Deoxynucleotidyl Transferase dUTP Nick End Research Articles

Sennoside A represses the malignant phenotype and tumor immune microenvironment of non-small cell lung cancer cells by inhibiting the TRAF6/NF-κB pathway.

Non-small cell lung cancer (NSCLC) is a prominent cause of cancer death worldwide. Sennoside A (SA) is the primary anthraquinone active component from Rheum officinale Baill and exerts antitumor functions in multiple human tumors. This research aimed to elucidate the function and mechanism of SA in NSCLC. SA functions in NSCLC were determined using Cell Counting Kit-8 (CCK-8) assay, Terminal deoxynucleotidyl transferase dUTP nick-end labeling analysis, Transwell assay, Enzyme-Linked Immunosorbent Assay (ELISA), and Western blot. Meanwhile, the SA mechanism in NSCLC was examined with Western blot, immunofluorescence assay, CCK-8 assay, Transwell analysis, and ELISA. Furthermore, SA functions in NSCLC growth in vivo were assessed by the establishment of a tumor xenograft model, hematoxylin-eosin staining, analysis of NSCLC tissue apoptosis, and immunohistochemistry assays. Functionally, less than 200µM SA had no significant effect on normal human bronchial epithelial cell BEAS-2B cell viability. Furthermore, H460 cell viability was decreased when the SA dose was greater than or equal to 25µM (IC50 = 53.34µM). A549 cell viability was reduced when the SA dose was greater than or equal to 12.5µM (IC50 = 48.21µM). Also, SA repressed NSCLC cell proliferation and boosted cell apoptosis. SA restrained NSCLC cell invasion and tumor microenvironment. Mechanically, SA weakened NSCLC cell proliferation, invasion, and tumor microenvironment, yet this impact was abolished after transfecting pcDNA3.1-TRAF6, which indicated that SA repressed NSCLC cell proliferation, invasion, and tumor microenvironment through inactivating TRAF6/NF-κB. Moreover, SA reduced subcutaneous tumor volume and promoted NSCLC tissue apoptosis in vivo. SA repressed NSCLC cell proliferation, invasion, and tumor microenvironment through inactivating TRAF6/NF-κB.

20-Hydroxyeicosatetraenoic Acid Regulates the Src/EGFR/NF-κB Signaling Pathway Via GPR75 to Activate Microglia and Promote TBI in the Immature Brain.

20-Hydroxyeicosatetraenoic acid (20-HETE) is associated with secondary damage in traumatic brain injury (TBI) of the immature brain. Microglial activation is pivotal in this process. However, the underlying mechanism of action remains unknown. While 20-HETE interacts with G protein-coupled receptor 75 (GPR75) in some pathological processes, their interaction in brain tissue remains uncertain. This study aimed to investigate whether 20-HETE can activate microglia by binding to GPR75 in TBI of the immature brain. Drug affinity responsive molecular target stability (DARTS) assays, cycloheximide (CHX) chase assays, and auto-dock assays were employed to analyze the interaction between 20-HETE and GPR75. The expression levels of cytochrome P450 4A (CYP4A) and GPR75 in activated microglia in an immature brain TBI model were observed by western blot and multiple immunofluorescence staining. The effects of different levels of 20-HETE expression and lentivirus-mediated GPR75 gene silencing on 20-HETE-induced inflammatory factor release from BV-2 cells were observed by enzyme-linked immunoassay (ELISA). The phosphorylation levels of the downstream Src kinase, epidermal growth factor receptor (EGFR), and nuclear factor (NF)-κB were assessed using western blot. Cell viability and apoptosis were detected by CCK-8 and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays. 20-HETE bound to the GPR75 protein and inhibited its degradation. GPR75 gene silencing reversed the 20-HETE-induced inflammatory activation of BV-2 cells, effectively inhibiting the activation of the Src/EGFR/NF-κB pathway and the effects of 20-HETE on cell viability and the apoptosis rate. In contrast, overexpression of GPR75 had the opposite effect. In addition, after immature brain TBI, the 20-HETE and GPR75 expression levels were upregulated in microglia, with significant activation of the Src/EGFR/NF-κB pathway. Inhibition of 20-HETE synthesis with N-hydroxy-N'-(4-n-butyl-2-methylphenyl) formamidine (HET0016) produced the opposite effect. 20-HETE regulates the Src/EGFR/NF-κB signaling pathway via GPR75 to activate microglia, promoting immature brain TBI. These findings offer a novel target for promoting the brain injury effect of 20-HETE.

Prunella vulgaris Extract Ameliorates Testosterone-Induced Benign Prostatic Hyperplasia by Regulating Androgen Levels, Cell Proliferation, and Apoptosis

Background/Objectives: Benign prostatic hyperplasia (BPH) is a prevalent urological condition affecting elderly men. Prunella vulgaris L. (PV), a perennial herbaceous plant native to Europe and Asia, has anti-inflammatory, antioxidant, and antimicrobial effects. In this study, we determined the effect of PV extract on the development of BPH. Methods: Rats were treated via a daily hypodermic injection of testosterone propionate (TP; 3 mg/kg) for 4 weeks. Groups of BPH rats were treated with or without PV (60 or 80 mg/kg) by oral gavage. Results: In BPH model rats, PV considerably reduced their relative prostate weight and serum concentrations of dihydrotestosterone (DHT) and testosterone. The TP-induced increases in epithelial thickness in the prostate, proliferating cell nuclear antigen (PCNA) expression, and cyclin D1 expression were remarkably reduced, whereas terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells and cleaved caspase-3 levels were increased, in PV-treated rats compared to BPH rats. The mRNA expression levels of growth factors, such as transforming growth factor-β (TGF-β), fibroblast growth factor (FGF), and insulin-like growth factor (IGF-2), were significantly reduced in PV-treated rats. Mechanistically, the TP-induced activation of c-Jun N-terminal kinase (JNK) was reduced by PV administration. Conclusions: These results designate that PV effectively ameliorates the development of testosterone-induced BPH through anti-androgenic, anti-proliferative, and pro-apoptotic activities, suggesting that it could be a potential therapeutic substance for BPH.

Embryotrophic effect of exogenous protein contained adipose-derived stem cell extracellular vesicles

BackgroundExtracellular vesicles (EVs) regulate cell metabolism and various biological processes by delivering specific proteins and nucleic acids to surrounding cells. We aimed to investigate the effects of the cargo contained in EVs derived from adipose-derived stem cells (ASCs) on the porcine embryonic development.MethodsASCs were isolated from porcine adipose tissue and characterized using ASC-specific markers via flow cytometry. EVs were subsequently extracted from the conditioned media of the established ASCs. These EVs were added to the in vitro culture environment of porcine embryos to observe qualitative improvements in embryonic development. Furthermore, the proteins within the EVs were analyzed to investigate the underlying mechanisms.ResultsWe observed a higher blastocyst development rate and increased mitochondrial activity in early stage embryos in the ASC-EVs-supplemented group than in the controls (24.8% ± 0.8% vs. 28.6% ± 1.1%, respectively). The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay of blastocysts also revealed significantly reduced apoptotic cells in the ASC-EVs-supplemented group. Furthermore, through proteomics, we detected the proteins in ASC-EVs and blastocysts from each treatment group. This analysis revealed a higher fraction of proteins in the ASC-EVs-supplemented group than in the controls (1,547 vs. 1,495, respectively). Gene analysis confirmed that ASC-EVs showed a high expression of tyrosine-protein kinase (SRC), whereas ASC-EVs supplemented blastocysts showed a higher expression of Cyclin-dependent kinase 1 (CDK1). SRC is postulated to activate protein kinase B (AKT), which inhibits the forkhead box O signaling pathway and activates CDK1. Subsequently, CDK1 activation influences the cell cycle, thereby affecting in vitro embryonic development.ConclusionASC-EVs promote mitochondrial activity, which is crucial for the early development of blastocysts and vital in the downregulation of apoptosis. Additionally, ASC-EVs supply SRC to porcine blastocysts, thereby elongating the cell cycle.

Icariin ameliorates Coxsackievirus B3-induced viral myocarditis by modulating the S100 calcium binding protein A6/β-catenin/c-Myc signaling pathway

BackgroundCoxsackievirus B3 (CVB3) is a leading cause of viral myocarditis and is currently lacking specific pharmacological treatments, highlighting the critical need for therapeutic development. Icariin (ICA), a prenylated flavonol glycoside, was previously found to exhibit several pharmacological effects, but its potential to combat CVB3 remains uninvestigated. PurposeThis study aimed to elucidate the anti-CVB3 efficacy of ICA and elucidate its molecular mechanisms. MethodsCVB3-infected HeLa cells, H9C2 cells and neonate rat ventricular cardiomyocytes (NRVCs) were selected as in vitro models, and were treated with ICA at 1 and 10 μM. Additionally, BALB/c mice that were infected with CVB3 via intraperitoneal injection were chosen as in vivo model and were treated with ICA or ribavirin over 3 days. The effect of ICA against CVB3 was determined by Cell Counting Kit-8 (CCK-8) assay, western blot, real-time fluorescence quantitative PCR (RT–qPCR), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC) and flow cytometry. ResultsIn this study, it was found that ICA is capable of reducing CVB3 viral load both in vitro and in vivo. Mechanistic studies suggested that ICA prevents cardiomyocyte apoptosis by attenuating the S100 calcium binding protein A6 (S100A6)/β-catenin/c-Myc signaling pathway. Additionally, ICA inhibits the secretion of proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-1beta (IL-1β) and CXC motif chemokine ligand 2 (CXCL2) in heart tissue, thereby mitigating CVB3-induced myocarditis. Moreover, ICA also regulates the immune response of CD4+ T, CD8+ T and Treg cells by changing the cells numbers in spleen tissue. Lastly, ICA can reduce the load of other enteroviruses (such as CVA6, CVA16 and EV71) in rhabdomyosarcoma (RD) cells as well. ConclusionOur findings indicate that ICA provides significant protection against CVB3 infection by modulating the S100A6/β-catenin/c-Myc signaling pathway, suggesting its potential use as a novel drug against CVB3 infection in clinical application.

Lysine Acetyltransferase TIP60 Restricts Nerve Injury by Activating IKKβ/SNAP23 Axis-Mediated Autophagosome-Lysosome Fusion in Alzheimer's Disease.

The hyperphosphorylation of Tau protein is considered an important cause of neuronal degeneration in Alzheimer's disease (AD). The disruption of neuronal histone acetylation homeostasis mediated by Tip60 HAT is a common early event in neurodegenerative diseases, but the deeper regulatory mechanism on β-amyloid peptide (Aβ)-induced neurotoxicity and autophagic function in AD is still unclear. AD models were established both in APP/PS1 mice and Aβ1-42-treated SH-SY5Y cells. The Morris water maze test (MWM) was performed to examine mouse cognitive function. Neurological damage in the hippocampus was observed by hematoxylin-eosin (H&E), Nissl's, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and NeuN staining. Autophagosome-lysosome fusion was detected by immunohistochemistry, immunofluorescence, and Lyso-Tracker Red staining. Cell viability and apoptosis were evaluated by CCK-8 assay and flow cytometry. The molecular interactions were verified by co-immunoprecipitation (Co-IP), dual luciferase assays, and ChIP detections. The RNA and autophagy-lysosome-related proteins were assessed by Western blot and RT-qPCR. TIP60 overexpression improved cognitive deficits and neurological damage and restored the impairment of autophagy-lysosomes fusion invivo. Similarly, the upregulation of TIP60 in Aβ1-42-treated SH-SY5Y cells suppressed neuronal apoptosis and tau phosphorylation through the activating autophagy-lysosome pathway. Mechanistically, TIP60 activated IKKβ transcription by promoting SOX4 acetylation, thus leading to the translocation of SNAP23 to STX17-contained autophagosomes. Moreover, the protective roles of TIP60 in neuron damage were abolished by the inhibition of SOX4/IKKβ signaling. Collectively, our results highlighted the potential of the TIP60 target for AD and provided new insights into the mechanisms underlying neuroprotection in this disorder.

Ovule abortion accompanied by programmed cell death in Castanea mollissima

Chinese chestnut (Castanea mollissima Blume.) ovary contains 12-18 ovules, but only one can develop and grow normally, which indicates that the abortive rate of ovules can go as high as 94%. In this study, we observed the developmental characteristics and cytological changes of abortive ovules and fertile ovules during development. Morphological analysis showed it would take about 15∼20 days from globular to cotyledon embryo in fertile ovules, which is accompanied with the formation and apoptosis of endosperm. But there were stark differences in the nutrient transport, microstructure and ultrastructure of fertile and abortive ovules. The fluorescence disodium fluorescein and FDA indicated that all ovules were viable at early stages, but some ovules became inactive at different times as the ovary developed. Fluorescence labelling and ultrastructure showed cell nuclei of abortive ovules were disintegrated at 25 days after anthesis (DAA). Compared with the fertile ovules, starch grains were synthesized at earlier periods, but disappeared immediately, and the cells were usually irregular in shape, and had folded cell membranes in the abortive ovules. Furthermore, the Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was carried out to assess programmed cell death in fertile and abortive ovules, results showed that DNA fragmentation was occurred in the cells of abortive ovules. In short, these results provide new insights into ovule abortion in the angiosperm.

Overexpression of Nfs1 Cysteine Desulphurase Relieves Sevoflurane-Induced Neurotoxicity and Cognitive Dysfunction in Neonatal Mice Via Suppressing Oxidative Stress and Ferroptosis.

Clinical evidence suggests that multiple exposures to sevoflurane in young people may be detrimental to cognitive development. Iron accumulation in the hippocampus is associated with sevoflurane-induced neurotoxicity and cognitive deficits. The cysteine desulphurase, Nfs1, the rate-limiting enzyme for the biosynthesis of iron-sulphur clusters, plays a role in cellular iron homeostasis. However, the impact of Nfs1-mediated ferroptosis on sevoflurane-induced neurotoxicity and cognitive impairments in neonatal mice remains undetermined. Neonatal mice at postnatal Day 6 received 3% sevoflurane daily for 3 consecutive days. Cognitive function was assessed using the Morris water maze test, and neurotoxicity was evaluated through terminal deoxynucleotidyl transferase dUTP nick end labeling and immunofluorescence staining. Here, HT22 hippocampal neurons were employed for in-vitro experiments, and Fe2+ accumulation was measured. Ferroptosis-related genes, including glutathione peroxidase 4 (GPX4), transferrin receptor 1 (TFR1) and ferritin, in the hippocampus and HT22 cells were observed, along with oxidative stress-related indicators such as reactive oxygen species (ROS), methionine adenosyltransferase (MAT), glutathione (GSH) and lipid peroxidation (LPO). Transmission electron microscopy was utilized to examine the mitochondrial microstructure. Sevoflurane exposure significantly decreased Nfs1 expression in the hippocampus of mice and HT22 cells. This exposure resulted in cognitive impairments and neuronal damage in the hippocampus, which were alleviated by overexpression of Nfs1. Intracellular and mitochondrial iron accumulation occurred in HT22 cells following sevoflurane treatment. Sevoflurane exposure also significantly reduced GSH levels and increased levels of malondialdehyde, ROS and LPO in the hippocampus or HT22 cells. Additionally, sevoflurane exposure decreased GPX4 expression but increased TFR1 and ferritin expression in the hippocampus or HT22 cells. Overexpression of Nfs1 reversed the sevoflurane-induced alterations in ferroptosis-related genes and oxidative stress-related indicators. Furthermore, overexpression of Nfs1 alleviated sevoflurane-induced mitochondrial dysfunction. However, Nfs1 knockdown alone did not result in cognitive impairments, ferroptosis or oxidative stress. The overexpression of Nfs1 mitigated sevoflurane-induced neurotoxicity and cognitive impairment by modulating oxidative stress and ferroptosis through the regulation of iron metabolism and transport.

Trifolirhizin targets PTK6 to induce autophagy and exerts antitumor effects in nasopharyngeal carcinoma.

Trifolirhizin, a natural flavonoid glycoside, has been proved to exert antitumor activities in various human malignant tumors. PTK6 was identified as a direct target of trifolirhizin based on public database SuperPred (https://prediction.charite.de/). Overexpressed PTK6 in a variety of tumors is closely associated with the malignant development of tumors. Herein, this present research was formulated to elaborate the effects of trifolirhizin on the biological behaviors of nasopharyngeal carcinoma (NPC) cells and to probe into the intrinsic mechanisms. The current study firstly elucidated the tumor-inhibiting functions of trifolirhizin in NPC malignant progression from the perspective of targeting inhibition of PTK6. In this work, CCK-8 for cell viability, EdU staining for cell proliferation, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining for cell apoptosis and immunofluorescence staining for LC3 expression were performed. Besides, levels of proliferation-related, apoptosis-related and autophagy-related proteins were detected by western blot analysis. Moreover, molecular docking of trifolirhizin with PTK6 was conducted to seek the compound-protein binding potential. It was demonstrated that trifolirhizin treatment inhibited the proliferation and promoted the apoptosis of NPC cells as well as strengthened autophagy in NPC cells. Furthermore, it was verified that trifolirhizin targeted PTK6 and negatively regulated PTK6 expression. The suppressive effects of trifolirhizin on the malignant behaviors of NPC cells and the enhancing effect of trifolirhizin on autophagy in NPC cells were partly abolished upon upregulation of PTK6. To conclude, findings suggested that trifolirhizin may downregulate PTK6 expression to induce autophagy and exert the antitumor activities in NPC.

The impact of crowding stress on growth and intestinal integrity in largemouth bass (Micropterus salmoides): Insights into ER stress, autophagy and apoptosis

Crowding stress is a common abiotic stressor in intensive aquaculture that negatively impacts fish species, leading to growth depression. This study primarily explored the effects of crowding stress on the growth and intestinal integrity of largemouth bass (Micropterus salmoides). A 10-week feeding experiment was conducted with two groups: a control group (0.55 kg/m³) and a crowding stress group (1.10 kg/m³). The results showed that crowding stress significantly impaired fish growth and compromised intestinal integrity. To further elucidate the underlying mechanisms, we investigated the effects of crowding stress on endoplasmic reticulum (ER) stress, autophagy and apoptosis. Our findings revealed an increased proportion of terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL)-positive cells and ER swelling in the intestines of stressed fish, along with a higher number of autophagosomes. Furthermore, there was a significant upregulation in the mRNA expression of genes related to ER stress, autophagy and apoptosis, including GRP78, eIF2α, IRE1, atg3, LC3-2, ulk1α, P62, and caspase-8. In conclusion, crowding stress negatively affects fish growth and intestinal integrity, potentially through the induction of apoptosis and autophagy associated with ER stress-mediated unfolded protein response (UPR). These findings provided critical insights into how intensive aquaculture disrupts intestinal integrity and inhibits growth in fish, offering a valuable reference for future research aimed at enhancing stress resistance in fish under intensive aquaculture conditions.

Anti-Alzheimer's Disease Target and Beneficial Effect in Formononetin

Introduction: Alzheimer's Disease (AD), a common neurodegenerative relevance of dementia, is spreading in the world. Hitherto, the pharmacological treatment for AD is prescribed limitedly in clinical application. Recently, it has been found that naturally occurring extracts possess promising anti-neurodegenerative properties, including AD. Method: Our previous study indicated that formononetin (FMN) exerts the anti-AD benefits based on bioinformatics analysis. However, the experimental validation for bioinformatics findings has not been conducted. In this study, we primarily applied the molecule docking analysis to ascertain the pharmacological targets, including cytochrome P450 19A1 (CYP19A1). Transgenic AD mice were used to validate the bioinformatics findings in vivo experimentally. Molecular docking data showed that FMN acted directly on CYP19A1 target protein with effective binding sites and potent combining affinity/energy. Results: Meanwhile, FMN intervention contributed to an increased trend of body weight in transgenic AD mice reduced hippocampal expression of Aβ1-42, and elevated content of CYP19A1. Additionally, FMN intervention showed reduced terminal deoxynucleotidyl Transferase dUTP Nick-End Labeling (TUNEL) expression and increased CYP19A1 and Ki67 expressions in hippocampal sections of transgenic AD mice. Conclusion: Collectively, FMN may be used for the prevention of AD, and the pharmacological activities are possibly related to reducing Aβ1-42, and TUNEL expressions to increase Ki67 and CYP19A1 activities.

Extracellular Vesicles Derived from Adipose-Derived Mesenchymal Stem Cells Alleviate Apoptosis and Oxidative Stress of Retinal Pigment Epithelial Cells Through Activation of Nrf2 Signaling Pathway.

Purpose: To examine the potential protective effects of adipose-derived mesenchymal stem cell-derived extracellular vesicles (ASC-EVs) on ARPE-19 cells exposed to hydrogen peroxide (H2O2) stress and to evaluate their ability to delay retinal degeneration in Royal College of Surgeons (RCS) rats. Methods: ARPE-19 cells were pre-treated with ASC-EVs for 24 h, followed by exposure to 200 μM H2O2 for an additional 24 h. RCS rats received an intravitreal injection of phosphate-buffered saline in one eye and ASC-EVs in the other eye. Results: ASC-EV pretreatment significantly protected against H2O2 in the Cell Counting Kit-8 assay and was also effective in the lactate dehydrogenase-release assay. It notably reduced early apoptosis (Annexin V-fluorescein isothiocyanate/propidium iodide assay) and late apoptosis (Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling assay), while significantly decreasing intracellular reactive oxygen species, glutathione levels, and superoxide dismutase activity. NFE2L2, HMOX1, and NQO1 mRNA levels, along with Nrf2, HO-1, and NQO1 protein levels, were significantly elevated with ASC-EV pretreatment. Compared with ARPE-19-derived EVs, 11 miRNAs were upregulated and 34 were downregulated in ASC-EVs. In RCS rats, intravitreal injections of ASC-EVs led to significant preservation of the outer nuclear layer and photoreceptor segments, along with increased nuclear Nrf2 expression and elevated HO-1 and NQO1 levels in the inner retina. Eyes that received intravitreal injections of ASC-EVs demonstrated significantly preserved electroretinography a- and b-wave amplitudes at 1 week post-injection, though this effect faded by 2 weeks. Conclusions: ASC-EVs mitigated apoptosis and oxidative stress in ARPE-19 cells subjected to H2O2 exposure and temporarily slowed retinal degeneration in RCS rats via Nrf2 pathway activation by miRNAs.

Role of BIX01294 in the intracranial inhibition of H3K9 methylation lessens neuronal loss in vascular dementia model.

Dementia develops as a result of multiple factors, including cerebrovascular disease which is called vascular dementia (VD). Histone-3 lysine-9 dimethylation (H3K9me2) broadly increases during VD and inhibits neuroprotective gene expressions. So, we aimed to determine how H3K9me2 inhibitor (BIX01294) affects neuronal damage in VD. An in vivo model of VD was used followed by BIX01294 treatment. Behavioral tests, hematoxylin, and eosin (H&E), Congo red, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were carried out. Hippocampal phosphorylated cyclic-AMP responsive element binding protein (p-CREB), c-fos, brain-derived neurotrophic factor (BDNF), and H3K9me2, were detected by western blot analysis technique. Neurological deficit and anxiety-related behavior significantly reduced in the treatment group compared to the VD group (p < 0.05). BIX01294 improved spatial and passive avoidance memory (p < 0.01 and p < 0.05, respectively) compared to the VD group. Treatment with BIX01294 restored the level of p-CREB/CREB ratio (p < 0.05), cfos (p < 0.01), BDNF (p < 0.01), and suppressed H3K9me2 (p < 0.001) when compared to the VD group. BIX01294 microinjection reduced the apoptosis level in TUNEL staining (p < 0.05), and raised neural cell count in H&E staining (p < 0.01); amyloid beta accumulation significantly decreased in the treatment group (p < 0.05) compared to the VD group. In conclusion, long-term treatment with a low dose of BIX01294 can prevent the progression of neuronal loss in VD model by raising the expression of neurotrophic factors, and reducing the apoptosis level.

Anticancer Effects of Weizmannia coagulans MZY531 Postbiotics in CT26 Colorectal Tumor-Bearing Mice by Regulating Apoptosis and Autophagy

Weizmannia coagulans has been shown to have anticancer properties. However, there is limited research on the effects of postbiotic W. coagulans on colorectal cancer cell proliferation. Additionally, the exact mechanisms through which it influences apoptosis- and autophagy-related signaling pathways are yet to be thoroughly elucidated. This study explored the role of W. coagulans MZY531 as a postbiotic in inhibiting tumor growth by modulating apoptosis and autophagy in tumor cells. During the experimental period in the model group, tumors proliferated, tumor markers increased significantly, and immunofluorescence results showed that caspase-3 and terminal deoxynucleotidyl transferase dUTP nick-end labeling were significantly decreased. Conversely, supplementation with W. coagulans MZY531 postbiotics significantly reduced the levels of tumor markers carcinoembryonic antigen, colon cancer antigen, and extracellular protein kinase A and promoted cell apoptosis by increasing the caspase-3-positive count and terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive cells in tumor tissue. Mechanistically, W. coagulans MZY531 postbiotics inhibit tumor growth through the modulation of the Bax/Bcl-2/caspase-3 and JAK2/STAT3 apoptosis pathways and PI3K/AKT/mTOR and TGF-β/SMAD4 cell autophagy pathways. W. coagulans MZY531 postbiotics had a more significant effect than that of W. coagulans MZY531 alone. Probiotics are expected to become effective natural functional foods for the treatment of colorectal cancer.

EGCG enhances antitumor effect of apatinib in nonsmall cell lung cancer by targeting VEGF signaling to inhibit glycolysis.

Nonsmall cell lung cancer (NSCLC), one of the most aggressive malignancies globally, is characterized by poor prognosis and limited life expectancy. Epigallocatechin-3-gallate (EGCG), a natural polyphenol found in green tea, has emerged as a promising anticancer agent due to its potent antitumor properties. However, the role and the underlying mechanisms of EGCG in NSCLC remain poorly understood. Hence, this research aimed to explore the effect of EGCG on the antitumor effect of apatinib in NSCLC throughvascular endothelial growth factor(VEGF)-regulated glycolysis. Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine staining, wound healing, transwell, terminal deoxynucleotidyl transferase dUTP nick-end labeling, and flow cytometry assays were carried out to evaluate the proliferation, migration, invasion, and apoptosis of H1299 cells, respectively. Furthermore, western blot analysis was used to detect the expressions of VEGF, p-vascular endothelial growth factor receptor-2, hypoxia-inducible factor 1α, neuropilin-1, phosphorylated-phosphatidylinositol 3-kinase, and phosphorylated-AKT. The transfection efficiency of H1299 cells with VEGF overexpression plasmid was also assessed by western blot analysis. Glycolysis was analyzed by estimating extracellular acidification rate, lactate concentration, glucose uptake, and the expressions of lactate dehydrogenase A, pyruvate kinase M2, and hexokinase 2. The results demonstrated that VEGF activated glycolysis in NSCLC cells. EGCG alone and apatinib alone or in combination inhibited cell viability, proliferation, invasion, migration, and glycolysis whereas promoted apoptosis in NSCLC cells. EGCG regulated glycolysis levels in NSCLC through VEGF overexpression, and enhanced the antitumor effect of apatinib in NSCLC through VEGF-regulated glycolysis. Taken together, EGCG strengthened the protective effects of apatinib in NSCLC through glycolysis mediated by VEGF.

Between Life and Death: Sea Urchin Embryos Undergo Peculiar DNA Fragmentation after Exposure to Vanadium, Cadmium, Gadolinium, and Selenium.

Exogenous DNA damage represents one of the most harmful outcomes produced by environmental, physical, or chemical agents. Here, a comparative analysis of DNA fragmentation was carried out on Paracentrotus lividus sea urchin embryos exposed to four common pollutants of the marine environment: vanadium, cadmium, gadolinium and selenium. Using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, fragmented DNA was quantified and localized in apoptotic cells mapping whole-mount embryos. This is the first study reporting how different chemicals are able to activate distinctive apoptotic features in sea urchin embryos, categorized as follows: (i) cell-selective apoptosis, showing DNA fragmentation restricted to a subset of extremely damaged cells, acting as an embryo survival mechanism; or (ii) total apoptosis, with fragmented DNA widespread throughout the cells of the entire embryo, leading to its death. Also, this is the first report of the effects of Se exposure on P. lividus sea urchin embryos. These data confirm the TUNEL assay as the most suitable test to study DNA fragmentation in the sea urchin embryo model system. Taken together, this research highlights embryos' ability to find alternative pathways and set physiological limits for development under stress conditions.

MiRNA506 Activates Sphk1 Binding with Sirt1 to Inhibit Brain Injury After Intracerebral Hemorrhage via PI3K/AKT Signaling Pathway.

Intracerebral hemorrhage (ICH) is an acute neurological disorder characterized by high mortality and disability rates. Previous studies have shown that 75% of patients who survive ICH experience varying degrees of neurological deficits. Sphk1 has been implicated in a multitude of phylogenetic processes, including innate immunity and cell proliferation. An in vivo rat model of ICH and an in vitro model of neuronal oxyhemoglobin (OxyHb) were constructed. The expression level of Sphk1 was assessed using western blotting and immunofluorescence, whereas cell death following ICH was evaluated using fluoro-Jade B and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Immunofluorescence facilitated the examination of microglial phenotypic alterations, while enzyme-linked immunosorbent assays were used to determine the concentrations of inflammatory markers. Behavioral assays were employed to assess the overall behavioral modifications of animals. Neuronal Sphk1/Sirt1 protein levels gradually increased following the induction of ICH. Elevated Sphk1 expression resulted in increased levels of anti-inflammatory microglia and reduced levels of pro-inflammatory factors. In contrast, suppression of Sphk1 expression resulted in an increased number of dead cells, thereby exacerbating neurological deficits. In vitro findings indicated that the levels of phosphorylated PI3K and AKT proteins increased in conjunction with Sphk1 expression. This study established that after ICH, Sphk1 interacts with Sirt1 to mitigate neuroinflammation, cell death, oxidative stress, and brain edema via the PI3K/AKT signaling pathway. Augmenting expression of Sphk1 significantly can ameliorate neurological impairments induced by ICH, offering novel targets and perspectives for therapeutic interventions in ICH treatment.

Membrane-bound receptor for advanced glycation end products (RAGE) is a stable biomarker of low-quality sperm

Abstract STUDY QUESTION Does receptor for advanced glycation end products (RAGE) on the surface membrane of the sperm cell function as a biomarker of low-quality sperm? SUMMARY ANSWER Membrane-bound RAGE at a cellular level directly correlates with low sperm motility, high cell permeability, decreased mitochondrial function, DNA fragmentation, and higher levels of apoptosis. WHAT IS KNOWN ALREADY RAGE has previously been measured by ELISA in low-quality sperm in diabetic men and has been shown to correlate with DNA fragmentation (terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay). STUDY DESIGN, SIZE, DURATION Semen samples were recovered from 60 non-obese, non-diabetic and non-smoking subjects, washed with fresh media, and analysed directly or purified further by differential gradient centrifugation (DGC) or fractionated by direct swim-up before being analysed for sperm motility and molecular health parameters, including cell membrane permeability, cell death, mitochondrial membrane potential, DNA fragmentation, and RAGE protein expression. PARTICIPANTS/MATERIALS, SETTING, METHODS Sperm motility assessments were carried out by computer-assisted sperm analysis (CASA) on 1000 spermatozoa for washed samples and 300 spermatozoa for purified samples. Molecular sperm health parameters were evaluated using flow cytometry with the use of the following markers: DAPI for cell membrane permeability, Annexin V/DAPI for cell death (apoptosis and necrosis), MitoTracker® Red CMXRos for mitochondrial membrane potential, TUNEL assay for DNA fragmentation and 8-hydroxy-2-deoxyguanosine for identification of oxidative damage to sperm DNA, and contrasted to membrane-bound RAGE expression levels, which were evaluated using an anti-RAGE monoclonal mouse antibody. MAIN RESULTS AND THE ROLE OF CHANCE RAGE protein was shown to be present on the acrosomal and equatorial regions of sperm, with the levels of membrane bound receptor strongly correlating with poor sperm health across all parameters tested; motility (R2 = 0.5441, P &amp;lt; 0.0001) and mitochondrial membrane potential (R2 = 0.6181, P &amp;lt; 0.0001) being of particular note. The analysis was performed at a single cell level thereby removing confounding complications from soluble forms of the RAGE protein that can be found in seminal plasma. The expression of the RAGE protein was shown to be stable over time and its levels are therefore not subject to variation in sample handling or preparation time. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION Inclusion criteria for this study were non-diabetic, non-obese and non-smoking participants to assess the distribution of RAGE expression in the general population, thereby excluding disease conditions that may increase RAGE expression in sperm or contribute to low sperm quality. The study does not address how RAGE expression may be affected in other patient subpopulations or disease states associated with male infertility. Sperm analysis by flow cytometry is not amenable to the study of males with a low sperm count. WIDER IMPLICATIONS OF THE FINDINGS Results of this study suggest that RAGE expression is a molecular maker of sperm cell health, which may be used for improvements in assisted reproduction through the removal of RAGE expressing sperm and facilitate in the diagnoses of unexplained infertility through its use as a biomarker of male infertility. STUDY FUNDING/COMPETING INTEREST(S) The study was funded by the Irish Research Council under the Government of Ireland Programme (GOIPG/2015/3729) and the Enterprise Ireland Innovation Partnership Programme (IP-2020-0952). All authors declare no competing interests.

Evaluation of Sperm DNA Fragmentation in Oligoasthenoteratozoospermia Patients Using Two Different Techniques: TUNEL and Sperm Chromatin Dispersion Assays

Background: Oligoasthenoteratozoospermia (OAT) is the most prevalent male infertility condition that is mainly caused by sperm DNA fragmentation (SDF). This study compared the sensitivity and effectiveness of two different approaches for analyzing SDF in patients with OAT: sperm chromatin dispersion (SCD) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Materials and Methods: In this study, which received ethical committee approval, participants were divided in to normal and OAT groups (n=20 for each). both TUNEL and SCD assays were used to analyze the sperm DNA fragmentation. And Malondialdehyde (MDA) levels was measured to determine levels of lipid peroxidation in the seminal plasma. Results: The TUNEL assay showed better ability to predict OAT patients than that of the SCD. For our patient population, the projected cut-off points for the DNA fragmentation index of 29% and 19% were reported using the TUNEL and SCD tests, respectively. Seminal levels of MDA were significantly higher in the OAT group (P=0.002) than that of control group. Conclusion: OAT patients showed higher MDA levels of seminal plasma and DNA fragmentation than the control group. Although sperm DNA fragmentation can be detected with high efficiency and sensitivity using both TUNEL and SCD assays, the TUNEL test was found to be a more accurate predictor for OAT patients.

Purified Astragalus Polysaccharide Combined with Inactivated Vaccine Markedly Prevents Infectious Haematopoietic Necrosis Virus Infection in Rainbow Trout (Oncorhynchus mykiss).

Rainbow trout (Oncorhynchus mykiss) is experiencing a catastrophic pandemic. In recent years, infectious hematopoietic necrosis virus (IHNV) has spread nationwide, resulting in significant mortality. Currently, there are no available treatments or vaccines for IHNV in China. Here, the Astragalus extract was purified and characterized. Then, we developed an inactivated IHNV vaccine with purified Astragalus polysaccharide (P-APS) as an adjuvant. Safety assays showed that IHNV was successfully inactivated. After a serious IHNV challenge, the cumulative mortality rates were 76.0, 38.0, and 22.1% in control, vaccine, and P-APS + vaccine groups, respectively. P-APS + vaccine was effective at reducing head kidney damage and apoptosis after IHNV challenge by histopathological and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analyses. The P-APS + vaccine group showed better results in enhancing specific antibodies (IgM) and immune enzyme activities (C3, LZM, GOT, and GPT). RNA-seq revealed that many immune-related pathways were significantly enriched. TLR2, TLR7, C3, IFN-γ, IgM, MHC1, MHC2, MX1, and VIG1 were identified as core genes based on RNA-seq and PPI networks. Mechanistic investigations showed that P-APS + vaccine activates the immune pathway by upregulating the expression of these genes. P-ASP+vaccine induced effective innate and adaptive immune responses that were stronger than single vaccines after vaccination and IHNV challenged. Our findings will provide a promising vaccine candidate against IHNV.