Free Radical Biology and Medicine最新文献

Nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy contributes to maintain intracellular iron balance by regulating ferritin degradation, which is essential for redox homeostasis. CXC-motif chemokine receptor 3 (CXCR3) is involved in the regulation of oxidative stress and autophagy. However, its role in modulating intestinal oxidative damage through ferritinophagy and the gut microbiota remains unclear. In this study, the impact of CXCR3 inhibition on intestine oxidative damage, ferritinophagy, and the gut microbiota, as well as mitochondrial quality control was investigated both in vivo and in vitro. The results show that CXCR3 inhibition by AMG487 relieves Diquat-induced intestinal damage, enhances the expression of tight junction proteins, and enhances antioxidant capacity in mice. Simultaneously, CXCR3 inhibition improves gut microbiota composition, and promotes NCOA4-mediated ferritinophagy. Mechanistically, the effects of CXCR3 inhibition on ferritinophagy are explored in IPEC-J2 cells. Co-localization and interaction between CXCR3 and NCOA4 were observed. Downregulation of NCOA4-medicated ferritinophagy leads to increase the expression of tight junction proteins, reduces iron levels, restricts ROS accumulation, and enhances GPX4 expression. Moreover, CXCR3 suppression facilitates mitochondrial biogenesis and mitochondrial fusion, increases antioxidative capacity, as well as resulting in elevation of tight junction proteins. These findings suggest that CXCR3 inhibition reverses Diquat-induced intestinal oxidative damage, enhances mitochondrial function, and improves gut microbiota composition by elevating NCOA4-medicated ferritinophagy, which implies that CXCR3 may serve as a potential therapeutic intervention targeting iron metabolism for treating intestinal diseases.

Recent studies have shown that neuroinflammation and heightened glial activity, particularly astrocyte overactivation, are associated with Alzheimer's disease (AD). Abnormal accumulation of amyloid-beta (Aβ) induces endoplasmic reticulum (ER) stress and activates astrocytes. Artemisinin (ART), a frontline anti-malarial drug, has been found to have neuroprotective properties. However, its impact on astrocytes remains unclear. In this study, we used Aβ_1-42 induced astrocyte cultures and 3 × Tg-AD mice as in vitro and in vivo models, respectively, to investigate the effects of ART on AD related astrocyte overactivation and its underlying mechanisms. ART attenuated Aβ_1-42-induced astrocyte activation, ER stress, and inflammatory responses in astrocyte cultures by inhibiting IRE1 phosphorylation and the NF-κB pathway, as evidenced by the overexpression of IRE1 WT and IRE1-K599A (kinase activity invalidated), along with application of activators and inhibitors related to ER stress. Furthermore, ART alleviated the detrimental effects and restored neurotrophic function of astrocytes on co-cultured neurons, preventing neuronal apoptosis during Aβ_1-42 treatment. In 3 × Tg-AD mice, ART treatment improved cognitive function and reduced astrocyte overactivation, neuroinflammation, ER stress, and neuronal apoptosis. Moreover, ART attenuated the upregulation of IRE1/NF-κB pathway activity in AD mice. Astrocyte-specific overexpression of IRE1 via adeno-associated virus in AD mice reversed the ameliorating effects of ART. Our findings suggest that ART inhibits astrocyte overactivation and neuroinflammation in both in vitro and in vivo AD models by modulating the IRE1/NF-κB signaling pathway, thereby enhancing neuronal functions. This study underscores the therapeutic potential of ART in AD and highlights the significance of modulating the ER stress-inflammatory cycle and normalizing astrocyte-neuron communication.

Proliferative vitreoretinopathy (PVR) is a major cause of rhegmatogenous retinal detachment repair failure. Despite many attempts to find therapeutics for PVR, no pharmacotherapy has been proven effective. Steroids, as the epitome, show uncertain clinical effectiveness, which lacks an explanation and hints at unappreciated mechanisms of PVR. In this study, we investigated the involvement of metabolic reprogramming, mitochondrial impairment, and their association with steroid effectiveness in PVR using dexamethasone (Dex) as an example. Proteomics of vitreous samples from PVR patients demonstrated an upregulation in the glycolysis pathway. Transcriptomics of PVR tissues (dataset GSE179603) revealed downregulations in oxidative phosphorylation (OXPHOS), mitochondrial respiration, and mitochondrial quality control-related pathways. Transcriptomics of TGFβ and TNFα (TNT)-induced retinal pigment epithelial (RPE) cell model (GSE176513) confirmed the changes in glycolysis, OXPHOS, and mitochondria and also revealed downregulation of Dex response pathway with increased duration of TNT exposure. Transcriptomics of mouse RPE/choroid following Dex intravitreal injections (GSE49872) showed that glycolysis decreased at 1-week postinjection but increased at 1-month postinjection; OXPHOS increased but gradually decreased with treatment duration. The dispase-induced mouse PVR model revealed that a simultaneous Dex injection could alleviate PVR severity rather than an injection 5 days after the PVR induction. The TGFβ2-induced RPE cell model demonstrated the enhancement of EMT, oxidative stress, and mitochondrial impairment, which could be alleviated by Dex: Cellular ROS were accumulated; the mRNA expressions of antioxidases (GPX, SOD1 and TXN2) were decreased; mitochondrial morphology and dynamics were impaired, exhibiting decreases in mitochondrial heterogeneity, mitochondrial length and MFN2 expression; Mitochondrial membrane potential showed an elevation; and mitophagy was decreased, related to reduced Parkin recruitment. These results demonstrate the essential roles of metabolic reprogramming and mitochondrial dysfunction in PVR pathology, which is associated with the therapeutic effect of steroids. Steroid intervention might benefit the treatment of PVR in the early rather than late stages.

Aims/hypothesis: Emerging evidence underscored the significance of leucine-rich repeat-containing G protein-coupled receptor (LGR) 4 in endocrine and metabolic disorders. Despite this, its role in LGR4 in hepatic glucose metabolism remains poorly understood. In this study we set out to test whether LGR4 regulates glucose production in liver through a specific signaling pathway.

Methods: Hepatic glucose production and gluconeogenic gene expressions were detected after silence of LGR4 in three obese mice models. Then, whole-body LGR4-deficient (LGR4 KO) mice, liver-specific LGR4 knockout (LGR4^LKO) mice, and liver-specific LGR4 overexpression (LGR4^LOV) mice were generated, in which we analyzed the effects of LGR4 on hepatic glucose metabolism upon HFD feeding, among which live imaging and quantitative analysis of hepatic phosphoenolpyruvate carboxykinase (PEPCK)-luciferase activity were conducted.

Results: LGR4 expression was significantly upregulated in the liver of three obese mouse models, and presented dynamic expression patterns in response to nutritional fluxes. We utilized global and liver-specific LGR4 knockouts (LGR4^LKO), along with adenoviral-mediated LGR4 knockdown in mice, to show improved glucose tolerance and decreased hepatic gluconeogenesis. Specifically, the expression of rate-limiting gluconeogenic enzymes, PEPCK was significantly downregulated. Conversely, mouse model with adenovirus-mediated LGR4 overexpression (LGR4^LOV) exhibited elevated gluconeogenesis and PEPCK expression and reversed the suppression observed in LGR4 knockout models. Notably, neither RANKL nor PKA signaling pathways, which were reported to take part in LGR4's function, were involved in the process of LGR4 regulating PEPCK. Instead, TopFlash reporter system and inhibitors application suggested that LGR4's influence on hepatic gluconeogenesis operates through the canonical Wnt/β-catenin/TCF7L2 signaling pathway.

Conclusions/interpretation: Overall, these findings underscore a novel mechanism by which LGR4 regulates hepatic gluconeogenesis, presenting a potential therapeutic target for diabetes management.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by cognitive decline and memory loss. A critical aspect of AD pathology is represented by oxidative stress, which significantly contributes to neuronal damage and death. Microglia and astrocytes, the primary glial cells in the brain, are crucial for managing oxidative stress and supporting neuronal function. Carnosine is an endogenous dipeptide possessing a multimodal mechanism of action that includes antioxidant, anti-inflammatory, and anti-aggregant activities. The present study investigated the effects of Aβ1-42 oligomers (oAβ), small aggregates associated with the neurodegeneration observed in AD, on primary rat mixed glia cultures composed of both microglia and astrocytes, focusing on the ability of these detrimental species to induce oxidative stress. We assessed intracellular reactive oxygen species (ROS) and nitric oxide (NO) levels as markers of oxidative stress. Exposure to oAβ significantly elevated both ROS and NO intracellular levels compared to control cells. However, this effect was completely inhibited by the pre-treatment of mixed cultures with carnosine, resulting in ROS and NO levels similar to those observed in untreated (control) cells. Single-cell analysis of cellular responses to oAβ revealed heterogeneous ROS production, resulting in two distinct clusters of cells, one of which was very responsive to the treatment. The presence of carnosine counteracted the overproduction of ROS, also leading to a single, homogeneous cluster, similar to that observed in the case of control cells. Interestingly, unlike ROS response, single-cell analysis of NO production did not show any distinct clusters. Overall, our findings demonstrated the ability of carnosine to mitigate Aβ-induced oxidative stress in mixed glia cells, by rescuing ROS and NO intracellular levels, as well as to normalize the heterogeneous response to the treatment measured in terms of clusters' formation. The present study suggests a therapeutic potential of carnosine in pathologies characterized by oxidative stress including AD.

The emergence of cuproptosis, a novel form of regulated cell death, is induced by an excess of copper ions and has been associated with the progression of multiple diseases, including liver injury, cardiovascular disease, and neurodegenerative disorders. However, there are currently no inhibitors available for targeting specific cuproptosis-related pathways in therapy. Here, the compound merestinib (MTB) has been identified as a strong inhibitor of cuproptosis through screening of a kinase inhibitor library. The results show that MTB effectively blocks elesclomol-CuCl₂ (ES-Cu) induced cuproptosis by preventing the aggregation of lipoylated proteins and the destabilization of Fe-S cluster proteins, thereby preventing proteotoxic stress and ultimately cell death. Mechanistically, MTB decreases oxidative stress levels by binding directly to NRF2. Additionally, it boosts the efficiency of the copper homeostasis and facilitates the exocytosis and transportation of copper ions, ultimately inhibiting cuproptosis. Furthermore, our research showed that MTB has the ability to alleviate cuproptosis-driven acute liver injury in mice. These findings suggest that MTB is a specific inhibitor of cuproptosis, presenting a hopeful option for therapeutic approaches in cuproptosis-related diseases.

Cancer remains as a global health threat, with the incidence of breast cancers keep increasing. Dis-regulated redox homeostasis has been considered with essential roles for tumor initiation and progression. Using triple negative breast cancers, the most malignant subtype of breast cancers, as the tumor model, we explored the roles of the anti-oxidant spermidine, the pro-oxidative tool cold atmospheric plasma (CAP), and their combined use in cancer growth, anti-oxidative ability and cell cycle. We also characterized the important roles of FTO in driving the redox modulatory functionalities of spermidine and CAP-activated medium (PAM) as well as their demonstrated synergy on breast cancer cells. We found that spermidine reversed the anti-cancer effect of PAM and stimulated outrageous progression of transformed cells to the level exceeding that treated with spermidine alone, and combined launch of spermidine and PAM enabled cancer cells with elevated anti-oxidant ability and enhanced survival in response to instant redox perturbation via transient stalk at the G₀/G₁ stage. We, in addition, identified the vital role of FTO in mediating the observed effect of spermidine, PAM and their synergy, on triple negative breast cancer cells. Our results reported the antagonism between PAM and anti-oxidants as represented by spermidine for cancer treatment, and implicated the differential responses of healthy and diseased individuals to anti-oxidants for improved design on redox-based anti-cancer regimen.

Manganese superoxide dismutase (MnSOD/SOD2) is an essential mitochondrial enzyme that detoxifies superoxide radicals generated during oxidative respiration. MnSOD/SOD2 lysine 68 acetylation (K68-Ac) is an important post-translational modification (PTM) that regulates enzymatic activity, responding to nutrient status or oxidative stress, and elevated levels have been associated with human illness. To determine the in vivo role of MnSOD-K68 in the heart, we used a whole-body non-acetylation mimic mutant (MnSOD^K68R) knock-in mouse. These mice exhibited several cardiovascular phenotypes, including lower blood pressure, decreased ejection fraction, and importantly, dilated cardiomyopathy, as evidenced by echocardiography at four months of age. In addition, both mouse embryo fibroblasts (MEFs) and cardiovascular tissue from MnSOD^K68R/K68R mice exhibited an increase in cellular senescence. Finally, MnSOD^K68R/K68R mouse hearts also showed an increase in lipid peroxidation. We conclude that constitutively active MnSOD detoxification activity, lacking the normal switch between non-acetylated and acetylated forms, dysregulates mitochondrial physiology during development, leading to dilated cardiomyopathy.

Inhaled nitric oxide (iNO) is a selective pulmonary vasodilator that is used as a treatment for persistent pulmonary hypertension in neonates (PPHN) with hypoxic respiratory failure. The generation of reactive oxygen and nitrogen species might induce oxidative/nitrosative damage to multiple organs. There is an increasing scientific and clinical interest in the determination of specific biomarkers to measure the degree of oxidative/nitrosative stress in non-invasively collected biofluids. A method for the simultaneous detection of a panel of oxidative and nitrosative stress-related biomarkers for quantifying damage to proteins and DNA/RNA in 20 μL of infant urine samples based on reversed-phase ultra-performance liquid chromatography coupled to tandem mass spectrometry operating in positive electrospray ionization mode (ESI⁺) was optimized and validated. Infant urine samples from two different studies were analyzed: (i) term and preterm infants from a nutrition study (Nutrishield, N = 50) and (ii) infants with respiratory insufficiency, including infants with PPHN (N = 16) that required iNO treatment and a control group without treatment (N = 14). Eleven of 14 metabolites were detected in >50 % of infant urine samples, with ranges between 0.008 and 1400 μmol/g creatinine. When comparing across groups, differences in samples collected after iNO treatment in comparison to the rest of the groups were found for m-tyrosine (m-Tyr and m-Tyr/Phe) and ortho-tyrosine (o-Tyr and o-Tyr/Phe) (p-values <0.001, Wilcoxon rank-sum test). Positive linear relationships were found with NO exposure corrected by infant weight for m-Tyr, m-Tyr/Phe, o-Tyr, o-Tyr/Phe and 3-nitrotyrosine. Future studies will focus on the evaluation of the impact of iNO treatment on health and oxidative/nitrosative stress-related morbidities associated with prematurity.

Iron accumulation and mitochondrial dysfunction in astroglia are reported in Parkinson's disease (PD). Astroglia control iron availability in neurons in which dopamine (DA) synthesis is affected in PD. Despite their intimate relationship the role of DA in astroglial iron homeostasis is limited. Here we show that DA degrades iron storage protein ferritin in astroglial cells involving lysosomal proteolysis. Lysosomal ferritinophagy is mainly associated with macroautophagy; however, we revealed the involvement of chaperone-mediated autophagy (CMA) in DA-induced ferritin degradation. In CMA, cytosolic proteins containing a specific pentapeptide motif bind with HSC70 to be transported to lysosome mediated by LAMP2A. We identified the conserved pentapeptide motif in ferritin-H (Ft-H), mutations of which resulted loss of its interaction with HSC70. Pharmacological inhibitors of HSC70 or LAMP2/2A knockdown blocks DA-induced Ft-H degradation. DA also induces cytosolic cargo NCOA4 for ferritinophagy. We further reveal that DA promotes cathepsin B to lysis ferritin within the lysosome. Inhibitor of cathepsin B, knocking down of LAMP2, or HSC70 inhibitor attenuate DA-induced elevated mitochondrial iron level. Our results establish a direct role of DA on astroglial iron homeostasis and novel involvement of CMA in ferritin degradation in response to a biological stimulus. These results also may help in better understanding iron dyshomeostasis and mitochondrial dysfunction reported in PD.