Deoxynucleotidyl Transferase dUTP Nick End Research Articles

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.

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.

COL6A1 Inhibits the Malignant Development of Bladder Cancer by Regulating FBN1.

Bladder cancer (BLCA) is a prevalent malignancy worldwide with a high recurrence rate. Collagen Type VI Alpha 1 (COL6A1) plays a key role in several cancer types. In this study, we aimed to explore the role of COL6A1 in BLCA. COL6A1 expression in BLCA was determined using The Cancer Genome Atlas database and real-time quantitative polymerase chain reaction (RT-qPCR). Counting Kit-8, wound-healing, and transwell assays were used to assess the effect of COL6A1 on T24 and 5637 cells. Apoptosis in BLCA cell lines was explored using western blotting and flow cytometry. Co-immunoprecipitation was performed to determine interactions between proteins. The role of COL6A1 in tumor growth in nude mice was evaluated by hematoxylin-eosin, immunohistochemical, and terminal deoxynucleotidyl transferase dUTP Nick-End Labeling. In BLCA, COL6A1 expression was downregulated. Moreover, the COL6A1 overexpression suppressed the viability, migration, and invasion, while promoting apoptosis of BLCA cell lines, with increased Caspase-3, Bax, and p53, and decreased Bcl-2. Conversely, silencing of COL6A1 promoted proliferation, migration, and invasion, while inhibiting apoptosis in BLCA cell lines. In vivo, COL6A1 inhibits tumor growth and progression. Fibrillin-1 (FBN1) was positively correlated with COL6A1 expression. COL6A1 could bind to FBN1 in BLCA cell lines. The expression of FBN1 in BLCA cell lines decreased after COL6A1 silencing, whereas COL6A1 overexpression upregulated FBN1 expression. COL6A1 was downregulated and exerted an inhibitory effect on the development of BLCA, and its expression was positively correlated with the expression of FBN1.

Short-Term Treatment of Melatonin Improves the Expression of Cell Adhesion Molecules in the Testis of the Mouse Cryptorchidism Model.

Melatonin plays a major role in regulating the sleep-wake cycle and enhancing testosterone production. We investigated the short-term effects of melatonin treatment for 14 consecutive days in the cryptorchidism model. We categorized experimental mice into Sham (S), Orchiopexy (O), Melatonin (Mel), and Orchiopexy + Melatonin (OMel) groups. Surgery involved inducing cryptorchidism in the left testis for seven days, followed by orchiopexy. The Mel group's testes did not descend, but they received melatonin injections after seven days of cryptorchidism. The OMel group underwent both orchiopexy and melatonin treatment. Both O and Mel groups exhibited decreased sperm and round-headed sperm in the epididymis. Significant increases were observed in the numbers of giant cells and negative Nectin-3 cells at p-value<0.05. The pattern of Cadm1 expression changed, and Nectin-2 and Nectin-3 co-expression was lacking in abnormal spermatids. Sertoli cell cytoplasm in both O and Mel groups exhibited autophagosomes and multivesicular bodies, which correlated with increased cyclooxygenase-2 expression. However, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cell numbers increased significantly in all treatment groups compared to the S group. Our study found that the combination of orchiopexy and melatonin positively influenced the expression of cell adhesion molecules (Cadm1, Nectin-2, and Nectin-3) involved in spermatogenesis, while reducing giant cells, autophagosomes, and apoptosis.

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.

Exosomal miR-142-3p from M1-polarized macrophages suppresses cell growth and immune escape in glioblastoma through regulating HMGB1-mediated PD-1/PD-L1 checkpoint.

Glioblastoma (GBM) is one of the most prevalent cancerous brain tumors. Former studies have reported that exosomes derived from M1-polarized macrophages (M1 exosomes) inhibit tumor occurrence and development through delivery of tumor suppressor genes. Also, microRNA-142-3p (miR-142-3p) has been verified to function as a tumor suppressor. GBM cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8), colony formation assay and 5-ethynyl-2'-deoxyuridine (EdU) assay; cell apoptosis was determined by flow cytometry analysis and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Mechanism investigations were conducted for analyzing the molecular mechanism by which miR-142-3p and M1 exosomes affect GBM progression. Upregulation of miR-142-3p expression was detected in M1-polarized macrophages and M1 exosomes. M1 exosomes inhibit GBM cell proliferation and trigger cell apoptosis. Functionally, miR-142-3p silencing promotes the proliferation and inhibits the apoptosis of GBM cells treated with M1 exosomes. As for molecular mechanism, miR-142-3p inhibits GBM cell growth via targeting high-mobility group box1 (HMGB1). In addition, miR-142-3p/HMGB1 axis affects GBM cell immune escape through modulation of programmed death-1/programmed death ligand-1 (PD-1/PD-L1) checkpoint. Our study demonstrated that exosomal miR-142-3p from M1-polarized macrophages suppresses cell growth and immune escape in GBM through regulating HMGB1-mediated PD-1/PD-L1 checkpoint.

Sinapic-Acid-Loaded Nanoparticles Optimized via Experimental Design Methods: Cytotoxic, Antiapoptotoic, Antiproliferative, and Antioxidant Activity.

Nanoparticles are frequently investigated as carrier systems that increase the biological activities of hydrophobic molecules, especially by providing them with water solubility. Sinapic acid (Sa), commonly found in plants, is a phenolic compound with a wide spectrum of biological activities and extensive pharmacological properties. The aim of this study was the synthesis/characterization of optimized sinapic-acid-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (SaNPs) to improve the solubility of sinapic acid (Sa) that limit its use in the biological system and investigate the biological activities of these nanoparticles in the breast cancer cell line. For this purpose, sinapic-acid-loaded PLGA nanoparticles were obtained and optimized by experimental design methods. Then, cytotoxic (MTT method), antiapoptotic (terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay), antiproliferative (immunocytochemically by PCNA assay), and antioxidant activities (superoxide dismutase (SOD) and catalase activities, glutathione, malondialdehyde (MDA), and caspase-3 levels) of optimized nanoparticles were examined comperatively with free drug on MCF-7 cells. The IC50 values of the SaNPs (170.6 ± 3.6 nm size) in MCF-7 cells were determined at 180, 168, and 145 μg/mL for 24, 48, and 72 h, respectively, and at these doses, the nanoparticles did not show any toxic effect on the MCF10A cell line. Treatment of Sa and SaNPs at doses of 24 and 48 h showed a statistically significant reduction in the PCNA level in MCF-7 cells, with an increase in the number of cells leading to apoptosis. In MCF-7 cells treated with SaNP at concentrations of 150 and 200 μg/mL for 24 h, MDA levels were significantly increased, SOD activities were significantly decreased, and reduced glutathione (GSH) and catalase levels were increased compared with control groups. The findings of this study indicate that polyphenolic compounds can contribute to the design of drugs for treatment by forming nanoparticle formulations. The developed nanoparticle formulation is thought to be a useful model for other hydrophobic biological active substances.

CD206 accelerates hepatocellular carcinoma progression by regulating the tumour immune microenvironment and increasing M2-type polarisation of tumour-associated macrophages and inflammation factor expression

ObjectiveThis study aims to investigate the effect of CD206 on the progression of hepatocellular carcinoma (HCC) and the regulation of the tumour immune microenvironment.MethodsA subcutaneous mouse model of HCC was established and treated with CD206-overexpressing adenovirus by tail vein injection or CD206 antibody C068C2 by intratumoral injection. The hepatocarcinoma-bearing mice were divided into four groups (IgG+ tail vein adenovirus group, IgG group, C068C2+ tail vein adenovirus group and C068C2 group) to observe the changes in tumour weight and volume with different expression levels of CD206. The proportion of M2-type tumour-associated macrophages (TAMs) was detected by flow cytometry and immunofluorescence. The apoptosis of tumour cells was detected using terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) staining, and inflammatory factors in serum and tissues were detected using the ENZYME-LINKED IMMUNOSORBENT ASSAY.ResultsCompared with the mice with low CD206 expression, the hepatocarcinoma-bearing mice with high CD206 expression in HCC exhibited faster tumour growth and more aggressive progression. Flow cytometry and immunofluorescence staining revealed that the expression level of CD206-positive M2-type TAMs was highest in the IgG + adenovirus group and lowest in the C068C2 group (p < 0.001). Compared with the IgG + adenovirus group, the proportion of TUNEL-positive cells in tumour cells was significantly reduced in the C068C2 group. The IgG + adenovirus group had the highest concentrations of transforming growth factor-β (TGF-β) and interleukin 6 (IL-6) in both serum and tumour tissues.ConclusionThe overexpression of CD206 accelerates the progression of HCC and changes the tumour immune microenvironment. The high expression of CD206 in HCC increases the M2-type polarisation of TAMs and induces the expression of both TGF-β and IL-6 in tumour tissues and serum, thereby promoting HCC progression.

Curculigoside Regulates Apoptosis and Oxidative Stress Against Spinal Cord Injury by Modulating the Nrf-2/NQO-1 Signaling Pathway In Vitro and In Vivo.

Spinal cord injury (SCI) is a severe neurological disorder that can lead to paralysis or death. Oxidative stress during SCI is a critical phase causing extensive nerve cell damage and apoptosis, thereby impairing spinal cord healing. Thus, a primary goal of SCI drug therapy is to mitigate oxidative stress. Curculigoside (CUR), a phenolic glucoside extracted from the dried root and rhizome of Curculigo orchioides Gaertn, possesses neuroprotective and antioxidant properties. This study aimed to investigate whether CUR effectively promotes the recovery of spinal cord tissue following SCI and elucidate its mechanism. We employed a hydrogen peroxide (H2O2)-induced PC12 cell model and an SCI rat model to observe the effects of CUR on oxidation and apoptosis. The results demonstrated that CUR significantly reduced the expression of apoptosis-related proteins (Bax and Caspase-3), Annexin V/propidium iodide (PI), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), while increasing the expression of the anti-apoptotic protein Bcl-2. Moreover, CUR effectively enhanced levels of antioxidants (glutathione [GSH)] and decreased reactive oxygen species (ROS) in vitro. Furthermore, CUR facilitated functional recovery through its anti-apoptotic and anti-oxidative stress effects on spinal cord tissues in SCI rats. These effects were mediated via the Nrf2/NQO1 signaling pathway. Therefore, our study showed that CUR acted as an anti-apoptotic and anti-oxidative stress agent, inhibiting astrocyte activation and promoting neuronal reconstruction and functional recovery. These findings may contribute significantly to the development of SCI treatments and advance the field of SCI drug therapy.

Inhibition of miR-194-5p avoids DUSP9 downregulation thus limiting sepsis-induced cardiomyopathy

Sepsis-induced cardiomyopathy (SIC) is described as a reversible myocardial depression that occurs in patients with septic shock. Increasing evidence shows that microRNA-194-5p (miR-194-5p) participates in the regulation of oxidative stress, mitochondrial dysfunction, and apoptosis and its expression is associated with the occurrence and progression of cardiovascular disease; however, the effects of miR-194-5p in SIC are still unclear. This study explores whether miR-194-5p could modulate SIC by affecting oxidative stress, mitochondrial function, and apoptosis. Experimental septic mice were induced by intraperitoneal injection of lipopolysaccharide (LPS) in C57BL/6J mice. The biological role of miR-194-5p in SIC in vivo was investigated using cardiac echocardiography, ELISA, western blot, qRT-PCR, transmission electron microscopy, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, bioinformatics analysis, and dual-luciferase reporter gene assay. Our major finding is that miR-194-5p antagomir mitigates sepsis-induced cardiac dysfunction, inflammation, oxidative stress, apoptosis and mitochondrial dysfunction in the hearts of septic mice, while miR-194-5p agomir triggers the opposite effects. Furthermore, dual-specificity phosphatase 9 (DUSP9) is a direct target of miR-194-5p and the cardioprotective effects of miR-194-5p antagomir on cardiac dysfunction, inflammation, apoptosis, mitochondrial dysfunction and oxidative stress are abolished through inhibiting DUSP9. Therefore, miR-194-5p inhibition could mitigate SIC via DUSP9 in vivo and the novel miR-194-5p/DUSP9 axis might be the potential treatment targets for SIC patients.

Hydrogen attenuates ischaemia–reperfusion injury in skeletal muscles post-limb replantation by activating the NRF2/HO-1 signalling pathway to reduce BAX expression

BackgroundIschaemia–reperfusion injury (IRI) is a critical complication post-limb replantation. The oxidative stress and cellular apoptosis due to IRI considerably hinder the healing process. This study aimed to investigate the modulatory effects of pre-perfusion with hydrogen-rich heparin sodium on the nuclear factor erythroid 2–related factor 2 (NRF2)/haeme oxygenase-1 (HO-1) pathway and its potential mechanisms in mitigating skeletal muscle IRI post-limb replantation. MethodsForty healthy Sprague–Dawley rats (250–300 g) were classified into five groups (n = 8 each): normal control, IRI + heparin sodium pre-perfusion (heparin group), IRI + hydrogen-rich heparin sodium pre-perfusion (hydrogen-rich heparin group), IRI + hydrogen-rich heparin sodium pre-perfusion + NRF2 inhibitor (hydrogen-rich heparin + all-trans retinoic acid [ATRA] group), and IRI + heparin sodium pre-perfusion + NRF2 inhibitor (heparin + ATRA group). The activation of the NRF2/HO-1 pathway in skeletal muscle IRI was evaluated based on HO-1 expression using western blotting and immunofluorescence. Furthermore, haematoxylin and eosin staining and transmission electron microscopy were employed to determine the histopathological characteristics. Additionally, superoxide dismutase and malondialdehyde levels in skeletal muscle tissue were measured to assess antioxidant capacity and the degree of oxidative stress damage. Tissue hypoxia was assessed based on hypoxia-inducible factor 1-alpha expression, whereas apoptosis markers BCL-2-associated X protein (BAX) and Caspase-3 in skeletal muscle tissues were analysed using western blotting with terminal deoxynucleotidyl transferase dUTP nick end labelling staining to quantify cell apoptosis. ResultsCompared with the control group, the heparin group exhibited significant pathological changes, including inflammatory infiltration and cellular hypertrophy, with increased apoptosis and oxidative stress. Notably, NRF2 suppression aggravated these effects. However, hydrogen-rich heparin sodium prominently activated the NRF2/HO-1 pathway, enhancing antioxidant defence and reducing BAX/Caspase-3-mediated apoptosis, thereby mitigating IRI-induced damage. The use of an NRF2 inhibitor to inhibit NRF2 excitation by hydrogen-rich heparin sodium notably weakened NRF2 activation and the antioxidant response, resulting in a substantial increase in cellular apoptosis. ConclusionPre-perfusion with hydrogen-rich heparin sodium markedly diminishes the BAX/Caspase-3-mediated apoptotic pathway in skeletal muscle tissues with IRI through the excitation of the NRF2/HO-1 pathway.

CCT2 Regulates ZEB1-Induced EMT Gene Transcription to Promote the Metastasis and Tumorigenesis of Papillary Thyroid Carcinoma.

Papillary thyroid carcinoma (PTC) is the most common malignant tumor of the thyroid, and its invasiveness and metastatic ability are closely related to patient prognosis. Chaperonin containing TCP1 subunit 2 (CCT2) is an important component of the molecular chaperone protein complex and has been shown to regulate cell proliferation and migration in various tumors. Epithelial-mesenchymal transition (EMT) is a critical process in tumor metastasis, and Zinc Finger E-Box Binding Homeobox 1 (ZEB1) is a core transcription factor that regulates EMT. This study aims to explore how CCT2 induces EMT gene transcription through ZEB1, thereby promoting the metastasis and tumorigenesis of PTC. CCT2 in PTC tissues was analyzed using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot. siRNA and overexpression vectors were used to silence and overexpress CCT2, respectively, and the effects on PTC cell migration, invasion, proliferation, and apoptosis were observed. Rescue experiments were used to investigate the effect of CCT2 on ZEB1 and EMT-related genes. Cell apoptosis was detected by Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) assay. Silencing ZEB1 was used to verify its effect on the oncogenic activity of CCT2. CCT2 was found to be highly expressed in PTC tissues (p < 0.01). In in vitro and in vivo experiments, silencing CCT2 inhibited the migration and invasion of PTC cells and their metastasis, while overexpression of CCT2 produced the opposite effect. Additionally, CCT2 promoted PTC cell proliferation and inhibited apoptosis (p < 0.01). Mechanistic studies revealed that CCT2 upregulated ZEB1 expression (p < 0.01), thereby inducing EMT gene transcription (p < 0.01). Silencing ZEB1 reduced the oncogenic effect of CCT2. This study first revealed the high expression of CCT2 in PTC and its essential role in the migration, invasion, proliferation, and anti-apoptosis of tumor cells. CCT2 promotes the metastasis and tumorigenesis of PTC by regulating ZEB1 and EMT-related genes. These findings provide new potential targets for molecular targeted therapy of PTC and explore new directions for future clinical treatment strategies.

Near-stop in spermatogonia progression and reduced sperm motility in two testis forms of captive North African catfish in Kalasin, Thailand

North African catfish (Clarias gariepinus) is integral to aquaculture, particularly in hybrid production with bighead catfish (Clarias macrocephalus). Recent declines in their production output have been linked to the quality of the parental sperm. This study aimed to evaluate the state of spermatogenesis in two morphological testis forms, normal white testes (CGA_A) and black-brown testes (CGA_B), in captive populations to understand their differences in the cytological processes of spermatogenesis and impact on sperm quality and fertility. We performed computer-assisted sperm analysis (CASA), meiotic chromosome analysis, gene expression profiling, histological examination, and identification of apoptotic cells using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Additionally, microsatellite genotyping was conducted to assess genetic diversity of the populations, showing that similar gene pool patterns were observed between the two populations with the CGA_A and the CGA_B testes. The average sperm motility was 11.15 %. Histologically, the CGA_A testes predominantly contained spermatozoa. By contrast, the CGA_B testes, characterized by black-brown color and harder texture, were composed mainly of spermatocytes and exhibited a higher incidence of apoptotic spermatogonia. Additionally, the CGA_B testes displayed reduced expression of genes essential for normal testicular development. Notably, the CGA_A testes showed significantly higher expression of GtH I, GtH II, Dazl, and Cxcr4b genes and lower levels of Caspase-3 and p53 (p < 0.05). The selective removal of deficient spermatogonia through early apoptosis in CGA_B testes suggest a mechanism contributing to male infertility due to imbalances between the number of germ cells and Sertoli cells. Despite the use of CGA_A testis for fertilization, a hatching rate in the captive populations was 36.43 % over the last three years (2021−2023), substantially lower than that previously reported, indicating the presence of genetic and environmental factors that influence reproductive success in North African catfish. Differences in spermatogenesis between testis forms highlight the need for genetic and environmental strategies to enhance reproductive outcomes in aquaculture.

Berberine Alleviates Sevoflurane Anesthesia-Induced Cognitive Dysfunction in Neonatal Mice via Regulating CREB1.

Sevoflurane has been shown to stimulate neurotoxicity and lead to cognitive impairment. Berberine is known for its role in regulating nervous system diseases, including cognitive dysfunction. This study aimed to investigate the effects of berberine on cognitive dysfunction induced by sevoflurane anesthesia and its potential mechanisms. In the in vivo study, neonatal mice were subjected to sevoflurane anesthesia to induce cognitive dysfunction. The cognitive function of the neonatal mice was evaluated using the Morris water maze test, open field test, and tail suspension test. Enzyme-linked immunosorbent assay (ELISA) was utilized to assess the levels of inflammatory factors. Immunohistochemistry (IHC) was conducted to detect ionized calcium-binding adaptor molecule 1 (IBA-1)-positive cells and cleaved caspase-3-positive cells in the hippocampus of the neonatal mice. Western blotting was used to measure the levels of cyclic adenosine monophosphate (cAMP) response element-binding protein 1 (CREB1) in hippocampal tissues and neurons. Hippocampal neurons were isolated from the hippocampus of neonatal mice. These neurons were treated with berberine or subjected to cell transfection. The cell counting kit-8 (CCK-8) assay and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay were conducted to measure cell viability and apoptosis of hippocampal neurons in vitro. Berberine significantly attenuated sevoflurane-induced cognitive impairment and inflammation in neonatal mice (p < 0.05 or p < 0.01). Additionally, berberine reduced sevoflurane-triggered neuronal apoptosis in the hippocampus of neonatal mice (p < 0.01). Sevoflurane markedly decreased CREB1 expression in the hippocampus of neonatal mice (p < 0.01), which was elevated by berberine treatment (p < 0.01). Mechanistically, sevoflurane significantly suppressed cell viability and promoted cell apoptosis of hippocampal neurons (p < 0.0001 or p < 0.01), which were mitigated by berberine (p < 0.05, p < 0.01, or p < 0.001). Furthermore, berberine significantly elevated CREB1 expression in sevoflurane-treated hippocampal neurons (p < 0.01). The beneficial effects of berberine on cell viability and apoptosis in sevoflurane-treated hippocampal neurons were blocked by CREB1 depletion (p < 0.001). Our results demonstrated that CREB1 was significantly decreased in the hippocampus of sevoflurane-treated neonatal mice in vivo and in sevoflurane-treated hippocampal neurons in vitro. This decrease was mitigated by berberine treatment. Moreover, berberine improved sevoflurane anesthesia-induced cognitive impairment in neonatal mice by attenuating neuronal inflammation and apoptosis in vivo. The inhibitory effects of berberine on sevoflurane-induced cell apoptosis were reversed by CREB1 downregulation. These findings indicate that berberine protects against sevoflurane anesthesia-induced cognitive impairment by reducing apoptosis of hippocampal neurons, partially through increasing CREB1 expression.

Imaging Hypoxia to Predict Primary Neuronal Cell Damage in Branch Retinal Artery Occlusion.

To develop a reliable method to generate a mouse model of branch retinal artery occlusion (BRAO) using laser-induced thrombosis of a major artery in the mouse retina. Also, to develop a reliable method to detect retinal hypoxia as predictive biomarker for the risk of neuronal cell damage in BRAO. A reliable and reproducible model of laser-induced BRAO was developed in mouse retina using Rose Bengal. To characterize retinal hypoxia in BRAO, pimonidazole immunostaining and HYPOX-4 molecular imaging methods were used. Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) was used to characterize neuronal cell damage in the BRAO retina. Expression of mRNA in retinal tissues from BRAO and age-matched control retinas were analyzed using qRT-PCR. Occlusion of a branch retinal artery near the optic nerve head (ONH) caused a pattern of retinal tissue hypoxia covering about 12.5% of the entire retina. TUNEL-positive cells were localized in all layers in BRAO retinal tissue cross sections. In addition, qRT-PCR data analysis suggests that BRAO is associated with both inflammation and hypoxia. This study provides a reliable method for BRAO in mouse retina and demonstrates the utility of molecular imaging method to detect retinal hypoxia as predictive biomarker for the risk of neuronal cell damage in BRAO. In addition, our data suggest that BRAO retinas are associated with inflammation and also associated with hypoxia-related neuronal cell damage. Imaging areas of retinal hypoxia may provide accurate diagnosis, evaluating retinal tissue injury from BRAO.

Oncolytic Virus Senecavirus A Inhibits Hepatocellular Carcinoma Proliferation and Growth by Inducing Cell Cycle Arrest and Apoptosis.

Hepatocellular carcinoma (HCC) is a highly aggressive tumor with limited treatment options and high mortality. Senecavirus A (SVA) has shown potential in selectively targeting tumors while sparing healthy tissues. This study aimed to investigate the effects of SVA on HCC cells in vitro and in vivo and to elucidate its mechanisms of action. The cell counting kit-8 assay and colony formation assay were conducted to examine cell proliferation. Flow cytometry and nuclear staining were employed to analyze cell cycle distribution and apoptosis occurrence. A subcutaneous tumor xenograft HCC mouse model was created in vivo using HepG2 cells, and Ki67 expression in the tumor tissues was assessed. The terminal deoxynucleotidyl transferase dUTP nick end labeling assay and hematoxylin and eosin staining were employed to evaluate HCC apoptosis and the toxicity of SVA on mouse organs. In vitro, SVA effectively suppressed the growth of tumor cells by inducing apoptosis and cell cycle arrest. However, it did not have a notable effect on normal hepatocytes (MIHA cells). In an in vivo setting, SVA effectively suppressed the growth of HCC in a mouse model. SVA treatment resulted in a significant decrease in Ki67 expression and an increase in apoptosis of tumor cells. No notable histopathological alterations were observed in the organs of mice during SVA administration. SVA inhibits the growth of HCC cells by inducing cell cycle arrest and apoptosis. It does not cause any noticeable toxicity to vital organs.

Euphorbia milii and propolis combination tea reduced hepatic steatosis and hepatocyte apoptosis in high-fat diet rat model

Background: Dyslipidemia can cause various organ disorders, such as fatty liver disease. Over time, fatty liver disease has become more commonplace worldwide and may cause mortality if the progression worsens. Natural components from Euphorbia milii (E. milii) and propolis (EMP) have been demonstrated as immunomodulators that reduce total cholesterol levels.Objective: To prove the effect of EMP tea on inhibiting fatty liver and hepatocyte apoptosis in high-fat diet (HFD)-induced rats.Methods: This study applied a post-test-only control group design using 18 Wistar rats, which were divided into three groups: K0 (received standard feed), KN (received HFD of 2 g/200 g Body weight (BW) in a day), and P (received HFD of 2 g/200 g BW in a day and EMP of tea 40 mg/100 g BW in a day). The interventions were conducted for 30 days, followed by termination on day 31 for liver tissue collection and analysis. We calculated the hepatic steatosis with the help of hematoxylin-eosin (HE) staining. Hepatocyte apoptosis was also determined with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining.Results: The K0 group had a lower hepatic steatosis percentage (17.87 ± 1.81) than KN (63.75 ± 15.88). We also found no hepatocyte apoptosis in the K0 but a high hepatocyte apoptosis index in the KN (3.98 ± 0.29). The combination of EMP tea in HFD-induced rats significantly reduced hepatic steatosis and apoptosis percentage (25.33 ± 1.17 and 0.91 ± 0.61, respectively). Conclusion: We demonstrated that combining EMP tea reduced hepatic steatosis and hepatocyte apoptotic index in HFD-induced rats, suggesting its potential as a treatment for these conditions.

Gasdermin D deficiency attenuates development of ascending aortic dissections in a novel mouse model.

Thoracic aortic dissection (TAD) is a silent killer. Approximately two-thirds of the cases occur in the ascending aorta (i.e. type A dissection) and majority of them are unrelated to genetic mutations. However, animal models of spontaneous type A dissection are not widely available. In the present study, a novel mouse TAD model was created. Further, the role of gasdermin D (GSDMD) in TAD development was evaluated. TADs were created by treating ascending aorta of adult mice (C57BL/6J) with active elastase (40.0 U/ml) and β-aminopropionitrile (Act E+BAPN). The temporal progress of the TAD pathology was rigorously characterized by histological evaluation and scanning electron microscopy, while potential mechanisms explored with bulk RNA sequencing of specimens collected at multiple timepoints. With this novel TAD model, further experiments were performed with Gsdmd -/- mice to evaluate its impact on TAD formation. The ascending aorta challenged with Act E+BAPN developed pathology characterized by an early onset of intimomedial tears (complete penetration) and intramural hematoma, followed by progressive medial loss and aortic dilation. Ingenuity Pathway Analysis and functional annotation of differentially expressed genes suggested that a unique inflammatory micro-environment, rather than general inflammation, promoted the onset of TADs by specifically recruiting neutrophils to the aortic wall, while the pathology at the advanced stage was driven by T-cell mediated immune injury. Gsdmd -/- attenuated medial loss, adventitial fibrosis, and dilation of TADs. This protective effect was associated with a reduced number of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) positive cells and T-cells in TADs. A novel mouse TAD model was created in the ascending aorta. It produces a unique microenvironment to activate different immune cell subsets, promoting onset and subsequent remodeling of TADs. Consistently, Gsdmd -/- attenuates TAD development, with modulation of cell death and T-cell response likely acting as the underlying mechanism.

Transcriptome and proteomic analysis reveal the protective mechanism of acupuncture on reproductive function in mice with asthenospermia

Acupuncture is an integral component of complementary and alternative medicine that has been reported to enhance sperm motility, improve semen quality, and consequently augment male fertility. However, the precise mechanisms of action and the underlying molecular pathways remain unclear. In the present study, we aimed to elucidate the potential mechanisms through which acupuncture improves reproductive function in a mouse model of cyclophosphamide-induced asthenozoospermia. We collected sperm from the epididymis for semen analysis, collected serum to determine gonadotropin and oxidative stress marker levels, conducted histological examination of testicular tissue using hematoxylin and eosin (HE) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and observed mitochondrial morphology using transmission electron microscopy (TEM). We also assessed oxidative stress levels and total iron content in testicular tissue and validated the proteomic and transcriptomic analysis results of testicular tissue using real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR), protein imprinting analysis, and immunohistochemistry (IHC). Our results indicate that acupuncture enhances sperm quality in asthenozoospermic mice; increases serum testosterone (T), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) levels; and attenuates oxidative damage, iron accumulation, and mitochondrial injury in mouse testicular tissues. Through protein and transcriptomic analyses, we identified 21 key genes, of which cytochrome b-245 heavy chain (CYBB), glutathione peroxidase 4 (GPX4), acyl-CoA synthetase long-chain family member 1 (ACSL1), and ferritin mitochondria (FTMT) were closely associated with ferroptosis. RT-qPCR, protein imprinting, and immunofluorescence (IF) analyses collectively indicated that acupuncture reduced ACSL1 and CYBB expression, and increased GPX4 and FTMT expression. Overall, the ferroptosis pathway associated with ACSL1/CYBB/FTMT/GPX4 represents a potential strategy through which acupuncture can improve the reproductive function in asthenozoospermic mice.