BACKGROUND AND PURPOSE:Mesenchyme homeobox 1 (MEOX1) has been characterised as a central transcriptional regulator of fibroblast activation. Ligustilide (LIG) has significant antifibrotic, anti-inflammatory and antioxidative activities. We investigated if LIG can ameliorate hepatic fibrosis by targeting MEOX1 and exploring the underlying mechanism. EXPERIMENTAL APPROACH:In vivo, carbon tetrachloride (CCl4)-induced mice were used to assess the antifibrotic effects of AAV9-pGFAP-sh-MEOX1 and LIG. In vitro, LX-2 cells, following transfection with siRNA or pcDNA or pretreatment with LIG, were incubated with TGF-β1 for the assessment. KEY RESULTS:Knockdown of MEOX1 mitigated fibrosis, both in vivo and in vitro. Furthermore, through a strategy involving Traditional Chinese Medicine (TCM) prescription screening, molecular docking, cellular thermal shift assay (CETSA), site-specific mutation and surface plasmon resonance (SPR), we identified LIG as a potential inhibitor of MEOX1. Subsequent validation confirmed that LIG exerted significant antifibrotic effects through MEOX1. Mechanistic studies revealed that MEOX1 promoted transcriptional enhanced associate domain factor 2 (TEAD2) transcription, by binding to the -988 to -982 nt region of the TEAD2 promoter, which increased the transcription of Hippo signalling targets and stimulated hepatic stellate cell (HSC) activation and proliferation. LIG can also bind to the HOX domain of MEOX1, thereby inhibiting its function and alleviating hepatic fibrosis. CONCLUSION AND IMPLICATIONS:The MEOX1-TEAD2 signalling axis is crucial for hepatic fibrosis. LIG attenuates hepatic fibrosis by targeting MEOX1 and inhibiting the downstream of the Hippo signalling pathway. Our results elucidated the mechanistic basis for developing LIG as a clinical antifibrotic agent.

Nasopharyngeal carcinoma (NPC) is a highly aggressive malignancy. Given conventional therapy limitations, exploring non-apoptotic pathways like ferroptosis is crucial. Tryptophan (Trp) metabolic reprogramming is a key mechanism by which NPC cells evade ferroptosis by maintaining redox homeostasis. In this study, we investigated the anti-NPC activity of rAnguillin, a novel recombinant peptide derived from Anguilla anguilla, and elucidated the molecular mechanisms by which it induces ferroptosis via the FOXM1/PABPC1L axis. rAnguillin (7009 Da) was prepared via prokaryotic expression, with its primary sequence verified by LC-MS/MS. Notably, rAnguillin significantly inhibited NPC cell proliferation (IC50 = 3.306 μM at 24 h) and suppressed xenograft tumor growth in vivo in a dose-dependent manner. Mechanistically, as validated by CETSA and site-directed mutagenesis, rAnguillin directly interacted with the transcription factor FOXM1, triggering its ubiquitin-proteasome-dependent degradation. The downregulation of FOXM1 inhibited the transcriptional activation of PABPC1L, subsequently leading to the inactivation of the JAK2-STAT1 signaling axis. This cascade significantly suppressed the expression of rate-limiting enzymes such as IDO1, thereby depleting the tryptophan metabolic flux and the antioxidant protective effects of its metabolite, 3-hydroxyanthranilic acid (3-HAA). Ultimately, this disruption of redox homeostasis triggered robust ferroptosis, as evidenced by TEM and inhibitor rescue experiments. Taken together, this study demonstrates that rAnguillin induces ferroptosis in NPC cells by targeting FOXM1 for degradation, which subsequently suppresses the FOXM1-PABPC1L-JAK2/STAT1-tryptophan metabolism axis. These findings provide a promising therapeutic strategy and a potent drug candidate for the treatment of NPC.

BACKGROUND:Intestinal ischemia/reperfusion (I/R) injury is a critical clinical condition associated with high mortality, in which the ubiquitin‒proteasome system (UPS) plays a pivotal pathogenic role. Cullin-associated and neddylation-dissociated 1 (CAND1), a critical regulator of cellular protein homeostasis, governs the ubiquitination and degradation of abnormal protein substrates by regulating the assembly of SKP1‒Cullin1‒F-box (SCF) E3 ubiquitin ligase complexes. However, the mechanisms by which CAND1 regulates SCF complex assembly and its potential therapeutic role in intestinal I/R injury remain unclear. OBJECTIVES:This study aims to elucidate the molecular mechanisms by which CAND1 mediates intestinal I/R injury and to identify potential therapeutic strategies targeting CAND1. METHODS:Intestinal I/R was induced by superior mesenteric artery (SMA) occlusion in mice. Four weeks before I/R challenge, AAV-CAND1 was injected into the mice via the tail vein. Evodiamine (evo) was administered daily via intraperitoneal injection for three days before I/R challenge. Caco-2 cells were subjected to hypoxia/reoxygenation (H/R) treatment in vitro to simulate intestinal I/R in mice. RESULTS:Excessive oxidative stress during intestinal I/R injury triggers mitochondrial fission and apoptosis. We identified CAND1 as a key regulator in this process, demonstrating upregulated expression during intestinal I/R injury. CAND1 knockdown attenuated reactive oxygen species (ROS) overproduction, mitochondrial fission, and apoptosis. Mechanistically, CAND1 inhibited Cullin1-FBXO6-PKM2 complex assembly and reduced PKM2 ubiquitination and degradation, thereby increasing PKM2 stability. Phosphorylated PKM2 formed dimers and translocated to mitochondria, where it activated Drp1-dependent fission pathway, worsening oxidative stress and apoptosis. Through molecular docking, evo was identified as a potential small-molecule candidate targeting CAND1. CAND1 may be inhibited by evo, thereby alleviating intestinal I/R injury. CONCLUSION:CAND1 suppresses Cullin1-FBXO6-PKM2 complex assembly and PKM2 ubiquitination, promoting PKM2 dimerization-mediated mitochondrial fission and apoptosis in intestinal I/R injury. CAND1 is likely inhibited by evo, thereby alleviating mitochondrial dynamic alterations and cell death during intestinal I/R injury.

Nasopharyngeal carcinoma-associated malignant epithelial models remain useful for exploring integrin-related therapeutic strategies. In this study, we evaluated the antitumor activity and potential mechanisms of rRGD3(mu), a recombinant peptide with a triple-RGD architecture. Using CNE2 cells as the primary experimental model, we evaluated cell viability, colony formation, migration, invasion, adhesion, apoptosis-related marker expression, and EMT-associated molecular changes. In vivo efficacy was assessed using a CNE2 cell-derived BALB/c nude mouse xenograft model. rRGD3(mu) inhibited CNE2 cell viability, clonogenic growth, migration, invasion, and adhesion in a dose-dependent manner and suppressed xenograft tumor growth under the tested dosing schedule. Mechanistically, rRGD3(mu) promoted mitochondria-associated apoptosis, as indicated by an increased Bax/Bcl-2 ratio and caspase-9/3 activation, and modulated the expression of EMT-associated markers, including E-cadherin, N-cadherin, vimentin, and MMP2. Bioinformatic analysis and experimental validation suggested that ITGB1-containing integrin complexes might serve as important mediators and putative cellular engagement sites of rRGD3(mu). rRGD3(mu) treatment reduced ITGB1 protein abundance and attenuated FAK/AKT signaling. ITGB1 knockdown partially mimicked the effects of rRGD3(mu) and reduced the additional cellular response to rRGD3(mu) treatment, supporting the substantial contribution of ITGB1-associated signaling. These findings provide preliminary mechanistic evidence that rRGD3(mu) suppresses malignant phenotypes in CNE2-based models, at least in part through modulation of ITGB1-associated FAK/AKT signaling.

BACKGROUND:Intestinal ischemia/reperfusion (I/R) injury is a prevalent pathophysiological occurrence that results in significant morbidity and mortality. Growth arrest and DNA damage-inducible β (GADD45B) mediates various cellular responses and is engaged in apoptosis, DNA demethylation and repair, and cell survival. Nonetheless, the role of GADD45B in intestinal I/R injury remains inadequately defined. This study aimed to elucidate whether GADD45B contributes to intestinal I/R injury through the regulation of apoptosis and DNA demethylation. METHODS:In this study, we established a mouse intestinal I/R model using mesenteric artery occlusion, and Caco-2 cells were used to create the in vitro hypoxia/reoxygenation (H/R) model. Using these models, the function of GADD45B in intestinal I/R injury was investigated by knockdown and overexpression approaches, combined with histological staining, immunohistochemistry, and Western blotting. Transcriptomic analyses were performed to identify downstream signaling pathways and molecular targets of GADD45B. The specific molecular mechanisms by which GADD45B regulates intestinal I/R injury were further elucidated using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR), methylation-specific polymerase chain reaction (MSP), co-immunoprecipitation (Co-IP), and chromatin immunoprecipitation (ChIP) assays. RESULTS:Our research revealed a notable increase in GADD45B in both H/R-induced Caco-2 cells and mouse models of intestinal I/R. GADD45B knockdown significantly mitigated intestinal barrier dysfunction and apoptosis resulting from H/R, whereas its overexpression had the opposite effect in vitro . GADD45B -knockdown mice were generated via the adeno-associated virus (AAV)-short hairpin (sh)-GADD45B and subjected to I/R. These findings indicated that GADD45B knockdown alleviated intestinal injury and reduced apoptosis triggered by intestinal I/R. Transcriptomic Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that GADD45B regulated apoptosis during intestinal I/R by activating the Hippo pathway, with mammalian sterile 20-like kinase 1 ( MST1 ) recognized as a downstream target gene of GADD45B. ChIP confirmed that GADD45B bound to the MST1 promoter and that ten-eleven-translocation 1 (TET1) occupied the MST1 promoter region following GADD45B overexpression. Co-IP confirmed that GADD45B interacts with TET1. MSP confirmed that TET1 knockdown impaired GADD45B-induced MST1 promoter DNA demethylation. Mechanistically, GADD45B interacted with TET1 to enhance MST1 expression through DNA demethylation, which subsequently activated the Hippo pathway and exacerbated apoptosis following intestinal I/R. CONCLUSION:GADD45B represents a pivotal regulatory factor in intestinal I/R injury, and targeting the GADD45B/TET1/MST1 axis might serve as a feasible treatment strategy.

Mitochondrial dysfunction is a central contributor to the pathogenesis of intestinal ischemia/reperfusion (I/R) injury. This dysfunction is closely linked to mitochondrial calcium overload and excessive reactive oxygen species (ROS) production, culminating in cellular apoptosis. Leucine Zipper And EF-Hand Containing Transmembrane Protein 1 (LETM1), a key regulator of mitochondrial permeability, is essential for cellular homeostasis and survival. However, the role and underlying mechanism of LETM1 in intestinal I/R injury remain poorly understood. Here, we observed that LETM1 expression was significantly downregulated in intestinal tissues following I/R. AAV9-mediated overexpression of LETM1 significantly alleviated mitochondrial dysfunction. We further found that the acetylation status at lysine 597 (K597) modulates the stability of LETM1 in Caco-2 cells. LETM1 was identified as a downstream target of mitochondrial deacetylase Sirtuin 3 (SIRT3), and its knockdown significantly impaired the protective effects of SIRT3 in vitro. Collectively, our findings provide the first evidence that LETM1 serves as a protective target against calcium overload-induced mitochondrial dysfunction and apoptosis during intestinal I/R injury. These findings highlight the therapeutic potential of targeting LETM1 deacetylation as a novel strategy for intestinal I/R injury prevention.

AIMS:Ferroptosis, the cell death induced by iron accumulation, contributes to the pathogenesis of Parkinson's disease (PD). While transient receptor potential vanilloid 2 (TRPV2) is known to mediate pathological processes in neurodegenerative diseases, its specific role in ferroptosis in PD remains largely unknown. This study aims to investigate the underlying mechanisms of TRPV2 in PD. MATERIALS AND METHODS:An MPTP-induced mouse model of PD and MPP+-induced SH-SY5Y cellular model were established. To investigate the role and mechanism of TRPV2 in PD, AAV2/9 TRPV2 was injected into the substantia nigra (SN) of mice. In parallel, SH-SY5Y cells were transfected with si-TRPV2, pc-TRPV2, TRPV2-S339A, or pc-PTEN-induced putative kinase 1 (PINK1) plasmids, or treated with ferroptosis inhibitor ferrostatin-1. KEY FINDINGS:Our results demonstrated that TRPV2 expression was dramatically decreased in PD, particularly in dopaminergic neurons. Notably, overexpression of TRPV2 obviously improved neurological impairment and ferroptosis, whereas TRPV2 knockdown strongly exacerbated these effects. Interestingly, ferrostatin-1 reversed the detrimental effect of TRPV2 knockdown in PD in vitro. Furthermore, bioinformatics analysis and our experimental results indicate that TRPV2 is phosphorylated by PINK1 at serine 339. Additionally, the protective roles of PINK1 overexpression in inhibiting ferroptosis were abolished by TRPV2 interference or TRPV2-S339A. SIGNIFICANCE:These findings implicate a neuroprotective role for TRPV2 in PD, potentially through a mechanism involving its regulation of ferroptosis via phosphorylation by PINK1.

Thyroid cancer (TC) dedifferentiation contributes to tumor progression, treatment resistance, and poor survival, yet the underlying molecular mechanisms remain elusive. Genetic alterations, copy number variations, and epigenetic modifications may contribute to this process. Here, we conducted an integrated analysis to screen for critical molecules involved in TC dedifferentiation, identifying nucleolar and spindle-associated protein 1 (NUSAP1) as a candidate. NUSAP1 is overexpressed in TC and associated with poor prognosis. Upregulation of NUSAP1 enhances TC cell proliferation, migration, and dedifferentiation, evidenced by the suppression of thyroid differentiation genes and the induction of epithelial-mesenchymal transition. Mechanistically, branched-chain amino acid (BCAA) catabolism is a potential mechanism of dedifferentiation in TC mediated by NUSAP1 through RNA sequencing enrichment analysis. Specifically, NUSAP1 forms a transcriptional complex with bromodomain containing 4 (BRD4), which activates branched-chain amino acid transaminase 1 (BCAT1) transcription and leads to accelerated BCAA catabolism and reduced α-ketoglutarate (α-KG) levels. This metabolic shift increases histone H3 lysine 27 trimethylation (H3K27me3) levels by impairing histone demethylation, thereby suppressing the expression of thyroid differentiation genes. Pharmacologic inhibition of NUSAP1 with entinostat suppresses downstream target levels, suggesting a novel treatment modality. In summary, NUSAP1 promotes dedifferentiation through the BCAT1/α-KG/H3K27me3 axis by forming a transcriptional complex with BRD4, which indicates that NUSAP1 is a potential therapeutic target in TC.

Liver fibrosis constitutes a central pathological hallmark of advanced chronic liver diseases. However, effective therapeutic targets and pharmacological interventions remain insufficiently defined. Mitochondrial oxidative stress plays a pivotal role in the pathogenesis of hepatic stellate cells (HSCs) activation during liver fibrosis. While erianin, a natural bibenzyl compound extracted from the stems of Dendrobium chrysotoxum Lindl., exerts antioxidant effects in various diseases, its protective effect against liver fibrosis remains elusive. Thus, this study aimed to evaluate the therapeutic potential of erianin against liver fibrosis and to explore the underlying molecular mechanisms. The results revealed that erianin markedly attenuated CCl4-and TGF-β1-induced HSCs activation and liver fibrosis. Molecular docking analyses identified PRDX3 as a specific binding target of erianin, with a subsequent increase in PRDX3 expression. However, PRDX3 knockdown abolished the protective effects of erianin on liver fibrosis. Mechanistically, RNA-seq and molecular assays demonstrated that PRDX3 alleviated liver fibrosis by activating NLRX1 and that erianin mediated its protection through the PRDX3/NLRX1 pathway. Collectively, these findings demonstrate that erianin alleviates liver fibrosis via the PRDX3/NLRX1 axis.

Ferroptosis, an iron-dependent type of cell death, has attracted significant attention in recent years. Accumulating evidence demonstrates that ferroptosis critically contributes to the pathogenesis of intestinal ischemia/reperfusion (I/R) primarily through triggering excessive lipid peroxidation, disrupting iron homeostasis, and disabling endogenous antioxidant system. Therefore, targeted inhibition of ferroptosis represents a promising therapeutic strategy to alleviate intestinal I/R damage. However, the upstream molecular regulatory networks governing ferroptosis in intestinal I/R, especially miRNA-mediated post-transcriptional regulatory mechanisms, are not fully understood. MiRNA microarray analysis revealed that the expression of miR-381-3p is upregulated following intestinal I/R insult. Bioinformatic prediction combined with dual-luciferase reporter assays confirmed that miR-381-3p directly targets and negatively regulates cAMP response element-binding protein 1 (CREB1), a key transcription factor closely implicated in ferroptosis modulation. Subsequent experiments confirmed that CREB1 specifically binds to the promoter region of peroxiredoxin 6 (PRDX6) to facilitate its transcriptional activation, as quantified by quantitative real-time PCR. Both in vivo intestinal I/R injury mouse models and an in vitro Caco-2 cell hypoxia/reoxygenation (H/R) model were utilized for functional verification. The results revealed that upregulation of the CREB1/PRDX6 axis significantly suppressed the expression of acyl-CoA synthetase long-chain family member 4 (ACSL4), which is a key marker of ferroptosis. Meanwhile, enhanced GPX4 activity and elevated intracellular glutathione (GSH) content were observed, accompanied by reduced intracellular iron overload and alleviated histological and cellular morphological damage. Conversely, inhibition of the CREB1/PRDX6 axis exacerbated ferroptosis, with corresponding adverse changes in these ferroptosis-related indicators. In conclusion, the miR-381-3p/CREB1/PRDX6 signaling axis protects against intestinal I/R by suppressing ferroptosis, providing novel molecular targets and a theoretical basis for the prevention of intestinal I/R injury.

AIMS:Mitochondrial oxidative damage is one of the factors that contributes to the pathological process of intestinal ischemia/reperfusion (II/R) injury. Glutaredoxin (GRX2), which serves as a crucial protein in maintaining mitochondrial redox homeostasis, affects the activity of downstream proteins through its deglutathionylation effect. Silent information regulator (SIRT3), a crucial deacetylase in mitochondria, has regulatory effects on the activity of various mitochondrial antioxidant enzymes. However, the precise regulatory mechanism underlying SIRT3 enzymatic activity is unknown. Our research is designed to explore GRX2-mediated SIRT3 deglutathionylation's role and mechanism in II/R injury. RESULTS:GRX2 levels decreased after II/R injury, and GRX2 overexpression alleviated II/R-induced intestinal mucosal injury, mitochondrial oxidative damage, damage to mitochondrial structure and function, remote organ injury, and the systemic inflammatory response. GRX2 overexpression substantially decreased the S-glutathionylation of SIRT3 and increased its activity. The results of the incubation of recombinant SIRT3 with glutathione and H2O2 indicated that the S-glutathionylation of SIRT3 inhibited SIRT3 activity. Subsequently, SIRT3 mutant plasmids with cysteine-to-serine substitutions were constructed to screen for the S-glutathionylation sites of SIRT3 among the four cysteine residues in its amino acid sequence. The results demonstrated that C280 and C283 are the SIRT3 S-glutathionylation sites. The results of experiments using ischemic intestines from clinical cases confirmed the relationship between GRX2 and SIRT3. INNOVATION:This study demonstrates that GRX2 alleviates mitochondrial oxidative damage following II/R by reversing the S-glutathionylation of SIRT3. CONCLUSION:GRX2 is an important protective factor against II/R injury, and GRX2-mediated deglutathionylation of SIRT3 alleviates II/R-induced mitochondrial injury and intestinal damage. Antioxid. Redox Signal. 00, 000-000.

Sepsis-induced liver injury is common in intensive care units and is associated with extremely low survival rates. Tripartite motif protein 21 (TRIM21) positively regulates Gasdermin D (GSDMD)-dependent pyroptosis via its PRY-SPRY domain; however, whether TRIM21 is involved in sepsis-induced liver injury is still unclear. In the present study, hepatic TRIM21 expression was dramatically upregulated in mice with LPS-induced liver injury. TRIM21 silencing in vivo alleviated pyroptosis and inflammation during sepsis-induced liver injury, as shown by a marked increase in GSDMD protein levels and a significant decrease in the protein levels of GSDMD N-terminal domain (GSDMD-N), IL-18 and IL-1β. A protein interaction screening revealed ubiquitin-specific peptidase 15 (USP15), a deubiquitinating enzyme, as a novel interactor of TRIM21. Notably, USP15 interacted with TRIM21 and maintained its stabilization through deubiquitination. In vivo, USP15 knockdown alleviated pyroptosis and inflammation in LPS-induced liver injury. Nevertheless, USP15 overexpression increased GSDMD-N, IL-18 and IL-1β protein expression and decreased GSDMD protein levels, and these effects were blocked by TRIM21 knockdown. In summary, TRIM21 induces GSDMD-N aggregation to increase pyroptosis and inflammation in LPS-induced liver injury, and USP15 serves as a critical regulator of TRIM21 by deubiquitinating and stabilizing the TRIM21 protein. Our results reveal the contribution of the USP15/TRIM21/GSDMD axis to cell pyroptosis in sepsis-induced liver injury, and this contribution could provide a novel approach for sepsis-induced liver injury treatment.

Excessive endoplasmic reticulum (ER) stress and neuronal apoptosis contribute to neurodegeneration in Parkinson’s disease (PD). However, the molecular mechanisms underlying these perturbations and how they are directly regulated remain unclear. B cell receptor-associated protein 31 (BAP31), which is highly expressed in the ER, has been shown to participate mainly in regulating ER stress and apoptosis. Here, our results showed that BAP31 expression was dramatically decreased in PD. Notably, overexpression of BAP31 exerted neuroprotective effects by inhibiting ER stress and apoptosis in vitro and in vivo, whereas BAP31 siRNA strongly abolished these effects. Interestingly, 4-phenylbutyric acid (4-PBA), the ER stress inhibitor, reversed the detrimental effect of BAP31 knockdown in vitro. Mutations in PTEN-induced putative kinase 1 (PINK1) are known to cause autosomal recessive early-onset PD. PINK1 has been implicated in protein phosphorylation pathways that are associated with ER stress and apoptosis. Bioinformatics analysis and our results demonstrated that PINK1 interacts with BAP31 and phosphorylates it at the Ser 142 residue. Furthermore, the protective effects of PINK1 overexpression against ER stress-mediated apoptosis were abolished by BAP31 interference or BAP31-S142A and strengthened by BAP31-S142E. Overall, the present study suggests that BAP31 overexpression exerts neuroprotective effects by inhibiting ER stress-induced apoptosis. Regulation of the PINK1/BAP31 pathway may be a beneficial strategy for PD.

Background and PurposeEndoplasmic reticulum (ER) stress is a crucial pathogenic mechanism in alcoholic liver disease (ALD). B-cell receptor-associated protein 31 (BAP31) can regulate ER homeostasis and anti-apoptosis, but the function and regulation of BAP31 in ALD are unclear. The purpose of this study is to investigate whether BAP31 deacetylation by sirtuin 2 could attenuate ER stress and apoptosis during ALD and to explore whether carnosol could alleviate ALD through the sirtuin 2/BAP31 pathway.Experimental ApproachA mouse model of ALD was established by feeding mice with alcoholic liquid chow. In vitro, AML-12 cells were stimulated with alcohol. The therapeutic efficacy of carnosol in protecting mice from ALD pathogenesis was evaluated.Key ResultsTreatment with carnosol protected mice against ALD and attenuated hepatocyte ER stress and apoptosis. Carnosol up-regulated sirtuin 2 expression, and sirtuin 2knockdown abolished the protective effect of carnosol during ALD. Moreover, sirtuin 2 knockdown reduced BAP31 expression. Carnosol-mediated BAP31 up-regulation was abolished upon knockdown of sirtuin 2. Mechanistically, sirtuin 2 selectively regulates the deacetylation of BAP31 at K158.Conclusion and ImplicationsTaken together, the present study shows for the first time that carnosol exerts its protective efficacy through facilitating sirtuin 2-mediated deacetylation of BAP31 at K158 to attenuate hepatocyte ER stress and apoptosis during ALD. These results provide new therapeutic targets and approaches for combating chronic ALD.

BACKGROUND:Alcohol-associated liver disease (ALD), one of the most frequent chronic liver diseases globally, is characterized by steatosis. HMG-CoA reductase-degrading protein 1 (HRD1) participates in the endoplasmic reticulum-associated protein degradation pathway through the recognition, translocation, and ubiquitination of substrate proteins. HRD1 is implicated in endoplasmic reticulum stress, oxidative stress and cell metabolism; however, the function of HRD1 in ALD remains unclear. AIMS:We aimed to explore the contribution and underlying molecular mechanism of HRD1 in alcoholic liver disease. METHODS:Mice were administered adeno-associated virus 9 encoding HRD1- or ACSL3-specific shRNA via intravenous injection, followed by feeding with a Lieber-DeCarli liquid diet containing 5 % ethanol. HepG2 cells were transfected with either HRD1 siRNA or HRD1 overexpression plasmids prior to ethanol exposure. RESULTS:Hepatic HRD1 expression was significantly increased under alcohol conditions. Hepatocyte-specific HRD1 knockdown markedly attenuated alcohol-induced hepatic injury, inflammation, oxidative stress and lipid metabolism disorders in vivo. Additionally, similar results were shown in vitro. Mechanistically, acyl-CoA synthetase long chain family member 3 (ACSL3), a key regulator known to ameliorate hepatic steatosis, was identified as a novel substrate of HRD1. HRD1 facilitates the ubiquitination and degradation of ACSL3. Interestingly, HRD1 knockdown significantly suppressed fatty acid synthesis and promoted fatty acid oxidation, which was reversed by ACSL3 silencing both in vivo and in vitro. CONCLUSION:In summary, HRD1 functions as a key mediator of ALD by ubiquitinating ACSL3, thereby promoting lipid dyshomeostasis, and aggravating ALD. Our findings reveal novel mechanistic insights into HRD1 and identify ACSL3 as a new downstream target of HRD1 to facilitate ALD treatment.

Overdosing on acetaminophen (APAP) is the primary cause of drug-induced liver injury. Recent studies have demonstrated that dysregulated lipid metabolism, particularly decreased fatty acid oxidation (FAO), is a key contributor to APAP-induced acute liver injury (AILI). OTU domain-containing ubiquitin aldehyde-binding protein 1 (OTUB1), a crucial member of the OTU deubiquitinase family, has been involved in the metabolic progression of multiple diseases. Nevertheless, its involvement in AILI as well as FAO remains unclear. Here, we aimed to elucidate the effects of OTUB1 on the regulation of FAO in AILI. Our investigation revealed decreased OTUB1 expression in AILI. OTUB1 overexpression not only alleviated liver injury but also improved FAO in vivo and in vitro. Conversely, opposite biochemical changes were observed in hepatocytes with OTUB1 knockdown. Mechanistically, long-chain acyl-CoA synthase 5 (ACSL5), which plays a crucial role in regulating FAO, was identified as a novel substrate of OTUB1 in AILI via mass spectrometry analysis. OTUB1 interacts with ACSL5 and promotes its deubiquitination and stability. Moreover, the protective effect of OTUB1 on FAO in AILI occurred via the deubiquitination of ACSL5. Overall, the present study revealed that the OTUB1-ACSL5 axis plays an essential role in regulating FAO during AILI progression and might be a novel target for therapeutic intervention.

Excessive intestinal ischemia/reperfusion (I/R)-induced epithelial cell apoptosis results in damage to the intestinal defense barrier. Circular RNAs (circRNAs) are functional RNA transcripts, and their functions as microRNA (miRNA) sponges and binding proteins have been well characterized. Recent evidence has indicated that some circRNAs encode functional proteins. However, whether protein-encoding circRNAs contribute to intestinal I/R remains undiscovered. Here, we identified a protein-encoding circRNA, circARHGAP12, that can significantly attenuate intestinal I/R-induced cell apoptosis. Moreover, circARHGAP12 can encode a 229-amino-acid (aa) protein, ARHGAP12-229aa. The expression of both circARHGAP12 and ARHGAP12-229aa was downregulated in intestinal I/R injury. Additionally, circARHGAP12 protected against intestinal I/R injury mainly by encoding ARHGAP12-229aa. We performed RNA sequencing (RNA-seq) analyses to identify downstream targets of ARHGAP12-229aa. The results revealed that overexpression of ARHGAP12-229aa led to increased expression of MDC1, a DNA damage-related protein, and that this increase was accompanied by a decrease in intestinal epithelial cell DNA damage and apoptosis. Furthermore, MDC1 silencing weakened the protective effect of ARHGAP12-229aa against intestinal I/R injury. Our findings suggest a novel perspective on circRNA function, providing an innovative therapeutic strategy for intestinal I/R.

BACKGROUND:Hepatic ischemia-reperfusion (I/R) injury related to liver transplantation and hepatic resection remains a challenge in clinical practice. Accumulating evidence indicates that mitochondrial dysfunction is a critical cause of I/R injury. The protein 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1) is involved in the regulation of mitophagy and the recruitment of autophagy receptor proteins independent of PTEN induced putative kinase 1. AIM:To clarify the protective mechanism of NIPSNAP1 against hepatic I/R, with a focus on mitophagy and mitochondrial dynamics, as well as the potential mechanism by which n6-methyladenosine (m6A) modification regulates NIPSNAP1. METHODS:Mice were administered an adeno-associated virus in vivo and a hepatic I/R model was established via portal vein interruption followed by reperfusion to explore the effect of NIPSNAP1 on hepatic I/R. HepG2 cells were subjected to hypoxia/reoxygenation treatment in vitro. RESULTS:We observed a significant downregulation of both NIPSNAP1 and insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) expression in vivo and in vitro. NIPSNAP1 knockdown impaired mitophagy and disrupted mitochondrial dynamics; in contrast, NIPSNAP1 overexpression resulted in the opposite effects. Further studies revealed that IGF2BP2 functions as an m6A reader that targets and binds NIPSNAP1, thereby regulating its mRNA stability. CONCLUSION:NIPSNAP1 prevents hepatic I/R injury by promoting mitophagy and maintaining mitochondrial homeostasis, serving as a novel target of the m6A reader IGF2BP2. Therefore, targeting the IGF2BP2/NIPSNAP1 axis may facilitate the development of better therapeutics for hepatic I/R.

Intestinal ischemia/reperfusion (I/R) is a severe pathophysiological process that occurs in a variety of clinical conditions and can trigger multiple life-threatening syndromes. Intestinal I/R is associated endoplasmic reticulum (ER) stress. Prolyl 4-hydroxylase subunit beta (P4HB) contributes significantly to maintaining ER redox homeostasis, which is affected by I/R injury. Nevertheless, the molecular mechanism of P4HB expression and function in intestinal I/R is still unknown. In our study, we discovered that the expression of P4HB was clearly downregulated in the intestine of mice at the reperfusion stage and in Caco2 cells at the reoxygenation stage. In addition, P4HB-knockdown mice exhibited clearly enhanced ER stress-mediated apoptosis of intestinal tissue under intestinal I/R, whereas P4HB overexpression in Caco2 cells alleviated ER stress-mediated apoptosis under HR. Furthermore, via bioinformatics screening of proteins that interact with P4HB, ubiquitin-specific protease 5 (USP5) was identified as a critical factor in the abnormal expression of P4HB. USP5 interacts with P4HB and remains stable by removing ubiquitin. In vivo, P4HB knockdown counteracted the effect of USP5 overexpression on alleviating ER stress-mediated apoptosis in response to intestinal I/R. In summary, this study revealed that P4HB plays a crucial role in regulating ER stress-mediated apoptosis and identified USP5, which is a novel mediator of P4HB, as a prospective target for the treatment of intestinal I/R.