The Mechanism Of Ferroptosis Mediated By PEBP1-GPX4 In Mice With Liver Depression And Spleen Deficiency And The Regulatory Effect Of Xiaoyaosa

BackgroundThe syndrome of stagnation of liver qi and spleen deficiency is a common clinical syndrome type caused by the dysfunction of liver controlling conveyance and dispersion with spleen in transportation.It mainly includes symptoms of liver qi depression such as hypochondriac fullness and pain,emotional anxiety or mental depression,as well as spleen deficiency symptoms such as loss of appetite,abdominal distension,abdominal pain and diarrhea.The syndrome of stagnation of liver qi and spleen deficiency is also one of the common syndrome types of depression.Many clinical and experimental studies have observed the high correlation between depression and the syndrome of stagnation of liver qi and spleen deficiency from the macro and micro perspectives.As a classic formula for the treatment of the syndrome of stagnation of liver qi and spleen deficiency,Xiaoyao Powder has the characteristics of multi-component,multi-target and multi-channel efficacy,which is also used in the treatment of depression and other physical and mental diseases.Ferroptosis is a way of regulating cell death discovered in recent years,which is mainly induced by the accumulation of reactive oxygen species produced by lipid peroxidation products and iron metabolism.It is significantly different from other forms of regulatory cell necrosis such as apoptosis,necrosis and autophagy at the morphological,biological and genetic levels.At present,with the in-depth study of the mechanism of ferroptosis,it is found that the pathogenesis of depression may be associated with GPX4 gene polymorphism,and PEBP1 may inhibit the expression of GPX4,while ERK pathway closely related to mental diseases such as depression may promote the occurrence of depression after being regulated by PEBP1.At the same time,ERK pathway is also considered to be closely related to the occurrence of ferroptosis.In addition,as a common clinical syndrome type of depression,the syndrome of stagnation of liver qi and spleen deficiency involves the changes of multiple systems and functions of the body,such as neuroendocrine,immunity,digestion and metabolism,the mechanism of which is also related to the pathogenesis of depression.Therefore,it can be speculated that the pathogenesis of depression and the syndrome of stagnation of liver qi and spleen deficiency may be related to the ferroptosis pathway related to PEBP1-GPX4.Studying the ferroptosis related mechanism regulated by PEBP1-GPX4 can provide new ideas for exploring the pathological mechanism of depression and the syndrome of stagnation of liver qi and spleen deficiency,which can also further expand the scientific connotation of the basis of the syndrome of stagnation of liver qi and spleen deficiency.ObjectiveThis study aims to explore whether the relevant mechanism of the syndrome of stagnation of liver qi and spleen deficiency and depression includes the ferroptosis pathway related to PEBP1-GPX4,and observe the possible pharmacodynamic mechanism of Xiaoyao Powder in the treatment of depression and syndrome of stagnation of liver qi and spleen deficiency through ferroptosis pathway,so as to provide a new reference for the basic research of the syndrome of stagnation of liver qi and spleen deficiency from the perspective of ferroptosis,and also provide new ideas for the application and pharmacological research of Xiaoyao Powder.Methods(1)The possible mechanism of the antidepressant effect of the active ingredient of Xiaoyao Powder through iron death was observed by using the research method of network pharmacology.Preliminarily discusses the action targets of the active components of Xiaoyao Powder,the target genes related to ferroptosis and depression,as well as the key targets and pathways of Xiaoyao Powder in the treatment of depression through ferroptosis pathway,and verifies the combination degree between the active components of Xiaoyao Powder and key targets by virtual molecular docking.(2)Chronic unpredictable mild stress(CUMS)was used to replicate the depression with syndrome of stagnation of liver qi and spleen deficiency mice model.The model was evaluated by observing the general state performance of mice,body weight,food intake,serum D-xylose,sucrose preference test,forced swimming test,novelty-suppressed feeding test and open field test.At the same time,the regulating effect of Xiaoyao Powder on the above behavioral changes of model mice was observed.(3)To detect the occurrence of PEBP1-GPX4 related ferroptosis pathway in the hippocampus of depression with syndrome of stagnation of liver qi and spleen deficiency model mice.The expression of key protein GPX4 in ferroptosis pathway was detected by qRT-PCR,Wes protein quantification and immunohistochemistry,and the mRNA expression of ferroptosis pathway related genes FTH1,ACSL4 and COX2 was detected by qRT-PCR.At the same time,the expression levels of PEBP1 and ERK1/2 were also detected.In addition,the regulatory effect of Xiaoyao Powder on the above indexes related to ferroptosis pathway was observed.(4)To detect the functional changes of hippocampal glial cells in the depression with syndrome of stagnation of liver qi and spleen deficiency model mice.The expressions of astrocyte marker GFAP and microglia marker IB A1 were detected by qRT-PCR,Wes protein quantification and immunohistochemistry,and the regulatory effects of Xiaoyao Powder on GFAP and IB A1 were also observed.Results(1)Network pharmacology results:Through the preliminary network pharmacological analysis of the potential target of Xiaoyao Powder in the treatment of depression through ferroptosis,a total of 143 active components were obtained,and then 941 single prediction targets of the active components of Xiaoyao Powder were obtained through target prediction.Through the analysis of ferroptosis targets related to depression,151 target genes were obtained,and then enriched and analyzed with Xiaoyao Powder target genes,40 key target genes of Xiaoyao Powder for the treatment of depression through ferroptosis pathway were obtained.After PPI network analysis of these target genes,10 core targets and 2 sub-networks were obtained.By further analyzing the biological functions and pathways involved in Xiaoyao Powder’s treatment of depression through ferroptosis,283 go enrichment functions and 121 KEGG pathways related to core targets were obtained.Through the virtual molecular docking study,it was found that isoquercetin,the active component of Xiaoyao Powder,had the strongest binding ability to GPX4,PEBP1 and MAPK3,acacetin had the strongest binding ability to COX2,and luteolin had the strongest binding ability to ACSL4.(2)Results of Experiment 1:General states:CUMS modeling led to the gradual mental depression of model mice,with symptoms such as curling up,dry and messy hair,fatigue and loose stools.The resistance was significantly weakened during the modeling process,and some mice even did not struggle.After the intervention of Xiaoyao Powder,the hair became glossy and smooth,the activity and reactivity increased,and the stool texture was moderate.CUMS model made the gap between the weight and food intake of normal mice and other groups of mice gradually increase.Compared with the normal group,the body weight of the model mice at the beginning of the first weekend was statistically different(P<0.05,P<0.01),and the food intake of the model mice at the beginning of the second weekend was significantly lower than that of the normal group(P<0.01);After drug intervention,the body weight and food intake of mice in Xiaoyao Powder treatment group and fluoxetine treatment group were significantly higher than those in CUMS model group(P<0.05,P<0.01).After 6 weeks of modeling,the level of serum D-xylose in CUMS model group was significantly lower than that in normal group(P<0.01),and the level of serum D-xylose in Xiaoyao Powder and fluoxetine treatment group was increased(P<0.05,P<0.01).Before the start of modeling,the sugar water preference rate of mice in each group was basically the same.After modeling,the sucrose preference ratio of mice in CUMS model group was significantly lower than that in normal group(P<0.01),and the sucrose preference ratio of mice in Xiaoyao Powder treatment group was significantly higher than that in CUMS model group(P<0.05,P<0.01);The results of forced swimming test showed that the immobility time of CUMS model group was significantly higher than that of normal group(P<0.01),and the immobility time of Xiaoyao Powder treatment group and fluoxetine treatment group was lower than that of CUMS model mice(P<0.05);The results of the novelty-suppressed feeding test were similar,the feeding latency of CUMS model group was significantly higher than that of the normal group(P<0.01),and Xiaoyao Powder and fluoxetine treatment group were lower than that of the CUMS model group(P<0.05,P<0.01).The results of open field test showed that the total distance of movement and the number of central area entry in CUMS model group were significantly higher than those in normal group(P<0.05,P<0.01);Compared with the CUMS model mice,the total distance of movement,the number of entry into the central area and the residence time in the central area in the Xiaoyao Powder treatment group were significantly increased(P<0.05,P<0.01),and the total distance of movement in the fluoxetine treatment group was significantly increased(P<0.01).(3)Results of Experiment 2:Compared with the normal group,the expression level of GPX4 in the hippocampus of CUMS model group was significantly decreased(P<0.01);and there were significant differences in the mRNA expressions of FTH1,ACSL4 and COX2 in the hippocampus of CUMS model group compared with normal mice(P<0.05,P<0.01).Compared with the CUMS model group,Xiaoyao Powder and fluoxetine can intervene the down-regulation of GPX4 and fthl and the up-regulation of ACSL4 and COX2 caused by stress(P<0.05,P<0.01).Immunohistochemical results also further showed that the MOD and positive area of GPX4 in CUMS model group decreased significantly in hippocampal CA1,CA3 and DG areas(P<0.01),and the treatment of Xiaoyao Powder and fluoxetine increased GPX4 to a certain extent(P<0.01).The expression of PEBP1 in the hippocampus of CUMS model mice was significantly up-regulated(P<0.05,P<0.01),while the expression of t-ERK1/2 was significantly decreased(P<0.01).The expression levels of PEBP1 and t-ERK1/2 in Xiaoyao Powder treatment group and fluoxetine treatment group were significantly increased(P<0.05,P<0.01).Meanwhile,CUMS model also decreased the level of p-ERK1/2 in mice hippocampus(P<0.01),while Xiaoyao Powder and fluoxetine significantly increased the expression of p-ERK1/2(P<0.01).In addition,the ratio of p-ERK1/2 and t-ERK1/2 in the hippocampus of CUMS model group was significantly higher than that of the normal group(P<0.01),while the ratio of the two treatment groups was significantly lower than that of CUMS model group(P<0.01).Compared with the normal group,the expression GFAP of CUMS model mice decreased significantly(P<0.05),while the level of GFAP in hippocampus of mice treated with Xiaoyao Powder and fluoxetine increased significantly(P<0.05,P<0.01).At the same time,the data also showed that the level of IBA1 in hippocampus of CUMS model group was significantly higher than that of normal group(P<0.05,P<0.01);Compared with the CUMS model group,the intervention of Xiaoyao Power or fluoxetine could significantly reverse the up regulation of IB A1 expression caused by CUMS(P<0.05,P<0.01).The expression and distribution of GFAP and IBA1 in mouse hippocampus were further observed by immunohistochemistry.In CUMS model group,the MOD of GFAP in hippocampal CA1 and DG was significantly decreased(P<0.05,P<0.01),while Xiaoyao Powder and fluoxetine could significantly increase the MOD of GFAP in hippocampal CA1,CA3 and DG areas(P<0.05,P<0.01);At the same time,the positive area of GFAP decreased significantly in hippocampal CA1,CA3 and DG areas(P<0.01),and Xiaoyao Powder could significantly increase the positive area of GFAP in hippocampal CA1,CA3 and DG areas(P<0.01),while fluoxetine could also significantly increase the positive area of GFAP in hippocampal CA3 and DG areas(P<0.01).The MOD of IBA1 in the hippocampus of CUMS model group was significantly up-regulated in CA1 and CA3 areas(P<0.05,P<0.01).Xiaoyao Powder and fluoxetine could also significantly inhibit the up-regulation of IBA1 in CA1,CA3 and DG areas(P<0.05,P<0.01);At the same time,the positive area of IB A1 was significantly up-regulated in hippocampal CA1,CA3 and DG areas(P<0.01),and Xiaoyao Powder could significantly down-regulate the positive area of IBA1 in hippocampal CA1,CA3 and DG areas(P<0.05,P<0.01),while fluoxetine could also significantly down regulate the positive staining area of IB A1 in hippocampal CA1 and CA3 areas(P<0.01).ConclusionFirstly,by means of network pharmacology,this study successfully demonstrated the key targets and pathways for the active components of Xiaoyao Powder to exert antidepressant efficacy through ferroptosis pathway,and verified the binding degree between the active components of Xiaoyao Powder and key targets through virtual molecular docking.Then,the depression with syndrome of stagnation of liver qi and spleen deficiency mice model was copied by the modeling method of CUMS,then the model was evaluated by behavioral evaluation.Meanwhile,it was found that PEBP1-GPX4 related ferroptosis pathway changed in the hippocampus of CUMS model mice,and the disorder of hippocampal glial cell function in model mice may be related to the occurrence of ferroptosis.Furthermore,the pharmacodynamic mechanism of Xiaoyao Power exerting therapeutic effect of dispersing stagnated liver qi for relieving qi stagnation may be achieved by regulating the expression of PEBP1,affecting the expression and phosphorylation level of ERK1/2,intervening in the ferroptosis process involved in GPX4,and improving the function of central glial cells.