Search Results (54)

Background: Small-cell lung cancer (SCLC) has a poor prognosis because of the development of drug resistance. Our previous studies showed that the plant steroid hormone, epibrassinolide (EB), interacts with human SCLC cells through inhibition of GSK3β-mediated signaling and overcomes drug resistance. However, the latter does not occur via membrane-mediated drug efflux, a commonly reported mechanism. Methods: We investigated a novel mechanism, ferroptosis, for drug resistance and the effects of EB. Pharmacological, cellular and molecular investigations were performed. We used drug-sensitive (NCI-H69) and multi-drug-resistant (VPA) SCLC cell lines. Results: 4-hydroxynonenal (HNE), a marker for lipid peroxidation and ferroptosis, was lower in drug-resistant VPA cells than drug-sensitive cells (H69) (22 vs. 60 pg/mg protein). Incubation with EB at a dose that reverses drug resistance in VPA cells increased HNE (94 pg/mg). Exogenously applied HNE increased intracellular HNE and reversed drug resistance (IC₅₀ for etoposide in VPA cells at 7.5 µM (untreated) vs. 1.0 µM (treated)). Ferrostatin, which inhibits ferroptosis, reduced HNE accumulation and increased drug resistance (IC₅₀ for etoposide at 5.5 µM (untreated) vs. 19 µM (treated)). As measured by phosphorylation, the signaling molecule Akt was more active in drug-resistant compared to drug-sensitive SCLC cells. This activation was accompanied by increased drug resistance. Both EB and a specific inhibitor of Akt phosphorylation/activation reversed this resistance. Akt activation was reflected in increased GSK3β activity. The latter was also inhibited by incubation in EB. The level of the GSK3β-controlled “antioxidant transcription factor” NRF2 was higher in drug-resistant VPA cells than drug-sensitive H69 cells. This increase was reversed in EB-treated cells. The protein level of the “antioxidant enzyme”, GPX4, which chemically reduces peroxidized lipids, was higher in VPA than H69 cells (21.2 vs. 10.1 pg/mg protein). Likewise, GPX4 enzyme activity was higher in drug-resistant than drug-sensitive cells. Incubation with EB reversed these increases. Incubation of SCLC cells with RSL3, a specific inhibitor of GPX4 activity, increased intracellular HNE and reversed drug resistance in VPA cells. Conclusions: These data indicate that ferroptosis is an important mechanism for drug resistance in SCLC cells and the plant steroid, EB, reverses drug resistance by interfering with the Akt signaling pathway and GPX4 anti-oxidant activity. Full article

Hypoxia-induced radioresistance remains a major obstacle in non-small cell lung cancer (NSCLC) radiotherapy. This study investigates whether artificially activating mitochondrial reverse electron transfer (RET) can enhance radiosensitivity in NSCLC by triggering oxidative stress. An in vitro hypoxia/reoxygenation (H/R) model was established in A549 cells to assess reactive oxygen species (ROS) levels, mitochondrial function, and metabolic alterations using fluorescence probes, flow cytometry, confocal microscopy, and targeted metabolomics. Mitochondrial complex inhibitors and dimethyl succinate (DM-S) were employed to validate the RET mechanism, and radiosensitivity was evaluated through clonogenic survival, apoptosis assays, and γ-H2AX staining. In vivo, A549 tumor-bearing mice received high oxygen (95% O₂) combined with DM-S and localized irradiation (4 Gy); tumor growth, histopathology, and immunohistochemistry were examined. H/R triggered substantial mitochondrial ROS production via complex I-mediated RET, dependent on a high mitochondrial membrane potential and electron transport chain imbalance, with succinate accumulation serving as a key metabolic switch. Exogenous DM-S exacerbated H/R-induced oxidative damage, DNA fragmentation (8-OHdG elevation, mtDNA integrity loss), and mitochondrial network disruption. H/R combined with DM-S significantly enhanced in vitro radiosensitivity, reducing clonogenic survival and increasing apoptosis to 53.4% ± 1.9% versus 10.3% ± 1.2% with irradiation alone. In vivo, the combination therapy markedly suppressed tumor growth, induced apoptosis and oxidative lipid damage (4-HNE), alleviated hypoxia (reduced HIF-1α), and showed no overt toxicity. These findings demonstrate that activating mitochondrial RET effectively enhances radiosensitivity in NSCLC. Succinate metabolism is a critical therapeutic target, and combining high oxygen with a succinate analog represents a promising radiosensitization strategy for hypoxic tumors. Full article

Background: Recruitment and activation of monocyte-derived macrophages (MoMFs) sustain cholangitis in primary biliary cholangitis (PBC), but whether MoMFs amplify inflammation through ferroptosis remains unclear. We defined ferroptotic programs in MoMFs and evaluated the calpain/ACSL4 axis as a regulatory and therapeutic node. Methods: We analysed a public human liver single-cell RNA sequencing (scRNA-seq) dataset and examined MoMF-associated ACSL4 and 4-hydroxynonenal (4-HNE) signals in CD11b⁺CD68⁺ cells by multiplex immunofluorescence. We used a 2OA–BSA-induced PBC-like mouse model to assess liver injury, inflammation and ferroptosis-related markers and tested Liproxstatin-1 (Lip-1), rosiglitazone (ROSI) or the calpain inhibitor PD150606. Bone marrow-derived macrophages (BMDMs) from control and PBC mice were profiled and challenged with RSL3, with or without Ferrostatin-1 (Fer-1), ROSI or PD150606. Results: MoMFs were expanded in PBC livers and showed the strongest induction of ferroptosis signatures, centered on ACSL4, with enhanced inflammatory crosstalk with cholangiocytes. Human PBC tissues showed increased CD11b⁺CD68⁺ cells positive for ACSL4 or 4-HNE. In PBC-like mice, malondialdehyde (MDA) increased and glutathione (GSH) decreased, and macrophages showed greater colocalization with ferroptosis markers; Lip-1, ROSI or PD150606 improved liver biochemistry, reduced inflammation scores and limited macrophage infiltration. PBC-derived BMDMs upregulated ACSL4 and CAPN1/2 and were more sensitive to RSL3; Fer-1, ROSI or PD150606 attenuated ferroptosis-associated molecular changes. Conclusions: MoMF ferroptosis is prominently engaged in PBC, and our findings implicate a pharmacologically tractable calpain/ACSL4 axis that may contribute to macrophage ferroptotic susceptibility and inflammatory liver injury. Full article

Human neutrophil elastase (HNE) is a key driver of inflammatory lung disorders, promoting extracellular matrix degradation and tissue damage. Although inhibitors such as Sivelestat and Alvelestat are clinically relevant, their performance within the oxidative microenvironment of diseased lungs remains poorly understood. Here, we employed an integrated in vitro and in silico approach to investigate their behavior under physiological and oxidative conditions and to provide a molecular-level interpretation. Under physiological conditions, enzymatic assays and steady-state kinetics confirmed that both compounds act as competitive inhibitors, with Sivelestat displaying higher baseline potency. Under oxidative stress, however, Sivelestat exhibited a marked reduction in inhibitory potency, whereas Alvelestat retained its efficacy. Molecular modeling and molecular dynamics simulations of native and oxidized HNE variants provided a structural rationale for this divergence. Alvelestat, as a non-covalent inhibitor, maintains stable binding despite increased flexibility of the active site, whereas Sivelestat, acting via a reversible covalent mechanism, requires a precise pre-acylation geometry. Oxidation-induced remodeling of the S1 pocket disrupts the near-attack configuration required for covalent bond formation, thereby impairing inhibition. Overall, these findings indicate that oxidative stress may selectively compromise covalent inhibition while preserving enzymatic activity, and suggest that context-dependent redox-related structural effects may represent a consideration for the design of next-generation HNE inhibitors. Full article

Human neutrophil elastase (HNE) is a central mediator of neutrophil-driven inflammation. Yet, despite decades of research and drug development, therapies targeting HNE have not consistently translated into clear clinical benefits. We suggest that this translational gap partly arises from how HNE has traditionally been conceptualized, as a single enzyme to inhibit. In biological systems, however, HNE operates within a complex and tightly regulated network of proteases and inflammatory mediators. This network is spatially compartmentalized and strongly influenced by local redox conditions, making HNE activity highly context-dependent. From a systems perspective, HNE acts as an amplifier of inflammation. Its extracellular activity connects several pathological processes, including activation of innate immunity, extracellular matrix degradation, disruption of epithelial and endothelial barriers, and the transition toward chronic inflammation. In this review, we integrate insights from enzymology, systems biology, and clinical research to reassess the development of HNE inhibitors, ranging from endogenous antiproteases to more recent reversible synthetic compounds. Despite their chemical and pharmacological diversity, many of these strategies have encountered similar limitations. We therefore argue that future therapeutic approaches should move beyond the inhibition of HNE as an isolated target and instead aim to modulate the broader protease network, with particular attention to drug–target kinetics and precise delivery to disease-relevant microenvironments. Full article

Activating PIK3CA mutations occur in approximately 40% of hormone receptor-positive (HR+)/HER2-negative breast cancers and represent a major driver of endocrine resistance. The PI3Kα-selective inhibitor alpelisib, in combination with fulvestrant, significantly improves progression-free survival in patients with PIK3CA-mutant disease, as demonstrated in the SOLAR-1 trial. However, this therapeutic strategy is frequently complicated by treatment-induced hyperglycemia, a metabolic disturbance that promotes oxidative stress, mitochondrial dysfunction, and inflammatory signaling, thereby increasing cardiovascular vulnerability. Sodium–glucose cotransporter-2 (SGLT2) inhibitors have emerged as cardiometabolic modulators with benefits extending beyond glucose lowering. In this study, we used a human cardiomyocyte in vitro model designed to recapitulate the hyperglycemic metabolic milieu observed in breast cancer patients receiving PI3Kα-targeted therapy, to investigate whether the SGLT2 inhibitor dapagliflozin directly protects cardiomyocytes from alpelisib- and fulvestrant-induced injury. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were cultured under hyperglycemic conditions (25 mM glucose) to mimic the metabolic environment associated with PI3Kα inhibitor-induced dysglycemia. Cells were exposed to alpelisib (100 nM) and fulvestrant (100 nM), alone or in combination, in the absence or presence of dapagliflozin (1 μM). Cardiomyocyte viability was assessed using the MTS assay, mitochondrial function by TMRM-based mitochondrial membrane potential (ΔΨm) measurements, and apoptosis by caspase-3 quantification. Cardiomyocyte injury was evaluated by release of cardiac troponin I and heart-type fatty acid binding protein (H-FABP). Lipid peroxidation markers (MDA and 4-HNE) were measured to assess oxidative membrane damage. Intracellular inflammasome-related signaling (NLRP3 and MyD88) and secreted inflammatory mediators (IL-1β, IL-18, IL-6, TNF-α, and CCL2) were quantified by ELISA. Exposure to alpelisib, particularly in combination with fulvestrant, significantly reduced cardiomyocyte viability, induced mitochondrial depolarization, and increased caspase-3-mediated apoptotic signaling. These alterations were accompanied by elevated lipid peroxidation (MDA and 4-HNE) and increased release of cardiac injury biomarkers (troponin I and H-FABP). Alpelisib-based treatments also activated inflammasome-related signaling, as indicated by increased intracellular NLRP3 and MyD88 levels and enhanced secretion of pro-inflammatory mediators (IL-1β, IL-18, IL-6, TNF-α, and CCL2). Co-treatment with dapagliflozin significantly attenuated these alterations, preserving mitochondrial membrane potential, reducing apoptotic signaling, limiting oxidative membrane damage, and suppressing inflammatory cytokine release. This study provides evidence that alpelisib-based therapy under hyperglycemic conditions is associated with oxidative, mitochondrial, and inflammatory stress responses in human cardiomyocytes, recapitulating key features of cardiometabolic stress relevant to PI3Kα-targeted therapy. Importantly, dapagliflozin markedly attenuated these alterations, supporting a potential cardioprotective role that may extend beyond glycemic control. These findings provide a mechanistic rationale for further investigation of SGLT2 inhibition as a cardiometabolic protective strategy in patients receiving PI3Kα inhibitor-based cancer therapy. Full article

Background: Recurrent vulvovaginal candidiasis (RVVC) is a chronic inflammatory disease primarily caused by Candida albicans (C. albicans). Its pathogenesis remains incompletely understood, and clinical management is challenged by recurrence and drug resistance. Ferroptosis, an iron-dependent form of programmed cell death driven by lipid peroxidation, has been implicated in various infectious and inflammatory diseases. However, its role in RVVC remains unclear, with a particular lack of evidence from clinical samples and animal experiments. Objective: This study aimed to investigate the association between RVVC and ferroptosis. First, we analyzed high-throughput sequencing data from human RVVC samples in the Gene Expression Omnibus (GEO) database to identify the expression profile of ferroptosis-related genes. Second, using an established murine model of chronic vulvovaginal candidiasis (CVVC), we validated changes in ferroptosis-related markers in vaginal tissues in vivo. Furthermore, an in vitro model of C. albicans-infected bone marrow-derived macrophages (BMDMs) was employed to explore the underlying mechanisms. This study provides experimental evidence for elucidating the pathogenesis of RVVC and exploring novel therapeutic strategies. Methods: The RVVC-related gene expression dataset GSE278036 was obtained from the GEO database. Differentially expressed genes (DEGs) were screened using the DESeq2 algorithm and intersected with ferroptosis-related genes from the FerrDb database to identify key targets. A protein–protein interaction (PPI) network was constructed using the STRING database and Cytoscape software, and hub genes were identified via the Betweenness centrality algorithm. Functional and pathway analyses, including gene set enrichment analysis (GSEA), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and WikiPathways, were performed. Immune infiltration analysis characterized the immune microenvironment in RVVC patients. A CVVC mouse model was established in vivo, and a C. albicans-BMDMs infection model was established in vitro. The ferroptosis inhibitor ferrostatin-1 (Fer-1) was administered to investigate the pathological function and regulatory mechanisms of ferroptosis in RVVC at the molecular, cellular, and tissue levels. Results: Differential analysis identified 3132 DEGs in RVVC, which intersected with ferroptosis-related genes to yield 194 key targets. Among them, 20 hub genes were identified, including ferroptosis regulators and inflammatory factors. Functional enrichment analysis confirmed that these shared targets regulate RVVC pathology through a “ferroptosis-inflammation-immunity” multi-pathway network. Immune infiltration analysis revealed a specific immune disorder in RVVC patients characterized by “activation of the pro-inflammatory innate immune axis and suppression of the adaptive immune axis,” which was closely associated with ferroptosis-related genes. In vivo and in vitro experiments confirmed that C. albicans infection induced ferroptosis in vaginal tissues and macrophages, as manifested by lipid ROS accumulation, Fe²⁺ overload, GSH depletion, downregulation of GPX4 and SLC7A11, upregulation of ACSL4, 4-HNE, and MDA, and mitochondrial structural damage. Macrophages were identified as key target cells for ferroptosis, and their ferroptosis led to impaired antifungal function. Fer-1 treatment significantly inhibited ferroptosis, reduced vaginal histopathological damage and inflammatory cell infiltration, decreased fungal burden, downregulated abnormally elevated inflammatory factors, and restored Th1/Th2 immune balance. Furthermore, Fer-1 preserved macrophage viability and enhanced their antifungal killing capacity. Conclusions: This study provides the first evidence linking RVVC to ferroptosis through a combination of clinical data analysis and experiments, suggesting that ferroptosis is involved in its pathological process. These findings offer a new perspective for elucidating RVVC pathogenesis and developing targeted therapeutic strategies. Full article

Diabetic peripheral neuropathy (DPN) is one of the most common chronic complications of diabetes mellitus, driven by oxidative stress, inflammation, and microvascular dysfunction. Dapagliflozin, a selective inhibitor of sodium–glucose cotransporter type 2 (SGLT2), is used in the treatment of type 2 diabetes and has pleiotropic antioxidant and anti-inflammatory effects. The aim of this study was to evaluate the neuroprotective effects of dapagliflozin in an experimental model of streptozotocin (STZ)-induced diabetic peripheral neuropathy in mice. C57BL/6 mice were divided into three groups: control (DM–), STZ-induced diabetes (DM+), and diabetes + dapagliflozin (DM + DAPA, 10 mg/kg/day, oral administration for 12 weeks). Clinical (glycemia, weight, diuresis), electrophysiological, and histopathological parameters were evaluated, and behavioral tests (Open Field, Von Frey, Hot Tail) were performed. Dapagliflozin significantly reduced hyperglycemia, limited weight loss and polyuria, and improved locomotor behavior and nociceptive sensitivity. Electrodiagnostically, the treatment increased the amplitude and reduced the duration of motor potentials, indicating improved nerve conduction. Histological analyses showed decreased hydroxynonenal (HNE) immunoreactivity, suggesting attenuation of oxidative stress, reduced perineural fibrogenesis, and maintained intraepidermal nerve fiber density. Dapagliflozin exerts significant neuroprotective effects in experimental diabetic peripheral neuropathy by reducing oxidative stress, inflammation, and fibrosis and maintaining the structural and functional integrity of peripheral nerves. Full article

Background: Lung ischemia reperfusion injury (LIRI) is a severe complication after lung transplantation (LT). Ferroptosis contributes to the pathogenesis of LIRI. Maresin1 (MaR1) is an endogenous pro-resolving lipid mediator that exerts protective effects against multiorgan diseases. However, the role and mechanism of MaR1 in the ferroptosis of LIRI after LT need to be further investigated. Methods: A mouse LT model and a pulmonary vascular endothelial cell line after hypoxia reoxygenation (H/R) culture were established in our study. Histological morphology and inflammatory cytokine levels predicted the severity of LIRI. Cell viability and cell injury were determined by CCK-8 and LDH assays. Ferroptosis biomarkers, including Fe²⁺, MDA, 4-HNE, and GSH, were assessed by relevant assay kits. Transferrin receptor (TFRC) and Acyl-CoA Synthetase Long Chain Family Member 4 (ACSL4) protein levels were examined by western blotting. In vitro, lipid peroxide levels were detected by DCFH-DA staining and flow cytometry analysis. The ultrastructure of mitochondria was imaged using transmission electron microscopy. Furthermore, the potential mechanism by which MaR1 regulates ferroptosis was explored and verified with signaling pathway inhibitors using Western blotting. Results: MaR1 protected mice from LIRI after LTx, which was reversed by the ferroptosis agonist Sorafenib in vivo. MaR1 administration decreased Fe²⁺, MDA, 4-HNE, TFRC, and ACSL4 contents, increased GSH levels, and ameliorated mitochondrial ultrastructural injury after LTx. In vitro, Sorafenib resulted in lower cell viability and worsened cell injury and enhanced the hallmarks of ferroptosis after H/R culture, which was rescued by MaR1 treatment. Mechanistically, the protein kinase A and YAP inhibitors partly blocked the effects of MaR1 on ferroptosis inhibition and LIRI protection. Conclusions: This study revealed that MaR1 alleviates LIRI and represses ischemia reperfusion-induced ferroptosis via the PKA-Hippo-YAP signaling pathway, which may offer a promising theoretical basis for the clinical application of organ protection after LTx. Full article

This study systematically investigated the inhibitory mechanism of Arctium lappa L. polyphenols (ALP) against human neutrophil elastase (HNE). Molecular docking techniques were employed to predict the binding patterns and inhibition types between polyphenolic components and HNE, complemented by in vitro enzymatic tests to validate inhibitory efficacy. Combination index (CI) analysis was applied to evaluate synergistic effects. Through preliminary in vitro screening, chlorogenic acid, quercetin, and isochlorogenic acid A were identified as key bioactive constituents. Experimental results demonstrated that the half-inhibitory concentration (IC₅₀) of individual compounds against HNE ranged from 46.4 to 203.3 μM, while ALP extract exhibited dose-dependent inhibition (IC₅₀ = 0.99 mg/mL). Drug combination ratios based on individual IC₅₀ values revealed synergistic effects (CI < 1) in chlorogenic acid-quercetin and isochlorogenic acid A-quercetin combinations, whereas antagonism (CI > 1) was observed in chlorogenic acid-isochlorogenic acid A pairs. The molecular docking results predicted that chlorogenic acid and isochlorogenic acid A competitively occupy the same binding site of the target protein (HNE) to exert inhibitory effects, thereby explaining the antagonism produced by their combination. In contrast, quercetin may inhibit HNE with a binding site different from that of chlorogenic acid or isochlorogenic acid A, which accounts for the observed synergistic effects. This study provides the first systematic elucidation of synergistic mechanisms of ALP as natural HNE inhibitors, providing theoretical foundations for developing novel natural HNE inhibitors with potential applications in acute lung injury, COVID-19-associated inflammatory conditions, and chronic inflammatory diseases. Full article

Anthracyclines and human epidermal growth factor receptor 2 (HER-2) inhibitors are cornerstone therapies for breast cancer but are associated with significant cardiotoxicity. While sodium–glucose cotransporter 2 (SGLT2) inhibitors such as dapagliflozin have demonstrated cardio–renal protective effects during anthracycline treatment, their efficacy in preventing cardiotoxicity from sequential anthracycline and HER-2 blockade remains poorly understood. This study investigates the cardioprotective role of dapagliflozin in a preclinical model of chemotherapy-induced cardiotoxicity. Female C57Bl/6 mice were divided into four groups and treated for 10 days as follows: (1) a normal control group receiving saline (sham); (2) a model control group receiving doxorubicin (2.17 mg/kg/day for 5 days) followed by HER-2-blocking monoclonal antibody (2.25 mg/kg/day for 5 days); (3) a dapagliflozin-only group (10 mg/kg/day via oral gavage); and (4) a treatment group receiving the combination of doxorubicin, HER-2 inhibitor, and dapagliflozin. Cardiac function was assessed using echocardiography (VEVO 2100). Biomarkers of myocardial injury and inflammation (NLRP3, MyD88, CXCR4, H-FABP, troponin-T, and cytokines) were quantified via ELISA and immunohistochemistry. Circulating markers such as mitofusin-2, cardiac myosin light chain, malondialdehyde (MDA), and 4-hydroxy-2-nonenal (4-HNE) were also measured. Dapagliflozin significantly preserved the ejection fraction and reduced both radial and longitudinal strain impairment in mice treated with the doxorubicin–HER-2 inhibitor combination (p < 0.001). Levels of myocardial NLRP3, MyD88, CXCR4, H-FABP, interleukin-1β, and troponin-T were significantly lower in the dapagliflozin-treated group compared to the chemotherapy-only group. Serum markers of oxidative stress and cardiac injury, including mitofusin-2, MDA, 4-HNE, BNP, and high-sensitivity C-reactive protein (hs-CRP), were also reduced by dapagliflozin treatment. Our findings demonstrate that dapagliflozin effectively mitigates early cardiac dysfunction and injury in a preclinical model of sequential doxorubicin and HER-2 inhibitor therapy. Full article

GATA-3 is a master regulator of preadipocyte differentiation and function. Pharmacological or genetic targeting of GATA-3 will allow us to understand the function of GATA-3 in regulating metabolism, insulin signaling, and inflammation. Pyrrothiogatain, a novel small molecule inhibitor of GATA family proteins, has emerged as a promising tool for modulating GATA-3 activity. This study aims to investigate the specificity of Pyrrothiogatain in regulating GATA-3-mediated preadipocyte differentiation and adipokine secretion under normal and pathological conditions. Wild-type and GATA-3 knockout 3T3-L1 cells were treated with different concentrations of Pyrrothiogatain in the presence and absence of 4-hydroxy-2-nonenal (4HNE), an inducer of oxidative stress and impairment of adipogenesis. As expected, GATA-3 knockout cells exhibited enhanced adipogenic capacity, characterized by increased cell and lipid droplet sizes, and upregulated expression of key adipogenic markers including CEBPβ, PPARγ, and PGC-1α. Pyrrothiogatain treatment reduced cell proliferation in both wild-type and GATA-3 knockout 3T3-L1 cells, but did not alter their adipogenic capacity. Furthermore, Pyrrothiogatain lowered secreted IL-6 levels and attenuated 4-HNE-induced TNF-α elevation in wild-type, but not in GATA-3 knockout cells. Co-treatment of 4-HNE and Pyrrothiogatain led to increased cell size, suggesting complex interactions between oxidative stress and GATA protein inhibition. This effect was similar to GATA-3 knockout cells, indicating Pyrrothiogatain’s potential to modulate cellular stress responses independently of GATA-3 inhibition. These results reveal that Pyrrothiogatain’s effects on adipocyte biology extend beyond simple GATA-3 inhibition. While GATA-3 knockout primarily affects adipogenesis, Pyrrothiogatain modulates inflammatory responses and potentially cellular stress mechanisms without directly impacting adipocyte differentiation. This study provides new insights into the multifaceted actions of Pyrrothiogatain and highlights its potential as a therapeutic agent for lowering inflammation and oxidative-stress-related aspects of metabolic disorders, distinct from the direct modulation of adipogenesis. Full article

Cisplatin-induced ototoxicity occurs in approximately half of patients treated with cisplatin, and pediatric patients are more likely to be affected than adults. The oxidative stress elicited by cisplatin is a key contributor to the pathogenesis of ototoxicity. Notoginsenoside R1 (NGR1), the main bioactive compound of Panax notoginseng saponins, has antioxidant and antiapoptotic effects. This study investigated the ability of NGR1 to protect against cisplatin-induced damage in auditory HEI-OC1 cells and neonatal murine cochlear explants. The viability of HEI-OC1 cells treated with NGR1 and cisplatin was greater than that of cells treated with cisplatin alone. The results of Western blots and immunostaining for cleaved caspase-3 revealed that the level of cleaved caspase-3 in the cells treated with cisplatin was repressed by NGR1. NGR1 attenuated cisplatin-induced cytotoxicity in HEI-OC1 cells. Intracellular reactive oxygen species (ROS) were detected with a DCFDA assay and immunostaining for 4-HNE. The result revealed that its expression was induced by cisplatin and was significantly reduced by NGR1. Moreover, NGR1 can promote heme oxygenase-1 (HO-1) expression at both the mRNA and protein levels. ZNPPIX, an HO-1 inhibitor, was administered to cisplatin-treated cells to investigate the role of HO-1 in the protective effect of NGR1. The suppression of HO-1 activity by ZNPPIX markedly abolished the protective effect of NGR1 on cisplatin-treated cells. Therefore, NGR1 protects cells from cisplatin-induced damage by activating HO-1 and its antioxidative activity. In cochlear explants, NGR1 protects cochlear hair cells and attenuates cisplatin-induced ototoxicity by inhibiting ROS generation. In the group treated with cisplatin alone, prominent loss of outer hair cells and severe damage to the structure of the stereociliary bundles of inner and outer hair cells were observed. Compared with the group treated with cisplatin alone, less loss of outer hair cells (p = 0.009) and better preservation of the stereociliary bundles of hair cells were observed in the group treated with cisplatin and NGR1. In conclusion, these findings indicate that NGR1 can protect against cisplatin-induced ototoxicity by inducing HO-1 expression and suppressing oxidative stress. Full article

Gastric cancer is one of the most common cancers worldwide, and new therapeutic strategies are urgently needed. Ferroptosis is an intracellular iron-dependent cell death induced by the accumulation of lipid peroxidation, a mechanism different from conventional apoptosis and necrosis. Therefore, induction of ferroptosis is expected to be a new therapeutic strategy. Glutathione peroxidase 4 (GPX4) and ferroptosis suppressor protein 1 (FSP1) have been identified as the major inhibitors of ferroptosis. Herein, we performed immunohistochemistry for GPX4, FSP1, and 4-HNE using tissues from patients with gastric cancer and investigated the relationship between these factors and prognosis. Patients with high GPX4 expression or high GPX4 expression and low 4-HNE accumulation tended to have a poor prognosis (p = 0.036, 0.023), whereas those with low FSP1 expression and high 4-HNE accumulation had a good prognosis (p = 0.033). The synergistic induction of cell death by inhibiting GPX4 and FSP1 in vitro was also observed, indicating that the cell death was non-apoptotic. Our results indicate that the expression and accumulation of lipid peroxidation-related factors play an important role in the clinicopathological significance of gastric cancer and that novel therapeutic strategies targeting GPX4 and FSP1 may be effective in treating patients with gastric cancer who have poor prognosis. Full article

Neutrophil elastase (HNE), like other members of the so-called GASPIDs (Granule-Associated Serine Peptidases of Immune Defense), is activated during protein biosynthesis in myeloid precursors and stored enzymatically active in cytoplasmic granules of resting neutrophils until secreted at sites of host defense and inflammation. Inhibitors thus could bind to the fully formed active site of the protease intracellularly in immature progenitors, in circulating neutrophils, or to HNE secreted into the extracellular space. Here, we have compared the ability of a panel of diverse inhibitors to inhibit HNE in the U937 progenitor cell line, in human blood-derived neutrophils, and in solution. Most synthetic inhibitors and, surprisingly, even a small naturally occurring proteinaceous inhibitor inhibit HNE intracellularly, but the extent and dynamics differ markedly from classical enzyme kinetics describing extracellular inhibition. Intracellular inhibition of HNE potentially affects neutrophil functions and has side effects, but it avoids competition of inhibitors with extracellular substrates that limit its efficacy. As both intra- and extracellular inhibition have advantages and disadvantages, the quantification of intracellular inhibition, in addition to classical enzyme kinetics, will aid the design of novel, clinically applicable HNE inhibitors with targeted sites of action. Full article