Combinatorial Chemistry & High Throughput Screening - Volume 28, Issue 11, 2025

Hydnocarpus anthelmintica (HA) has been traditionally used for treating leprosy and is known for its antioxidant and anti-inflammatory activities. The aim of this study was to investigate the active compounds and targets of HA extracts, involved in oxidative stress and skin aging. The active compounds and targets of HA extracts were identified using network pharmacology.

The pathway study was conducted using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. HA semen was measured for its in-vitro antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and anti-aging activities using collagenase, elastase, and tyrosinase assays. A total of 21 intersecting core targets were identified from 8 compounds, 51 action targets, and 1810 skin aging and oxidative stress-associated target genes.

A compound-target network was constructed, and 3 compounds (luteolin, beta-carotene and genkwanin), and 4 hub genes (TP53, HSP90AA1, JUN, and MAPK1) were identified. The KEGG pathway study revealed that the compounds were correlated with PI3K-Akt, p53, HIF-1, and MAPK signaling.

The results of in-vitro experiments showed the effect of HA extract on oxidative stress reduction and collagenase inhibition. We discovered two main active compounds, luteolin and β-carotene, that may be involved in p53 and MAPK signaling, and showed HA extract activity against oxidative stress and collagenase.

To date, disease-modifying antirheumatic drugs (DMARDs) are widely used as the primary first-line treatment option for patients with rheumatoid arthritis (RA), and the curative effect of methotrexate (MTX) and leflunomide (LEF; MTX + LEF) is greater than that of single-agent MTX therapy, but the synergistic mechanism of MTX + LEF is unclear.

First, we explored the mechanism of action of MTX + LEF in RA through network pharmacology and molecular docking. Venn diagram analysis revealed 97 overlapping gene targets of MTX + LEF-RA and STRING, along with Cytoscape plug-in MOCDE and cytoHubba; and GO enrichment analysis revealed that the functions of 97 synergistic targets were related to 123 molecular functions (MF), 63 cell components (CC), and 1,068 biological processes (BP). The Cytoscape plug-in ClueGO demonstrated that these targets were enriched in KEGG pathways of 52 terms, whereas 9 pivotal genes were mainly involved in the signaling pathways of estrogen, Ras, Rap1, PI3K-Akt, relaxin, TNF, AMPK, FoxO, prolactin, IL-17, and adherens junction. Finally, CETSA and DARTS validated the direct binding of MTX or LEF to the selected target proteins EGFR, PPARG, MMP9, and SRC in RAW264.7 cells.

We identified 292 MTX targets and 247 LEF targets from 7 databases. Furthermore, 2,814 potential targets of RA were identified by merging 1,925 targets from 7 databases and 999 differentially expressed genes (DEGs) between normal controls and patients with RA extracted from 5 GEO databases. Nine pivotal genes, ESR1, ALB, CASP3, EGFR, HSP90AA1, SRC, MMP9, PPARG, and IGF1, were identified. Molecular docking verified that both MTX and LEF strongly bind to most of the 9 pivotal proteins except ESR1 and IGF1.

These results contribute to our understanding of the enhancement mechanism of MTX combined with LEF and provide a targeted basis for the clinical treatment of RA.

Sageretia theezans is one of the classic medicines in ancient times, which is commonly used to treat scabies, lacquer sores, acute and chronic pharyngitis, Tonsillitis, Cholecystitis, secondary infection of hemorrhoids, and other symptoms. However, the potential molecular mechanism of Sageretia theezans is still unclear. In this study, we explored the active compounds of Sageretia theezans in the treatment of hemorrhoids (HD), predicted the potential targets of drugs, and verified their functions through network pharmacology and in vivo and in vitro experiments.

First, we identified the active compounds and key targets of Sageretia theezans in treating HD through network pharmacology. The key signaling pathways related to the role of Sageretia theezans were analyzed. HUVEC Human umbilical vein endothelial cells were used to study the function of Sageretia theezans and its target in vitro. In addition, we also used the SD rat hemorrhoid model to explore the efficacy of Sageretia theezans in HD in vivo.

A total of 159 drug targets were obtained from the TCMSP, ETCM, and PubChem databases. Constructing a drug component target network; differential analysis using sequencing data identified 1046 differentially expressed genes. Intersecting drug targets and differentially expressed genes obtained four intersection targets (GOT1, SLC25A10, SUCLG1, CLEC4E). Perform single gene GSEA functional enrichment analysis on intersection targets, select KEGG and GO of the top 10 for display, and merge the results. In order to investigate the interaction between intersecting genes and differentially expressed genes, we conducted a PPI protein interaction analysis on 1046 differentially expressed genes. Finally, a network of Chinese medicine active molecule intersection genes was proposed, and the genes and their corresponding active molecules (Successful acid, Taraxerone, Taraxerol) were Macromolecular docking, respectively. The results showed that these four genes could be successfully docked with the responsive active molecules and had high binding affinity. In vivo, the low-dose treatment group of Sageretia theezans, the medium-dose treatment group of Sageretia theezans, and the high-dose treatment group of Bromelia can inhibit the proliferation of HUVECs cells. In vitro, the middle dose of Sageretia theezans has the best therapeutic effect on hemorrhoids, and the treatment of Sageretia theezans on hemorrhoids is correlated with the expression of GOT1, SLC25A10, SUCLG1, and CLEC4E.

To sum up, Sageretia theezans can alleviate the symptoms of hemorrhoids and is related to the expression of GOT1, SLC25A10, SUCLG1, and CLEC4E.

Diffuse Large B-Cell Lymphoma (DLBCL) is the most common B-cell lymphoma type. Detoxification and tumor elimination formula, a herbal compound, can potentially treat lymphoma. In this study, network pharmacology and molecular docking approaches were utilized to reveal the potential mechanism of the Jiedu Xiaoliu formula (JDXLF) against DLBCL.

Active compounds and targets of JDXLF were obtained from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. Targets related to DLBCL were retrieved from GeneCards and Online Mendelian Inheritance in Man (OMIM) databases. Protein-Protein Interaction (PPI) networks were established to screen core targets. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using R 4.2.2. Model interactions between potential disease targets and pharmacologically active compounds were determined by molecular docking.

Screening of 14 herbal active ingredients yielded 129 active compounds and 1414 disease targets for DLBCL. GO annotations showed that the effects of JDXLF were related to protein phosphorylation and reactive oxygen species response. KEGG pathway enrichment analysis indicated that the detoxification and elimination of tumors formula mainly regulated apoptosis pathways. Nobiletin showed good interaction with AKT1, TP53, and CASP3, and the cell counting kit-8 (CCK-8) assay confirmed that nobiletin inhibited the proliferation of SU-DHL-4 cells. Western blot analysis showed that nobiletin downregulated the expressions of p-PI3K, p-AKT, and BCL-2 proteins and upregulated those of cleaved-caspase3 and BAX.

Our findings preliminarily suggested that the active ingredient of JDXLF, nobiletin, may induce apoptosis in Diffuse Large B-Cell Lymphoma SU-DHL-4 cells by regulating the PI3K/AKT signaling pathway.

This study aims to investigate the effects and mechanism of Morinda Officinalis How (MOH), a lianoid shrub with potential therapeutic properties, on Metabolism-Associated Fatty Liver Disease (MAFLD).

The objective of this study was to construct a MOH-MAFLD network prediction model and explore the effect of MOH on MAFLD and its underlying mechanism in vivo.

Screening of MAFLD targets was performed using the DisGeNET database. Venny database was used to establish the MOH-MAFLD interaction network map, while the STRING database was applied to assess the Protein-Protein Interaction (PPI) network. The central target gene was screened using Gene Ontology (GO) function analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway.

GO function enrichment analysis revealed that MOH affected MAFLD through apoptosis and estrogen-related pathways. KEGG pathway enrichment and PPI network analysis indicated that MOH might mitigate MAFLD by reducing apoptosis and improving lipid metabolism. Additionally, 6 weeks of MOH treatment in rats decreased caspase-3 levels and increased Bcl-2, Estrogen receptor α(Esr1), and JUN proteins, thus ameliorating MAFLD progression.

MOH could delay the progression of MAFLD by affecting estrogen-related pathways, reducing cell stress, and inhibiting apoptosis.

According to the 2022 Global Cancer Statistics, lung cancer is the leading cause of cancer-related mortality worldwide. Lung adenocarcinoma (LUAD), which is a histological subtype of Non-Small Cell Lung Cancer (NSCLC), accounts for 40% of primary lung cancer. Therefore, there is an urgent need to identify new prognostic markers as clinical predictive markers for LUAD.

This study aimed to investigate the role of Keratin 80 (KRT80) in the prognosis of LUAD and its underlying mechanisms.

Bioinformatics analysis was conducted using data retrieved from The Cancer Genome Atlas (TCGA) databases. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were employed to predict the involved biological processes and signaling pathways, respectively. The LinkedOmics database was utilized to identify differentially expressed genes (DEGs) correlated with KRT80. Nomograms and Kaplan-Meier plots were constructed to evaluate the survival outcomes of patients diagnosed with LUAD. Moreover, TIMER was employed to conduct correlation analyses between KRT80 expression and immune cell infiltration, shedding light on the intricate interplay between KRT80 and the tumor microenvironment in LUAD. To ascertain the RNA and protein expression levels of KRT80 in LUAD and adjacent normal tissues, Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR) and immunohistochemistry techniques were employed, respectively.

Scrutiny of the TCGA dataset revealed KRT80 up-regulation across pan-cancer tissues, notably elevated in LUAD compared to healthy lung tissues. This finding was validated in our clinical samples, where Kaplan-Meier survival curves indicated poorer survival rates for high KRT80 expression in LUAD. A positive correlation was found between the transcription level of KRT80 in LUAD samples and clinical parameters, such as lymph node metastasis stage, distant metastasis, and pathological stage. Survival, logistic regression, and Cox regression analyses emphasized the clinical prognostic significance of high KRT80 expression in LUAD. Nomogram results underscored the robust predictive potential of KRT80 for the survival of LUAD patients. Gene functional enrichment analyses mainly associated KRT80 with cytokine-cytokine receptor interactions, cell cycle, apoptosis, and chemokine signaling pathways. Based on the results of the immune infiltration analysis, it can be found that the expression of KRT80 is related to the immune cell subsets and survival rate of patients with LUAD.

Our research revealed a significant upregulation of KRT80 in LUAD, with heightened KRT80 expression correlating with unfavorable prognosis. This study represents a comprehensive and systematic evaluation of KRT80 expression in LUAD, encompassing its prognostic and diagnostic significance, as well as underlying mechanisms. Our findings suggest that KRT80 may emerge as a novel prognostic and predictive biomarker in LUAD.

The incidence of dyslipidemia increases after menopause. Electroacupuncture (EA) has been recommended for menopause-related disease. However, the positive effect on lipid metabolism disorders is still unclear.

To investigate the underlying mechanism of EA treatment on lipid metabolism disorders through ONT full-length transcriptome sequencing

Adult female SD rats were randomly divided into Ctrl, sham operation+high-fat feed(Sham+HFD), Ovariectomized+high-fat feed (OVX+HFD), Ovariectomized+high-fat feed + Atorvastatin (OVX+HFD+ATO) and OVX+HFD+EA groups. Periovarian adipose tissue around the bilateral ovaries of rats in the Sham+HFD group was resected. Rats in the OVX+HFD, OVX+HFD+ATO and OVX+HFD+EA groups were subjected to bilateral oophorectomy to prepare the ovariectomized rat model. Treatment was applied to rats in the OVX+HFD+EA group. ST36, PC6, SP6, BL18 and ST40 were the selected acupoints. Daily food intake and body weights of rats were recorded. The samples were collected 30 days after treatment. The serum levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein (HDL-C) were detected to assess the improvement of lipid metabolism disorders. HE and oil red O staining were used to stain the liver tissues. Total RNA was extracted from liver tissues, and its transcriptional changes were determined by high-throughput sequencing. Additionally, RT-qPCR and immunofluorescence staining were used to verify the crucial signal pathway screened by the ONT full-length transcriptome sequencing.

EA treatment resulted in a lowered weight of perirenal fat and liver and a significant improvement in the color of the liver. In addition, EA could improve the lipid profile and hepatic steatosis in OVX+HFD rats. According to full-length transcriptome sequencing, 2292 genes showed differential expression in the OVX+HFD group; of these, 1121 were upregulated and 1171 down-regulated. 609 DEGs were found in the OVX+HFD+EA group compared to the OVX+HFD group; 235 up-regulated and 374 down-regulated. We also found that 77 genes are significantly upregulated after EA intervention through Venn map analysis (including Agtr1a, Pdia3, etc.), which may be the targeted genes for EA treatment of lipid metabolism disorders. Finally, we verified the expression of Pdia3, Perk and Qrich1 levels in liver tissues. HFD feeding could increase the expression of Pdia3 and its downstream signal pathways molecular Perk and Qrich1. But these effects were reversed by EA treatment, the results demonstrated that the expression of pdia3, Perk, as well as Qrich1 of OVX+HFD rats had a decreasing trend after EA treatment.

EA could ameliorate lipid metabolic disorder in OVX+HFD rats. The Pdia3/Perk/Qrich1 signal pathway may play crucial roles in the improvement of lipid metabolism disorder of OVX+HFD rats after EA treatment.

Excessive vascular smooth muscle cell (VSMC) proliferation and migration are the main contributors to the symptoms of lower-extremity arteriosclerosis obliterans (ASO). Previous studies suggested that microRNAs (miRNAs) regulate VSMC activity. Nevertheless, the molecular mechanisms by which they do so are unclear.

The present study aimed to identify the biological processes accounting for the effects of miR-140-3p on VSMCs in ASO.

The expression levels of miR-140-3p in clinical samples were analyzed by real-time polymerase chain reaction. An ASO cell model was established to investigate the expression of miR-140-3p on VSMCs. The transwell^® assays and MTT assays were used to assess migration and proliferation. The interaction between RhoA and miR-140-3p was verified using the Dual-luciferase reporter assay. Western blot technique was used to identify RhoA, RhoA-associated protein kinase 1 (ROCK1), and ROCK2.

We discovered that miR-140-3p inhibited the proliferation, migration, and invasion but promoted the apoptosis of VSMCs, and RhoA was its downstream target gene. RhoA, ROCK1, and ROCK2 were upregulated in vascular tissues damaged by ASO compared to normal, healthy arteries. MiR-140-3p also decreased RhoA, ROCK1, and ROCK2 mRNA and protein expression.

Overall, the present work partially elucidated the mechanism by which miR-140-3p regulates VSMC function and offered novel insights into potential therapeutic approaches for patients with lower-extremity arteriosclerosis obliterans.

This study aimed to examine the associations of FTO expression with prognosis, tumor microenvironment (TME), immune cell infiltration, immune checkpoint genes, and relevant signaling pathways in GC. Furthermore, the relationship between FTO and TGF-β was studied in GC.

The mRNA expression and clinical survival data of GC samples were obtained from The Cancer Genome Atlas Stomach Adenocarcinoma (TCGA-STAD). TIMER2, TNM plot, and GEPIA database were used to analyze FTO expression. The associations of FTO with prognosis and clinicopathologic features were assessed using the Kaplan-Meier plotter and UALCAN database, respectively. The R software was employed to analyze its related signaling pathways and the associations with TME, immune cell infiltration, and immune checkpoint genes. GEPIA and ENCORI were used to examine the association of FTO with TGF-β expression. The SRAMP website was utilized to predict m6A modification of TGF-β. IHC, Western blot, and qPCR were used to analyze the expression levels of FTO and TGF-β in clinical gastric cancer tissue samples or gastric cancer cell lines. In addition, a m6A RNA methylation assay kit was used to determine m6A levels in gastric cancer cells.

FTO mRNA and protein levels were significantly elevated in GC compared to normal gastric tissues. Kaplan-Meier survival analysis suggested that upregulated FTO was associated with a worse prognosis in GC. Upregulated FTO was markedly correlated with differentiation degree, lymph node metastasis, and clinical TNM stage. GO and KEGG pathway analyses revealed that FTO-associated molecules were enriched in neuroactive ligand-receptor interaction, calcium signaling, PI3k-Akt signaling, cAMP signaling pathways, and TGF-β signaling pathways, among others. The TME score was remarkably higher in the high-FTO group than in the low-FTO group. Furthermore, FTO expression had positive correlations with different types of immune cells and immune checkpoint genes. Moreover, FTO may regulate TGF-β in an m6A RNA modification manner in GC.

FTO may become an independent predictive prognostic biomarker correlating with TME, immune cell infiltration, and immune checkpoint genes in gastric cancer and might influence GC progression by regulating TGF-β expression.

The purpose of this study was to analyze the relationship between serum indicators and high-throughput drug screening (HDS) results, aiming to achieve specific therapy for hepatocellular carcinoma (HCC).

This study recruited patients with HCC who underwent surgical resection at the Hepatobiliary Surgery Center of the First Affiliated Hospital of Chongqing Medical University from December 2019 to December 2021. HCC tissues were obtained from patients during surgery and subjected to in vitro cell culture, and then HDS testing was performed on the cultured tissue samples. We used Spearman's correlation analysis to examine the relationships between drug sensitivity results for anti-hepatocellular carcinoma drugs, other antitumor drugs, and serological indicators, the Neutrophil Lymphocyte Ratio (NLR), Platelet Lymphocyte Ratio (PLR), Systemic Immune Inflammatory Index (SII), Systemic Inflammatory Response Index (SIRI), Prognostic Nutritional Index (PNI), and Lymphocyte Monocyte Ratio (LMR). A significant correlation was considered when P<0.05 and |r|>0.40. Furthermore, linear regression analysis was conducted to elucidate the relationship between serological indicators and drug susceptibility, with significant results indicated by P<0.05 and R²≥0.50.

In this study, 82 patients with HCC who had undergone hepatectomy and completed in vitro cell culture and HDS testing were evaluated. Using Spearman's correlation with a significance threshold of P<0.05 and |r|>0.40, we identified significant associations between serological indicators and specific drug regimens: NLR correlated with 5-Fluorouracil, 5-Fluorouracil+Calcium folinate (FOLFOX4), and Capecitabine + Cisplatin (XP); PLR with FOLFOX4; SII with XP, FOLFOX4, Doxorubicin + Oxaliplatin (ADM+L-OHP); and SIRI with XP and FOLFOX4. No correlations were found between PNI or LMR and any drug inhibition rates. A comprehensive evaluation using linear regression analysis—which included variables such as sex, age, hepatitis B virus and liver cirrhosis status, size and number of lesions, alpha-fetoprotein, total bilirubin, albumin, alanine aminotransferase, aspartate aminotransferase, and prothrombin time, alongside NLR, PLR, SII, and SIRI was conducted in relation to drug regimens. This analysis revealed that NLR, SII, and SIRI are significant predictors of FOLFOX4 inhibition rate, while NLR predicts the inhibition rate of XP effectively. However, no significant links were established between molecular targeted drugs, other antitumor drugs, and serological indicators.

NLR, SII, and SIRI were correlated with FOLFOX4, and the higher the values of NLR, SII, and SIRI, the higher the in vitro inhibition of FOLFOX. Also, NLR was correlated with XP, and the higher the value of NLR, the higher the in vitro inhibition of XP.

Obstructive Jaundice (OJ) is a common clinical condition with potential outcomes, including hepatocyte necrosis, bile duct hyperplasia, significant cholestatic liver fibrosis, and, in severe cases, liver failure. Resveratrol (RES), a polyphenol present in grapes and berries, has demonstrated efficacy in improving OJ. However, the precise mechanism of its action remains unclear.

In this study, we employed network pharmacology to investigate the underlying molecular mechanism of RES in the treatment of OJ. The targets of RES were identified using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), SuperPred, and SwissTargetPrediction database. The targets related to OJ were gathered from the DisGeNET, GeneCards, DrugBank, and Online Mendelian Inheritance in Man (OMIM) databases, and the intersection of these targets was determined using Venny2.1.0. Subsequently, an active component-target network was constructed using Cytoscape software. The Protein-Protein Interaction (PPI) network was generated using the String database and Cytoscape software. Following this, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted using the Bioconductor platform. Finally, quantitative Real-Time PCR (qRT-PCR), Western Blotting (WB), and Enzyme-Linked Immunosorbent Assay (ELISA) were employed to assess RNA and protein expression levels in related pathways.

The findings revealed a selection of 56 potential targets for RES, and a search through the online database identified 2,742 OJ-related targets with overlapping in 27 targets. In the PPI network, mTOR, CYP2C9, CYP1A1, CYP3A4, AHR, ESR1, and HSD17B1 emerged as core targets. KEGG analyses demonstrated that the primary pathways of RES in treating OJ, particularly those related to lipid metabolism, include linoleic acid metabolism, arachidonic acid metabolism, metabolism of xenobiotics by cytochrome P450, lipid and atherosclerosis, tyrosine metabolism, steroid hormone biosynthesis, and pentose and glucuronate interconversions signaling pathways.

Furthermore, in vivo experiments indicated that RES significantly ameliorated liver injury induced by Common Bile Duct Ligation (CBDL) in rats with OJ. It lowered serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, reduced liver tissue MDA levels, increased glutathione (GSH) content, and enhanced activity of superoxide dismutase (SOD), alleviating liver damage. Metabolomics analysis revealed that the therapeutic effect of RES in OJ involved alterations in lipid metabolic pathways, hinting at the potential mechanism of RES in treating OJ. ELISA, qRT-PCR, and WB analyses confirmed lower expression levels of mTOR, CYP1A1, and CYP2C9 in the RES group compared to the model group, validating their involvement in the lipid metabolism pathway.

In conclusion, RES exhibited a protective effect on liver function in rats with OJ. The underlying mechanism appears to be linked to antioxidant activity and modulation of lipid metabolism pathways.

Disulfidptosis is a new metabolic-related regulated cell death associated with cancer growth. This study aimed to investigate the molecular mechanisms associated with disulfidptosis in skin cutaneous melanoma (SKCM) and establish a disulfidptosis-related gene signature for prognostic prediction in SKCM.

Disulfidptosis-associated genes were identified from RNA-seq data of SKCM. A risk score signature was developed and validated through univariate Cox and LASSO analyses. Additionally, the immune microenvironment related to the risk score signature was investigated. Finally, a disulfidptosis-related genes-transcription factor -miRNA network was developed, and the expression levels of five disulfidptosis-related genes were initially verified in SKCM cell lines.

A total of 107 disulfidptosis-related differentially expressed genes in SKCM samples were identified. A ten-disulfidptosis-gene signature was established, including BIN2, CCL3L3, CCL8, CD79A, CIITA, CXCR3, DEFB1, GPR171, IL2RB, and SOCS1. The SKCM samples were divided into high- and low-risk groups, of which samples in the low-risk group showed better survival performance. The receiver operating characteristic curve analysis confirmed the good potency of the disulfidptosis-related gene prognostic model. Except for DEFB1, the other nine genes were positively related with T cell CD8+, T cell CD4+ memory activated, T cell gamma delta, NK cell activated, and macrophage M1, and they were all negatively related with NK cell resting, macrophage M0, macrophage M2, and mast cell activated. Finally, we verified downregulated levels of SOCS1 and DEFB1 and upregulated CXCR3, BIN2, and CCL3L3 in A875 and A375.

We successfully established ten disulfidptosis-related genes' prediction prognostic signatures for SKCM patients.

Variants in the PRRT2 gene are associated with paroxysmal kinesigenic dyskinesia and other episodic disorders. With the employment of variant screening in patients with episodic dyskinesia, many PRRT2 variants have been discovered. Bioinformatics tools are becoming increasingly important for predicting the functional significance of variants. This study aimed to evaluate the performance of six in silico tools for PRRT2 missense variants.

Pathogenic PRRT2 variants were retrieved from the Human Gene Mutation Database (HGMD) and literature from the PubMed database. The benign set of non-deleterious variants was retrieved from the Genome Aggregation Database (gnomAD). The overall accuracy, sensitivity, specificity, positive predictive values, and negative predictive values of SIFT, PolyPhen2, MutationTaster, CADD, Fathmm, and Provean were analyzed. The MCC score and ROC curve were calculated. The GraphPad Prism 8.0 software was used to plot ROC curves for the six bioinformatics software.

A total of 45 missense variants with confirmed pathogenicity were used as a positive set, and 222 missense variants were used as a negative set. The top three tools in accuracy are Fathmm, Provean, and MutationTaster. The top three predictors in sensitivity are SIFT, PolyPhen2, and CADD. Regarding specificity, the top three tools were Provean, Fathmm, and MutationTaster. In terms of the MCC and F-score, the highest degree was observed in Fathmm. Fathmm also had the highest AUC score. The cutoff values of Fathmm, CADD, PolyPhen2, and Provean were between the median prediction scores of the positive and negative sets. In contrast, the cutoff value of SIFT was below the median prediction score of the positive and negative sets. Fathmm had the highest accuracy.

The prediction performance of six in silico tools differed among the parameters. Fathmm had the best prediction performance, with the highest accuracy and MCC/F-score for PRRT2 missense variants.

Antidepressants have adverse effects and induce drug resistance when used excessively or frequently. Therefore, adjuvants are needed to reduce the use of antidepressants during treatment. Traditional Chinese medicine (TCM) is an important adjunctive approach to depression with safety, environmental protection, and low toxicity. Glycyrrhizaglabra (licorice, GG) is a plant commonly used in various herbal remedies.

To explore the potential antidepressant-related targets of Glycyrrhizaglabra (GG) and its underlying mechanisms, we utilized a combination of animal behavioral experiments, molecular biology, and network pharmacology to analyze the antidepressant effects of GG. Initially, we conducted behavioral assays to verify the capacity of GG to mitigate depressive-like behaviors in mice. Subsequently, we selected 56 active compounds and 695 target compounds of licorice from TCMSP. The PPI network screened 80 core targets for enrichment analysis. Lastly, Western blot and ELISA techniques were utilized to authenticate and corroborate the predicting outcomes of PPI and enrichment analysis.

GG extracts reversed lipopolysaccharide (LPS)-induced depression-like behavior in behavioral tests. The results of enrichment analysis showed that,GG significantly affected neurodegeneration pathways, neuroactive ligand-receptor interaction, cAMP signaling pathway, serotonergic synapse, dopaminergic synapse, and MAPK signaling pathway. Mechanistic studies showed that GG reduced IL-1β, IL-6, and TNF-α levels, 5-HTRA1 expression, and GSK3β phosphorylation in mouse hippocampus. It also increased BDNF and DRD1 expression and CREB and ERK1/2 phosphorylation.

Our experimental results demonstrate that GG targets multiple proteins associated with depression, influencing diverse pathways and consequently regulating depressive-like behaviors in mice.