Background: Fibrosis and inflammation in the renal tubular epithelial cells (TECs) are key contributors to the pathology of diabetic kidney disease (DKD). Nevertheless, the precise triggers of these processes remain unclear. This study aimed to explore the role of interferon-stimulated gene 15 (ISG15) in the injury of TECs induced by high glucose (HG) conditions and its implications for the development of DKD.

Methods: ISG15 knockout (ISG15 KO) mice injected with streptozotocin-treated mice on a high-fat diet were used to investigate its role in DKD. Cellular models with ISG15 knockdown were exposed to HG conditions to assess the effects of ISG15 on cellular responses. Subsequently, we evaluated the impact of ISG15 on pyroptosis, a form of programmed cell death, to understand its potential role in DKD pathology. Furthermore, RNA sequencing (RNA-seq) and molecular biology techniques were employed to explore the signalling pathways potentially regulated by ISG15.

Results: We first confirmed an up-regulation of ISG15 within the renal tubule in DKD. The deletion of ISG15 alleviated renal functional damage, fibrosis and inflammation, which correlated with reduced ISGylation levels. Mechanistic investigation revealed that HG stimulation in TECs disrupted the mtDNA–cGAS–STING signalling, which exacerbates the DKD through the NLRP3–CASP1–GSDMD axis. Furthermore, we uncovered a bidirectional regulatory loop between STING and ISG15, with STING enhancing ISG15 expression upstream and ISG15 modulating STING expression through ISGylation.

Conclusion: ISG15–mtDNA–STING emerges as a critical hub that integrates the processes of pyroptosis, fibrosis and inflammation. Therapeutic interventions that target this signalling network at various levels may pave the way for innovative treatments for DKD.

Background: Neutrophil extracellular traps (NETs) are pivotal in the metastasis of non-small cell lung cancer (NSCLC). Our previous research demonstrated that NETs facilitate NSCLC metastasis by triggering the stimulation of the NOD-like receptor protein 3 (NLRP3) inflammasome, which is mediated through the suppression of the long non-coding RNA MIR503HG. However, the precise molecular mechanisms linking MIR503HG to NLRP3 are still not fully understood.

Methods: By employing protein mass spectrometry and the Human TFDB database, key molecules involved in NLRP3 regulation were identified. The involvement of CCAAT enhancer binding protein beta (C/EBPβ) in NSCLC metastasis was examined in both cellular and animal models. Dual-luciferase and CUT&RUN assays confirmed the mechanism by which C/EBPβ controls NLRP3. The regulatory relationship between MIR503HG and C/EBPβ was explored through RNA pulldown, RNA immunoprecipitation and coimmunoprecipitation assays. Additionally, methylation-specific PCR and other studies revealed that NETs suppress MIR503HG via DNA methylation.

Results: We found that C/EBPβ mediates the regulation of NLRP3 by MIR503HG. Further investigation confirmed that C/EBPβ promotes the migration and invasion of NSCLC both in vivo and in vitro and is highly expressed in NSCLC tissue. Mechanistically, C/EBPβ binds to the NLRP3 promoter to promote NLRP3 expression. Conversely, MIR503HG suppressed C/EBPβ expression by facilitating C/EBPβ interaction with the E3 ubiquitin ligase RNF43, which in turn reduced NLRP3 expression and NSCLC metastasis. Meanwhile, we investigated the mechanism by which NETs inhibit MIR503HG expression and found that DNA methylation is involved in the suppression of MIR503HG by NETs. Additionally, reversing this methylation partially restored MIR503HG and NLRP3 expression and mitigated the metastatic effects of NETs in NSCLC.

Conclusions: This study emphasises the critical roles of C/EBPβ and DNA methylation in NETs-mediated NSCLC metastasis. These findings unveil C/EBPβ and DNA methylation as potential novel targets for NSCLC with high NETs expression.

Background and Aims: Inflammageing represents both a critical pathophysiological hallmark and independent risk factor for myocardial infarction (MI), with age-related increases observed in MI incidence and severity of post-MI ventricular remodelling. Novel therapeutic strategies targeting inflammageing-driven mechanisms are urgently required to attenuate adverse ventricular remodelling following MI. This investigation was designed to elucidate the impact of fibroblast-specific p16^INK4a on inflammageing-associated ventricular remodelling after MI and to develop a targeted nanotherapy to mitigate this process.

Methods and Results: We found that p16-mediated inflammageing positively correlated with the severity of post-infarction ventricular remodelling in patients. POSTN-driven p16^INK4a knockout improved cardiac function, and reduced ventricular remodelling, myocardial inflammation and NLRP3 signalling activation following MI through downregulating STAT3-mediated NLRP3 inflammasome and upregulating glutathione metabolism pathway in fibroblasts. P16^INK4a overexpression induced NLRP3 signalling activation through upregulating NLRP3 transcribed by STAT3 in fibroblasts. In terms of mechanisms, p16^INK4a interacted with STAT3, which depended on the SH2 domain of STAT3; P16^INK4a promoted the interaction of EZH2 and STAT3, increased the di-methylation on K49 and phosphorylation on Y705 of STAT3 by EZH2, and promoted NLRP3 transcription through regulating histone modification in the NLRP3 promoter by interfering the formation of Bmi-1-EZH2 or Bmi-1-BCL6 complex in fibroblasts. Injection of p16^INK4a-accumulated ageing cardiac fibroblasts, or p16^INK4a overexpression adenovirus aggravated profibrosis and proinflammation in MI area. However, a novel FH peptide ‘FHKHKSPALSPV’-neutrophil membrane proteins (NMPs)-artificial lipid (Li) membranes-mesoporous silica nanoparticle (MSN) core (FNLM)-nanocaged p16^INK4a-siRNA, as a newly constructed nanomaterial drug, could prevent post-infarction ventricular remodelling through inhibiting NLRP3 transcription in targeted cardiac fibroblasts and ameliorating proinflammation and profibrosis.

Conclusions: P16^INK4a drives inflammageing-mediated post-MI ventricular remodeling by activating STAT3/NLRP3 signaling in fibroblasts. Targeting p16^INK4a via FNLM-siRNA nanotherapy represents a novel strategy to ameliorate adverse cardiac remodelling, offering translational potential for clinical intervention.

Progress in living conditions and medical technology have extended the human life span such that population aging, and thus the development of multi-system degenerative diseases, has become a major problem in many countries. Bone is a metabolically dynamic tissue and bone aging is closely related to a shift in the balance between bone resorption and bone formation. The resulting loss of bone mass and bone mechanical properties in older adults place them at risk of injury and premature death. Cellular senescence occurs in response to endogenous and exogenous stresses that lead to permanent cell cycle arrest and, thus, to tissue degeneration and dysfunction. Senescence in the bone microenvironment, as occurs during aging, induces a decline in bone formation. Research into the treatment of bone aging has therefore focused on the senescence process. This review begins with a summary of the key events in cellular senescence and bone aging and then examines recent progress in the targeting of cellular senescence, both to treat aging-related bone diseases. Novel therapeutic agents, natural products, and innovative biomedical materials are considered. Our discussion concludes by considering areas of future research.

Background: Herpes simplex virus-1 (HSV-1) infections are lifelong and linked to neurological diseases such as multiple sclerosis (MS), yet the underlying mechanisms in the host remain poorly understood.

Methods and Results: This study investigates new molecular dynamics following HSV-1 infection, uncovering the pivotal role of the mixed lineage kinase domain-like (MLKL) protein. Beyond its known function in necroptosis, MLKL was found to control HSV-1 transport into the nucleus, tightly regulated by Optineurin (OPTN). We evidenced an essential regulatory interaction between MLKL and OPTN, governing MLKL's activity in both necroptosis-dependent and independent pathways. In vivo, studies using Optn knockout mice demonstrated how this MLKL-OPTN axis contributes to demyelination and neurological symptoms mimicking MS. This axis critically prevents oligodendrocyte death and the associated demyelination during HSV-1 infection. Furthermore, pharmacological interventions with Necrosulfonamide (NSA), an MLKL inhibitor, showed therapeutic potential in preserving myelin integrity and reducing neurological deficits in HSV-1-infected models, suggesting a viable strategy for managing virus-induced neurodegeneration.

Conclusion: Our findings highlight the significant role of MLKL in HSV-1 pathogenesis and suggest that MLKL dysregulation is a key mechanism behind severe neurological damage.

Background: Temozolomide (TMZ), which is an alkylating agent, is the standard chemotherapeutic drug used for glioma treatment. However, the development of resistance to TMZ limits its efficacy. Thus, identifying novel therapeutic targets is necessary.

Methods: In this study, the levels of midkine (MDK) and c-Myc expression in glioma patient samples downloaded from TCGA were analyzed. Their interactions were also demonstrated through microthermometry and immunocoprecipitation. Furthermore, proteomics technology and Western blot showed that MDK interacted with c-Myc and influenced its ubiquitination, thereby activating a prosurvival signalling pathway and epithelial–mesenchymal transition mechanism, which contributed to TMZ resistance. To target the MDK/c-Myc complex, we screened for a small-molecule inhibitor (ACT001) that specifically disrupts the interaction between MDK and c-Myc. Treatment with ACT001 greatly sensitized TMZ-resistant glioma cells to TMZ, promoting cell death and inhibiting cell proliferation. Moreover, combination therapy with ACT001 and TMZ showed synergistic effects that inhibit tumour growth in glioma xenograft models and glioma in situ models.

Results: ACT001 facilitated the degradation of c-Myc by focusing on the MDK/c-Myc complex and controlled the Wnt/β-catenin signalling pathway via MDK, ultimately halting the advancement of glioma. When combined with TMZ, ACT001 showed good therapeutic potential for the treatment of glioma.

Conclusion: Focusing on the MDK/c-Myc complex could be an effective approach to combat resistance to TMZ in glioma. Therapy with ACT001 may be a novel approach to improve the efficacy of TMZ-based chemotherapy in patients with glioma. Further preclinical and clinical studies are warranted to validate the therapeutic potential of targeting the MDK/c-Myc complex in glioma treatment.

Aims: Adventitial remodelling in hypertension is characterized by a transformation of adventitial fibroblasts (AFs) into myofibroblasts. Previous studies have highlighted the crucial role of discoidin domain receptor 2 (DDR2) in vascular remodelling. Since DDR2-sustained tyrosine phosphorylation activates PI3K, which may inhibit autophagy through the mTOR signalling pathway, we aimed to investigate whether DDR2 contributes to mTOR-mediated autophagy suppression and subsequently promotes AFs transformation and adventitial remodelling.

Methods and results: Single-cell RNA sequencing revealed that DDR2 was upregulated in adventitial fibroblasts (AFs) in angiotensin II (Ang II, 1000 ng/min/kg) administrated wild-type (WT) mice. In AFs, rapamycin, an autophagy agonist, significantly attenuated Ang II-induced autophagy suppression and phenotype switching, whereas the autophagy inhibitor chloroquine (CQ) exacerbated these effects. DDR2 inhibition significantly alleviated PI3K/Akt/mTOR pathway-mediated autophagy suppression and subsequently inhibited AFs phenotypic switching. Conversely, DDR2 overexpression aggravated autophagy suppression and AFs phenotypic switching. Consistent with the cellular findings, prophylactic administration of rapamycin (4 mg/kg/d) or conditional knockout of Ddr2 in mice ameliorated autophagy suppression, AFs differentiation and adventitial remodelling in vivo.

Conclusion: DDR2 serves as a critical mediator of autophagy suppression during Ang II-induced phenotypic transformation of AFs and adventitial remodelling. Targeting DDR2 signalling attenuates autophagy dysfunction and inhibits AFs activation, thereby mitigating pathological adventitial remodelling. These findings highlight DDR2 as a potential therapeutic target for preventing conditions driven by aberrant adventitial remodelling.

Neutrophil extracellular traps (NETs) are reticular ultrastructures released by activated neutrophils. As the reaction products of neutrophils, NETs have been identified as crucial effectors in pathogen defence and autoimmune diseases. Recently, increasing evidence suggest that this process also occurs in cancer. The formation and clearance of NETs are dynamically influenced by the tumour microenvironment, while NETs reciprocally play a dual role in either promoting or inhibiting tumour progression through their DNA scaffold, proteases and other granule-derived proteins. Given the interplay between NETs and tumours, active exploration is currently underway to harness their potential as tumour biomarkers and therapeutic targets. Here, we delve into the biochemical and immunological mechanisms underlying NETs formation within the tumour microenvironment, along with recent advances elucidating their multifaceted roles in tumourigenesis, metastasis and tumour-associated co-morbidities. Furthermore, we present emerging strategies for NETs-based tumour diagnostic approaches and therapeutics, with a special focus on the challenging questions that need to be answered within this field.

Background: Alternative splicing (AS) plays a crucial role in regulating gene expression and governing proteomic diversity by generating multiple protein isoforms from a single gene. Increasing evidence has highlighted the regulation for pre-mRNA splicing of the splicing factors (SFs). This review aims to examine featured mechanisms and examples of SF regulation by AS, focusing on paradigmatic feedback loops and their biological implications.

Main Body of the Abstract: We specifically focus on the autoregulation and inter-regulation of SFs through AS machinery. These interactions give rise to a feedback system, where the negative feedback loops aid in maintaining cellular homeostasis, and the positive feedback loops play roles in triggering cellular state transitions. We examine the growing evidence highlighting the specific mechanisms employed by SFs to autoregulate their own splicing, including AS-coupled nonsense-mediated mRNA decay (AS-NMD), nuclear retention, and alternative 3'UTR regulation. We showcase the influence of AS feedback in amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and cancer. Furthermore, we discuss how master splicing factors can dominantly orchestrate splicing cascades, leading to widespread impacts in cellular processes. We also discuss how non-coding RNAs, particularly circular RNAs and microRNAs, engage in the splicing regulatory networks. Lastly, we showcase how negative and positive feedback loops can collaboratively achieve remarkable biological functions during the cell fate decision.

Short Conclusion: This review highlights the regulation of SFs by AS, providing enriched information for future investigations that aim at deciphering the intricate interplay within splicing regulatory networks.

Objective: We aim to investigate the spatiotemporal dynamics of intervertebral disc (IVD) cell subpopulations in IVD degeneration (IVDD).

Methods: To gain combined spatial and transcriptomic insights into IVDD, we employed both spatial transcriptomic sequencing (stRNA-seq) and single nucleus RNA sequencing (snRNA-seq) in a rat puncture-induced IVDD model. The findings were verified in rat and human IVD by immunostaining and qRT-PCR. Tamoxifen-administered Pdgfra^CreERT2;R26^tdTomato mice were adopted to track platelet-derived growth factor receptor alpha (Pdgfra) positive cells.

Results: Puncture response regions were revealed on day 1 post-puncture, for which oxidative stress emerged as a prominent pathway in a Stress Zone consisting of lipocalin-2 (Lcn2)+ annulus fibrosus (AF) cells (AFC), which propagated and migrated into nucleus pulposus (NP), playing a key role in delivering injury signals and triggering pathological processes, including ferroptosis, fibrosis, and immune reactions. In the NP, Collagen 3-high (Col3hi) NP cells (NPC) were another induced population demonstrating a fibrochondrocyte-like phenotype and high epithelial–mesenchymal transition activation, an important pathway involved in tissue fibrosis. Crucially, lineage tracing experiments in Pdgfra^CreERT2;R26^tdTomat mice revealed the significant migration and proliferation of Pdgfra+ AFCs from the AF into the NP following puncture. These findings provide direct evidence that both Pdgfra+ AFCs and Col3hi NP cells may contribute to NP fibrosis.

Conclusion: Puncture-induced oxidative stress in a stress zone is the primary reaction playing an important role in initiating IVDD. Several puncture-induced cell subpopulations were identified, including Lcn2+ AFC, Col3hi NPC, and Pdgfra+ AFC. Lcn2+ AFC plays a pivotal role in connecting oxidative stress with other pathological processes. Our results clarified the dual origin of Pdgfra+ cells, highlighting the contribution of AF-derived cells to the NP during degeneration and emphasizing the complexity of cellular changes underlying NP fibrosis. Further investigation into the specific contributions of Pdgfra+ cells from different origins to fibrosis is warranted.

Background: Phosphoglycerate kinase 1 (PGK1) serves as a critical metabolic enzyme in the process of glycolysis and has many nonmetabolic functions in tumour progression. One of the most prevalent malignant tumours is still esophageal squamous cell carcinoma (ESCC), with high recurrence rates, high probabilities of metastasis, and poor prognoses. However, the molecular mechanisms and physiological contribution of PGK1 to ESCC carcinogenesis remain largely elusive.

Methods: Esophageal cancer bioinformatics analysis and tissue microarray analysis were employed to elucidate the aberrant expression of PGK1 during ESCC progression. The carcinogenic effect of PGK1 was examined using cell proliferation, migration and sphere formation assays. Mass spectrometry analysis, immunoprecipitation, ChIP and luciferase assays, hypoxia assays and in vitro and in vivo experiments were used to clarify the mechanism of the PGK1‒MYH9 interaction in the β-catenin/c-Myc signalling pathway.

Results: We clarified that in patients with ESCC, elevated PGK1 levels were linked to poor survival, tumour size, lymph node metastatic status, and TNM stage. In vivo and in vitro experimental analyses revealed that PGK1 promoted ESCC cell tumour stemness and EMT both in vivo and in vitro. Mechanistically, we discovered that PGK1 interacts with myosin-9 (MYH9), leading to MYH9-mediated ubiquitination-mediated degradation of GSK-3β, thereby triggering the β-catenin signalling pathway and transcriptionally increasing c-Myc expression. In addition, we found that hypoxic conditions upregulated PGK1, with HIF-1α transactivating PGK1 expression, further promoting the PGK1-MYH9 interaction and PGK1/MYH9/β-catenin/c-Myc axis activation.

Conclusions: PGK1 promotes ESCC tumourigenicity and migratory capacity by facilitating β-catenin-dependent c-Myc transcription. Under hypoxic conditions, the PGK1‒MYH9 interaction is strengthened, and HIF-1α-mediated transcription increases PGK1 expression, thereby activating the β-catenin/c-Myc signalling pathway. Taken together, PGK1 holds promise as a potential biomarker for predicting postoperative prognosis and recurrence in patients with ESCC.

Background: Destruction of the blood–spinal cord barrier (BSCB) following spinal cord injury (SCI) can result in various harmful cytokines, neutrophils, and macrophages infiltrating into the injured site, causing secondary damage. Growing evidence shows that M2 macrophages and their small extracellular vesicles (sEVs) contribute to tissue repair in various diseases.

Methods and Results: In our previous proteomics-based analysis of protein expression profiles in M2 macrophages and their sEVs (M2-sEVs), the proteoglycan perlecan, encoded by HSPG2, was found to be upregulated in M2-sEVs. Perlecan is a crucial component of basement membranes, playing a vital role in stabilising BSCB homeostasis and functions through its interactions with other matrix components, growth factors, and receptors. Here, we verified the high levels and remarkable therapeutic effect of M2-sEV-derived perlecan on the permeability of spinal cord microvascular endothelial cells exposed to oxygen glucose deprivation and reoxygenation in vitro. We also decorated the surface of M2-sEVs with a fusion protein comprising the N-terminus of Lamp2 and arginine glycine aspartic acid (RGD) peptides, which have an affinity for integrin αvβ3 and are primarily present on neovascular endothelium surfaces. In SCI model mice, these RGD-M2-sEVs accumulated at injured sites, promoting BSCB restoration. Finally, we identified M2-sEV-derived perlecan as a key player in regulating BSCB integrity and functional recovery post-SCI.

Conclusion: Our results indicate that RGD-M2-sEVs promote BSCB restoration by transporting perlecan to neovascular endothelial cells, representing a potential strategy for SCI treatment.

Background: OXA1L is crucial for mitochondrial protein insertion and assembly into the inner mitochondrial membrane, and its variants have been recently linked to mitochondrial encephalopathy. However, the definitive pathogenic link between OXA1L variants and mitochondrial diseases as well as the underlying pathogenesis remains elusive.

Methods: In this study, we identified bi-allelic variants of c.620G>T, p.(Cys207Phe) and c.1163_1164del, p.(Val388Alafs*15) in OXA1L gene in a mitochondrial myopathy patient using whole exome sequencing. To unravel the genotype–phenotype relationship and underlying pathogenic mechanism between OXA1L variants and mitochondrial diseases, patient-specific human-induced pluripotent stem cells (hiPSC) were reprogrammed and differentiated into myotubes, while OXA1L knockout human immortalised skeletal muscle cells (IHSMC) and a conditional skeletal muscle knockout mouse model was generated using clustered regularly interspaced short palindromic repeats/Cas9 genomic editing technology.

Results: Both patient-specific hiPSC differentiated myotubes and OXA1L knockout IHSMC showed combined mitochondrial respiratory chain defects and oxidative phosphorylation (OXPHOS) impairments. Notably, in OXA1L-knockout IHSMC, transfection of wild-type human OXA1L but not truncated mutant form rescued the respiratory chain defects. Moreover, skeletal muscle conditional Oxa1l knockout mice exhibited OXPHOS deficiencies and skeletal muscle morphofunctional abnormalities, recapitulating the phenotypes of mitochondrial myopathy. Further functional investigations revealed that impaired OXPHOS resulting of OXA1L deficiency led to elevated reactive oxygen species production, which possibly activated the nuclear factor kappa B signalling pathway, triggering cell apoptosis.

Conclusions: Together, our findings reinforce the genotype–phenotype association between OXA1L variations and mitochondrial diseases and further delineate the potential molecular mechanisms of how OXA1L deficiency causes skeletal muscle deficits in mitochondrial myopathy.