| 1 BackgroundOxidative stress was proposed as a biological and medical concept of redox in 1985.It refers to the highly active molecules such as reactive oxygen species(ROS)and active nitrogen(ROS)in the body when the body is subjected to harmful stimulation,reactive nitrogen species(RNS)generates too much free radicals,which exceeds the body's ability to scavenge oxides,resulting in a dynamic imbalance between the oxidation system and the antioxidant system.This imbalance can cause oxidative damage to intracellular lipids,proteins and nucleic acids Eventually cause tissue damage.To maintain the body's redox balance,when there are too many free radicals,the body will spontaneously generate a defense reaction to remove the oxidation products.Endogenous antioxidant signaling pathways in the body:Kelch's ECH-related protein 1(Keap1)-nuclear factor erythrocyte 2 related factor 2(Nrf2)-antioxidant response element(ARE)pathway,which can regulate the transcription of many antioxidant genes,these Antioxidant genes can maintain the homeostasis of cells,while detoxification genes can process and eliminate carcinogens and toxins before they cause damage.They play a very important role in the body's defense against various foreign damage,so it is considered to be The most important endogenous antioxidant signaling pathway in the body.Under normal physiological conditions,Keap1 binds to the Neh2 domain of Nrf2 through its DGR domain,thereby restricting the Keap1-Nrf2 heterodimer to the actin skeleton in the cytoplasm,and stress stimulation causes the conformation of the ?R domain to change,thereby Nrf2 is uncoupled from Keapl,activated Nrf2 is transferred from the cytoplasm to the nucleus,and is combined with the Maf protein to form a heterodimer and then combined with ARE.thereby promoting the expression of cell protective target genes,including regulation of phase ? metabolic enzymes,Transcriptional activity of antioxidant enzymes or drug transporters,thereby exerting anti-oxidative damage.Studies have shown that depression is usually accompanied by inflammation and oxidative stress.The level of oxidative stress in patients with depression increases,and the anxiety state of depression can easily lead to various degrees of neuronal damage.However,the body's resistance Oxidative defense ability is reduced,causing oxidative damage to proteins,lipids and nucleic acids in the body.Under this pathological condition,oxidative stress will eventually lead to neurodegeneration through various biological pathways such as apoptosis,cell excitotoxicity,and axon damage.At the same time,oxidative stress is closely related to the occurrence of liver diseases.Studies have shown that activation of the Nrf2 signaling pathway by enhancing the pharmacological pathway or genetic pathway can protect the liver in different oxidative stress models.The Nrf2-ARE signaling pathway is used to prevent and treat various livers.Play an important role in the disease process,such as viral hepatitis,alcoholic and non-alcoholic hepatitis,liver fibrosis,liver cirrhosis,liver cancer,cholestatic liver injury and liver injury caused by drugs or chemicals.At the same time,the liver is the main organ attacked by ROS.When ROS are excessive,the homeostasis will be disturbed,resulting in oxidative stress.Excessive ROS and oxidative stress will induce cell death through necrosis or apoptosis mechanism,and seriously damage lipids and proteins.And DNA,which leads to damage to cells and tissues.This plays a key role in liver diseases and other chronic and degenerative diseases[],suggesting that oxidative stress is closely related to the occurrence of liver diseases."Liver storing blood,and controlling conveyance and dispersion" is the premise and foundation of the normal physiological function of the body,the normal discharge of qi movement,promote blood circulation in the body,blood to support the whole body.In syndrome of stagnation of liver qi and spleen deficiency,the liver's conveyance and dispersion are inhibited,the qi movement is not smooth,and it stays in the body or viscera,which affects the whole body's qi and blood disorders and affects other visceral diseases.Studies have shown that CUMS-induced stress depression can impair the function of rats and cause liver parenchyma damage.Therefore,we can use the 6-week CUMS modeling method to build a mouse model of syndrome of stagnation of liver qi and spleen deficiency,using the Nrf2/ARE signal pathway as an entry point,observe the liver oxidative stress level and damage degree of syndrome of stagnation of liver qi and spleen deficiency,and explore whether Xiaoyaosan can eliminate the free radicals by activating the anti-oxidative stress of the Nrf2/ARE signaling pathway to repair liver damage caused by the syndrome of stagnation of liver qi and spleen deficiency,thereby further improving the syndrome.2 MethodsThis research is mainly divided into three parts:Part I:use 6-week CUMS to establish a combined mouse model of syndrome of stagnation of liver qi and spleen deficiency,and evaluate the characteristics of "stagnation of liver qi"syndrome by means of mouse mental state,open field behavior,forced swimming,sugar water preference,etc.The body weight,food intake and changes in stool morphology were observed to evaluate the characteristics of "spleen deficiency" syndrome.Xiaoyaosan is a representative compound for the treatment of syndrome of stagnation of liver qi and spleen deficiency.The model is evaluated by Xiaoyaosan's prescription.At the same time,fluoxetine,an antidepressant widely used in clinic,is used as a positive control drug to compare its efficacy.Part ?:On the basis of successfully replicating the mouse model of depression,syndrome of stagnation of liver qi and spleen deficiency,the mouse liver tissue HE was stained and observed small Pathological structure of mouse liver tissue,and detection of ALT,AST content in mouse serum,SOD activity and MDA content in mouse liver tissue,compare the differences between mice in each group,and observe whether the model mice have abnormal liver function and liver parenchymal damage.Changes in the level of oxidative stress in the liver.Part ?:Taking the body's important endogenous antioxidant pathway Nrf2/ARE signaling pathway as the observation object,combined with real-time fluorescent quantitative PCR,immunoblotting and immunohistochemistry to detect the liver and liver levels in mice The expression of pathway-related genes and proteins,combined with the liver function and the degree of parenchymal injury in mice with depression,syndrome of stagnation of liver qi and spleen deficiency,were observed the changes of Nrf2/ARE signaling pathway and the regulatory effect of Xiaoyaosan.In order to understand the biological basis of liver stagnation and spleen deficiency syndrome in a more comprehensive manner,and to further explore the biological mechanism of Xiaoyaosan on syndrome of stagnation of liver qi and spleen deficiency,and to provide experimental basis for the scientific connotation between stagnation of liver qi and spleen deficiency and liver injury.3 Results(1)Evaluation of the 6-week CUMS preparation of a mouse model of depression,stagnation of liver qi and spleen deficiency:At the end of the 6th week of modeling,the food intake of the model group was significantly lower than that of the contorl group(P<0.01),and in Xiaoyaosan group,the food intake of mice was significantly higher than that of the model group(P<0.01).The mean food intake of mice in the fluoxetine group was slightly higher than that of the model group,but there was no significant difference(P>0.05);The weight of mice in the model group was significantly lower than that in the control group(P<0.01),and the weight of mice in the Xiaoyaosan group and fluoxetine group were significantly higher than the model group(P<0.01);Open field experiments showed that the stay time in the central area of the model group was higher than that of the contorl group(P<0.05),the number of central entry and the total movement distance in the open field box of the model group were significantly lower than the control group(P<0.01),The distance of the total movement of the mice in the Xiaoyaosan group and the fluoxetine group in the open field box was significantly higher than the model group(P<0.01);The sugar water preference experiment suggested that the sugar water preference rate of the mice in each group changed with the modeling time,and the sugar water consumption rate of the model group mice was significantly lower than the control group(P<0.01),the sugar consumption rate of mice in Xiaoyaosan group and fluoxetine group was significantly higher than that in model group(P<0.01);The forced swimming test results showed that the forced swimming time of the model group mice was significantly higher than that of the control group(P<0.01);The new environment inhibition feeding experiment found that the eating time of the model group mice was significantly higher than control group(P<0.01).(2)Changes in liver oxidative stress level and injury in mice with depression,stagnation of liver qi and spleen deficiency:Observation of the pathological structure of the mouse liver showed that the liver lobule structure of the conrtol group of mice was clear,the hepatocytes were arranged radially around the central vein,and the size of the morphology fofliver cells is normal.no degeneration and necrosis are seen,and the liver sinus is visible;A small amount of inflammatory cells in the junction area is infiltrated,and the small bile duct is as usual.In the model group,hepatic lobular structure was disordered,hepatocytes were enlarged,watery degeneration,hepatic sinusoidal compression and atrophy;The diversion of the confluent area,and mild hyperplasia of small bile ducts.The indicators reflecting the liver function of the mice showed that the ALT and AST contents in the serum of the model group were higher than those in the control group,and the difference was significant(P<0.01).The content of the ALT and AST in the serum of the mice in the Xiaoyaosan group and fluoxetine group was higher than that in the control group,which was significantly different from the model group and had statistical significance(P<0.01).Detection of oxidative stress in the liver of mice revealed that compared with the control group,the SOD activity of the liver tissue of the model group,Xiaoyaosan group,and fluoxetine group decreased,and the MDA content increased,and the difference is significant(P<0.01);Compared with the model group,the intervention of Xiaoyaosan and fluoxetine increased the SOD activity of the mouse liver tissue and reduced the content of MDA(P<0.01 or P<0.05).(3)Expression of genes related to Nrf2/ARE signaling pathway in the liver of mice with depression,stagnation of liver qi and spleen deficiency:real-time fluorescence quantitative PCR was used to detect,compared with the control group,The expressions of the genes of Nrf2 and HO-1 in the liver tissue of the model group mice were decreased(P<0.01),and Keap1 gene expression in liver tissue increased to some extent(P<0.05).Compared with the model group,the gene expressions of Nrf2 and HO-1 in the liver tissues of mice in Xiaoyaosan group and fluoxetine group were all increased(P<0.01),and the gene expression of Keap1 was decreased,the difference was statistically significant(P<0.05)),(4)Expression of Nrf2/ARE signal-related proteins in the liver of mice with depression,stagnation of liver qi and spleen deficiency:The results of immunohistochemical methods showed that the changes in protein levels were basically consistent with the results measured by immunoblotting,compared with the control group.The expression ofNrf2 and HO-1 protein in the liver of the model group was significantly reduced,with statistical difference(P<0.01),while the expression of Keapl in the liver was significantly increased(P<0.01);After intervention by Xiaoyaosan,compared with the model group,liver Nrf2 and HO-1 levels increased in the Xiaoyaosan group(P<0.05),and keap1 protein expression decreased(P<0.05).The results showed that the expression of Nrf2,Keap1,and HO-1 in the liver tissue of mice with depression,stagnation of liver qi and spleen deficiency were changed regardless of gene or protein levels.4 Conclusion(1)At 6 weeks,CUMS can successfully prepare a combined mouse model of depression,liver depression and spleen deficiency syndrome.(2)Modeling can cause the imbalance of oxidative stress level in the liver of mice,and abnormal liver function and liver tissue damage.(3)Modeling can cause the down-regulation of Nrf2,HO-1 protein and mRNA expression in mouse liver tissue and the up-regulation of Keapl protein and mRNA,which inhibits the transduction of this signaling pathway.(4)Xiaoyaosan can play an anti-oxidative stress role by activating the Nrf2/ARE signaling pathway,reduce liver oxidative stress and improve liver damage. |
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