免疫染色通透液(Triton X-100)

Bioengineered tracheas have shown considerable potential in tracheal injury repair; however, their practical value is limited by challenges in tracheal cartilage regeneration, and postoperative tracheal stenosis remains a common issue. Here, inspired by the 3-layer structure of the trachea and the multi-segmental characteristics of its cartilage, a multilayered bioengineered tracheal scaffold (named Sd@d-ECM/BMSCs/SilMA) with a microgroove structure is designed in this study to repair tracheal defects. In this design, the microgrooved surface of the methacrylated silk fibroin (SilMA) hydrogel provides spatial guidance for the directional growth of bone marrow mesenchymal stem cells (BMSCs) and enhances their adhesion and proliferation. The extracellular matrix of the decellularized cartilage scaffold offers the necessary microenvironment and mechanical support for BMSCs to differentiate into cartilage. Under the influence of the dual-layer structure (inner and outer), the middle-layer BMSCs can undergo stable chondrogenic differentiation without any inducing agents. Sd@d-ECM/BMSCs/SilMA effectively promotes tracheal cartilage formation in a rabbit defect model, reduces the incidence of tracheal stenosis, and substantially improves respiratory function. Sd@d-ECM/BMSCs/SilMA not only confirms the successful construction of microgroove structures on the surface of the SilMA hydrogel and the effective loading of BMSCs but also demonstrates significant experimental value in tracheal cartilage repair and regenerative medicine.

Background. Asthma treatment is difficult due to disease heterogeneity and comorbidities. In addition, the development of drugs targeting the underlying mechanisms of asthma remains slow. We planned to identify the most upregulated differentially expressed long noncoding RNA in asthma to explore its regulatory patterns and pathways in asthma. Methods. We sensitized mice using a mixture of ovalbumin, house dust mites, and lipopolysaccharide to establish an asthma mouse model. We also sensitized asthma cells with TGF-β1 in an in vitro model. We performed a microarray analysis to identify the lncRNA with the differential expression level in model mice. We applied hematoxylin and eosin and Masson’s trichrome stainings to mouse tissues to quantify the tissue damage extent. Next, we assess the levels of lncRNA CRNDE, miR-29a-3p, TGF-β1, MCL-1, E-cadherin, vimentin, and snail. We counted the percentages of Th17 cells using flow cytometry. Finally, we performed a dual-luciferase reporter assay to assess the association between lncRNA CRNDE and miR-29a-3p. Results. We successfully established asthma mouse/cell models and selected the lncRNA CRNDE for our study. Transfection of si-CRNDE reduced the degree of injury and inflammation in the mouse model and reversed the TGF-β1-induced epithelial-mesenchymal transition (EMT) in the cell model. Moreover, the E-cadherin level was upregulated, and the levels of IL-17A, vimentin, snail, and α-SMA were downregulated. We also discovered that lncRNA CRNDE negatively regulated miR-29a-3p and that this one in turn inhibited MCL-1 in mice. After lncRNA CRNDE expression downregulation, the level of miR-29a-3p was increased, and we detected reduced levels of MCL-1 and EMTs. Conclusions. lncRNA CRNDE expression downregulation led to reduced inflammation and reduced lung damage in mice with induced asthma, it inhibited the EMTs of lung epithelial cells via the miR-29a-3p/MCL-1 pathway, and it reduced the levels of Th17/IL-17A cells to reduce asthma signs.

Background and aims Increasing evidence indicates that modulating pyroptosis in endothelial cells (ECs) can alleviate atherosclerosis (AS) progression; however, despite reports that nucleolin (NCL) regulates vascular smooth muscle cell proliferation in AS, the potential mechanism by which cell surface NCL mediates pyroptosis in ECs during AS remains poorly understood. Methods AS was induced in ApoE -/- mice by feeding a high-fat diet, after which aortic lesions were evaluated. Pyroptosis, inflammatory status, and NCL expression in ECs of the aortic root were then assessed. The effects of NLRP3 inflammasome inhibition and NCL modulation on atherosclerotic lesion severity in AS mice, as well as on pyroptosis in ox-LDL-stimulated ECs, were systematically investigated. In addition, the mechanistic role of NCL in AS was further explored using approaches including immunoprecipitation-mass spectrometry (IP-MS). Results AS model mice developed severe aortic lesions accompanied by pronounced EC pyroptosis and inflammation, together with elevated NCL expression in ECs of the aortic root. Both inhibition of NLRP3 and NCL knockdown alleviated atherosclerotic lesion severity in ApoE -/- mice and attenuated ox-LDL-induced EC pyroptosis. Mechanistically, cell-surface NCL interacted with RASSF2 via its RNA-binding domain, and suppression of NCL decreased nuclear RASSF2 expression. NCL facilitated the translocation of RASSF2 into the nucleus, thereby exacerbating EC pyroptosis and amplifying inflammatory responses. Conclusions This study demonstrates that, in AS, NCL exacerbates EC pyroptosis and promotes disease progression by facilitating nuclear transport of RASSF2. This study defines the mechanistic roles of NCL in AS, thereby identifying a new molecular pathway and suggesting potential therapeutic targets.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease that lacks effective treatment modalities. Once patients are diagnosed with IPF, their median survival is approximately 3–5 years. PPARγ is an important target for the prevention and treatment of pulmonary fibrosis. Asarinin is a lignan compound that can be extracted from food plant Asarum heterotropoides . In this study, we investigated the therapeutic effects of asarinin in a pulmonary fibrosis model constructed using bleomycin in mice and explored the underlying mechanisms. Intraperitoneal administration of asarinin to mice with pulmonary fibrosis showed that asarinin effectively attenuated pulmonary fibrosis, and this effect was significantly inhibited by the PPARγ inhibitor GW9662. Asarinin inhibited TGF-β1-induced fibroblast-to-myofibroblast transition in vitro , while GW9662 and PPARγ gene silencing significantly inhibited this effect. In addition, asarinin inhibited not only the canonical Smad pathway of TGF-β but also the non-canonical AKT and MAPK pathways by activating PPARγ. Our study demonstrates that asarinin can be used as a therapeutic agent for pulmonary fibrosis, and that PPARγ is its key target.

Background Alzheimer’s disease (AD) is characterized by cognitive decline and neuronal loss, with cellular senescence emerging as a key driver. The insulin-like growth factor-1 receptor (IGF1R) pathway is implicated in aging and AD pathology. IGF2 mRNA binding protein 2 (IGF2BP2) can stabilize IGF1R mRNA, but its role in AD-associated neuronal senescence remains unclear. Methods We established AD mouse models and H 2 O 2 -induced senescent neuronal cell models to explore the impact of IGF2BP2 in neuronal senescence and cognitive deficits. RNA pull-down assays, methylated RNA immunoprecipitation (MeRIP)-qPCR, and behavioral tests were used to elucidate the molecular mechanisms and therapeutic potential of targeting IGF2BP2. Results IGF2BP2 was significantly up-regulated in the hippocampal neurons of AD mice. This upregulation correlated with increased β-amyloid (Aβ) deposition, neuronal damage, and cognitive impairments. In vitro , IGF2BP2 knockdown in H 2 O 2 -induced senescent neurons reduced IGF1R expression and alleviated neuronal senescence, as evidenced by decreased senescence-associated secretory phenotype factors and improved cell viability. Mechanistically, IGF2BP2 stabilized IGF1R mRNA through m6A modification, enhancing its expression. Knockdown of IGF2BP2 decreased IGF1R mRNA stability and expression, thereby mitigating neuronal senescence. In AD mice, IGF2BP2 knockdown improved cognitive function, reduced Aβ deposition, and delayed neuronal senescence. Conclusion IGF2BP2 contributes to neuronal senescence and cognitive deficits in AD by regulating IGF1R expression through m6A modification.

Oral submucous fibrosis (OSF) is a precancerous condition primarily caused by arecoline in betel nuts. The transforming growth factor (TGF)-β/mothers against decapentaplegic homolog 3 (Smad3) signaling pathway plays a pivotal role in its pathogenesis. This study explores the interaction between tropomyosin-1 (TPM1) and STIP1 homology and U-Box-containing protein 1 (STUB1) in regulating this pathway and its impact on OSF progression. We found that arecoline dose and time-dependently upregulates the expression of TPM1 mRNA and protein in human oral fibroblasts (HOrF). Treating HOrF with 20 μg/mL arecoline for 48 h could effectively drive fibroblast proliferation, fibrosis, migration and invasion. TPM1 knockdown reversed these effects. Mechanistically, arecoline upregulates TPM1 by activating the TGF-β/Smad3 signaling pathway. Notably, TPM1 overexpression rescued TGF-β/Smad3 activity even in the presence of the TGF-β inhibitor SB431542, revealing a positive feedback loop. Additionally, Western blotting and co-immunoprecipitation (Co-IP) analyses showed that arecoline downregulates STUB1, thereby inhibiting the ubiquitination of TGF-β/Smad3 in HOrF. Meanwhile, TPM1 competitively binds to STUB1, blocking its interaction with TGF-β/Smad3 and thereby stabilizing the pathway, which exacerbates fibrosis. Statistical analysis (one-way ANOVA with Tukey's HSD post-hoc test or independent-samples t-test) confirmed the significance of all major findings ( p  < 0.05). In conclusion, activation of the TGF-β/Smad3 pathway upregulates TPM1, which in turn blocks STUB1-mediated ubiquitination of the same cascade, establishing a positive-feedback loop that exacerbates arecoline-induced OSF. These findings improve our understanding of OSF's molecular pathogenesis and offer a potential target for its prevention and treatment.

Immunoglobulin A (IgA) nephropathy (IgAN) is characterized by the deposition of IgA1 in the glomerular mesangium, which induces secondary glomerular and tubulointerstitial inflammation and subsequently leads to podocyte apoptosis and fibrosis. This condition often progresses to end-stage renal disease and lacks effective targeted treatment. Our study aimed to explore the role of M2 macrophage-mediated Ubiquitin C-terminal hydrolase L1 (UCHL1) expression in podocytes and its potential impact on the progression of IgAN. This study established an IgAN cellular model by exposing podocytes to aggregated IgA1 (aIgA1)-treated glomerular mesangial cells supernatants and assessed the impact of M2 macrophage polarization on UCHL1 expression and podocyte apoptosis. Additionally, we utilized siRNA technology and overexpression constructs to investigate the direct effects of UCHL1 modulation on podocyte apoptosis. The supernatant from aIgA1-treated glomerular mesangial cells significantly induced apoptosis in podocytes. Based on this, M2 macrophage polarization was induced using interleukin (IL)-4. The results showed that M2 macrophages (CD163 + ) effectively alleviated podocyte apoptosis by reducing the secretion of inflammatory cytokines IL-6, tumor necrosis factor (TNF)-α, and IL-1β, as well as downregulating the expression of apoptosis-related proteins. Notably, M2 macrophages (CD163 + ) inhibited the expression of UCHL1 in podocytes. Blockade of UCHL1 promoted podocyte proliferation, reduced apoptosis, and downregulated the protein expression of the fibrotic markers vascular endothelial growth factor and collagen type IV. Overexpression of UCHL1 reversed the protective effects of M2 macrophages on podocyte apoptosis. M2 macrophage (CD163 + )-mediated UCHL1 downregulation in podocytes presents a potential therapeutic approach for IgAN by alleviating apoptosis.

Cisplatin-based chemotherapy is the first-line treatment for lung cancer. However, cisplatin resistance (CR) remains a major challenge, leading to treatment failure. A key driver of CR is enhanced DNA damage repair. Although males absent on the first (MOF) participate in DNA repair, their specific role in mediating CR remains unclear. In this study, CR models were established in PC9 and A549 lung cancer cell lines. Our results showed that high expression of Williams syndrome transcription factor (WSTF) in lung cancer cells was associated with CR. WSTF knockdown inhibited proliferation and promoted apoptosis, DNA damage, and γ-H2AX levels in CR cells. Moreover, MOF was highly expressed in lung cancer cells and regulated by WSTF acetylation. Furthermore, MOF knockdown downregulated H4K16ac levels in CR cells. MOF overexpression significantly upregulated H4K16ac levels, enhanced proliferation, and suppressed apoptosis in CS cells, concomitant with DNA damage repair and reduced γ-H2AX expression. Notably, transfection with the K46R attenuated these MOF-mediated effects in CS cells. Collectively, our study demonstrates that MOF promotes DNA damage repair and enhances CR in lung cancer cells via H4K16ac-mediated WSTF acetylation. These findings provide valuable insights for overcoming chemoresistance and improving patient outcomes.