| Depression is a mental disorder characterized by persistent loss of interest and pleasure.World Health Organization projected that by 2030,in addition to cardiovascular disease,depression will become the main killer of human health in the 21 st century,which is a public health problem that seriously affects the quality of human life and the development of social economy.The pathogenesis of depression is extremely complicated and remains still unclear.Using computed tomography,MRI and other imaging techniques,the hippocampus of patients with depression was found the same overall morphological changes as chronic stress induced rats,which were manifested as hippocampal neuron loss including hippocampal neuron necrosis,hippocampal neuron apoptosis,and hippocampal volume reduction.Therefore,neuronal injury plays a vital role in the pathogenesis of depression,and antidepressants are generally believed to exert antidepressant effects through neuroprotective effects.Comprised by Fructus Gardeniae and Semen sojae praeparatum,Zhi-zi-chi decoction(ZZCD)is chronicled in Shang Han Lun.Increasing TCM clinical studies have shown that ZZCD can significantly improve the depressive symptoms in patients with depression.However,the antidepressant mechanism of ZZCD remains unknown.In this study,PC12 cells were treated with high concentrate of glutamate to simulate the pathological state of glutamate accumulation when chronic stress causes neuronal injury.The method of combining chronic unpredictable mild stress(CUMS)and separate breeding was used to establish an animal model of depression.Consist of the overall metabolic profiles and biomarkers based on metabolomics,a comprehensive drug efficacy evaluation system was established at the level of cell and animal to reveal the antidepressant mechanism of ZZCD based on neuroprotection,and the pharmacodynamic substance basis of ZZCD was confirmed.The details are as follows:1.PC12 cells treated with high concentration of glutamate were used as a cell model of depression with neuronal injury.The antidepressant compatibility mechanism of ZZCD based on neuroprotection was revealed from the perspectives of pharmacodynamics and molecular biology,and the pharmacodynamic substance basis of ZZCD was confirmed.(1)The neuroprotective effects between single herbs(Fructus Gardeniae and Semen sojae praeparatum)and formula of ZZCD were compared by the detection of pharmacodynamic indicators related to neuronal injury and depression.Results showed that ZZCD exerted significant neuroprotection by not only increasing cell survival rate and theactivity of GR and SOD,but also reducing apoptosis rate and the level of LDH and ROS.And the efficacy of ZZCD was stronger than that of single herb.At the same time,the antidepressant target of ZZCD was found by detecting the expression level of depression-related proteins.Results showed that ZZCD significantly increased the expression of CREB and Bcl-2 protein,and reduced the expression of Bax protein.The antidepressant compatibility mechanism of ZZCD based on neuroprotection was revealed from the perspective of pharmacodynamics and molecular biology,which provided a high-throughput screening in vitro model for the study of compatibility mechanism of other antidepressant Chinese medicines.(2)Ultra high performance liquid chromatography-quadrupole/time-of-flight-mass spectrometry(UHPLC-Q/TOF-MS)and reversed-phase high performance liquid chromatography(RP-HPLC)method were used to characterize and quantify the pharmacodynamic substance basis of ZZCD.25 substances were identified and 6 components were quantified in ZZCD,including Genipin-1-β-D-gentiobioside,Geniposide,Daidzin,Glycitin,Genistin,and Daidzein,which confirmed the pharmacodynamic substance basis of ZZCD.2.PC12 cells treated with high concentration of glutamate were used as a cell model of depression with neuronal injury.The cell metabolomics based on gas chromatography-mass spectrometry(GC-MS)and UHPLC-Q/TOF-MS were used to reveal the antidepressant mechanism of ZZCD based on neuroprotection at the level of cell.(1)The metabolic profiles of PC12 cells were investigated based on GC-MS.Univariate analysis and multivariate analysis were preformed to screen differential metabolites related to neuroprotection and 40 differential metabolites were screened.Then enrichment analysis and topological analysis of differential metabolites were conducted on Metaboanalyst to screen differential metabolic pathways related to the antidepressant effects of ZZCD based on neuroprotection.(2)The metabolic profiles of PC12 cells were investigated based on UHPLC-Q/TOF-MS.Univariate analysis and multivariate analysis were preformed to screen differential metabolites related to neuroprotection and 49 differential metabolites were screened.Then enrichment analysis and topological analysis of differential metabolites were conducted on Metaboanalyst to screen differential metabolic pathway related to the antidepressant effect of ZZCD based on neuroprotection.(3)The differential metabolites and metabolic pathways were compared,and resultsshowed that the antidepressant effects of ZZCD based on neuroprotection were related to the regulation of cell energy metabolism,amino acid metabolism,and lipid metabolism.3.CUMS-induced rats were used as an animal model of depression with neuronal injury.The metabolomics based on gas chromatography-mass spectrometry(GC-MS)and UHPLC-Q/TOF-MS were used to reveal the antidepressant mechanism of ZZCD based on neuroprotection at the level of animal.(1)The method of combining CUMS and separate breeding was used to establish an animal model of depression.The behavior tests were conduct to evaluate the antidepressant effects of ZZCD.Results showed that ZZCD exerted significant neuroprotection by not only increasing the sugar preference rate of rats,but also reducing the immobility time in forced swimming test and tail suspension test.(2)The brain metabolic profiles of CUMS-induced rats were investigated based on UHPLC-Q/TOF-MS.26 differential metabolites were screened by data analysis.Then metabolic pathway analysis revealed that antidepressant effects of ZZCD was associated with the regulation of glutathione metabolism,phenylalanine,tyrosine and tryptophan biosynthesis,phenylalanine metabolism,lysine degradation,cysteine and methionine metabolism,tyrosine metabolism,and tryptophan metabolism.4.The metabolomic results between glutamate-induced PC12 cells and CUMS-induced rats were compared to reveal the correlations between the two models.The metabolomic results showed that 10 common differential metabolites and 5 common metabolic pathways existed in glutamate-induced PC12 cells and CUMS-induced rats,including glutathione metabolism,phenylalanine,tyrosine and tryptophan biosynthesis,phenylalanine metabolism,cysteine and methionine metabolism,and tyrosine metabolism.Glutamate-induced PC12 cells have a strong correlation with CUMS-induced rats in revealing the antidepressant effects and mechanism,which can provide technical references for the study of the mechanism of other antidepressants.In conclusion,glutamate-induced PC12 cells and CUMS-induced rats were used as model of depression with neuronal injury in this study.Consist of the overall metabolic profiles and biomarkers based on metabolomics,a comprehensive drug efficacy evaluation system was established from the level of cell and animal to reveal the antidepressant mechanism of ZZCD based on neuroprotection,and the pharmacodynamic substance basis of ZZCD was confirmed.The above research results not only provided a high-throughput screening in vitro model for the study of the compatibility mechanism of other antidepressantChinese medicines and a theoretical basis for the secondary development of ZZCD and antidepressant treatment,but also provided technical references for the study of the mechanism of other antidepressants. |
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