The Protective Effect And Mechanism Of Compound Chaigui Fang On CORT-induced Poorly Differentiated PC12 Cells Injury

Rationales:Depression is a common affective disorder,the incidence of the disease to individuals,families and society has brought huge economic burden and loss,and the currently antidepressants exist some problems such as low efficacy and adverse reactions,it's urgent need to develop new antidepressant drugs.Xiaoyaosan,from the "Tai Ping Hui Min He Ji Ju Fang",has been shown an antidepressant effects.Our group previously researched the antidepressant components and chemical composition of Xiaoyaosan.According to change the prescription and extraction process,obtaining the Compound Chaigui Fang,which is consisted of 95%alcohol extraction part and dregs with water extract and alcohol precipitate part from Radix bupleuri,Angelica,Atractylodes,Radix Paeoniae Alba,Fried licorice and Mint.It's confirmed Compound Chaigui Fang exerts a good antidepressant effect,and is superior to Xiaoyaosan.However,the antidepresant mechanism of Compound Chaigui Fang is still unclear.In order to elucidate the antidepressant mechanism of Compound Chaigui Fang,the study choose the in vitro depressed model,namely CORT-induced PC 12 injury,which model could simulate the hyperactivities of hypothalamic-pituitary-adrenal(HPA)in depression and has advantages of easy acquistion,convenience and easy intervention.However,it's not clear that the differentiated type of PC 12 cell by CORT-induced injury in depressed research,and not clarify the metabolism correlation between CORT-induced PC 12 cells and depressed patients.In this paper,the differentiated type of PC 12 cells is identified for stimulating the depression after CORT treatment,and the conducting the metabolism analysis,which research provided theoretical support in the study of depression for application of this model;the protective effect of Compound Chaigui Fang on PC 12 cells after CORT-treated is investigated,which study supplement and perfect the antidepressant efficacy of drug;the protective mechanism is clarified,which would be helpful to lay the foundation for the development of new drugs.Objective:To evaluated the suitable differentiated type of PC12 cells after CORT stimulating that was used for the depression research,and analyzed the disordered metabolism.To investigated the protective effect and discussed the protective mechanism of Compound Chaigui Fang on CORT-induced PC 12 injury.Methods:MTT assay is used for determining the sensitivity of the distinct differentiated PC 12 cells to CORT,and the 1H-NMR metabolomics of cells and medium coupled with network analysis are applied for investigating the differential metabolites and pathways between before and after CORT stimuling.Cell viability,lactate dehydrogenase release rate,aptoptosis,mitochondrial membrane potential and the content of calcium are measured in the normal control group,the CORT-stimulated model group and the Compound Chaigui Fang-treated group.Collect cells in the different treatment group to conduct the transcriptomic analysis.Ultra high performance liquid chromatography with fluorescence detector(UPLC-ED)is used for determining the monoamine neurotransmitters and its metabolites of cells and medium.Results:1.High concentration of CORT could induce the three differentiated PC 12 cells,and the higher the degree of differentiation,the higher the sensitivity to CORT-stimulated.The intracelluar and extracelluar metabolism are disordered in PC 12 cells after CORT-treated,and 11/18?15/22 and 15/22 differentisl metabolites are identified in undifferentiated,poorly differentiated and well differentiated PC 12 cells/medium after CORT-treated.11 common metabolism pathways are disordered,but there are 5 different disordered pathways in three distinct differentiated PC 12 cells after CORT treatment.According to network analysis 'in 5 different pathways,the study predicate the 18 proteins,which are related to depression and all appear in CORT-induced poorly differentiated PC 12 cells,so the model of CORT-induced poorly differentiated PC 12 cells is considered as the more suitable in vitro depressed model.2.A significant decreased cell viability coupled with the significant increased lactate dehydrogenase release rate,apoptosis rate,mitochondrial membrane potential and intracellular calcium content are observed in poorly differentiated PC 12 cells after CORT treatment,but Compound Chaigui Fang could regulate the above indexes levels to the normal control group.3.118 and 277 genes are increased and decreased in CORT-stimulated model group cells compared to normal control group,and 398 and 30 genes are increased and decreased in Compound Chaigui Fang-treated group compared to model group.According to GO and KO analysis,it's observed that Compound Chaigui Fang may exert the protective effect through regulating the neurotransmitters metabolism,cell growth and death,signal transduction,cell communication and stress response pathways.Additionally,the levels of DOPA,DA,DOPAC,HVA and 5-HT are significantly decreased,and the levels of Tyr and Trp are significantly increased in model groups.Compound Chaigui Fang could significantly regulate the intracellular levesl of DOPA,DA,DOPAC,5-HT,Tyr and Trp,but there are no regulated effect on intracellular HVA level and extracellular DOPA,DOPAC,HVA,Tyr and Trp levels.Conclusion:It's similar between depression metabolism and CORT-induced poorly differentiated PC 12 cells,which model may be more suitable for the study of depression.Compound Chaigui Fang paly a significant protective effect on CORT-induced poorly differentiated PC12 cells injury,and the protective effect mechanism of drug may be associated with cell growth and death,signal transduction,cell communication,stress response and monoamine neurotransmitters metabolism.