Combinatorial Chemistry & High Throughput Screening - Online First

DNA microarray synthesis enables the large-scale and precise generation of DNA sequences for genomic research, data storage, and synthetic biology. However, the order of nucleotide addition significantly affects synthesis efficiency and accuracy. This study aims to model DNA microarray in situ synthesis as a traveling salesman problem (TSP) and to develop an optimized synthesis strategy.

A mathematical model for in situ microarray synthesis was established, and both greedy algorithms and a simulated annealing algorithm were applied to optimize the nucleotide addition order. The performance of these approaches was evaluated by comparing the number of synthesis cycles required at different sequence scales, ranging from 10 × 10 nt to 10000 × 120 nt arrays.

The optimized synthesis schemes effectively reduced the total number of synthesis cycles. At the 10 × 10 nt scale, simulated annealing reduced cycles by 40.65% compared to the traditional scheme and by 8.52% compared to the greedy algorithm. At larger scales (100 × 100 nt to 10000 × 120 nt), cycle reductions ranged from 33.80% to 37.26%, with simulated annealing outperforming the greedy algorithm by 2.68% to 3.42%. These reductions translated into significant savings in synthesis time, reagent consumption, and overall cost.

The simulated annealing–based optimization strategy demonstrates clear advantages in improving DNA microarray synthesis efficiency while reducing material usage and waste, thereby enhancing cost-effectiveness. Such improvements offer practical benefits for applications, including gene editing, drug development, and DNA data storage.

Endometriosis (EMs), a prevalent disorder characterized by pelvic pain and infertility, affects numerous bodily systems and markedly diminishes life quality. Yiqi Huoxue formula (YQHXF) has demonstrated promising therapeutic efficacy. However, its active substances and underlying mechanisms remain ambiguous.

An innovative methodological framework incorporating NP, transcriptomics, proteomics, and molecular biology was utilized to investigate the active components and mechanisms of YQHXF. A network pharmacological analysis was conducted to identify the targets, biological processes, and pathways associated with YQHXF’s effects on EMs. Additionally, transcriptomics, proteomics, and molecular biology techniques were applied for further mechanistic exploration at both the gene and protein levels.

The findings suggest that YQHXF prevents EMs by modulating critical cellular processes—proliferation, invasiveness, adhesion, and apoptosis—within ectopic endometrial cells. The integration of network pharmacology, multi-omics, and molecular biology confirmed that this regulation occurs via key targets (EGFR, AKT1, FOS, MAPK3, NFKB1) and associated pathways.

This study employed an integrated approach combining transcriptomics, proteomics, and molecular biology to analyze the effects of YQHXF on EMs. A total of 180 direct targets, 128 indirect targets, and 19 pathways related to YQHXF’s anti-EMs effects were preliminarily identified. Based on these findings, it is proposed that YQHXF potentially achieves its remedial outcomes by influencing the proliferation, invasiveness, adhesion, and apoptosis of ectopic endometrial cells through the regulation of EGFR, AKT1, FOS, MAPK3, and NFKB1.

The findings suggest that YQHXF prevents EMs by regulating critical cellular processes, including the proliferation, invasiveness, adhesion, and apoptosis of ectopic endometrial cells.

Damp retention in the middle-jiao syndrome (DRMS), a common manifestation in Traditional Chinese Medicine (TCM), results from stagnation of damp pathogens in the middle jiao and impaired transport of food and fluids. Given the complex pathogenesis of DRMS, this study aimed to investigate its biological mechanisms using an advanced analytical approach.

A DRMS rat model was established based on three etiological factors: dietary disorders, depletion of vital qi, and excessive external dampness. Model validity was assessed via small intestinal carbon propulsion rate and histopathological examination. Urine metabolomics, using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS), systematically characterized metabolic profile changes and potential biomarkers.

DRMS rats exhibited significantly reduced small intestinal propulsion, along with varying degrees of edema, disorganized tissue structures, and inflammatory cell infiltration in gastric, renal, and small intestinal tissues. Metabolomic analysis identified 52 differential metabolites as potential DRMS biomarkers, primarily involved in phenylalanine, tyrosine, and tryptophan biosynthesis, phenylalanine and tyrosine metabolism, the citrate cycle, and cysteine and methionine metabolism pathways. Metabolic correlation networks further validated the model’s accuracy.

The identified metabolites and pathways provide insight into the mechanisms underlying DRMS, complement existing TCM research, and offer a foundation for further studies. However, the findings are currently limited to the rat model and require human validation.

This study successfully established a DRMS animal model under clinically relevant TCM conditions and demonstrated the utility of metabolomics in elucidating DRMS mechanisms, providing experimental evidence for TCM syndrome characterization and advancing understanding of its etiology.

Huoxue San (HXS) is a traditional Chinese medicinal formulation widely used to treat bone fractures and blood stasis. Comprising seven herbs—Siphonostegia chinensis Benth, Kochia scoparia (L.) Schrad, Scutellaria barbata D.Don, Polygonum cuspidatum Sieb. et Zucc, Arisaema erubescens (Wall.) Schott, Phellodendron chinense Schneid, and Eupolyphaga sinensis Walker—HXS has been administered at Nanjing Chinese Medicine Hospital for over 50 years. It is effective in promoting fracture healing, supporting soft tissue repair, and rarely causing adverse reactions such as skin allergies. The present study aimed to elucidate the molecular mechanisms underlying HXS’s therapeutic effects.

Ultrahigh-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (UHPLC–Q-TOF MS) was used to identify HXS components absorbed into the bloodstream. Network pharmacology, molecular docking, and molecular dynamics simulations were then conducted to explore the active ingredients and their regulatory mechanisms in fracture healing and blood stasis.

Transdermal absorption tests identified 20 active compounds from HXS. Network pharmacology analyses using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform highlighted vanillic acid, demethyleneberberine, palmatine hydrochloride, luteolin, apigenin, and wogonin as key active ingredients. Molecular dynamics simulations further validated the stability, conformational changes, and interactions of these compounds with their target proteins.

Analysis of the transdermal absorption samples revealed 291 potential active targets for HXS in treating fractures and blood stasis, of which 159 were common to both conditions. Protein–protein interaction (PPI) network analysis identified core targets including AKT1, ALB, EGFR, STAT3, and CTNNB1. Molecular docking confirmed strong binding interactions between HXS compounds and these core targets, while molecular dynamics simulations validated the stability and mechanistic plausibility of these interactions.

This study provides a systematic elucidation of HXS’s molecular mechanisms in fracture healing and blood stasis. Identification of active compounds, core targets, and their interactions offers a scientific basis for the therapeutic effects of HXS and supports the rational development of herbal-medicine-based interventions for fracture management and blood stasis treatment.

This study aimed to develop and validate a Cuproptosis-Related Gene (CRG) signature for predicting Biochemical Recurrence-Free Survival (BCRFS) and characterizing the Tumor Immune Microenvironment (TIME) in Prostate Cancer (PCa).

Transcriptomic and clinical data were collected from TCGA (n=405) and GEO (GSE70770, n=203). Consensus clustering based on 10 CRGs defined molecular subtypes. Differentially expressed genes between clusters were subjected to LASSO Cox regression to construct a prognostic signature in the TCGA cohort, followed by validation in GEO and combined cohorts. Quantitative real-time polymerase chain reaction (qRT-PCR) and Immunohistochemistry (IHC) were conducted for experimental validation.

Two CRG-based subtypes were identified, characterized by distinct clinicopathological features, immune checkpoint expression, and BCRFS. A six-gene signature (CALML5, MMP11, UBE2C, ANPEP, TMEM59L, COMP) stratified patients into high- and low-risk groups with significantly different BCRFS (log-rank P<0.001). The model showed good predictive accuracy (AUCs 0.717–0.837 at 1 year, 0.728–0.771 at 3 years, 0.683–0.695 at 5 years) and remained independent of clinicopathological factors. High-risk patients exhibited elevated immune/stromal scores, altered immune infiltration, and higher immune checkpoint expression. qRT-PCR confirmed upregulation of CALML5, MMP11, UBE2C, and COMP in PCa cell lines, while IHC validated differential protein expression of all six genes between PCa and BPH tissues (all P<0.05).

This six-gene CRG signature predicts BCRFS and reflects immune heterogeneity in PCa. Its integration into prognostic models may guide personalized management and inform immunotherapy strategies, warranting further validation in prospective clinical studies.

This study initially identified two cuproptosis-related molecules based on the expression patterns of cuproptosis-related genes. In addition, we developed a new cuproptosis-related molecular signature with great predictive performance for BCRFS and tumor immune environment using six DERRGs (including CALML5, MMP11, UBE2C, ANPEP, TMEM59L, COMP). These findings would be conducive to a deeper cognition of the potential mechanism of cuproptosis of PCa.

Icteric hepatitis remains a significant clinical challenge. The integration of Chinese Herbal Medicine (CHM) with conventional Western Medicine (WM) is becoming increasingly common; however, its overall efficacy and safety profile requires systematic evaluation. This study aims to conduct a meta-analysis to assess the safety and effectiveness of using CHM in conjunction with WM to treat icteric hepatitis.

We conducted a systematic search of PubMed, EMBASE, Cochrane Library, Scopus, Web of Science, and major Chinese databases (CNKI, Wanfang, VIP, CBM) from inception to December 2023 for Randomized Controlled Trials (RCTs) comparing CHM plus WM with WM alone for icteric hepatitis. Two reviewers independently performed study selection, data extraction, and quality assessment using the Cochrane Risk of Bias 2.0 tool. Meta-analysis was performed using RevMan 5.4 software.

Eight RCTs involving 645 participants were included. The combined therapy group demonstrated a significantly higher clinical efficacy rate (RR = 1.22, 95% CI [1.11, 1.34], P < 0.0001) compared to the WM alone group. The combined therapy also resulted in greater improvements in liver function, with significant reductions in ALT (WMD = -58.33 U/L, 95% CI [-87.75, -28.91]), AST (WMD = -47.11 U/L, 95% CI [-69.83, -24.39]), TBIL (WMD = -48.27 μmol/L, 95% CI [-67.48, -29.06]), and DBIL (WMD = -31.30 μmol/L, 95% CI [-45.16, -17.44]). Furthermore, the integrated approach led to lower levels of inflammatory markers (IL-6, CRP, TNF-α) and faster symptom resolution. There was no significant difference in the incidence of adverse events between the two groups (RR = 0.83, 95% CI [0.44, 1.57], P = 0.56).

The pooled evidence suggests that adding CHM to standard WM treatment can enhance therapeutic outcomes by improving liver function and reducing systemic inflammation. The synergistic effects may be attributed to the multi-target pharmacological properties of the herbs used. However, the findings are limited by the high risk of bias and significant heterogeneity across the included studies.

The adjunctive use of CHM with WM appears to be an effective and safe strategy for treating icteric hepatitis. Nonetheless, due to methodological weaknesses in the primary studies, these results should be interpreted cautiously. High-quality, rigorously designed RCTs are needed to confirm these findings.

Metabolic Dysfunction-Associated Steatohepatitis (MASH) is a growing global health concern, with only one FDA-approved therapy currently available. Acetyl-CoA carboxylase (ACC) inhibition has emerged as a promising strategy, yet effective and clinically translatable inhibitors remain limited. This study aimed to identify potential ACC inhibitors for MASH via drug repurposing.

A small-molecule library was screened using structure-based virtual screening, and candidate compounds were validated in a free fatty acid-induced MASH cell model. Intracellular triglyceride (TG) and aspartate aminotransferase (AST) levels were measured, while quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to evaluate lipid metabolism-related gene expression. Molecular dynamics simulations were conducted to further evaluate binding stability.

Lanatoside C was identified as the most potent candidate. In vitro studies revealed significant reductions in TG and AST levels, downregulation of lipogenesis-related genes (SREBP1, FASN, ACC), and upregulation of fatty acid oxidation genes (CPT1A, ACOX1, FABP1). Molecular dynamics simulations confirmed the stable binding of Lanatoside C to ACC.

These findings indicate that Lanatoside C exerts dual regulatory effects on lipid metabolism by suppressing fatty acid synthesis and enhancing oxidation. As an FDA-approved cardiac glycoside, Lanatoside C’s known pharmacological profile supports its potential repositioning for MASH, although further in vivo studies and mechanistic validation are warranted.

Lanatoside C demonstrates promise as a repurposed ACC inhibitor for MASH treatment, offering a cost-effective repurposing strategy to advance therapeutic options for MASH.

The key toxicological constituents and mechanisms of Epimedii Folium and its formulations, such as Xianling Gubao Capsules (XLGB) and Zhuanggu Guanjie Pills (ZGGJ), remain insufficiently understood, particularly when used in combination. The objective of this study is to investigate the hepatotoxic effects and mechanisms of Epimedii Folium and its formulations, XLGB and ZGGJ, using network toxicology, molecular docking, and in vitro validation.

Potential hepatotoxic components and targets of Epimedii Folium, XLGB, and ZGGJ were screened from multiple databases. PPI networks were constructed, and GO/KEGG enrichment analyses were performed. Molecular docking was used to assess the binding affinities between key components and core targets. In vitro validation was conducted using HepG2 cells to assess cell viability and ROS levels through CCK-8 and HCS assays, respectively.

This study confirms that Sagittatoside A, Epimedin B, and Icariside I are the primary hepatotoxic constituents of Epimedii Folium, capable of targeting core pathways involving KDR, AR, PTGS2, F7, and DPP4. Furthermore, Sagittatoside A and Icariside I significantly elevated ROS levels. The toxic constituents of XLGB and ZGGJ overlapped with those of Epimedii Folium, and Bavachinin and Neobavaisoflavone from PCL were found to exert synergistic hepatotoxic effects. Neobavaisoflavone enhanced the hepatotoxicity of Epimedin B and Icariside I, while Bavachinin showed synergistic toxicity when combined with Sagittatoside A.

Molecular docking confirmed strong binding affinities between these compounds and their targets. In vitro experiments demonstrated that Sagittatoside A and Icariside I significantly increased ROS levels. The compound formulations XLGB and ZGGJ shared similar hepatotoxic components and mechanisms. Additionally, Bavachinin and Neobavaisoflavone from PCL synergistically enhanced the hepatotoxicity of Epimedii Folium monomers, providing a modern scientific basis for evaluating compatibility principles in traditional Chinese medicine.

This study comprehensively elucidates the hepatotoxicity and synergistic toxic effects of Epimedii Folium and its formulations XLGB and ZGGJ, offering a modern scientific rationale to guide the safe formulation and compatibility of traditional Chinese medicine.

Longlutong Decoction (LLTD) is a Chinese traditional prescription used for coronary heart disease (CHD). The present study aimed to illuminate the mechanisms of LLTD treatment on CHD.

The therapeutic effect of LLTD on CHD was investigated using a CHD rat model. The chemical components of LLTD were identified, following which network pharmacology approaches were utilized to identify active components and disease-related targets. GO and KEGG analyses were conducted to explore potential molecular mechanisms. Finally, the molecular mechanism of LLTD treatment of CHD was verified.

Histopathological assessment revealed markedly attenuated myocardial injury severity in the medicated groups when compared to the model group. Moreover, 81 potential active ingredients were identified in LLTD, with 645 overlapping targets between component targets and disease targets. Network analysis identified Pinocembrin, Magnoflorine, Jatrorrhizine as key active ingredients, and AKT1, TNF, IL-6, STAT3, and Bcl-2 as primary core targets. A total of 1792 biological processes were affected according to GO analysis, and 187 pathways were identified through KEGG analysis. Finally, molecular docking and experimental results validated that LLTD could alleviate cardiomyocyte injury in CHD by regulating the primary core targets.

This study indicates that LLTD may achieve systematic modulating of the signaling network through a “network pharmacology” model, which provides valuable insights for the development of multi-target therapies targeting the complex pathological mechanism underlying CHD.

LLTD may exert cardioprotective effects by regulating inflammatory responses, apoptosis, and oxidative stress.

One of the most dangerous illnesses in the world today is hepatitis B virus (HBV) infection, which mainly affects the liver and can cause cirrhosis, hepatocellular carcinoma (HCC), and chronic infection. The primary goal of this study was to determine whether genotype polymorphisms at particular locations of the IL-18 promoter region may affect the host susceptibility to HBV infection in the North Indian population.

Genetic polymorphism of the IL-18 gene in the promoter region at positions -607 and -137 was performed in the North Indian population (100 controls and 100 HBV patients) using the PCR-RFLP method. Genotypic, allelic, and haplotype frequencies were compared using SHEsis software.

There were no significant differences in individual genotype or allele frequencies at positions -607 and -137 between HBV patients and controls. However, there were statistically significant differences with high frequencies of the -607A/-137C haplotype in HBV patients (p=0.010), whereas the -607C/-137C haplotype was more prevalent in controls (p=0.001), indicating a protective effect.

The individual SNPs did not show significant association, but specific haplotypes of the IL-18 promoter region may influence the risk of HBV infection. These results align partially with previous studies and suggest that haplotype-based analysis provides improved insight into genetic susceptibility.

The present study indicates that a double mutation (polymorphism) -607A/-137C in the IL-18 gene promoter region may contribute to the onset of HBV infection, while a single nucleotide polymorphism (-607C/-137C) may provide less susceptibility to HBV and may have a protective impact.

Migraine, a primary headache disorder, is the third disabling disease of neurological disorders worldwide. The pathological mechanism underlying migraine remains poorly understood. Lipid metabolism may be related to migraine pathophysiology. We aimed to investigate the causal relationship between plasma lipids and migraine or its subtypes, including migraine with aura (MA) and migraine without aura (MO), and explore whether the circulating cytokines serve as mediators in the pathway from plasma lipids to migraine.

A two-step Mendelian randomization (MR) approach was used to assess the mediating effect. The summary genetic data for 179 lipid species were obtained from were derived from a genome-wide association studies (GWAS) summary dataset encompassing 7,174 individuals. The summary genetic data for 91 circulating cytokines were obtained from genome-wide pQTL mapping data. The summary genetic data of GWAS related to migraine and its subtypes were derived from the FinnGen Release 10 database. The MR Analysis methods included the inverse-variance-weighted (IVW), MR-Egger, and weighted median.

The risk of migraine was reduced mediated by CD5 with phosphatidylinositol (18:1_18:2), sphingomyelin (d34:1), and sphingomyelin (d38:1). The risk of migraine and MA was reduced mediated by CD6 with sphingomyelin (d40:1) and sphingomyelin (d42:2). The risk of migraine and MA was increased mediated by CD6 with phosphatidylethanolamine (O-16:1_18:2).

CD5 and CD6 were found to be related to migraine. CD5 and CD6 may affect migraine by immunological dysregulation-induced neuroinflammation. Six plasma lipids are associated with two cytokines, indicating that lipid metabolism participates in neuroinflammation, and T cells may be part of it. Plasma levels of lipids were associated with the risk of migraine. The circulating cytokines may serve as mediators in the pathway from plasma lipids to migraine.

CD5 and CD6 appeared to mediate the causal relationship between plasma lipids and migraine or MA, including four kinds of sphingomyelin, one phosphatidylinositol, and one phosphatidylethanolamine.

Sleep disorders (SD) affect approximately 25% of the global population. Traditional Chinese Medicine (TCM) formulas have been shown to alleviate SD by modulating endogenous melatonin. This study used data mining, network pharmacology, and meta-analysis to identify key herbal pairs from TCM formulas and the mechanism of action.

Literature was retrieved from PubMed, Web of Science, Embase, Cochrane Library, CNKI, Wanfang Data Information Site, China Science and Technology Journal Database, and SinoMed. R was used for frequency and association rule analysis, SPSS for clustering, and Cytoscape, STRING, Gene Ontology, and KEGG enrichment analyses were utilized to explore targets, protein-protein interactions, and pathways. A meta-analysis using the Metan command was performed to assess the optimal herbal pairs for SD treatment.

Data mining identified 77 commonly used herbs, revealing four advantageous herbal pairs: PAEONIAE RADIX ALBA (PRA)-BUPLEURI RADIX (BR), COPTIDIS RHIZOMA (CR)-CINNAMOMI CORTEX (CC), PORIA (PA)-BUPLEURI RADIX (BR), and ZIZIPHI SPINOSAE SEMEN (ZSS)-MARGARITIFERA CONCHA (MC). Network pharmacology showed that (PRA-BR)-SD, (PA-BR)-SD, (CR-CC)-SD, and (ZSS-MC)-SD targeted CACNA1D, GRIN2A, AGT, and ATP1A1 via prion diseases, nicotine addiction, neuroactive ligand-receptor interaction, and cardiac muscle contraction pathways, respectively.

Research shows that CACNA1D could regulate Ca²⁺ inward flow, avoid mitochondrial dysfunction in prion diseases, and reduce ROS generation, thus indirectly maintaining MT levels and sleep. GRIN2A as an amygdala hub gene closely related to daily smoking, combining brain transcriptome analysis and tobacco consumption GWAS data. The sleep regulation mechanism of MT relies on the neuroactive ligand-receptor pathway. As a neuroactive ligand, MT triggers sleep-promoting physiological responses by activating the G-protein-coupled receptors MT1 and MT2 and transmitting “night” signals to the relevant neural networks. Insufficient MT secretion or circadian rhythm disruption might lead to abnormal blood pressure rhythms accompanied by sympathetic overactivation, increasing the risk of insomnia and cardiovascular disease. ATP1A1 is a key molecule in the maintenance of electrochemical gradients in cardiac myocytes through the modulation of the Na⁺/K⁺ homeostasis affects myocardial excitability, calcium kinetics, and contractile function.

Meta-analysis and network pharmacology suggest that the PA-BR pair might offer superior efficacy by modulating membrane potential and nicotine addiction pathways, targeting GRIN2A, GRIN1, GRIN3A, and GRIN2B to regulate melatonin levels.

Observational studies have linked diabetes with cataracts, but they cannot fully elucidate the underlying causes and mechanisms. This investigation aims to evaluate the causal relationship between genetically predicted diabetes and cataract risk utilizing Mendelian randomisation (MR) techniques.

We identified single nucleotide polymorphisms (SNPs) with a significant threshold of P < 5×10^-8 as instrumental variables from genome-wide association study datasets pertaining to Type 1 (finn-b-E4_DM1, n=189,113), Type 2 diabetes (finn-b-E4_DM2, n=215,654), and cataract (ukb-b-8329, controls=136,388, cataract=14,254). Various Mendelian randomisation methods were employed, including inverse-variance weighted (IVW), MR-Egger, weighted median, simple mode (SM), and weighted mode analyses. Additionally, sensitivity analyses were conducted to assess the robustness of the findings, encompassing tests for heterogeneity, pleiotropy, and leave-one-out assessments. A multivariable (MVMR) approach was used to account for potential confounders, such as obesity (IEUA-92, controls = 47468, obesity = 2896), smoking (ukb-a-16, n = 337030), and alcohol consumption (IEUA-1283, n = 112,117).

The analysis included 12 SNPs, which were derived from loci specifically associated with Type 1 diabetes and known to govern immune-inflammatory and metabolic pathways. The genetically-predicted Type 1 diabetes was found to elevate cataract risk significantly (OR=1.003, 95% CI: 1.001–1.005, P=0.001). The results of the sensitivity analyses corroborated the robustness of these findings, showing no significant heterogeneity (Cochran Q, P value = 0.73) or pleiotropy (MR-Egger intercept, P value = 0.38). Furthermore, multivariable MR demonstrated that the impact of diabetes on cataract risk remained significant after adjustment for multiple lifestyle factors.

We provide novel MR evidence that Type 1 diabetes causally increases the risk of cataract through the synergistic activity of immune dysregulation, chronic inflammation, and metabolic disturbance, with immune-metabolic crosstalk as the primary driver.

T1D causally increases the risk of cataract through the disruption of immune-inflammatory and metabolic pathways. Targeting immune-metabolic interactions may offer novel therapeutic strategies for preventing diabetic cataracts.

This study investigated the anti-ovarian cancer (OC) effects of Shuangzi Powder (SZP) and its regulatory impact on the tumor microenvironment.

This study employed systems biology approaches, integrating molecular docking and experimental validation, to explore the pharmacological mechanisms of SZP in OC treatment. To identify potential bioactive compounds and target genes of SZP, network pharmacology, protein–protein interaction network analysis, Gene Ontology (GO) analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were conducted.

Among the 11 bioactive ingredients identified in SZP, 1,767 potential therapeutic targets were predicted, while 2,637 differentially expressed genes were found to be associated with OC. KEGG pathway analysis revealed significant enrichment in pathways related to cancer, apoptosis, the PI3K-Akt signaling pathway, and the PD-L1/PD-1 checkpoint pathway. Treatment of A2780 cells with β,β-Dimethylacrylshikonin (DMAS) inhibited cell viability, migration, and invasion. Moreover, DMAS downregulated the expression of cell cycle- and apoptosis-related genes (CCNB1, CHEK1, CCNE1, and PARP1) and upregulated the immune checkpoint gene PD-L1.

These findings indicate that multiple components, targets, and pathways are involved in OC treatment by SZP.

DMAS, one of the bioactive ingredients of SZP, was predicted and preliminarily validated to exert inhibitory effects on OC cells, mainly through the regulation of the cell cycle, apoptosis, and immune response, as demonstrated by molecular docking and experimental analyses.

Y-box binding protein 1 (YBX1), an RNA-binding protein capable of recognizing the 5-methylcytosine (m5C), plays a role in the development and progression of various cancers. In this study, we aim to investigate the functional mechanism of YBX1-mediated m5C modification in Bladder Cancer (BCa).

The impact of YBX1 on glycolysis and biological functions in BCa cells was evaluated through a set of in vitro experiments. The underlying mechanisms involving YBX1, Transmembrane 4 L six family 1 (TM4SF1), and β-catenin/C-myc in BCa and their relationship were investigated using RNA immunoprecipitation (RIP), m⁵C-RIP, Actinomycin D, and luciferase reporter gene assays.

BCa cells exhibited elevated expression levels of YBX1 compared to human transitional bladder epithelial cells. YBX1 knockdown inhibited BCa cell proliferation, migration, and invasion while also attenuating glycolytic activity, as evidenced by reduced glucose uptake, lactic acid production, and ATP synthesis. Mechanically, we found that YBX1-dependent m⁵C modification promoted the stability of TM4SF1 mRNA, thereby upregulating TM4SF1 expression and subsequently activating the β-catenin/C-myc signaling. Furthermore, we discovered that overexpression of β-catenin could reverse the inhibitory effects of TM4SF1 silencing on proliferation and glycolysis in BCa cells.

This study has refined the mechanism of BCa progression, but the clinical significance and in vivo functions of the YBX1/TM4SF1 axis still require further verification.

YBX1 stabilizes TM4SF1 mRNA via m⁵C modification in BCa, activating β-catenin/c-Myc signaling to drive tumor growth and glycolysis. This reveals a novel therapeutic target for BCa.

Barberry Root (Sankezhen, SKZ), a traditional Uyghur herb from Xinjiang, China, has been shown to alleviate diarrhea-predominant irritable bowel syndrome (IBS-D); however, its molecular mechanisms remain unclear. This study aimed to systematically predict SKZ’s therapeutic targets and pathways for IBS-D using computational and experimental integration.

Active SKZ compounds and targets were sourced from TCM-Suite, BATMAN-TCM, and related databases. IBS-D targets were identified via DisGeNET and GeneCards, etc. Protein-Protein Interaction (PPI) networks, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed. Molecular docking and 100-ns Molecular Dynamics (MD) simulations validated compound-target stability. In vitro (LPS-induced RAW264.7 macrophages) and in vivo (IBS-D model rats, isolated intestinal segments) experiments verified SKZ’s effects.

Fifteen bioactive compounds and 85 overlapping targets were identified, with four key compounds [(R)-Reticuline, Ferulic acid 4-O-glucoside, Magnoflorine, SW 7] and 15 hub targets (e.g., ESR1, EGF, ALB) prioritized. Enrichment analyses linked targets to inflammation and intestinal motility pathways. Docking showed strong binding affinities (<-8.0 kcal/mol), and MD simulations confirmed stability. SKZ suppressed inflammatory mediators, downregulated CHAT/C-FOS/5-HT3R/5-HT4R mRNA, and antagonized acetylcholine/barium chloride-induced intestinal contractions.

The findings highlight SKZ’s synergistic role in ameliorating IBS-D via multi-pathway regulation, consistent with existing research on inflammation and neurotransmission, though limitations include the need for further validation of individual compounds.

SKZ exerts synergistic therapeutic effects on IBS-D by ameliorating inflammation and regulating neurotransmission and intestinal motility, potentially via NF-κB/MAPK, COX-2/PGE2, cholinergic/5-HT, and calcium/potassium channel pathways, forming a multidimensional network.

Sarcopenia (Sar) is an age-related loss of muscle mass and function. Propolis, a natural product with anti-inflammatory properties, may help prevent Sar, but its active components and mechanisms remain unclear.

Network pharmacology identified intersecting targets of propolis ethanol extract (PEE) and Sar. PPI and CTP networks highlighted key compounds and targets, verified by molecular docking. In vitro, apigenin (Ap), the predicted main compound, was tested on D-galactose-induced senescent C2C12 myoblasts via cell viability and Western blotting.

Twelve overlapping targets were identified between PEE and Sar, with TNFα and IL6 highlighted as hub targets. Network analysis determined Ap as the main active compound. Molecular docking revealed strong binding affinities of Ap with TNFα and IL6. In vitro experiments demonstrated that Ap significantly enhanced the viability and differentiation of senescent C2C12 cells, downregulated TNFα and IL6 expression, and inhibited JAK2 and STAT3 phosphorylation, indicating suppression of the JAK-STAT signaling pathway.

The findings suggest that PEE, primarily through Ap, alleviates Sar by targeting inflammatory pathways and suppressing JAK-STAT signaling, thereby promoting muscle regeneration. The integration of network pharmacology, molecular docking, and in vitro validation provides mechanistic insights supporting the therapeutic potential of PEE in Sar. Limitations include the absence of in vivo confirmation, which warrants further animal and clinical studies to validate these effects and explore translational applications.

This study identifies Ap as the key active compound in PEE that alleviates Sar by downregulating TNFα and IL6 and inhibiting the JAK-STAT pathway. The results provide a molecular basis for the use of propolis as a natural intervention for Sar and support its development as a functional food or therapeutic agent targeting age-related muscle degeneration.

Hua-Zhuo-Ning-Fu decoction (HZD) is a traditional Chinese medicine prescription that has been clinically used by Chinese medical master Wang Xinlu for treating psoriasis. However, the specific molecular mechanisms remain unclear.

To identify the effective compounds of HZD and psoriasis-related genes, we conducted comprehensive searches in public databases, including TCMSP, SwissTargetPrediction, Gene Cards, and OMIM. Based on the degree values, core genes of HZD against psoriasis were determined. Furthermore, the affinity energy between the active compounds of HZD and their core targets was validated via molecular docking. Finally, the anti-psoriasis effects and potential mechanisms of HZD were examined in M5-stimulated HaCaT cells in vitro and IMQ-induced psoriasis mice in vivo.

Network pharmacological analysis of HZD for psoriasis treatment identified 43 active components and 243 targets. Topological and molecular docking analyses identified interleukin (IL)-6 and tumor necrosis factor-α (TNF-α) as core targets for its anti-psoriasis effects. Specifically, the docking energy of isovitexin with IL-6 was lower (-7.30 kcal/mol), and that of baicalin with TNF-α was lower (-6.70 kcal/mol). KEGG analysis revealed that the main pathway was the PI3K/AKT pathway. HZD inhibited cell viability, inflammation, and oxidative stress in M5-induced HaCaT cells. Animal experiments demonstrated that HZD alleviated psoriatic dermatitis, histopathological features, and inflammation in IMQ-induced mice with psoriatic plaques. Notably, HZD inhibited the expression of TNF-α and IL-6 and the activation of the PI3K/AKT pathway both in vivo and in vitro.

Specific upstream/downstream regulators of the PI3K/AKT axis regulated by HZD still need to be explored. Further investigation is essential to clarify the functional relationship between the predicted targets and active components.

In summary, HZD potentially mitigated inflammatory responses by targeting the TNF-α and IL-6 proteins, interfered with the PI3K/AKT pathway, and consequently drove the anti-psoriatic effect in IMQ-induced mice. Our findings provide a theoretical basis for HZD’s clinical use in psoriasis treatment.

The comorbidity of myocardial ischemia reperfusion injury (MIRI) and depression (DEP) may worsen the prognosis of coronary heart disease surgery. Currently, research on medications and therapeutic mechanisms for MIRI combined with DEP is still insufficient. This study aims to explore the relationship between DEP and MIRI, and the therapeutic effects and mechanisms of Baihe Dihuang Danshen decoction (BDDSD) on DEP combined with MIRI.

SD rats were assigned to a final experimental framework of six groups (Sham, MIRI, DEP+MIRI, BDDSD, DEP drug control, MIRI drug control). DEP was induced via 6-week chronic unpredictable mild stress (CUMS), with BDDSD administered during the final 2 weeks. MIRI was then induced by 30-minute coronary artery ligation and 2-hour reperfusion. DEP severity was assessed using behavioral tests (open field, elevated plus maze, sucrose preference, forced swimming). MIRI outcomes were evaluated via infarct size, histopathology, serum markers (LDH, IL-6, IL-1β), myocardial oxidative stress (MDA, GSH, SOD, Fe²⁺), and NADPH/FSP1/CoQ10 pathway proteins (FSP1, CoQ10, FTL, NOX2, NOX4, COX2).

Compared with the MIRI group, DEP significantly exacerbated MIRI, manifested by increased serum IL-6 and IL-1β levels, enlarged infarction area, and aggravated oxidative damage (elevated MDA/Fe²⁺, decreased SOD/GSH). Compared with the DEP+MIRI group, BDDSD intervention relieved DEP of rats, and subsequently reduced infarction area; decreased serum LDH, IL-6, and IL-1β; lowered myocardial MDA and Fe²⁺ while increasing SOD and GSH; upregulated FSP1/CoQ10/FTL; and downregulated NOX2/NOX4/COX2 expression.

DEP can aggravate inflammation and oxidative stress, promoting cardiac ferroptosis, thereby exacerbating MIRI. Our results demonstrate that BDDSD alleviates MIRI-DEP comorbidity through a dual mechanism, mitigating depressive symptoms and inhibiting myocardial ferroptosis via modulation of the NADPH/FSP1/CoQ10 pathway. Although the efficacy of BDDSD is encouraging, its dose-effect relationship and long-term safety require further study.

BDDSD effectively treats DEP-MIRI comorbidity through its dual mechanism, mitigating DEP and protecting against myocardial ferroptosis. Our study not only offers a novel therapeutic strategy for patients with DEP requiring coronary heart disease surgery but also provides new targets for developing drugs to treat MIRI combined with DEP.

Chronic atrophic gastritis (CAG) is an important stage in the occurrence and development of gastric cancer, and the morbidity of CAG is increasing year by year. Qilianshupi Decoction (QLSP) is a Chinese herbal compound which has been proved to reverse CAG, but its mechanism remains unknown. We wanted to identify the main components of QLSP by mass spectrometry and liquid phase analysis, and investigate their potential pathways for CAG treatment in combination with network pharmacology.

The main active components of QLSP were identified by liquid chromatography and mass spectrometry. Combined with network pharmacology, the targets where the drugs may act were identified and verified by animal experiments. Rats were randomly divided into control group, model group, QLSP low-dose group, QLSP medium-dose group, QLSP high-dose group and Weifuchun group. Rat CAG model was prepared by “N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) + ethanol intragastric + ranitidine feed”. After the test, gastric tissues were taken for pathological staining and immunohistochemistry.

We identified 51 prototype components of QLSP and found that QLSP treatment of CAG was closely related to p53. In animal experiments, CAG results in the decrease of E-cadherin and the increase of N-cadherin, Vimentin, p53, SMAD2 and TGF-β (p<0.05). Both QLSP and Weifuchun can increase E-cadherin and decrease N-cadherin, Vimentin, p53, SMAD2 and TGF-β (p<0.05).

QLSP, a traditional Chinese medicine formula with multi-component and multi-target characteristics, has been shown in our study to effectively regulate key EMT (epithelial-mesenchymal transition) markers and their upstream/downstream regulators. In animal experiments, QLSP successfully reversed the EMT process in CAG model rats. This finding provides new therapeutic targets for CAG treatment, though several challenges remain in clinical translation: First, rat CAG models differ from human CAG in pathological features and disease progression, and species-specific physiological and metabolic variations may limit the extrapolation of these findings. Second, network pharmacology analysis identified IL-6, alongside TP53, as another critical target of QLSP in CAG intervention. Therefore, future studies should further clarify the molecular mechanisms by which QLSP modulates EMT via IL-6-related pathways and validate its efficacy through well-designed clinical trials, ultimately providing a comprehensive understanding of QLSP's therapeutic potential in CAG.

QLSP inhibits epithelial-mesenchymal transition (EMT) in gastric mucosal epithelial cells and prevents CAG, possibly by regulating p53/TGF-β signaling pathway.

Metabolic syndrome (MetS) is a metabolic disorder characterized by the accumulation of various risk factors, including obesity, dyslipidemia, hypertension and so on. Songyang Duanwu Tea (SYT) has a high value in nutrition and health care, and it is widely used in traditional Chinese medicine for weight loss. Nevertheless, the mechanisms of SYT improving MetS remain to be elucidated. The objective of this study was to investigate the molecular targets and potential mechanisms by which SYT may improve MetS based on animal experiments and bioinformatics.

MetS model mice were established by a high-fat, high-sugar, high-salt diet (HFSSD). Obesity, dyslipidemia, hypertension, hyperuricemia and non-alcoholic fatty liver disease (NAFLD) of MetS model mice were evaluated to assess the effect of SYT on the treatment effects of MetS. The bioactive components in SYT were identified by bioinformatics and verified by HPLC-QTOF-MS. The possible molecular targets and mechanisms of action were predicted and verified using bioinformatics.

SYT (1.2 g/kg) ameliorated obesity, dyslipidemia, hypertension, hyperuricemia and NAFLD in HFSSD-induced mice. Bioinformatics results suggested that the major bioactive components in SYT include the flavonoid components apigenin, kaempferol, luteolin and quercetin, and the polyphenolic component eugenol. HPLC-QTOF-MS further validated the presence of apigenin, kaempferol, luteolin and quercetin. These 4 bioactive components are involved in the regulation of SYT to improve MetS by regulating metabolism and attenuating inflammation, and the key targets include peroxisome proliferator-activated receptor gamma (PPARG), tumor necrosis factor alpha (TNFα), interleukin 1beta (IL1B) and interleukin 6 (IL6).

SYT effectively improved the MetS model mice induced by HFSSD. The potential mechanism may regulate PPARG and attenuate inflammatory targets: TNFα, IL1B and IL6 through 4 flavonoid components: apigenin, kaempferol, luteolin and quercetin.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a global health concern, even among lean individuals. The Gan-Jiang-Ling-Zhu decoction (GZD), a traditional Chinese medicine formula, shows therapeutic potential against MASLD. This study investigated the efficacy of GZD in lean MASLD and explored its mechanisms of action.

A lean MASLD mouse model was established using C57BL/6 mice fed with a cholesterol-rich Paigen’s diet (PD). Following successful modeling, mice were administered GZD (1.8, 3.6, or 7.2 g/kg) or vehicle control. Body weight, food intake, and liver weight were monitored. Hepatic steatosis and lipid accumulation were assessed via H&E and Oil Red O staining, while serum enzymes were quantified biochemically. Gut microbiota composition was analyzed by 16S rRNA gene sequencing, and bile acid (BA) profiles in feces and serum were measured using UPLC-TQMS.

Twelve weeks of PD feeding induced a lean MASLD phenotype characterized by reduced body weight alongside hepatic steatosis and dyslipidemia. The GZD treatment dose-dependently ameliorated liver steatosis and lipid accumulation, with the highest dose (7.2 g/kg) showing superior efficacy. GZD restored gut microbiota balance by reducing pathogenic bacteria and enriching taxa involved in BA metabolism, leading to increased fecal excretion of secondary BAs. Conversely, serum levels of secondary BAs were significantly reduced after GZD treatment.

Our study highlights the promising role of GZD in lean MASLD, the involvement of gut microbiota and related BA metabolism that aligns with emerging evidence that gut dysbiosis and disrupted BA homeostasis are central to MASLD pathogenesis, even in lean individuals. However, the mechanistic links between specific microbial changes, BA pool composition, and hepatic outcomes remain to be elucidated.

GZD ameliorates hepatic steatosis in lean MASLD mice, an effect associated with modulation of gut microbiota composition and increased fecal excretion of secondary BAs. These findings suggest the potential of GZD as a therapeutic option for lean MASLD through gut-liver axis regulation.

Bladder cancer (BLCA) is a highly aggressive malignancy with poor prognosis. DNA replication stress-related genes (DRSGs) hold prognostic significance in multiple cancers, and their expression patterns in BLCA may reveal novel biomarkers and therapeutic targets.

This study was designed using a public database and the Cancer Genome Atlas (TCGA). Genes associated with DNA replication stress in BLCA were discovered by analyzing data from the TCGA and GEO databases using bioinformatics tools. The prognostic gene expression profiles in BLCA cell lines were analyzed using Western blotting (WB). The motility capacity of BLCA cells was evaluated using the wound healing and Transwell migration assays, while cell growth was ascertained with the CCK-8 assay.

Five DRSGs with prognostic significance were identified, and a risk score model was constructed using univariate Cox regression and the Least Absolute Shrinkage and Selection Operator (LASSO) regression algorithm. Kaplan-Meier (KM) analysis showed worse Overall Survival (OS) in the high-risk group (P < 0.05). Gene Set Enrichment Analysis (GSEA) indicated involvement in tumor-related pathways. The nomogram effectively predicted OS in both training and validation cohorts. WB and functional assays confirmed gene expression and effects on BLCA cell proliferation and migration.

This study first validates DRSGs’ prognostic value in bladder cancer, highlighting potential biomarkers and targets. Limitations include reliance on public data and in vitro tests. Future research should use multicenter cohorts and animal models to confirm clinical relevance.