This study aimed to investigate the effect of peimisine on diabetic nephropathy (DN) and explore its potential mechanisms in a mouse model of high-fat diet and streptozocin-induced DN and cell model of high glucose (HG)-treated MPC5 cells. The beneficial effects of peimisine in DN mice were analyzed via determining body weight, fasting blood glucose, plasma total cholesterol, plasma triglyceride, serum creatinine, blood urea nitrogen, pathological changes, oxidative stress markers, inflammatory cytokines, and ferroptosis-related indicators. Furthermore, CCK-8 assay, flow cytometry, inflammatory cytokines, mitochondrial membrane potential (MMP) detection, DCFH-DA probe, BODIPY 581/591 C11 probe, oxidative stress markers, and ferroptosis-related indicators were conducted to measure the beneficial effects of peimisine on HG-treated MPC5 cells. qRT-PCR and western blotting were employed for determining Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway-associated genes expression. The potential mechanism was further investigated through rescue experiments using RO8191 (a JAK activator). We discovered that peimisine could ameliorate metabolic dysfunction, and the impairment of renal function and structure in DN mice. Moreover, peimisine could ameliorate inflammation and ferroptosis in DN mice. Similarly, peimisine was also demonstrated to ameliorate inflammation and ferroptosis in HG-treated MPC5 cells. Mechanistically, peimisine could inhibit the JAK/STAT pathway in vitro and in vivo. The inhibitory effects of peimisine on inflammation and ferroptosis in HG-treated MPC5 cells were reversed by RO8191. Our findings demonstrated that peimisine could ameliorate DN by attenuating inflammatory responses and ferroptosis through inhibition of the JAK/STAT signaling.

Sepsis-associated acute liver injury (SALI) is an independent risk factor that significantly worsens sepsis prognosis, underscoring the need for mechanistic insights to guide targeted therapy. Accordingly, we established a sepsis mouse model via cecum ligation and puncture (CLP) and performed in vitro experiments using murine hepatic Kupffer cells stimulated with lipopolysaccharide (LPS) and adenosine triphosphate (ATP). Here, we found that growth arrest and DNA damage 45A (GADD45A) was highly expressed in liver tissues of CLP-induced septic mice, and its knockdown ameliorated liver injury and systemic inflammation. Mechanistically, GADD45A knockdown upregulated ATP synthase F1 subunit alpha (ATP5A1) expression by disrupting the interaction between tripartite motif-containing 25 (TRIM25) and ATP5A1, thereby suppressing pyroptosis and pro-inflammatory cytokine secretion in LPS + ATP-stimulated murine Kupffer cells. Additionally, Aly/REF export factor (ALYREF), a 5-methylcytosine (m[5]C) reader, was upregulated in septic liver tissues and LPS + ATP-treated Kupffer cells. ALYREF likely stabilized GADD45A mRNA in an m[5]C-dependent manner, thereby promoting its expression. ALYREF knockdown inhibited NLRP3 inflammasome-dependent macrophage pyroptosis and alleviated SALI, likely by modulating the GADD45A/TRIM25/ATP5A1 axis. Notably, the increased expression of ALYREF in SALI appeared to be driven by activated STAT3 signaling. In conclusion, ALYREF, likely driven by activated STAT3 signaling, appears to activate the GADD45A/TRIM25/ATP5A1 axis in an m[5]C-dependent manner to promote NLRP3 inflammasome-dependent macrophage pyroptosis, thereby contributing to the development of SALI and suggesting a promising therapeutic strategy for targeted SALI intervention.

Keloids are pathological scars characterized by excessive collagen deposition and fibroblast hyperactivity, yet effective therapies remain limited due to incomplete understanding of their molecular drivers. Emerging evidence implicates N[6]-methyladenosine (m[6]A) RNA modification in fibrotic diseases, but its role in keloid pathogenesis is unclear. Here, we investigated the function and mechanism of the m[6]A methyltransferase METTL3 in keloid fibrosis. RNA sequencing of human keloid and normal skin tissues revealed significant upregulation of METTL3 and enrichment of extracellular matrix-related pathways. METTL3 was consistently overexpressed in keloid tissues and primary keloid fibroblasts (KF), which exhibited enhanced proliferation, migration, and resistance to apoptosis. Knockdown of METTL3 in KF suppressed these pro-fibrotic phenotypes and markedly reduced expression of α-SMA, TGF-β1, and collagen type I alpha 1 chain (COL1A1). Mechanistically, METTL3 deposited m[6]A modifications on the coding sequence of COL1A1 mRNA, enhancing its stability. YTHDF1 recognized these m[6]A marks and promoted COL1A1 mRNA translation. RNA pull-down and gene-specific m[6]A-PCR confirmed direct binding of YTHDF1 to m[6]A-modified COL1A1; loss of YTHDF1 reduced COL1A1 protein levels without affecting its mRNA abundance. Rescue experiments in which COL1A1 overexpression restored the fibrotic phenotypes suppressed by METTL3 knockdown established COL1A1 as a critical downstream effector of METTL3. Importantly, intradermal delivery of METTL3 shRNA in a rat tension-driven excisional scar model significantly attenuated scar formation, collagen accumulation, and fibrotic marker expression. Our findings establish METTL3 as a central epitranscriptomic regulator of keloid fibrosis by stabilizing COL1A1 mRNA and enabling YTHDF1-mediated translational enhancement, forming an m[6]A-YTHDF1-COL1A1 axis, highlighting its potential as a novel therapeutic target for pathological scarring.

Maternal malnutrition is a major risk factor for adverse health outcomes in offspring, including cardiovascular disorders. According to the Developmental Origins of Health and Disease (DOHaD) concept, adverse conditions during critical developmental windows can induce long-lasting physiological adaptations that increase disease susceptibility later in life. Among the experimental models used to investigate these mechanisms, maternal protein restriction (MPR) has been associated with metabolic, cardiovascular, and hypertensive phenotypes in offspring, some of which emerge early in life. Therefore, this study evaluated the effects of MPR on the cardiovascular morphophysiology of female offspring at postnatal day 21 (PND21). Pregnant rats were allocated to either a Control group (CTR), receiving a normoprotein diet (17% protein), or a Gestational and Lactational Low-Protein group (GLLP), receiving a low-protein diet (6% protein) throughout gestation and lactation. At PND21, female offspring underwent electrocardiographic assessment and were subsequently euthanized for morphological, molecular, and oxidative stress analyses. Female offspring from the GLLP group exhibited reduced body weight and body length, whereas absolute heart weight was preserved, resulting in increased relative heart weight and suggesting disproportionate somatic growth. Electrocardiographic analysis revealed a shortened P-R interval and decreased heart rate, indicating early alterations in cardiac electrical activity. Morphologically, cardiomyocyte architecture was preserved; however, reduced nuclear width and increased collagen and elastin deposition were observed, indicating early extracellular matrix remodeling without significant changes in reticular fibers. At the molecular level, GLLP offspring exhibited reduced PRDX3 expression, accompanied by decreased catalase (CAT) activity, reduced glutathione (GSH) levels, and lower lipid peroxidation (TBARS), suggesting alterations in redox regulation and antioxidant homeostasis during early cardiac development. Collectively, these findings demonstrate that MPR induces early structural, molecular, oxidative, and electrophysiological alterations in the hearts of female offspring. These adaptations may represent early programming events that increase susceptibility to cardiovascular dysfunction later in life, reinforcing the importance of maternal nutrition in shaping cardiovascular health across generations.

Lung cancer poses a severe global health burden with limited effective therapeutic strategies, and dysregulated aerobic glycolysis and cuproptosis are closely linked to its progression. This study aimed to explore the role and underlying mechanism of the AP2α/PDHA1 signaling axis in lung cancer. In this research, lung cancer cell lines with stably altered AP2α and PDHA1 expression were constructed via lentivirus and shRNA transfection; rescue experiments were performed using the glycolysis inhibitor 2-deoxyglucose (2-DG) and copper chelator tetrathiomolybdate (TTM), and nude mouse xenograft models were established for in vivo verification. The results showed that AP2α was highly expressed in lung cancer, while PDHA1 was lowly expressed in lung cancer. High AP2α and low PDHA1 expression predicted poorer overall survival in lung cancer patients. Moreover, AP2α- and PDHA1-correlated genes were enriched in ATP-related biological processes, which were closely associated with aerobic glycolysis. Downregulation of AP2α suppressed cell viability, migration and invasion while inducing apoptosis, reduced aerobic glycolysis, elevated intracellular copper content and regulated key cuproptosis-related markers. Moreover, AP2α directly bound to the PDHA1 promoter. Further rescue assays confirmed that PDHA1 mediated the oncogenic effects of AP2α, and 2-DG could reverse the phenotypic changes caused by PDHA1 knockdown. In vivo experiments also validated that the AP2α/PDHA1 axis facilitated tumor growth via modulating glycolytic metabolism. Collectively, the AP2α/PDHA1 pathway may accelerate lung cancer progression by promoting aerobic glycolysis and restraining cuproptosis. This finding reveals a novel molecular mechanism of lung cancer development and provides promising targets for combined antitumor therapy.

The DNA binding domains of transcription factors are structurally well-defined and their presence in novel genome sequence can be reliably detected via sequence homology. In contrast, regions of transcription factors that stimulate or repress mRNA synthesis by RNA polymerase II are more enigmatic and difficult to identify with bioinformatic tools. Experimental discovery of transactivation (or repression) domains typically makes use of a reporter gene transcription assay in one or more cell types and the progressive removal of protein sequence from the transcription factor under study. By and large, this reveals one or more minimal subdomains that are required for most of the transcription activity for the wild type protein. In applying this approach to the experimental identification of minimal transactivation domains of the chicken DLX5 and DLX6 paralogues, we find no obvious subdomain organization but, instead, show redundant transactivation activity that is distributed throughout each protein. We also reveal that DLX domains can cooperate in trans in homo- or heterotypic reactions with DLX domains that are DNA-bound via the homeodomain.

Herpes simplex virus 1 (HSV-1) is a neurotropic enveloped DNA virus that latently infects sensory ganglia cell bodies. HSV-1 causes cold sores, keratitis, meningitis, and encephalitis. During the HSV-1-replicating phase (lytic phase), viral proteins and genomes are produced in the host cell and assemble into virions. Mature virions egress from the cell, leading to cell lysis. Here, we show that HSV-1-replicating neuroblastoma cells generate cytotoxic aggregates that are detected in the extracellular medium. We initially examined the intracellular and extracellular levels of polyubiquitin conjugates in SH-SY5Y cells undergoing HSV-1 replication. The dynamics of ubiquitination were altered within cells during the lytic phase. The accumulation of poly-ubiquitin conjugates and HSV-1 membrane proteins (gB and gD) was mainly detected at the juxtanuclear cytoplasm in HSV-1-replicating cells. Moreover, poly-ubiquitin conjugates, HSP90, gB, gD, alpha-synuclein, and cytotoxic molecules were detected in the culture supernatant. Co-immunoprecipitation assays revealed that polyubiquitin conjugates in the supernatant contained aggregated gD and alpha-synuclein. Furthermore, poly-ubiquitin conjugates and alpha-synuclein in the culture supernatant contributed to its cytotoxic effects. Our results indicate that HSV-1-replicating SH-SY5Y cells produce not only virions but also cytotoxic protein aggregates containing poly-ubiquitin conjugates and alpha-synuclein. These findings may provide insight into mechanisms underlying virus-induced protein aggregation and cytotoxicity, which may be relevant to neurodegenerative processes.

Gastric cancer (GC) is a prevalent and life-threatening malignancy worldwide, with its complex pathogenesis remaining incompletely understood. Identifying novel molecular mechanisms underlying gastric cancer development is crucial for improving diagnosis and treatment strategies. In this study, we investigated the role of miR-1281 and its target gene LMX1B in gastric cancer progression. We employed a series of experimental techniques, including real-time quantitative polymerase chain reaction (RT-qPCR) to measure the expression levels of miR-1281 in gastric cancer cell lines and clinical tissue samples. Luciferase reporter assays were performed to validate the direct binding between miR - 1281 and the 3'-untranslated region (3'-UTR) of LMX1B mRNA. Functional assays such as cell proliferation, invasion and migration assays were carried out to evaluate the effects of miR-1281 and LMX1B on gastric cancer cell behaviors. The results showed that miR-1281 was significantly downregulated in gastric cancer tissues and cell lines compared with normal counterparts. Overexpression of miR-1281 remarkably inhibited gastric cancer cell proliferation, invasion and migration. Mechanistically, we demonstrated that miR-1281 directly targeted the 3'-UTR of LMX1B mRNA, leading to decreased LMX1B expression. Conversely, knockdown of LMX1B phenocopied the effects of miR-1281 overexpression, while restoration of LMX1B expression reversed the inhibitory effects of miR-1281 on gastric cancer cells. In addition, clinical correlation analysis revealed that low miR-1281 expression was associated with poor prognosis in gastric cancer patients, while high LMX1B expression showed an opposite correlation. In conclusion, our findings suggest that miR-1281 functions as a tumor suppressor in gastric cancer by downregulating LMX1B expression. The miR - 1281/LMX1B axis may serve as a potential therapeutic target and prognostic biomarker for gastric cancer, providing new insights into the molecular mechanisms of gastric cancer development and offering novel strategies for its treatment.

An important hallmark of cancer cells is altered glycosylation of cell surface molecules in comparison with normal or differentiated cells. Terminal galactose residues represent important elements of cellular glycocalyx, but their regulation and functions remain not fully elucidated. Here, we used lectin-induced aggregation assay to analyze changes in the expression of galactose residues on the surface of live human leukemia HL-60 cells in association with their neutrophilic differentiation induced by all-trans retinoic acid (ATRA). We found that live HL-60 in suspension expressed multiple types of glycans on the cell surface recognized by plant lectins with different glycan-binding specificity. Detailed analysis of cell aggregation responses induced by the galactose-binding Ricinus communis agglutinin (RCA) demonstrated that this activity was enhanced by a sialyltransferase inhibitor P-3FAX-Neu5Ac and suppressed by an inhibitor of N-glycosylation tunicamycin. Following the ATRA-induced differentiation (1 μM ATRA, 3 days), RCA-induced aggregation was elevated, implying the increase in the level of terminal galactose residues on the cell surface. Bioinformatics analysis of available GEO datasets showed that this change might be associated with the metabolic reprogramming of sialyltransferases and neuraminidases, both controlling the availability of galactose-terminated glycans in cells. Collectively, these findings demonstrate that ATRA-induced neutrophilic differentiation of promyelocytic leukemia HL-60 cells is accompanied by elevated cell surface galactosylation, suggesting new directions for future investigations into the cell biological mechanisms of cancer differentiation therapy.

Diabetic nephropathy (DN) is one of the most common microvascular complications of diabetes mellitus, without suitable therapies, ultimately leading to end-stage renal disease. Podocyte loss serves as an initial insult during DN progression, yet the endogenous protective mechanisms remain elusive. Our single-nucleus RNA sequencing data revealed a large group of differentially expressed lncRNAs in renal tissues of DN. In this study, we found that Gm13561 was a podocyte-specific lncRNA predominantly localized in the nucleus and significantly downregulated in both DN mouse renal tissues and high-glucose-induced mouse podocytes. Gain- and loss-of-function studies demonstrated that overexpression of Gm13561 attenuated high-glucose-induced podocyte apoptosis, ROS accumulation, and oxeiptosis, while knockdown of Gm13561 aggravated these phenotypes under normal glucose conditions. Mechanistically, RNA pull-down coupled with mass spectrometry was performed and identified that Gm13561 interacts with the splicing factor Hnrnpa1. RNA immunoprecipitation assay further validated their binding association. Intriguingly, Hnrnpa1 knockdown abolished the protective effects of Gm13561 against apoptosis and oxeiptosis, and vice versa. Furthermore, RNA sequencing revealed that Gm13561 and Hnrnpa1 co-regulated alternative splicing of 446 overlapping genes, with 25 genes enriched in ROS-related and apoptotic pathways. These results together suggested a novel regulatory axis between Gm13561 and Hnrnpa1 that safeguards podocytes from apoptosis and oxeiptosis via modulating alternative splicing, and may serve as a potential therapeutic target for DN.