Free Radical Biology and Medicine最新文献

Elevated synovial expression of the triggering receptor expressed on myeloid cells 1 (TREM1) has been identified as a significant biomarker for assessing disease activity in rheumatoid arthritis (RA). The upregulated expression of TREM1, induced by inflammatory mediators in infiltrating macrophages, plays a critical role in synovitis and joint destruction in RA. Our previous sequencing data linked TREM1 activation to aberrant mitophagy. Thus, we explored the efficacy of targeting TREM1 in treating experimental arthritis and its regulatory effect on mitophagy. TREM1 signalling activation was assessed via TREM1, DAP12, and p-SYK levels, and mitophagy was measured through PINK1, PARKIN, and LC3A/B levels. In vitro, TREM1-overexpressing RAW264.7 cells were generated, and the differences in expression and pathways were analyzed via RNA-seq. Changes in the number and morphology of mitochondria and mitophagy in TREM1-overexpressing RAW264.7 cells and normal control were observed via transmission electron microscopy, MitoTracker confocal microscopy and mitochondrial membrane potential analysis. The promotion of TOMM40 gene transcription by TREM1-activated E2F1 was determined via ChIP-PCR and E2F1 siRNA. We found that TREM1 was highly expressed and activated in the synovial tissues of CIA mice concomitant with abnormal mitophagy. The mitochondrial outer membrane transporter TOMM40 was upregulated in experimental arthritis, and the protein levels of PINK1 and LC3B were decreased. RNA-seq analysis indicated that mitophagy-related proteins were extensively downregulated and that the transcription factor E2F1 and the mitochondrial outer membrane transporter TOMM40 were significantly upregulated in TREM1-overexpressing cells. ChIP-PCR revealed that TREM1 overexpression significantly promoted the interaction between E2F1 and TOMM40 gene in RAW264.7 cells. E2F1 knockdown markedly reversed TOMM40 upregulation, mitophagy injury and ROS production in TREM1-overexpressing macrophages but not in control cells. Our study provides preliminary evidence that E2F1 regulates TOMM40 transcription and disrupts mitophagy flux in TREM1-activated macrophages. Inhibiting TREM1 effectively mitigated experimental arthritis by restoring macrophage mitophagy and reducing intracellular ROS levels.

Cutaneous melanoma is a highly invasive, heterogeneous and treatment resistant cancer. It's ability to dynamically shift between transcriptional states or phenotypes results in an adaptive cell plasticity that may drive cancer cell invasion or the development of therapy resistance. The expression of peroxidasin (PXDN), an extracellular matrix peroxidase, has been proposed to be associated with the invasive metastatic melanoma phenotype. We have confirmed this association by analysing the transcriptomes of 70 metastatic melanoma cell lines with variable levels of PXDN expression. This analysis highlighted a strong association between high PXDN expression and the undifferentiated invasive melanoma phenotype. To assess the functional role of PXDN in melanoma invasion, we performed a knockout of PXDN in a highly invasive cell line (NZM40). PXDN knockout decreased the invasive potential by ∼50% and decreased the expression of epithelial-mesenchymal transition and invasive marker genes as determined by RNAseq and substantiated by proteomics analysis. Bioinformatics analysis of differentially expressed genes following PXDN knockout highlighted decreases in genes linked to extracellular matrix formation, organisation and degradation as well as signalling pathways such as the WNT pathway. This study provides compelling evidence that PXDN plays a functional role in melanoma invasion by promoting an invasive, mesenchymal-like transcriptional phenotype.

Coenzyme Q₁₀ (CoQ₁₀) is a critical component of the mitochondrial respiratory chain. CoQ₁₀ deficiencies often cause a variety of clinical syndromes, often involving encephalopathies. The heterogeneity of clinical manifestations implies different pathomechanisms, reflecting CoQ₁₀ involvement in several biological processes. One such process is cholesterol homeostasis, since CoQ₁₀ is synthesized through the mevalonate pathway, which also produces cholesterol. To elucidate the role of lipid dysfunction in the pathogenesis of CoQ₁₀ deficiency, we investigated lipid metabolism in human CoQ₁₀ deficient iPSCs-derived neurons, and in SH-SY5Y neurons after pharmacological manipulation of the mevalonate pathway. We show that CoQ₁₀ deficiency causes alterations in cholesterol homeostasis, fatty acids oxidation, phospholipids and sphingolipids synthesis in neurons. These alterations depend on the molecular defect, and on the residual CoQ₁₀ levels. Our results imply that CoQ₁₀ deficiencies can induce pathology by altering lipid homeostasis and the composition of cellular membranes. These findings provide further understanding of the mechanisms underlying CoQ₁₀ deficiency and point to potential novel therapeutic targets.

The lack of effective protection against UVB radiation, that severely disrupts the metabolism of keratinocytes, underlines the search for bioactive compounds that would provide effective protection without causing side effects. Therefore, the aim of the study has been to assess the effect of two compounds, that are different in terms of structure and properties: 3-O-ethyl ascorbic acid-EAA (a stable derivative of vitamin C) and cannabigerol-CBG, used separately or concurrently, on the metabolism of keratinocytes previously exposed to UVB. The obtained results indicate diverse, yet mutually reinforcing localization of the tested compounds, both within the membrane structures and cytosol. When used concurrently, EAA + CBG effectively prevent modifications of the structure of cell membranes, particularly the increase in their fluidity and permeability caused by UVB. It promotes cell survival and enhances the expression of membrane transporters, especially BCRP. Moreover, the concurrent use of both compounds, by reducing the level of ROS and regulating the expression of both Nrf2 activators (p62, MAPK) and inhibitors (Keap1, Bach1, PAGM5), supports the antioxidant efficiency of cells, visible in the increased activity of antioxidant enzymes (SOD1/2, CAT) and the effectiveness of GSH- and Trx-dependent antioxidant systems. Consequently, oxidative modifications of lipids (assessed as 4-HNE and isoprostanes) and proteins (measured as 4-HNE-protein adducts and carbonyl groups) are reduced. The tested compounds also reveal anti-inflammatory effects by modifying the expression of the activator (p62) and inhibitors (IKKα, IKKβ) of NFκB. The observed EAA + CBG effect in preventing changes in the structure and functionality of keratinocyte membranes, maintaining redox balance, and mitigating inflammatory effects caused by UVB provides the basis for further research.

Enzymatically formed side-chain oxysterols function as signaling molecules regulating cholesterol homeostasis and act as intermediates in the biosynthesis of bile acids. In addition to these physiological functions, an imbalance in oxysterol homeostasis has been implicated in pathophysiology. Cholesterol 25-hydroxylase (CH25H) and its product 25-hydroxycholesterol (25-OHC), also formed by autoxidation, are associated with amyotrophic lateral sclerosis. However, the effects of 25-OHC on cell viability in glial cells remain unclear. This study demonstrates that 25-OHC induces ferroptosis, an iron-dependent programmed cell death, in mouse Schwann IMS32 cells. Mechanistically, 25-OHC suppressed the expression of selenoprotein glutathione peroxidase 4 (GPX4) at both the transcriptional and translational levels by inhibiting the processing of sterol regulatory element-binding proteins (SREBPs). In addition, 25-OHC upregulated the expression of NADH-cytochrome b5 reductase 1 (CYB5R1) and NADPH-cytochrome P450 reductase (POR), enzymes that promote lipid peroxidation. We further found that 25-OHC increases the expression of glutathione-specific gamma-glutamylcyclotransferase 1 (CHAC1) and decreases glutathione levels. Importantly, non-cytotoxic concentrations of 25-OHC enhanced cellular sensitivity to ferroptosis inducers by downregulating GPX4 expression. These findings reveal a multifaceted approach whereby 25-OHC induces ferroptosis through SREBP pathway suppression and redox imbalance in mouse Schwann IMS32 cells.

Cell death under stress conditions like hypoxia, involves multiple interconnected pathways. In this study, a stable dihydroorotate dehydrogenase (DHODH) knockdown human corneal epithelial cell line was established to explore the regulation of hypoxic cell death, which was mitigated by various cell death inhibitors, particularly by a lipid peroxyl radical scavenger liproxstatin-1 (Lip-1), suggesting that hypoxic cell death involves crosstalk of ferroptosis and PANoptosis. We discovered that both DHODH and Glutathione peroxidase 4 (GPX4) protected cells from hypoxic death by inhibiting lipid peroxidation, mitochondrial reactive oxygen species (ROS) and maintaining mitochondrial membrane potential. However, upregulation of DHODH suppressed GPX4 upstream, exhibiting a trade-off in the expression levels between DHODH and GPX4 under hypoxia, with DHODH exerting a more decisive impact on cell survival. DHODH knockdown under hypoxia did not significantly alter lipid peroxidation levels, demonstrating the balance between DHODH and GPX4 expression finely regulated cellular ferroptosis homeostasis. This study highlights the complex interplay between ferroptosis and PANoptosis in hypoxic cell death, particularly the dual role of DHODH in regulating both pathways. DHODH is not merely maintaining the quantity of mitochondria but is promoting the selection of mitochondria favorable to cell survival. These findings not only deepen our understanding of cell death but also suggest potential therapeutic strategies for diseases involving oxidative stress and mitochondrial dysfunction.

Autologous fat grafting is a widely used technique in plastic and reconstructive surgery, but its efficacy is often limited by the poor survival rate of transplanted adipose tissue. This study aims to enhance the survival of fat grafts by investigating the role of thymosin beta-4 (Tβ4) in facilitating mitochondrial transfer from adipose-derived stem cells (ADSCs) to adipocytes and newly formed blood vessels within the grafts via tunneling nanotubes (TNTs). We demonstrate that Tβ4 upregulates the Rac/F-actin pathway, leading to an increased formation of TNTs and subsequent transfer of mitochondria from ADSCs. This process mitigates oxidative stress, reduces apoptosis, and promotes revascularization, thereby improving the quality and volume retention of fat grafts. Our findings provide a novel mechanistic insight into the enhancement of fat graft survival and suggest that mitochondrial transplantation and Tβ4 are potential therapeutic strategies to improve clinical outcomes in autologous fat transfer procedures.

Perioperative neurocognitive disorders (PND) are common complications following surgery and anesthesia, especially in the elderly. These disorders are associated with disruptions in neuronal energy metabolism and mitochondrial function. This study explores the potential of intranasal insulin administration as a therapeutic strategy to prevent PND by targeting the calcium transport protein complex IP3R/GRP75/VDAC1 on mitochondria-associated endoplasmic reticulum membranes (MAMs).

Methods: Male C57BL/6J mice underwent partial hepatectomy to induce PND and were subsequently treated with either intranasal insulin or saline. Cognitive function was evaluated using the Morris water maze test, and hippocampal tissue was analyzed for calcium transport protein complex IP3R/GRP75/VDAC1 expression and apoptosis markers. In vitro, HT22 and BV2 cell co-cultures were utilized to simulate surgical injury, with IP3R knockdown employed to assess its effects on oxidative stress and apoptosis.

Results: Intranasal insulin effectively alleviated cognitive impairment as demonstrated by improved performance in the Morris water maze. It significantly reduced neuronal apoptosis and modulated the expression of the IP3R/GRP75/VDAC1 complex, enhancing mitochondrial ATP production and stabilizing MAMs. Furthermore, insulin administration also increased PI3K/AKT signaling, counteracting the impact of surgical stress. In vitro experiments confirmed that IP3R knockdown mitigated inflammation-induced oxidative stress and neuronal apoptosis, while insulin's beneficial effects were blocked by inhibition of the PI3K/AKT pathway.

Conclusion: Intranasal insulin mitigates PND by modulating the IP3R/GRP75/VDAC1 complex and enhancing mitochondrial function through the PI3K/AKT signaling pathway. This study supports the potential of intranasal insulin as a promising therapeutic strategy for preventing and managing PND, potentially leading to improved surgical outcomes for elderly patients.

Previous studies have demonstrated that high-mobility group box protein 1(HMGB1) was increased and released to the extracellular and participated in the pathogenesis of steroid-insensitive asthma induced by toluene diisocyanate (TDI). Mitochondrial dysfunction of bronchial epithelia is a critical feature in TDI asthma. However, whether mitochondrial dysfunction regulated HMGB1 release in asthma remains unknown. The aim of this study was to explore whether phosphoglycerate mutase family member 5 (PGAM5), a mitochondrial protein, can regulate HMGB1 release in TDI-induced asthma. The gene expression data series (GSE) 67472 from gene expression omnibus (GEO) database was analyzed to compare the levels of PGAM5 in airway epithelial cells from asthma patients and healthy individuals. Male C57BL/6J mice were sensitized and challenged with TDI and treated with the PGAM5 inhibitor LFHP-1c. In vitro, human bronchial epithelial cells(16HBE) were stimulated by TDI-human serum albumin (HSA) and pretreated with PGAM5 siRNA. In this study, we observed PGAM5 expression was notably increased in airway epithelial cells of asthma patients and TDI-induced asthma mice. In vivo, inhibition of PGAM5 significantly ameliorated airway inflammation, airway hyperresponsiveness (AHR) and mucus hypersecretion, coupled with the decrease of pulmonary HMGB1 expression and release in TDI-exposed mice. In vitro, inhibition of PGAM5 improved mitochondrial dysfunction, decreased the production of reactive oxygen species (ROS) in mitochondrial. Knockdown of PGAM5 reduced the release of cytochrome C (cyt c) and HMGB1 release in TDI-induced asthma. Mechanistically, PGAM5 in bronchial epithelial cells treated by TDI-HSA significantly increased the dephosphorylation of Bax at the S184 residue, promoted the translocation of Bax to mitochondria, and contributed to the activation of mitochondrial-dependent apoptosis in TDI-induced asthma. Based on these findings, we uncovered a novel regulatory mechanism by which high PGAM5 expression promotes airway inflammation by mediating HMGB1 release in TDI-induced asthma, identifying the therapeutic effects of targeting PGAM5 in steroid-insensitive asthma model.

We found recently that a C-C bonding phenyl-quinone product was produced with high yield (96 %) from the reaction between 2,5-dichloro-1,4-benzoquinone (DCBQ) and N-phenylbenzohydroxamic acid (N-PhBHA) via an unusual Claisen rearrangement mechanism, accompanied with the concurrent formation of the minor byproducts amide (N-phenylbenzamide, N-PhBA; only 2 % yield) and hydroxychloroquinone (2 % yield). Surprisingly, when DCBQ was replaced with its reduced form 2,5-dichloro-1,4-hydroquinone (DCHQ), no C-C bonding product was detected, whereas N-PhBA (83 % yield) and hydroxychloroquinone (80 % yield) became the predominant products, indicating a dramatic mechanistic shift. The ascorbate reduction experiment suggested that it was not DCHQ itself, but its corresponding semiquinone radical, that directly reacts with N-PhBHA. Analogous results were observed when N-PhBHA was substituted with its N-methylated analog (N-methyl Benzohydroxamic acid, N-MeBHA), and when DCHQ was replaced with other halohydroquinones. Taking advantage of the relative stability of the N-MeBHA-quinone conjugate intermediate, we demonstrated that this quinone conjugate was capable of being reduced to its semiquinone form by DCHQ. Taken together, we proposed an unusual semiquinone-mediated self-catalysis redox mechanism for the reaction between halohydroquinones and N-substituted hydroxamic acids.