| BackgroundDepression is caused by many factors,such as biology,psychology and society.It is mainly manifested by depression,slow thinking and decreased function of will activity.Depression often has the characteristics of high recurrence rate,high disability rate and death rate,which brings adverse effects and heavy burden to the family and the whole society.According to the latest WHO report,there are about 322 million of the global depressive patients,and the prevalence rate of depression is 4.4%.The incidence rate of depression in China is about 4.2%.The incidence rate of depression is the highest in mental disorders,which seriously affects the physical and mental health of patients.With the development of research on depression,the study of cell signaling pathway has been widely studied in the pathogenesis of depression in recent years,and its role in the treatment of antidepressants has also received more and more attention.JNK signaling pathway,which is closely related to the pathogenesis of depression,is activated by extracellular stimulation.The activation of JNK signaling pathway can cause atrophy,inflammatory response and apoptosis.A large number of studies have shown that in recent years,the use of JNK inhibitors or gene knockout and other means have proved that the symptom regulation of depression is closely related to JNK.At the same time,some antidepressants are involved in the regulation of JNK activity,which has initially established the relationship between depression and JNK signaling pathway.Depression belongs to the category of depression syndrome in traditional Chinese medicine.Liver depression and spleen deficiency syndrome is one of the common syndrome types.Therefore,based on ask1-mkk4/7-jnk signal transduction pathway,it may be used as the research pathway of the pathogenesis of depression of liver depression and spleen deficiency syndrome ObjectiveIn this study,six week chronic unpredictable mild stimulation animal model of liver depression and spleen deficiency was selected.The main effect factors closely related to depression and stress on behavioral and askl-mkk4/7-jnk signaling pathway,and caspase-9 cell apoptosis were used as the main observation indexes to observe the expression changes of key effect molecules on ask1-mkk4/7-jnk signaling pathway in the model of liver depression and spleen deficiency Based on ask1-mkk4/7-jnk signal pathway,the effectiveness of Xiaoyaosan in antidepressant effect.MethodSixty SPF male C57BL/6J mice were used in the experiment.They were 10-12 weeks old and 20 ± 3g in weight.The animals were fed in SPF animal room for 7 days in a single cage.Then 60 mice were randomly divided into four groups:normal group,model group,Xiaoyao Powder group and fluoxetine group.The conditions of feeding environment should be(21±2)? at room temperature,30%-40%relative humidity.The drinking water for feeding mice should be deionized water,and the light time and dark time should be 12h every day and kept relatively quiet.In this experiment,6 weeks of chronic unpredictable mild stimulation was used to establish the model of depression of liver depression and spleen deficiency.The animal model was evaluated by macroscopical characterization,food intake,body weight and a series of behavioral studies(including sugar water preference experiment,open field experiment,etc.).Mice were anesthetized by intraperitoneal injection.After anesthesia,the hippocampus was isolated on ice.Based on the ask1-mkk4/7-jnk signal pathway,Western blot and real-time fluorescence quantitative PCR,the protein content and mRNA content of key effectors and the gene expression of caspase-9 were detected.ResultBased on the ask 1-mkk4/7-jnk signal pathway,the mRNA levels of ASK1,MKK4,MKK7 and JNK in mouse hippocampus were detected by real-time fluorescence quantitative PCR.Compared with the normal group,the expression of ASK1 group increased significantly(P<0.01);the expression of Xiaoyaosan group and fluoxetine group was significantly lower than that of the model group(P<0.05)Compared with the normal group,the expression of MKK4 in the model group increased,and there was a statistical difference(P<0.01);the expression of Xiaoyaosan group was significantly lower than that in the model group,and there was a statistical difference(P<0.01),while the expression of fluoxetine group decreased,but there was no statistical difference(P>0.05).Compared with the normal group,the expression of MKK7 in the model group increased and had statistical difference(P<0.05);compared with the model group,the expression of Xiaoyaosan group and fluoxetine group decreased and had statistical difference(P<0.01).Compared with the normal group,the expression of JNK in the model group increased and had statistical difference(P<0.05);the expression of Xiaoyaosan group and fluoxetine group decreased and had statistical difference(P<0.05).The gene expression of caspase-9 in mouse hippocampus was detected by real-time fluorescence quantitative PCR.Compared with the normal group,the expression of caspase-9 gene in the model group increased significantly and had statistical difference(P<0.05);compared with the model group,the expression of caspase-9 gene in the Xiaoyaosan group and fluoxetine group decreased significantly and had statistical difference(P<0.05).Based on askl-mkk4/7-jnk signal pathway,Western blot was used to detect the protein content of ASK1,MKK4,MKK7,JNK and p-JNK in mouse hippocampus Compared with the normal group,the expression level of ASK1 in the hippocampus of the model group was significantly higher(P<0.01);compared with the model group,the content of ASK1 in the hippocampus of the Xiaoyao Powder group and fluoxetine group was lower(P<0.05).Compared with the normal group,the content of MKK4 in the hippocampus of the model group increased significantly(P<0.01);compared with the model group,the content of MKK4 in the hippocampus of the Xiaoyaosan group and fluoxetine group decreased significantly(P<0.01).Compared with the normal group,the content of MKK7 in the hippocampus of the model group was significantly higher,with significant statistical difference(P<0.01);compared with the model group,the content of MKK7 in the hippocampus of the Xiaoyaosan group was decreased,but there was no statistical difference(P>0.05),while the content of MKK7 in the hippocampus of the fluoxetine group was significantly lower,with significant statistical difference(P<0.05)Conclusion(1)Through the observation of macroscopical characterization and behavioral results of experimental mice,it is suggested that 6 weeks of chronic unpredictable mild stimulation can successfully and effectively prepare the model of liver depression and spleen deficiency syndrome in mice,and Xiaoyaosan can improve the behavior change of model mice and effectively reduce their depressive behavior.(2)Based on the ask1-mkk4/7-jnk signaling pathway,the expression of ASK1,MKK4,MKK7,JNK mRNA in the whole hippocampus homogenate of the model mice with liver depression and spleen deficiency syndrome was significantly higher than that of the other three groups,and the protein expression of ASK1,MKK4,MKK7,JNK,p-JNK in the hippocmpus of the model mice was also significantly higher than that of the other three groups,which suggested that the biological mechanism of the model might be related to ask1-mkk4/7-jnk Activation of signaling pathways is correlated(3)Based on the ask1-mkk4/7-jnk signal pathway,Xiaoyaosan can regulate the key effector molecules in the signal pathway from the gene and protein level,which may be one of the biological basis of Xiaoyaosan in the prevention and treatment of liver depression and spleen deficiency syndrome.Ask1-mkk4/7-jnk is the signal pathway of its action dependence(4)Xiaoyaosan has certain protective effect on nerve cells of model mice.The action ways are as follows:1.Xiaoyaosan can effectively inhibit the activity of JNK signal pathway in the hippocampus of model mice,significantly reduce the phosphorylation level of JNK,and thus inhibit the apoptosis of nerve cells.2?Xiaoyaosan can effectively inhibit caspase dependent apoptosis,significantly reduce the expression of caspase-9 gene in the hippocampus of model mice,and then play a protective role on neurons. |
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