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.

Quercetin and Kaempferol inhibit HMC-1 activation via SOCE/NFATc2 signaling and suppress hippocampal mast cell activation in lipopolysaccharide-induced depressive mice.

OBJECTIVE AND DESIGN: Mast cells (MCs), as the fastest immune responders, play a critical role in the progression of neuroinflammation-related diseases, especially in depression. Quercetin (Que) and kaempferol (Kae), as two major diet-derived flavonoids, inhibit MC activation and exhibit significant antidepressant effect due to their anti-inflammatory capacity. The study aimed to explore the mechanisms of inhibitory effect of Que and Kae on MC activation, and whether Que and Kae suppress hippocampal mast cell activation in LPS-induced depressive mice. SUBJECTS AND TREATMENT: In vitro assays, human mast cells (HMC-1) were pretreated with Que or Kae for 1 h, then stimulated by phorbol 12-myristate 13-acetate (PMA) and 2,5-di-t-butyl-1,4-benzohydroquinone (tBHQ) for 3 h or 12 h. In vivo assays, Que or Kae was administered by oral gavage once daily for 14 days and then lipopolysaccharide (LPS) intraperitoneally injection to induce depressive behaviors. METHODS: The secretion and expression of TNF-α were determined by ELISA and Western blotting. The nuclear factor of activated T cells (NFAT) transcriptional activity was measured in HMC-1 stably expressing NFAT luciferase reporter gene. Nuclear translocation of NFATc2 was detected by nuclear protein extraction and also was fluorescently detected in HMC-1 stably expressing eGFP-NFATc2. We used Ca2+ imaging to evaluate changes of store-operated calcium entry (SOCE) in HMC-1 stably expressing fluorescent Ca2+ indicator jGCamP7s. Molecular docking was used to assess interaction between the Que or Kae and calcium release-activated calcium modulator (ORAI). The  hippocampal mast cell accumulation and activation  were detected by toluidine blue staining and immunohistochemistry with ß-tryptase. RESULTS: In vitro assays of HMC-1 activated by PtBHQ (PMA and tBHQ), Que and Kae significantly decreased expression and secretion of TNF-α. Moreover, NFAT transcriptional activity and nuclear translocation of NFATc2 were remarkably inhibited by Que and Kae. In addition, the Ca2+ influx mediated by SOCE was suppressed by Que, Kae and the YM58483 (ORAI inhibitor), respectively. Importantly, the combination of YM58483 with Que or Kae had no additive effect on the inhibition of SOCE. The molecular docking also showed that Que and Kae both exhibit high binding affinities with ORAI at the same binding site as YM58483. In vivo assays, Que and Kae significantly reversed LPS-induced depression-like behaviors in mice, and inhibited hippocampal mast cell activation  in LPS-induced depressive mice. CONCLUSIONS: Our results indicated that suppression of SOCE/NFATc2 pathway-mediated by ORAI channels may be the mechanism of inhibitory effect of Que and Kae on MC activation, and also suggested Que and Kae may exert the antidepressant effect through suppressing hippocampal mast cell activation.

Combination of paeoniflorin and calycosin-7-glucoside alleviates ischaemic stroke injury via the PI3K/AKT signalling pathway.

CONTEXT: Paeoniflorin (PF) and calycosin-7-glucoside (CG, Paeonia lactiflora Pall. extract) have demonstrated protective effects in ischaemic stroke. OBJECTIVE: To investigate the synergistic effects of PF + CG on ischaemia/reperfusion injury in vivo and in vitro. MATERIALS AND METHODS: Male Sprague-Dawley rats were subjected to the middle cerebral artery occlusion/reperfusion (MCAO/R). After MCAO/R for 24 h, rats were randomly subdivided into 5 groups: sham, model (MCAO/R), study treatment (PF + CG, 40 + 20 mg/kg), LY294002 (20 mg/kg), and study treatment + LY294002. Males were given via intragastric administration; the duration of the in vivo experiment was 8 days. Neurologic deficits, cerebral infarction, brain edoema, and protein levels were assessed in vivo. Hippocampal neurons (HT22) were refreshed with glucose-free DMEM and placed in an anaerobic chamber for 8 h. Subsequently, HT22 cells were reoxygenated in a 37 °C incubator with 5% CO2 for 6 h. SOD, MDA, ROS, LDH and protein levels were measured in vitro. RESULTS: PF + CG significantly reduced neurobehavioral outcomes (21%), cerebral infarct volume (44%), brain edoema (1.6%) compared with the MCAO/R group. Moreover, PF + CG increased p-PI3K/PI3K (4.69%, 7.4%), p-AKT/AKT (6.25%, 60.6%) and Bcl-2/BAX (33%, 49%) expression in vivo and in vitro, and reduced GSK-3ß (10.5%, 9.6%) expression. In vitro, PF + CG suppressed apoptosis in HT22 cells and decreased ROS and MDA levels (20%, 50%, respectively). CONCLUSIONS: PF + CG showed a synergistic protective effect against ischaemic brain injury, potentially being a future treatment for ischaemic stroke.

Pharmacokinetic Analysis of Huangqi Guizhi Wuwu Decoction on Blood and Brain Tissue in Rats with Normal and Cerebral Ischemia-Reperfusion Injury by Microdialysis with HPLC-MS/MS.

OBJECTIVE: The aim of our research was to analyze and compare the pharmacokinetics of paeoniflorin, calycosin, calycosin-7-o-ß-d-6-glucoside, and 6-gingerol in the blood and brain tissue of normal and cerebral ischemia-reperfusion injury rats by HPLC-MS/MS method. METHODS: The blood and brain tissue samples of normal and middle cerebral artery occlusion (MCAO) rats were compared. The blood and brain tissue samples were collected by using microdialysis technique. The concentrations of paeoniflorin, calycosin, calycosin-7-o-ß-d-6-glucoside, and 6-gingerol in blood and brain tissues were determined by the HPLC-MS/MS internal standard method. RESULTS: Compared with the normal group, the model group after the administration of the Huangqi Guizhi Wuwu Decoction showed that Cmax blood, AUC0-t blood, and AUC0-inf blood of paeoniflorin were increased, CLblood, t1/2 brain, and Vbrain of paeoniflorin were decreased; Cmaxblood, AUC0-tblood, AUC0-infblood, and average residence time (MRTbrain) of calycosin-7-o-ß-d-6-glucoside were decreased and the CLblood and Cmax brain of calycosin-7-o-ß-d-6-glucoside were increased; Cmax blood of calycosin was decreased, Vblood and Vbrain of calycosin were increased; Cmax blood, AUC0-t blood, AUC0-inf blood, and MRTbrain of 6-gingerol were decreased, CLblood of 6-gingerol was increased. CONCLUSION: This method is simple, rapid, and sensitive. It is suitable for the pharmacokinetic study of Huangqi Guizhi Wuwu Decoction in the blood and brain tissue of rats. Cerebral ischemia-reperfusion injury increased the content of paeoniflorin, calycosin, calycosin-7-o-ß-d-6-glucoside, and 6-gingerol in the blood, affecting the clearance rate of paeoniflorin in the brain, the detention time of calycosin-7-o-ß-d-6-glucoside and the 6-gingerol in the brain. In normal and cerebral ischemia-reperfusion rats, the content of paeoniflorin and 6-gingerol in the blood was higher than that in brain tissue, while the content of calycosin, calycosin-7-o-ß-d-6-glucoside in the brain tissue was higher than that in blood, suggesting that calycosin and calycosin-7-o-ß-d-6-glucoside have brain targeting properties.

Calycosin-7-O-ß-D-glucoside Attenuates OGD/R-Induced Damage by Preventing Oxidative Stress and Neuronal Apoptosis via the SIRT1/FOXO1/PGC-1α Pathway in HT22 Cells.

Neuronal apoptosis induced by oxidative stress is a major pathological process that occurs after cerebral ischemia-reperfusion. Calycosin-7-O-ß-D-glucoside (CG) is a representative component of isoflavones in Radix Astragali (RA). Previous studies have shown that CG has potential neuroprotective effects. However, whether CG alleviates neuronal apoptosis through antioxidant stress after ischemia-reperfusion remains unknown. To investigate the positive effects of CG on oxidative stress and apoptosis of neurons, we simulated the ischemia-reperfusion process in vitro using an immortalized hippocampal neuron cell line (HT22) and oxygen-glucose deprivation/reperfusion (OGD/R) model. CG significantly improved cell viability and reduced oxidative stress and neuronal apoptosis. In addition, CG treatment upregulated the expression of SIRT1, FOXO1, PGC-1α, and Bcl-2 and downregulated the expression of Bax. In summary, our findings indicate that CG alleviates OGD/R-induced damage via the SIRT1/FOXO1/PGC-1α signaling pathway. Thus, CG maybe a promising therapeutic candidate for brain injury associated with ischemic stroke.

Cell Chromatography-Based Screening of the Active Components in Buyang Huanwu Decoction Promoting Axonal Regeneration.

Buyang Huanwu decoction (BHD), a popular formulation prescribed in traditional Chinese medicine (TCM) for the treatment of ischemic stroke, has been reported to have a potential role in promoting axonal regeneration. The purpose of the study was to screen and identify bioactive compounds from BHD using live PC12 cells coupled with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Using this approach, we successfully identified six bioactive components from BHD. These components have protective effects on oxygen-glucose deprivation/reperfusion (OGD/R) injury to PC12 cells. Furthermore, calycosin-7-d-glucoside (CG) and formononetin-7-O-ß-d-glucoside (FG) could upregulate the protein expression of growth-associated protein 43 (GAP-43) and brain-derived neurotrophic factor (BDNF). This study suggests that living cells combined with HPLC-MS/MS can be used for the screening of active ingredients in TCMs.

Erythrocyte membrane affinity chromatography, solid-phase extraction and UPLC-QTOF-MS/MS to screen active ingredients of Buyang Huanwu decoction.

Buyang Huanwu decoction (BHD) is a well-known traditional Chinese medicine that has long been used to treat ischemic brain damage which is associated with hemorheology. To screen active ingredients in BHD responsible for reducing blood viscosity by reducing red blood cell (RBC) lesions to treat ischemic stroke, a method involving RBC membrane binding and solid-phase extraction (SPE) was developed in this study. The components of BHD interacting with RBC were analyzed by mass spectrometry and four compounds, calycosin, paeoniflorin, 6-hydroxy behenol-3,6-di-O-glucoside and calycosin-7-O-ß-d-glucoside, showed binding affinity to RBCs. An erythrocyte activity assay revealed that the identified ingredients promoted the activities of Na+-K+-ATPase, sialic acid and superoxide dismutase and reduced the content of cholesterol on the RBC membrane, suggesting a mechanism underlying their anti-erythrocyte aggregation activity. Based on these results, the RBC membrane binding assay combined with SPE and mass spectrometry is a novel and effective approach for screening potentially anti-erythrocyte lesion constituents in traditional Chinese medicines.