European Journal of Histochemistry最新文献

The cardiac stem cells (CSCs) are essential in improving myocardial infarction (MI). Although miR-199a-5p and hypoxia-inducible factor-1 alpha (HIF-1α) were proven to participate in the process of heart repair, the related mechanisms are still unclear. This study aimed to explore the effects of miR-199a-5p and HIF-1α on c-kit+ cells and their regulatory mechanisms. After isolating, purifying, and identifying CSCs (c-kit+ cells) from mice, they were subjected to a hypoxia model. After the c-kit+ cells were transfected with corresponding transfectants, the CCK-8, EdU staining, and wound healing approaches were used to evaluate their cell viability, proliferation, and migration. The targeted relation between miR-199a-5p and HIF-1α was determined using a dual-luciferase reporter. Immunofluorescence staining, RT-qPCR, and Western blot approaches were employed to determine Nkx2.5, CD31, α-SMA, miR-199a-5p, and HIF-1α expression. Overexpressing miR-199a-5p and knocking down HIF-1α both inhibited the cell viability (p<0.01), reduced the proliferation (p<0.05), suppressed the migration (p<0.001), and down-regulated the Nkx2.5, CD31, and α-SMA expression of c-kit+ cells (p<0.05). Overexpressing HIF-1α effectively reversed the effects of overexpressing miR-199a-5p on c-kit+ cells (p<0.05). Taken together, miR-199a-5p negatively targeted HIF-1α to inhibit the proliferation, migration, and differentiation of c-kit+ cells.

Cervical cancer is a serious gynecological malignancy, and the specific mechanisms of miR-129-5p remain unclear. This study aims to investigate the mechanism by which miR-129-5p regulates the high mobility group box 1 (HMGB1/receptor for advanced glycation end-products (RAGE) axis to inhibit pyroptosis and ameliorate cervical epithelial cell deterioration. Using RT-qPCR and Western blotting, we detected significantly downregulated miR-129-5p and upregulated HMGB1 in cervical cancer cells. To establish a deterioration model, we stimulated cervical epithelial cells with lipopolysaccharide (LPS). Further results revealed that miR-129-5p overexpression markedly reduced HMGB1 expression, suppressed RAGE activation, and decreased pyroptosis executer GSDMD-N production. Additionally, we conducted miR-129-5p overexpression and knockdown experiments to verify its regulatory effects on the HMGB1/RAGE axis and downstream pathways. Caspase-1 activity assays confirmed reduced pyroptosis upon miR-129-5p overexpression. Cell viability and proliferation were assessed using EdU incorporation assays and colony formation experiments. Our data demonstrated significant downregulation of miR-129-5p in cervical cancer cells. Overexpression of miR-129-5p substantially reduced HMGB1 expression and inhibited RAGE activation, thereby decreasing production of the pyroptosis executer GSDMD-N. LPS stimulation potently activated the HMGB1/RAGE axis and induced pyroptosis, while miR-129-5p overexpression inhibited these processes and ameliorated in vitro cervical epithelial cell deterioration. Cells overexpressing miR-129-5p exhibited attenuated caspase-1 activity with enhanced survival and proliferation following LPS treatment. Collectively, these in vitro findings indicate that miR-129-5p suppresses HMGB1/RAGE-mediated pyroptosis and cellular deterioration and also provide new mechanistic insights for cervical cancer therapeutics.

Oxidative stress is a major contributor to male infertility, particularly oligoasthenozoospermia. This study aimed to investigate the cytoprotective mechanism of Guilu Erxian Oral Liquid (GLEX) against H₂O₂-induced oxidative damage in spermatogonial cells, focusing on miR-6739-5p regulation and activation of the PI3K/AKT pathway using histocytochemical approaches. An oxidative stress model was established in rat spermatogonial stem cells (SSCs) with 250 µM H₂O₂. Cell proliferation, apoptosis, reactive oxygen species (ROS) accumulation, and DNA oxidative damage were assessed using EdU incorporation, flow cytometry, immunofluorescence, and 8-hydroxy-2'-deoxyguanosine (8-OHdG) ELISA. Expression of miR-6739-5p and Phosphatidylinositol 3-Kinase/Protein Kinase B (PI3K/AKT) pathway components (PIK3CA, p-PI3K, p-AKT) was evaluated by RT-qPCR and Western blotting. The interaction between miR-6739-5p and PIK3CA was confirmed via dual-luciferase reporter assay. The cytoprotective effects of GLEX were examined through pre-treatment and quantified using histochemical and cytological markers. H₂O₂ treatment significantly impaired cell viability, increased apoptosis and ROS production, and upregulated miR-6739-5p. Overexpression of miR-6739-5p exacerbated damage, while silencing reversed it and restored PI3K/AKT signaling. GLEX pretreatment effectively reduced miR-6739-5p expression, restored cell viability, suppressed oxidative and inflammatory markers (ROS, 8-OHdG, TNF-α, IL-1β), and enhanced PI3K/AKT activation. These effects were comparable to PI3K pathway activation. GLEX confers histocytochemical protection to spermatogonial cells under oxidative stress by downregulating miR-6739-5p and activating the PI3K/AKT pathway. This study highlights a novel regulatory mechanism and supports GLEX as a potential therapeutic agent for oxidative stress-associated male infertility.

Myocardial ischemia-reperfusion injury (MIRI) induces severe inflammatory damage to cardiac tissue, leading to structural impairment and functional decline. Maresin 1 (MaR1) is an anti-inflammatory lipid mediator derived from macrophages that has shown protective effects in various inflammatory conditions. This study investigated the anti-inflammatory properties and underlying mechanisms of MaR1 in the context of MIRI, both in vivo and in vitro. A rat model of MIRI was established, and MaR1 was administered subcutaneously once daily for one week prior to model induction. Cardiac function was monitored intraoperatively, and serum and myocardial tissue samples were collected postoperatively for analysis. Structural alterations, myocardial injury biomarkers, and inflammatory cytokines were evaluated. In vitro experiments using H9c2 rat cardiomyocytes assessed the effects of MaR1 on cell viability and proliferation. MaR1 treatment significantly improved cardiac function impaired by MIRI, preserved myocardial architecture, and reduced serum and tissue levels of creatine kinase, lactate dehydrogenase, cardiac troponin I, and pro-inflammatory cytokines (IL-1β, IL-6, IL-8, MCP1, and TNF-α). In contrast, MaR1 enhanced the expression of the anti-inflammatory cytokine IL-10. In cultured cardiomyocytes, MaR1 promoted viability and proliferation. Collectively, these findings demonstrate that MaR1 confers protection against MIRI by attenuating inflammation, preserving myocardial structure, improving cardiac function, and enhancing cardiomyocyte survival, underscoring its potential as a therapeutic agent for ischemic cardiac injury.

Store-operated calcium channels (SOCCs) are involved in the process of cell proliferation; however, their expression levels differ among cell types and information on their effects in different cells is lacking. This study aimed to compare the differing effects of SOCCs on the proliferation of vascular smooth muscle cells (VSMCs) and vascular endothelial cells (VECs), and the repair ability of SOCC after vascular endothelial injury. Rat primary coronary VSMCs and VECs were cultured in vitro and expression levels of SOCC molecules were detected by western blotting and quantitative polymerase chain reaction. Various molecules were selected and transfected into VSMCs and VECs using an adenovirus vector, and cell proliferation, the cell cycle, and intracellular Ca2+ were then detected. We also established a rat carotid artery endothelial injury model to verify the results of the in vitro experiments. Expression levels of transient receptor potential canonical (TRPC) 3 and TRPC5 were higher in VSMCs than in VECs. Silencing TRPC3/5 significantly inhibited cell proliferation and Ca2+ influx in VSMCs, but not in VECs. Silencing TRPC3/5 after rat carotid artery endothelial injury inhibited neointima formation, with a better reparative effect on the endothelial cell layer than rapamycin. TRPC3/5 participates in the proliferation of VSMCs via SOCCs, and silencing its expression inhibits the formation of neointima after endothelial injury. However, this effect was not significant in VECs, suggesting that other compensatory pathways may have emerged.

Tanshinone IIA is derived from Salvia miltiorrhiza and has multiple therapeutic targets and functions. The exact therapeutic effects on liver fibrosis as well as the underlying hepatoprotective mechanisms are still lacking. A liver fibrosis model was established via ligation of the common bile duct ligation (BDL). The mice were intraperitoneally administered different concentrations of tanshinone IIA (4 mg/kg, 8 mg/kg) for 2 weeks. Liver function was assessed through hematoxylin and eosin and Sirus red staining. Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), glutathione (GSH) and malondialdehyde (MDA) were quantified by enzyme-linked immunosorbent assay (ELISA), via microplate reader. The total iron content of the liver was quantified via Triple Quad-ICP-MS. TGFβ-induced hepatic stellate cells (HSCs), a cell model of liver fibrosis, were treated with tanshinone IIA at different concentrations (10 mM, 20 mM, 30 mM, 40 mM). The combination of tanshinone IIA with YAP agonists was applied in activated HSCs and animal models. Tanshinone IIA treatment relieved BDL-induced liver fibrosis; mitigated histological liver damage; lowered the serum ALT and AST levels; reduced macrophage infiltration and the MDA and iron contents; and increased the GSH and GPX4 levels by inhibiting YAP signaling. tanshinone IIA also suppressed the activation of HSCs and collagen production through blocking the YAP signaling pathway. The YAP agonist reversed the therapeutic effect of tanshinone IIA on activated HSCs and BDL-induced liver fibrosis. Tanshinone IIA inhibited HSC activation and oxidative stress and alleviated liver fibrosis by inhibiting the YAP signaling pathway.

Musk is a biologically valuable secretion from the musk gland of male musk deer, with significant economic and medicinal importance. Due to severe decline and depletion of wild musk deer population, captive breeding of musk deer has become the primary approach for sustainable musk production. So far, the histological structure and secretion mechanism of the musk gland remain incompletely understood. In this study, we employed histological and immunohistochemical (IHC) techniques, along with three-dimensional (3D) tissue reconstruction, to systematically analyze the cellular composition and secretory functions of the musk gland in forest musk deer (Moschus berezovskii). Our results revealed that the musk gland was primarily composed of acinar structures containing two distinct glandular cell (GC) types based on the histological observation. IHC results showed type I glandular cells (GCIs) predominantly expressed GALNT7 while type II glandular cells (GCIIs) mainly expressed BMP6. The 3D reconstruction demonstrated structural heterogeneity along the gland's longitudinal axis, with the proportion of the acinar area varying between 40% and 65%. This is the first time that a detailed 3D view of musk gland in forest musk deer has been shown, which provides essential histological insights into musk gland function in this species. These preliminary observations may provide useful groundwork for future investigations into the regulatory mechanisms of musk secretion.

The bone morphogenetic protein (BMP) pathway, which plays a crucial role in the control of intestinal epithelial cell homeostasis, has been studied in mice and humans, leading to an understanding of its involvement in several intestinal pathologies. However, the expression and localization of the various actors (ligands, antagonists, receptors) of this pathway remain unknown in the rat intestine, although this species is widely used in pathophysiology studies. Here, we aimed to determine the expression and localization of the various players in the BMP pathway in the jejunum and colon of the rat using RT-qPCR and immunohistochemistry. BMP2, mainly localized in epithelial cells, was the most expressed ligand in the jejunum and colon in comparison with BMP4, BMP6 and BMP7. We showed for the first time that BMP7 was highly expressed in epithelial cells in both tissues. BMP2, BMP6 and BMP7 ligands were also present in the enteric nervous plexuses, as the BMP receptors and antagonists Noggin and Chordin-like 1. The expression of BMP antagonists and ligands in enterocytes and mature colonocytes could suggest a paracrine or autocrine feedback modulation at the cellular level. Finally, all the studied BMP actors were present in colonic vessel walls including GREM1, a BMP antagonist described as pro-angiogenic and also being a ligand for VEGFR receptors. These data provided a good correlation between the observations in rats compared to those in humans and highlighted the importance of the BMP pathway not only in the intestinal epithelium, but also in both the enteric nervous system and vascular system. Our work lays the foundations for further studies on the involvement of the BMP pathway in rat models of intestinal pathophysiology.

Endometriosis is a benign disease with similar characteristics to tumors. Recent studies have found that the erythropoietin-producing hepatoma receptor A2 (EphA2) has the dual effect of promoting tumor and inhibiting tumor. The objective of this study was to explore the specific regulatory mechanism of EphA2 in endometriosis. The expression level of Eph protein family in endometriosis was analyzed by bioinformatics method. At the clinical level, qPCR, Western blot and immunohistochemistry were used to verify the correlation between increased EphA2 levels and endometriosis. The effects of blocking EphA2 on cell migration, invasion, proliferation and apoptosis of primary eutopic endometriotic stromal cells were explored in vitro. Our study indicated that EphA2 expression was elevated in endometriosis patients, and blocking EphA2 in vitro inhibited cell proliferation, migration and invasion through AMPK signaling pathway. Targeting EphA2 can inhibit the progression of endometriosis through the AMPK signaling pathway.

Bladder cancer (BC) is a prevalent and aggressive malignancy with high recurrence. Autophagy plays a dual role in cancer, acting as a tumor suppressor early on and promoting survival in later stages. NR4A3, a nuclear receptor with tumor-suppressive effects in other cancers, has not been explored in BC. NR4A3 expression was analyzed using TCGA data and validated in clinical BC samples via immunohistochemistry and RT-qPCR. NR4A3-overexpressing BC cell lines (5637, T24) were created using lentiviral vectors. Cell viability, proliferation, migration, and invasion were assessed through CCK-8, EdU, and Transwell assays. Autophagy was measured by microtubule-associated protein 1A/1B-light chain 3 (LC3), autophagy-related protein 5 (ATG5), Beclin-1 and p62 expression via immunofluorescence and Western blotting. The phosphoinositide 3-kinase (PI3K) / protein kinase B (AKT) / mammalian target of rapamycin (mTOR) pathway was examined by assessing phosphorylation levels. It was found that NR4A3 was significantly downregulated in BC tissues. Overexpression of NR4A3 inhibited BC cell proliferation, migration, and invasion, while promoting apoptosis. NR4A3 overexpression increased autophagy markers and suppressed PI3K/AKT/mTOR signaling. Autophagy inhibition reversed these effects. In conclusion, NR4A3 suppresses BC progression by promoting autophagy via the PI3K/AKT/mTOR pathway. Targeting NR4A3-mediated autophagy may provide a novel therapeutic strategy for BC.