Network Pharmacology And Experimental Mechanism Study Of Xiaoyaosan In The Treatment Of Parkinson's Disease With Depressio

Xiaoyaosan has been a classic prescription for treating liver depression and spleen deficiency in antiquity.In modernity,it is used to treat depression and other diseases accompanied by depression.However,due to the complexity of compound composition,it is challenging to study the specific mechanism of Xiaoyaosan compound.Parkinson’s disease and depression are both neurological diseases,and some of the same regulatory mechanisms can work together with these two diseases,such as oxidative stress and disorders of monoamine neurotransmitters.However,there is no relevant study to explain the effect and mechanism of Xiaoyaosan and Parkinson’s disease with depression.To explain these problems,this study analyzed the network interaction relationship of "F(formulas)-H(herbs)-C(chemical compounds)-T(targets)".We first explored the relationship between Xiaoyaosan and Parkinson’s disease with depression through network pharmacology,and carried out animal and cell experiments for further verification.The first part of the network pharmacology study aimed at the mechanism of Xiaoyaosan on Parkinson’s disease with depression,analyzed the relationship among each herb,compound and target in Xiaoyaosan.We found Bupleurum chinensis DC.and Glycyrrhiza uralensis Fisch.had the most common targets.In the identified seven major compounds,quercetin and kaempferol had the most common targets,and these two compounds were similar in structure,only differ a hydroxyl group.The most significant co-regulatory pathway of quercetin and kaempferol is oxidative stress,so these two compounds were verified at the cellular level in the third part.In addition,the study also found that different Chinese medicines and different compounds,respectively,have different regulatory pathways.Moreover,some traditional Chinese medicines and compounds have similar regulatory effects on the same pathway.Therefore,it can be inferred that the clinical addition and subtraction of TCM according to patients’ syndrome type and drug flavor classification may be to increase the number of effective drugs for patients’ disease pathways,so as to achieve the purpose of precision medicine.The enrichment analysis of Xiaoyaosan and Parkinson’s disease with depression found that 20 representative KEGG pathways are as follows:Pathways in cancer;Pathways of neurodegeneration-multiple diseases;Lipids and atherosclerosis;AGE-RAGE signaling pathway in diabetic complications;MicroRNAs in cancer;Human T cell leukemia virus type 1 infection;Chemical carcinogenesis-receptor activation;cAMP signaling pathway;HIF-1 signaling pathway;Tuberculosis;Non-alcoholic fatty liver disease;Calcium signaling pathway;Platinum resistance;p53 signaling pathway;Neuroactive ligand-receptor interaction;Necrotic apoptosis;Serotonergic synapses;Cytokine-cytokine receptor interaction;Malaria;Dopaminergic synapses.According to the results of the first part,the cAMP signaling pathway of Xiaoyaosan and Parkinson’s disease with depression was selected for verification.Firstly,the mouse model of Parkinson’s disease was induced by injection of 6-OHDA striatum,which is commonly used in Parkinson’s disease,and the open field experiment showed that the motor function of the model mice was impaired.Then,the suspended tail experiment was conducted in Parkinson’s model mice.According to the parameters of normal mice,mice with depression-like behavior in Parkinson’s model mice were selected as mice with Parkinson’s depression for the following research.Behavioral tests were performed on mice and samples were taken three weeks after administration.Behavioral tests showed that Xiaoyaosan and kaempferol had significant therapeutic effects on depression-like behavior in mice with Parkinson’s disease,but had no therapeutic effects on Parkinson-like behavior.It was found that Xiaoyaosan and kaempferol could increase the concentration of cAMP and lead to the change of cAMP signal cascade and its downstream targets p-CREB and BDNF,thus playing an antidepressant role in mice with Parkinson’s disease and depression.The third part of the cell experiment was based on the results of the first part,mainly studied whether the flavonoids quercetin and kaempferol in Xiaoyaosan had the synergistic effect of anti-depression through regulating oxidative stress.In this part of the study,different concentrations of quercetin and kaempferol were used to treat HT-22 cells with glutamateinduced nerve injury,and the optimal concentration of quercetin and kaempferol with neuroprotective effect was screened out for this cell model.Then the HT-22 cells with glutamate-induced nerve injury were applied with quercetin and kaempferol respectively and in combination.The results showed that quercetin and kaempferol,respectively and in combination,could reduce the increase of reactive oxygen species induced by glutamate in HT22 model cells,and the effect of quercetin and kaempferol in combination was better than kaempferol alone.Determination of cellular protein levels showed that quercetin and kaempferol,respectively and in combination,reduced pAKT level and increased pJNK level in HT-22 cells,but had no effect on AKT and JNK expression.Therefore,it can be concluded that quercetin and kaempferol alone or in combination regulate oxidative stress in HT-22 cells by regulating JNK/AKT phosphorylation signaling pathway.This study focusing on Xiaoyaosan,from "F(formulas)-H(herbs)-C(chemical compounds)-T(targets)" network relationship to illustrate its working mechanism,and experiments verification found Xiaoyaosan may perform effectively because similar functional role of a class of compounds(such as flavonoids of quercetin,kaempferol,etc.).Through network pharmacology analysis,we found the mechanism of Xiaoyaosan’s multi-target effect.In animal experiments,we proved that Xiaoyaosan and kaempferol,have therapeutic effects on Parkinson’s disease and depression,and this therapeutic effect is activated through cAMP signaling pathway.Finally,we demonstrated through cell experiments that quercetin and kaempferol can simultaneously regulate the JNK/AKT phosphorylation signaling pathway to achieve the regulation of oxidative stress in HT-22 cells,and the combination of the two is better than kaempferol alone.This indicates that the action mechanism of Xiaoyaosan may be related to the synergistic effect of multiple components in the compound,and the combined use of a class of small molecules with similar effects may make the effect on the same pathway better.