Cerebral ischemia/reperfusion (I/R) injury is a major cause of neurological disability. This study investigated the role of lncRNA PVT1 in I/R-induced cerebral injury and its clinical relevance. MCAO/reperfusion mouse and OGD/R neuronal cell models were established. Neurological function, infarct volume, brain edema, and neuronal apoptosis were assessed. For clinical validation, 50 acute ischemic stroke patients and 50 healthy controls were enrolled. Serum PVT1/miR-187-3p were measured by qRT-PCR; plasma MyD88 and inflammatory cytokines (TNF-α, IL-6, IL-18) by ELISA. Machine learning (Random Forest) and correlation analyses were performed to evaluate diagnostic utility and relationships with clinical severity (NIHSS score, infarct volume). PVT1 was significantly upregulated under I/R both in vivo and in vitro. Clinically, AIS patients showed markedly elevated PVT1 (3.92 vs. 1.02, p < 0.0001) and reduced miR-187-3p (0.42 vs. 1.01, p < 0.0001), with increased MyD88 and inflammatory cytokines (all p < 0.001). The Random Forest model achieved AUC = 0.92, with PVT1 as the top predictor. PVT1 correlated positively with MyD88 (r = 0.68, p < 0.001) and NIHSS score (r = 0.61, p < 0.001). PVT1 silencing in MCAO mice significantly improved neurological function, reduced infarct volume and brain edema, and attenuated neuronal apoptosis (all p < 0.01). Mechanistically, PVT1 sponged miR-187-3p to derepress MyD88/TLR4 signaling, which was reversed by miR-187-3p restoration. In vivo rescue experiments confirmed that miR-187-3p antagomir reversed the protective effects of PVT1 silencing across multiple endpoints. PVT1 inhibition protects against cerebral I/R injury by restoring miR-187-3p and blocking the MyD88/TLR4 pathway. PVT1 and its downstream axis are associated with stroke diagnosis and severity, and represent promising therapeutic targets and diagnostic biomarkers for ischemic stroke.
Tendon stem/progenitor cells (TSPCs) play essential roles in tendon homeostasis and repair, but their regenerative capacity is compromised by oxidative stress-induced senescence and the associated loss of stemness. Here, we identified PEP472, a previously uncharacterized micropeptide encoded by a small open reading frame (smORF) within the long non-coding RNA (lncRNA) MSTRG.44472.1, as a regulator of TSPC function under oxidative stress. The expression of MSTRG.44472.1 decreased in an H₂O₂-induced TSPC senescence model, and the coding potential of its smORF and translation into PEP472 were subsequently validated. Functional analyses demonstrated that PEP472 attenuated H₂O₂-induced senescence, preserved TSPC stemness, and promoted tenogenic differentiation. Mechanistically, PEP472 increased SIRT1 protein abundance by suppressing proteasome-dependent SIRT1 degradation and enhancing SIRT1 stability. Consistent with a role for SIRT1 in mediating the protective effects of PEP472, pharmacological inhibition of SIRT1 with EX-527 markedly attenuated the anti-senescence effects of PEP472. Together, these findings identify a previously unrecognized MSTRG.44472.1-PEP472-SIRT1 regulatory axis in TSPCs and demonstrate that PEP472 attenuates oxidative stress-induced senescence and preserves TSPC stemness through SIRT1 stabilization. This study highlights PEP472 as a promising micropeptide-based biologic candidate for preventing senescence-associated TSPC dysfunction and supporting tendon regenerative repair.
Objective. This study aimed to investigate the protective effect of Calycosin, a major bioactive flavonoid derived from Astragalus membranaceus, on TGF-β1-induced pulmonary fibrosis in human embryonic lung fibroblast (MRC-5) cells and to elucidate the underlying mechanisms. Methods. Cell viability was assessed using the MTT assay to determine non-cytotoxic concentrations of CA for subsequent experiments. TGF-β1-stimulated MRC-5 cells were treated with varying doses of CA. The anti-fibrotic effects were evaluated by measuring the expression of fibrosis markers (α-SMA and Collagen I) via Western blotting and immunofluorescence. Intracellular reactive oxygen species (ROS) levels and the activities of antioxidant enzymes were measured to assess oxidative stress. Cell migration was monitored using a scratch wound assay. The involvement of the TLR4/MyD88/NF-κB signaling pathway and the expression of downstream inflammatory cytokines (IL-17A, IL-6, TNF-α) were analyzed by Western blot and quantitative real-time PCR (qRT-PCR). The TGF-β receptor inhibitor SB-431542 was used as a comparative control. Results. Calycosin treatment significantly attenuated TGF-β1-induced activation of MRC-5 cells, as evidenced by the marked downregulation of α-SMA and Collagen I expression. CA also ameliorated oxidative stress by reducing ROS generation and restoring glutathione (GSH) levels and total superoxide dismutase (T-SOD) activity. Furthermore, CA inhibited the enhanced migratory capacity of fibroblasts induced by TGF-β1. Mechanistically, CA downregulated the protein expression of key components (TLR4, MyD88, phospho-NF-κB p65, phospho-IκBα) in the TLR4/MyD88/NF-κB pathway and reduced the mRNA and protein levels of the pro-fibrotic inflammatory cytokines IL-17A, IL-6, and TNF-α. The efficacy of CA was comparable to that of SB-431542. Conclusion. Calycosin effectively attenuates TGF-β1-induced fibroblast activation, oxidative stress, and ECM remodeling in pulmonary fibroblasts. Its anti-fibrotic effects are primarily mediated through the suppression of the TLR4/MyD88/NF-κB signaling pathway and the reduction of the pro-inflammatory microenvironment. These findings suggest that Calycosin holds promising potential as a therapeutic agent for silicosis and other pulmonary fibrotic diseases.
Functional reconstruction of calvarial defect is a considerable clinical challenge. Many studies have emphasized the osteogenic abilities of stem cells for tissue regeneration. Ttricalcium phosphate (TCP) has been employed successfully as a synthetic graft material in bone defect repair. However, the lack of osteogenic and osteoinductive properties of this substitute invariably results in bone regeneration of low quality and quantity. The purpose of this study was to determine whether loading NR4A1 gene-modified bone marrow mesenchymal stem cells (BMSCs) onto TCP promoted bone regeneration and osteointegration of calvarial defect in mice. Alizarin red staining and qPCR were performed to investigate the influences of NR4A1 on osteogenic differentiation of BMSCs and MC3T3-E1 cells. The effects of NR4A1 overexpressed BMSCs/TCP scaffold on osteogenic differentiation of BMSCs were assessed using Alizarin red staining, ALP staining and qPCR. To further evaluate the roles of NR4A1 overexpressed BMSCs/TCP scaffold in osteogenesis in vivo, we conducted animal experiments in mice. New bone formation was assessed using micro-CT and histological methods. In the present study, we found that NR4A1 promotes osteogenic differentiation of BMSCs and MC3T3-E1 cells in vitro. Besides, NR4A1 overexpressed BMSCs/TCP scaffold showed stronger osteogenic differentiation ability than TCP scaffold in vitro. Finally, In vivo studies have shown that micro-CT and histology revealed significantly greater volume of new bone formation in the NR4A1 overexpressed BMSCs/TCP scaffold group than in the model group. HE staining of lung, liver, kindey and heart tissues showed that the TCP scaffold was not toxic to mice. These results demonstrated that combinations of TCP scaffolds and NR4A1 gene-modified BMSCs could be used to construct tissue-engineered bone to enhance mineralization of the regenerated bone and osseointegration in mice.
Chronic sleep deprivation (CSD) is known to impair cognitive function, but the molecular mechanisms involved remain poorly understood. In this study, we explored the role and regulatory mechanism of DNM1 in CSD-induced cognitive deficits. Using CSD mouse models, we performed behavioral tests and examined hippocampal DNM1 expression, GABAergic function, and lactylation levels. In parallel, HT-22 cells were exposed to glutamate to mimic GABAergic dysfunction, and treated with sodium lactate together with 2-deoxyglucose (2-DG) to assess the impact of lactylation on DNM1 expression. Our results showed that DNM1 was downregulated in the hippocampus of CSD mice, concurrent with elevated lactate levels and disrupted GABA signalling. Overexpression of DNM1 in HT-22 cells effectively suppressed glutamate-induced neuronal GABAergic dysfunction and apoptosis. Moreover, sodium lactate increased DNM1 lactylation and reduced its expression, whereas the glycolysis inhibitor 2-DG decreased DNM1 lactylation and upregulated its expression. In CSD mice, 2-DG administration similarly lowered DNM1 lactylation and restored DNM1 levels in the hippocampus. Notably, DNM1 overexpression and 2-DG treatment acted synergistically to ameliorate both GABAergic dysfunction and cognitive impairment in the CSD model. Collectively, our findings suggest that CSD elevates hippocampal lactate, which may promote lactylation-mediated downregulation of DNM1, thereby contributing to GABAergic dysfunction and cognitive decline. These observations provide new insights into the interplay between sleep, cognition, and the GABAergic system, and highlight DNM1 as a potential candidate gene worthy of further investigation.
