Modified Danzhi XiaoyaoSan inhibits neuroinflammation via regulating TRIM31/NLRP3 inflammasome in the treatment of CUMS depression.

The NLRP3 inflammasome is critically involved in the development of depression. The E3 ubiquitin ligase TRIM31 negatively regulates this process by promoting the degradation of NLRP3 through the ubiquitin-proteasome pathway. Modified Danzhi Xiaoyaosan (MDZXYS) has shown good therapeutic effect in both preclinical and clinical depression treatments, yet the underlying mechanisms of its antidepressant effects are not fully understood. In the present study, we aimed to explore the antidepressant mechanisms of MDZXYS, focusing on NLRP3 activation and ubiquitin-mediated degradation. We employed rats with depression induced by chronic unpredictable mild stress (CUMS) and conducted various behavioral tests, including the sucrose preference, forced swimming, and open field tests. Neuronal damage in CUMS-treated rats was assessed using Nissl staining. We measured proinflammatory cytokine levels using ELISA kits and analyzed NLRP3/TRIM31 protein expression via Western blotting and immunofluorescence staining. Our results disclosed that MDZXYS reversed CUMS-induced depression-like behaviors in rats, reduced proinflammatory cytokine levels (IL-1ß), and ameliorated neuronal damage in the prefrontal cortex. Additionally, CUMS activated the NLRP3 inflammasome in the prefrontal cortex and upregulated the protein expression of TRIM31. After MDZXYS administration, the expression of NLRP3 inflammasome-associated proteins was reduced, while the expression level of TRIM31 was further increased. Through co-localized immunofluorescence staining, we observed a significant elevation in the co-localization expression of NLRP3 and TRIM31 in the prefrontal cortex of the MDZXYS group. These findings suggest that inhibiting NLRP3 inflammasome-mediated neuroinflammation by modulating the TRIM31signaling pathway may underlie the antidepressant effects of MDZXYS, and further support targeting NLRP3 as a novel approach for the prevention and treatment of depression.

Effect of Danzhi Xiaoyaosan on Phosphorylation of Tau Protein in Rats with Alzheimer's Disease Based on PP2A and GSK-3β / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Danzhi Xiaoyaosan on the phosphorylation of tau protein and different sites of glycogen synthase kinase-3β （GSK-3β） and phosphoseryl/suanyl phosphate protein phosphatase 2A （PP2A） in the hippocampus of rats with Alzheimer's disease （AD） and its mechanism. MethodThe rat model of AD was established by injecting okadaic acid into the bilateral hippocampus of 90 male Wistar rats in SPF grades. The rats with successful modeling were selected and randomly divided into model group， aricept group （0.5 mg·kg-1）， and Danzhi Xiaoyaosan high， medium， and low groups （17.55， 8.77， and 4.38 g·kg-1）， and then gavaged for 42 d， once a day. Morris water maze was used to detect the learning and memory ability of rats， Nissl's staining was used to observe the morphological structure of neurons in the hippocampus， and Real-time polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression levels of tau protein， GSK-3β， and PP2A. Western blot was used to determine the protein expression levels of tau protein， GSK-3β， and PP2A. ResultAs compared with the control group， the learning and memory abilities of the rats in the model group were significantly decreased （P<0.01）， and the hippocampal CA3 region cells had abnormal structure， disorderly arrangement， and decreased number. The expression levels of GSK-3β mRNA， GSK-3β， p-GSK-3β-Tyr216， p-PP2A， and p-tau were increased in the model group as compared with the control group （P<0.01）， and those of p-GSK-3β-Ser9 and PP2A decreased significantly （P<0.01）. As compared with the model group， the learning and memory ability of the Aricept group and the Danzhi Xiaoyaosan groups were improved （P<0.05， P<0.01）， and the cell morphology and the number of hippocampal CA3 regions were better. The mRNA expression levels of PP2A and tau in the Aricept group were significantly up-regulated （P<0.05）， the mRNA expression level of GSK-3β was significantly down-regulated （P<0.01）， and the protein expression levels of GSK-3β， p-GSK-3β-Tyr216， and p-PP2A were down-regulated （P<0.05， P<0.01）， and the protein expression level of PP2A was significantly up-regulated （P<0.01）. As compared with the model group， the mRNA expression level of PP2A in the high-dose Danzhi Xiaoyaosan group was significantly up-regulated （P<0.01）， and that of GSK-3β was significantly down-regulated （P<0.01）， whereas the protein expression levels of p-PP2A， p-GSK-3β-Tyr216， and p-tau were down-regulated （P<0.05， P<0.01）， and the protein expression level of PP2A was significantly up-regulated （P<0.01）. As compared with the model group， the mRNA expression level of GSK-3β was significantly down-regulated in the medium-dose Danzhi Xiaoyaosan group （P<0.01）， the protein expression levels of GSK-3β， p-GSK-3β-Tyr216， and p-tau were down-regulated （P<0.05， P<0.01）， and the protein expression level of PP2A was significantly up-regulated （P<0.01）. As compared with the model group， the mRNA expression level of PP2A was significantly up-regulated in the low-dose Danzhi Xiaoyaosan group （P<0.01）， and that of GSK-3β was significantly down-regulated （P<0.01）， whereas the protein expression levels of GSK-3β and p-GSK-3β-Tyr216 were down-regulated （P<0.05， P<0.01）， and those of p-GSK-3β-Ser9 and PP2A were significantly up-regulated （P<0.01）. ConclusionDanzhi Xiaoyaosan can improve the learning and memory ability of rats with AD， and its mechanism may be related to the regulation of the activities of GSK-3β and PP2A protein-related sites and the phosphorylation of tau protein.

Danzhi Xiaoyaosan-containing Serum Reverses Energy Metabolic Reprogramming of MDA-MB-231 Breast Cancer Cells / 中国实验方剂学杂志

ObjectiveTo observe the effect of Danzhi Xiaoyaosan-containing serum on MDA-MB-231 breast cancer cells， and to find out whether the action mechanism is related to its intervention in energy metabolism. MethodThirty six-week-old male SD rats were randomly divided into the blank group， Danzhi Xiaoyaosan （8.99 g·kg-1） group， and Xihuangwan （0.55 g·kg-1） group. The serum was isolated after drug intervention for seven days. The cell viability was detected by methyl thiazolyl-tetrazolium （MTT） assay， the cell cycle by flow cytometry， and the apoptosis by Annenxin V/propidium iodide （PI） double staining. Following the determination of intracellular glucose content using the glucose testing kit， the expression of glucose transporter 1 （GLUT1） was measured by immunofluorescence staining. Seahorse XFe cell energy metabolism analyzer was used to detect the extracellular acidification rate （ECAR） and oxygen consumption rate （OCR）. The expression levels of hexokinase 2 （HK2）， pyruvate kinase M2 （PKM2）， and lactate dehydrogenase A （LDHA） were assayed by Western blot. ResultCompared with the blank group， Danzhi Xiaoyaosan-containing serum inhibited the proliferation of MDA-MB-231 cells， with the best effect observed after intervention with 15% Danzhi Xiaoyaosan-containing serum for 48 h （P<0.01）， blocked the MDA-MB-231 cells in G0/G1 phase（P<0.01）， and down-regulated the GLUT1 expression， basal glycolysis， glycolysis capacity， glycolytic reserve， basal respiration， adenosine triphosphate （ATP） production， spare respiratory capacity（P<0.01）， as well as the protein expression of HK2， PKM2， and LDHA in MDA-MB-231 cells（P<0.05，P<0.01）. ConclusionDanzhi Xiaoyaosan-containing serum inhibits MDA-MB-231 cell proliferation， promotes apoptosis， and induces cell cycle arrest， which may be related to its reversal of energy metabolic reprogramming in MDA-MB-231 cells.

Effect and Mechanism of Modified Danzhi Xiaoyaosan in Treating Post-stroke Depression / 中国实验方剂学杂志

Objective: To observe the clinical efficiency of the liver soothing method(modified Danzhi Xiaoyaosan) in the treatment of post-stroke depression(PSD), and explore its mechanism.Method: A total of 148 patients with PSD were randomly divided into study group and control group, with 74 cases in each group. Control group received Escitalopram Oxalate, 10 mg·d-1. Study group received Shugan granule 3 g, twice a day, in addition to the therapy of control group. One course of treatment of 4 weeks. Hamilton depression scale (HAMD) score, neurotransmitter power, 5-hydroxytryptamine(5-HT), noradrenalin(NE) levels and inflammatory factors were compared between the two groups.Result: The clinical efficacy of study group was significantly better than that of control group(Z=2.97, Pχ2=4.40, PPPPPPPPα(TNF-α), interleukin-1 beta(IL-1β) and interleukin-6(IL-6) levels in both groups decreased in the process of treatment (Pα, IL-1β and IL-6 levels in study group were decreased more significantly than those in control group (PConclusion: Modified Danzhi Xiaoyaosan combined with traditional antidepressants has a more significant curative effect in the treatment of PSD. The mechanism may be related to the inhibition of the expressions of inflammatory factors. Further studies shall be conducted.