Application of glyyrrhizin chalcone A in preparation of medicine for treating depression

Abstract

The application relates to the technical field of medicines, in particular to application of glycyrrhiza chalcone A in preparation of medicines for treating depression. The application experiment shows that the glycyrrhiza chalcone A can significantly improve the CUMS-induced depression-like behavior of mice, reduce the hippocampal neuron damage, inhibit the neuroinflammatory reaction, and restore the 5-HT level, and the anti-depression mechanism may be closely related to multiple mechanisms such as down-regulation of MAPK / JNK path activity, inhibition of inflammatory factor expression and neurotransmitter regulation.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of glycyrrhizin A in the preparation of drugs for treating depression. Background Technology

[0002] Depression is a persistent and severe mental illness, with main symptoms including sleep disturbances and anhedonia. Current research has proposed multiple hypotheses regarding the pathogenesis of depression, including factors such as the monoamine neurotransmitter system, the hypothalamic-pituitary-adrenal (HPA) axis, neuroplasticity, and neurotrophic factors. However, the efficacy of these antidepressants is limited; one-third of patients exhibit treatment-resistant depression (TRD) and elevated levels of pro-inflammatory cytokines (PICs). The response rate to existing medications in patients with treatment-resistant depression is less than 50%, and these medications are often accompanied by slow onset of action and various side effects. These issues suggest that traditional neurotransmitter hypotheses may be insufficient to fully explain the complex pathogenesis of depression, necessitating the exploration of new therapeutic targets and mechanisms of action.

[0003] Licorice is a commonly used ingredient in traditional Chinese medicine for treating depression. It is a core ingredient in antidepressant compound formulas such as Xiaoyao San and Ganmai Dazao Tang. However, its specific active ingredients are unknown. Whether glycyrrhizin A, as an active ingredient of glycyrrhizin flavonoids, has an antidepressant effect is still unknown. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides the application of glycyrrhizin A in the preparation of drugs for treating depression.

[0005] Application of glycyrrhizin A in the preparation of drugs for treating depression.

[0006] This invention has found that glycyrrhizin chalcone A can significantly improve CUMS-induced depressive-like behavior in mice, reduce hippocampal neuronal damage, inhibit neuroinflammatory responses, and restore 5-HT levels. Therefore, the application of glycyrrhizin chalcone A in the preparation of drugs for treating depression is proposed.

[0007] Preferably, the drug has glycyrrhizin A as its sole active ingredient.

[0008] Preferably, the drug is used to improve hippocampal structural damage caused by depression.

[0009] Preferably, the drug is used to relieve neuroinflammation caused by depression.

[0010] Preferably, the drug is an oral or injectable form.

[0011] Preferably, the oral dosage form includes granules, tablets, powders, soft capsules, capsules, soft capsules, drop pills, and solutions. Preferably, the drug comprises a pharmaceutically acceptable carrier.

[0012] Preferably, the pharmaceutically acceptable carrier includes a diluent.

[0013] Preferably, the diluent is composed of BE-β-CD physiological saline and dimethyl sulfoxide in a volume ratio of 9 to 10:1.

[0014] Preferably, the purity of the glycyrrhizin A is ≥98%.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides the first systematic evaluation of the antidepressant effect of glycyrrhizin chalcone A in a CUMS mouse model. Combining network pharmacology and experimental validation methods, its potential mechanism of action was clarified. Results showed that glycyrrhizin chalcone A significantly improved CUMS-induced depressive-like behavior in mice, reduced hippocampal neuronal damage, inhibited neuroinflammatory responses, and restored 5-HT levels. Its antidepressant mechanism may be closely related to multiple mechanisms, including downregulation of MAPK / JNK pathway activity, inhibition of inflammatory cytokine expression, and neurotransmitter regulation. Attached Figure Description

[0016] Figure 1 This diagram illustrates the effect of glycyrrhizin chalcone A on depressive-like behavior in mice. A represents a schematic diagram of the experimental procedure; B represents the sucrose preference rate in the SPT (Self-Test); C represents the resting time in the TST (Self-Test); and D represents the resting time in the FST (French-Test). All data are expressed as SD ± mean. n =6.

[0017] Figure 2 The study showed that glycyrrhizin chalcone A reduced CUMS-induced neuronal damage and neuroinflammation. Nissl staining was performed on hippocampal sections, with black arrows indicating necrotic cells. The scale line length in the section image is 50 micrometers, and the overall magnification is 500 times.

[0018] Figure 3 The study showed that glycyrrhizin chalcone A reduced CUMS-induced neuroinflammation, where A represents the effect of glycyrrhizin chalcone A on 5-HT levels in the hippocampus of CUMS-treated mice. n =6), B represents the effect of glycyrrhizin A on the IL-6 content in the hippocampus of CUMS-treated mice ( n =6), C represents the effect of glycyrrhizin A on IL-1β content in the hippocampus of CUMS-treated mice ( n =6), D represents the effect of glycyrrhizin A on the IL-18 content in the hippocampus of CUMS-treated mice ( ). n =6), all values ​​are expressed as SD ± average.

[0019] Figure 4Venn diagram showing glycyrrhizin chalcone A and its target for depression.

[0020] Figure 5 A visual PPI network diagram for glycyrrhizin-A-depression targets. The area of ​​each node in the PPI network diagram represents its degree. The larger the area, the darker the color, and the more important the node.

[0021] Figure 6 GO-BP, CC, and MF analyses were performed.

[0022] Figure 7 For KEGG analysis. The color of the column indicates its count.

[0023] Figure 8 This shows the protein levels of PJNK / JNK (A, B, P < 0.0001) and P38 / PP38 (C, D, P < 0.0001) in the hippocampus of mice with the glycyrrhizin A P38MAPK / JNK signaling pathway, n = 3 mice per group.

[0024] Note: * in the image p <0.05,** p <0.01, *** p <0.001, # p<0.05, ## p<0.01, ### p<0.001. Detailed Implementation

[0025] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0026] Sucrose (cane sugar, GC205014), RIPA lysis buffer (RIPA lysate, G2002), phosphatase inhibitors (G2007), protease inhibitors (PMSF, G2008), Nissl staining kit (100 ml, G1086), electrode buffer (Tris-Glycine SDS-PAGE SWE High Resolution Fast Running Buffer, G2152), transfer buffer (Ice-free rapid transfer buffer, G2154), protein-free rapid blocking solution (G2052), Tris-buffered saline (TBS, G0001-2L), and dimethyl sulfoxide (DMSO, biochemical grade, GC203002) were all purchased from Solarbio Science & Technology Co. Ltd. (Wuhan, China). 4% paraformaldehyde (M247512), sodium sulfobutylether-β-cyclodextrin (M207869), and Tween 20 (M214475) were all purchased from MREDA, Ltd. (Beijing, China). Sodium chloride (500g, S805275) was purchased from Macklin, Ltd. (Shanghai, China). IL-18 kit (Mouse; E-HSEL-M0006), IL-1β kit (Mouse; E-EL-M0037), IL-6 kit (Mouse; E-EL-M0044), and 5-HT kit (E-EL-0033) were all purchased from Elabscience Biotechnology Co., Ltd. (Wuhan, China). The BCA Protein Assay Kit (PC0020) was purchased from Solarbio, Ltd. (Beijing, China). JNK (17572-1-AP, 1:2000), Phospho-JNK (80024-1-RR, 1:2000), P38 (66234-1-Ig, 1:4000), Phospho-P38 (28796-1-AP, 1:2000), and GAPDH (60004-1-Ig, 1:100000) were purchased from Proteintech.

[0027] Licorice chalcone A (licochalcone-A, purity ≥98%) was provided by Gansu Fanzhi Pharmaceutical Co., Ltd. The positive control drug fluoxetine (batch number 52423005A) was purchased from Suzhou Zhonghua Pharmaceutical Industry Co., Ltd.

[0028] Thirty 8-week-old male C57BL / 6J mice (weighing 20-25 g) were used in the experiment and purchased from the Experimental Animal Center of Lanzhou University (License No.: SYXK(Gan)2023-0004). The mice were acclimatized for 7 days in a clean-grade animal laboratory environment, maintained at a temperature of 22±2℃, a relative humidity of 40%±10%, and a 12-hour light / dark cycle, with free access to food and water. The experimental protocol was approved by the Experimental Animal Ethics Committee of Lanzhou University (Ethics No.: MECI20250001) and followed relevant ethical principles for animal experiments, minimizing the number of animals used and their suffering.

[0029] 1. Experimental Design Mice were randomly assigned to 5 groups (n=6): control group (Con), model group (CUMS), medium-dose glycyrrhizin A group (AZ, 80 mg / kg), high-dose group (AG, 100 mg / kg), and fluoxetine group (Flu, 5 mg / kg). Except for the control group (Con), all other groups received 6 weeks of Chronic Unpredictable Mild Stress (CUMS) to establish a depression model. The stress treatment included eight stimuli (such as reversed day / night cycle, food / water deprivation, wet bedding, ice water swimming, tail clamp stimulation, electric shock, noise stimulation, and tilted cage housing), with 1–2 stimuli randomly selected daily. Each stimulus was repeated 2–3 times throughout the experimental period (see details for operation). Figure 1 A).

[0030] Starting from week 4, mice in each treatment group were administered the corresponding drugs via gavage daily for a total of 3 weeks. Glycyrrhizin chalcone A was dissolved in a diluent prepared from SBE-β-CD saline and DMSO (dimethyl sulfoxide) at a volume ratio of 9:1. SBE-β-CD saline was an isotonic solution prepared by dissolving sulfobutyl ether-β-Cyclodextrin (SBE-β-CD, trade name Captisol®) in 0.9% sodium chloride injection (physiological saline). The SBE-β-CD content in the saline was 20%. Different doses were administered via gavage. Fluoxetine was dissolved in saline. The model group was administered an equal volume of the solvent via gavage. Behavioral testing was performed at the end of week 6. After the behavioral tests, mice were anesthetized, and brain tissue and hippocampus were extracted via saline infusion and stored at -80℃ for later use.

[0031] To evaluate depressive-like behavior in mice, a combination of sucrose preference test (SPT), forced swimming test (FST), and tail suspension test (TST) was used. All tests were performed blinded to avoid experimenter bias.

[0032] 2. Behavioral tests (1) Sucrose Preference Test (SPT) All mice underwent a 3-day acclimatization training period before the experiment to ensure they became familiar with the experimental environment. On Day 1, each mouse was housed individually, with two water bottles containing 2% sucrose solution placed in each cage. On Day 2, two identical individual water bottles were simultaneously placed in each cage, one containing 2% sucrose solution and the other containing distilled water, ensuring the mice could choose their drinking water independently without intervention. The bottles were switched every 4 hours to eliminate interference from positional preferences. On Day 3, the mice were weighed and subjected to fasting and water restriction. The formal experiment was conducted at 10:00 AM on Day 4. Each mouse was simultaneously given a fixed amount of 2% sucrose solution and one bottle of distilled water. The bottles were switched every 3 hours for 6 consecutive hours. After 6 hours, the bottles were removed and the mice were weighed. The intake of sucrose solution and distilled water for each group was recorded, and the percentage of sucrose preference was calculated to assess the mice's anhedonia state. Sucrose preference index = (1% sucrose consumption) / (1% sucrose consumption + water consumption) × 100%.

[0033] (2) Tail Suspension Test (TST) During the experiment, the mouse tail was fixed with tape about 3 cm from the tip and suspended on a horizontal bar at a height of at least 10 cm above the table. The total suspension time was 6 minutes. The cumulative time the mouse remained still within the last 4 minutes was recorded to assess the animal's behavioral despair level.

[0034] (3) Forced swimming test (FST) The experiment was conducted in a cylindrical water tank with a diameter of 5 cm, a height of 25 cm, a depth of 15 cm, and a temperature controlled at 22±2℃. Mice were placed in the water and observed for 6 minutes. After the initial 2-minute acclimatization period, the cumulative time the mice remained motionless over the subsequent 4 minutes was recorded to evaluate behavioral despair. Immediately after the experiment, the mice were removed, dried, and their fur was blow-dried before being returned to their cages. To avoid interactions between different behavioral experiments, a one-day interval was set between the FST and TST experiments.

[0035] 3. Nissl staining After behavioral testing, three mice from each group were randomly selected, anesthetized, and fixed with 4% paraformaldehyde via cardiac perfusion. Brain tissue was then collected, embedded in paraffin, and serial sections were prepared with a thickness of 5 μm. After dewaxing and hydration, Nissl staining was performed to observe the morphology and arrangement of neurons in the CA1, CA3, and dentate gyrus (DG) regions of the hippocampus. Images were acquired using a PANNORAMIC DESK / MIDI / 250 / 1000.

[0036] 4. Measurement of IL-6, IL-18, 5-HT and IL-1β levels (ELISA) After the behavioral tests, the mouse hippocampus was quickly isolated, weighed, and homogenized in pre-cooled PBS buffer. The homogenate was centrifuged at 10,000×g for 10 min at 4°C, and the supernatant was collected for later use. The levels of each indicator in the mouse hippocampus were measured using a commercially available IL-6, IL-18, 5-HT, and IL-1β ELISA kit (Elabscience Biotechnology Co. Ltd., Nanjing, China), strictly following the manufacturer's instructions. The relevant data were recorded for statistical analysis.

[0037] 5. Network pharmacology analysis (1) Screening of potential targets for glycyrrhizin A Using "Licochalcone-A" as the keyword, potential targets for glycyrrhizin A were extracted from the HERB database (https: / / herb.ac.cn / ). Simultaneously, the smiles structure of glycyrrhizin A was obtained from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ). Using this as input, the Swiss Target Prediction database (http: / / www.swisstargetprediction.ch / , data as of June 7, 2025) was further used to predict potential targets for this compound. Finally, targets with a probability greater than 0 were selected as candidate targets for further analysis.

[0038] (2) Screening of depression-related targets Using "depression" as the keyword and specifying "Homo sapiens" as the species, a systematic search was conducted on June 7, 2025, in the following six authoritative databases to obtain molecular targets related to depression: DrugBank (https: / / www.drugbank.ca / ), Online Human Mendelian Inheritance Database (OMIM, http: / / www.omim.org / ), Human Genome Database (GeneCards, https: / / www.genecards.org / ), Pharmacogenomics Database (PharmGkb, https: / / www.pharmgkb.org / ), Gene Expression Database (GEO, https: / / www.ncbi.nlm.nih.gov / geo / ), and Therapeutic Target Database (TTD, http: / / db.idrblab.net / ttd / ). After deduplication and normalization of the targets retrieved from these databases, a standardized list of depression-related targets was obtained.

[0039] (3) Intersection analysis of drug-disease targets The intersection analysis of the obtained potential targets of glycyrrhizin A and depression-related targets was performed using Venny 2.1.0 software (https: / / bioinfogp.cnb.csic.es / tools / venny / ) to identify the core targets with common effects. The results were visualized using Venn diagrams.

[0040] (4) Protein-protein interaction (PPI) network analysis The protein-protein interaction (PPI) relationships between the aforementioned drug-disease intersection targets were analyzed using the STRING database (https: / / string-db.org / , data as of June 20, 2025), with the analysis conditions set to medium confidence (confidence score ≥ 0.4). Subsequently, the obtained interaction data were imported into Cytoscape 3.7.1 software, and the Network Analyzer tool was used to calculate the topological parameters of each node, including degree, betweenness centrality, and closeness centrality. Furthermore, the MCODE algorithm was used to analyze the sub-network structure of the PPI network to identify core functional modules and key node targets.

[0041] (5) Enrichment analysis of GO and KEGG pathways The selected drug-disease intersection targets were uploaded to the DAVID database (https: / / david.ncifcrf.gov / ) for Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis to determine the possible biological processes and related signaling pathways of glycyrrhizin A's antidepressant effect. GO enrichment analysis selected the top 10 significant entries for display, while KEGG pathway enrichment analysis used P<0.05 as the significance criterion and excluded pathways unrelated to the disease. Finally, a network diagram of "glycyrrhizin A-target-signaling pathway" was constructed using Cytoscape 3.7.1 software and visualized.

[0042] 6. Western blot detection Hippocampal tissue was homogenized in RIPA lysis buffer containing a mixture of protease and phosphatase inhibitors, and then quantified by BCA assay according to the manufacturer's protocol. Quantified proteins (40 μg / lane) were isolated on 10% SDS-PAGE and then blotted onto a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% skim milk for 2 hours at room temperature, the membrane was incubated overnight at 4°C with primary antibodies: P38, P-P38, JNK, P-JNK, and GADPH in 5% skim milk in TBST buffer (TBS and Tween 20). After incubation with a secondary antibody linked to horseradish peroxidase for 2 hours, immunoreactive proteins were detected using a chemiluminescence detection system (FUSIONSOLO 6S.EDGE, VILBER, France). Bands were compared on an image analyzer, and relative quantification was performed using an Image-J digital imaging system (Bio-Rad, Hercules, California). Gel imaging and relative quantification were performed using the Image-J digital imaging system.

[0043] 7. Statistical Analysis Statistical analysis was performed using IBM SPSS 26.0 and GraphPad Prism 10.1.2 software. Data are expressed as mean ± standard deviation (SD). Two-group comparisons were performed using independent samples t-tests, and multiple group comparisons were performed using one-way ANOVA, followed by Tukey's method for multiple comparisons. Statistical significance was defined as P < 0.05.

[0044] result 1. Glycyrrhizin chalcone A attenuates CUMS-induced depressive-like behavior in mice. This invention establishes a mouse model of depression using chronic unpredictable mild stress (CUMS), and evaluates the antidepressant effects of glycyrrhizin chalcone A at different doses (80, 100 mg / kg) using the sucrose preference test (SPT), forced swimming test (FST), and tail suspension test (TST). Figure 1 A). Compared with the control group, the CUMS model group mice exhibited typical depressive-like behaviors, including a significant decrease in sucrose preference and a significant increase in immobility time (both p<0.001), indicating successful modeling from a behavioral perspective. After intervention with glycyrrhizin A, the AG group (100 mg / kg) showed a significant increase in sucrose preference during SPT (p<0.001). Figure 1 B); The AG and AZ dosage groups (100, 80 mg / kg) showed significantly shorter immobility times in FST and TST (p<0.001). Figure 1 CD). Compared with the fluoxetine hydrochloride group (5 mg / kg) as a positive control, it significantly improved the target indicators, and the difference was statistically significant compared with the blank control group (P<0.001). Compared with the fluoxetine hydrochloride group, the medium and high doses of glycyrrhizin A showed no significant difference in the improvement effect on sucrose preference and selection in the forced swimming test (P>0.05). However, in the forced swimming test, the high dose of glycyrrhizin A showed a significant difference compared with fluoxetine hydrochloride (P<0.01), suggesting that the high dose of glycyrrhizin A was slightly better than the fluoxetine hydrochloride group, and the medium dose of glycyrrhizin A may have an effect comparable to that of fluoxetine hydrochloride. In behavioral studies, compared with the fluoxetine hydrochloride group, the high dose group showed no significant difference in the improvement effect on sucrose preference and selection in the forced swimming test (P>0.05). The results suggest that glycyrrhizin chalcone A not only has antidepressant effects comparable to fluoxetine hydrochloride at medium and high doses, but also shows better results on some key behavioral indicators at high doses. Furthermore, based on its natural origin, it may have better safety and fewer side effects, making it worthy of further in-depth research and development.

[0045] 2. Alleviating CUMS-induced hippocampal nerve damage in mice The hippocampus plays a crucial role in mood regulation, and its damage is closely related to depression. Nissl staining results showed that neurons in the CA1, CA3, and DG regions of the hippocampus in the control group mice were neatly arranged and structurally intact, while neurons in the CUMS model group were disordered and their nuclear staining was lighter, indicating significant neurological damage. Medium and high doses of glycyrrhizin chalcone A and fluoxetine significantly improved hippocampal structural damage, manifested as neat neuronal arrangement and enhanced nuclear staining, indicating their neuroprotective effects. Figure 2 Compared to fluoxetine hydrochloride, the high-dose glycyrrhizin A group showed a greater number and more evenly distributed neurons in the overall brain tissue, demonstrating superior neuronal protection compared to fluoxetine hydrochloride. The medium-dose glycyrrhizin A group showed only minor shrinkage and deep staining of neurons in the brain tissue, which was comparable to the effect of fluoxetine hydrochloride.

[0046] Further analysis of 5-HT levels in the hippocampus revealed a significant decrease in 5-HT levels in the model group mice (p<0.01). This decrease was significantly reversed in the glycyrrhizin chalcone A intervention group, and the 5-HT levels in the hippocampus of the medium and high dose groups were superior to those in the fluoxetine hydrochloride group (p<0.01, P<0.01). Figure 3 (A) It was also found that 5-HT levels increased with increasing dose. In summary, the results suggest that glycyrrhizin A may exert its antidepressant effect by protecting the structural integrity of hippocampal tissue and restoring 5-HT levels in the hippocampus, and this effect may be dose-dependent.

[0047] 3. Glycyrrhizin chalcone A alleviates neuroinflammation in a CUMS-induced mouse model. Neuroinflammation is one of the important pathological mechanisms of depression. This invention detected the expression levels of pro-inflammatory cytokines IL-1β, IL-6, and IL-18 in hippocampal tissue to evaluate the anti-neuroinflammatory effect of glycyrrhizin chalcone A. The results showed that, compared with the control group, the levels of IL-1β, IL-6, and IL-18 in the hippocampus of mice in the CUMS model group were significantly increased (all p < 0.01), suggesting that CUMS successfully induced a significant neuroinflammatory response. After treatment with medium-dose (80 mg / kg), high-dose (100 mg / kg), or fluoxetine, the levels of IL-1β, IL-6, and IL-18 in the mouse hippocampus were significantly decreased (all p < 0.01). Compared with the fluoxetine hydrochloride group, the high- and medium-dose glycyrrhizin chalcone A groups showed a greater effect on interleukin-6 (IL-6, IL-6, IL-18). Figure 3 B) Interleukin-1β (IL-1β, Figure 3 The inhibitory effect of glycyrrhizin A on interleukin-18 (IL-18) was superior compared to that of fluoxetine hydrochloride group. Figure 3 D) The inhibitory effect is better. The high-dose glycyrrhizin A group and the fluoxetine hydrochloride group have similar effects. This result may be related to the bidirectional regulation or feedback mechanism of drug action. This suggests that there may be an optimal dose window for the regulation of IL-18 by glycyrrhizin A. The specific mechanism is worth further investigation.

[0048] The above results indicate that glycyrrhizin chalcone A can effectively inhibit CUMS-induced neuroinflammatory responses, suggesting that its antidepressant effect may be related to reducing hippocampal inflammatory factor levels, and its anti-inflammatory effect generally shows a dose-dependent trend.

[0049] 4. Network pharmacology analysis predicts the potential mechanism of glycyrrhizin chalcone A in the treatment of depression. To clarify the potential mechanism by which glycyrrhizin chalcone A exerts its antidepressant effect, this invention employs network pharmacology to predict the potential mechanism of glycyrrhizin chalcone A in treating depression. First, potential targets for glycyrrhizin chalcone A were predicted using the Swiss Target Prediction and HERB databases, respectively. A total of 88 potential targets were predicted from the Swiss Target Prediction database, 123 from the HERB database, and 32 from the TCSMP database. After merging the targets predicted from the two databases and removing duplicates, a total of 160 potential active targets for glycyrrhizin chalcone A were finally identified.

[0050] Secondly, to obtain targets related to depression, the system searched six databases: GeneCards, OMIM, DisGeNET, PharmGkb, TTD, and DrugBank, obtaining 3949, 560, 1719, 24, 92, and 137 relevant targets, respectively. After normalization and deduplication of these targets, a total of 5101 targets related to depression were finally identified.

[0051] Then, using Venny 2.1.0 software, an intersection analysis was performed on 160 potential targets of glycyrrhizin A and 5101 targets of depression, revealing 89 common targets of glycyrrhizin A and depression. Figure 4 This suggests that glycyrrhizin A may exert its antidepressant effect through these common targets.

[0052] Furthermore, the protein-protein interaction (PPI) relationships of the 89 intersection targets were analyzed using the STRING database, constructing a PPI network of potential targets for glycyrrhizin A in the treatment of depression, containing 86 nodes and 778 edges. Cytoscape 3.7.1 software was used to calculate the topological parameters of each node (including degree, betweenness, and closeness), and a visualization of the network was generated. Figure 5Network topology analysis revealed a positive correlation between node size and color intensity and degree value; higher degree values ​​correspond to larger nodes and darker colors. Furthermore, the MCODE algorithm identified three functional modules within the PPI network. The core module, with the highest score, comprised 27 nodes and 572 edges. Key nodes within this module included JAK2, PPARG, HDAC1, CXCL8, AGER, HIF1A, HDAC4, BRAF, SLC2A1, APP, AR, ESR1, BCL2, MMP9, ESR2, HSP90AB1, PTGS2, GSK3B, MAPK14, NFE2L2, TGFB1, EGFR, MAPK1, STAT3, ACE, MDM2, and CCND1. These nodes may be key targets for the antidepressant effects of glycyrrhizin A.

[0053] Finally, to clarify the biological processes and signaling pathways involved in these potential targets, GO and KEGG enrichment analyses were performed on the 89 common targets using the DAVID database, yielding 565 GO annotation entries and 133 KEGG pathway entries. Using an FDR < 0.05 as the criterion, GO analysis identified 474 significantly enriched biological process entries. Figure 6 Enrichment results showed that multiple targets were significantly enriched in depression-related biological processes and pathways, including neuroinflammation regulation, apoptosis, and neuroprotection, demonstrating the multi-target and multi-pathway synergistic antidepressant effects of glycyrrhizin chalcone A. Further analysis of KEGG enrichment results (…) Figure 7 The study found that glycyrrhizin A's potential targets are significantly enriched in the MAPK signaling pathway, with key targets MAPK14 (p38) and MAPK1 (ERK2) playing important roles in this pathway. Simultaneously, the core regulators of the JNK signaling pathway, MAPK8 / JNK1, were also identified as important targets. These results suggest that glycyrrhizin A may exert its antidepressant effect by directly regulating the MAPK and JNK signaling pathways, inhibiting the abnormal activation of stress and inflammation-related kinases, thereby alleviating neuroinflammation and neuronal damage.

[0054] 5. Glycyrrhizin A reduces CUMS-induced neuroinflammation by regulating the MAPK / JNK pathway. To further elucidate the molecular mechanism of the antidepressant effect of glycyrrhizin A, this invention used Western blot to detect changes in the expression of key proteins in the MAPK / JNK pathway in mouse hippocampus. p38 MAPK is an important member of the MAPK family, and its activated form, p-p38, is significantly upregulated under various stress conditions. Similarly, JNK, also a core member of the MAPK family, shows the same upregulation trend after phosphorylation to p-JNK and undergoes nuclear translocation to initiate the transcription of downstream inflammatory genes; both are key regulators of neuroinflammatory responses. Therefore, this invention selected the ratio of p-p38 / p38 to p-JNK / pJNK to assess the activation status of this pathway under CUMS induction and to further observe whether glycyrrhizin A can exert antidepressant and anti-inflammatory effects through this pathway.

[0055] The results showed that, compared with the control group, the expression of p-P38 and p-JNK proteins in the hippocampus of mice in the CUMS model group was significantly increased, with the p-P38 / P38 ratio increasing by about 2.3 times (p<0.001) and the p-JNK / PJNK ratio increasing by about 1.6 times (p<0.001), indicating that the MAPK / JNK pathway was significantly activated.

[0056] After intervention in the high-dose (GAG) and medium-dose (GAZ) glycyrrhizin A groups, the p-PJNK levels and ratios decreased significantly (p<0.001). After intervention in the high-dose (GAG) glycyrrhizin A group, the p38 levels and ratios decreased significantly (p<0.01). After intervention in the medium-dose (GAZ) glycyrrhizin A group, the p38 levels and ratios were downregulated compared with the model group, but there was no significant difference between the groups. The results show that it is still dose-dependent.

[0057] Compared with the fluoxetine (Flu) group, the medium-dose glycyrrhizin A (GAZ) group had relatively higher PP38 / P38 and PJNK / JNK phosphorylation levels, while the high-dose group (GAG) showed no difference from the Flu group. This suggests that the high-dose glycyrrhizin A group is comparable in efficacy to the fluoxetine-positive control group, both effectively downregulating model-induced hyperphosphorylation of the p38 and JNK pathways.

[0058] These results indicate that glycyrrhizin A exerts its anti-inflammatory and antidepressant effects by downregulating the phosphorylation level of the MAPK P38 / JNK pathway. This signaling pathway may be one of the key molecular mechanisms by which glycyrrhizin A treats depression.

[0059] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0060] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0061] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Application of glycyrrhizin A in the preparation of drugs for treating depression.

2. The application according to claim 1, characterized in that, The drug uses licorice chalcone A as its sole active ingredient.

3. The application according to claim 1, characterized in that, The drug is used to improve hippocampal structural damage caused by depression.

4. The application according to claim 1, characterized in that, The drug is used to relieve neuroinflammation caused by depression.

5. The application according to claim 1, characterized in that, The drug is available in oral or injectable form.

6. The application according to claim 5, characterized in that, The oral preparations include granules, tablets, powders, soft capsules, capsules, soft capsules, drop pills, and solutions.

7. The application according to claim 5, characterized in that, The drug includes a pharmaceutically acceptable carrier.

8. The application according to claim 7, characterized in that, Pharmaceutically acceptable carriers include diluents.

9. The application according to claim 8, characterized in that, The diluent is composed of BE-β-CD physiological saline and dimethyl sulfoxide in a volume ratio of 9 to 10:

1.

10. The application according to claim 1, characterized in that, The purity of the glycyrrhizin A is ≥98%.

Images