JC-1 fluorescence is widely used to assess mitochondrial membrane potential (ΔΨm) and is typically summarized as a mean red/green ratio, which may not fully capture spatial variability within images. However, ΔΨm is spatially heterogeneous at the subcellular level, raising the possibility that mean-based analysis alone may overlook differences between cellular states. JC-1 imaging was performed in undifferentiated and differentiated SH-SY5Y cells. Mean JC-1 red/green fluorescence ratios and the coefficient of variation (CV) were calculated from identical image fields. Differentiation induced a neuronal-like state characterized by tyrosine hydroxylase (TH) positivity (95.33
Osteosarcoma (OS) is the most common and lethal primary bone malignancy in children and adolescents. Although YTHDF2 is known to modulate OS progression via m6A-modified mRNAs, whether H3K18 lactylation drives OS malignancy by activating YTHDF2 transcription remains unclear. This study investigated the role of histone lactylation in regulating YTHDF2 expression and OS cell progression. OS cells were treated with LDHA/LDHB interference plasmids, the glycolysis inhibitor 2-deoxy-D-glucose (2-DG), or the lactate donor sodium lactate (NaLa). Cellular functions were evaluated via CCK-8, colony formation, and Transwell assays, while ChIP and dual-luciferase reporter assays assessed YTHDF2 promoter regulation. Results revealed marked elevation of histone lactylation, particularly at H3K18 (H3K18la), in OS cells. Silencing LDHA and LDHB significantly reduced lactylation and suppressed OS cell proliferation, migration, and invasion, effects partially reversed by NaLa. Pharmacological inhibition with 2-DG decreased H3K18la enrichment at the YTHDF2 promoter, suppressing YTHDF2 transcription. Crucially, overexpression of YTHDF2 counteracted the suppressive effects of 2-DG on OS malignant behaviors. In conclusion, elevated histone lactylation in OS cells enhances YTHDF2 transcription, thereby promoting tumor cell proliferation, migration, and invasion, highlighting a novel epigenetic pathway driving osteosarcoma progression.
Hair follicles (HF) are the crucial components of mammalian skin, and their regeneration is stimulated by dermal papilla cells (DPCs). Cell proliferation and differentiation are modulated by the connector enhancer of kinase suppressor of Ras2 (CNKSR2), which is a key scaffold protein in the Ras/MAPK pathway. Although CNKSR2 is expressed in the skin, its function in DPCs remains undetermined. This study evaluated how CNKSR2 regulates DPC proliferation. The CNKSR2 gene’s complete coding sequence (CDS) was acquired, and bioinformatics methods were employed for a preliminary systematic analysis of its predicted protein structure and functional domains. Furthermore, RT-qPCR analysis revealed that the regulation of CNKSR2 expression significantly altered the transcriptional levels of HF growth and development-related genes, including CCND1, BMP4, BCL2, SFRP2, and TGF-β1. Moreover, proliferating cell nuclear antigen (PCNA) immunostaining, EdU incorporation, and CCK-8 proliferation assays revealed that CNKSR2 overexpression substantially enhanced DPC proliferation, whereas its knockdown had a marked inhibitory effect. The results demonstrated that CNKSR2 exerts a positive regulatory effect on the proliferation of rabbit DPCs, providing preliminary experimental evidence for subsequent research on its potential role in HF biology.
Sepsis-associated acute lung injury (SALI) is a severe complication of sepsis with high mortality, and the molecular mechanisms underlying its pathogenesis remain incompletely elucidated. This study aimed to investigate the regulatory role and molecular mechanism of miR-125b-5p in SALI by targeting protein tyrosine phosphatase 1B (PTP1B). The rat alveolar macrophage cell line NR8383 was stimulated with 1 μg/mL lipopolysaccharide (LPS) to establish an in vitro SALI cell model. High-throughput transcriptome sequencing was used to screen differentially expressed miRNAs associated with PTP1B, and qRT-PCR was performed for validation. The direct binding between miR-125b-5p and PTP1B was verified by a dual-luciferase reporter assay in 293 T cells. A rat model of SALI was established by cecal ligation and puncture (CLP) in 6–8 week-old male SD rats, which were randomly divided into the sham operation group (Sham), SALI model group (Model), Model + miR-125b-5p agonist negative control group (Model + NC), and Model + miR-125b-5p agonist group (Model + Agonist). The pathological changes of lung tissues were observed by HE staining. The mRNA expressions of miR-125b-5p, PTP1B, IL-1β, and IL-18 were detected by qRT-PCR. The protein expressions of Cleaved-Caspase-1, CHOP, GSDMD, Cleaved-GSDMD, and PTP1B were measured by Western blotting (WB). High-throughput sequencing and qRT-PCR validation identified five miRNAs with potential interaction with PTP1B, among which miR-125b-5p and miR-185-3p were significantly downregulated, while miR-351-5p, miR-125a-5p, and miR-345-3p were significantly upregulated in the LPS-induced NR8383 cell model (all P < 0.05). The dual-luciferase reporter assay confirmed that miR-125b-5p directly bound to the 3’UTR of PTP1B, and this binding significantly reduced the luciferase activity of the wild-type PTP1B reporter vector ( < 0.05). In the in vivo SALI model, HE staining showed severe lung tissue damage in the Model group, including thickened alveolar walls, massive inflammatory cell infiltration, and pulmonary interstitial hemorrhage, while the Model + Agonist group exhibited significantly alleviated pathological damage with no obvious lesions. qRT-PCR results showed that compared with the Sham group, the mRNA expressions of PTP1B, IL-1β, and IL-18 were significantly increased, and miR-125b-5p was significantly decreased in the Model group (all P < 0.05); these changes were significantly reversed in the Model + Agonist group compared with the Model + NC group (all P < 0.05). WB results demonstrated that the protein levels of Cleaved-Caspase-1, CHOP, GSDMD, Cleaved-GSDMD, and PTP1B were significantly elevated in the Model group relative to the Sham group (all P < 0.05), and miR-125b-5p agonist treatment markedly downregulated the expression of these proteins (all P < 0.05 vs. Model + NC group). MiR-125b-5p is a protective miRNA in SALI, and its agonist can effectively alleviate CLP-induced lung tissue damage in rats. The underlying mechanism may be that miR-125b-5p directly targets PTP1B to inhibit the activation of the endoplasmic reticulum stress-pyroptosis axis, thereby reducing inflammatory response and pyroptotic cell death in lung tissues. This study identifies a novel miR-125b-5p/PTP1B regulatory axis, providing a potential molecular target for the clinical treatment of SALI.
Hyperuricemia-associated acute kidney injury (HUA-AKI), a frequent complication of chronic hyperuricemia, is driven by oxidative stress, inflammation, and pyroptosis in renal tubular epithelial cells. Ginsenoside Rg3, a principal bioactive compound derived from Panax ginseng, possesses anti-inflammatory and metabolic regulatory activities; however, its therapeutic effects and underlying mechanisms in HUA-AKI remain elusive. In this study, an HUA-AKI mouse model was established via intraperitoneal injection of potassium oxonate combined with oral hypoxanthine, and an in vitro model using uric acid (UA)-stimulated HK-2 cells. Rg3 treatment markedly improved renal function, reduced tubular injury, and alleviated oxidative stress without altering serum UA levels. Transcriptomic profiling identified 229 differentially expressed genes (DEGs) reversed by Rg3, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed significant inhibition of the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome and pyroptosis pathways, alongside upregulation of the carnitine palmitoyltransferase 1A (CPT1A)-mediated fatty acid oxidation pathway. Western blotting, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) confirmed that Rg3 upregulated CPT1A expression and suppressed activation of NLRP3, cleaved caspase‑1, gasdermin D N‑terminal fragment (GSDMD‑NT), as well as the release of interleukin (IL)-1β and IL-18 in both mouse kidney tissues and HK-2 cells. Notably, co-administration of the CPT1A inhibitor sodium taurocholate (STC) partially abolished the renoprotective effects of Rg3, as demonstrated by restored cell viability, elevated lactate dehydrogenase (LDH) release, re‑elevation of pyroptosis-related proteins and cytokines, and reappearance of membrane pore formation and rupture under transmission electron microscopy. Collectively, our findings suggest that Rg3 may ameliorate HUA‑AKI, at least in part, through CPT1A upregulation and subsequent suppression of the NLRP3/caspase-1/GSDMD axis, positioning Rg3 as a promising therapeutic candidate for HUA-AKI.
