Histochemistry and Cell Biology最新文献

Salamander Pleurodeles waltl is an emerging animal model for developmental and regenerative biology studies. However, the exploration of skeletal muscle regeneration has been hindered by the absence of suitable in vitro cell systems for in-depth mechanism research. In this study, we established a protocol for the cultivation of muscle stem cells derived from Pleurodeles waltl for cell biology experiments. Trunk and limb muscles were minced and digested with collagenase. Cells with a high nucleoplasmic ratio were isolated from the muscle tissue. Immunofluorescence and RT-PCR analysis revealed that these proliferating cells expressed the typical muscle stem cell markers. Furthermore, these cells demonstrated effective myogenic differentiation in vitro, as evidenced by the expression of the myogenic differentiation marker protein, myosin heavy chain. Additionally, it was observed that cultured myotubes derived from these cells initiated DNA synthesis and upregulate cell cycle genes upon stimulation with a high concentration of serum. Notably, the muscle stem cells (Pw-1) maintained a steady proliferation rate even after undergoing 35 subcultures. In conclusion, this study has successfully established a method for isolating and cultivating muscle stem cells from salamanders, confirming the dedifferentiation potential of the myotubes derived from these cells. This methodology provides a valuable tool for exploring the molecular mechanisms that govern skeletal muscle regeneration.

Pulmonary fibrosis (PF) is an insidious, progressive, and fatal age-associated disease that occurs primarily in older adults and has a poor prognosis. Alveolar epithelial cell (AEC) senescence is the critical pathological mechanism of PF. The accumulation of oxygen radicals, commonly referred to as reactive oxygen species (ROS), strongly contributes to cellular senescence. The triggering receptor expressed on myeloid cells-1 (TREM-1) is a pattern recognition receptor. Triggering via TREM-1 results in ROS, leading to the amplification of inflammation. However, whether TREM-1 is involved in PF by inducing oxidative stress to exacerbate AEC senescence remains unclear. We first observed that blockade of TREM-1 during the fibrotic phase attenuated bleomycin (BLM)-induced PF in mice, with decreased expression of senescence-related proteins, including p16, p21, p53, and γ-H2AX, in the lung tissue. Moreover, TREM-1 blockade during the fibrosis stage restored antioxidant levels by increasing the percentage of Nrf2- and HO-1-positive cells in mice with PF. Notably, TREM-1 was highly expressed in surfactant-associated protein (SPC)-positive AECs in mice with PF. In vitro, blocking TREM-1 activated Nrf2 antioxidant signaling, thereby decreasing intracellular ROS levels and diminishing BLM-induced senescence in AECs. Furthermore, inhibition of Nrf2/HO-1 partially counteracted the anti-senescence effect of blocking TREM-1 in BLM-treated AECs. In this study, we reported that TREM-1 stimulated the senescence of AECs, induced ROS and exacerbated PF. We also provide compelling evidence suggesting that the Nrf2/HO-1 signaling pathway underpins TREM-1-triggered senescence. Therefore, our findings provide new insights into the molecular mechanisms associated with TREM-1 and AEC senescence in the pathogenesis of PF.

Peroxisomes have gained increasing attention and are now considered vital players in normal physiological functions. To gain further insight into how peroxisomal defects influence cellular functions, we developed BV-2 microglial models featuring CRISPR/Cas9 gene-edited mutations in peroxisomal Acox1 or Abcd1 and Abcd2 genes. The Acox1^-/- BV-2 cell line we generated lacks acyl-CoA oxidase 1, the key enzyme that initiates peroxisomal β-oxidation. In contrast, the double mutant Abcd1/d2^-/- BV-2 cell line carries mutations in the genes encoding the membranous ABC transporters ABCD1 and ABCD2, which are responsible for transporting fatty acyl-thioesters inside peroxisome. Here, for the first time, we used analytical fractionation to compare these three genotypes. Through flow cytometry, we observed an increase in cell granularity in these mutant cells, which could be associated with alterations in peroxisome distribution and mitochondrial dynamics. Additionally, the analysis of organelle markers in microglial cells, employing differential centrifugation, exhibited an enrichment of peroxisomes particularly in both L and P fractions of these BV-2 cell line models. The use of an isopycnic Nycodenz density gradient showed that peroxisomes sedimented with a median density of 1.18 g/ml. Notably, our results revealed no significant differences in the distribution profiles of organelles when comparing microglial BV-2 Wt cells with deficient Acox1^‒/‒ or Abcd1/d2^-/‒ BV-2 cells, which lack peroxisomal fatty acid beta-oxidation. Our study is the first to report on the fractionation of brain-derived microglial cells, laying valuable groundwork for future proteomic and/or metabolomic analyses of peroxisome fractions.

Cholesterol is involved in the regulation of various signaling processes, and oxysterols are essential lipid messengers. The cholesterol derivative 25-hydroxycholesterol (25-HC) is overproduced by muscle macrophages in sarcopenia, myasthenia, and neurodegenerative diseases. Herein, we examined the effects of 25-HC on Ca²⁺ signaling and contractions of the mouse diaphragm, the main respiratory muscle. We found that 25-HC increased resting levels of cytosolic Ca²⁺ in muscle fibers. This effect was dependent on estrogen receptor α (ERα) and was mediated by Ca²⁺-efflux from intracellular stores via dantrolene-insensitive and TMB-8-sensitive channels, presumably inositol trisphosphate receptors (IP₃Rs). In addition, 25-HC suppressed diaphragm contractile responses to direct stimulation of the muscle fibers. The negative effect of 25-HC on contraction force was inhibited by blockers of ERα and Ca²⁺ mobilization. Thus, 25-HC may suppress diaphragm muscle contractility due to activation of an ERα/IP₃R/Ca²⁺_in axis in muscle fibers. At the same time, 25-HC did not significantly modify the contractions elicited by phrenic nerve stimulation and respiratory activity in vivo. We discuss that the previously found enhancement of neuromuscular transmission mediated by 25-HC in the diaphragm can compensate for the reduction in the muscle contractions in the case of phrenic nerve activation.

Prostate cancer is a frequently diagnosed cancer in men, but today the prognosis of local cancer and its metastasis has improved markedly owing to multiple treatment options. In therapy, various molecular entities that induce the reversion of malignancy with the restoration of mature cells of the same histological lineage are still under development. The antler of the red deer is a large and easily available source of stem cells, rich in products with promising applications in cancer treatments. Here, for the first time, antler stem cell culture medium was used for the treatment of prostate cancer cells in vitro to determine the cellular and molecular effects. Our data revealed that antler stem cell culture medium promotes pronounced changes in the expression of the vaspin receptor GRP78, intercellular adhesion molecule 1 (ICAM-1), androgen receptor (AR), leptin, vaspin, and SRC and AKT kinase signaling pathways (connected with autophagy and cell cycle inhibition and apoptosis induction). Moreover, the inhibition of cell migration was also revealed. These results indicate useful properties of red deer antler stem cell culture medium that can control molecular and cellular mechanisms that halt carcinogenesis. All these can be considered targets for further cancer stem cell treatment or cancer diagnostic improvements.

This study aimed to quantitatively analyze subchondral vascularization during the progression of osteoarthritis (OA) in the elderly, particularly regarding the timing of initial blood vessel emergence and when their density peaks. A total of 129 independent areas from 43 human femoral heads, obtained through arthroplasty for OA or hemiarthroplasty for fractures, were analyzed. The femoral heads were grouped by Kellgren-Lawrence (KL) grades: KL 1 (6 heads), KL 2 (14 heads), KL 3 (10 heads), and KL 4 (13 heads), and the Mankin score was assessed. Quantitative measurements of blood vessel length 1 mm below the tidemark, cartilage volume and thickness, chondrocyte volume, ECM volume, subchondral bone volume, and bone marrow volume were performed using stereology and immunohistochemistry. The most substantial increase in the characteristics of blood vessels within the subchondral region began at KL 3 and peaked at KL 4. Blood vessel volume increased from 6.71 ± 5.84 mm³ in group KL 1 to 156.21 ± 138.67 mm³ in group KL 4 (p < 0.001). Blood vessel surface area showed an increase from 14.78 ± 9.89 cm² (group KL 1) to 125.20 ± 93.18 cm² (group KL 4) (p < 0.001). Likewise, blood vessel length grew from 27.53 m (IQR 13.70-65.41 m) in group KL 1 to 112.03 ± 76.07 m in group KL 4 (p = 0.001). This study offers deeper insights into the role of vascularization in OA pathophysiology, quantifying subchondral blood vessel characteristics in the femoral head across different OA stages.

Fibroblasts, the most abundant cell type in the human body, play crucial roles in biological processes such as inflammation and cancer progression. They originate from the mesoderm or neural-crest-derived ectomesenchyme. Ectomesenchyme-derived fibroblasts contribute to facial formation and do not express HOX genes during development. The expression and role of the HOX genes in adult fibroblasts is not known. We investigated whether the developmental pattern persists into adulthood and under pathological conditions, such as cancer. We collected adult fibroblasts of ectomesenchymal and mesodermal origins from distinct body parts. The isolated fibroblasts were characterised by immunocytochemistry, and their transcriptome was analysed by whole genome profiling. Significant differences were observed between normal fibroblasts from the face (ectomesenchyme) and upper limb (mesoderm), particularly in genes associated with limb development, including HOX genes, e.g., HOXA9 and HOXD9. Notably, the pattern of HOX gene expression remained consistent postnatally, even in fibroblasts from pathological tissues, including inflammatory states and cancer-associated fibroblasts from primary and metastatic tumours. Therefore, the distinctive HOX gene expression pattern can serve as an indicator of the topological origin of fibroblasts. The influence of cell position and HOX gene expression in fibroblasts on disease progression warrants further investigation.

Cutaneous wounds represent a significant health concern, and effective treatment strategies are crucial for optimal healing. This study investigates the therapeutic potential of Luetzelburgia auriculata lectin (LAA), a plant-derived protein, in accelerating wound closure. Excisional wounds were created on the backs of mice, which were subsequently treated topically with LAA solutions at two concentrations (100 µg/mL and 200 µg/mL) or saline control. Wound healing was assessed through clinical observations, including wound area measurement and inflammatory score, as well as histopathological analysis and measurement of inflammatory cytokines. LAA significantly accelerated wound closure, reduced inflammation, and promoted tissue regeneration. Histological analysis revealed enhanced re-epithelialization, increased fibroblast proliferation, and improved collagen deposition in LAA-treated wounds compared with the control group. Furthermore, LAA treatment significantly reduced the levels of proinflammatory cytokines in wound tissues (interleukin-6, tumor necrosis factor-alpha, and monocyte chemoattractant protein-1). These findings suggest that LAA exerts its beneficial effects through a multifaceted mechanism, likely involving anti-inflammatory properties and stimulation of cellular processes crucial for tissue repair. This study provides preliminary evidence for the therapeutic potential of LAA in wound healing and warrants further investigation into its underlying mechanisms and clinical applications.