats received CUMS for 6 weeks.The rats in the Xiaoyao Powder group and fluoxetine group received gastric perfusion intervention respectively,and the rats in the normal group and model group received deionized water of equal volume.3.The general situation of rats was observed and recorded,including macroscopic characterization,body weight and food intake.4.Behavioral experiment:open field experiment,forced swimming experiment and sugar water preference experiment were used to evaluate the degree of anxiety and depression in rats.5.The changes of D-xylose in the serum of rats were detected by ELISA to further verify whether the model has spleen deficiency syndrome.6.HE staining,PAS staining and ultrastructural pathology were used to observe the pathological changes of the colon in rats.7.Immunohistochemistry,Western blot and PCR were used to detect the expression of ZO-1,Occludin and Claudin-1 in the colon of depression rats with the syndrome of stagnation of liver Qi and spleen deficiency.8.Immunohistochemistry and Western blot were used to detect the phosphorylation of p38,ERK and JNK in rat colon.9.The changes of 5-HT,SP and VIP in the hypothalamus and colon of depression model rats before and after the intervention of Xiaoyao Powder were observed by ELISA.Research Results1.General observation results of rats:the model group rats were in poor mental state,tired and prone to lying down,liked to curl up and pile up,their hair was disorderly,dry and yellow,the skin color of auricle and nose became light and matte,the color of mucous membrane of eye crack was light,there were obvious secretions in the canthus,the voice was weak,the phenomenon of arching back disappeared,the feces were dry and wet,and loose stools appeared.Compared with the model group,the general state of rats in the Xiaoyao Powder group and fluoxetine group was significantly improved.Weight change of rats in each group:compared with the normal group,the weight of rats in the model group decreased significantly from the second weekend to the sixth weekend?P<0.01?;compared with the model group,the rats in the Xiaoyao Powder group began to show the drug effect at the third weekend and increased significantly at the sixth weekend?P<0.01?;compared fourth weekend?P<0.05?.At the end of the sixth week,the body weight increased significantly?P<0.01?.Changes of food intake in each group:compared with the normal group,the food intake in the model group decreased significantly from the third week?P<0.01?;compared with the model group,the food intake in the Xiaoyao Powder group increased significantly from the fourth week?P<0.01?,the food intake in the fluoxetine group increased significantly from the third week?P<0.05?,and the food intake increased significantly from the fourth week?P<0.01?.2.Behavioral test results:the results of open field experiment showed that:compared with the normal group,the total distance of movement,the number of times of entering the central area,the time of staying in the central area,the total number of wearing and standing in the model group were significantly reduced?P<0.01?;compared with the model group,the total distance of movement,the number of times of entering the central area,the time of staying in the central area,the total number of wearing and standing in the Xiaoyao Powder group and the fluoxetine group were significantly reduced?P<0.01?The times of standing increased significantly?P<0.01?.However,there was no significant difference in the modification times among the four groups.Comparison of immobility time within 5 minutes of forced swimming:Compared with the normal group,the immobility time in the model group increased significantly?P<0.01?;compared with the model group,Xiaoyao Powder could significantly reduce the immobility time?P<0.01?;although the slight decrease was observed in the fluoxetine group,there was no statistical significance?P>0.05?.The results of the sugar water preference experiment comparison:?1?There was no difference among the rats in each group before modeling?P>0.05?.?2?At the end of modeling,compared with the normal group,the preference of sugar and water in the model group was significantly reduced?P<0.01?.Compared with the model group,the sugar water preference of Xiaoyao Powder group and fluoxetine group increased significantly?P<0.01?.3.Results of serum D-xylose:compared with the normal group,the content of serum D-xylose in the model group decreased significantly?P<0.01?.Compared with the model group,the content of D-xylose in the Xiaoyao Powder group was significantly increased?P<0.01?,while that in the fluoxetine group was significantly increased?P<0.05?.4.Histopathological changes of the colon in rats:?1?HE staining results:the structure of colon in normal group was intact and the arrangement of intestinal glands was good.In the model group,the epithelial structure was destroyed,and the intestinal glands were reduced or even disappeared.Compared with the model group,the Xiaoyao Powder group and fluoxetine group reduced the infiltration of inflammatory cells and the degree of gland damage.?2?PAS staining results:after six weeks of CUMS,the number of goblet cells in the colon decreased significantly compared with the normal group?P<0.01?.Compared with the model group,the number of goblet cells in the fluoxetine group increased significantly?P<0.05?.?3?The results of transmission electron microscopy showed that the normal group showed regular columnar cells,the intestinal epithelium was complete,the microvilli were neat and closely connected.In the model group,the distribution of microvilli was sparse and disordered,the intercellular junction complex was destroyed,the intercellular space was widened,and the top part of the tight junction was destroyed.In the Xiaoyao Powder group,the size and quantity of microvilli increased,and the arrangement of microvilli was denser and tidier than that in the model group.In the fluoxetine group,the size,number and arrangement of microvilli were also improved,but the damage of the intercellular junction complex and the damage of tight junction protein were not significantly improved.5.The results of immunohistochemistry were as follows:?1?the OD values of ZO-1,Occludin and Claudin-1 in colon of rats decreased significantly after modeling,which was significantly different from that of normal group?P<0.01?;the OD values of ZO-1 in colon of Xiaoyao Powder group increased significantly compared with that of model group?P<0.05?,while the OD values of Occludin and Claudin-1 in colon of rats increased significantly compared with that of model group?P<0.01?;there was no significant difference between fluoxetine group and model group.The OD values of Occludin and Claudin-1 were significantly higher than those of the model group?P<0.05?.?2?The expression of p-p38,p-ERK and p-JNK in colon of model group was significantly higher than that of normal group?P<0.01?;compared with model group,the expression of p-p38and p-JNK in Xiaoyao Powder group was significantly lower?P<0.01?,and the expression of p-ERK was significantly lower?P<0.05?;compared with model group,the expression of p-p38,p-ERK?P<0.05?and p-JNK?P<0.01?in fluoxetine group was significantly decreased.6.The results of Western Blot showed that:?1?Compared with the normal group,the expression of ZO-1 and Claudin-1 in the colon of the model group was significantly reduced?P<0.01?,and the expression of Occludin was significantly reduced?P<0.05?; compared with the model group,the expression of ZO-1 and Claudin-1in Xiaoyao Powder group and fluoxetine group increased significantly?P<0.01?,and the expression of Occludin in Xiaoyao Powder group increased significantly?P<0.05?,but there was no statistical significance in the expression of Occludin in fluoxetine group?P>0.05?.?2?Compared with the normal group,p-p38 and p-JNK in the colon of the model group increased significantly?P<0.01?;compared with the model group,p-p38 and p-JNK in the Xiaoyao Powder group decreased significantly?P<0.01?,p-p38 and p-JNK in the fluoxetine group decreased significantly?P<0.05?.There was no significant difference in colon p-ERK between each group?P>0.05?.7.PCR results:?1?ZO-1 mRNA expression:compared with the normal group,the model group decreased significantly?P<0.05?;compared with the model group,the Xiaoyao Powder group and fluoxetine group increased significantly?P<0.05?.?2?Occludin mRNA expression:compared with the normal group,the model group decreased significantly?P<0.01?;compared with the model group,the Xiaoyao Powder group and fluoxetine group increased significantly?P<0.01?.?3?Claudin-1 mRNA expression:compared with the normal group,the model group decreased significantly?P<0.05?;compared with the model group,the Xiaoyaosan group increased significantly?P<0.01?,and the fluoxetine group also increased significantly?P<0.05?.8.The results of ELISA were as follows:?1?comparison of 5-HT and VIP contents:compared with normal group,the contents of 5-HT and VIP in hypothalamus and colon in model group decreased significantly?P<0.01?;compared with the model group,the content of 5-HT and VIP in the hypothalamus and colon of Xiaoyao Powder group increased significantly?P<0.01?,the content of 5-HT in the hypothalamus and colon of fluoxetine group increased significantly?P<0.01?,the content of VIP in the hypothalamus and colon of fluoxetine group increased significantly?P<0.05?.?2?SP content comparison:compared with normal group,SP content in hypothalamus and colon of model group increased significantly?P<0.01?;compared with model group,SP content in hypothalamus and colon of Xiaoyao Powder group decreased significantly?P<0.01?,SP content in hypothalamus and colon of fluoxetine group decreased significantly?P<0.05?.Research Conclusion1.Six weeks of CUMS was used to construct the depression model with the syndrome of stagnation of liver Qi and spleen deficiency in rats.The model was tested by macroscopical characterization of animals,various behavioral experiments,D-xylose content in serum and the regulating effect of Xiaoyao Powder.The model was evaluated.The animal model of syndrome of stagnation of liver Qi and spleen deficiency in depression was successfully reproduced in this study.2.According to the changes of colonic histopathology,the expression of ZO-1,Occludin and Claudin-1 in depression model rats with the syndrome of stagnation of liver Qi and spleen deficiency,the damage of colonic mucosal barrier function and the increase of intestinal permeability can be comprehensively determined.Xiaoyao Powder could regulate the changes of colonic permeability in depression rats with syndrome of stagnation of liver Qi and spleen deficiency.3.According to the results of detecting the phosphorylation level of related protein kinases?p38,ERK and JNK?in the MAPK signaling pathway,which suggested that the protective effect of Xiaoyao Powder on intestine was related to the MAPK signal pathway of colon.4.Based on the observation of the changes of 5-HT,SP and VIP in the hypothalamus and colon of depression model rats before and after the intervention of Xiaoyao Powder,the relationship between the intestine and brain in the syndrome of stagnation of liver Qi and spleen deficiency in depression and the mechanism of the antidepressant action of Xiaoyao Powder were indicated. |
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