Research On The Treatment Of Depression And The Protection Of Vascular Endothelium With Yiqi Jianpi Method

Research on the Treatment of Depression and the Protection of Vascular Endothelium with Yiqi Jianpi MethodObjective Depression is a common psychiatric disorders in clinical and has been listed as one of the independent risk factors for cardiovascular disease.Antidepressants commonly used in clinical practice can cause side effects such as arrhythmia and hypotension.Therefore,it is urgent to develop a safe and effective drug to treat depression and protect cardiovascular.Traditional Chinese medicine(TCM)emphasizes the concept of the unity of body and spirit,and has accumulated rich clinical experience in the treatment of psycho-cardiology disease,which is of great value to the development of more effective and safe drugs.Guided by indepth scientific thinking,this research is carried out from three aspects:theoretical research,clinical research and experimental research.Through the sorting of ancient books,clinical investigation,meta-analysis,data mining and Delphi expert correspondence,it is initially clear that Jianpi Tongmai Formula(JPTMF),the core prescription for treating depression based on "treating from Xu" had the scientificity and rationality.The targets and potential mechanisms of JPTMF in the treatment of depression were predicted by network pharmacology,and the effects of JPTMF in treating depression and protecting vascular endothelium were verified through animal experiments.Methods1.Theoretical research:In the library of Zhejiang Chinese Medical University and the"Chinese Medical Classic",the ancient books of TCM from the pre-Qin period to the Ming and Qing dynasties were retrieved by using the search terms "stagnation" "stagnation syndrome" "Qingzhi" "Qiqing",and "sorrow".Through the research method of TCM literature,the origin,evolution and connotation of "stagnation" theory are sorted out and analyzed,and the theoretical deduction is carried out from the perspective of logical relationship.Therefore,the treatment prescriptions for "sorrow" the core emotional symptom of depression in ancient books,were sorted out,and the basic treatment methods and medication characteristics were studied through frequency statistics and association rule analysis.2.Clinical investigation:2.1 This study collected clinical symptom data of depression patients through clinical investigation,conducted unsupervised clustering analysis with machine learning algorithm,and discussed the correlation between the clustering results and TCM syndrome types.2.2 Evidence-based medicine(EBM)was used to clarify the efficacy and safety of Chinese herbal formulas for treating depression.Randomized controlled trials(RCT)studies were retrieved and collected from Chinese and English databases,and the studies that met the criteria were screened and data extracted according to the inclusion and exclusion criteria and quality evaluation.Revman5.3 software and Statal3.0 software were used to combined the effect size with.The outcomes included the clinical efficacy of tonic prescriptions in the treatment of depression,Hamilton Depression Scale(HAM-D)score,TCM syndrome score,Treatment Emergent Symptom Scale(TESS)score and adverse events.2.3 TCM data mining was performed on the tonic formulas included in the meta-analysis through frequency analysis,association rules and cluster analysis.Based on the data mining results,a Delphi expert questionnaire was constructed to collect expert opinions.Combined with the results of expert opinions and weight analysis,on the basis of the previous research of this research group,it was clear that JPTMF was one of the core prescriptions for "treating from Xu" on depression.3.Experimental study:3.1 Network pharmacology prediction:The databases combined with literatures were used to obtain the potential targets of JPTMF in the treatment of depression.Through protein-protein interaction(PPI)network analysis,Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis,predicted the core targets and pharmacological mechanisms of JPTMF in the treatment of depression.3.2 The animal model of depression-like behavior was induced by chronic unpredictable mild stress(CUMS).(1)The effect of JPTMF and synergistic component quercetin in improving depression-like behavior in CUMS model mice was verified by behavioral tests,neurotransmitters(5-HT,NE)and inflammatory factors in the hippocampus of mice were determined by ELISA(TNFa,IL-1β,IL6)levels,and serum cytokines(TNFα,IL-1β,IL6)levels,Western blot was used to verify glial cell marker proteins(GFAP,Iba1)in the hippocampus,the expression of inflammatory factors(TNFα,IL-1β),and synaptic plasticity protein(PSD95,Arc).(2)The levels of NO,plasma ET-1,aortic inflammatory factors(TNFα,IL-1β,IL6)and oxidative stress(SOD,MDA)in the thoracic aorta and serum of mice were measured by ELISA,HE staining was used to observe the mice The pathological changes of thoracic aortic endothelial cells,the ultrastructural changes of mouse thoracic aortic endothelial cells were observed by transmission electron microscopy,the expressions of eNOS,VCAM-1 and ICAM-1 in mouse thoracic aorta were detected by immunohistochemistry.Results1.Theoretical research:Through the sorting and analysis of ancient books,it is found that the connotation of "stagnation" has experienced an evolution process from image to concrete.Neither the broad meaning stagnation or the narrow meaning stagnation cannot completely correspond with depression in symptoms and logic.Depression should belong to the category of TCM emotional disease,and its core emotional symptom is most similar to"sorrow".By analyzing the prescriptions for treating "sorrow" recorded in ancient books,it is found that these prescriptions can be divided into four aspects:treatment from "Xu",treatment from "Shi",treatment from "the mixed Xu and Shi",and "Others".Among them,the treatment from "Xu" is the most categories,mainly focuses on nourishing the heart,lung and spleen,and nourishing qi and blood.The treatment from "Shi" mainly includes:wind,cold,heat,phlegm,and blood stasis.The pathogenesis of "the mixed Xu and Shi" is complicated,mainly including Rongwei disorder,wind and cold two senses with three energizer qi deficiency,the lung meridian syndrome(wind and cold),unsteady heart qi and deficiency of five internal organs(wind and heat).Through the statistical analysis of the ancient Chinese prescriptions for treating "sorrow",it is found that the usage of qiinvigorating herbs and yang-boosting herbs are the highest.The results of association rule analysis found that the qi-invigorating and damp-removing herbs had the highest correlation with other types of TCM herbs.2.Clinical investigation:2.1 Research on TCM syndromes of depression in the real world:The cluster analysis results based on machine learning found that the symptoms of patients with depression can be divided into five clusters.The main emotional symptoms of cluster 1 are irritability,depression,anxiety,and the main physical symptoms are feeling of foreign body in the throat,stomach bloating,acid reflux,bitter mouth,constipation,which are in line with the syndrome of liver stagnation transforming into fire.The main emotional symptoms of cluster 2 are anxiety,depression,nervousness and cranky thinking,but compared with cluster 1,the severity of emotional symptoms is less severe and the frequency of attacks is lower.The main physical symptoms of cluster 2 are dry mouth,inactivity,fatigue,chest tightness,sighing.The cluster 2 are in line with the syndrome of liver stagnation and spleen deficiency.The main emotional symptoms of cluster 3 are reduced the interest,depression,timidity and hypochondriasis,and the main physical symptoms are loose stools,poor appetite,inactivity,dizziness,fatigue,shortness of breath,which are in line with the spleen deficiency syndrome.The main emotional symptoms of cluster 4 are depression,reduced the interest and interest,low self-esteem,and the main physical symptoms are memory loss,lack of energy,backache,weak legs,hair loss,dry mouth,dizziness,which are in line with the syndrome of the kidney deficiency and brain dystrophy.The above studies shows that Xu syndrome exists objectively in clinical depression patients.2.2 EBM study on the treatment of depression with tonic prescriptions:The results of the meta-analysis including 37 studies showed that tonic TCM prescription can significantly improve the efficiency of depression(OR=2.03,95%CI[1.73,2.38]),reduce depression score(SMD=0.97,95%CI[-1.32,-0.62])and TCM syndrome score(SMD=-1.76,95%CI[-2.58,-0.94]),compared with control group.Meanwhile,the Treatment Emergent Symptom Scale(TESS)score was lower(SMD=-1.63,95%CI[-2.66,-0.60])and fewer adverse events occurred(OR=0.28,95%CI[0.19,0.40])in tonic TCM prescription treatment groups.2.3 JPTMF is one of the core prescriptions for the treatment of depression from tonifying deficiency:through data mining of the effective tonifying prescriptions in the meta-analysis,it is found that the high-frequency tonifying Chinese medicines mainly include Radix Astragali,Poria,Radix Glycyrrhizae,Fructus Zizyphi Jujubae,Radix Angelicaesinensis,、Polygala tenuifolia、Rhizoma Atractylodis macrocephalae,and Radix Codonopsispilosulae.The analysis of the characteristics of TCM herbs showed that the herbs with warm nature,sweet taste and entering the spleen meridian were used most frequently,and the top 20 high-frequency TCM herbs had the most qi-invigorating TCM herbs.The results of the Delphi expert’s questionnaire found that the frequency of the average score and full score was the highest for tonifying spleen qi,which indicated that experts had more approval for tonifying spleen qi in the treatment of depression,which was more consistent with the formulation idea of JPTMF.And the coefficient of variation of the expert questionnaire and Kendall’s The coordination coefficients are all low,indicating that the degree of coordination of expert opinions is high.The weight analysis results show that experts believe that Radix Astragali、Radix Codonopsispilosulae、Poria are the core tonic TCM herbs for the treatment of depression,which is basically the same as the herb composition of JPTMF.The above results indicate that JPTMF is one of the core prescriptions for the treatment of depression.3.Experimental study:3.1 Network pharmacology predicted that the hub targets of JPTMF in the treatment of depression included TNF,AKT1,IL6,TP53,IL1β,VEGFA,JUN,CASP3,MAPK3 and PTGS2.GO analysis mainly pointed to chemical synaptic transmission(postsynaptic)and G protein-coupled amine receptor activity.The KEGG pathways mainly involved neuroactive ligand-receptor interaction,serotonergic synapse,TNF signaling pathway,and PI3K-Akt signaling pathway.3.2 JPTMF improved the depression-like behavior of mice induced by CUMS.(1)After exposure to CUMS for 8 weeks,we evaluated depression-like behaviors.The SPT of CUMS mice was significantly lower than that of control,and treatment with JPTMF(MD and HD)significantly reversed this phenomenon(p<0.05).There was a significantly increase in the immobility time in TST and FST in the CUMS group as compared to the control group.Treatment with JPTMF(MD and HD)significantly shortened the immobility time.(2)We also assessed the levels of neurotransmitters in the hippocampus.Compared with control group,the levels of both 5hydroxytryptamine(5-HT)and norepinephrine(NE)in the hippocampus in the CUMS group were decreased.By contrast,5-HT and NE levels increased significantly after JPTMF(MD and HD)treatment.(3)The inflammatory cytokine levels both in serum and hippocampus were significantly increased in CUMS group compared to controls.After administration of JPTMF(HD),their levels all decreased(p<0.05).These results were in agreement with results from WB experiments about the protein expression of TNFa and IL1β.Moreover,as specifically activated biomarkers for astrocytes and microglia,GFAP and Ibal in the CUMS hippocampus were significantly increased,while JPTMF administration(HD)reversed this phenomenon(p<0.05).(4)The expressions of PSD95,Arc and BDNF in CUMS group were significantly decreased compared with controls,whereas treatment with JPTMF(HD)led to a significant increase(p<0.05).3.3 The vascular endothelium of CUMS mice was protected by JPTMF.(1)Compared with the control group,the levels of NOx in serum and aortic tissue of mice after 8 weeks of CUMS modeling decreased,and the level of plasma ET-1 increased(p<0.05).After JPTMF(HD)treatment,the levels of NOx in serum and aorta tissue of mice increased,and the level of ET-1 in plasma decreased(p<0.05).At the same time,compared with the control group,the expression level of eNOS in thoracic aorta tissue of mice in the CUMS group decreased(p<0.05),the expression level of eNOS increased after JPTMF(HD)treatment.(2)HE staining results of aortic tissue showed that small amounts of endothelial cells were shed in the CUMS group,and the damage was not obvious,the endothelial cells of control group and JPTMF treatment group were complete,all layers were clearly visible,and vascular endothelial cells were completely covered.(3)The results of TEM showed that the morphology of vascular endothelial cells and smooth muscle cells in the control group was normal,and the structure of endothelial elastic layer was complete.The vascular endothelial cells in CUMS modeling showed swelling of cells.The endothelial cells of the mice in the JPTMF(HD)group were normal.(4)Compared with the control group,the levels of TNFα,IL1β and IL6 in the thoracic aorta tissue of the mice in the CUMS group increased,and the levels of inflammatory factors decreased after the treatment with JPTMF(HD)(p<0.05).(5)Compared with the control group,the expression of vascular endothelial cell adhesion molecules(VCAM-1,ICAM-1)increased(p<0.05)in CUMS group,and the expressions decreased after treating with JPTMF(HD).(6)Compared with the control group,the MDA content in the thoracic aorta tissue of the mice in the CUMS group was significantly increased(p<0.05).And the detection results of SOD activity in thoracic aorta tissue of CUMS group was significantly decreased(p<0.05)compared with the control group.The JPTMF(HD)treatment reversed these phenomena.Conclusion1.Depression belongs to the category of TCM emotional diseases in TCM,and it is scientific and reasonable to study the TCM treatment of depression from the perspective of TCM emotional "sorrow".The prescriptions for treating "sorrow" in ancient books and documents can be mainly divided into four aspects:treatment from "Xu",treatment from"Shi",treatment from "the mixed Xu and Shi",and "Others".Among them,the prescription of "Treatment from Xu" is the highest,and the tonic method is one of the main treatments for emotional "sorrow".The analysis of the characteristics of the prescriptions found that the TCM herbs for invigorating qi is the main category,and it is also one of the most commonly used basic compatibility for treating sorrow.The theoretical research provides the theoretical basis of TCM for the treatment of depression with tonifying deficiency.2.According to clinical research,spleen qi deficiency syndrome exists objectively in in depression patients in the real world.EBM analysis showed that tonic prescriptions can improve the effective rate of depression treatment,reduce HAM-D score and TCM syndrome score,and has good safety.Among the effective tonic prescriptions proven by EBM,Chinese herbs with warm in nature,sweet in taste,and enter the spleen meridian were most frequently used.The results of the Delphi expert questionnaire showed that experts recognized the invigorating spleen qi method for the treatment of depression.Radix Astragali and Radix Codonopsispilosulae were regarded as valuable herbs for the treatment of depression.This clinical research provides a clinical basis for the treatment of depression with tonic method,and found that JPTMF is one of the core prescriptions of tonic for depression.3.Network pharmacology study predicted that the treatment of depression with JPTMF may be associated with improved neurotransmitter levels and inflammatory responses.Experimental research confirmed that JPTMF can improve the depression-like behaviors of mice induced by CUMS,and the mechanisms are related to increasing the level of neurotransmitters in the hippocampus of mice,inhibiting glial cell activation,decreasing levels of inflammatory cytokines and improving synaptic plasticity.Meanwhile,JPTMF can regulate the vasomotor function of thoracic aorta in CUMS model mice,inhibit the activation of vascular endothelial cells,reduce the level of inflammatory factors in aortic tissue and oxidative stress response,so as to protect the vascular endothelium.This experimental research provides scientific verification for the core prescription of tonifying deficiency,JPTMF,in the treatment of depression,and preliminarily discusses the protective effect on vascular endothelium.