Lidocaine is widely used for perioperative pain management, but repeated exposure may cause neurotoxicity, including neurological deficits. This study investigates mechanisms underlying cognitive decline induced by repeated lidocaine exposure. Eighteen-month-old mice received repeated clinically relevant lidocaine infusions over 3 days. Cognitive function was assessed by Morris water maze, Y-maze and open field tests. Hippocampal pathology was examined via TEM, Nissl staining, immunofluorescence for astrocyte polarisation and Aβ deposition, and western blot for tau, BDNF, TrkB, mTOR and autophagy proteins. The TrkB agonist 7,8-DHF was used to modulate BDNF/TrkB/mTOR signalling. Repeated lidocaine exposure impaired cognition and induced Alzheimer's-like hippocampal pathology, as evidenced by increased accumulation of Aβ and tau toxic proteins, along with neuronal death. It reduced BDNF expression, inhibited TrkB phosphorylation, and activated mTOR signalling, leading to autophagy inhibition and pathological protein accumulation. Lidocaine shifted astrocytes towards the neurotoxic A1 phenotype, decreasing neuroprotective A2 astrocytes and BDNF synthesis. TrkB agonist treatment restored signalling, enhanced autophagy and improved cognitive deficits and pathology. Repeated lidocaine exposure promotes A1 astrocyte increase and A2 decrease, inhibiting autophagy via the BDNF/TrkB/mTOR pathway, resulting in toxic protein deposition and Alzheimer's-like cognitive impairment.

This study aims to elucidate the correlation between the lactylation-related genes and the progression of knee osteoarthritis (KOA). Integrating genome-wide association study data and expression quantitative trait locus data, Mendelian randomisation (MR) and summary-data-based MR analyses assessed the correlation between lactylation-related genes and KOA, validated candidates in chondrocytes by RNA-seq, western blotting (WB) and quantitative reverse transcriptase PCR (qRT-PCR). Moreover, CCK-8, transwell migration and scratch assays were conducted to assess the proliferation/migration. Finally, mediation analysis was utilised to explore the downstream mechanisms. LDHA and LDHC expression was significantly downregulated in the KOA group, as confirmed by RNA-seq, WB and qRT-PCR analyses. Functional experiments also confirmed that the down-regulation of LDHA or LDHC expression could produce a chondrocyte proliferation and migration inhibitory effect similar to that of the OA group, while restoring the expression of these two genes could significantly reverse this phenotype. Furthermore, mediation analysis revealed that CD38 on IgD + CD24- B lymphocytes mediated the protective effect of these genes against KOA, with CD25+ memory B cells also serving as mediators for LDHC's impact on KOA. LDHA and LDHC are identified as therapeutic targets for KOA, providing promising targets and ideas for the development of new treatment strategies.

Lung cancer prognosis varies significantly among patients, highlighting the need for accurate prediction tools. Emerging evidence suggests that the immune microenvironment plays a crucial role in lung cancer progression and treatment response. We collected RNA expression profiles and clinical data of lung cancer patients from TCGA and GEO databases. Differential expression analysis identified 276 lung cancer-associated genes using strict statistical criteria (logFC > 1, FDR < 0.05). Unsupervised consensus clustering divided patients into 'lung cancer-related' and 'non-lung cancer-related' subgroups. We evaluated 10 machine learning algorithms and 101 algorithmic combinations for prognostic model development. Single-cell RNA sequencing data were analysed using Seurat and CellChat to investigate immune cell interactions within the lung cancer microenvironment. Our prognostic model demonstrated excellent predictive performance with AUC values of 0.874, 0.891 and 0.925 at 1, 2 and 3 years, respectively (C-index = 0.874). Six key immune genes (TLR2, TLR4, CCR7, IL18, TIRAP and FOXP3) showed cell-type specific expression patterns in the lung cancer microenvironment. Intercellular communication analysis revealed complex signalling networks between B cells, T cells, NK cells and dendritic cells. CIBERSORT and ESTIMATE analyses confirmed significant differences in immune infiltration between high-risk and low-risk patients, with distinct patterns of T cell subsets, macrophages and dendritic cells. This study provides a reliable prognostic tool for lung cancer and offers insights into the critical role of the immune microenvironment in lung cancer pathogenesis. Our findings may guide the development of personalised immunotherapy strategies for lung cancer patients.

Prostate cancer (PCa) is the second most prevalent malignancy in men, and therapeutic options become severely limited once androgen deprivation therapy (ADT) fails. This study evaluated the antitumor activity of Annonacin, a natural acetogenin, alone or in combination with docetaxel (DTX) in PCa. The antitumor effects and underlying mechanisms of Annonacin and/or DTX were investigated in DU145 cells and a xenograft mouse model by assessing proliferation, migration, apoptosis, colony formation, DNA damage and FAK expression and distribution. Through an integrated strategy combining network pharmacology and a series of in vitro assays, the findings demonstrated that Annonacin exerts significant antitumor activity by inducing DNA damage and downregulating FAK expression and localisation. Co-treatment with DTX further enhanced these effects, with combination index (CI) values < 1, indicating strong synergism. In vivo, the combination therapy achieved more than 74% tumour growth inhibition (p < 0.0001), accompanied by increased tumour cell death, reduced Ki-67 expression and elevated γ-H2AX levels. Collectively, these findings demonstrate that Annonacin exerts potent antitumor activity and synergistically enhances DTX efficacy by promoting DNA damage and suppressing FAK signalling, supporting its potential as a promising adjuvant candidate for PCa treatment.

Considering the distinct etiological pathways and molecular characteristics of different lung cancer subtypes, it is crucial to develop subtype-specific prevention strategies and therapeutic targets. This study aimed to identify protein biomarkers and potential therapeutic targets for specific subtypes of lung cancer by integrating population-based observational studies and Mendelian randomisation (MR) analyses. The cohort study was conducted in the UK Biobank, including about 47,000 participants whose blood samples were measured for 2,923 unique proteins and who were followed for the development of lung cancer. Two-sample MR was performed leveraging publicly available data from genome-wide association studies (GWAS) and protein quantitative trait loci (pQTL). Proteins were prioritised based on consistent associations across logistic regression, MR, transcriptomic validation and sensitivity analyses. Tier 1 proteins passed all evaluations, including GP1BA (squamous cell carcinoma) and ACADSB (small cell carcinoma). Tier 2 proteins, supported by transcriptomic evidence but not sensitivity analyses, included AGRN, ITGB2, SEPTIN3 (adenocarcinoma) and DPP10 (squamous cell carcinoma). Tier 3 proteins, supported by logistic regression and MR only, included CD5L, GNPDA, ACAN, C7, DMP1, HEPH, CEACAM6, COX6B1, CPXM2 and IL12RB2. Druggability evaluation suggests that existing drugs targeting ITGB2, GP1BA, ACADSB and COX6B1 could potentially be repurposed for the treatment of specific lung cancer subtypes.

The underlying mechanisms of sepsis-induced myocardial dysfunction (SIMD) remain elusive, and no targeted therapies currently exist. This study aimed to explore the expression features and functional effects of cholesterol 25-hydroxylase (CH25H) in SIMD in vitro. CH25H was identified as an upregulated gene related to SIMD through bioinformatics analysis. Its upregulation was validated in the myocardial tissue of SIMD mice as well as in lipopolysaccharide (LPS)-induced primary cardiomyocytes and AC16 cells. CH25H overexpression elevated 25-hydroxycholesterol levels and aggravated oxidative stress, mitochondrial dysfunction, apoptosis, and NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome and NF-κB pathway activation in AC16 cells. The effect of CH25H overexpression was similar to that induced by LPS treatment. Conversely, silencing CH25H attenuated these LPS-induced injuries. Furthermore, CH25H overexpression exacerbated oxidative stress, mitochondrial dysfunction, and apoptosis in LPS-stimulated AC16 cells, and these effects of CH25H overexpression can be counteracted by the NLRP3 inhibitor. In conclusion, CH25H may promote LPS-induced cardiomyocyte injury through NLRP3/NF-κB pathway activation.

Dyslipidaemia has been implicated in osteonecrosis through some clinical studies. However, a direct causal relationship between hyperlipidaemia and osteonecrosis remains unconfirmed, and whether lipid-lowering agents could be used to treat osteonecrosis remains unclear. This study aimed to investigate the causal role of lipid traits in osteonecrosis using Mendelian randomisation (MR) analysis, assess the potential effects and mechanisms of lipid-lowering drug targets on osteonecrosis risk and validate these findings through experimental approaches. Genome-wide association study (GWAS) data were used to analyse lipid traits, drug targets and FinnGen osteonecrosis. Statin effects were further studied in a rat model of steroid-induced osteonecrosis and in vitro cell models. MR analysis revealed a significant association between LDL-C and increased osteonecrosis risk. Genetic mimicry of HMGCR inhibitors was associated with reduced osteonecrosis risk, which was validated through colocalisation. Stem cell growth factor-β (SCGF-β) was identified as a mediator of 21.3% of HMGCR inhibitors' effect on osteonecrosis risk. Further studies confirmed simvastatin's alleviating effect on SONFH, suggesting that simvastatin promotes osteogenesis and inhibits adipogenesis of mesenchymal stem cells (MSCs), partly mediated by SCGF-β upregulation, which activates the Wnt signalling pathway. Our findings supported dyslipidaemia as a causal factor for osteonecrosis, highlighting HMGCR as a promising therapeutic target.

Sjögren's Syndrome (SS) and Type 1 Diabetes (T1D) are autoimmune disorders that can co-occur in patients, leading to complex clinical presentations. Despite observational evidence of their co-occurrence, the underlying genetic mechanisms remain poorly understood. To investigate the shared genetic factors and pathways between SS and T1D, we conducted a comprehensive analysis using multiomic approaches. Conditional and conjunctional false discovery rate analyses were performed to identify genetic polygenicity and overlap between the two diseases. Functional annotation and pathway analysis identified SNPs with regulatory potential. Furthermore, Mendelian Randomization (MR) analyses were employed to investigate causal associations between gene expression and disease risk. Single-cell differential gene expression analysis was also employed to validate the associations of risk genes with T1D and SS. Our analysis identified 36 shared loci, revealing common genetic enrichment between SS and T1D. Functional annotation and pathway analysis revealed 52 credible genes involved in cysteine-related processes, apoptotic signalling and immune responses. MR analyses revealed that AC007283.5 was positively linked with both SS and T1D, while PLEKHM1 and CRHR1-T1 were negatively associated. Additionally, CERS2 was positively associated with SS, DEF6 was positively associated with T1D, and KANSL1-AS1 was negatively associated with T1D, indicating the presence of complex regulatory mechanisms. Moreover, Single-cell differential gene expression analysis confirmed the dysregulation of risk genes in SS and T1D. This study identified shared genetic factors and pathways underlying SS and T1D, highlighting cysteine-related processes and apoptotic signalling. The findings underscore the complex interplay of autoimmunity and the need for targeted treatments addressing their common mechanisms.

Long noncoding RNAs (lncRNAs) are emerging as key regulators in cardiovascular diseases. This study investigated the role of lncRNA LINC01605 in aortic dissection (AD) pathogenesis through its effects on vascular smooth muscle cells (VSMCs). Bioinformatics analysis of GEO datasets (GSE107844, GSE147026) identified LINC01605 as differentially expressed in AD. Its expression was validated in human aortic tissues and VSMCs using RT-qPCR and FISH. Functional assays (CCK-8, Transwell, Western blot) assessed VSMC proliferation, migration, phenotypic switching and autophagy. SGK1 was predicted as a target via bioinformatics and confirmed by RIP assays. Ang II-induced AD mice with LINC01605 knockdown were used for in vivo validation. LINC01605 was significantly upregulated in AD aortic tissues and VSMCs. Functional studies demonstrated that LINC01605 promoted VSMC proliferation, migration, invasion, phenotypic switching and autophagy, particularly under Ang II stimulation. Mechanistically, LINC01605 targeted SGK1 to regulate VSMC function. Knockdown of LINC01605 alleviated AD pathology in mice, modulating synthetic phenotype and autophagy markers. LINC01605 plays an important role in AD. It regulates the function of VSMCs by targeting SGK1 and promotes the pathological process of AD. LINC01605 may be a potential target for AD treatment, providing new directions for the mechanism research and treatment strategies of AD.

High-risk human papillomavirus (HPV) is a central factor in cervical cancer development, largely due to its E6 and E7 oncoproteins that disrupt normal cellular regulation. This study explored the influence of high-risk HPV on the expression of PI3K and the long non-coding RNAs (lncRNAs) MALAT1, H19 and LINC00460 in cervical cells. Using a case-control design, cervical liquid samples from 50 HPV-positive patients and 20 healthy controls were analysed via quantitative real-time PCR, with statistical methods employed to assess correlations between viral oncoproteins and target gene expression. Results demonstrated a significant upregulation of PI3K (24.59-fold change, p < 0.036), MALAT1 (9.75-fold change, p < 0.005), LINC00460 (1.15-fold change, p < 0.013) and H19 (7.1-fold change, p < 0.018) in HPV-infected samples, indicating their potential role in HPV-mediated oncogenesis. Although correlation analysis revealed trends between E6/E7 and certain lncRNAs, these were not statistically significant. Overall, these findings deepen our understanding of the molecular changes linked to high-risk HPV infections and identify PI3K, MALAT1 and H19 as promising biomarkers and therapeutic targets for cervical cancer. Future studies should further investigate these interactions to enhance early detection and improve treatment strategies for HPV-associated malignancies.

Pyroptosis is a gasdermins-dependent programmed cell death (PCD) characterised by progressive cellular swelling and plasma membrane rupture (PMR). This process releases intracellular contents that amplify inflammatory cascades and immune activation, involving the pathogenesis of various disorders such as tumours, heart and vascular diseases, diabetic complications and inflammatory/infectious disorders. With the advancement of research, the regulatory role of noncoding RNA (ncRNA) in the pyroptosis pathway was delineated. Among, studies have demonstrated that circular RNAs (circRNAs) regulate the pyroptosis cascade mainly through three principal mechanisms: functioning as miRNA sponges, modulating protein activity and encoding functional polypeptides. Numerous circRNAs regulating pyroptosis have been characterised, indicating their significant role in this process and associated disease progression. This review systematically summarised current knowledge on the regulatory mechanisms of circRNAs in canonical, noncanonical and caspase-3/8-mediated pyroptosis pathways. We further discussed their pathophysiological roles in disease development and potential clinical applications, aiming to advance mechanistic understanding, facilitate clinical translation and inform diagnostic and therapeutic strategies.

Head and neck squamous cell carcinoma (HNSCC) is a highly aggressive cancer with restricted therapeutic options and unfavourable survival outcomes. To identify novel prognostic biomarkers and therapeutic targets, we investigated the role of macrophage polarisation in HNSCC progression. Using integrative computational approaches, including biological network algorithms and molecular subtyping, we established a robust gene signature associated with M1/M2 macrophage balance, which exhibited significant prognostic value in HNSCC patients. Further analysis employing multi-model machine learning algorithms pinpointed ZBP1 as the pivotal gene, linking it to key clinical and immunological features, including disease progression, immune microenvironment remodelling, tumour mutational burden, and response to immune checkpoint inhibitors. Mechanistic studies confirmed ZBP1's tumour-suppressive function, demonstrating its ability to inhibit HNSCC cell proliferation and migration in vitro. Moreover, macrophage co-culture assays revealed that ZBP1 modulates immune regulation by restricting macrophage recruitment and altering polarisation dynamics. Collectively, our findings highlight ZBP1 as a promising prognostic biomarker and a potential immunotherapeutic target in HNSCC. This study not only enhances our understanding of macrophage-mediated tumour immunity but also provides mechanistic insights into how ZBP1 integrates tumour-intrinsic and immune-regulatory pathways to influence HNSCC progression. These discoveries may contribute to the development of more precise therapeutic strategies for this aggressive malignancy.

Acute myeloid leukaemia (AML) with isolated trisomies (ITs) represents a distinct cytogenetic subgroup with heterogeneous clinical behaviour and incompletely defined molecular features. To explore its genomic and transcriptomic landscape, we performed next-generation sequencing (NGS) on 14 AML patients harbouring isolated trisomies of chromosomes 8, 9, 10, 13, 14, 21 and 22. RNA sequencing (RNA-Seq) was conducted on 15 samples, including 12 with IT and 3 cytogenetically normal AML cases (normal karyotype, NK-AML) serving as controls. Trisomy 8 was most frequent, followed by chromosomes 13, 14 and 21. Recurrent mutations were identified in epigenetic regulators (DNMT3A, IDH1/2, ASXL1, TET2). Transcriptomic profiling stratified cases into IT-8, IT-21 and IT-13+22 subgroups. Gene set enrichment analysis (GSEA) revealed shared downregulation of cell cycle-related pathways (e.g., G2M checkpoint) and subgroup-specific patterns: oxidative stress and unfolded protein response in IT-8; epithelial-mesenchymal transition and oxidative phosphorylation in IT-21; inflammatory signalling (IL-6/JAK/STAT, TNF-α/NF-κB) in IT-13+22. A core set of 60 differentially expressed genes (DEGs) was shared, with nine hub genes related to cell cycle (MCM4, CDC7, CDC25A, DHFR), proteostasis (HSPA5, DNAJC3, CALR, HSP90B1) and inflammation. Drug sensitivity profiling revealed subgroup-specific vulnerabilities: IT-8 to DNA damage checkpoint inhibitors, IT-21 to PLK/mTOR inhibitors and IT-13+22 to BRAF/EGFR-targeted agents. These findings highlight AML-IT heterogeneity and therapeutic potential.

Long non-coding RNAs (lncRNAs) play important roles in colorectal cancer (CRC) progression. However, the biological function and regulatory mechanism of small nucleolar RNA host gene 26 (SNHG26) in CRC remain largely unexplored. SNHG26 expression was analysed in CRC tissues and cell lines using quantitative real-time PCR (qRT-PCR). The biological functions of SNHG26 were investigated through loss- and gain-of-function approaches. Interaction between SNHG26 and CDKN2A was examined by RNA immunoprecipitation, RNA pull-down and RNA stability assays. The effects of the SNHG26-CDKN2A axis on Cu + ELES(copper plus elesclomol)-induced cuproptosis and CD8+ T cell-mediated anti-tumour immunity were evaluated through cell viability, apoptosis, co-culture cytotoxicity and migration assays. SNHG26 was significantly upregulated in CRC tissues and cell lines, with high expression showing trends toward poor prognosis. SNHG26 knockdown suppressed CRC cell proliferation and enhanced apoptosis. Additionally, it increased sensitivity to Cu + ELES-induced cuproptosis. Mechanistically, SNHG26 directly interacted with CDKN2A mRNA, promoting its degradation. CDKN2A, which exhibits context-dependent effects in CRC, was post-transcriptionally regulated by SNHG26. Rescue experiments demonstrated that CDKN2A knockdown partially reversed the oncogenic effects of SNHG26 overexpression, including enhanced proliferation, reduced apoptosis and increased resistance to cuproptosis. Furthermore, the SNHG26-CDKN2A axis modulated the tumour immune microenvironment by regulating CD8+ T cell cytotoxicity and chemokine expression, specifically downregulating CXCL9 and CXCL10, which are critical for T cell recruitment. Our findings reveal a novel regulatory axis whereby SNHG26 promotes CRC progression by destabilising CDKN2A mRNA, resulting in enhanced cell proliferation, cuproptosis and immune evasion. This study provides new insights into the molecular mechanisms underlying CRC development.

Ovarian cancer (OC) is a high-mortality gynaecological malignancy, and the role of PANoptosis, a comprehensive cell death mechanism, in its prognosis remains unexplored. This study aims to clarify it, potentially guiding OC diagnosis and treatment. We analysed the ovarian data from TCGA and GTEx, and the GSE184880 scRNA-seq dataset from GEO. Spatial data and pathological images were sourced from the 10X Genomics website and GDC Portal. Features were extracted using CellProfiler and ResNet-50, and a PANoptosis-related pathomics prognostic model (PANPM) powered by deep learning was developed. The PANoptosis-related hub gene STAT4 potentially served as a protective factor for patients with OC. A better prognosis in OC was found linked to higher PANoptosis. The PANPM, manifesting distinct advantages for clinical application by accurately extracting pathological features, performed excellently in validation and the high-risk group indicated a poor prognosis. Additionally, STAT4 T cells may inhibit OC, by activating the PANoptosis of epithelial cells through TNFSF12-TNFRSF12A and TNF-TNFRSF1A, which sheds light on potential therapeutic interventions involving STAT4 T cells.

Amoxicillin (Amx), a β-Lactam antibiotic frequently used to treat bacterial infections, has been linked to neurological effects, including anxiety, hyperactivity, ambiguity, seizures, and behavioural changes. We examined the neurotoxic effects of Amx in zebrafish and investigated the potential of liposome-encapsulated melatonin (L-Mel) as a therapeutic intervention. Computational studies have indicated that Amx and Mel interact with GABA receptors, suggesting the potential of L-Mel in mitigating Amx-induced neurological changes. Our findings demonstrated that the nanoformulated L-Mel showed reduced toxicity in zebrafish larvae. Administration of L-Mel to Amx-affected zebrafish brain tissue significantly lowered the levels of reactive oxygen species, antioxidants (catalase, superoxide dismutase, and nitric oxide), and proinflammatory cytokines (TNF-α, IL-1β, and NF-kB), based on the fixed EC-50. Behavioural assessments revealed that L-Mel treatment notably enhanced the immobility time and swimming performance, improving the movement abilities of zebrafish with Amx-induced neuroinflammation. Moreover, the GABA/glutamate levels in the neural tissues exhibited significant recovery in the L-Mel group. Gene and protein analysis showed substantial increases in BDNF, CREBBP, ASCL, NF-κB and GABA-A R γ2 in L-Mel treated subjects. Histopathological evaluation revealed that L-Mel treatment markedly attenuated Amx-induced neurotoxicity, as evidenced by reduced neuronal degeneration and necrosis in the brain tissue, indicating a pronounced neuroprotective effect. In conclusion, our research suggests that L-Mel is a promising therapeutic agent for mitigating Amx-induced neurotoxicity.

Kidney renal clear cell carcinoma (KIRC) is associated with abnormal ribosome production (RiboSis), but how this affects tumour growth and response to immunotherapy is still unclear. In this study, we analysed large-scale multi-omics data using machine learning. Using single-cell RNA sequencing and gene network analysis (hdWGCNA), we found a key RiboSis-related gene group. We then classified KIRC tumours into two subtypes based on RiboSis activity. Patients with subtype 1 lived significantly longer, and this group showed activation of tumour-promoting pathways. Using machine learning, we identified PTGER4 as a potential tumour suppressor. Higher PTGER4 levels were linked to better survival in multiple patient groups. Tumours with high PTGER4 also had stronger immune cell activity and higher levels of immunotherapy-related markers, suggesting they may respond better to immune-based treatments. PTGER4 also predicted better outcomes with certain chemotherapy drugs. Further analysis confirmed that PTGER4 is involved in immune-related pathways and is often reduced in tumours, supporting its role in slowing cancer progression. Lab experiments confirmed that PTGER4 helps block tumour growth. These findings suggest PTGER4 plays a central role in KIRC progression and treatment response. Targeting RiboSis-related mechanisms and PTGER4-related pathways could lead to better therapies for KIRC patients.

Asherman Syndrome (AS) is caused by injury to the endometrium leading to uterine scarring, decreased menstruation and infertility; it typically occurs after surgical curettage of the uterus. AS is treated surgically albeit with limited success. Administration of bone marrow-derived mesenchymal stem cells (MSCs) has recently been demonstrated to restore uterine function in AS; however, there is no data available on the role of the estrous cycle phase on outcomes. Here, we describe endometrial injury during estrus or diestrus, its differential effect on fertility, and its response after bone marrow MSC treatment to reverse the infertility in a murine model. Endometrial injury in the estrus phase did not affect fertility outcomes whereas injury in the diestrus phase resulted in infertility. Bone marrow (BM)-derived MSC treatment without injury in the estrus or diestrus phase did not affect the pregnancy outcomes. BM MSC treatment following endometrial injury in the diestrus phase restored fertility. Immunofluorescence analysis revealed that vimentin or cytokeratin-positive BM-derived cells in the uterus were extremely rare. BM MSC treatment after injury increased CD45 cells, indicating a role for immunomodulation in endometrial repair. Finally, qRT-PCR showed that Ccl3, Il-1β and Mmp3 gene expression was significantly higher in the endometrium of the injury + BM MSC group than in other groups. In summary, injury to the endometrium during the diestrus phase results in infertility that can be restored by the treatment of BM MSCs. The therapeutic effect of BM MSCs on the endometrium appears to be mediated primarily by immunomodulation rather than BM MSC engraftment.

Clostridioides difficile (C. difficile ) is a Gram-positive anaerobic bacillus that causes intestinal disorders. Toll-like receptor 4 (TLR4) plays a key role in innate immunity. This study examines the role of TLR4 in the response to C. difficile toxins, which induce cell death and inflammatory responses in enteric glial cells (EGCs). Male C57BL/6 mice were infected with C. difficile, and cecum samples were analysed 3 days post-infection for TLR4 expression. In vitro, EGCs were exposed to C. difficile toxins with or without C34, a TLR4 antagonist, or pre-exposed to TLR4-specific 21-nt small interfering RNAs (siRNA). TLR4 expression was assessed by immunocytochemistry, immunofluorescence, qPCR, and Western blotting. NFκB p65, TNF-α, IL-6, cleaved caspase-3, and phosphatidylserine binding to annexin-V were evaluated. TLR4 expression increased in infected intestinal tissue and toxin-exposed EGCs. TLR4 antagonist or TLR4 knockdown reduced NFκB p65 nuclear translocation and TNF-α expression but did not affect IL-6 upregulation. Additionally, TLR4 antagonist or TLR4 knockdown mitigated toxin-induced cell death, as shown by decreased cleaved caspase-3 and phosphatidylserine binding. These findings suggest that TLR4 contributes to C. difficile pathogenesis and that its inhibition reduces inflammation and prevents cell death in EGCs.

Quinovic acid is a key constituent of cat's claw (Uncaria tomentosa) extract and exhibits antioxidant and anti-inflammatory activities. In this study, we investigated the potential of quinovic acid to enhance natural killer (NK) cell activity by using the KHYG-1 cell line. Our data indicated that quinovic acid increased the expression levels of cytolytic molecules, including perforin, granzymes A and B, Fas ligand, and granulysin, and induced the phosphorylation of the transcription factors CREB and STAT4, thereby enhancing cytotoxic activity against K562 cells. Furthermore, when KHYG-1 cells were cocultured with K562 cells in the presence of quinovic acid, we observed an increase in the expression of t-Bid, cleaved caspases 3, 8, and 9, and PARP, promoting apoptosis in K562 cells. Quinovic acid also reduced the expression of SET, Ape1, and HMGB2, effectively inhibiting the DNA repair mechanism in target cells. Similar results were observed in other cancer cell lines. In addition, quinovic acid induced interferon-gamma secretion by upregulating the Ras/MAPK and PI3K/AKT/mTOR signalling pathways through the activation of NKG2D and other NK receptors. These effects were observed not only in KHYG-1 cells but also in NK cells derived from adult patients with head and neck squamous cell carcinoma. Our findings suggest that quinovic acid enhances NK cell cytotoxicity, showing promise as a potential therapeutic against various cancer cell types.