INTRODUCTION:Mitochondrial oxidative stress is a key driver of inflammation-induced chondrocyte dysfunction and cartilage degeneration. However, the molecular regulators linking inflammatory signaling to mitochondrial impairment in chondrocytes remain incompletely understood. MATERIAL AND METHODS:Public transcriptomic datasets were analyzed using differential expression analysis, weighted gene co-expression network analysis (WGCNA), and screening for oxidative stress-related genes to identify osteoarthritis (OA)-associated hub genes. An IL-1β-induced CHON-001 chondrocyte injury model was established, and the GJB2 gene, which encodes connexin 26, was silenced using siRNA. Cell viability, oxidative stress, apoptosis, Nrf2/HO-1 signaling, and mitochondrial membrane potential were evaluated. RESULTS:Integrated bioinformatics analysis of multiple GEO datasets identified GJB2 as a key oxidative stress-related hub gene. IL-1β stimulation significantly increased GJB2 expression and induced oxidative stress, apoptosis, and mitochondrial dysfunction. GJB2 knockdown reduced reactive oxygen species (ROS) accumulation, attenuated inflammatory responses, inhibited apoptosis, restored Nrf2 nuclear translocation and HO-1 expression, and preserved mitochondrial membrane potential. CONCLUSIONS:GJB2 promotes inflammation-associated oxidative injury and mitochondrial dysfunction in chondrocytes. Silencing GJB2 activates the Nrf2/HO-1 antioxidant pathway and confers cytoprotective effects, identifying GJB2 as a critical regulator and highlighting connexin-mediated redox signaling as a potential therapeutic target for cartilage degeneration.
INTRODUCTION:. Osteosarcoma (OS) is a highly vascularized malignant tumor whose growth and metastasis depend on angiogenesis. Tumor-suppressing STF cDNA 3 (TSSC3) has been identified as a tumor suppressor in OS, but its role and underlying mechanisms in regulating angiogenesis remain unclear. MATERIALS AND:METHODS: . Immunohistochemistry was used to assess the expression of TSSC3, VEGF-A, and CD31 in OS tissues. OS cells that stably overexpressed TSSC3 were established via lentiviral transduction, and conditioned medium from these cells was used to culture human umbilical vein endothelial cells (HUVECs). The proliferation, migration, and tube formation of HUVECs were evaluated using CCK-8, wound healing, Transwell, and tube formation assays. qRT-PCR and Western blotting were conducted to assess VEGFA mRNA and VEGF-A protein levels in TSSC3-overexpressing cells. A xenograft model in nude mice was used to evaluate angiogenesis in vivo, and changes in the Src/ERK pathway were examined by Western blotting. RESULTS:. TSSC3 was downregulated, and VEGF-A was upregulated in OS tissues, both of which were associated with prognosis. Conditioned medium from the TSSC3-overexpressing cells significantly inhibited HUVEC proliferation, migration, and tube formation. In vivo, TSSC3 overexpression led to reduced tumor weight, VEGF-A expression, and microvessel density. Moreover, TSSC3 suppressed VEGF-A synthesis and secretion in a Src/ERK-dependent manner. CONCLUSIONS:. TSSC3 inhibits angiogenesis in OS by downregulating VEGF-A via the Src/ERK pathway, providing a theoretical and experimental basis for anti-angiogenic therapies targeting TSSC3.
INTRODUCTION:Keloids are fibroproliferative scars characterized by persistent fibroblast activation and excessive extracellular matrix deposition. Tectochrysin is a natural flavone with anti-inflammatory and anti-oxidative activities, but its effects on keloid fibroblasts remain unclear. MATERIAL AND METHODS:Human keloid fibroblasts (HKFs) and human skin fibroblasts (HSFs) were exposed to tectochrysin. Cell viability was assessed by CCK-8. HKF apoptosis, intracellular reactive oxygen species (ROS), wound closure, vimentin immunofluorescence, profibrotic protein expression, and nuclear/cytoplasmic p65 distribution were evaluated. TGF-β1-stimulated HSFs were used as a controllable profibrotic activation model. RESULTS:Tectochrysin preferentially decreased HKF viability at 5 and 10 μg/mL while exerting weaker effects on HSFs, and significantly increased HKF apoptosis. It reduced intracellular ROS, delayed wound closure, altered vimentin-positive cytoskeletal organization in adherent HKFs, and decreased α-smooth-muscle-actin, collagen I, and fibronectin expression in HKFs. Tectochrysin also attenuated TGF-β1-induced upregulation of these markers in HSFs. Nuclear p65 abundance was reduced, whereas cytoplasmic p65 changed only modestly. CONCLUSIONS:Tectochrysin suppresses the profibrotic phenotype of keloid fibroblasts and is associated with reduced nuclear p65 accumulation, supporting further evaluation as a candidate anti-keloid agent.
INTRODUCTION:Periodontitis, a chronic inflammatory disease that severely compromises oral health and is linked to systemic disorders, has attracted increasing research attention. Kangfuxin (KFX), an ethanol extract derived from Periplaneta americana (L.) with documented anti-inflammatory and tissue-regenerative properties, remains unexplored for localized periodontal therapy. This study investigated whether KFX extended-release gel (KFX-ERG) could inhibit the progression of periodontal disease and provide potential support for the treatment of periodontitis. MATERIAL AND METHODS:We selected the optimal concentration from different component ratios of KFX-ERG for subsequent experiments. In vivo, a periodontitis model was established in rats, and pathological changes in periodontal tissue were examined using histological and micro-computed tomography (micro-CT) analyses. RESULTS:Micro-computed tomography revealed that KFX-ERG reduced alveolar bone resorption in experimental periodontitis in rats. Histopathological analysis further revealed markedly reduced inflammatory cell infiltration and concurrently elevated osteoblast density in periodontal tissues of the KFX-ERG group. CONCLUSIONS:This study establishes a theoretical foundation for the therapeutic application of KFX-ERG in mitigating periodontitis progression and demonstrates its significant potential to promote periodontal tissue regeneration, thereby offering promising translational prospects for clinical regenerative periodontal therapy.
INTRODUCTION:The widely used inhalational anesthetic sevoflurane increases susceptibility to postoperative cognitive dysfunction (POCD), especially among the elderly. Esculetin, a natural coumarin compound derived from Cortex Fraxini, possesses anti-inflammatory and neuroprotective activities. This study aimed to determine whether esculetin mitigates sevoflurane-induced POCD and to clarify its underlying mechanisms. MATERIAL AND METHODS:POCD was induced in aged male C57BL/6J mice by 3% sevoflurane inhalation. Esculetin was administered 1 h before each exposure. Learning, memory, and locomotion were evaluated by Morris water maze and open field tests. Hippocampal apoptosis was detected by TUNEL staining and western blotting (WB) of apoptosis-associated proteins. Neuroinflammation was assessed through Iba-1 immunofluorescence, ELISA, WB, and RT-qPCR. NF-κB and NLRP3 inflammasome pathways were analyzed by WB and immunofluorescence. In vitro, HT22 neurons were treated with sevoflurane and/or esculetin, and microglia-mediated neuroinflammation was examined using a BV2-HT22 co-culture system. RESULTS:In aged mice, esculetin alleviated sevoflurane-induced spatial learning and memory impairments. Hippocampal neuronal apoptosis was reduced, as indicated by fewer TUNEL-positive cells and restored expression of Bcl-2, Bax, and cleaved caspase-3. Esculetin also inhibited microglial activation, along with the sevoflurane-induced elevation of proinflammatory cytokines (TNF-α, IL-1β, IL-6) and M1 microglial markers (iNOS, CD68). Mechanistically, esculetin inhibited sevoflurane-triggered phosphorylation of IκBα and p65, as well as NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), and caspase-1 upregulation. In vitro, esculetin dose-dependently protected HT22 cells against sevoflurane-induced apoptosis. In the co-culture system, esculetin attenuated microglia-driven neuroinflammation and NF-κB/NLRP3 inflammasome activation in HT22 cells. CONCLUSIONS:Esculetin ameliorates sevoflurane-induced cognitive deficits in aged mice by inhibiting hippocampal neuroinflammation and neuronal apoptosis through NF-κB/NLRP3 inflammasome inhibition.
INTRODUCTION:Long non-coding RNA (lncRNA) IGFL2-AS1 has been implicated in oral squamous cell carcinoma (OSCC) progression, whereas its relationship to ferroptosis remains unclear. Erastin is a small-molecule ferroptosis inducer that inhibits system Xc-, reduces cystine uptake and glutathione availability, and thereby promotes lipid peroxide accumulation. We investigated whether IGFL2-AS1 modulates erastin-induced ferroptosis-associated changes through the miR-181a-5p/SLC7A11 axis. MATERIAL AND METHODS:Correlations among IGFL2-AS1, miR-181a-5p, and SLC7A11 were analyzed in public HNSC datasets using online bioinformatic tools. RNA immunoprecipitation (RIP)-qPCR and dual-luciferase assays were used to validate the interactions. Erastin-treated OSCC cell lines (Cal-27 and SCC15) were subjected to IGFL2-AS1 knockdown/overexpression or miR-181a-5p modulation for CCK-8 assays, and Cal-27 cells were further used for MDA, GSH, and Fe2+ measurements. Fluorescence staining and immunofluorescence analyses of ROS, SLC7A11, and FTH1 were further performed in Cal-27 cells. In vivo, tumor growth was monitored in xenografts derived from Cal-27 cells with stable IGFL2-AS1 knockdown and treated with erastin or antagomiR-181a-5p. RESULTS:IGFL2-AS1 knockdown or miR-181a-5p overexpression increased the sensitivity of OSCC cells to erastin, as indicated by reduced viability. In Cal-27 cells, this was accompanied by increased MDA and Fe2+ levels and depleted GSH. Mechanistically, IGFL2-AS1 acted as a competing endogenous RNA for miR-181a-5p and regulated the expression of the ferroptosis-related protein SLC7A11. In Cal-27 cells, IGFL2-AS1 knockdown further enhanced erastin-associated ROS fluorescence and reduced SLC7A11 and FTH1 immunoreactivity, whereas miR-181a-5p inhibition partially reversed these changes. In vivo, IGFL2-AS1 silencing enhanced erastin-associated tumor suppression and reduced SLC7A11 expression, while antagomiR-181a-5p partly attenuated these effects. CONCLUSIONS:The present data support that IGFL2-AS1 knockdown enhances erastin-induced ferroptosis-associated changes in OSCC, at least in part through the miR-181a-5p/SLC7A11 axis.
INTRODUCTION:MutL homolog 1 (MLH1) loss is a defining molecular feature of endometrial cancer (EC) and a principal driver of microsatellite instability (MSI). Ishikawa cells harbor intrinsic MLH1 promoter hypermethylation, resulting in reduced but not abolished MLH1 expression and placing these cells in a vulnerable, partially compromised mismatch repair state. This study explores the effects of MLH1 knockdown (MLH1-KD) on MSI, cellular functions, signaling pathways, and tumor growth in Ishikawa EC cells. MATERIAL AND METHODS:Using CRISPR/Cas9, we created an MLH1-KD Ishikawa EC cell line, validated through Sanger sequencing, qRT-PCR, western blotting, comet assays, and γ-H2AX analysis. Functional assays assessed proliferation, migration, and cell cycle progression and apoptosis. RNA sequencing identified global transcriptomic changes, and Wnt/β-catenin pathway activity was measured by a dual-luciferase reporter assay. A xenograft model evaluated tumor growth in vivo. RESULTS:MLH1-KD cells showed MSI-H characteristics, increased DNA damage, and downregulation of key EC-related genes. Functionally, MLH1-KD led to significant reductions in cell proliferation and migration, which was accompanied by cell cycle arrest and a marked increase in apoptosis. RNA sequencing revealed profound alterations in the Wnt signaling pathway. Crucially, this was confirmed by a dual-luciferase reporter assay, which showed a significant inhibition of Wnt/β-catenin signaling activity. In vivo, MLH1-KD significantly decreased tumor weight and size in nude mice. CONCLUSIONS:In EC cells with pre-existing MLH1 promoter methylation, MLH1-KD leads to MSI-H, enhances genomic instability, disrupts Wnt signaling, impairs cellular functions, and inhibits tumor growth, highlighting Wnt signaling and MSI-H as potential therapeutic targets in EC.
INTRODUCTION:Temozolomide (TMZ) resistance in glioblastoma (GBM) involves dynamic crosstalk of cancer cells with tumor-associated microglia, but the role of exosomal long non-coding RNAs (lncRNAs) remains poorly defined. MATERIAL AND METHODS:We isolated exosomes from TMZ-sensitive (U251) and TMZ-resistant (U251TR) GBM cells. Lnc-DLK1-35 expression was modulated by overexpression/knockdown and validated by RT-qPCR. Exosome internalization by microglia (HMC3 cells) was tracked via PKH67 labeling. Microglial polarization was assessed by studying morphology, marker expression (CD16/CD32/iNOS/Arg-1/CD206/CD163), and cytokine secretion (ELISA: IL-6, TNF-α, TGF-β, IL-10, CXCL13). Functional impact on GBM cells by indirect co-culture with IL4-activated HMC3 cells was examined using CCK-8 and Transwell assays. RESULTS:Lnc-DLK1-35 was enriched in U251TR cells and their exosomes. GBM exosomes delivered Lnc-DLK1-35 to microglia, inducing a unique GBM-educated phenotype: amoeboid morphology, elevated type 1 macrophage (M1) markers (CD16, iNOS) but suppressed immunoregulatory factors (IL-10, CXCL13). This reprogramming required exosomal transfer. In addition, indirect co-culture with IL4-activated HMC3 cells enhanced GBM malignancy, reduced TMZ sensitivity and promoted migration and invasion. CONCLUSIONS:Exosomal Lnc-DLK1-35 reprograms cultured microglia cells into a pro-tumorigenic state via a non-canonical cytokine signature, driving TMZ resistance and invasion. Targeting this axis disrupts microglia-GBM communication, revealing a novel therapeutic strategy.
INTRODUCTION:Mitochondrial open reading frame of the 12S rRNA type-c (MOTS-c), a 16-amino acid mitochondrial-derived peptide, regulates cellular metabolism through AMPK and mTOR signaling and exerts protective effects across multiple endocrine tissues. However, its role in adrenal physiology remains unexplored. We hypothesized that MOTS-c establishes "steroidogenic readiness" by priming metabolic pathways rather than directly activating hormone synthesis. MATERIAL AND METHODS:Adult male Wistar rats (n = 16) received continuous MOTS-c (0.1 μmol/24 h) or saline via subcutaneous micro-osmotic pumps for 24 hours. Adrenal tissues were analyzed using qRT-PCR, immunohistochemistry, ELISA, and RNA-sequencing. RESULTS:MOTS-c showed significantly higher expression in ZF/ZR vs. ZG. MOTS-c treatment did not alter classical steroidogenic genes or circulating corticosterone and aldosterone levels. RNA-seq identified 39 differentially expressed genes, notably upregulation of purinergic receptor P2ry4 (4.3-fold, P < 0.05) - a novel MOTS-C target enhancing calcium signaling. Additional changes included upregulation of Apoc4 and downregulation of stress markers Bag3 and Smurf2, mitochondrial carrier Slc25a30, and peroxisomal factor Pex11a. Gene Set Enrichment Analysis revealed inhibition of cAMP response, mitophagy, and histone deacetylation pathways, alongside activation of cell proliferation, indicating metabolic reprogramming without steroidogenic activation. CONCLUSIONS:MOTS-c functions as a metabolic conductor that primes adrenocortical cells for enhanced steroidogenic responsiveness without stimulating basal hormone synthesis. By upregulating calcium signaling, modulating lipid metabolism, downregulating stress-response proteins, and inhibiting mitophagy, MOTS-c establishes a preparatory metabolic state optimized for subsequent ACTH or stress stimulation. These findings reveal a novel preparatory mechanism in adrenal physiology and identify MOTS-c as a potential therapeutic target for HPA axis disorders requiring enhanced adrenal reserve capacity without basal hypercortisolemia.
INTRODUCTION:This study investigated the expression of brain-derived neurotrophic factor (BDNF) signaling components (BDNF-TrkB-AKT1) and apoptosis-related factors (Bcl-2 and Bax) in yak brain regions at different altitudes. MATERIAL AND METHODS:The cerebral cortex, cerebellum, hippocampus, thalamus, and medulla oblongata were collected from 3-year-old yaks living at low and high altitudes. The relative mRNA expression of BDNF, TrkB, AKT1, Bcl-2, and Bax was assessed by qRT-PCR. Protein abundance and cellular localization of BDNF, TrkB, AKT1, Bcl-2, and Bax were evaluated by Western blotting and immunohistochemistry, with immunoreactivity quantified by optical density analysis. RESULTS:Within each altitude group, BDNF, TrkB, AKT1, and Bcl-2 mRNA expression and the corresponding protein levels (BDNF, TrkB, AKT1, and Bcl-2) were significantly higher in the cerebral cortex and hippocampus than in the cerebellum, thalamus, and medulla oblongata (P < 0.05). In contrast, Bax mRNA and Bax protein levels did not differ significantly among the five regions. Compared with low-altitude yaks, high-altitude yaks showed significantly higher BDNF, TrkB, AKT1, and Bcl-2 mRNA expression and higher BDNF, TrkB, AKT1, and Bcl-2 protein levels in brain tissues (P < 0.05), whereas Bax protein expression did not differ between altitude groups. Immunohistochemistry revealed immunoreactivity for BDNF, TrkB, AKT1, Bcl-2, and Bax in both altitude groups, with prominent labeling in cortical pyramidal neurons and across the pyramidal cell layer in the hippocampal CA region. Immunoreactivity was also detected in large neurons of the thalamus and medulla oblongata. In the cerebellum, labeling was strongest in Purkinje cells, with weaker signals in the granule cell layer and molecular layer. CONCLUSIONS:BDNF-TrkB-AKT1 pathway components and Bcl-2 showed relatively higher expression in the cerebral cortex and hippocampus within each altitude group, whereas Bax expression did not vary across regions. These patterns are consistent with an association between BDNF-TrkB-AKT1 signaling and increased Bcl-2 expression without a corresponding increase in Bax, which may support neuronal adaptation in the cerebral cortex and hippocampus. Elevated expression of BDNF, TrkB, AKT1, and Bcl-2 at high altitude suggests enhanced adaptation to hypoxia in high-altitude yaks; the underlying mechanisms require further investigation.
INTRODUCTION:Cardiac fibrosis is a major pathological feature of multiple cardiovascular diseases and an important risk factor for heart failure. Ginsenoside Rg3 (Rg3), a natural triterpenoid saponin extracted from Panax ginseng, has been shown to exert cardioprotective effects. In this study, we assessed the effects of Rg3 on angiotensin II (Ang II)-induced cardiac fibrosis in both cellular and animal models and investigated the underlying mechanisms. MATERIAL AND METHODS:For the cellular experiments, primary mouse cardiac fibroblasts (CFs) were treated with Ang II (1 μM) and Rg3 (25, 50, or 100 μM) for 24 h to assess the effects of Rg3 on cardiac fibrosis in vitro. The glucagon-like peptide-1 receptor (GLP-1R) antagonist exendin-3 (9-39) (1 μM) was used to validate the role of GLP-1R signaling in the anti-fibrotic effects of Rg3 in vitro. A CCK-8 assay was performed to assess cell viability. For the animal experiments, male C57BL/6J mice were divided into 4 groups (6 mice per group): Sham, Ang II, Ang II + Rg3 (50 mg/kg), and Ang II + Rg3 (100 mg/kg). Collagen deposition in mouse cardiac tissues was assessed by picrosirius red staining. The expression of fibrosis-related proteins (MMP-2, MMP-9, α-SMA, collagen I, collagen III, and fibronectin), GLP-1R, phosphorylated Smad2/3, total Smad2/3, RhoA, and ROCK2 in CFs and mouse cardiac tissues was examined by RT-qPCR, western blotting, and immunofluorescence staining. RESULTS:Rg3 treatment reversed the Ang II-induced upregulation of MMP-2, MMP-9, α-SMA, collagen I, collagen III, p-Smad2/3, RhoA, and ROCK2 and the downregulation of GLP-1R in CFs. Exendin-3 (9-39) antagonized the effects of Rg3 on the expression of fibrosis-related proteins, GLP-1R, p-Smad2/3, RhoA, and ROCK2 in CFs. Furthermore, Rg3 administration suppressed collagen deposition, reduced the expression of MMP-2, MMP-9, α-SMA, collagen I, collagen III, p-Smad2/3, RhoA, and ROCK2, and increased GLP-1R levels in the cardiac tissues of Ang II-infused mice. CONCLUSIONS:Rg3 exerts an anti-fibrotic effect in cardiac fibrosis models by inhibiting activation of the RhoA/ROCK and Smad2/3 pathways through upregulation of GLP-1R.
INTRODUCTION: . Typhoid fever, a disease resulting from an infection with Salmonella Typhi (S. Typhi) remains widespread in economically disadvantaged regions, where it continues to be a critical public health concern. As the symptoms and signs are non-specific, they are difficult to diagnose directly based on the clinical picture. Therefore, laboratory examinations are essential for diagnosis. MATERIAL AND METHODS: . This research introduces a fast and equipment-independent approach for detecting S. Typhi by employing CRISPR/Cas12a-based technology. The optimized CRISPR/Cas12a system achieved a detection limit of 103 copies/μL of S. Typhi DNA per reaction, with the entire assay completed within 60 min. RESULTS: . Four clinical isolates cultured from patients with typhoid fever were collected and evaluated using our CRISPR/Cas12a-based detection system. The assay results demonstrated that all four samples were accurately identified as positive. CONCLUSIONS: . We showed that the developed CRISPR/Cas12a-based detection method provides a promising alternative for the on-site and simple detection of S. Typhi.
A laboratory toolbox for detecting and quantifying DNA breaks in animal cells and tissues includes various methods that employ biochemical tests, DNA sequencing, or imaging approaches. Various methods based on microscopy were developed to detect DNA breaks in fixed and live cells, including the nick translation assay, TUNEL, STRIDE, and detection methods based on imaging of histone modifications (γH2A.X), recruitment of repair factors (XRCC1, PCNA, 53BP1, Rad51) or poly-ADP-ribosylation. This review discusses the advantages and limitations of various microscopy-based methods for the detection and quantification of single- and double-strand DNA breaks.
INTRODUCTION:Lung cancer remains the leading cause of cancer-related mortality worldwide, with non-small cell lung cancer (NSCLC) representing the majority of cases. Molecular profiling of NSCLC has identified multiple genomic alterations, including BRAFV600E mutation, which occurs in 1-5% of patients, predominantly in adenocarcinomas. Detection of this mutation is clinically relevant due to the availability of targeted therapies. Immunohistochemistry using the VE1 antibody offers a rapid and practical screening method, although interpretation criteria and methodological variability remain challenging. MATERIAL AND METHODS:This study represents the first systematic evaluation of BRAFV600E immunohistochemical (IHC) and molecular status in the Montenegrin population. A total of 135 patients undergoing surgical resection for primary lung adenocarcinoma were analyzed retrospectively. Clinicopathological data were collected, and tissue microarrays (TMA) were constructed. Immunohistochemical staining was performed with the VE1 monoclonal antibody, and results were scored based on cytoplasmic staining intensity and homogeneity. Positive cases, along with matched negative controls, were further analyzed using fully automated Biocartis Idylla real-time polymerase chain reaction (PCR). RESULTS:VE1 IHC positivity was observed in 7 patients (5.2%), with strong, homogeneous staining (3+) in 2 cases. BRAFV600E mutations were confirmed by PCR exclusively in these 3+ cases, giving a prevalence of 1.5% overall and 28.6% relative to IHC-positive cases. Statistical analysis demonstrated perfect concordance between strong, homogeneous VE1 staining and PCR results. Weak or focal staining presented interpretative challenges, highlighting the need for standardized criteria and molecular confirmation. CONCLUSIONS:Immunohistochemistry using VE1 antibody is a reliable and accessible initial screening tool for BRAFV600E mutation in lung adenocarcinoma. Strong, homogeneous cytoplasmic staining predicts mutation status with high accuracy, while weak or heterogeneous staining requires confirmatory molecular testing. The present findings provide essential local epidemiological data and support universal IHC testing to guide personalized therapy in NSCLC.
INTRODUCTION:This study aimed to provide a comprehensive histopathological and immunohistochemical analysis of Meibomian adenomas and adenocarcinomas. MATERIAL AND METHODS:A total of 80 tumours were included in the study: 33 adenomas and 47 Meibomian adenocarcinomas. Ki-67, cytokeratin-7 and von Willebrand factor antibodies were used for the immunohistochemical staining. RESULTS:Detailed characteristics of inflammatory infiltration depending on tumor type and damage to surrounding tissue by tumour cells are presented. A close correlation was found between the structure and intensity of blood vessel proliferation and the level of factor VIII expression. CONCLUSIONS:The presented detailed histopathological analysis, combined with the results of immunohistochemical studies, now allows for better treatment planning and prognosis assessment for patients.
INTRODUCTION:Diabetic retinopathy involves retinal pigment epithelium (RPE) dysfunction. Tectochrysin is a natural flavonoid and antioxidant. However, its effects on retinal pigment epithelial cells remain unveiled. In this study, the investigation undertook the question of whether tectochrysin could protect human adult RPE cells ARPE-19 from high-glucose (HG) damage. Also the potential regulatory mechanism was explored. MATERIAL AND METHODS:Cell counting assay 8 (CCK8) was used to measure cell viability in ARPE-19 cells with tectochrysin treatment for 72 h to confirm the potential cytotoxicity. Furthermore, cells were treated by HG (40 mM glucose) for 48 h with or without tectochrysin treatment (5 or 10 μg/mL). Then cell viability, apoptosis and Reactive Oxygen Species (ROS) levels were evaluated using CCK8, flow cytometry and DCFH-DA fluorescence methods. Migration capacity was evaluated using wound healing and Transwel assays. In addition, malondialdehyde (MDA) content, glutathione (GSH), and Fe2+ levels were also examined with kits. Protein expression of ferroptosis markers (ACSL4, GPX4), EMT-related proteins (E-cadherin, vimentin, Snail), and Nrf2/HO-1 were determined using western blotting. RESULTS:In ARPE-19 cells tectochrysin (5 or 10 μg/mL) significantly reversed HG-induced cytotoxicity, reducing apoptosis, ROS, MDA and Fe2+ levels, and increasing GSH. Tectochrysin reversed HG-induced ferroptosis-related protein ACSL4 and restored HG-inhibited GPX4 expression. In addition, tectochrysin inhibited HG-induced cell migration and vimentin and Snail levels and restored expression of HG-inhibited mesenchymal marker E-cadherin. Moreover, tectochrysin activated the Nrf2/HO-1 pathway. CONCLUSIONS:The findings demonstrate that tectochrysin protects ARPE-19 cells from HG-induced injury by inhibiting apoptosis, ferroptosis and EMT while activating Nrf2/HO-1 signaling, suggesting its potential as a therapeutic agent for diabetic retinopathy.
Papillary thyroid carcinoma (PTC) constitutes the predominant subtype among thyroid malignancies. Despite its generally favorable prognosis, certain aggressive subtypes, along with recurrent and metastatic manifestations, substantially affect patient survival outcomes. Recent advancementsin the diagnostic and therapeutic strategies for PTC have ushered in a new era, characterized by the integration of molecular mechanisms and imaging-based evaluations. This review offers an integrated perspective of the clinicopathological features, molecular genetic characteristics, epigenetic regulation, and the contribution of the immune microenvironment to the aggressiveness of PTC. Primary inve stigation targets include BRAF/RAS/RET-related molecular mechanisms and the functional significance of non-coding RNAs [especially long non-coding RNAs (lncRNAs) and microRNAs (miRNAs)] in molecular regulation. Additionally, the impact of clinical factors such as age, sex, obesity, and comorbidity with Hashimoto's thyroiditis on the aggressiveness of PTC is thoroughly examined. Furthermore, this review systematically synthesizes the clinical advances in the early detection and risk assessment of aggressive PTC by emerging imaging modalities such as conventional ultrasound, interventional ultrasound, ultrasound elastography, contrast-enhanced ultrasound, and artificial intelligence-assisted analysis. Looking ahead, multidisciplinary collaborations integrating pathology, genomics, and imaging are anticipated to enhance the precise evaluation of PTC aggressiveness and facilitate the development of individualized treatment strategies. This review serves as a comprehensive reference for mechanistic exploration and clinical translation in the study of PTC aggressiveness, and provides guidance for the progression of precision medicine and management models for PTC patients.
INTRODUCTION:Papillary thyroid carcinoma (PTC) is the most common thyroid malignancy, encompassing distinct histological variants and a wide spectrum of clinical behaviors. Advances in molecular diagnostics have identified key genetic alterations - particularly BRAFV600E, RAS mutations, RET/PTC fusions, and TERT promoter mutations - that are strongly linked to tumor aggressiveness and prognosis. This study aimed to determine the prevalence of these alterations and evaluate their clinicopathological significance in a Saudi Arabian patient cohort. MATERIALS AND METHODS:A retrospective analysis was conducted on 114 formalin-fixed paraffin-embedded (FFPE) PTC samples diagnosed between 2019 and 2023. Targeted next-generation sequencing (NGS) was used to detect BRAF, NRAS, KRAS, HRAS, RET/PTC fusions, and TERT promoter mutations. Immunohistochemistry (IHC) for BRAFV600E, TTF-1, CK19, HBME-1, and galectin-3 was performed using automated staining systems. Associations between genetic alterations and clinicopathological parameters - including tumor size, histological subtype, lymph node metastasis, and extrathyroidal extension - were analyzed statistically. RESULTS:BRAFV600E mutations were identified in 39.5% of cases and were significantly associated with larger tumor size (P = 0.01), extrathyroidal extension (P = 0.04), and lymph node metastasis (P = 0.03). RET/PTC fusions were detected in 15.8% of patients, predominantly younger individuals, and correlated with multifocal tumors and lymphovascular invasion. RAS mutations (19.3%) were more frequent in the follicular variant but showed no significant association with adverse features. TERT promoter mutations were present in 10.5% of cases, significantly correlating with older age and advanced tumor stage. IHC profiles demonstrated strong concordance with molecular findings. CONCLUSIONS:In this Middle Eastern cohort of PTC patients, BRAFV600E and TERT promoter mutations were significantly associated with aggressive clinicopathological features, while RET/PTC fusions and RAS mutations demonstrated distinct demographic and histological distributions. The integration of NGS and IHC enhanced diagnostic accuracy and supports personalized risk stratification in PTC management.
INTRODUCTION:This study investigated the therapeutic mechanisms of electroacupuncture (EA) in chronic unpredictable stress (CUS)-induced depression rat model. Since SIRT1 plays in oligodendrocyte differentiation and neuroprotection, we hypothesize that it may mediate the effect of electroacupuncture (EA) on myelin regeneration in depression. MATERIAL AND METHODS:Sixty adult male Sprague-Dawley rats were divided into control, CUS, EA + CUS and CUS + EA + SIRT1-specific inhibitor - EX527 (EX) groups. We established a CUS-induced depression rat model by subjecting rats to 4-week CUS paradigm. Four weeks post-modeling, EA treatment and intraperitoneal administration of EX527 were applied. Behavioral tests including open field test, forced swim test and sucrose preference test were performed to assess the depressive-like state. Immunohistochemistry and stereological analysis for quantification of oligodendroglial cell populations was performed. Immunohistochemical staining and transmission electron microscope were performed for evaluation of myelination. Western blot and qRT-PCR analyses were performed to detect the mRNA and protein expression of SIRT1 in the prefrontal cortex (PFC) of rats in each group. RESULTS:Four weeks of EA intervention significantly alleviated depressive-like behaviors in CUS rats, as evidenced by increased sucrose preference (P < 0.05), enhanced exploratory activity (P < 0.01), and reduced immobility time (P < 0.05) compared to the CUS group. Histopathological and ultrastructural analyses demonstrated that EA restored myelin integrity in the PFC, with myelin basic protein immunoreactivity significantly higher in the EA+CUS group than in untreated CUS rats (P < 0.05). Electroacupuncture promoted oligodendrocyte differentiation, reversing the chronic stress-induced reduction in CC1+/Olig2+/BrdU+ progenitor cells (31.5 ± 3.1% vs. 3.23 ± 1.4% in CUS + EA + EX group, P < 0.01). Mechanistically, EA upregulated SIRT1 mRNA and protein expression in the PFC (P < 0.05 vs. CUS), while pharmacological inhibition of Sirt1 with EX-527 abolished these effects, reducing Sirt1 mRNA and SIRT1 protein expression (P < 0.01, P < 0.01, respectively) and Olig2 expression (P < 0.05). EX-527 treatment also blocked EA-induced behavioral improvements and myelin regeneration, confirming the critical role of the Sirt1. CONCLUSIONS:The findings indicate that EA ameliorates depression-like behavior by enhancing oligodendrocyte maturation and myelin repair via activating SIRT1 signaling. These findings provide novel mechanistic insights into non-pharmacological interventions for depression.
