Ischemic stroke induces severe neuronal injury after ischemia/reperfusion (I/R), with oxidative disturbance and ferroptotic death emerging as critical contributors. Although GAS6-AS1 has been associated with ischemic stroke progression, the molecular mechanisms underlying its function in neuronal injury remain to be elucidated. An in vitro ischemic model was established by exposing SH-SY5Y neuronal cells to oxygen–glucose deprivation/reperfusion (OGD/R) conditions, followed by genetic modulation of GAS6-AS1 to assess its functional role. Neuronal damage, iron accumulation, lipid oxidation, glutathione homeostasis, and ferroptosis-associated proteins were examined by biochemical assays and Western blotting. Molecular interactions among GAS6-AS1, miR-370-3p, and TFRC were validated by luciferase reporter, RNA immunoprecipitation, and qRT-PCR assays. In vivo effects were evaluated in MCAO rats receiving AAV-sh-GAS6-AS1. The clinical association of these molecules was further examined in plasma samples from 35 patients with ischemic stroke and matched controls. GAS6-AS1 levels showed consistent upregulation across OGD/R-treated neuronal cells, MCAO brain tissues, and plasma samples from patients with ischemic stroke, accompanied by reduced miR-370-3p and increased TFRC levels. GAS6-AS1 overexpression aggravated ferroptotic alterations and disrupted Nrf2/ARE-related antioxidant responses. Conversely, miR-370-3p restoration, TFRC suppression, or Fer-1 treatment attenuated ferroptosis-associated changes and partially recovered antioxidant capacity. GAS6-AS1 silencing alleviated neurological deficits, reduced infarct injury, and improved ferroptosis-related abnormalities in MCAO rats. In patients, GAS6-AS1 and TFRC levels positively correlated, whereas miR-370-3p levels inversely correlated with NIHSS scores. Our data indicate that GAS6-AS1 facilitates ischemic neuronal injury through modulation of the miR-370-3p/TFRC pathway and ferroptosis-associated responses, together with changes in Nrf2/ARE antioxidant activity. These results support the involvement of GAS6-AS1 in ischemic stroke pathology and suggest its potential value for future therapeutic exploration.
Long noncoding RNAs (lncRNAs) represent a significant avenue for investigating tumor molecular mechanisms and identifying potential therapeutic targets. It was aimed to explore the expression and prognostic significance of lncRNA TRHDE-AS1 in glioma, and clarify its ceRNA-mediated regulation of miR-22-5p/DLG3 axis in promoting glioma progression in vitro. Using the TCGA database and clinical glioma tissue samples (n = 150), qRT-PCR was employed to detect the expression of TRHDE-AS1. Its prognostic value was assessed through Kaplan–Meier analysis and Cox regression analysis. The targeting interaction between TRHDE-AS1, miR-22-5p and DLG3 was confirmed by luciferase reporter assay and RNA Immunoprecipitation (RIP) experiments. Using CCK-8 and Transwell assays, the impact of TRHDE-AS1 on glioma cells’ malignant phenotypes was evaluated. To assess the influence on epithelial-mesenchymal transition (EMT), the protein levels of N-cadherin, Snail, and MMP-2 were measured via Western blot. Low expression of TRHDE-AS1 in glioma tissues correlated with patients’ malignant features, consistent with findings from the TCGA database. It was independently related to reduced overall survival. Upon TRHDE-AS1 upregulation, miR-22-5p levels significantly decreased while downstream target DLG3 expression increased in both U251 and LN229 cells. The proliferation, invasive metastatic ability and EMT of both cells were significantly reduced, which was offset by miR-22-5p overexpression. TRHDE-AS1/miR-22-5p mediated cellular phenotype alterations were restored by DLG3 again in both U251 and LN229 cells. Downregulation of TRHDE-AS1 was closely related to glioma patients’ poor prognosis. It may regulate miR-22-5p/DLG3 axis through the ceRNA mechanism to promote the malignant activity of glioma cells.
Pregnant women increasingly use cannabidiol (CBD) isolates to alleviate symptoms such as depression, anxiety, vomiting, or nausea. However, the effects of gestational CBD exposure on postnatal reproductive capacity remain poorly characterized. Since CBD interacts with the endocannabinoid system, which is critically involved in regulating male reproduction, we investigated the impact of prenatal CBD exposure on postnatal spermatogonial differentiation and adult reproductive parameters. Pregnant female mice were administered a daily oral dose of either vehicle (DMSO) or CBD (5 or 20 mg/kg/day) from embryonic day (E) 6.5 to E15.5. Male offspring gonads were analyzed at 5 days postpartum (DPP), when spermatogonia are in an undifferentiated state, and in adulthood, using morphological, cellular, and transcriptomic approaches. Prenatal CBD exposure did not affect litter parameters or overall testicular morphology in 5 DPP males but significantly altered spermatogonial homeostasis. CBD promoted the commitment of GFRA1-positive undifferentiated spermatogonia toward a differentiated state characterized by the expression of SOX3 and RARG. These effects were associated with alterations in CB1 expression and changes in histone marks, including an increase in H3K4me3 and a decrease in H3K27me3 in the prepubertal testis. Long-term analyses revealed that gestational CBD exposure impaired sperm production and modified the adult testicular transcriptome, notably through the downregulation of kallikrein genes encoding peptidases involved in semen quality. This study demonstrates that prenatal exposure to CBD can disrupt spermatogonia differentiation after birth and negatively affect spermatogenesis in adulthood. 1. Prenatal CBD exposure altered neonatal spermatogonial homeostasis, reducing stem-like (GFRA1 +) cells and increasing differentiation-committed (SOX3/RARG +) cells. 2. CBD exposure was associated with reduced CB1 protein and altered H3K4me3/H3K37me3 levels in testis. 3. Ex vivo PND5 testis cultures showed a shift toward differentiation commitment, supporting in vivo findings. Adult offspring displayed reduced sperm count, meiotic alterations, and testicular transcriptomic dysregulation.
Perioperative neurocognitive disorders (PND) is a common surgical complication. Abnormal cholesterol metabolism may link to neuronal apoptosis and cognitive decline in PND. This study explored whether esketamine (ESK) improves PND by promoting cholesterol synthesis and its molecular mechanism. Mouse PND models and in vitro neuronal PND models were constructed. Open field test (OFT), fear conditioning test (FCT), and odor test were used to evaluate cognitive function in mice. HE, Nissl, and TUNEL staining were performed to detect neuronal damage. Molecular levels were assessed by RT-qPCR and western blot. Neuronal biological activities were detected using CCK-8, EdU, and flow cytometry. TC, FC, and CE level were examined using commercial kits. The m6A level of 24-dehydrocholesterol reductase (DHCR24) was detected by MeRIP. The binding of methyltransferase 3 (METTL3) to DHCR24 mRNA was verified by RIP and luciferase activity assays. ESK significantly improved cognitive function, reduced neuronal apoptosis, and promoted hippocampal cholesterol synthesis in PND mice by upregulating DHCR24. In vitro experiments showed that ESK enhanced cholesterol synthesis and inhibited neuronal apoptosis by upregulating the expression of METTL3 and DHCR24. Mechanistically, ESK stabilized DHCR24 mRNA through METTL3-mediated m6A methylation modification, thereby promoting DHCR24 expression. ESK improved PND by promoting cholesterol synthesis and inhibiting neuronal apoptosis through the METTL3/DHCR24 axis. This study provides a new potential therapeutic target for PND.
L-menthol (LM), as the primary active component of peppermint, has been demonstrated to exhibit pharmacological effects like anti-inflammatory and antioxidant properties. However, its neuroprotective effects following intracerebral hemorrhage (ICH) and the underlying mechanisms remain unclear. In vivo experiments employed collagenase injection to establish an ICH mouse model, with mice divided into sham, ICH, and LM (5, 10, 20 mg/kg) treatment groups. Following neurological and behavioral assessments, pathological staining was performed to examine brain tissue lesions in the mice. Inflammatory cytokines, microglial polarization, the cGAS-STING signaling, and pyroptosis-related protein levels were assessed using ELISA, immunofluorescence, and Western blot. Pathway specificity was validated via intracerebroventricular injection of cGAMP (a STING agonist). In vitro experiments established a damage model by stimulating BV2 microglia with hemin, establishing treatment groups with different concentrations of LM (0.5, 1, 2 μM) and a cGAMP intervention group to verify pathway specificity. Cell viability, apoptosis, pyroptosis, and related pathway protein expression were assessed through Cell Counting Kit-8, flow cytometry, lactate dehydrogenase (LDH) assays, and Western blot. LM improved neurological deficits in ICH mice, reduced brain water content and pathological brain tissue damage, and diminished neuronal degeneration and apoptosis. Simultaneously, LM inhibited microglia activation, neutrophil infiltration, and the release of pro-inflammatory mediators, while downregulating the cGAS-STING pathway and NLRP3 inflammasome protein expression. cGAMP intervention reduced the aforementioned neuroprotective effects of LM. In vitro experiments demonstrated that LM at 0.5–2 μM did not affect BV2 cell viability. Furthermore, LM enhanced cell viability following hemin treatment while reducing both apoptosis and pyroptosis rates. Additionally, LM reduced inflammatory cytokine levels, and suppressed the cGAS-STING pathway, but cGAMP intervention attenuated these effects of LM. LM mitigates brain injury in ICH mice by suppressing the cGAS-STING signaling in microglia, thereby inhibiting neuroinflammation and pyroptosis.
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality worldwide, yet the contribution of histone lactylation to its progression remains unclear. Histone H3K9 lactylation (H3K9la) has recently emerged as a metabolic–epigenetic mark linking glycolytic reprogramming to transcriptional regulation. We integrated histone modification profiling, transcriptome assays, RNA–protein interaction analyses, and in vitro and in vivo functional experiments to investigate the role of H3K9la in HCC. Molecular and cellular assays included qPCR, Western blotting, ChIP-qPCR, RIP, RNA stability analysis, and functional rescue. Xenograft mouse models were used to validate mechanistic findings. H3K9la levels were elevated in HCC tissues and cell lines and positively correlated with expression of the RNA-binding protein LUC7L2. Inhibition of glycolysis or lactylation suppressed LUC7L2 expression and reduced tumor cell proliferation, migration, and invasion, while promoting apoptosis. Mechanistically, H3K9la activated LUC7L2 transcription, which in turn bound to and destabilized SPINK13 pre-mRNA, repressing its tumor-suppressive function. Overexpression of SPINK13 inhibited HCC cell growth by inducing G1-phase arrest and reducing AKT phosphorylation, whereas its knockdown rescued the inhibitory effects of LUC7L2 silencing. In vivo, blockade of lactylation suppressed tumor growth and downregulated LUC7L2, an effect reversed by SPINK13 silencing or LUC7L2 overexpression. Our findings establish a novel H3K9la–LUC7L2–SPINK13 regulatory axis that links lactate metabolism to post-transcriptional gene silencing and malignant progression in HCC. Targeting this pathway provides a promising epigenetic-metabolic therapeutic strategy.
Immune checkpoint blockade (ICB) has brought transformative advances to lung cancer management; nevertheless, immunosuppressive tumor microenvironment (TME)-mediated therapeutic resistance constitutes a prominent unsolved bottleneck, mainly originating from the sustained buildup of tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs). Among its key regulators, TAMs and MDSCs play pivotal roles in dampening antitumor immunity through activation of the STAT3/NF-κB signaling axis that structurally locks myeloid cells into an immunosuppressive and therapy-resistant state. This axis integrates cytokine, metabolic, and stress cues to promote chronic inflammation, immunosuppressive gene expression, and immune checkpoint upregulation. Crosstalk between STAT3 and NF-κB amplifies IL-6, IL-10, and TNF-α loops, sustaining TAM and MDSC recruitment, polarization, and suppressive activity. Furthermore, this pathway drives angiogenesis and immune exclusion, contributing to poor immunotherapy responses. By shifting the focus from isolated molecular targets to this integrated inflammatory-metabolic network, we outline emerging therapeutic strategies, such as small-molecule inhibitors and combination regimens with immune checkpoint inhibitors, that hold the potential to dismantle this central inflammatory hub of lung cancer and restore durable antitumor immunity. A deeper understanding of STAT3/NF-κB-driven myeloid reprogramming may open new avenues for restoring effective antitumor immunity in lung cancer in this mechanistic review.
APY0201 is a phosphatidylinositol kinase containing an FYVE finger structure (PIKfyve) inhibitor that has been reported to inhibit the malignant progression of several cancers. N6-methyladenosine (m6A) methylation is the most abundant RNA modification in cancer development, including esophageal cancer (EC). This study aimed to investigate whether APY0201 played an anti-tumor role in EC, and whether its function was associated with m6A methylation. Cell viability, apoptosis, invasion, and stemness were determined using MTT assay, flow cytometry, transwell assay, and sphere formation assay for cell stemness. Ferroptosis was assessed by detecting the associated indicators. The mRNA and protein expression were examined using real-time quantitative PCR and Western blot. Methylated RNA immunoprecipitation (MeRIP) assay was performed to affirm m6A modification of ALOX5 by METTL16. RIP assay and dual-luciferase reporter assay were used to indicate the interaction between METTL16 and ALOX5. Xenograft tumor assay was conducted to explore APY0201 function in vivo. In EC cells, APY0201 suppressed cell viability, invasion, and stemness, and promoted ferroptosis by regulating arachidonate 5-lipoxygenase (ALOX5). Methyltransferase-like 16 (METTL16) catalyzed m6A methylation to affect ALOX5 mRNA stability. Functionally, METTL16 facilitated EC cell development and inhibited ferroptosis via downregulating ALOX5. Additionally, METTL16 overexpression could partly attenuate the repression role of APY0201 treatment on ALOX5 expression in EC cells. In vivo, APY0201 also reduced tumor growth by targeting the METTL16/ALOX5 axis. These functional studies demonstrate that APY0201 impeded cell malignant behaviors and induced ferroptosis in EC through METTL16-mediated ALOX5 m6A methylation.
Rapid and early radiation biodosimetry is essential for post-exposure medical triage and timely intervention. Owing to their high radiosensitivity, lipids are promising biodosimetry biomarkers. However, radiation-induced structural alterations, particularly changes in carbon–carbon double bond (C=C) location isomers, remain poorly characterized. Here, the Paternò-Büchi reaction coupled with liquid chromatography-tandem mass spectrometry (LC-PB-MS/MS) was employed to quantify unsaturated C=C location isomers with minimal interindividual variation. These isomers served as the basis for developing biodosimetry. Plasma structural lipidomic profiling was performed in C57BL/6 mice at 4 h and 24 h following irradiation at doses of 0, 2, and 6 Gy using LC-PB-MS/MS. Least absolute shrinkage and selection operator (LASSO) regression was applied to filter dose-dependent lipids for constructing multivariate regression models, validated by receiver operating characteristic analysis curves and bootstrap optimism correction. Mechanistic investigations were conducted using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, quantitative real-time polymerase chain reaction, Western blotting, and publicly available transcriptomic databases. Eighty-three species-level lipids and 311 C=C location isomers in phosphatidylcholine and phosphatidylethanolamine were identified in plasma samples. LASSO regression selected 11 isomers at 4 h and 8 lipids at 24 h. Optimized three-feature biodosimetry models demonstrated good performance across both sexes (R2 > 0.8; optimism-corrected area under the curve = 0.8224‒1.0000). Profiling of Δ9 monounsaturated isomer together with KEGG analysis suggested dysregulation of fatty acid desaturation, accompanied by reduced expression of stearoyl-CoA desaturase 1 in AHH-1 cells and murine models. C=C location isomers exhibited favorable dose classification performance in murine plasma. These promising biomarkers provide a foundation for developing translational approaches for acute radiation biodosimetry. Integrating structural lipidomics with acute radiation dose assessment for the first time; Developing a promising, C=C-based dose classification model; Linking SCD1 reduction in murine blood and bone marrow to Δ9 isomer depletion.
Per- and polyfluoroalkyl substances (PFAS), including perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), are persistent environmental contaminants. Epidemiological studies have associated PFAS exposure with several cancers, but TNBC-specific molecular evidence remains limited. We integrated computational target prediction, network analysis, bulk transcriptomic and microarray cohorts, single-cell RNA sequencing, spatial transcriptomics, prognostic modeling, molecular docking, molecular dynamics simulation, and expression assessment in 15 paired TNBC and adjacent tissues. The public patient cohorts and tissue specimens did not contain measured PFAS exposure, so all PFAS-related interpretations were prespecified as associative and hypothesis-generating. Results The intersection of 654 predicted PFAS targets, 3,944 TNBC-associated genes, and 27,529 TNBC differentially expressed genes yielded 197 candidates. A hypergeometric test indicated that this overlap exceeded chance expectation (observed/expected = 3.22, P = 3.65 × 10 − 52), although the permissive DEG set and heterogeneous disease-gene sources remain important limitations. Six candidate genes (CDK9, PF4, VEGFC, FABP7, NR2F6, and PLAU) formed the prognostic signature. The 2-year training-set AUC of 0.999 was interpreted as probable optimism rather than clinical-level performance. RT-qPCR showed significant differences for CDK9, PF4, VEGFC, FABP7, and NR2F6, whereas PLAU was directionally increased but not significant. Docking and simulation supported computational compatibility for selected protein-PFAS complexes but did not demonstrate cellular target engagement. The findings define a TNBC gene signature that overlaps with predicted PFAS targets and provide a testable computational hypothesis. Causal inference requires PFAS-stratified patient cohorts, direct exposure transcriptomics, dose–response experiments, and independent prospective validation.
Chemoresistance worsens the prognosis of hepatocellular carcinoma (HCC). This study focuses on how methyltransferase-like protein 6 (METTL6)/DALR anticodon binding domain containing 3 (DALRD3) regulates cell cycle progression and impacts HCC chemoresistance. HCC cell lines with relatively low or high METTL6 expression were intervened with cisplatin (DDP) to construct DDP-resistant cell models, and later transfected with overexpression (oe)-METTL6 plasmid, oe-DALRD3 plasmid, small interfering RNA (si)-METTL6, and/or si-DALRD3. A battery of cellular biological functional experiments and DDP drug sensitivity tests were performed to characterize the modulatory effects of METTL6 or DALRD3 on HCC chemoresistance. The interaction between METTL6 and DALRD3 was validated using coimmunoprecipitation. 3-methylcytosine (m3C) modification and expression were further detected using dot blot and Northern blot assays. A subcutaneous xenograft mouse model was established with METTL6- or DALRD3-deficient HCC cells, which were exposed to DDP for experimental validation of in vitro findings. METTL6 and DALRD3 were upregulated in DDP-resistant HCC cells, and their upregulation in HCC tissues correlated with poor prognosis. Re-expression of METTL6 or DALRD3 led to strengthened cell proliferation, aggravated cell cycle progression, and augmented DDP chemoresistance. The validated METTL6-DALRD3 interaction increased m3C modification. Northern blot further validated serine transfer RNA-m3C at position 32 (tRNASer-m3C32) modification mediated by the METTL6-DALRD3 complex. Rescue experiments unveiled that DALRD3 overexpression reversed METTL6 knockdown-mediated HCC phenotypes. Either METTL6 or DALRD3 knockdown potentiated DDP chemosensitivity in HCC xenograft mice. METTL6/DALRD3 mediated tRNASer-m3C32 modification may participate in DDP resistance in HCC cells by regulating cell cycle progression.
The lung tumor microenvironment (TME) is shaped by reciprocal interactions among cancer-associated fibroblasts (CAFs), extracellular matrix (ECM), immune cells, endothelial cells, and tumor cells. Available evidence supports an integrated sequence in which tumor-derived factors, chronic tissue injury, inflammatory cytokines, and mechanical stress generate context-dependent CAF states. Matrix-producing CAFs subsequently deposit, align, and crosslink fibrillar collagen, whereas inflammatory and antigen-presenting CAF programs regulate chemokine gradients, myeloid-cell recruitment, and local T-cell responses. These structural and biochemical changes converge to reduce vascular perfusion, restrict lymphocyte movement from stromal regions into malignant-cell nests, and promote functional immune suppression, thereby contributing to resistance to immune checkpoint blockade. The supporting evidence has been obtained from complementary systems, including single-cell and spatial profiling of human lung tumors, live imaging of human lung tumor slices, primary fibroblast-tumor cell co-cultures, three-dimensional collagen or organoid models, orthotopic xenografts, and immune-competent murine lung cancer models. Because each platform captures only part of the CAF-ECM-immune axis, conclusions should distinguish clinical association from causal evidence and specify the biological system in which a mechanism was demonstrated. Stromal signatures, collagen architecture, and spatial immune-exclusion patterns may complement tumor-intrinsic biomarkers, but their utility for treatment selection remains investigational and requires analytical standardization and prospective validation. Therapeutic development may therefore prioritize phenotype-selective CAF reprogramming and functional matrix normalization over indiscriminate fibroblast depletion, while recognizing that no single stromal feature is currently sufficient to direct clinical management. Importantly, CAF activation and ECM deposition are not uniformly tumor promoting: depending on disease stage, fibroblast state, collagen topology, and immune context, selected stromal programs may physically contain early lesions or support local immune organization. This context dependence further supports cautious, biomarker-informed evaluation of stromal interventions rather than generalized depletion. Throughout this review, spatial co-occurrence and neighborhood enrichment are treated as hypothesis-generating associations unless they are supported by temporally resolved, cell-specific perturbation and rescue experiments.
As a major viral oncogenic factor, the hepatitis B virus (HBV) X protein (HBx) critically contributes to the malignant progression of liver cancer (LC). Although cytoskeletal proteins are implicated in HBx-mediated tumorigenesis, their specific functions and underlying mechanisms in HBx-associated LC remain largely underexplored. Here, we identified that tropomyosin 1 (TPM1), an actin-binding cytoskeletal protein, is significantly overexpressed in HBx-positive LC cells and HBV-associated tumor tissues. Both in vitro and in vivo experiments demonstrated that TPM1 contributes to HBx-induced growth and migration of LC cells. Furthermore, HBx can stabilize TPM1 by disrupting its interaction with SYVN1, an E3 ubiquitin ligase. Additionally, aldehyde dehydrogenase 2 (ALDH2) was identified as a key downstream effector of TPM1. TPM1 markedly downregulates ALDH2 expression, leading to cholesterol accumulation. Specifically, TPM1 binds to histone deacetylase 2 (HDAC2), an epigenetic coregulator, and represses ALDH2 transcription by reducing histone acetylation at the ALDH2 promoter region. Interestingly, HBx enhances the interaction of TPM1with HDAC2, resulting in greater downregulation of ALDH2. Through high-throughput virtual screening and experimental validation, dihydroergotamine mesylate was further identified as a specific TPM1 inhibitor. This compound exerted potent antitumor effects by counteracting HBx-induced cholesterol accumulation and oncogenic phenotypes in LC cells. Collectively, these results delineate a mechanism by which the cytoskeletal protein TPM1 facilitates HBx-induced hepatocarcinogenesis and highlight TPM1 as a promising therapeutic target for HBV-positive LC. suppressing histone acetylation. DHE, a newly identified inhibitor of TPM1, restrains HBx-mediated LC cell biological behaviors. A schematic illustration of HBx upregulating TPM1 to inhibit ALDH2 and increase cholesterol accumulation, LC cell proliferation, and migration. HBx promotes TPM1 stabilization by inhibiting SYVN1-mediated ubiquitination-dependent degradation of TPM1. HBx enhances TPM1’s interaction with HDAC2 to suppress ALDH2 gene transcription by suppressing histone acetylation. DHE, a newly identified inhibitor of TPM1, restrains HBx-mediated LC cell biological behaviors.
Prolyl 4-hydroxylase subunit alpha 1 (P4HA1) stabilizes the collagen triple helix and is increasingly implicated in non-canonical metabolic processes, though the structural basis of these roles remains poorly understood. We combined single-cell RNA sequencing (scRNA-seq) with high-dimensional weighted gene co-expression network analysis (hdWGCNA) to identify P4HA1 in microglia with high STING activity. Functional validation was performed in MPTP mice and MPP⁺-stimulated BV2 microglia using AAV-shRNA-mediated knockdown, Seahorse metabolic flux analysis, and assays for oxidative stress and polarization. Druggability was evaluated by virtual screening of an FDA-approved drug library, and the top complex underwent 100 ns molecular dynamics (MD) simulations assessing RMSD, Rg, SASA, and free energy landscape (FEL). P4HA1 was upregulated in Parkinson’s disease (PD) models, promoting a glycolytic shift—evidenced by increased ECAR and suppressed mitochondrial OCR—that drove ROS production, mitochondrial depolarization, and M1 microglial polarization. P4HA1 knockdown attenuated dopaminergic neuron loss and improved motor and cognitive deficits in MPTP mice. Virtual screening identified Exatecan as a high-affinity P4HA1 inhibitor (−12.2 kcal/mol). MD simulations confirmed complex stability and revealed anisotropic compaction toward a global free energy minimum. Our findings establish a structural and dynamic basis for the non-canonical immunometabolic function of P4HA1, supporting its potential as a therapeutic target in PD.
The safety of drug candidates is routinely evaluated using animal models; however, physiological differences between species can lead to different responses. In vitro safety testing with cells derived from preclinical species can help support mechanistic investigations and assess the relevance, or lack thereof, of in vivo preclinical toxicity findings to human. To that end, liver microtissues (LiMTs) from human (Homo sapiens), rat (Rattus norvegicus), dog (Canis familiaris), and monkey (Macaca fascicularis) were used to investigate the species-specific responses to fialuridine, a nucleoside analogue, that resulted in fatal hepatotoxicity in human clinical trials. The morphology and functionality of these models was assessed over seven days in culture by monitoring ATP levels, albumin production, and cytochrome P450 (CYP450) activity. Fialuridine is markedly more cytotoxic to human LiMTs than to their animal counterparts. Moreover, whole genome transcriptomic analysis showed that fialuridine elicits distinct gene expression responses in human versus animal LiMTs, including the upregulation DNA damage repair pathways and induction of apoptosis. Overall, these findings are consistent with historical clinical and preclinical observations, showing these species-specific LiMTs provide a physiological, scalable, and reproducible in vitro platform for investigating interspecies differences in drug metabolism and hepatotoxicity. Integrating these models into preclinical safety assessment prior to first-in-human studies offers a robust, weight-of-evidence approach to understand species-specific responses and refine human risk assessment.
Intracerebral hemorrhage (ICH) is a neurological disorder with high morbidity and mortality. PAR4 has been implicated in ICH and neuroinflammation. The rat model of intracerebral hemorrhage (ICH) was established by injecting collagenase into the brain. TTC staining, H E staining, and immunofluorescence were used to evaluate the brain tissue. In vitro, rat primary microglia (RPMCs) were isolated and the rat microglial cell line HAPI and the hippocampal neuronal cell line H19-7 were cultured. RNA-seq analysis, Western blot, co-immunoprecipitation, cell immunofluorescence, RIP assay, m6A Dot Blot Analysis were performed to investigate the role of PAR4 and its downstream signaling pathways. In the ICH rat model, treatment with the PAR4 antagonist inhibited neuronal death and reversed the M1-dominant shift of microglia. PAR4 regulated m6A methylation in microglia via KIAA1429-mediated recruitment of methyltransferase complexes. PAR4AP increased JAK2 mRNA m6A modification, mRNA and protein levels, and enhanced STAT3 protein phosphorylation. It also increased the expression of inflammatory factors and inflammasome components, with a stronger effect on AIM2 expression. PAR4AP enhanced AIM2 expression in microglial cells through mechanisms dependent on the JAK2/STAT3 signaling pathway and m6A modification. PAR4AP-activated microglia induce neuronal PANoptosis via exosome-mediated transfer of AIM2, promoting Pyrin-ZBP1 complex formation. In a rat model of ICH, lentivirus-delivered shRNAs targeting KIAA1429, FMR1, and AIM2, as well as PAR4 inhibition, suppressed PANoptosis in neuronal cells. This study provides insights into the role of PAR4 in ICH and its downstream signaling pathways, suggesting potential therapeutic targets for ICH treatment.
Mesenchymal stem cells (MSCs) within the shoulder joint serve as a critical progenitor pool for regenerating the fibrocartilaginous enthesis during rotator cuff repair. However, the chondrogenic potential of MSCs across distinct rotator cuff sites, along with the underlying molecular mechanisms, remains to be elucidated. We performed a comparative analysis of human subacromial bursa-derived MSCs (sMSCs) and rotator cuff enthesis-derived MSCs (rMSCs). By integrating functional chondrogenic assays with longitudinal bulk and single-cell RNA sequencing, we reconstructed source-specific differentiation trajectories and identified key regulatory drivers. rMSCs exhibited superior chondrogenic potency compared to sMSCs. Transcriptional profiling identified a region-specific pro-chondrogenic module in rMSCs driven by the transcription factor SIX2. Single-cell atlas construction revealed that tissue origin dictates lineage fate: rMSCs were enriched for a high-potency SIX2+ progenitor population that differentiated into metabolic-active S100A2+ chondrocytes. Conversely, sMSCs were dominated by DPP4+ progenitors that preferentially bifurcated into an aberrant CXCL8+ inflammatory trajectory. Mechanistically, we demonstrated that SIX2 acted as a competency factor, coordinating early proliferative expansion with late-stage matrix assembly and, crucially, active suppression of inflammatory signaling. Accordingly, lentiviral overexpression of SIX2 in sMSCs was sufficient to rescue their chondrogenic defects and rewire their trajectory toward a regenerative phenotype. Our findings define the transcriptional hierarchy of shoulder-resident progenitors, identifying SIX2 as a master regulator that couples chondrogenesis with immune evasion. This establishes a molecular framework for precision cell sourcing and rational design of lineage-specific therapies.
Osteoarthritis (OA) is closely associated with mitochondrial dysfunction, but whether mitochondrial dynamics restoration can serve as an effective therapeutic entry point remains unclear. This study investigated the mitochondrial fusion promoter M1 (MFPM1) as a modulator of mitochondrial dynamics in OA and explored its target-associated mechanism. Bioinformatic and transcriptomic analyses linked OA progression and MFPM1 activity to mitochondrial dynamics, oxidative stress, inflammatory activation, apoptosis, and extracellular matrix (ECM) homeostasis. In OA-like chondrocytes, MFPM1 restored the balance between DNM1L/DRP1-associated fission signaling and MFN2/OPA1-associated fusion machinery, accompanied by recovery of mitochondrial energy metabolism and redox homeostasis and attenuation of inflammatory and apoptotic injury. Target prediction, molecular docking, molecular dynamics simulation, and CETSA identified GSK3B as a prioritized MFPM1 target, while GSK3B overexpression weakened the mitochondrial and cartilage-protective effects of MFPM1, supporting its functional involvement. MFPM1 further preserved ECM homeostasis and alleviated OA progression. These findings identify mitochondrial dynamics remodeling as a mechanistic entry point for OA intervention and support MFPM1 as a mitochondria-centered therapeutic candidate.
The mitochondrial transporter SLC25A39 mediates GSH transport from the cytosol to mitochondria and is essential for maintaining mitochondrial oxidative homeostasis and function. However, its exact molecular mechanism in ovarian cancer has not yet been elucidated. In the present work, protein and mRNA expression levels were measured using database analysis, IHC, IF, Western blot, and RT-qPCR. Cell proliferation, apoptosis, and migration capabilities were assessed by functional assays. Mitochondrial function was assessed by measuring glutathione reductase activity, ROS, MMP, and mPTP opening status. Energy metabolism was quantified through ATP content, oxygen consumption rate (OCR), and extracellular acidification rate (ECAR). Transcriptomics, metabolomics and proteomics analyses combined with ChIP and experiments on gain and loss of function were carried out to explore associated signaling pathways, with final validation conducted in mouse models. The results show that SLC25A39 expression was upregulated in ovarian cancer tissues. Ovarian cancer cell proliferation and migration were markedly enhanced, while apoptosis was suppressed, upon SLC25A39 overexpression. Furthermore, SLC25A39 overexpression drove significant metabolic reprogramming, characterized by elevated GSH levels, reduced ROS production, increased MMP, closed mPTP, augmented ATP generation, and concurrently enhanced oxidative phosphorylation (OCR) and glycolytic flux (ECAR). Mechanistically, SLC25A39 potentiated the RXFP1/cAMP/PKA/CREB signaling axis by remodeling the intracellular redox and energy microenvironment, thereby driving metabolic reprogramming and promoting cell survival in ovarian cancer. Critically, CREB specifically bound to the SLC25A39 promoter, establishing a reciprocal reinforcing transcriptional regulatory circuit. These findings were further validated in vivo through animal experiments. In conclusion, SLC25A39 orchestrates metabolic reprogramming and promotes tumor growth by potentiating the RXFP1/cAMP/PKA/CREB axis, therefore positioning it as a promising therapeutic target for ovarian cancer. The graphical abstract was obtained from Stock (https://www.storkapp.me/aifigure/app.php).
Juvenile sepsis-associated brain injury (SABI) lacks effective targeted therapies, and ferroptosis has emerged as a critical pathogenic mechanism. N⁶-methyladenosine (m⁶A) modification is increasingly recognized as a key epitranscriptomic regulator in inflammatory and neurological disorders, yet its role in ferroptosis regulation during SABI remains unexplored. Here, we identify a novel METTL3–PCBP1–ferroptosis axis that drives hippocampal neuronal injury in SABI. Using a cecal ligation and puncture (CLP)-induced rat SABI model and an in vitro co-culture system of lipopolysaccharide (LPS)-stimulated microglia with primary hippocampal neurons, we demonstrate that SABI significantly upregulates methyltransferase-like 3 (METTL3) expression and global m⁶A modification levels in the hippocampus. MeRIP-seq reveals that poly(rC)-binding protein 1 (Pcbp1) mRNA—encoding an iron chaperone that maintains cellular iron homeostasis—exhibits the most pronounced increase in m⁶A modification among cell death-related genes, with site-specific methylation at position 1151 (GGACA motif). This modification recruits the m⁶A reader YTH domain-containing family protein 2 (YTHDF2), promoting Pcbp1 mRNA decay and subsequent PCBP1 protein downregulation. Loss of PCBP1 reduces glutathione peroxidase 4 (GPX4) expression—likely through impaired mRNA binding—and disrupts intracellular Fe2⁺ homeostasis, ultimately triggering ferroptosis characterized by lipid peroxidation and mitochondrial damage. Notably, METTL3 knockdown restores PCBP1 expression, normalizes Fe2⁺ levels, upregulates GPX4, and attenuates ferroptosis and hippocampal injury; however, concurrent PCBP1 knockdown reverses these protective effects. Furthermore, PCBP1 overexpression in SABI rats rescues ferroptosis and neurological deficits. Collectively, our findings establish that METTL3-mediated m⁶A modification of Pcbp1 mRNA exacerbates ferroptosis and SABI pathogenesis, identifying the METTL3/PCBP1 axis as a promising therapeutic target for juvenile SABI.
