Inhibition Of Signaling Pathway Research Articles

Adiponectin regulates proliferation and differentiation of chicken skeletal muscle satellite cells via ERK1/2 and p38 signaling pathways.

Skeletal muscle satellite cells (SMSCs) are critical for postnatal skeletal muscle growth and regeneration. Adiponectin plays a pivotal role in regulating muscle glucose uptake and fatty acid metabolism. However, its function in the proliferation and differentiation of chicken SMSCs remains poorly understood. In this study, we investigated the effects of adiponectin on the proliferation and differentiation of in vitro cultured chicken SMSCs. Our results demonstrated that adiponectin promoted SMSCs proliferation while inhibiting myogenic differentiation and inducing adipogenic differentiation. RNA-seq analysis revealed enrichment of the MAPK signaling pathway, suggesting its potential involvement in the regulation of adiponectin on SMSCs activity. Western blot analysis revealed that adiponectin activated ERK1/2 phosphorylation and inhibited p38 phosphorylation during the process of the inhibition on myogenic differentiation in chicken SMSCs. Furthermore, suppression of ERK1/2 signaling with U0126 or activation of p38 signaling with SSK1 reversed the downregulated expression of myogenic differentiation marker MyHC, MyOD1, and MyOG induced by adiponectin. These findings validated that adiponectin impeded myogenic differentiation through activation of ERK1/2 and inhibition of p38 signaling pathways. Additionally, activation of p38 signaling pathway reduced the increased percentage of EdU-positive cells induced by adiponectin. Collectively, these findings demonstrated that adiponectin impedes myogenic differentiation of SMSCs through activating ERK1/2 and inhibiting p38 signaling pathways, while promoting proliferation by inhibiting p38 signaling pathway.

OTUD4-mediated inhibition of YAP1 signaling pathway in ovarian cancer: Implications for macrophage polarization and recruitment.

Ovarian cancer is a malignancy gynecologic oncology with high incidence and high mortality rate. M2-like tumor-associated macrophages promote cancer cell migration and metastasis. Ovarian tumor family deubiquitinase 4 (OTUD4) belongs to deubiquitinating enzyme family. The roles of OTUD4 in tumor microenvironments in ovarian cancer remains unknow. In this work, OTUD4 was overexpressed or knocked down in high-grade serous ovarian cancer cells OVCAR8 and CAOV3. Ovarian cells were co-cultured with THP-1 macrophages to simulate the tumor microenvironment. We found that OTUD4-expressed ovarian cells inhibited macrophage chemotaxis and M2 polarization. Besides, in ovarian tumor-bearing mouse model, OTUD4 suppressed tumor metastasis and remodeling tumor-associated macrophages phenotype (pro-tumor M2 to anti-tumor M1). In mechanism, OTUD4 protein bound to YAP1 protein, and downregulation of OTUD4 enhanced K63 ubiquitination and nuclear translocation of YAP1, thus increasing CCL2 transcription and subsequent macrophage recruitment. OTUD4 might inhibit CCL2 expression to regulate tumor-associated macrophages in ovarian tumor microenvironment. Those findings present a potential therapeutic strategy for ovarian cancer.

The role and molecular mechanism of GDF5 in regulating cell migration and angiogenesis via the Hippo-YAP signaling pathway in colorectal cancer.

292 Background: Distant metastasis is a leading cause of poor prognosis and mortality in colorectal cancer (CRC) patients, with angiogenesis playing a crucial role in tumor progression. The TGF-β superfamily has been shown to significantly influence tumor angiogenesis, yet the biological function and molecular mechanism of Growth Differentiation Factor 5 (GDF5), a member of this superfamily, remain unexplored in CRC. Given that the efficacy of current anti-angiogenic therapies is limited to a subset of patients, identifying new therapeutic targets involved in tumor angiogenesis is of great clinical importance. GDF5, with its potential role in angiogenesis regulation, presents a promising candidate to bridge this therapeutic gap. Methods: In this study, we analyzed GDF5 expression in CRC tissue samples using both the TCGA dataset and samples from our center, revealing significantly lower GDF5 expression in tumors compared to adjacent normal tissues. Moreover, GDF5 downregulation correlated with lymph node metastasis and disease recurrence. Functional assays in vitro demonstrated that GDF5 overexpression reduced tumor cell migration and invasion, while its knockdown produced the opposite effect. Co-culture assays showed that GDF5 knockdown significantly enhanced endothelial cell migration and angiogenesis, whereas overexpression diminished these effects. Additionally, F-actin cytoskeletal staining indicated that GDF5 overexpression led to cytoskeletal remodeling in endothelial cells. Mechanistically, we found that GDF5 overexpression in tumor cells downregulated the epithelial-mesenchymal transition (EMT) marker Slug and upregulated E-cadherin, while downregulating N-cadherin. In endothelial cells, GDF5 inhibited the Hippo-YAP signaling pathway, increasing YAP phosphorylation and reducing nuclear YAP levels. Conversely, GDF5 knockdown suppressed YAP phosphorylation and promoted its nuclear translocation. Results: Our findings demonstrate that GDF5 functions as a tumor suppressor in CRC by regulating cell migration and angiogenesis. This study reveals that GDF5 exerts its anti-cancer effects by modulating both EMT in tumor cells and cytoskeletal changes in endothelial cells, leading to the inhibition of the Hippo-YAP signaling pathway and reduced angiogenic potential. These results identify GDF5 as a promising new target for anti-angiogenic therapy in CRC, offering potential avenues for improving treatment strategies in patients resistant to current therapies. Conclusions: GDF5 acts as a tumor suppressor in colorectal cancer by inhibiting cell migration and angiogenesis through modulation of the Hippo-YAP signaling pathway, making it a promising therapeutic target for anti-angiogenic treatment in patients resistant to current therapies.

Pinocembrin alleviates renal ischemia-reperfusion injury/unilateral ureteral obstruction (UUO)-generated renal fibrosis by targeting the CYP1B1/ROS/MAPK axis.

In our research, we constructed models of renal ischemia-reperfusion (I/R)-exposed acute kidney injury (AKI) and unilateral ureteral obstruction (UUO)-stimulated renal fibrosis (RF) in C57BL/6 mice and HK-2 cells. We firstly authenticated that oral pinocembrin (PIN) administration obviously mitigated tissue damage and renal dysfunction induced by I/R injury, and PIN attenuated UUO-caused RF, as confirmed by the reduced expression of fibrotic markers as well as hematoxylin-eosin (H&E), Sirius red, immunohistochemistry, and Masson staining. Meanwhile, the beneficial role of PIN was again demonstrated in HK-2 cells with hypoxia-reoxygenation (H/R) or transforming growth factor beta-1 (TGF-β1) treatment. Importantly, the "ingredient-target-pathway-disease" network was established through bioinformatics analysis and molecular docking, which showed that PIN may target cytochrome P450 1B1 (CYP1B1) and modulate the mitogen-activated protein kinase (MAPK) pathway to exert its impact during injury. Furthermore, experiments confirmed that PIN usage remarkably constrained CYP1B1 expression, reactive oxygen species (ROS) production, MAPK-pathway-associated inflammation, or apoptosis during I/R injury or UUO exposure. PIN also ameliorated the elevated protein phosphorylation of MAPK pathway components [p38, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase 1 (JNK ERK and JNK)], which validated the PIN-induced inhibition of the MAPK signaling pathway in renal I/R or UUO injury. Moreover, the AAV9 (adeno-associated virus 9)-packed CYP1B1 or pcDNA-CYP1B1 overexpression plasmid was utilized to treat C57BL/6 mice or HK-2 cells to overexpress CYP1B1, respectively. Notably, CYP1B1 overexpression considerably abolished PIN's restriction impact on ROS generation and MAPK pathway activation. In conclusion, via bioinformatics analysis, molecular docking, animal model, and cellular experiments, we proved that PIN alleviates renal I/R injury/UUO-generated renal fibrosis through regulating the CYP1B1/ROS/MAPK axis.

GDF15-mediated enhancement of the Warburg effect sustains multiple myeloma growth via TGFβ signaling pathway

The Warburg effect, characterized by the shift toward aerobic glycolysis, is closely associated with the onset and advancement of tumors, including multiple myeloma (MM). Nevertheless, the specific regulatory mechanisms of glycolysis in MM and its functional role remain unclear. In this study, we identified that growth differentiation factor 15 (GDF15) is a glycolytic regulator, and GDF15 is highly expressed in MM cells and patient samples. Through gain-of-function and loss-of-function experiments, we demonstrated that GDF15 promotes MM cell proliferation and inhibits apoptosis. Moreover, GDF15 enhances Warburg-like metabolism in MM cells, as evidenced by increased glucose uptake, lactate production, and extracellular acidification rate, while reducing oxidative phosphorylation. Importantly, the tumor-promoting effects of GDF15 in MM cells are fermentation-dependent. Mechanistically, GDF15 was found to promote the expression of key glycolytic genes, particularly the glucose transporter GLUT1, through the activation of the TGFβ signaling pathway. Pharmacological inhibition of the TGFβ signaling pathway effectively abrogated the oncogenic activities of GDF15 in MM cells, including cell proliferation, apoptosis, and fermentation. In vivo experiments using a subcutaneous xenotransplanted tumor model confirmed that GDF15 knockdown led to a significant reduction in tumor growth, while GDF15 overexpression promoted tumor growth. Overall, our study provides insights into the molecular mechanisms underlying MM pathogenesis and highlights the potential of targeting GDF15-TGFβ signaling -glycolysis axis as a therapeutic approach for future therapeutic interventions in MM.

Abstract B011: Adenosine signaling and immune modulation in the tumor microenvironment: insights from lung cancer murine model of radiation therapy

Abstract Introduction: Radiation therapy (RT) induces adenosine signaling by releasing ATP in the tumor microenvironment (TME), promoting immune suppression and tumor progression. This study investigates how RT impacts adenosine signaling and immune cell infiltration in a preclinical model of non-small cell lung cancer (NSCLC). Using immunophenotyping of tumor-infiltrating immune cells in LLC1 murine model, we aim to explore the potential of targeting the adenosine pathway in combination with RT to enhance antitumor immunity and clinical outcomes. Methods: LLC1 tumor cells (5 x 105) were implanted subcutaneously in the flanks of C57BL/6 mice. After 12 days, mice received RT (4 Gy x 3 or 8 Gy x 3). Tumor volumes were measured thrice a week and harvested 5- and 10-days post-RT. We performed immunophenotyping of tumor tissue to assess changes in immune (CD45+) and CD45- compartments. The expression of adenosine signaling pathway members was quantified in distinct immune cell populations. Results: RT caused significant inhibition in tumor growth in a dose- and time-dependent manner. 5-days post-RT, CD73 (P< 0.01) and CD39 expressions (P< 0.01) were significantly upregulated in the immune compartment, while A2BR (P< 0.01) and A2AR (P< 0.05) were decreased in the non-immune compartment. RT decreased T cell populations, including CD4+ and CD8+ T cells. Further, CD4+ T cells downregulate adenosine signaling markers, particularly CD73 (P< 0.05) and ENPP1 (P< 0.05), while CD8+ T cells decreased in CD73 expression (P< 0.01). Myeloid subtype abundance was unchanged, but A2AR, A2BR, CD39, and CD73 expressions increased in the M2-like macrophage compartment (F4-80+MHCII-). 10 days post-RT, all adenosine signaling pathway markers significantly increased in the immune compartment and significantly decreased in the non-immune compartment. T cells (TCRb+) and macrophages (F4-80+) increased in abundance, including immuno-suppressive Arg+ macrophages (P< 0.05). Macrophage population that expressed the adenosine signaling markers increased by 10-15% (P< 0.05). While the proportion of CD4+ T cells and Tregs (CD4+FoxP3+) that expressed CD39 and CD73 also increased (P< 0.05). Conclusions: Our study revealed that RT promotes an immunosuppressive TME, in part through adenosine signaling. 5 days post-RT, we observed lymphocyte depletion and upregulation of the adenosine signaling pathway in M2-like macrophages. 10 days post-RT, adenosine signaling significantly increased across the immune compartment and decreased in the non-immune compartment, suggesting that the increase in adenosine signaling post-RT is immunologically mediated and largely driven by the myeloid compartment. These data lead to the hypothesis that inhibition of the adenosine signaling pathway may partially mitigate RT-induced immune suppression and improve RT efficacy. Ongoing work includes quantification of adenosine by LC-MS, extension to a preclinical SCC model of NSCLC, and evaluating adenosine pathway inhibition in combination with RT as a therapeutic strategy to improve antitumor immune responses. Citation Format: Krishan K. Saini, Samanta Sarti, Chelsea L. Rahiman, Brian E. Ragaishis, Patrick J. McCann, Shruti Bansal, Jason Cham, Julia An, Rosa Martin, Ariel Chen, Catherine S. Spina. Adenosine signaling and immune modulation in the tumor microenvironment: insights from lung cancer murine model of radiation therapy. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr B011.

Lipopolysaccharide (LPS) induces sclerostin secretion by extracellular vesicle via TLR4/miR-92a-3p/PTEN/NF-κB signalling pathway in murine macrophage.

Sclerostin (SOST) is traditionally regarded as an osteocyte-derived secreted glycoprotein that regulates bone mineralization. Recent studies reported that SOST is also released from non-skeletal sources, especially during inflammation. However, the cellular source and regulatory mechanisms governing SOST generation in inflammation remain unclear. This study investigated whether macrophages produce SOST in response to inflammatory stimuli and determined associated regulatory pathways. The effect of lipopolysaccharide (LPS)-induced inflammation in SOST generation and its underlying regulatory mechanism was examined on mouse macrophage RAW 264.7 by western blot and immunofluorescent staining. Transfection with miR-92a-3p mimic and inhibitor were used to validate its role in SOST production. The role of NF-κB and TLR4 were studied using pharmacological inhibitors BAY 11-7085 and TAK242, respectively. The involvement of NF-κB and TLR4 in LPS-induced SOST production was further validated through nuclear NF-κB p65 immunoprecipitation and TLR4 small interfering RNA (siRNA) experiments, respectively.GW4869 and manumycin A (extracellular vesicles (EV) biogenesis inhibitors) were used to examine the associated of SOST and EV. Finally, SOST expression and characteristics of the isolated EV were assessed by Western blot and nanoparticle tracking analysis (NTA). LPS significantly induced SOST protein expression and secretion in RAW 264.7. MiR-92a-3p was upregulated by LPS stimulation in macrophages. Transfection of miR-92a-3p mimic increased SOST generation in RAW 264.7. Inhibition of TLR4 and NF-κB signalling pathways using pharmacological inhibitors significantly suppressed LPS-induced SOST in RAW 264.7. Similarly, TLR4 siRNA effectively suppressed LPS-induced SOST level. However, the LPS-induced upregulation of miR-92a-3p was only regulated by TLR4, but not by NF-κB. NF-κB was found to directly bind to the mouse sost promoter, thereby activating sost transcription. Additionally, SOST secretion was found predominantly associated with EV from LPS-stimulated cells, and inhibition of EV biogenesis suppressed SOST production in RAW 264.7 cells. In conclusion, our study showed, for the first time, that LPS induced SOST generation and secretion via TLR4/miR-92a-3p/PTEN/NF-κB singling pathway in murine macrophage RAW 264.7 cells. Moreover, we showed that SOST is secreted from the RAW 264.7 cells in the form of extracellular vesicle. This study identified macrophage as a novel source of SOST, highlighting its potential role in inflammatory diseases.

Subacute PM2.5 Exposure Induces Hepatic Insulin Resistance Through Inflammation and Oxidative Stress.

Epidemiological studies prove that type II diabetes, characterized by insulin resistance (IR), may be caused by fine particulate matter 2.5 (PM2.5). However, underlying mechanisms whereby PM2.5, particularly during short-term exposure, induces liver dysfunction leading to IR remains poorly understood. In the present study, HepG2 cells and the BALB/c mouse model were used to explore how PM2.5 affects insulin sensitivity. The effects of subacute PM2.5 exposure on glucose metabolism were examined using commercial kits. Oxidative stress and inflammation were detected by fluorescent staining and RT-qPCR. The phosphorylation of PI3K/AKT was examined by Western blot. Subacute PM2.5 exposure induced IR, as reflected by increased glucose levels in cell supernatants, elevated insulin levels, and the impaired intraperitoneal glucose tolerance test in mice. PM2.5 induced oxidative stress, as evidenced by increased reactive oxygen species, cytochrome P450 2E1, and malondialdehyde, along with reduced superoxide dismutase 1/2 and silent information regulator 1. IL-6 and TNF-α were found to be upregulated using RT-qPCR. Western blot showed that PM2.5 inhibited the PI3K-AKT signaling pathway, indicated by the decreased phosphorylation of PI3K/AKT in HepG2 cells. Additionally, H&E staining showed only mild hepatic injury in mice liver. These results firmly suggest that subacute PM2.5 exposure induces insulin resistance through oxidative stress, inflammation, and the inhibition of the PI3K-AKT signaling pathway.

Baicalin Mitigates Cardiac Hypertrophy and Fibrosis by Inhibiting the p85a Subunit of PI3K.

Background: Heart failure (HF) is a serious public health concern. Baicalin is one of the major active ingredients of a traditional Chinese herbal medicine, Huang Qin, which is used to treat patients with chest pain or cardiac discomfort. However, the underlying mechanism(s) of the cardioprotective effect of baicalin are still not fully understood. Methods: Isoprenaline injection or transverse aortic constriction-induced animal models and isoprenaline or angiotensin 2 administration-induced cell models of heart failure were established. Baicalin (15 mg/kg/day or 25 mg/kg/day) was administered in vivo, and 10 μM baicalin was administered in vitro. Potential pharmacological targets of baicalin and genes related to heart failure were identified via different databases, which suggested that PI3K-Akt may be involved in the effects of baicalin. Molecular docking was carried out to reveal the effect of baicalin on p85a. Results: We observed significant antihypertrophic and antifibrotic effects of baicalin both in vivo and in vitro. The mean cross-sectional area of cardiomyocytes recovered from 390 μm2 in the HF group to 195 μm2 in the baicalin-treated group. The area of fibrosis was reduced from 2.8-fold in the HF group to 1.62-fold in the baicalin-treated group. Baicalin displayed a significant cardioprotective effect via the inhibition of the PI3K signaling pathway by binding with five amino acid residues of the p85a regulatory subunit of PI3K. The combination treatment of baicalin and an inhibitor of PI3K p110 demonstrated a stronger cardioprotective effect. The mean ejection fraction increased from 54% in the baicalin-treated group to 67% in the combination treatment group. Conclusions: Our work identified baicalin as a new active herbal ingredient that is able to treat isoprenaline-induced heart dysfunction and suggests that p85a is a pharmacological target. These findings reveal the significant potential of baicalin combined with an inhibitor of PI3K p110 for the treatment of heart failure and support more clinical trials in the future.

Qige Decoction attenuated non-alcoholic fatty liver disease through regulating SIRT6-PPARα-mediated fatty acid oxidation.

Sirtuin 6 (SIRT6), a potential therapeutic target for non-alcoholic fatty liver disease (NAFLD), has been shown to regulate fatty acid oxidation (FAO) by interacting with peroxisome proliferator-activated receptor α (PPARα). However, the impact of SIRT6-PPARα pathway on NAFLD phenotype has not yet been reported. Qige decoction (QG), a traditional Chinese medicine (TCM) formula, is widely applied to treat disorders of glycolipid metabolism. Our previous experiments showed that QG reduced hepatic steatosis and provided preliminary evidence that QG may promote FAO. However, a thorough understanding of molecular mechanisms by which QG regulates FAO requires further investigation. To investigate the role of SIRT6-PPARα signalling pathway on NAFLD phenotype and explore the mechanism by which QG improves NAFLD and its relationship with FAO regulated by SIRT6-PPARα signalling pathway. In vivo study, NAFLD mice induced by high fat diet (HFD) were divided into two parts. The first part involved four groups: control (CON), model (MOD), PPARα agonist (WY-14,643, WY), and SIRT6 inhibitor (OSS-128,167, OS) groups. The second part involved five groups: CON group, MOD group, positive drug (POS) group, low dose QG (QGL) group, and high dose QG (QGH) group. Widely-targeted lipidomic were performed by UHPLC-QTOF/MS to analyse differential lipids (DELs) in the liver, while differentially expressed genes (DEGs) were analysed by transcriptome analysis on the Illumina sequencing platform. In vitro study, co-immunoprecipitation and dual luciferase assay were employed to further identify the molecular mechanisms of SIRT6-PPARα interaction. The lentiviral vector, TG assay, and acetyl-CoA assay were used to clarify the indispensable role of the SIRT6-PPARα signalling pathway on QG amelioration of lipid accumulation in vitro. Down-regulation of SIRT6 inhibited PPARα-mediated FAO and aggravated lipid accumulation in hepatocytes both in vivo and in vitro. SIRT6 bound to PPARα in HepG2 cells; however, SIRT6 activation of the PPARα promoter was not detected. Along with QG reduced hepatocyte lipid accumulation, SIRT6-PPARα signalling pathway was upregulated in vivo and in vitro. However, the alleviating effect of QG on lipid accumulation was blocked by SIRT6 silencing in vitro. This study verified that SIRT6-PPARα signalling pathway inhibition exacerbated NAFLD dyslipidaemia and hepatic steatosis. In addition, this study provided the first in-depth analysis of the molecular mechanisms by which QG ameliorates NFALD, involving promotion of FAO through activation of the SIRT6-PPARα signalling pathway. Our study offers significant insights for the clinical application of QG.

Activation of the cGAS-sting Pathway Mediated by Nanocomplexes for Tumor Therapy.

cGAS (cyclic GMP-AMP synthase)-STING (stimulator of interferon genes) pathway is an natural immune response signaling pathway in the human body that is essential for sensing abnormal DNA aggregation in the cell. When the cGAS protein senses abnormal or damaged DNA, it forms a second messenger called cyclic dinucleotide (cGAMP). The cycled dinucleotide will activate the downstream STING protein, thereby inducing the expression of inflammatory cytokines such as type I interferon, which binds to receptors on its own cell membrane and ultimately initiates multiple immune response pathways. This signaling pathway plays an important immune role in antimicrobial and antitumor functions, etc. so the development of drugs targeting this signaling pathway has important clinical application value. In recent years nanocomplexes based cGAS-STING signaling pathway activation and inhibition treatments have been gradually developed. In this review, on the basis of elaborating the main activation mechanism of the cGAS-STING pathway, we further introduced the nanocomplexes that effectively activate the cGAS-STING pathway, focusing on the composition, types and applications of the nanocomplexes. In addition, we discussed the key challenges and future research directions of the way that stimulating the cGAS-STING signaling pathway in the form of nanocomplexes to activate immuno-tumor therapy. Our work aims to provide a better understanding of the progress of nanotherapeutics in the cGAS-STING pathway, providing a promising anti-tumor therapeutic strategy.

Prostate cancer cells elevate glycolysis and G6PD in response to caffeic acid phenethyl ester-induced growth inhibition

BackgroundCaffeic acid phenethyl ester (CAPE) is the main bioactive component of poplar type propolis. We previously reported that treatment with caffeic acid phenethyl ester (CAPE) suppressed the cell proliferation, tumor growth, as well as migration and invasion of prostate cancer (PCa) cells via inhibition of signaling pathways of AKT, c-Myc, Wnt and EGFR. We also demonstrated that combined treatment of CAPE and docetaxel altered the genes involved in glycolysis and tricarboxylic acid (TCA) cycle. We therefore suspect that CAPE treatment may interfere glucose metabolism in PCa cells.MethodsSeahorse Bioenergetics platform was applied to analyzed the extra cellular acidification rate (ECAR) and oxygen consumption rate (OCR) of PCa cells under CAPE treatment. UPLC-MSMS with Multiple Reaction Monitoring (MRM), PCR, and western blot were used to analyze the effects of CAPE on metabolites, genes, and proteins involved in glycolysis, TCA cycle and pentose phosphate pathway in PCa cells. Flow cytometry and ELISA were used to determine the level of reactive oxygen species in PCa cells being treated with CAPE.ResultsSeahorse Bioenergetics analysis revealed that ECAR, glycolysis, OCR, and ATP production were elevated in C4-2B cells under CAPE treatment. Protein levels of glucose-6-phosphate dehydrogenase (G6PD), phosphogluconate dehydrogenase (PGD), glutaminase (GLS), phospho-AMPK Thr172 as well as abundance of pyruvate, lactate, ribulose-5-phosphate, and sedoheptulose-7-phosphate were increased in CAPE-treated C4-2B cells. ROS level decreased 48 h after treatment with CAPE. Co-treatment of AMPK inhibitor with CAPE exhibited additive growth inhibition on PCa cells.ConclusionsOur study indicated that PCa cells attempted to overcome the CAPE-induced stress by upregulation of glycolysis and G6PD but failed to impede the growth inhibition caused by CAPE. Concurrent treatment of CAPE and inhibitors targeting glycolysis may be effective therapy for advanced PCa.

The impact of signaling pathways on the desmosome ultrastructure in pemphigus

IntroductionThe autoantibody-driven disease pemphigus vulgaris (PV) impairs desmosome adhesion in the epidermis. In desmosomes, the pemphigus autoantigens desmoglein 1 (Dsg1) and Dsg3 link adjacent cells. Dsgs are clustered by plaque proteins and linked to the keratin cytoskeleton by desmoplakin (Dp). The aim of this study was to identify the impact of several PV-related signaling pathways on desmosome ultrastructure.MethodsSTED microscopy, Dispase-based dissociation assay.ResultsAs observed using STED microscopy, pemphigus autoantibodies (PV-IgG) reduced desmosome number, decreased desmosome size, increased plaque distance and thickness and caused loss of adhesion. Decreased desmosome number, increased plaque distance and thickness and loss of adhesion correlate with features found for newly assembled immature desmosomes, observed after Ca2+ depletion and repletion. This was paralleled by plaque asymmetry, keratin filament retraction and fragmentation of Dsg1 and Dsg3 immunostaining. Inhibition of each individual signaling pathway investigated here prevented the loss of adhesion and ameliorated keratin retraction. In addition, inhibition of p38MAPK or PLC completely rescued all parameters of desmosomes ultrastructure and increased desmosome number under basal conditions. In contrast, inhibition of MEK1/2 was only partially protective for desmosome size and plaque thickness, whereas inhibition of Src or increase of cAMP decreased desmosome size but increased the desmosome number even in the presence of PV-IgG. DiscussionAlterations of the desmosomal plaque ultrastructure are closely related to loss of adhesion and regulated differently by signaling pathways involved in pemphigus pathogenesis. This insight may allow identification of novel treatment options targeting specific steps of desmosome turn-over in the future.

The microenvironment cell index is a novel indicator for the prognosis and therapeutic regimen selection of cancers

BackgroundIt is worthwhile to establish a prognostic prediction model based on microenvironment cells (MCs) infiltration and explore new treatment strategies for triple-negative breast cancer (TNBC).MethodsThe xCell algorithm was used to quantify the cellular components of the TNBC microenvironment based on bulk RNA sequencing (bulk RNA-seq) data. The MCs index (MCI) was constructed using the least absolute shrinkage and selection operator Cox (LASSO-Cox) regression analysis. Single-cell RNA sequencing (scRNA-seq), spatially resolved transcriptomics (SRT), and multiplex immunofluorescence (mIF) staining analyses verified MCI. The mechanism of action of the MCI was investigated in tumor-bearing mice.ResultsMCI consists of the six types of MCs, which can precisely predict the prognosis of the TNBC patients. scRNA-seq, SRT, and mIF analyses verified the existence and proportions of these cells. Furthermore, combined with the spatial distribution characteristics of the six types of MCs, an MCI-enhanced (MCI-e) model was constructed, which could predict the prognosis of the TNBC patients more accurately. More importantly, inhibition of the insulin signaling pathway activated in the cancer cells of the MCIhigh the TNBC patients significantly prolonged the survival time of tumor-bearing mice.ConclusionsOverall, our results demonstrate that MCs infiltration can be exploited as a novel indicator for the prognosis and therapeutic regimen selection of the TNBC patients.

Infection-induced lysine lactylation enables herpesvirus immune evasion.

Aerobic glycolysis is a hallmark of many viral infections, leading to substantial accumulation of lactate. However, the regulatory roles of lactate during viral infections remain poorly understood. Here, we report that human cytomegalovirus (HCMV) infection leverages lactate to induce widespread protein lactylation and promote viral spread. We establish that lactyllysine is enriched in intrinsically disordered regions, regulating viral protein condensates and immune signaling transduction. Dynamic lactylation of immune factors suppresses immunity, a feature we show to be shared for HCMV and herpes simplex virus 1 infections, through regulation of RNA binding protein 14 and interferon-γ-inducible protein 16 (IFI16). K90 lactylation of the viral DNA sensor IFI16 inhibits recruitment of the DNA damage response kinase DNA-PK, preventing IFI16-driven virus gene repression and cytokine induction. Together, we characterize global protein lactylation dynamics during virus infection, finding that virus-induced lactate contributes to its immune evasion through direct inhibition of immune signaling pathways.

Effects of Different Levels of Lycium barbarum Flavonoids on Growth Performance, Immunity, Intestinal Barrier and Antioxidant Capacity of Meat Ducks.

Background: In vitro findings on the biological functions of Lycium barbarum flavonoids (LBFs) as feed additives are limited. This study aimed to explore the effects of different concentrations of LBFs on the growth performance, immune function, intestinal barrier, and antioxidant capacity of meat ducks. A total of 240 one-day-old male meat ducks were randomly allocated to four groups, each receiving a basal diet supplemented with 0 (control), 250, 500, or 1000 mg/kg of LBFs for 42 d. Results: The results showed that dietary supplementation with 500 mg/kg of LBFs resulted in a significant increase in average daily feed intake, body weight, average daily gain, and feed conversion ratio. Dietary supplementation with 500 or 1000 mg/kg of LBFs resulted in significant decreases in serum levels of D-lactic acid and lipopolysaccharide. Dietary supplementation with 500 mg/kg LBFs significantly decreased diamine oxidase activity and enhanced the activities of catalase, total antioxidant capacity, and glutathione peroxidase in the jejunal mucosa, as well as the activity of total superoxide dismutase and the content of glutathione in the ileal mucosa, while significantly lowering the content of malondialdehyde in the ileal mucosa. Dietary supplementation with 500 mg/kg LBFs significantly up-regulated the mRNA expression of genes associated with intestinal barrier function and antioxidant capacity in the jejunal and ileal mucosa, as well as the protein expression of these antioxidant genes, and led to a significant reduction in the mRNA expression of pro-apoptotic and inflammatory-related genes. Conclusions: The addition of LBFs to the diet improved the growth performance, intestinal barrier function, immune response, and antioxidant capacity of the ducks, which may be closely associated with the activation of the Nrf2 signaling pathway and the inhibition of the NF-κB signaling pathway. The optimal dietary inclusion level of LBFs in ducks was 500 mg/kg.

Inhibition of CCR2 attenuates NLRP3-dependent pyroptosis after myocardial ischaemia-reperfusion in rats via the NF-kB pathway.

Myocardial infarction (MI) is a leading cause of mortality worldwide, contributing significantly to long-term cardiac dysfunction and heart failure. Effective therapeutic strategies are urgently needed to mitigate the extensive damage caused by MI and subsequent ischemia-reperfusion (I/R) injury. This study investigates the role of the Chemokine receptor 2 (CCR2) in regulating NLRP3-dependent cardiomyocyte pyroptosis following myocardial ischemia-reperfusion (MIR), elucidating its molecular mechanisms. A myocardial ischemia-reperfusion model was established using 124 Sprague-Dawley rats by ligating the left coronary artery, inducing 30min of ischemia. Following ischemia, RS504393, a selective CCR2 antagonist, was administered intraperitoneally one hour after reperfusion. To further explore the underlying mechanisms, the NF-κB pathway agonist Phorbol 12-myristate 13-acetate (PMA) was administered 1h post-MIR. The results showed a marked increase in CCR2 expression in the heart, peaking on the first day of reperfusion. Treatment with RS504393 significantly improved short-term cardiac function and reduced myocardial infarction size, decreased myocardial pyroptosis and suppressed the expression of NLRP3, GSDMD, Caspase-1, IL-1β, and IL-18 through inhibition of the NF-κB signaling pathway. This effect was reversed with the administration of PMA. In summary, the inhibition of CCR2 shows potential in mitigating myocardial injury following MIR by modulating the NF-κB signaling pathway. These findings highlight CCR2 as a promising therapeutic target for myocardial ischemia-reperfusion injury.

Longikaurin A, a natural ent-kaurane, suppresses proliferation, invasion and tumorigenicity in oral squamous cell carcinoma cell by via inhibiting PI3K/Akt pathway in vitro and in vivo.

Background: Longikaurin A (LK-A), a naturally occurring ent-kaurane diterpenoid, has been identified as a promising anti-cancer agent. This study aims to elucidate the anti-tumorigenic effects of LK-A on oral squamous cell carcinoma (OSCC) cells and to unravel its underlying mechanisms. Methods: In vitro assays, including CCK-8 and EdU, were performed to assess cell viability and proliferation. Transwell migration and invasion assays evaluated cell mobility and invasive potential. Apoptotic effects were analyzed using Annexin V-FITC/PI staining and TUNEL assays. Western blot analysis was conducted to examine protein expression related to cell cycle, apoptosis, and the PI3K/Akt signaling pathway. In vivo experiments involved treating mouse xenograft models with LK-A and evaluating tumor growth and signaling pathway inhibition through immunohistochemistry and Western blot assays. Results: LK-A significantly suppressed cell viability and proliferation in a dose-dependent manner, with IC50 values of 4.36 μM and 4.93 μM at 24 h, and 1.98 μM and 2.89 μM at 48 h for CAL27 and TCA-8113 cells, respectively. EdU assays revealed a reduction in the EdU positive rate, and cell cycle analysis showed G2/M phase arrest. Western blot analysis confirmed decreased expression of CyclinB1 and Cdc2. LK-A significantly inhibited OSCC cell mobility and invasive potential, with downregulation of MMP-2 and MMP-9 expression. Apoptotic effects were confirmed by increased apoptosis, upregulation of Bax and cleaved caspase-3, and downregulation of Bcl-2. LK-A suppressed the PI3K/AKT signaling pathway, as evidenced by reduced phosphorylation of PI3K, AKT, and mTOR. The AKT activator SC79 reversed the antiproliferative and pro-apoptotic effects of LK-A. In vivo, LK-A significantly inhibited tumor growth in mouse xenograft models, with reduced tumor weights and volumes, and no significant loss in body weight. Immunohistochemistry and Western blot assays confirmed the inhibition of p-Akt and Ki-67 expression. Conclusion: These findings suggest that LK-A exerts potent antiproliferative, anti-migratory, and pro-apoptotic effects on OSCC cells through the suppression of the PI3K/AKT signaling pathway, demonstrating its potential as a therapeutic agent for OSCC.

Inactivated SARS-CoV-2 induces acute skeletal muscle damage in human K18-hACE2 transgenic mice.

The pandemic due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) resulted in over 7 million global fatalities and billions of individuals diagnosed with COVID-19. Acute and chronic muscle impairment associated with SARS-CoV-2 infection affected a substantial number of patients, leading to the development of symptoms such as fatigue, muscle pain, and exercise intolerance. Our study introduces an animal model to improve understanding of the pathogenicity caused by SARS-CoV-2 in human skeletal muscle. In this investigation, human angiotensin-converting enzyme 2 under a cytokeratin 18 promoter transgenic mice were subjected to intratracheal instillation with either inactivated SARS-CoV-2 or the virus-free culture medium, with or without pre-treatment with the P2X receptor inhibitor Brilliant Blue G (BBG). Muscle strength, morphology, and inflammatory mediators were measured. Inactivated SARS-CoV-2 induced a significant decrease in mice muscle strength, accompanied by histopathological changes in gastrocnemius and diaphragm muscles, including leukocytic infiltrates, cytoplasmic vacuoles, and centralized nuclei. Also, a notable increase in caspase 3 amount was observed, suggesting muscle apoptosis. Significant elevations were noted in inflammatory mediators in the muscle of inactivated SARS-CoV-2 mice, including high mobility group box-1, tumor necrosis factor-alpha, phospho-nuclear factor kappa B, caspase 11, and pannexin-1. On the other hand, pyroptosis markers such as caspase 1, interleukin-1β, and gasdermin D remained unaltered in all experimental groups. Treatment with BBG mitigated the observed effects, indicating that inhibition of purinergic signaling pathways protects muscles from the inflammatory impact induced by inactivated SARS-CoV-2. This study emphasizes the potential efficacy of purinergic inhibition in ameliorating SARS-CoV-2-induced muscular impairments.

Neuroprotective Effects of Eugenol Acetate Against Ischemic Stroke.

To explore the neuroprotective effect of Eugenol Acetate (EA) on post-stroke neuroinflammation and investigate the underlying mechanisms. For in vitro experiments, primary microglia were pre-incubated with EA for 2hours, followed by lipopolysaccharide (LPS) stimulation for 24hours or Oxygen-Glucose Deprivation (OGD) treatment for 4hours. Real-time quantitative PCR, enzyme-linked immunosorbent assay (ELISA) and Western blot were performed to examine the expression levels of inflammatory cytokines in primary microglia. The activation of NF-κB signaling pathway was evaluated by immunofluorescence staining and Western blot. For in vivo experiments, middle cerebral artery occlusion (MCAO) was constructed to mimic ischemic brain injury on 8-week-old male C57BL/6J mice. The mice were continuously injected intraperitoneally with EA or vehicle after MCAO. Neurobehavioral tests and TTC staining were conducted to estimate the neurological deficits and infarct area. Moreover, the white matter integrity after MCAO was observed via immunofluorescence staining. EA significantly reduced the expression of pro-inflammatory cytokines in LPS or OGD treated primary microglia, and inhibited LPS-induced activation of the NF-κB signaling pathway. In addition, EA alleviated ischemic brain injury and improved neuromotor function of MCAO mice. Furthermore, long-term neurological deficits and white matter integrity were improved by EA treatment after MCAO. EA alleviated ischemic injury and restored white matter integrity in MCAO mice, which might be associated with the inhibition of NF-κB signaling pathway in microglia. Therefore, EA might be a promising candidate for the treatment of ischemic stroke.