The Mechanism Of Liver Energy Metabolism Disorder In Rats With Liver Depression And Spleen Deficiency Syndrome And The Effect Of Xiaoyaosan

1 BackgroundLiver depression and spleen deficiency syndrome is related to more than 40 diseases,including depression,anxiety,gastritis and irritable bowel syndrome.Xiaoyaosan is a classic prescription for the treatment of liver depression and spleen deficiency syndrome.Now,the biological basis of liver depression and spleen deficiency syndrome is not fully understood,and the mechanism of Xiaoyaosan for treating this syndrome,is not fully understood.Chronic stress could induce anxiety,depression and other diseases.Based on the procedural and phasic features of chronic stress,it has a high degree of consistency and correlation with the etiology of liver depression and spleen deficiency syndrome.Relevant studies have proved that chronic stress could be used for animal models of liver depression and spleen deficiency syndrome.Based on the above reasons and our previous work,we utilized chronic restraint stress to replicate the rat model of liver depression and spleen deficiency.Previous studies have confirmed that there are multiple systemic disorders in patients with liver depression and spleen deficiency syndrome,including neuro-endocrine system,immune system,digestive system,hypothalamus,pituitary/adrenal gland/gonadal gland/thyroid axis.There were obvious abnormal food intake,changes in the growth rate of weight,loose stools/constipation,thin/obesity in the animal models of liver depression and spleen deficiency syndrome.Studies have shown that chronic restraint stress could affect the absorption function of the stomach and intestinal microflora through the brain-gut axis.PI3K/Akt signaling pathway is the classical pathway for insulin regulation in glycolipid metabolism.PI3K/Akt downstream target proteins are involved in cell growth,apoptosis,tumor metabolism and even neural signal transduction.Among them,downstream GLUTs are PI3K/Akt signaling pathways that mediate glycolipid metabolism;SHIP2 is a classical inhibitor of the PI3K/Akt signaling pathway,which inhibits the activation of Akt.So,all of the above proteins are involved in the regulation of energy metabolism.Patients with liver depression and spleen deficiency are often accompanied by abnormal energy metabolism such as abnormal appetite,weight ’loss or obesity.Therefore,in this experiment,the chronic stress method was adopted to replicate the rat model of liver depression and spleen deficiency syndrome;Insulin/PI3K/Akt/GLUTs signaling pathway as the entry point,is observed to explore the biological basis of the abnormal energy metabolism of liver depression and spleen deficiency syndrome and the treatment mechanism of Xiaoyao San.2 Objective(1)To observe the expression of proteins related to PI3K/Akt/GLUT2 signaling pathway in rats with liver depression and spleen deficiency syndrome,and to explore the molecular biological basis of abnormal energy metabolism of liver depression and spleen deficiency syndrome.(2)To explore the biological mechanism of Xiaoyaosan in the treatment of liver depression and spleen deficiency syndrome,and to provide experimental evidence for liver stagnation and spleen deficiency syndrome in clinical prevention and its treatment.3 MethodsThis experiment is mainly composed of four parts.(1)A 21-day chronic restraint stress method was applied to replicate the rat model with liver depression and spleen deficiency.Xiaoyaosan was used’ as a therapeutic drug,Fluoxetine and Rosiglitazone were used as the positive controls.General conditions,the open-field tests,the forced-swimming tests,the sugar preference tests,the gastrointestinal motility tests,the defecation tests,the corticosterone levels in peripheral blood and the therapeutic effects of Xiaoyao San were applied to confirm that the rats model were successfully replicated.(2)The concentrations of insulin and blood lipids in peripheral blood were tested to explore the relationship between abnormal energy metabolism and glycolipid metabolism.(3)The RT-qPCR was used to study the expression of P85,Akt and SHIP2 mRNA in rat liver.(4)The expression of P85,Akt,GLUT2 and SHIP2 proteinS in rat liver were detected by Western blot and immune-histochemical method.4 Results(1)Rats in the model group showed that chronic restraint stress gradually.decreased food consumption,slower the ponderal growth,induced dry or wet stools.Behavioral studies found that number of grid crossing and total moving distance in the open-field test significantly increased in the model group,the suger preference decreased,the stopping time in the forced swimming test was significantly increased,the gastric emptying.capacity.and the small intestine propulsion rate were significantly declined;the serum corticosterone concentration increased significantly;while Xiaoyaosan,Fluoxetine and Rosiglitazone were able to improve the above phenomena and indexes in different degrees.(2)Peripheral concentrations of the insulin and triglyceride in the model group were significantly lower than those in the normal group;but there was no significant difference in the content of LDL,HDL and cholesterol between the two groups.Xiaoyao powder,Fluoxetine and Rosiglitazone regulated their expression.(3)The expression of P85 and Akt mRNA in the liver of rats with liver depression and spleen deficiency was decreased,and the expression of SHIP2 mRNA was increased.Compared with the model group,the,expression of P85 and.Akt mRNA increased significantly in the Xiaoyaosan group,but there was no significant difference in the expression of SHIP2 mRNA.The expression of SHIP2 mRNA in Fluoxetine group was significantly decreased,and the expression of P85 and Akt mRNA were increased.The expression of P85,Akt and SHIP2 mRNA in Rosiglitazone group was significantly higher than that in model group but lower than Xiaoyaosan group.(4)It was showed that compared with the normal group,the rats in the model group had higher P85 and SHIP2 protein content,lower Akt and GLUT2 protein content.Compared with the model group,the content of P85 in Xiaoyao San group was lower,and the protein content of Akt and GLUT2 was increased.However,there was no statistical difference between the SHIP2 protein content and the model group.The P85 and Akt protein content were significantly reduced after Fluoxetine intervented,but compared with the model group,the contents of SHIP2 and GLUT2 protein were not significantly different.The liver protein levels of P85,Akt,GLUT2 and SHIP2 in the Rosiglitazone group were higher than those in the model group.5 Conclusions(1)Combining general conditions,behavioral experiment results and the serum CORT concentration,it was proved that the method of chronic restraint stress in this experiment succeeded in replicating the rat model with liver stagnation and spleen deficiency.In addition,the experiment found that Xiaoyao powder and Fluoxetine had a regulatory effect on the abnormalities of the rats;but the effect of Fluoxetine was better than that of Xiaoyaosan in the regulation of emotions,and Rosiglitazone did not show significant changes.Three groups of drugs had effects on the food intake,body weight and gastrointestinal function;however,the effect of Rosiglitazone was better than that of Xiaoyao San,Xiaoyao San was more effective than fluoxetine.(2)The content of insulin and lipids in peripheral blood of rats decreased significantly,indicating that the loss of appetite,slower ponderal growth,and gastrointestinal dysfunction in rats with liver depression and spleen deficiency syndrome are related to the abnormal energy metabolism.(3)The abnormal mechanism of energy metabolism of liver-depression and spleen-deficiency syndrome is related to the low expression of Insulin/PI3K/Akt/GLUT2 signaling pathway and the inhibition of SHIP2.The regulation of the insulin/PI3K/Akt/GLUT2 signaling pathway mabe the treatment mechanism of Xiaoyaosan,but it has no intervention effect of SHIP2.Fluoxetine could regulate the expression of PI3K/Akt Proteins,but the downstreams still unknown,and it has no intervention effect of SHIP2.Rosiglitazone could increase the expression of insulin/PI3K/Akt/GLUT2 signaling pathway by increasing the sensitivity of insulin and PI3K/Akt signaling pathway,but it also has no intervention effect of SHIP2,which indicating that Rosiglitazone may has no therapeutic effect on liver depression and spleen deficiency syndrome.(4)This study showed that Xiaoyao has obvious therapeutic effects on the emotional changes and abnormal energy metabolism in rats with liver depression and spleen deficiency,Fluoxetine has a significant regulating effect on the abnormal emotions and energy metabolism of rats,but their intervention mechanism in energy metabolism are not completely consistent.Rosiglitazone can affect energy metabolism through its sensitizing effect on insulin and PI3K/Akt signaling pathway,however,there is no therapeutic effect in situations such as depressed mood of liver depression and spleen deficiency syndrome.This study could provide a basis for the mechanism of energy metabolism disturbance of liver depression and spleen deficiency syndrome and the treatment mechanism of Xiaoyaosan.