DAB辣根过氧化物酶显色试剂盒

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.

Exposure to fine particulate matter (PM 2.5 ) represents a critical environmental health threat, with growing evidence linking it to accelerated chronic kidney disease (CKD) progression. However, the underlying mechanism of this toxicity remains poorly understood. This study investigated whether PM 2.5 exposure induces renal tubular cell senescence and explored the molecular basis of this process. We found that PM 2.5 exposure caused kidney injury in mice and upregulated senescence markers in both mice and human kidney proximal tubule epithelial (HK-2) cells. Mechanistically, PM 2.5 downregulated FOXP1 expression, relieving its transcriptional repression of CDKN1A (encoding P21), leading to P21 upregulation and subsequent cell cycle arrest. Overexpressing FOXP1 or treating with quercetin mitigated PM 2.5 -induced senescence in HK-2 cells. Our findings demonstrate that reduced FOXP1 drives cellular senescence in PM 2.5 -induced renal injury and identify quercetin as a potential therapeutic agent that activates FOXP1 and alleviates PM 2.5 nephrotoxicity.

Background Mitochondrial dysfunction affects the development of ovarian cancer (OC). ETV4 is involved in mitochondrial fusion. The regulatory pathways of ETV4 in OC cells have not been further investigated. In this study, we aimed to explore the effects of ETV4 on OC development and analyze the downstream regulatory pathways of ETV4. Methods The expression of ETV4 in OC cell lines (SK-OV-3, HEY, A2780, and OVCAR-3) was verified. After silencing ETV4, indicators related to mitochondrial function, including ATP level, mitochondrial membrane potential, mitochondrial DNA (mtDNA), and mitochondrial ROS (mtROS), were analyzed. The expression of mitochondrial fission/fusion-related markers (Mfn1, Mfn2, OPA1, DRP1, MFF, and FIS1) was detected. In vivo experiments were used to verify the effect of ETV4 on OC development. Results The TCGA-OV data indicated that ETV4 was highly expressed in OC. Silencing ETV4 inhibited the proliferation of OC cells. Mitochondrial membrane potential and ATP levels increased after ETV4 silencing, while mtDNA and mtROS levels decreased. ETV4 silencing promoted Mfn2 protein expression but did not affect Mfn2 mRNA level. Mfn2-associated E3 ubiquitin ligase MARCH9 was targeted and regulated by ETV4. MARCH9 overexpression alleviated the regulation of ETV4 silencing on mitochondrial function in OC cells. Lysosomal inhibitor CQ blocked the degradation of ubiquitinated Mfn2 protein. MARCH9 was found to mediate robust ubiquitination of Mfn2 via the K63-linked ubiquitination. Conclusions ETV4 was highly expressed in OC and involved in the regulation of mitochondrial function. ETV4 regulated Mfn2 ubiquitination linked by K63 by regulating MARCH9.

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: Intervertebral disc degeneration (IDD) significantly contributes to low back pain (LBP), yet effective treatment options are scarce. BSHXF, a classical traditional Chinese medicine formula, demonstrates dual pharmacological actions: tonifying kidneys, strengthening bones, activating blood circulation, and resolving stasis. It has been widely used in IDD management. Given its potential, combining BSHXF with miRNA regulation and stem cell therapy may enhance therapeutic outcomes by targeting molecular and cellular pathways underlying IDD pathogenesis.Aim of the study: IDD is recognized as one of the primary causes of low back pain, yet effective therapeutic interventions for this condition remain limited. This study explores the role of BSHXF drug-containing serum combined with adipose-derived stem cells (ADSCs) in slowing IDD progression via the miR-199a-3p/TGF-β/Smad signaling pathway. By comprehensively investigating the synergistic effects of this combination therapy, we aim to propose a novel multi-target strategy that addresses the complex pathogenesis of IDD.Materials and Methods: This study employed a combination of in vivo and in vitro models. An IDD model was induced in rat caudal intervertebral discs through needle puncture, while an oxidative stress-induced ADSCs injury model was created in vitro using tert-butyl hydroperoxide (T-BHP). Cell viability was measured with the CCK-8 assay. Cell cycle distribution and mitochondrial reactive oxygen species (ROS) levels were assessed using flow cytometry. Cellular senescence was assessed using SA-β-galactosidase staining. Lactate dehydrogenase (LDH) activity was quantified to evaluate cellular damage. Differentiation into nucleus pulposus-like cells was assessed using immunofluorescence double staining for CD73 and COL2A1. ELISA was used to measure inflammatory cytokines (TNF-α, IL-1β, IL-4, IL-10) in cell supernatants. miR-199a-3p expression was determined using RT-qPCR. Western blotting was employed to quantify COL2A1, SOX9, and ACAN protein levels, reflecting nucleus pulposus-like differentiation and extracellular matrix (ECM) synthesis capacity. Western blotting was employed to assess pathway activity by analyzing the protein expressions of TGF-β1, Smad2, Smad3, and their phosphorylated forms, P-Smad2 and P-Smad3. In vivo experiments assessed histopathological degeneration through hematoxylin-eosin (HE) and Safranin O-Fast Green staining. Immunohistochemistry (IHC) analyzed COL1A1 and COL2A1 expression levels. RT-qPCR quantified miR-199a-3p expression. Western blotting was employed to assess the expression levels of TGF-β1, Smad2, Smad3, P-Smad2, and P-Smad3 for pathway regulation evaluation.Results: Our experimental results demonstrated that serum containing BSHXF significantly alleviated T-BHP-induced oxidative stress, improved the cellular microenvironment, promoted ADSCs proliferation, and decelerated cellular senescence. Further mechanistic analysis revealed that BSHXF significantly activated the TGF-β/Smad signaling pathway, driving the differentiation of ADSCs into nucleus pulposus-like cells and restoring normal cell cycle progression. Overexpression of miR-199a-3p inhibited the TGF-β/Smad pathway, leading to ECM degradation and elevated expression of inflammatory factors (TNF-α, IL-1β). In contrast, BSHXF restored TGF-β/Smad pathway activity by downregulating miR-199a-3p expression. In vivo experiments demonstrated that miR-199a-3p overexpression exacerbated IDD, characterized by reduced COL2A1 expression, elevated COL1A1 levels, and increased disc fibrosis. BSHXF intervention markedly attenuated IDD progression by downregulating miR-199a-3p expression, reducing disc fibrosis, and effectively restoring collagen expression.Conclusion: BSHXF activated the TGF-β/Smad pathway to promote the differentiation of ADSCs into nucleus pulposus-like cells. It exerted protective effects by alleviating oxidative stress damage, improving the microenvironment, delaying senescence, and enhancing cellular functions. This study is the first to reveal that miR-199a-3p overexpression exacerbates intervertebral disc fibrosis and degeneration. BSHXF restored TGF-β/Smad pathway activity by downregulating miR-199a-3p expression, thereby improving disc structure and function. This integrated approach offers a novel multi-target intervention strategy for IDD, demonstrating significant therapeutic potential.

The role of E3 ubiquitin ligases in cellular mechanisms and cancer progression is critical. In this study, our primary objective was to elucidate the functional consequences of F-box only protein 32 (FBXO32) in the progression and metastasis of hepatocellular carcinoma (HCC) and to clarify the signaling networks. FBXO32 was highly expressed, and T-cell acute lymphocytic leukemia protein 1 (TAL1) was poorly expressed in HCC cells relative to THLE-2 cells. FBXO32 interacted with the TAL1 protein and degraded TAL1. Knocking down FBXO32 suppressed epithelial-mesenchymal transition and the PTEN/PI3K/AKT signaling in MHCC-97H cells, while knocking down TAL1 reversed this effect. Similarly, overexpression of FBXO32 in SNU-398 cells promoted HCC progression, and reactivation of TAL1 also reversed this trend. Importantly, HCC patients with high FBXO32 or low TAL1 expression were both associated with poor prognosis. Our study has shown that FBXO32 facilitates HCC growth and metastasis via the PTEN/PI3K/AKT signaling through ubiquitination of TAL1. Consequently, FBXO32 emerges as a promising target for therapeutic intervention in the treatment of HCC.

Obesity, characterized by excessive body fat, is a leading preventable cause of death globally and represents one of the most critical public health challenges of the 21st century. This study aimed to investigate the action of tangeretin on gut microbiota metabolism and inflammation in high-fat diet (HFD)-induced obese mice. A model of obesity was established using 6-week-old male C57BL/6J mice fed with HFD, which were then used for the treatment with tangeretin (20 mg/kg/mice/day) or antibiotic (Abx). The results showed that the tangeretin intervention alleviated fat deposition and disorder of cellular structural integrity in the model group. The obese mice showed a significant increase in the levels of lipid (glycerol, triglyceride, and total cholesterol), inflammatory factors (IL-6 and TNF-α), and F4/80 expression in both serum and adipose tissues. Following tangeretin treatment, the levels of lipid, inflammatory factors, and the ratio of F4/80 + CD206 + macrophages were decreased in both serum and adipose tissue. 16S rRNA sequencing and LC-MS/MS analysis revealed that tangeretin decreased obesity in HFD-induced obese mice by interacting with gut microbiota, particularly influencing Parabacteroides , Blautia , and Parasutterella , and amino acids such as threonine, isoleucine, leucine, phenylalanine, arginine, glutamine, L-tryptophan, and tyrosine. Abx-mediated clearance of gut microbiota blocked the HFD-induced obesity and abrogated the therapeutic effects of tangeretin in obese mice. Fecal microbiota transplantation (FMT) proved that clearing gut microbiota with Abx blocked the beneficial effects of FMT HFD+Tangeretin intervention. These findings suggested that tangeretin improved HFD-induced obesity by regulating lipid metabolism and modulating F4/80 macrophage activation via gut microbiota.

Ferroptosis is a critical contributor to ischemia-reperfusion (I/R) injury and subsequent organ failure. While ENPP2 has been implicated in regulating ferroptosis in cardiomyocytes, its specific role in myocardial I/R injury remains unclear. This study aims to elucidate the function of ENPP2-mediated ferroptosis in myocardial ischemia-reperfusion injury (MI/RI), to provide novel insights into potential therapeutic strategies. A mouse model of MI/RI was established and subjected to interventions with ENPP2 overexpression and/or SIRT1 knockdown. In vitro, cardiomyocytes were treated with palmitate and subjected to hypoxia/reoxygenation (H/R) to simulate I/R injury. These cells received treatments with ENPP2 overexpression (oe-ENPP2), SIRT1 overexpression (oe-SIRT1), PGC-1α silencing (si-PGC-1α), and/or SIRT1 knockdown (sh-SIRT1). Additionally, Erastin-induced ferroptosis in cardiomyocytes was used to assess the protective effects of oe-ENPP2. Ferroptosis was assessed through the lipid peroxidation (MDA, 4-HNE), iron and Fe 2+ assays, GPx4 and SLC7A11 expression, and transmission electron microscope. Overexpression of ENPP2 significantly alleviated myocardial infarction in MI/RI mice, as indicated by the upregulation of GPx4 and SLC7A11 protein levels. In cardiomyocytes subjected to hypoxia/reoxygenation (H/R) or erastin-induced ferroptosis, oe-ENPP2 reduced apoptosis rates, preserved Fe 2+ content, and restored GPx4 and SLC7A11 expression. Silencing PGC-1α blocked the protective effect of oe-ENPP2 against H/R-induced ferroptosis in HL-1 cells. Additionally, SIRT1 overexpression inhibited PGC-1α acetylation, whereas SIRT1 knockdown similarly reversed the anti-ferroptotic effects of oe-ENPP2 in H/R-treated HL-1 cells. SIRT1 silencing blocked the protective effects of oe-ENPP2 against myocardial infarction and fibrosis in MI/RI mice via the PGC-1α/NRF1 pathway. ENPP2 overexpression protects the mouse myocardium from I/R-induced ferroptosis injury via the SIRT1/PGC-1α/NRF1 pathway. These findings suggest a novel gene therapy strategy for mitigating myocardial I/R injury.