Medication Ideas for Depression from Wenyang Jieyu Prescription / 中国实验方剂学杂志

Depression is a complex emotional and mental disorder. The traditional Chinese medicine （TCM） methods for treating depression mainly include soothing the liver and relieving depression. Our research team proposes that depression is caused by Yang Qi deficiency and obstructed Qi movement， which are closely related to neurological and psychological changes induced by early traumatic experiences. Therefore， we suggest that the treatment should focus on warming Yang， replenishing Qi， and promoting Qi movement and have formulated Wenyang Jieyu prescription based on Erxiantang for warming yang and Xiaoyaosan for relieving depression. The experiment with the mouse model of early trauma induced by maternal separation showed that Wenyang Jieyu prescription significantly improved the mouse activity and environmental exploration， reduced the immobility time in forced swimming and tail suspension tests， alleviated the behaviors such as aversion to darkness and fear of open space， enhanced social interaction and social cognitive abilities， altered decision-making biases， reduced depression-like behaviors， and improved the decision-making patterns. Additionally， the prescription lowered the serum level of cortisol， inhibited the cortisol surge in the dexamethasone/corticotropin-releasing hormone （Dex/CRH） test， up-regulated the expression of mineralocorticoid receptor （MR） and 11β-hydroxysteroid dehydrogenase 2 （11β-HSD2） in the hippocampus， down-regulated the expression of glucocorticoid receptor （GR） and corticotropin-releasing hormone receptor 1 （CRHR1）， inhibited the methylation of GR exon 1 and the expression of DNA methyltransferase 1 （DNMT1）， and restored the negative feedback of the hypothalamic-pituitary-adrenal （HPA） axis. Furthermore， Wenyang Jieyu prescription up-regulated the protein level of brain-derived neurotrophic factor （BDNF）， elevated the levels of postsynaptic density protein 95 （PSD95） and synaptophysin （Syn）， decreased the cell apoptosis index and B-cell lymphoma （Bcl-2）-associated X （Bax）/Bcl-2 ratio， suppressed the expression of Caspase-3， and enhanced the neuroplasticity and anti-apoptotic capacity in the hippocampus. Considering the research results， related articles， and clinical experience， we conclude that depression should be treated with liver-soothing and depression-relieving herbs， which can be supplemented with spleen-invigorating and Qi-regulating herbs to alleviate depressive symptoms. The Yang-warming and kidney-tonifying herbs can be used to eliminate the root cause and prevent relapse. Additionally， the wind-dispersing herbs can be supplemented to regulate the Qi movement throughout the body， thereby enhancing the efficacy of depression-relieving treatment.

Wenyang Jieyu Prescription Regulates Hippocampal Neuron Apoptosis and Improves Synaptic Plasticity in Depressed Mice via BDNF/Akt/mTOR Pathway / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of Wenyang Jieyu prescription in regulating hippocampal neuron apoptosis and improving synaptic plasticity in the mouse model of depression induced by maternal separation combined with restraint stress. MethodThe mice on postnatal day 0 （PD0） were randomly assigned into a control group （n=10） and a modeling group （n=50）. Maternal separation combined with restraint stress was adopted to establish the mouse model of depression， and the modeled mice were randomized into model， Wenyang prescription， Jieyu prescription， Wenyang Jieyu prescription， and fluoxetine groups （n=10） on the weaning day （PD21）. From PD21 to PD111， the mice were fed with the diets mixed with corresponding medicines. The sucrose preference test， open field test， O-maze test， and novel object recognition test were then conducted to evaluate the depression， memory， and learning abilities of mice. Immunohistochemistry （IHC） was employed to measure the atomic absorbance （AA） of postsynaptic density protein 95 （PSD95） in the hippocampus. Terminal-deoxynucleoitidyl transferase-mediated nick-end labeling （TUNEL） was employed to detect the apoptosis of hippocampal neurons. Western blot was employed to determine the protein levels of brain-derived neurotrophic factor （BDNF）， phosphorylated tyrosine kinase receptor B/tyrosine kinase receptor B （p-TrkB/TrkB）， phosphorylated protein kinase B/protein kinase B （p-Akt/Akt）， phosphorylated mammalian target of rapamycin/mammalian target of rapamycin （p-mTOR/mTOR）， B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X （Bax）， cysteinyl aspartate-specific proteinase-3 （Caspase-3）， synaptophysin （Syn）， and PSD95. ResultCompared with the control group， the modeling decreased the sucrose preference rate， time spent in central zone within 5 min， total movement distance， time spent in the open arm， and cognition index （P<0.01）. Furthermore， it decreased the expression of PSD95， increased the neuron apoptosis in the hippocampus （P<0.01）， down-regulated the protein levels of BDNF， p-TrkB/TrkB， p-Akt/Akt， p-mTOR/mTOR， Bcl-2， PSD95， and Syn （P<0.01）， and up-regulated the protein levels of Bax and Caspase-3 （P<0.05） in the hippocampus. Compared with the model group， Wenyang Jieyu prescription and fluoxetine increased the sucrose preference rate， time spent in central zone within 5 min， total movement distance， time spent in the open arm， and cognition index （P<0.05， P<0.01）. Moreover， the drugs increased the expression of PSD95， reduced the neuron apoptosis （P<0.01）， up-regulated the protein levels of BDNF， p-TrkB/TrkB， p-Akt/Akt， p-mTOR/mTOR， Bcl-2， PSD95， and Syn （P<0.01）， and down-regulated the protein levels of Bax and Caspase-3 （P<0.01）. ConclusionWenyang Jieyu prescription outperformed Wenyang prescription and Jieyu prescription in the treatment of the depressive behavior induced by maternal separation combined with restraint stress in mice. It exerted the therapeutic effect by reducing the hippocampal neuron apoptosis and improving the synaptic plasticity via the BDNF/Akt/mTOR pathway.

Wenyang Jieyu Prescription Regulates Hippocampal Neural Plasticity in Depressed Mice via NLRP3/Caspase-1/IL-1β Pathway / 中国实验方剂学杂志

ObjectiveTo explore the effects of Wenyang Jieyu prescription （WJP） on neuroinflammation and synaptic plasticity in the mouse model of depression induced by maternal separation combined with restraint stress. MethodThe mice on postnatal day 0 （PD0） were randomized into a control group and a modeling group. Maternal separation combined with restraint stress was employed to establish the mouse model of depression. After the removal of female mice， the modeled mice were randomized into model， Wenyang prescription （5.85 g·kg-1）， Jieyu prescription （12.03 g·kg-1）， WJP （16.71 g·kg-1）， and fluoxetine （2.6 mg·kg-1） groups on the weaning day （PD21）， with 15 mice in each group. The mice were administrated with corresponding drugs mixed with the diet from PD21 to PD111. The sucrose preference test， open field test， O-maze test， and novel object recognition test were then carried out to evaluate the depression state， memory， and learning ability of the mice. Immunohistochemistry （IHC） was employed to observe the ionized calcium-binding adapter molecule-1 （Iba-1） in hippocampal microglia. High performance liquid chromatography （HPLC） was employed to measure the content of noradrenaline （NE） and epinephrine （E） in the hippocampus. Enzyme-linked immunosorbent assay （ELISA） was employed to determine the content of interleukin （IL）-18 and IL-1β in the hippocampus. Western blot was employed to determine the protein levels of NOD-like receptor protein 3 （NLRP3）， apoptosis-associated speck-like protein containing a CARD （ASC）， cysteine aspartate-specific protease-1 （Caspase-1）， IL-1β， synaptophysin （Syn）， and postsynaptic density 95 （PSD95）. ResultCompared with control group， the model group showed decreased sucrose preference rate， time spent in central zone within 5 min， total movement distance， time spent in the open arm， and cognition index （P<0.05， P<0.01）. The microglia in the model group presented amoeba-like appearance， the Iba1 increased. Moreover， the model group showed decreased content of NE and E （P<0.01）， elevated levels of IL-1β and IL-18 （P<0.01）， down-regulated protein levels of PSD95 and Syn （P<0.05， P<0.01）， and up-regulated protein levels of NLRP3， ASC， Caspase-1， and IL-1β （P<0.05， P<0.01）. Compared with model group， WJP and fluoxetine increased the sucrose preference rate， time spent in central zone within 5 min， total movement distance， time spent in the open arm， and cognition index （P<0.05， P<0.01）. They recovered the microglia and the Iba1 decreased. Moreover， the drugs increased the content of NE and E （P<0.05， P<0.01）， lowered the levels of IL-1β and IL-18 （P<0.01）， up-regulated the protein levels of PSD95 and Syn （P<0.01）， down-regulated the protein levels of NLRP3， ASC， Caspase-1， and IL-1β （P<0.05， P<0.01）. ConclusionWJP can treat the depressive behavior induced by maternal separation combined with restraint stress in mice， with the performance outperforming Wenyang prescription and Jieyu prescription. It may alleviate the neuroinflammation induced by microglia and improve the synaptic plasticity by regulating the NLRP3 pathway and increasing neurotransmitters in the hippocampus.

Regulatory Effect of Wenyang Prescription， Jieyu Prescription， and Wenyang Jieyu Prescription on Pain Sensitivity and Depression-like Behaviors in Mice Induced by Maternal Separation and Chronic Neuropathic Pain / 中国实验方剂学杂志

ObjectiveTo observe the behavioral and pain threshold alterations， as well as the changes in indexes related to depression and pain in the serum and central system in mice stressed by maternal separation and chronic neuropathic pain， and explore the underlying mechanism of Wenyang prescription （WY）， Jieyu prescription （JY）， and Wenyang Jieyu prescription （WYJY） in improving depression and pain sensitivity. MethodThe birth date of mice was recorded as PD0. After birth， the mice were divided into a blank group and an experimental group. The neonatal mice in the experimental group underwent maternal separation in PD5-14 at 8 h·d-1. After ablactation， the mice were divided into a maternal separation group， a WY group （Erxian decoction， 5.84 g·kg-1）， a JY group （Xiaoyaosan， 12.00 g·kg-1）， a WYJY group （16.68 g·kg-1）， and a fluoxetine group （2.60 mg·kg-1）， with 15 mice in each group. Meanwhile， 15 male mice of the same age without maternal separation were assigned to the normal control group. Mice in the blank group and the maternal separation group were fed on a regular chow diet in PD21-PD90， while the remaining groups were fed on the corresponding drugs. In PD91， sciatic nerve ligation was performed to induce a model of maternal separation and chronic neuropathic pain. The open field test was used to observe the depression-like behaviors of mice in each group， and the mechanical and temperature pain thresholds were measured to detect the pain sensitivity of mice in each group. The serum levels of corticosterone （CORT）， substance P， and β-endorphin （β-EP） were determined by enzyme-linked immunosorbent assay （ELISA）， and the expression of the glucocorticoid receptor （GR） in the amygdala and β-EP protein in the hypothalamus was detected by immunohistochemistry. The mRNA expression levels of amygdala GR gene （Nr3c1）， FK506 binding protein 5 gene （FKBP5）， metabolic glutamate receptor 5 gene （GRM5）， and brain-derived neurotrophic factor （BDNF） were detected by real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）. ResultCompared with the blank group， the maternal separation group showed reduced stay time and total distance traveled in the 5-min open field test （P<0.01）， reduced mechanical pain threshold （P<0.01）， increased serum CORT and β-EP （P<0.01）， declining FKBP5 mRNA expression （P<0.01）， and increased hypothalamic β-EP expression （P<0.05）. Compared with the maternal separation group， the groups with drug intervention showed prolonged stay time （P<0.05， P<0.01） and up-regulated pain thresholds to different degrees. The total distance traveled in the 5-min open field test increased in the WY group， the WYJY group， and the fluoxetine group （P<0.05， P<0.01）. The JY group showed decreased serum CORT （P<0.01）， reduced β-EP ， and increased BDNF mRNA （P<0.01）. Nr3c1 and GRM5 mRNA decreased in the WY group （P<0.05， P<0.01）. The WYJY group showed decreased serum CORT （P<0.05）and decreased Nr3c1， GRM5， and BDNF mRNA （P<0.05， P<0.01）. The levels of β-EP expression were elevated to different degrees in the groups with drug intervention， but the differences were not significant. The levels of GR expression in the WY group， the JY group， and the WYJY group increased （P<0.05）. ConclusionWYJY can inhibit central pain sensitization and regulate hypothalamic-pituitary-adrenal gland （HPA） axis function by enhancing the expression of GR in the amygdala and inhibiting neuroplasticity and excitability in the amygdala to relieve depression-like behaviors and improve somatic hyperalgesia.

Changes of Microglia in Hippocampus of Mice Induced by Maternal Separation with Restraint Stress and Regulatory Effect of Wenyang Jieyu Prescription / 中国实验方剂学杂志

Objective:To observe the activation of microglia in hippocampus of depressed and anxious mice induced by maternal separation with acute restraint stress and the expression of interleukin-1<italic>β</italic>（IL-1<italic>β</italic>），interleukin-6（IL-6），tumor necrosis factor-<italic>α</italic>（TNF-<italic>α</italic>）， investigating the mechanism of Wenyang Jieyu prescription in treating anxiety and depression. Method:Eighty four male C57BL offspring were randomly divided into control group， acute restraint stress group and model group on postnatal day 0（PD0）. Maternal separation combined with acute restraint stress was used to prepare anxious and depressed model mice， dividing the model mice into model group， Wenyang， Jieyu， Wenyang Jieyu and fluoxetine group according to random number table method. During the period of PD21-PD90， the control， acute restraint stress and model mice were fed with normal diet， with the other groups fed with corresponding medicine mixed diet. The Wenyang， Jieyu and Wenyang Jieyu groups were given 5.85， 12.03 and 16.71 g·kg^-1·d^-1 respectively. The fluoxetine group was given 2.60 mg·kg^-1·d^-1. Open field， zero maze test and social interaction tests were used to evaluate the anxiety and depression of model mice. The expression of Iba-1 in hippocampal microglia was detected by immunohistochemistry（IHC）. The mRNA expression of IL-1<italic>β</italic>， IL-6， TNF-<italic>α</italic>， Iba-1 and glucocorticoid receptor（GR）were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. Result:Compared with the control group， total movement distance and time spent in central zone in 5 min of the model mice significantly decreased（<italic>P</italic><0.01）， time spent in opened arm and total movement distance decreased significantly（<italic>P</italic><0.05，<italic>P</italic><0.01）， investigation time during testing and training increased significantly（<italic>P</italic><0.01）. The expression of Iba-1 protein and mRNA，IL-1<italic>β</italic>，IL-6，TNF-<italic>α</italic> mRNA significantly increased（<italic>P</italic><0.01）， the expression levels of GR mRNA significantly decreased（<italic>P</italic><0.01）. The result of IHC staining showed that microglia were over activated. Compared with the model group， total movement distance and time spent in central zone in 5 min of mice in the Wenyang Jieyu and fluoxetine group significantly increased（<italic>P</italic><0.05，<italic>P</italic><0.01）.Time spent in opened arm significantly increased（<italic>P</italic><0.01）. Investigation time during testing and training significantly decreased（<italic>P</italic><0.05，<italic>P</italic><0.01）. The expression of Iba-1 protein and mRNA，IL-1<italic>β</italic>，IL-6，TNF-<italic>α</italic> mRNA significantly decreased（<italic>P</italic><0.05，<italic>P</italic><0.01）. The expression of GR mRNA increased significantly（<italic>P</italic><0.05，<italic>P</italic><0.01）. IHC staining showed the microglia recovered. Time spent in opened arm of mice in the Wenyang group and Jieyu group significantly increased （<italic>P</italic><0.01）， time spent investigating during testing decreased significantly （<italic>P</italic><0.05）， the expression levels of Iba-1 protein and mRNA，IL-6 mRNA significantly decreased （<italic>P</italic><0.05，<italic>P</italic><0.01）. The expression of GR mRNA of mice in the Wenyang group significantly increased （<italic>P</italic><0.05）， the expression of TNF-<italic>α </italic>mRNA significantly decreased （<italic>P</italic><0.05）. Total movement distance of mice in the Jieyu group increased significantly （<italic>P</italic><0.01）， time spent investigating during training decreased significantly （<italic>P</italic><0.05），the expression level of IL-1<italic>β </italic>mRNA significantly decreased （<italic>P</italic><0.05）. IHC staining showed that microglia recovered partly in both groups. Conclusion:The comprehensive curative effect and pharmacological action of Wenyang Jieyu prescription were better than Wenyang prescription and Jieyu prescription. Wenyang Jieyu prescription can treat anxiety and depression in maternal separation and acute restraint stress mice， its possible mechanism may be related to the decreased activation of microglia， down-regulation of IL-1<italic>β</italic>，IL-6，TNF-<italic>α</italic> expression and up-regulation of GR expression.