| Research Background:Depression is a common mental illness,and its main feature is the significant and lasting depression.It seriously disturbs people’s daily work,study and life,and also causes a lot of burden and unstable factors to the society.Among the TCM syndromes of depression,the syndrome of liver depression and spleen deficiency is the most common.The syndrome of liver depression and spleen deficiency mainly includes mood-related symptoms caused by stagnation of the liver,gastrointestinal disorders and disorders of material and energy metabolism caused by spleen deficiency.Among them,the pathogenesis of liver depression is related to the central nervous endocrine immune network,and the metabolism of material and energy is closely related to the pathological manifestations of spleen deficiency.Many years of research in this research group have found that the disorder of material and energy metabolism in the syndrome of liver depression and spleen deficiency is mainly manifested as a disorder of glucose metabolism,but the central mechanism is not yet fully understood.Studies have found that autophagy is involved in the energy metabolism process of liver-stagnation and spleen-deficiency syndrome.Glucose transporter-4(GLUT4)is the main regulatory protein for the body’s uptake of peripheral glucose and can play a role in the hypothalamic neurons to maintain the body’s glucose homeostasis.Rab8 and Rab10,members of the Ras GTPase superfamily,can jointly regulate the maturation of autophagy and the transport of GLUT4 vesicles,and play an important role in the regulation of GLUT4 translocation by autophagy.Studies have found that Xiaoyaosan can improve depression-like behaviors caused by CUMS,but whether it can regulate hypothalamic neuronal autophagy and further affect glucose metabolism in the syndrome of liver depression and spleen deficiency by adjusting the level of GLUT4 is not clear.Therefore,starting from the related index of autophagy and glucose metabolism-GLUT4,it is helpful to understand the modern pathogenesis of liver depression and spleen deficiency syndrome,and provide new scientific basis for Xiaoyaosan to treat depression.Objective:Through chronic unpredictable mild stress(CUMS),a mouse model of depression with liver depression and spleen deficiency syndrome was established,and autophagy blocking was set up as a control.Through the use of behavioral testing,transmission electron microscopy,immunofluorescence,western blotting,RT-q PCR and other modern technology,to explore the changes of hypothalamic neuron autophagy and its effect on GLUT4 in depression mice with liver depression and spleen deficiency syndrome,and to explore the regulatory effect of Xiaoyaosan on it during this process.In order to provide a new scientific basis for the biological basis of liver depression and spleen deficiency syndrome and the antidepressant effect of Xiaoyaosan.Methods:This experiment can be divided into two parts.1.Choose 60 SPF-grade C57BL/6J male mice,6-8 weeks old,weighing about 18-25 g,adapted to feeding for 7 days,and weighing.Exclude mice that are too active and too quiet(according to the open field test),as well as mice with slow and fast weight gains.The mice were numbered and randomly divided into 4 groups: normal,CUMS,Xiaoyaosan and fluoxetine.15 mice were included in each group,and were fed in 3 cages.The mice in the normal group eat and water freely without stress.The CUMS,Xiaoyaosan and fluoxetine groups received 6 weeks of continuous unpredictable mild stress modeling and intragastric administration of different drugs.From the 4th week of stress,intragastric administration was given.The normal group and the CUMS group were given intragastric administration with deionized water,the Xiaoyaosan group and the fluoxetine group were given intragastric administration with Xiaoyaosan and fluoxetine respectively for intervention.The general conditions of the mice(macro characteristics,body weight and food intake)are observed and recorded,the model is evaluated by behavioral tests such as open field test,tail suspension test,and sugar preference test.Later,intraperitoneal injection of glucose tolerance test(IPGTT)was used to detect the blood glucose of mice,the change of serum insulin level in mice was detected by ELISA method,the changes of autophagy in the mouse hypothalamus were observed by transmission electron microscope(TEM),and then immunofluorescence and other biological methods were used.Western Blot and RT-q PCR were used to detect the changes in the expression of proteins and genes of hypothalamic autophagy related indexes——LC3,p62,GLUT4,Rab8,Rab10 in depressed mice with liver depression and spleen deficiency syndrome.2.Through the first part of the experimental verification,it is known that the autophagy level of depression mice with liver depression and spleen deficiency syndrome decreases and glucose metabolism is disordered.In order to further study the role of autophagy in it,on this basis,we selected the second batch of C57BL/6J 80 male mice,6-8 weeks old,weighing about 18-25 g,adapted to feeding for 7 days,and weighed.Exclude mice that are too active and too quiet(according to the open field experiment),as well as mice with slow and fast weight gains.The mice were numbered and randomly divided into 4 groups: normal,sham,3-MA and Xiaoyaosan according to their body weight,with 20 mice in each group,and were fed in 4 cages.The mice in each group were free to eat and drink without stress.Mice in the sham group were injected with saline into the brain ventricle,and mice in the 3-MA and Xiaoyaosan group were injected with the autophagy inhibitor 3-Methyladenine(3-MA).After the operation recovered,mice in the normal group,sham group and 3-MA were given gastric deionized water,and the Xiaoyaosan group was given Xiaoyaosan for intervention.The general conditions of the mice(macro characteristics,body weight and food intake)are observed and recorded,behavioral tests such as open field test,forced swimming test and sugar preference test are used to evaluate the anxiety and depression of each group,especially the 3-MA. Evaluation.Other subsequent detection methods are the same as the first part of the experiment.Results:1.The results of the first part of the experiment(1)Model evaluation results:(1)General situation: CUMS caused the mice in the CUMS group to gradually show symptoms such as poor mental state,curling up,languidness,dry and messy hair,loose stools,etc.And the resistance was significantly weakened during the process of modeling stress,and some did not even struggle.After the intervention of Xiaoyaosan,the mental state gradually improved,the hair gradually became shiny,the degree of mobility and responsiveness increased,and the stool was formed.In terms of body weight,each group of mice began to grow steadily.From the3 rd weekend to the 6th week,except for the normal group,the weight of the mice in the other 3 groups decreased,and compared with the normal group,there was a statistical difference(P<0.01 or P<0.05).The other three groups of mice have little difference in body weight(P>O.O5).Although the daily food intake of mice in each group fluctuates,the overall food intake of mice in the CUMS group is less than that of the other three groups,which is not statistically significant(P>O.O5).(2)Behavioral results: The total distance of the mice in the open field test showed that compared with the normal group,the total distance of the mice in the CUMS group was reduced in the open field(P<0.05).The total movement distance of mice in the Xiaoyaosan group and the fluoxetine group was greatly increased compared with the CUMS group(P<0.05 or P<0.01);The results of time spent in open area of the open field test of each group of mice showed that the time spent in open area of the open field box in the CUMS group was significantly less than that of the normal group(P<0.01).Compared with the CUMS group,the mice in the Xiaoyaosan group and the fluoxetine group spent more time in the central area(P<0.05).The results of the immobility time of the tail suspension experiment of mice in each group showed that the immobility time of the tail suspension of the CUMS group was significantly longer than that of the normal group(P<0.01).The reduction of immobility time of mice in the Xiaoyaosan group and the fluoxetine group was shorter than that of the CUMS group(P<0.05 or P<0.01).The results of the sucrose preference test of mice in each group showed that the 6-week CUMS modeling reduced the sucrose consumption rate of mice in the CUMS group(P<0.01).The sucrose consumption of mice in the Xiaoyaosan group and fluoxetine group was significantly higher than that of the CUMS group(P<0.01).(2)Changes in the level of autophagy:(1)By taking transmission electron micrographs of the hypothalamus in each experimental group,we found that compared with the normal group,the number of hypothalamic autophagosomes in the CUMS group was significantly reduced(P<0.01).Compared with the CUMS group,the autophagosomes in the two medication groups of Xiaoyaosan and fluoxetine were significantly increased(P<0.01).(2)The WB results of each group of mice showed that in the protein expression of hypothalamic LC3,the LC3 level of the CUMS group was lower than that of the normal group,especially LC3 II decreased significantly,and the value of LC3II/LC3 I was lower than that of the normal group(P<0.05).After administering Xiaoyaosan and fluoxetine to the CUMS group,the values of LC3II/LC3 I increased(P<0.05 or P<O.O1).In the protein expression of p62 in the hypothalamus,the expression of the CUMS group was higher than that of the normal group(P<0.05),Xiaoyaosan and fluoxetine reduced the protein expression(P<0.05).(3)The RT-q PCR results of each group of mice showed that in the hypothalamic LC3 gene expression,the LC3 level of the CUMS group was lower than that of the normal group(P<0.05),while the expression of Xiaoyaosan and fluoxetine group was higher than that of the CUMS group(P<0.05).In the hypothalamic p62 gene expression,the CUMS group was higher than the normal group(P<0.05),and the Xiaoyaosan group and fluoxetine group were lower than the CUMS group(P<0.05).(3)Changes in GLUT4 and other related indicators of glucose metabolism:(1)From the immunofluorescence results of GLUT4,the expression of GLUT4 in the dorsal medial hypothalamic nucleus and paraventricular nucleus of the CUMS group was significantly lower than that of the normal group(P<0.01).The expression of nucleus GLUT4 was significantly higher than that of the CUMS group(P<0.01).(2)The results of serum insulin in each group showed that the serum insulin level of the CUMS group was significantly lower than that of the normal group(P<0.01),and the Xiaoyaosan group and fluoxetine group were significantly higher than that of the CUMS group(P<0.01).(3)By performing blood glucose detection at 0,15,30,60,and 120 minutes in each group of mice,the IPGTT results of each group of mice showed that the blood glucose level of the CUMS group mice at each time point was higher than that of the normal group and two therapy group.The blood glucose level of mice in the CUMS group at 60 min was significantly different from that of the Xiaoyaosan group(P<0.01),and at 120 min,it was significantly higher than that of the normal,Xiaoyaosan and fluoxetine groups (P<0.01,P<0.01 or P<0.05).(4)The WB results showed that the expression of GLUT4 in the CUMS group was lower than that in the normal group(P<0.01),and the Xiaoyaosan and fluoxetine groups were higher than the CUMS group(P<0.05);in the hypothalamic Rab8 and Rab10 protein expression,the CUMS group was lower than The normal group(P<0.05),the Xiaoyaosan group and the fluoxetine group were higher than the CUMS group(P<0.05).(5)RT-q PCR results of mice in each group showed that the gene expression of hypothalamic GLUT4 in the CUMS group was lower than that of the normal group(P<0.05),and the Xiaoyaosan and fluoxetine groups were higher than the CUMS group(P<0.05).Coming to Rab8 and Rab10 gene expression in the hypothalami,the expression of the CUMS group was lower than that of the normal group(P<0.05),and the expression of Xiaoyaosan group and fluoxetine group was higher than that of the CUMS group(P<0.01 or P<0.05).2.The results of the second part of the experiment(1)Behavioral evaluation results:(1)General situation: the mice in the normal group,the sham group and the Xiaoyaosan group are in good mental state,with bright hair,high responsiveness to external activities,and moderate dryness,wetness,and hardness of feces;Compared with the other three groups,the condition of mice in the 3-MA group is slightly worse,the hair is bright,the reaction sensitivity is slightly lower,and the stool is basically normal.In terms of body weight,the weight of mice in each group showed a steady increase.The weight of the normal group was the highest,the sham group and the Xiaoyaosan group were similar,and the weight of the 3-MA group was slightly lower than the other three groups.But there was no significant difference(P>0.05).The food intake results of mice in each group at different time points showed that the food intake of mice in the 3-MA group was always at the lowest level,and the difference in food intake between the other three groups was not significant(P>0.05).(2)Behavioral results:The total distance of the mice in the open field test showed that the total distance of the mice in the 3-MA group was significantly lower than that of the normal group(P<0.01),and the mice in the sham group had no significant changes compared with the normal group(P>0.05).The total movement distance of the mice in the sham group and the Xiaoyaosan group was significantly greater than that of the3-MA group(P<0.01).The results of the immobility time in the central area of the open field test of each group showed that the immobility time of 3-MA mice was significantly shorter than that of the normal group(P<0.01),and there was no significant difference between the sham group and the normal group(P>0.05),and the sham group and Xiaoyaosan group were significantly longer than the 3-MA group(P <0.01,P<0.05).The results of the immobility time of the tail suspension test showed that compared with the normal group,the immobility time of the mice in the 3-MA group was significantly prolonged during the tail suspension test(P<0.05),and there was no significant difference between the sham group and the normal group(P >0.05).Compared with the 3-MA group,the immobility time of the mice in the sham group and Xiaoyaosan group was significantly shorter(P<0.05).The results of the sucrose preference test are as follows.The sucrose consumption of the mice in the 3-MA group and the sham group is significantly lower than that of the normal group(P<0.01),and there is little difference between the sham group and the normal group(P>0.05).The sucrose consumption of Xiaoyaosan group was significantly higher than that of the 3-MA group(P<0.01).(2)Changes in the level of autophagy:(1)Through taking the hypothalamic transmission electron micrographs of each experimental group,we found that the number of autophagosomes in the 3-MA group was significantly less than that in the normal group and the sham group(P<0.01).The there was no significant difference between normal group and sham 3-MA group(p>0.05).And the number of hypothalamic autophagosomes in Xiaoyaosan group was significantly higher than that in the 3-MA group(P<0.01).(2)Through the WB results,we can find that considering the hypothalamic LC3 protein expression,compared with the normal group and the sham 3-MA group,the expression of LC3Ⅱ in the 3-MA group was significantly lower,and the expression of p62 was increased(P<0.05);While there was no significant difference between the sham 3-MA group and the normal group(P>0.05).After gavage with Xiaoyaosan,the expression of LC3Ⅱ increased significantly,while the expression of p62 decreased significantly(P<0.05).(3)Observing the results of RT-q PCR shows that in the gene expression of hypothalamic LC3,the level of LC3 in the 3-MA group was lower than that in the normal group(P<0.05),and the sham 3-MA group was slightly higher,but it was not statistically significant(P> 0.05).Compared with the 3-MA group,the expression of LC3 in the Xiaoyaosan group was higher(P<0.05).Regarding the gene expression of p62 in the hypothalamus,the 3-MA group was significantly higher than the normal group and the sham 3-MA group(P<0.01),there was no difference between the sham 3-MA group and the normal group(P>0.05),and the Xiaoyaosan group was lower than the 3-MA group (P<0.05).(3)Changes in glucose metabolism-related indicators such as GLUT4:(1)The immunofluorescence results of GLUT4 in each group of mice are as follows.The expression of GLUT4 in the dorsal medial hypothalamic nucleus and paraventricular nucleus of mice in the normal group was significantly higher than that of the mice in the3-MA group(P<0.01)The expression of GLUT4 in the hypothalamic dorsal nucleus of the sham group was significantly higher than that of the 3-MA group(P<0.01);the sham group and the normal group had less expression than the paraventricular nucleus,and there was no significant difference in the expression of the dorsal medial nucleus(P>0.05).Compared with the 3-MA group,the expression of GLUT4 in the dorsal medial hypothalamus and paraventricular nucleus of mice in the Xiaoyaosan group was significantly increased(P<0.01).(2)In terms of the results of serum insulin,compared with the normal group and the sham group,the 3-MA group had a significant decrease in serum insulin level(P<0.01).There was no difference between the sham group and the normal group(P>0.05).Compared with the 3-MA group,the serum insulin level of the Xiaoyaosan group was significantly increased(P<0.01).(3)The IPGTT results showed that the blood glucose level of the mice in the 3-MA group was higher than the other three groups at all time points,and the blood glucose level of the mice in the 3-MA group was significantly higher than that of the normal group and the sham group at 15,30,and 60 minutes(P< 0.01),the blood glucose level of mice in the Xiaoyaosan group was significantly lower than that of the 3-MA group at 15 and 60minutes(P<0.05).(4)In terms of the WB results,in the protein expression of GLUT4 in the hypothalamus,the 3-MA group was significantly lower than the normal group and the sham group(P<0.01 or P<0.05),and there was no significant difference between the sham group and the normal group(P>0.05)),the Xiaoyaosan group was significantly higher than the 3-MA group(P<0.05).Redarding the protein expression of Rab8 and Rab10 in the hypothalamus,compared with the normal group,the expression of the 3-MA group was significantly reduced(P<0.01);compared with the sham group,the expression of the 3-MA group was also reduced,and the reduction rate of Rab8 was greater than that of Rab10(P <0.01 or P<0.05);there was no difference between the sham group and the normal group(P>0.05);the expression of these two proteins in the hypothalamus of mice in the Xiaoyaosan group was higher than that in the 3-MA group(P<0.01 or P< 0.05).(5)In terms of the hypothalamic GLUT4 gene expression,the 3-MA group was significantly lower than the normal group and the sham group(P<0.05),there was no significant difference between the sham group and the normal group(P>0.05),and the expression level of the Xiaoyaosan group was significantly higher than the 3-MA group(P<0.05).Coming to the expression of Rab8 and Rab10 in the hypothalamus,the 3-MA group was significantly lower than the normal group(P<0.05),and at the same time,it was lower than the sham group,but the expression of Rab8 was not significantly different(P>0.05),and the difference in Rab10 expression was statistically significant(P<0.05);There was no significant difference in the expression of the two genes between the sham group and the normal group(P>0.05);the expressions of Rab8 and Rab10 in the Xiaoyaosan group were higher than those in the 3-MA group(P<0.05 or P< 0.01).Conclusion:1.The 6-week CUMS was used to construct the depression mouse model of liver depression and spleen deficiency syndrome.The animal model of liver depression and spleen deficiency syndrome was successfully constructed by observing the animal macro characterization and behavioral tests,and the regulatory effect of Xiaoyaosan was used to evaluate the model.2.According to the experimental results of transmission electron microscope,WB and RT-q PCR,the autophagy level of hypothalamus in mice with liver depression and spleen deficiency syndrome was changed,and Xiaoyaosan has a regulatory effect on it.3.According to the mouse serum insulin and fasting blood glucose levels,as well as the experimental results of WB and RT-q PCR,combined with the changes in autophagy levels,it is suggested that autophagy in hypothalamic neurons mediates Rab8 and Rab10 to change the expression of GLUT4,which affects glucose metabolism.4.Through intracerebroventricular injection of autophagy inhibitors,it is clear from both positive and negative aspects that Xiaoyaosan affects the glucose metabolism of liver depression and spleen deficiency syndrome by regulating the autophagy of hypothalamic neurons to mediate the expression of GLUT4. |
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