Ashwagandha (W. somnifera), known for its broad health benefits, has shown potential in cancer prevention and treatment. The aim of this study was to investigate the potential effect of ashwagandha aqueous extract (ASH-AE) on cell proliferation and therapy resistance markers in hepatocellular carcinoma (HCC). An in vitro study was conducted using the HepG2 cell line. The HepG2 cells were divided into 4 groups according to the treatment regimen received. ASH-AE was extracted from the whole plant and characterized by GC-MS and HPLC. HepG2 cell viability was determined for all groups. The protein expression of cluster of differentiation 90 (CD90) was determined by flow cytometry technique. Also, the gene expression of Sonic Hedgehog (SHH), Patched 1(PTCH1) and ATP-binding cassette subfamily C1 (ABCC1) was assayed by qRT-PCR. The protein expression and localization of glioma-associated oncogene 1 (Gli1) in HepG2 cells were determined by immunocytochemistry (ICC) assay. The results indicated that ASH-AE either alone or in combination with sorafenib (SOR) significantly reduced HepG2 cell viability in a concentration dependent manner (P ˂0.001) with IC50 was 6.65 mg/ml for ASH-AE, 11.3 µM for SOR, and 5.6 mg/ml + 18.6 µM for ASH-AE in combination with SOR. Moreover, SOR significantly increased the percentage of CD90+ cells and Gli1 protein expression and nuclear translocation as well as ABCC1 gene expression compared to untreated cells. On the other hand, ASH-AE either alone or in combination with SOR significantly decreased the percentage of CD90+ cells and Gli1 expression and nuclear translocation as well as SHH, PTCH1 and ABCC1 gene expression compared to untreated cells and that treated with SOR. We concluded for the first time that the combination of SOR and ASH-AE generates antagonistic antitumor effect in HepG2 cells. Moreover, ASH-AE can inhibit proliferation of HepG2 cells and mitigate sorafenib-induced resistance-associated markers in HepG2 cells by targeting CD90+ cells via Hedgehog pathway modulation.
Endoplasmic reticulum (ER) stress is the accumulation of misfolded or defective proteins in the ER. ER stress is capable of inducing both anti-apoptotic and pro-apoptotic cellular response, and at the same time plays an important role in metabolism and progression of many types of tumors. Current understanding of the role of ER stress in changing functional parameters of normal and tumor cells is lacking. This study investigated how ER stress inducers bortezomib, dithiothreitol, and tunicamycin influence proliferation, cell cycle, and changes in ploidy of normal and tumor cells of epidermal origin HaCaT and A431 in vitro following incubation with the agent as well as after its removal from the culture medium. Bortezomib caused a cell cycle arrest in the G2 phase in HaCaT cells, as well as polyploidization in both cell lines. Dithiothreitol induced apoptosis in HaCaT cells. Tunicamycin caused a decrease in proliferative index, cell cycle arrest, as well as apoptosis and necrosis in the A431 cells. In conclusion, induction of ER stress by different mechanisms has different effects on normal and tumor cells and can lead to both polyploidization and, presumably, cell differentiation or senescence.
Immunophenotyping of tumor-infiltrating immune cells is increasingly important for understanding the tumor microenvironment (TME), particularly in the diagnosis and treatment of skin cancer. CD1a+ dendritic cells (DC) initiate T cell activation by presenting tumor antigens, while T cells directly target tumor cells. Analyzing their spatial distribution in different types of skin cancer can provide insights into immune response patterns. To characterize the immune cell composition within the TME, we performed immunofluorescence staining for CD1a and CD3 on formalin-fixed, paraffin-embedded (FFPE) samples from actinic keratoses (n = 18), squamous cell carcinoma (n = 23), basal cell carcinoma (n = 19), and melanoma (n = 22), with nevi (n = 16) and healthy skin (n = 9) as controls. Immune cells were quantified across four tumor compartments: intratumoral, tumor margin, intraepidermal, and intradermal. Both CD1a+ DC and CD3+ T cells were detected across all tumor entities, displaying distinct spatial distribution patterns. DC were enriched intratumorally and within the epidermis, whereas T cells predominantly accumulated at the tumor margin (main effect of region, p < 0.001). Melanoma exhibited significantly fewer DC at the tumor margin while maintaining strong T cell infiltration. Overall, the immune architecture of skin tumors is highly compartmentalized, characterized by region-specific DC and T cell distributions. These findings underscore the relevance of spatial immune profiling for understanding immune escape mechanisms and informing immunotherapeutic strategies.
Impaired wound healing in type 2 diabetes is largely attributed to dysregulated cellular responses, defective extracellular matrix (ECM) remodeling, insufficient angiogenesis, and a prolonged inflammatory microenvironment. Understanding how these processes can be modulated at the cellular and tissue levels remains essential for improving diabetic wound repair. In this study, we examined the effects of a decellularized dermal scaffold (DDS) combined with photobiomodulation therapy (PBM), applied as an adjunct biophysical stimulus, on angiogenic, inflammatory, and matrix remodeling responses in a type 2 diabetic rat wound model. Full-thickness excisional skin wounds were created and assigned to control, DDS-treated, PBM-treated, or combined DDS + PBM groups. Wound tissues were harvested on days 8 and 16 post-injury for macroscopic evaluation, biomechanical testing, histological and histochemical analyses, and cytokine quantification. Morphometric assessment revealed that wounds receiving the combined intervention exhibited significantly accelerated wound contraction compared with single-modality and untreated groups at both timepoints. Biomechanical analyses demonstrated improved tissue integrity in treated wounds, with the combined group showing the highest values of tensile strength, maximum force, energy absorption, and bending stiffness, indicative of enhanced structural organization. Histological evaluation showed that DDS combined with PBM markedly increased fibroblast density and neovascularization while reducing inflammatory cell infiltration. Histochemical staining further demonstrated more advanced and organized collagen deposition in the combined group, reflecting accelerated ECM maturation and remodeling. At the molecular level, treated wounds displayed elevated levels of pro-regenerative mediators, including transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor (VEGF), with maximal expression observed in the combined group. Conversely, the expression of proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) was significantly attenuated. Collectively, these findings indicate that integrating a decellularized dermal scaffold with adjunct photobiomodulation effectively modulates cellular behavior, angiogenic signaling, inflammatory responses, and ECM remodeling in diabetic wounds. This study provides mechanistic insight into scaffold-based microenvironmental regulation of impaired wound healing under diabetic conditions.
The development of immunohistochemical and immunohistofluorescence assays is essential for investigating the tissue and cellular distribution of target proteins. In this study, we identify specific anti-ESR1 antibodies against rodent ESR1 proteins and evaluate their applicability for immunohistochemistry and dual immunohistofluorescence with the specific anti-ESR2 antibody PPZ0506. We assessed the specificity and cross-reactivity of six commercially available anti-ESR1 antibodies (Clones MC-20, C1355, E115, H4624, SP1, and F-10) against mouse and rat ESR1 proteins using immunoblotting and immunocytofluorescence assays. Among them, MC-20, C1355, E115, and H4624 exhibited specific immunoreactivity to mouse and rat ESR1 proteins. These four antibodies were subsequently applied to paraffin-embedded ovarian and uterine sections from mice and rats. Heat-induced antigen retrieval and an appropriate antibody dilution were required to obtain specific and adequate signals. MC-20 and E115 were suitable for immunohistochemical detection of ESR1 proteins, while C1355 was effective for uterine tissue staining. H4624 showed utility only in mouse tissues. Furthermore, rabbit-derived MC-20 and E115 antibodies were successfully employed in dual immunohistofluorescence assays with the mouse monoclonal PPZ0506 antibody, enabling simultaneous visualization of ESR1 and ESR2 proteins in paraffin-embedded ovarian sections. Notably, little cellular co-localization of ESR1 and ESR2 proteins was observed in mouse and rat ovarian sections. These findings provide a validated set of antibodies for ESR1 immunohistochemical detection and demonstrate their compatibility with ESR2 co-labeling, facilitating detailed analysis of estrogen receptor distribution in rodent tissues.
High-intensity electric fields (EFs) are increasingly encountered in occupational and environmental settings, raising concerns regarding their potential biological effects on hormonally responsive organs. However, the cellular and molecular responses of female reproductive tissues to short-term exposure to high-voltage EFs remain insufficiently characterized. This study aims to investigate EF-associated histopathological and immunohistochemical alterations in major female reproductive organs. Forty adult female Wistar albino rats were randomly assigned to five experimental groups (n = 8 per group): a control group (0 min) and four EF-exposed groups subjected to a nominal electric field intensity of 10 kV/m EF for 1, 5, 15, or 30 min using a custom-designed parallel-plate exposure system. The field intensity was verified at predefined measurement points within the exposure setup. Ovaries, uterus, and uterine tubes were examined using hematoxylin-eosin staining and semi-quantitative histopathological scoring. Immunohistochemical analyses were performed to evaluate the expression of tumor necrosis factor-α (TNF-α), vascular endothelial growth factor (VEGF), and osteonectin as markers of inflammation, angiogenesis, and extracellular matrix remodeling. Exposure to the electric field was associated with time-dependent histopathological alterations, including tissue edema, hemorrhage, epithelial degeneration, and leukocyte infiltration. The most pronounced ovarian and uterine changes were observed in the 30-min exposure group, whereas the uterine tubes exhibited comparatively milder structural alterations. Immunohistochemical analysis demonstrated increased TNF-α and VEGF expression in higher-exposure groups, suggesting activation of inflammatory and angiogenic pathways. Osteonectin expression was elevated in all examined reproductive tissues and was also detected in regions without overt morphological damage, indicating its potential sensitivity to early tissue stress responses associated with EF exposure. Significant correlations were observed between histopathological injury scores and immunohistochemical marker expression levels. Overall, short-term exposure to high-intensity electric field was associated with inflammatory signaling, angiogenic responses, and extracellular matrix remodeling in female reproductive tissues in this experimental model. These findings provide histological and immunohistochemical evidence of tissue responses to EF exposure and underscore the need for further studies incorporating comprehensive exposure characterization and additional molecular endpoints to better define potential reproductive health implications to high-voltage electric fields.
Achilles tendon ruptures are recognized as one of the most widespread musculoskeletal injuries. Tendon injuries are a notable clinical challenge, primarily due to the restricted regenerative capacity of the tissue and the associated risks of fibrosis and incomplete functional recovery. Recent studies suggest that cell-free therapies, including stem cell-derived secretomes, may facilitate tendon regeneration. Additionally, mechanical stimulation through exercise can enhance tissue remodeling. This study aimed to investigate the combined effects of tendon-derived stem cell (TDSC) secretome and treadmill-based rehabilitation on Achilles tendon regeneration in a rat model. TDSCs were isolated from rat Achilles tendon and characterized using morphology, cytochemical staining, and flow cytometry. In vitro scratch assays were performed to assess cell migration and wound healing in a laboratory setting. The secretome was collected from fourth-passage TDSCs and incorporated into a collagen-based, injectable hydrogel. A total of 42 adult female Wistar rats were categorized into eight distinct experimental groups, including injury-only, treadmill-only, secretome-only, and treadmill + secretome groups. A surgical procedure was performed to induce a partial rupture of the Achilles tendon, followed by the injection of a secretome-loaded hydrogel at the site of injury. Subsequently, a structured treadmill training program was initiated post-surgery. Regenerative outcomes were evaluated using footprint analysis, histological staining (hematoxylin and eosin, periodic acid-Schiff, and Masson's trichrome), and biomechanical testing. In vitro scratch assays demonstrated that TDSCs treated with secretome exhibited enhanced migratory capabilities. Flow cytometry confirmed the identity of these mesenchymal stem cells (MSCs). In vivo studies showed that the combination therapy group (secretome-loaded hydrogel and treadmill training) achieved superior histological recovery. This group exhibited organized collagen bundles, aligned spindle-shaped tenocytes, minimal inflammation, and restored extracellular matrix integrity. PAS staining indicated reduced glycosaminoglycan degradation, while Masson's trichrome staining revealed partial collagen maturation. Additionally, footprint analysis showed improved functional performance, with the combination group achieving significantly higher Achilles functional index scores. Biomechanical testing confirmed enhanced tensile strength and elastic modulus, approaching values comparable to those of intact tendon healing. The synergistic application of TDSC-derived secretome along with treadmill training significantly improved tendon regeneration, matrix remodeling, and functional recovery. This cell-free, bioactive approach offers a promising therapeutic alternative for enhancing tendon recovery.
Mesenchymal stem cells (MSCs) are widely utilized in regenerative medicine owing to their differentiation potential and paracrine effects. Although numerous tissues have been identified as sources of MSCs, the search for novel, noninvasive sources continues. To date, the stem cell content of colostrum has not been investigated. To address this gap, this study aimed to comparatively evaluate the biological properties, proliferative dynamics, and functional potential of cell populations derived from colostrum and mature milk. Cells isolated from both sources (n = 3) were morphologically assessed under in vitro culture conditions and were induced to undergo multilineage differentiation. Phenotypic characterization was performed by flow cytometric analysis. Proliferation capacity was assessed by determining population doubling time (PDT) and performing colony formation assays, while cell viability was evaluated using the methyl thiazolyl tetrazolium (MTT) assay. Cells initially exhibited an epithelial-like morphology and adopted a fibroblast-like phenotype after passaging. Colostrum-derived and mature milk-derived cells displayed multilineage differentiation potential, and flow cytometric profiling confirmed the presence of cells positive for CD73, CD90, and CD105 in both sources. Colostrum-derived cells exhibited higher cell densities; however, population doubling times showed no statistically significant difference between the groups. MTT analysis demonstrated a progressive increase in metabolic activity in both groups, with colostrum-derived cells exhibiting significantly higher optical density values from day 2 onward. No statistically significant difference in colony-forming efficiency was observed between the groups. Consequently, these preliminary findings suggest that colostrum may harbor cell populations exhibiting MSC-like properties and could represent a potential area of interest for MSC research.
This investigation sought to determine whether the formation and progressive buildup of advanced glycation end products (AGEs) within hyperglycemic environments impairs the biological functions of stem cells from human exfoliated deciduous teeth (SHEDs) through a mechanism mediated by the receptor for advanced glycation end products (RAGE), endoplasmic reticulum (ER) stress, and oxidative dysregulation. An in vitro hyperglycemic model was established by culturing SHEDs under high glucose concentrations, AGE exposure, or their combination. Multiple experimental approaches were employed to assess cellular behaviors: proliferative capacity was measured via cell counting kit-8 (CCK-8) assay, migratory ability was evaluated through scratch wound assays, the proportion of apoptotic cells was quantified through annexin V/propidium iodide (PI)-based flow cytometry, while senescence was assessed using senescence-associated β-galactosidase (SA-β-Gal) staining. Critical molecular mediators encompassing RAGE/nuclear factor-κB (NF)-κB signaling components, ER stress biomarkers, oxidative stress parameters, stemness regulators, and osteogenic differentiation markers were quantified through quantitative real-time polymerase chain reaction (qRT-PCR), immunoblotting, immunofluorescence microscopy, and enzyme-linked immunosorbent assay (ELISA) methodologies. Concurrent treatment with high glucose and AGEs substantially suppressed SHED proliferative and migratory capacity while simultaneously enhancing apoptotic cell death and cellular senescence. These functional impairments correlated with elevated RAGE expression, NF-κB pathway engagement, and intensified ER stress responses, as evidenced by augmented GRP78 and CHOP abundance. Oxidative stress parameters were similarly elevated, manifesting as increased reactive oxygen species (ROS) generation and malondialdehyde (MDA) accumulation alongside glutathione (GSH) depletion. The expression of the stemness-associated factors SOX2 and OCT4, together with osteogenic markers RUNX2 and OCN, exhibited marked downregulation. Notably, pharmacological RAGE blockade using FPS-ZM1 successfully reversed these pathological alterations, attenuating ER stress responses, oxidative dysregulation, and functional deterioration triggered by the combined treatment regimen. Our investigation demonstrates that AGEs exacerbate SHED dysfunction under high-glucose conditions by triggering RAGE-dependent ER stress and oxidative stress cascades. This pathological sequence ultimately compromises cellular self-renewal capacity and differentiation potential. Consequently, therapeutic targeting of the RAGE signaling axis represents a promising approach for maintaining dental stem cell functionality and promoting regenerative outcomes in pediatric populations with high-sugar dietary habits.
Assessing the biological activities of some potential drugs and comparing their suitability for in vitro and in vivo combination therapy or in silico drug repositioning against important targets is essential for minimizing labor, costs, and time in drug development. Herein, dose- and time-dependent in vitro anticancer activity studies of anti-inflammatory drugs, including celecoxib (C), indomethacin (I), and meloxicam (M), in combination with natural products (taxifolin (T), quercetin (Q), and rutin (R)) and doxorubicin (Dox), were carried out in MDA-MB-231, BT-20, MCF-7, and HT-29 human cancer cell lines. Drug C demonstrated significant anticancer activity in cancer cells with natural products (< 40 µM) and Dox (< 5 µM). The antiulcerative effect of the most promising drug C in combination with T and R in rats was carried out. The histopathological analysis suggests that the substitution of R with T, when combined with drug C, leads to a statistically significant improvement in the amelioration of gastric mucosal injury. Additionally, in silico studies have been conducted against the important cancer drug target sphingosine kinase 1 (SphK1). The obtained results highlight that drug C and T may be potential inhibitor candidates as SphK1 inhibitors for targeted cancer therapy. Overall, the combination of drug C with T has shown promising results, anticancer effects in breast and colon cancer cells and antiulcerative effects in rats.
Phosphoinositides are low-abundance regulatory lipids that control a broad range of cellular processes, from membrane trafficking and cytoskeletal remodeling to transcriptional regulation and RNA processing. These lipids are distributed across distinct subcellular compartments, where they carry out compartment-specific regulatory functions. Dysregulation of phosphoinositide metabolism is associated with cancer, neurodegenerative diseases, and immune dysfunction. However, their roles remain difficult to investigate owing to technical limitations in lipid detection and manipulation. This review outlines current strategies for modulating, visualizing, and quantifying phosphoinositide pools, including genetic manipulation techniques such as RNA interference, clustered regularly interspaced short palindromic repeats (CRISPR)-based approaches, and optogenetics. It also evaluates visualization tools such as fluorescent biosensors and live-cell imaging techniques, including superresolution microscopy. In parallel, quantitative methods such as thin-layer chromatography and mass spectrometry for profiling phosphoinositide species, including isomer- and acyl-specific variants, are discussed. By comparing the strengths and limitations of these approaches and highlighting how they can be combined, this review provides a practical framework for dissecting phosphoinositide function in defined subcellular contexts.
Renal tubular damage and interstitial fibrosis are highly linked to diabetic kidney disease (DKD) progression. Ferroptosis in renal tubular epithelial cells has emerged as one of the key mechanisms of DKD. Spermidine/spermine N1-acetyltransferase 1 (SAT1) knockdown has been found to alleviate repetitive low-dose cisplatin-induced kidney damage and fibrosis, and importantly, SAT1 silencing represses cellular sensitivity to ferroptosis. However, the effect of SAT1 on DKD-associated ferroptosis and its potential mechanism remain understood. In this study, we constructed a high-fat diet/streptozotocin-induced DKD mouse model and a high glucose (HG)-injured HK-2 cell model with the aim of verifying whether SAT1 silencing attenuates DKD tubular damage by regulating ferroptosis. We found that SAT1 was upregulated in DKD mouse kidneys and HG-treated HK-2 cells. Significant tubular damage, fibrosis, ferroptosis, and oxidative stress were observed in DKD mouse kidneys. In an in vitro loss-of-function assay, SAT1 silencing suppressed HG-induced HK-2 cytotoxicity, extracellular matrix (ECM) synthesis, and inflammation. Additionally, SAT1 silencing decreased HG-activated MDA and 4-HNE production, while restoring GSH levels. SAT1 silencing also abrogated HG-activated ferroptosis in HK-2 cells, as evidenced by a reduction in iron overload, inhibition of lipid peroxidation, and upregulation of ferroptosis-related protein (SLC7A11, GPX4, and TFR1) expression. Mechanistically, SAT1 silencing facilitated nuclear translocation and expression of NRF2. Impairment of NRF2 function abrogated the inhibitory effects of SAT1 silencing on HG-stimulated HK-2 cytotoxicity, ferroptosis, and ECM accumulation. Overall, the SAT1/NRF2 axis is a critical regulator of tubular damage in DKD, and suppression of SAT1 may be an underlying target for DKD treatment.
In humans, testicular peritubular cells (TPCs) form a small compartment surrounding the seminiferous tubules and, as shown previously, undergo age-related changes. How they may contribute to the age-associated decline of testicular functions is not well known. Likewise, the mechanisms of testicular aging in humans are not well examined. This is in part due to the lack of appropriate cellular models. We aimed to establish an aging model of TPCs, which is based on immortalized nonhuman primate (NHP) peritubular cells (iMKTPCs) from Callithrix jacchus. As shown previously by a comprehensive proteomic approach, they strongly resemble human TPCs but lack the human cell-intrinsic heterogeneity. Cellular senescence was robustly induced within 10 days upon exposure to 25 µg/mL bleomycin for 24 h. This resulted in increased β-galactosidase activity, enlarged cells, and elevated transcript levels of senescence-associated secretory phenotype (SASP) molecules (IL1b, TNFa, CCL2). The ability to contract upon a stimulus was reduced, as shown in live cell imaging studies. Reduced proliferation among others was indicated by a decreased abundance of proliferating cell nuclear antigen (PCNA), evident in a proteomic analysis, which also revealed massive changes (341 significantly increased and 372 decreased proteins). Bioinformatics analysis indicated a marked reduction in several cell-motility-associated proteins, whereas proteins linked to extracellular exosomes, the cytoskeleton, and lysosomal pathways were increased in abundance. To conclude, bleomycin treatment causes a rapid and robust induction of cellular senescence in a translational testicular cell model of peritubular cells. This model will enable future studies aiming to explore mechanisms and consequences of testicular aging.
This study investigated whether ovarian adipokines exhibit uniform or stage-specific expression patterns across different follicular stages under hyperandrogenic conditions using a letrozole-induced polycystic ovary syndrome (PCOS) mouse model. Adult female mice received oral letrozole treatment for 21 days to induce hyperandrogenism, and ovarian tissues were analyzed by immunohistochemistry and western blot to examine the localization and expression of adiponectin (ADPN), adipoR1, adipoR2, leptin (Ob), leptin receptor (ObR), apelin (APLN), apelin receptor (APJ), chemerin, CMKLR1, and visfatin. Intense immunostaining for Ob, ObR, APJ, APLN, adipoR2, and visfatin was observed in primary, secondary, and Graafian follicles, whereas ADPN, adipoR1, and CMKLR1 showed reduced reactivity. In follicular cysts, adipoR2, APLN, APJ, and Ob were markedly upregulated compared with the corpus luteum of control ovaries, whereas ADPN, adipoR1, chemerin, CMKLR1, and ObR were downregulated. These findings indicate that hyperandrogenism disrupts adipokine signaling in a follicle-dependent manner, with differential expression patterns contributing to altered follicular maturation and cyst formation. The enhanced activation of adiponectin, apelin, and leptin signaling observed in cystic follicles may indicate disrupted adipokine-mediated regulation of ovarian physiology in letrozole-induced PCOS. Given the established roles of these adipokines in folliculogenesis and steroidogenesis, their dysregulation may contribute to follicular arrest and impaired ovarian function. These alterations are likely reflective responses to an altered endocrine and metabolic environment rather than direct causal mechanisms. Nonetheless, they may participate in the pathophysiological processes underlying cyst formation in PCOS.
Endothelial cell senescence represents a critical mechanistic driver in the initiation and progression of cardiovascular diseases. Senescent endothelial cells exhibit characteristic features, including cell cycle arrest-mediated primarily through the p53/p21 and p16 pathways-morphological transformations such as increased cell volume, elevated caveolin-1 expression, and loss of LaminB1, as well as activation of the senescence-associated secretory phenotype (SASP). The SASP facilitates the secretion of numerous inflammatory cytokines and chemokines, thereby fostering a state of chronic inflammation and contributing to tissue dysfunction. Key molecular regulators of endothelial senescence include transcription factors such as NF-κB and p53, along with the p38 MAPK signaling pathway, which collectively modulate inflammatory responses, cell cycle progression, and stress adaptation. This review offers a comprehensive and integrative perspective on endothelial senescence as a central element in cardiovascular pathophysiology. Its novelty stems from a systematic synthesis of classical pathways, including p53/p21 and p16, with more recently implicated players such as mammalian target of rapamycin (mTOR) signaling and associated microRNAs (miRNAs), accompanied by a focused examination of the SASP as a core pathological mechanism in chronic inflammation and vascular impairment. Moving beyond singular pathways, this work constructs a multidimensional framework that integrates cell cycle arrest, morphological changes, SASP activation, and transcriptional regulation to delineate a cohesive pathological sequence through which endothelial senescence promotes cardiovascular disease.
The adrenal glands are essential endocrine organs whose cortex and medulla maintain systemic homeostasis and mediate stress responses via steroid hormone and catecholamine secretion. Despite anatomical and functional similarities between human and mouse adrenal glands, notable species-specific differences exist. Here, we leveraged spatial transcriptomics (10× Genomics Visium) to comprehensively map gene expression in adult human and mouse adrenal glands, aiming to identify canonical marker genes conserved across species. The analysis was based on a 31-year-old female human sample (GEO dataset) and four 10-week-old male CD-1 mice. Human adrenal sections were processed using optimal cutting temperature (OCT) embedding, whereas mouse adrenal sections were processed as formalin-fixed paraffin-embedded (FFPE) samples, highlighting differences in sample preparation. Using unsupervised clustering of spatial gene expression data, we delineated distinct adrenal cortex and medulla zones in both species, confirming known zonation patterns. Our cross-species analysis revealed highly conserved spatial expression of key known marker genes characteristic of the adrenal cortex (e.g., CYP11B2 for ZG, CYP11B1 for ZF) and medullary chromaffin cells (e.g., TH), as well as a core set of additional marker genes previously less characterized in adrenal biology. By integrating transcriptional profiles, we generated a catalogue of conserved canonical marker genes that define adrenal zonation and function in both humans and mice. These results highlight the fundamental molecular conservation of adrenal gland organization and support the translational value of mouse models in adrenal research. Our findings provide new insights into the evolutionary preservation of adrenal function and a valuable resource for studies on adrenal physiology and disease.
Salt stress significantly reduces plant growth and yield, which has led to extensive research on the mechanisms underlying plant salinity tolerance. Carrot (Daucus carota ssp. sativus) is a glycophyte highly sensitive to soil salinity. We investigated root and leaf anatomical, histological, and immunohistological alterations in two carrot accessions, previously identified as salt-sensitive (DH1) and salt-tolerant (DLBA), growing under control and salt stress conditions. The results demonstrate that the salt-tolerant DLBA growing under control conditions has trichome-rich leaves, high starch reserves and a hydraulically safer root xylem. Under salt stress, DLBA maintains mesophyll integrity, and increases the number of vessels and deposition of highly esterified pectins, hemicelluloses and spatially regulated AGPs in cell walls. In contrast, DH1 develops thinner, trichome-free leaves, and roots almost free of starch with fewer cambial cells and vessels. Salt stress induces overexpansion of palisade parenchyma, excess starch accumulation, loss of arabinan epitopes, disappearance of extensins in vascular bundles, and changes in hemicellulose and AGP distribution. These findings indicate that salt tolerance of DLBA plants results from the combination of constitutive anatomical characteristics and adaptive responses that together support tissue hydration, wall elasticity and stable water transport when plants are growing in saline soil.
In type 2 diabetes mellitus (T2DM), skeletal muscle is a major site of metabolic and microvascular dysfunction, yet most human data derive from large locomotor muscles, whereas postural and respiratory muscles remain less well characterised. We examined whether T2DM alters fibre morphology, intramyocellular lipid (IMCL) content, and three-dimensional (3D) capillary architecture across functionally distinct muscles. Postural (splenius capitis [SC]), respiratory (diaphragm [DIA]; external intercostal [EXT]), and locomotor (vastus lateralis [VL]) muscles from adult male individuals (T2DM versus control, n = 24/group) were sampled < 24 h post-mortem. Analysis included myosin heavy chain fibre typing, Sudan Black B intramyocellular lipid (IMCL) quantification, and 3D capillary morphometry (length, tortuosity, anisotropy, branching density). Groups were age-matched (T2DM 70.8 ± 7.4 versus 69.7 ± 11.8 years; p = 0.684), but body mass index (BMI) was higher in T2DM (31.9 ± 4.7 versus 24.8 ± 2.7 kg m-2; p < 0.0001). Fibre-type profiles were similar, except for elevated 2a/2x hybrids in T2DM VL (p = 0.014). Mean fibre diameters were preserved, though type 1 fibres were larger in T2DM SC (p = 0.0238). IMCL was higher in T2DM SC and EXT (p < 0.05), with non-significant differences in VL and DIA. Type 1 and 2a fibres had higher IMCL than glycolytic fibres, with no group-by-fibre-type interaction. BMI strongly predicted VL IMCL (p < 0.0001), while age was negatively associated with IMCL in respiratory muscles (p ≤ 0.05). Capillary length per fibre volume was selectively reduced in DIA (p = 0.0115); other indices were preserved, except for higher anisotropy in EXT (p = 0.0495). Overall, these functionally diverse muscles showed subtle muscle-specific remodelling in T2DM, with adiposity-linked IMCL accumulation and reduced DIA capillary supply, although findings should be interpreted in the context of the post-mortem study.
Hirschsprung's disease (HSCR) is a congenital disorder of the distal intestine characterized by aganglionosis of the enteric nervous system. While defective migration of neural crest-derived precursors is a well-established hallmark, how the intestinal microenvironment contributes to impaired neuronal support remains poorly understood. Here, we combined histochemical analyses of human HSCR colon with functional assays to investigate the role of muscularis macrophages (MMs) and a miR-93-5p-AHNAK pathway in regulating enteric neurons. Using immunohistochemistry and immunofluorescence on ganglionic and aganglionic segments, we mapped the density, spatial distribution, and phenotype of MMs, and quantified the expression of miR-93-5p and its predicted target AHNAK. In vitro, polarized M2-like macrophages were cocultured with enteric neuronal cell lines to assess neuronal migration, proliferation, and apoptosis. Gain-of-function and loss-of-function approaches for miR-93-5p, together with a dual-luciferase reporter assay, were used to validate AHNAK as a direct target. M2-like MMs (CD163+/CD206+) were abundant around myenteric ganglia in ganglionic colon but reduced in aganglionic segments, where AHNAK expression was increased. M2 macrophages enhanced neuronal migration and proliferation and protected against apoptosis, whereas disruption of the miR-93-5p-AHNAK axis impaired these neuro-supportive effects. Together, our data identify a muscularis macrophage-miR-93-5p-AHNAK axis that supports enteric neurons and demonstrates that loss of M2-like MMs and dysregulated miR-93-5p-AHNAK signaling compromise neuronal homeostasis in HSCR. These findings add a histochemical microenvironment perspective to HSCR pathogenesis and support a candidate neuroimmune pathway for restoring immune-neuronal balance, pending in vivo and clinical validation.
Small tissue biopsies, including renal core biopsies, bone marrow trephines, gastrointestinal endoscopic samples, prostate needle cores, liver biopsies, and skin punch or shave specimens, are fundamental to contemporary diagnostic pathology. Their limited volume, focal sampling, and susceptibility to technical artifacts impose distinct interpretive challenges, often requiring semi-quantitative assessment within a restricted architectural context. Although artificial intelligence (AI) has rapidly expanded in digital pathology, most models have been developed using large surgical resection specimens, with comparatively limited attention to small biopsy material. This minireview examines current and emerging applications of AI in small biopsy diagnostics across renal, hepatic, gastrointestinal, hematopathological, dermatopathological, and urological pathology. Reported applications include glomerular segmentation and fibrosis quantification in renal biopsies; automated cellularity, blast detection, and fibrosis grading in bone marrow trephines; dysplasia and microorganism detection in gastrointestinal biopsies; quantitative steatosis and fibrosis assessment in liver samples; tumor detection and grading in prostate cores; and neoplastic pattern recognition in skin specimens. Despite encouraging performance in research settings, substantial barriers to routine clinical implementation remain, including limited dataset size, class imbalance, pre-analytical variability, inter-institutional heterogeneity, and insufficient external validation. We discuss methodological considerations relevant to diagnostic practice, including multi-institutional validation, stain normalization, multimodal integration with histochemistry and ancillary testing, explainability, and regulatory oversight. In the context of small biopsies, AI should be regarded as a quantitative adjunct to morphological interpretation rather than an autonomous diagnostic system. Careful integration within established histopathological workflows is essential to ensure reproducibility, safety, and clinical accountability.