免疫组化二抗试剂盒（小鼠/兔超敏聚合物法检测系统）

Background Ambra1 has recently been identified as a key regulatory factor in the progression of mantle cell lymphoma (MCL). The objective of this study was to investigate the biological role and molecular mechanism of Ambra1 in MCL. Methods The m6A modification level of Ambra1 was detected by MeRIP-qPCR. Wild-type and mutant Ambra1 plasmids were constructed to verify the direct regulation of Ambra1 by METTL3-mediated m6A modification. The influence of METTL3/m6A/YTHDF2/Ambra1 on the viability, proliferation, migration, apoptosis, and cell cycle of MCL cells was evaluated by standard in vitro assays. RIP and RNA pull-down assays were performed to validate Ambra1 as a downstream target of YTHDF2. Xenograft tumor models were established using BALB/c nude mice to confirm the in vivo phenotype of METTL3 and Ambra1 silencing. Results Ambra1 was downregulated in MCL cells by METTL3-mediated m6A modification. Furthermore, knocking down METTL3 in the MCL cells inhibited their proliferation, migration, and invasion through the upregulation of Ambra1, while METTL3 overexpression had the opposite effect. The m6A reader protein YTHDF2 downregulated Ambra1 expression by binding to Ambra1-m6A. YTHDF2 knockdown inhibited the growth of MCL cells through Ambra1, while YTHDF2 overexpression had the opposite effect. Mechanistically, METTL3 downregulated Ambra1 in the MCL cells in an m6A-YTHDF2-dependent manner to inhibit apoptosis. Finally, METTL3 knockdown inhibited MCL progression in vivo by inducing Ambra1 expression. Conclusion METTL3 promotes MCL progression through YTHDF2-mediated degradation of Ambra1 mRNA, suggesting that the METTL3/YTHDF2/Ambra1 may serve as a potential therapeutic target for MCL.

Background PARP inhibitor (PARPi) maintenance therapy significantly extends progression-free survival of patients with homologous recombination repair deficiency (HRD) or BRCA mutations in ovarian cancer. However, more than 50% of patients lack HRD, highlighting the need to expand PARPi use for homologous recombination -proficient patients. In this study, the efficacy of GX15-070 combined with niraparib in ovarian cancer was evaluated. Methods Based on the core regulators of genome stability and homologous recombination (HR) repair pathway, a compound library was constructed. The effect of candidate drugs on niraparib sensitivity were measured using CCK-8 in ovarian cancer cell lines. Immunofluorescence and non-homologous end joining repair (NHEJ) assay were conducted to examine HR and NHEJ activity. Co-immunoprecipitation was used to investigate the interaction between Mcl1 and Ku70. BH3 domain deletion mutant of Mcl1 was generated to elucidate the structural basis of the interaction between Mcl1 and Ku70. Additionally, cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models were established to evaluate the efficacy of GX15-070 combined with niraparib in vivo. Results We constructed a compound library based on the core regulators of genomic stability and HR repair. Through high-throughput drug screening, GX15-070, a Mcl1 inhibitor, was identified as a synergist of niraparib, independent of BRCA status. Inhibition of Mcl1 expression significantly impaired HR activity and potentiated niraparib sensitivity. High expression of Mcl1 was associated with a wore prognosis in ovarian cancer patients treating PARPi maintenance therapy. Mechanistically, Mcl1 directly interacts with Ku70 protein via its BH3 domain, serving as a functional switch in selecting between HR and NHEJ. GX15-070 disrupts the interaction by displacing Ku70, promoting a shift in DNA repair pathways from HR to NHEJ. Furthermore, the synergistic efficacy of the combination treatment was further validated in CDX and PDX models. Conclusions The study demonstrated that the combination of GX15-070 with niraparib might be a promising therapeutic strategy for ovarian cancer patients with limited PARPi response.

Breast cancer is associated with a higher incidence of depression and decreased quality of life. Previous studies have indicated that quercetin can mitigate the advancement of breast cancer-related depression (BCRD); however, the specific mechanism by which quercetin affects BCRD is yet to be determined. In this study, we aimed to examine the effect of quercetin on BCRD and explore the underlying mechanisms. We established a mouse model of BCRD and administered quercetin. LC–MS was used to analyze and determine distinct alterations in metabolites in mouse tumor samples. Polymorphonuclear neutrophils (PMNs) were extracted from mouse femurs and treated with PMA and quercetin/Sphingosine 1-phosphate (S1P). Mouse breast cancer cells 4 T1 were treated with lipopolysaccharides (LPS), neutrophil extracellular traps (NETs) and S1P. Neuronal cells were treated with LPS, NETs, S1P, and Corticosterone. Pearson's correlation coefficient was used to evaluate the relationship between differential metabolites and NETs. Quercetin inhibited NET formation in BCRD mice. In vitro, quercetin reversed NET-induced 4 T1 cell proliferation, migration, and ROS production. Quercetin also reversed the effects of NET-induced 4 T1 cells on neuronal cells. LC–MS analysis demonstrated that quercetin ameliorated the metabolic abnormalities in the tumors of BCRD mice. Pearson's correlation analysis showed that S1P, Oleoyl glycine, N-Arachidonoylglycine, 2, 3-butanediol apiosylglucoside, and tetracosatetraenoyl carnitine levels positively correlated with MPO DNA levels. Furthermore, in vitro, S1P enhanced NET-induced 4 T1 cell proliferation, migration, and ROS production, as well as enhanced NET-induced 4 T1 cell damage to neuronal cells. Quercetin alleviated BCRD by inhibiting NETs via inhibition of the S1P/S1PR axis.

Introduction: Knee osteoarthritis (KOA) is a degenerative joint disease characterized by the progressive deterioration of cartilage and synovial inflammation. A critical mechanism in the pathogenesis of KOA is impaired efferocytosis in synovial tissue. The present study aimed to identify and validate key efferocytosis-related genes (EFRGs) in KOA synovial tissue by using comprehensive bioinformatics and machine learning approaches.Methods: We integrated three datasets (GSE55235, GSE55457, and GSE12021) from the Gene Expression Omnibus database to screen differentially expressed genes (DEGs) associated with efferocytosis and performed weighted gene co-expression network analysis. Subsequently, we utilized univariate logistic regression analysis, least absolute shrinkage and selection operator regression, support vector machine, and random forest algorithms to further refine these genes. The results were then inputted into multivariate logistic regression analysis to construct a diagnostic nomogram. Public datasets and quantitative real-time PCR experiments were employed for validation. Additionally, immune infiltration analysis was conducted with CIBERSORT using the combined datasets.Results: Analysis of the intersection between DEGs and EFRGs identified 12 KOA-related efferocytosis DEGs. Further refinement through machine learning algorithms and multivariate logistic regression revealed UCP2, CX3CR1, and CEBPB as hub genes. Immune infiltration analysis demonstrated significant correlations between immune cell components and the expression levels of these hub genes. Validation using independent datasets and experimental approaches confirmed the robustness of these findings.Conclusions: This study successfully identified three hub genes (UCP2, CX3CR1, and CEBPB) with significant expression alterations in KOA, demonstrating high diagnostic potential and close associations with impaired efferocytosis. These targets may modulate synovial efferocytosis-related immune processes, offering novel therapeutic avenues for KOA intervention.

Background: While programmed death-ligand 1 (PD-L1)-targeted immunotherapy represents an advancement in non-small cell lung cancer (NSCLC), patient outcomes remain suboptimal. Aberrant activation of the cyclic adenosine monophosphate (cAMP) response element binding protein (CREB)-regulated transcription coactivator (CRTC) is linked to malignant proliferation and functionality in lung cancer cells. This study investigates the involvement of CRTC1 in tumor immunity.Methods: CRTC1 and Notch1 expression were regulated in A549 and NCI-H1299 NSCLC lines through plasmid-mediated overexpression/silencing to assess their effects on cell viability, apoptosis, migration, and invasion. CRTC1/Notch1-dysregulated Lewis lung carcinoma (LLC) cells were co-cultured with T cells to evaluate T cell activation and function. The efficacy of combined CRTC1 knockdown/overexpression and atezolizumab (anti-PD-L1) was tested in an LLC xenograft mouse model.Results: CRTC1 promoted cell viability, migration, and invasion while suppressing apoptosis across NSCLC models. In LLC cells, CRTC1 upregulated tumor cell PD-L1 expression, suppressed T cell-derived IFN-γ and IL-2 production, diminished endogenous CXCL10/11 secretion, and impaired T cell proliferation and cytotoxicity. Mechanistically, CRTC1 interacted with Notch1 to activate the Notch1/Akt pathway, stimulating PD-L1 upregulation, thereby facilitating tumor immunosuppression and growth. Notably, CRTC1 overexpression reversed the protective effects of atezolizumab on tumor growth. Combining CRTC1 knockdown with atezolizumab synergistically enhanced anti-tumor T cell immunity, achieving the most significant tumor regression in xenografts.Conclusion: These findings indicate that CRTC1 in tumor cells suppresses PD-L1-mediated anti-tumor immunity and promotes tumorigenesis via the Notch1/Akt signaling axis. Dual targeting of CRTC1 and PD-L1 demonstrates therapeutic synergy, suggesting CRTC1 pathway inhibition could optimize immunotherapy outcomes in NSCLC patients.

Pancreatic cancer is highly challenging, with most patients developing intrinsic or acquired resistance to first-line chemotherapy drug gemcitabine (GEM). Although Matrix Metalloproteinase 28 (MMP28) is upregulated in pancreatic cancer and predicts a poor prognosis, its role in GEM resistance and molecular mechanism remain unclear. Here, we aimed to investigate the role of MMP28 in GEM resistance and molecular mechanism. First, differentially expressed genes in pancreatic cancer were identified through bioinformatics and validated in clinical samples and cells. MMP28 was significantly overexpressed in pancreatic cancer tissues and Capan-1 and PANC-1 cells, correlating with poor prognosis. Then, MMP28 knockdown was performed in Capan-1 and PANC-1 cells, followed by GEM treatment. Furthermore, in vivo experiments evaluated GEM sensitivity after MMP28 knockdown. The results showed that MMP28 knockdown enhanced GEM sensitivity both in vitro , reducing cell proliferation and survival, and in vivo , where tumor growth was significantly suppressed. Additionally, glycolysis-related changes were assessed. We revealed that glycolysis was implicated as a key pathway in this process, with reduced glucose uptake and lactate production observed after MMP28 knockdown. Protein-protein interaction analysis identified Staphylococcal nuclease domain-containing protein 1 (SND1) as a key interactor, and SND1 expression was upregulated in pancreatic cancer tissues. Moreover, MMP28 interacted with SND1 to regulate SND1′s recruitment of HK2 mRNA to promote glycolysis. However, overexpression of SND1 reversed the effects of MMP28 knockdown, restoring glycolysis and GEM resistance. In conclusion, MMP28 promoted tumor growth and GEM resistance in pancreatic cancer by regulating glycolysis via interaction with SND1.

Background Osteosarcoma (OS) is a bone tumor characterized by a high recurrence rate and poor prognosis. Arjunolic acid (AA), the most abundant triterpene component in Cyclocarya paliurus, is reported to have anti-tumor effects. Its specific role in OS is still unknown, which we aim to investigate in our study.Methods An OS mouse model was established to investigate the effects of AA. Subsequently, M2 macrophages and M0 macrophages pretreated with AA were co-cultured with OS cells. The impact of AA on OS cell behavior (proliferation, apoptosis, migration, and invasion) was evaluated via EdU staining, flow cytometry, and Transwell assays. Concurrently, the expression of M1- and M2-associated genes (CD86, CD163, IL-6, Arg1) was quantified to assess AA’s regulatory role in macrophages within the tumor microenvironment (TME). Knockdown or overexpression of Wnt3a in AA-treated M0 macrophages to determine whether AA modulates Wnt3a-mediated M2 polarization, which was further validated in vivo.Results In vivo, AA inhibited tumor progression in OS mice. Concurrently, AA-treated macrophages inhibited OS cell malignant behavior, and AA inhibited OS cell-mediated macrophage M2-type polarization. Mechanistically, AA inhibits the malignant behavior of OS cells and inhibits tumor progression in OS mice by suppressing Wnt3a-mediated macrophage M2 polarization. Additionally, AA-induced macrophage conversion to a pro-inflammatory phenotype in the TME of OS mice.Conclusion Our experiment demonstrated that AA from Cyclocarya paliurus inhibits Wnt3a-mediated M2 macrophage polarization to suppress the progression of osteosarcoma, providing a pharmacological foundation for developing therapies against OS.

Biofilm formation in Mycobacterium tuberculosis (MTB) enhances antibiotic resistance by impeding drug penetration and evading host immunity. This poses a significant challenge to conventional drug therapies, highlighting the urgent need for novel treatment strategies to overcome MTB's biofilm-mediated resistance. This study introduces the development of low-intensity ultrasound-mediated levofloxacin (LEV) and catalase (CAT) -loaded PEG-PLGA nanoparticles (LEV@CAT-NPs) for antimicrobial sonodynamic therapy (aSDT), offering an innovative strategy to combat BCG biofilm infection, by utilizing BCG as a model for MTB. N -acetylcysteine (NAC) was supplemented during the latter stages of the treatment process of anti-infection therapy to facilitate the transformation of macrophages to the M2 phenotype and to promote tissue repair. Ultrasound-mediated LEV@CAT-NPs, along with the subsequent addition of NAC not only enhanced repair at the infection site but also led to a progressive resolution of the inflammatory response in tissues. The treatment regimen induced a shift in macrophage polarization towards the M2 phenotype and modulated cytokine expression, decreasing pro-inflammatory while increasing anti-inflammatory cytokines, which contributed to the restoration of redox balance in the infected tissues. This study proposes a novel therapeutic strategy that not only targets drug-resistant MTB but also promotes tissue repair, highlighting its dual role in infection management.

Background The probiotic E. coli Nissle 1917 (EcN) alleviates the progression of various diseases, including colitis and tumors. However, EcN has not been studied in atherosclerosis. The study investigated the effects of EcN on atherosclerosis model mice and the potential mechanisms. Methods Mice in the high-fat diet (HFD) model were given EcN (1 × 10 9 CFU/g) or homocitrulline (150 mg/L) by oral administration for 12 weeks. The EcN + antibiotic group was set up to investigate the effects of EcN combined with antibiotics on gut microbiota. The control group was utilized as the negative control. Atherosclerosis status, pyroptosis, gut microbiota, and serum metabolites of mice were examined. Results EcN treatment alleviated HFD-caused atherosclerotic plaque and lipid droplet production. EcN treatment reversed HFD-induced increases in total cholesterol, triglycerides, and low-density lipoprotein levels and decreases in high-density lipoprotein levels. EcN inhibited the HFD-caused rise in the expression of pyroptosis-related indicators (cleaved Caspase 1, GSDMD-N, NLRP3, IL-18, and IL-1β). The antibiotics partially reversed the effects of EcN on the model mice, suggesting that EcN regulated pyroptosis in the model mice through gut microbiota. Probiotic bacteria, such as Lactobacillus and Muribaculum , were mainly enriched in the EcN and EcN + antibiotic groups, while Helicobacter , Alistipes , and Rikenella were depleted, suggesting that EcN and EcN + antibiotics could alleviate disorders of gut microbiota in the model mice. EcN reversed the trend of HFD-induced decrease of some metabolites, such as 2-methyl-5-nitroimidazole-1-ethanol, methionine sulfoxide, and shikimate 3-phosphate, and inhibited the increase of some metabolites, such as kynurenine, oxoadipate, and homocitrulline. In addition, homocitrulline showed the opposite effects of EcN in the model mice. Homocitrulline could bind to pyroptosis-related proteins to aggravate ox-LDL-induced endothelial cell pyroptosis. Conclusion EcN could alleviate atherosclerosis development by ameliorating HFD-induced disorders of gut microbiota and serum metabolites (such as homocitrulline) to alleviate pyroptosis, which may be associated with homocitrulline/Caspase 1/NLRP3/GSDMD axis. Our study lays the foundation for the development of promising drugs for atherosclerosis in the future.

We designed our study with the aim of conducting a comprehensive analysis of the role that GDF-15 plays in pan-cancer. Multiple databases were used to obtain GDF-15 expression. XIANTAO Academic and Sangerbox were utilized to conduct the diagnostic significance of GDF-15 expression across pan-cancer. The relationships between GDF-15 expression and clinical data, tumor stemness, genomic instability, and tumor prognosis in pan-cancer were evaluated. The interaction between molecules and RNA methylation phenotypes related to GDF-15 were explored. The correlations between GDF-15 expression and immune cell infiltration, immune-related genes, as well as immune checkpoints were also under investigation. Besides, immunohistochemistry (IHC)-based tissue microarray analysis of LUAD validated GDF-15 protein expression and its correlation with survival prognosis. GDF-15 exhibited differential expression across various cancers, being upregulated in BRCA, CHOL, COAD, HNSC, LIHC, LUAD, READ, STAD, THCA, and UCEC, but downregulated in KIRC and LUSC. Its expression correlated with clinical parameters and GDF-15 expression had prognostic value in some cancers. Notably, it had a remarkable diagnostic value in CHOL, COAD, ESCC, GBM, LAML, READ and THCA. GDF-15 was involved in influencing tumor stemness, genomic instability. Besides, GDF-15 interacted with molecules involved in iron ion homeostasis and acute-phase response, receptor activator, ligand activities and cellular senescence. It was intertwined with key tumor processes. Moreover, GDF-15 expression further influenced immune cell infiltration, related immune genes, and immune checkpoints. More importantly, IHC staining results of tissue microarrays confirmed its differential protein expression in LUAD tissues, where elevated levels were significantly associated with poorer overall survival, underscoring its clinical relevance. GDF-15 functioned as a prospective indicator for both the diagnosis and prognosis in various cancers.