Combinatorial Chemistry & High Throughput Screening - Volume 28, Issue 13, 2025

Oral cancer poses a significant threat to public health worldwide. In addition, because many chemotherapy treatments have negative side effects, natural herbs may be beneficial for oral cancer therapy. Achyranthes aspera (AA), a potential medicinal herb, exerts various pharmacological and biochemical activities.

The present study aimed to predict the anti-oral cancer potential of AA using in silico tools and cell death by in vitro testing.

A total of fourteen bioactive constituents from AA herb were selected using phytochemical databases. The toxicity of AA herb extract was analysed through MTT assay against oral carcinoma A253 cell line. The binding activities of the phytocomponents against serine/threonine-specific protein kinases isoforms, namely Akt1 (PDB ID: 3qkk) and Akt2 (PDB ID: 2jdo) proteins, were analysed using Discovery Studio 2021 and PyRx docking software.

Cell viability data revealed that AA extract decreased the viability and reduced the number of live cells of the oral carcinoma A253 cell line in a dose-dependent manner. The half-maximal concentration (IC50) value of AA was assessed as 204.74 µg/ml. Based on binding affinity, saponin C (-CDOCKER energy = -77.9862 Kcal/mol), oleanolic acid (-CDOCKER energy = -49.4349 Kcal/mol), spinasterol (-CDOCKER energy = -38.1246 Kcal/mol), 36,47-dihydroxyhenpentacontan-4-one (-CDOCKER energy = -32.4386 Kcal/mol), and 20-hydroxyecdysone (-CDOCKER energy = -31.9138 Kcal/mol) were identified as the best compounds against Akt1, while, compounds saponin C (-CDOCKER energy = -134.412 Kcal/mol), oleanolic acid (-CDOCKER energy = -90.0846 Kcal/mol), spinasterol (-CDOCKER energy = -78.3213 Kcal/mol), 20-hydroxyecdysone (-CDOCKER energy = -80.1049 Kcal/mol), and ecdysone (-CDOCKER energy = -73.3885 Kcal/mol) were identified as Akt2 inhibitors. These top compounds fulfilled drug score values, pharmacokinetic and physicochemical characteristics, and drug-likeness parameters.

The present findings reveal that the lead phytomolecules of AA could be effective and developed as a prospective drug against oral cancer.

Autoimmune thyroiditis (AIT) is the most common autoimmune thyroid disease. In recent decades, its incidence and prevalence have sharply increased. Yiqi Huatan Huoxue recipe is a traditional Chinese medicine formula we use to treat AIT. Its clinical efficacy is clear, but the specific mechanism remains unclear. This study aims to explore whether pyroptosis mediated by the SIRT1/NF-κB/NLRP3 Pyroptosis signaling pathway is one of the therapeutic mechanisms of Yiqi Huatan Huoxue recipe.

Forty 8-week-old female NOD.H-2h4 mice were randomly divided into four groups: the normal group (NG), model group (MG), Yiqi Huatan Huoxue recipe group (YG), and western medicine group (selenium yeast tablet, SeG). The normal group was gavaged with distilled water, while the remaining groups were gavaged with 0.05% sodium iodide (NaI) solution for 8 weeks. After the AIT animal model formed naturally, the mice were euthanized by gavage after 8 weeks. Hematoxylin-eosin staining was used to observe thyroid tissue changes, and enzyme-linked immunosorbent assay (ELISA) was used to detect serum anti-thyroglobulin antibodies (TGAb) and mouse anti-thyroid peroxidase antibodies (TPOAb). Real-time quantitative PCR (qRT-PCR), Western blot, and immunohistochemistry were used to detect the expression of sirtuin 1 (SIRT1), nuclear factor κB p65 (NF-κB p65), nod-like receptor protein 3 (NLRP3), Absent in melanoma 2(AIM2)、 apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), Cysteinyl Aspartate-Specific Protease-1(caspase-1), gasdermin D (GSDMD), interleukin (IL)-1β 、BCL2- Associated X(Bax)、B-cell lymphoma-2(Bcl2)and Caspase-3 in thyroid tissue.

Compared with NG group, the thyroid structure of MG group rats was severely damaged, lymphocyte penetration was obvious, serum TGAb and TPOAb levels were significantly increased, the expression levels of NF-kB p65, NLRP3, AIM2, ASC, Caspase-1, GSDMD, IL-1β, Bax, Caspase-3 mRNA and protein were significantly increased, and the expression levels of SIRT1 and Bcl2 mRNA and protein were significantly decreased. Compared with the MG group, the thyroid structural damage and lymphocyte invasion of rats in each treatment group were improved. The serum mRNA and protein expression levels of TGAb, TPOAb, NF-kB p65, NLRP3, AIM2, ASC, Caspase-1, GSDMD, IL-1β, Bax, Caspase-3 were significantly reduced, and the expression levels of SIRT1 and Bcl2 mRNA and protein were significantly increased

Yiqi Huatan Huoxue recipe can reduce thyroid structural damage and apoptosis in AIT mice. The mechanism may be related to the up-regulation of SIRT1 and NF kB deethylation, and the inhibition of NLRP3 and AIM2-mediated pyroptosis.

Current evidence highlights clear cell renal carcinoma (ccRCC) as the most prevalent form of kidney cancer despite ongoing challenges in treating advanced-stage disease. Integrin subunit beta 3 (ITGB3) has recently emerged as a critical player in tumorigenesis, prompting our investigation into its role in ccRCC. This study aimed to elucidate the mechanisms responsible for ITGB3 downregulation and evaluate its clinical significance, particularly regarding its impact on the immune landscape within ccRCC.

We first conducted analyses utilizing data from both TCGA and GEO datasets to explore ITGB3 expression in ccRCC tissues. Subsequently, we evaluated the association between ITGB3 expression levels and patient prognosis and pathological staging. Pathway and functional enrichment analyses were performed to assess correlations between ITGB3 and immune and methylation-related pathways. Additionally, we examined the relationship between ITGB3 transcriptional expression and DNA hypermethylation. A prognostic risk model was developed using LASSO-based analysis on selected ITGB3-associated DNA methylation probes. Immunohistochemistry (IHC) analysis, alongside TIMER and ssGSEA results, was utilized to investigate ITGB3 expression and its association with immune cell infiltration.

Our analyses revealed significant downregulation of ITGB3 mRNA expression in ccRCC tissues compared to other members of the ITGB family, consistent across TCGA and GEO datasets. Higher ITGB3 expression correlated with improved prognosis and lower pathological stage in ccRCC patients. Pathway and functional enrichment analyses demonstrated positive correlations between ITGB3 and immune and methylation-related pathways, while ITGB3 transcriptional expression showed a negative correlation with DNA hypermethylation. The established prognostic risk model identified a high-risk group with poorer survival probabilities than the low-risk group. Immunohistochemical quantification revealed a positive correlation between CD4⁺ and CD8⁺ immune cell infiltration and ITGB3 expression.

Overall, our study provides compelling evidence supporting the significant role of ITGB3 in ccRCC immunity. The downregulation of ITGB3, coupled with its association with better prognosis and immune activation, suggests its potential as a therapeutic target and prognostic marker for this patient population.

Bushen Zhuyun Decoction (BSZY), a traditional Chinese herbal prescription has shown promising effects on gynecological infertility, but the mechanism for endometrial receptivity is still unclear. This study aimed to investigate the regulatory effects of BSZY on endometrial receptivity, which plays a key role in colonization of embryo, and its regulatory mechanisms associated with NF- κB/NLRP3 pathway.

SD rats at reproductive age with affected endometrial receptivity was established using mifepristone (RU486), and the regulatory effects of BSZY on endometrial receptivity were evaluated by H&E staining, and changes in sex hormones by ELISA and Western blot. Moreover, human endometrial RL95-2 cells were treated with H2O2, and inflammatory cytokines in rats and RL95-2 cells were analyzed by ELISA. The activation of NF-κB/NLRP3 signaling pathway in RL95-2 cells were characterized using immunofluorescence and Western blot. Mitochondrial morphology and function in RL95-2 cells were observed by transmission electron microscope and cell mitochondrial stress test.

BSZY increased uterine endometrial thickness and attenuate histopathological changes induced by RU486. BSZY can regulate endometrial estrogen receptor and progesterone receptor, and the levels of sex hormones and inflammatory cytokines in pregnant rats. BSZY-containing serum also showed strong anti-inflammatory and cytoprotective effects in vitro. In addition, BSZY-containing serum inhibited the activation of NF-κB/NLRP3 signaling pathway, and improve mitochondrial morphology and function in RL95-2 cells.

BSZY can improve endometrial receptivity, potentially by improving mitochondrial morphology and function to inhibit the activation of NF-κB/NLRP3 signaling pathway in endometrial cells, thus regulate inflammation to improve endometrial receptivity.

Zinc finger C3H1-type containing (ZFC3H1) might regulate RNA processes. However, research lacks the prognostic value of ZFC3H1 in hepatocellular carcinoma (HCC).

The study analyzed ZFC3H1 expression in HCC cells and its correlation with patient prognosis using transcriptomics, immunohistochemistry, and quantitative real-time reverse transcription PCR, as well as single-cell RNA expression data. Additionally, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were used to investigate the potential ZFC3H1-related cellular functions and signaling pathways. The impact of ZFC3H1 expression on the tumor microenvironment and tumor mutational burden (TMB) was assessed using the ESTIMATE algorithm. Cell-based assays, including cell counting kit 8, proliferation, colony formation, cell cycle, wound healing, and Transwell assays, were conducted to evaluate the influence of ZFC3H1 on hepatocellular carcinoma proliferation and migration.

ZFC3H1 is upregulated in HCC and linked to tumor progression. High ZFC3H1 expression is a prognostic risk factor for HCC, according to Kaplan-Meier and Cox regression analyses. ESTIMATE analysis suggested that ZFC3H1 reduces immune cell infiltration and increases the TMB. Patients with low ZFC3H1 expression might respond better to immunotherapy. High ZFC3H1 expression is associated with increased half-maximal inhibitory concentration (IC50) of sorafenib. Functional experiments demonstrated that reducing ZFC3H1 expression inhibited HCC cell proliferation and migration.

ZFC3H1 is upregulated in HCC, promoting the proliferation and migration of liver cancer cells, impacting the prognosis of HCC patients and the effectiveness of immunotherapy. ZFC3H1 might serve as a therapeutic target and biomarker for HCC.

Increasing evidence indicates that microRNAs (miRNAs) play a crucial role in modulating tumor growth. This study is centered on investigating the contribution of miR-25 to the progression of Renal Cell Carcinoma (RCC).

The investigators examined the expression levels of miR-25 and ADAMTS16 in RCC samples and cell lines. The association between miR-25 and ADAMTS16 was validated via a luciferase reporter assay. Cell viability, apoptosis, migration, and invasion were evaluated utilizing CCK-8 and flow cytometry techniques, while the expression levels of ADAMTS16,β-catenin, GSK-3β, and p-GSK-3β were assessed through western blot analysis.

The investigation revealed elevated expression levels of miR-25 in RCC tissues. Subsequently, ADAMTS16 was identified as a target of miR-25. Increased miR-25 levels were associated with decreased expression of ADAMTS16, resulting in enhanced cell viability and diminished apoptosis. Conversely, inhibition of miR-25 led to decreased cell viability, proliferation, and migration. Additionally, the researchers observed that miR-25 triggered the phosphorylation of GSK-3β and β-catenin while leaving the total GSK-3β level unaffected.

This study suggests that miR-25 regulates the expression of ADAMTS16 through the Wnt/β-catenin signaling pathway, providing new insights into the cause and potential treatment of RCC.

Premature ovarian insufficiency [POI] is a disease characterized by a premature decline in ovarian function before the age of 40. In China, Ligustrum lucidum [FLL] has long been used to improve ovarian function and treat POI.

This study aims to verify the effect of FLL on POI through network pharmacology, molecular docking, and in-vitro cell experiments.

A total of 13 active substances were screened in FLL, including including quercetin, taxifolin, luteolin, kaempferol, and beta-sitosterol. Then, network analysis found that FLL may exert effects on POI through 10 targets, including AR, ESR1, ESR2, KDR, CYP19A1, CLPP, GC, MMP3, PPARG, and STS. According to GO and KEGG enrichment analysis, FLL is associated with mechanisms related to estrogen, including steroid hormone biosynthesis, ovarian steroidogenesis, and the estrogen signaling pathway. Molecular docking confirms the interaction between the active ingredients of FLL and CYP19A1, which encodes aromatase. CCK8 experiment confirmed that quercetin and taxifolin can enhance the proliferation of KGN granulosa cells, while quercetin, taxifolin, and kaempferol can inhibit the apoptosis of KGN granulosa cells. ELISA experiments have confirmed that quercetin, taxifolin, luteolin, and kaempferol can increase the synthesis of estradiol in KGN granulosa cells. WB confirms that quercetin can increase the expression level of CYP19A1 in KGN cells.

FLL can improve the proliferation, apoptosis, and synthesis of estradiol in ovarian granulosa cells, and has the potential to treat POI.

According to current worldwide cancer data, Prostate Cancer (PC) ranks as the second most common type of cancer and is the fifth leading cause of cancer-related mortality among men worldwide. PC in China has the 10th highest number of new cases and the 13th highest fatality rate, both of which show an ongoing annual increase. One of the significant challenges with prostate cancer is the difficulty in early detection, often resulting in diagnosis at intermediate or late stages, complicating treatment. Although hormonal therapy is initially successful in controlling the progression of prostate cancer, almost all tumors that respond to hormones eventually transform into Castration-resistant Prostate Cancer (CRPC) within 18-24 months of hormonal therapy. This poses clinical difficulties due to an absence of successful therapeutic approaches. Therefore, understanding the fundamental mechanisms of prostate cancer development, identifying effective therapeutic targets, and discovering reliable molecular biomarkers are crucial objectives.

CircRNA expression in plasma was assessed in 4 samples obtained from patients with Benign Prostatic Hyperplasia (BPH), and PC was detected through microarray probes. Statistical analysis of the expression of circDUSP22 and clinicopathological features was conducted. The investigation of target genes was conducted using luciferase reporter assays and bioinformatics analysis. The expression levels of circDUSP22, miR-18a-5p, and Solute Carrier Family 7 member 11 (SLC7A11) were assessed using a quantitative Real-time Polymerase Chain Reaction (qRT-PCR) assay. Cell invasion, migration, colony formation, and proliferation were evaluated using Transwell, wound healing, colony formation, and CCK-8 assays, respectively. RNA Immunoprecipitation (RIP) and dual-luciferase reporter assays were used to examine the connections among circDUSP22, miR-18a-5p, and SLC7A11. The impact of circDUSP22 on the expression of ferroptosis-related proteins, specifically SLC7A11, as well as its effects on Fe2+ and ROS were also examined.

In both plasma samples and PCa cell lines, there was a substantial elevation of circDUSP22 and SLC7A11 expression and a decline in miR-18a-5p expression. Suppression of circDUSP22 significantly impeded the migration, invasion, and proliferation of PC cells in vitro. The target gene of miR-18a-5p, SLC7A11, was found to be upregulated as an effect of circDUSP22's competitive binding to miR-18a-5p. Cellular experiments demonstrated that interference with circDUSP22 expression in DU145 and PC-3 cells led to increased ferroptosis and decreased SLC7A11 expression. The modulation of prostate cancer cell proliferation was reversed by either overexpressing SLC7A11 or inhibiting miR-18a-5p in response to the silencing of circDUSP22.

The circDUSP22 has been found to have a substantial effect on the development of ferroptosis in PC. It has been observed to influence the formation and evolution of this disorder by affecting the miR-18a-5p/SLC7A11 signaling pathway.

Dispel-Scar Ointment is used in Traditional Chinese Medicine to treat scarred tissue and increasing evidence has shown that DSO is a potent therapeutic; however, its exact mechanism remains unexplored.

This study explored the molecular mechanisms of action of DSO in scarring using network pharmacology, molecular docking, and experimental validation.

Public databases were applied to predict the bioactive ingredients and putative targets of DSO against scars. A compounds-targets network was constructed using the Cytoscape software. Molecular docking was performed to verify the correlation between the major positive ingredients and hub targets, visualised using PyMol 2.3. Enrichment analysis was implemented using ClueGo and FunRich to specify the biological capabilities and related pathways of hub targets. SwissADME software was used to predict the ADME capabilities of the protein-related active compounds between DSO and scar in order to analyse the absorption and permeation across cell membranes of DSO. We assessed the skin sensitizer potential of the bioactive compounds of DSO using Pred-Skin computational tool. Experimental validations were conducted to elucidate the influence of DSO on keloid fibroblast cells using the CCK-8, wound-scratch, cell reactive oxygen species, and western blot assays.

Network pharmacological analysis of DSO for scar treatment identified 146 ingredients and 1078 gene targets. Major targets included prostaglandin-endoperoxide synthase 2 matrix metallopeptidases and nitric oxide synthase 2. Molecular docking showed MMP2-flavoxanthin, MMP9-luteolin and MMP-9-kaempferol bound best to DSO. ClueGo analysis revealed 29 pathways (p<0.05) and FunRich 345 pathways (p<0.05), mainly toll-like receptor, TGF-β, interleukin-4/13, glypican, and tumour necrosis factor-related apoptosis-inducing ligand pathways. The results valued by the SwissADME and PreADMET tools illustrated that 12 compounds in DSO were almost permeable through the skin. Pred-Skin computational tool represented that these 12 bioactive compounds reflected skin sensitizer potential. Experimental analysis revealed that DSO could restrain the proliferation and migration of scar fibroblasts and facilitate their apoptosis in a concentration-dependent manner. DSO also decreased TGF-β1, -βR2, pSMAD2, pSMAD3, SMAD4, CoL1a1, and MMP2 expression.

Network pharmacology, molecular docking, and experimental validation showed DSO's feasibility in scar therapy. It may restrain scars through the TGF-β1/SMADs/MMPs signalling pathway, providing a basis for DSO's scar treatment application.

Although constitutive ginsenosides are credited with ginseng's remarkable anti-aging efficacy, the mechanism of action and bioactive components of ginsenosides are unclear.

The goal of the study was to examine the effect of ginsenosides on D-galactose (D-gal)-induced aging in rats and to figure out the underlying molecular mechanism using serum pharmacochemistry and network pharmacology.

Using behavioral, biochemical indexes, and histological analysis, ginsenosides were evaluated for their anti-aging effects in rats induced by D-gal, and effective ingredients absorbed in the blood were examined by ultra-performance liquid chromatography quadrupole time of flight coupled with mass spectrometry (UPLC-Q/TOF-MS) before being subjected to network pharmacology analysis.

As well as improving spatial learning and memory skills, Ginsenosides are known to regulate malondialdehyde (MDA), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) activity. In addition, it improved the ultrastructure of neurons in D-gal-induced rats' hippocampus. Seventy-four absorption components and metabolites of ginsenosides were identified in aging rat serum. According to a network pharmacology study, ginsenosides have anti-aging properties by modulating the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) and mitogen-activated protein kinases (MAPK) signaling pathways.

The potential mechanisms of the anti-aging effect of ginsenosides involve multiple components, targets, and pathways. These findings serve as a foundation for further research into the processes behind ginsenoside's anti-aging impact.

v-RAF murine sarcoma viral homolog B1 (BRAF) is one of the most frequently mutated kinases in human cancers. BRAF exhibits three classes of mutations: Class I monomeric mutants (BRAFV600), class II BRAF homodimer mutants (non-V600), and class III BRAF heterodimers (non-V600).

In this paper, the protein-ligand interaction site of all three mutants: BRAF monomer, BRAF homodimer BRAF2:14-3-32, and BRAF heterodimer BRAF:14-3-32:MEK (Mitogen extracellular Kinase) has been discussed. FDA-approved drugs still have limitations against all three classes of mutants, especially against the second and third classes. Using the DesPot grid model, 1114 new compounds were designed. Using virtual screening, the three PDB Ids 4XV2 for monomers, 7MFF for homodimers, and 4MNE for heterodimers were used for 1114 newly designed compounds.

Dabrafenib, encorafenib, sorafenib and vemurafenib were included as standard drugs. The top 10 hit molecules were identified for each protein. Additional binding studies were performed using molecular docking studies on the protein-ligand site of each PDB identifier. Absorption, distribution, metabolism, excretion (ADME) and toxicity studies were also performed.

It was identified that top-hit molecules had better binding and interaction activity than standard in all three classes of mutants.