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Phosphocreatine (PCr), a high-energy phosphate donor with established cytoprotective and antioxidative properties, is known to support endothelial function. However, its role in the regulation of cardiac fibrogenesis remains poorly defined. This study aimed to determine whether PCr attenuates isoproterenol (ISO)-induced cardiac fibrosis and to examine the potential involvement of the nuclear factor erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) signaling pathway. A murine model of cardiac fibrosis was established by subcutaneous administration of ISO. Mice were treated with PCr, and cardiac tissues were analyzed for histopathological alterations, collagen deposition, and markers of oxidative stress, endothelial integrity, and fibroblast activation. The expression and nuclear translocation of Nrf2 and its downstream antioxidant enzymes, including heme oxygenase-1 (HO-1) and superoxide dismutase (SOD), were evaluated by immunoblotting and immunofluorescence. Serum creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) levels were measured to assess myocardial injury. ISO administration induced marked myocardial fibrosis characterized by excessive collagen accumulation, oxidative damage, myofibroblast activation, and endothelial disruption. PCr treatment significantly preserved myocardial architecture and reduced interstitial collagen deposition. PCr treatment is associated with enhanced Nrf2 nuclear translocation and upregulation of HO-1 and SOD, while reducing lipid peroxidation, as indicated by decreased malondialdehyde (MDA) levels. Associated with downregulation of α-smooth muscle actin (α-SMA) and collagen type I, these findings highlight the therapeutic potential of PCr in preventing cardiac fibrosis and adverse myocardial remodeling. PCr also maintained endothelial integrity (increased CD31 expression) and reduced serum CK-MB and LDH levels, indicating attenuation of ISO-induced cardiac injury. PCr confers robust antifibrotic and antioxidant protection and is associated with activation of the Nrf2/ARE signaling pathway, suppression of fibroblast activation, and preservation of endothelial function. These findings highlight the therapeutic potential of PCr in preventing cardiac fibrosis and adverse myocardial remodeling.
Atherosclerosis (AS) is a leading global cause of cardiovascular mortality. Gynostemma pentaphyllum (GP), a well-recognized traditional Chinese medicine with a long history of medicinal application, has emerged as a promising candidate for anti-atherosclerotic intervention due to its well-documented potential in regulating cardiovascular homeostasis. This study aimed to systematically elucidate the anti-atherosclerotic mechanisms of GP using metabolomics, network pharmacology, molecular docking, molecular dynamics (MD) simulations, and in vitro validation. Leaf and root samples from five-leaf and seven-leaf gynostemma were analyzed by UHPLC-MS/MS for metabolite profiling. Active components and targets of GP were retrieved from TCMSP and PharmMapper, while AS-related targets were collected from GeneCards, OMIM, DrugBank, and DisGeNET. PPI networks were constructed using Cytoscape, and functional enrichment was analyzed via GO and KEGG. Molecular docking and MD simulations assessed binding affinities between GP components and core targets. Based on the bioinformatics analysis, PPARγ was chosen as a key therapeutic target for further in vitro experimental validation. Metabolomics identified 1898 compounds, with 208 differentially accumulated metabolites between GP varieties. Network pharmacology revealed 24 active components, 168 potential targets, and 69 AS-overlapping targets. Twelve core genes were identified, including AKT1, ALB, PPARγ, ESR1, CASP3, MMP9, EGFR, SRC, MMP2, MAPK1, MPO, and MAPK8. Enrichment analysis linked these targets to lipid metabolism, efferocytosis, and inflammation pathways. Molecular docking and MD simulations confirmed strong and stable binding of Gypenoside XL to PPARγ. Gypenoside A, one of the relatively abundant constituents among gypenosides (GPs), was also directly bound to PPARγ. In vitro, GPs reduced lipid accumulation, upregulated PPARγ and LXRα, suppressed NF-κB phosphorylation and nuclear translocation and attenuated oxidative stress. These coordinated regulations on lipid metabolism, inflammation, and oxidative stress collectively mitigate key pathological processes of AS, including foam cell formation and atherosclerotic plaque progression. GP exerts anti-atherosclerotic effects through a multi-component, multi-target mechanism, with activation of the PPARγ-LXRα pathway and inhibition of NF-κB driven inflammation being central to its therapeutic action.
The flavonoid cirsimaritin has pharmacological activities that potentially in modulate inflammatory responses, tumor progression, and glucolipid metabolism. However, its influence on cytochrome P450 (P450) enzyme activity remains unexplored. This study examines the influence of cirsimaritin on P450 activity, aiming to offer further guidance for its clinical use. The effect of cirsimaritin on key P450 isoform activities was investigated in pooled human liver microsomes (HLMs) using specific substrates. The half-maximal inhibitory concentrations (IC50) for the inhibition of P450 isoforms were determined using different doses of cirsimaritin. The type of inhibition was further assessed using different concentrations of substrates. Furthermore, time-dependent experiments were performed to obtain the corresponding kinetic parameters. Cirsimaritin inhibited the activity of CYP1A2, CYP3A4, and CYP2C9 with significant concentration-dependent changes, and the IC50 values were 18.4 ± 2.7, 15.3 ± 2.6, and 9.67 ± 1.9 μM, respectively. Cirsimaritin exhibited non-competitive inhibition of CYP3A4, with the inhibition constant (Ki) value of 7.81 ± 0.79 μM. Cirsimaritin was a competitive inhibitor of CYP1A2 and CYP2C9, with Ki values of 9.33 ± 0.65 μM and 4.81 ± 0.51 μM, respectively. In addition, the inhibitory effect of cirsimaritin on CYP3A4 was further found to be time-dependent, with the maximal rate of enzyme inactivation (kinact) value of 0.035 ± 0.005 min-1 and the concentration of cirsimaritin at half of the kinact (KI) value of 6.52 ± 0.81 μM. The observed inhibition of CYP1A2, CYP3A4, and CYP2C9 by cirsimaritin indicates its potential to cause herb-drug interactions with drugs metabolized by these isoforms. Further in vivo studies are needed to confirm such interactions.
Selenoproteins are critical regulators of redox homeostasis, protein folding, and metabolism, and their dysregulation has been implicated in cancer biology. Among them, selenoprotein F (SELENOF) has been reported to be tumor suppressive, whereas the RNA-binding protein EIF4A3, a component of the exon junction complex, has been implicated in post-transcriptional repression of selenoproteins. The regulatory and clinical significance of this interaction in colorectal adenocarcinoma (COAD) remains unclear. We performed an integrative analysis of transcriptomic data from The Cancer Genome Atlas (TCGA), proteomic data from the Clinical Proteomic Tumor Analysis Consortium (CPTAC), and patient tissue microarrays. Western blotting, qRT-PCR, and immunofluorescence staining were used to examine SELENOF, GPX1, and EIF4A3 expression in colon cancer cell lines and tumor tissues. Correlation, regression, and survival analyses were conducted, and pathway enrichment was assessed using gene set enrichment analysis (GSEA) of RNA and proteome correlation profiles. Motif discovery and translational efficiency analyses were performed to identify 3'-UTR features associated with EIF4A3 repressive activity. SELENOF and EIF4A3 showed inverse, stage-dependent protein expression patterns in COAD in the CPTAC cohort. Survival analyses demonstrated that SELENOF alone was not prognostic but acquired significance in EIF4A3-high tumors, where low SELENOF was associated with poor outcomes. Motif analyses identified enriched 3'UTR elements in SELENOF, suggesting that EIF4A3 represses translation through non-SECIS motifs positioned near canonical SECIS elements. Our findings explore a novel EIF4A3-SELENOF regulatory axis in colorectal cancer. SELENOF acquires conditional prognostic significance only in the context of elevated EIF4A3, highlighting the importance of molecular interaction specificity in biomarker discovery.
Polyphyllin VII is a major active component extracted from the traditional Chinese medicinal plant Paris polyphylla. It exhibits antitumor and anti-inflammatory activities, positioning it as a promising drug candidate. Nevertheless, its potential to induce drug-drug interactions remains unknown. The effects of polyphyllin VII on eight key cytochrome P450 (CYP450) isoenzymes were assessed using human liver microsomes, providing insights to inform its drug development and clinical use. Using specific probe substrates, the effects of polyphyllin VII on CYP1A2, 2A6, 2C9, 2D6, 2C19, 2C8, 2E1, and 3A4 were evaluated in human liver microsomes. The inhibition patterns were characterized through Lineweaver-Burk plots to determine the corresponding kinetic parameters. Polyphyllin VII suppressed the activity of CYP2C19, 2D6, 2E1, and 3A4. The inhibition of CYP2C19, 2D6, 2E1, and 3A4 by polyphyllin VII was concentration-dependent, with IC50 values of 21.41 ± 2.59, 18.88 ± 2.20, 9.83 ± 1.85, and 12.54 ± 1.93 μM, respectively. Polyphyllin VII was a competitive inhibitor of CYP2C19, 2D6, and 2E1, and a noncompetitive inhibitor of CYP3A4, with Ki values of 10.70, 9.52, 5.28, and 6.39 μM, respectively. Additionally, the inhibitory effect of polyphyllin VII on CYP3A4 was time-dependent, with KI and Kinact values of 5.60 μM and 0.038 min-1. This study highlights the inhibitory characteristics of polyphyllin VII on the activity of CYP2C19, 2D6, 2E1, and 3A4. Although these findings require further in vivo studies and clinical validation, polyphyllin VII has the potential to interact with other drugs metabolized by CYP2C19, 2D6, 2E1, and 3A4.
Benzene is a prevalent and potent carcinogenic air pollutant that poses significant risks to the hematopoietic system, even at low-level exposure. Although the blood toxicity of benzene has been widely confirmed, the potential molecular mechanisms of benzene poisoning have not yet been elucidated. In this study, we established an in vitro injury model by treating human chronic myeloid leukemia K562 cells with 1,4-benzoquinone (1,4-BQ), the primary toxic metabolite of benzene. We utilized molecular biology experimental methods such as immunofluorescence, qRT PCR, and Western blotting to quantitatively detect the expression levels of NLRP3, caspase-1, GSDMD, and related inflammatory cytokines. Furthermore, we evaluated the protective effect of MCC950 by detecting changes in the expression levels of various key proteins after intervention with NLRP3 specific inhibitor MCC950, elucidating the molecular regulatory pathway of benzene induced pyroptosis toxicity, and identifying early biomarkers of benzene toxicity. 1,4-BQ induced a dose-dependent and time-dependent reduction in K562 cell viability, accompanied by statistically significant increase in lactate dehydrogenase (LDH) release and morphological features characteristic of pyroptosis, such as cell swelling and membrane rupture. Exposure to 1,4-BQ markedly upregulated the expression of the NLRP3 inflammasome, activated caspase-1, and the N-terminal fragment of GSDMD. This was consistent with an increased release of pro-inflammatory cytokines (IL-1β and IL-18) and a decrease in the anti-inflammatory cytokine IL-10. Notably, intervention with MCC950 significantly attenuated these pyroptotic markers and mitigated the inflammatory response. Our findings demonstrate that benzene metabolites trigger cell pyroptosis via the canonical NLRP3/Caspase-1/GSDMD signaling pathway, which also enhances and enriches the molecular regulatory network of benzene induced hematopoietic toxicity In addition, we found that targeting NLRP3 may provide a promising prevention and treatment strategy for benzene induced hematopoietic injury, and provide potential molecular targets for the development of preventive drugs and early intervention agents for benzene poisoning, which has important research significance.
Esophageal cancer (ESCA) is a common gastrointestinal tumor with high incidence and metastatic potential. Cystatin 2 (CST2) has been identified as a carcinogenic factor that regulates the progression of esophageal squamous cell carcinoma (ESCC). However, its role in ESCA progression and the underlying molecular mechanisms require further investigation. Data from several databases were analyzed to examine gene expression and correlations, to confirm the correlation of gene expression with clinical pathological parameters and prognosis of ESCA patients, and to screen for transcription factors. CST2 and Spi-1 proto-oncogene (SPI1) expression was detected by quantitative real-time PCR (qRT-PCR) and Western blot. Cell functions were assessed using cell counting kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU) staining, colony formation, flow cytometry, wound healing, transwell, and tube formation assays. In vivo, xenograft tumor models were established to investigate the effect of CST2 knockdown on ESCA tumor growth. Mechanically, the binding between SPI1 and the CST2 promoter was confirmed by chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays. CST2 was up-regulated in ESCA cells and tissues, and its expression was associated with clinical pathological features. Knockdown of CST2 inhibited ESCA cell proliferation, migration, invasion, and angiogenesis, while inducing apoptosis. In vivo, CST2 down-regulation suppressed ESCA tumor growth. Moreover, SPI1 was identified as an upstream transcription factor of CST2, and its expression was positively correlated with CST2 expression. Clinically, the expression of SPI1 was also correlated with clinical pathological features in ESCA patients. Mechanically, SPI1 promoted ESCA malignant progression by transcriptionally activating CST2. In conclusion, SPI1 transcriptionally activates CST2 to promote ESCA cell proliferation, metastasis and angiogenesis. Moreover, the SPI1/CST2 axis is associated with aggressive clinical pathological features and poor prognosis in ESCA patients, highlighting its potential as a prognostic biomarker and therapeutic target for this malignancy.
The widespread use of UV filters and their release into the environment raise concerns due to their potential harmful effects on living organisms. Consequently, the toxicity and bioaccumulation capacity of UV filters are currently the subject of an intensive study. This work investigates the effect of two UV filters, Oxybenzone (Oxy) and Avobenzone (Avo), on the monolayers formed by the lipids typical for bacteria membranes, as well as on the mixed lipid films imitating E. coli and S. aureus membranes. The studied lipids differed in their hydrophobic and hydrophilic parts, enabling discussion of the effect of lipid structure and monolayer composition on UV filter affinity for the model systems. The experiments involved surface pressure/area measurements, penetration studies, and Brewster angle microscopy investigations. The in vitro tests on various bacteria strains were also performed. The studied UV filters showed varying affinity for the lipids and membrane models tested. Avo modified the properties of the investigated systems more strongly than Oxy. However, the most important conclusion was that filters' affinity for lipid systems depended on both the structure of the lipids, which determines monolayer properties, and the monolayer's organization. The presence of condensed domains dispersed in a more fluid matrix was a particularly important factor in enhancing the incorporation of Avo and its fluidizing effect. The toxic effects of Avo and Oxy were not directly linked to their interactions with lipids. Nevertheless, the affinity of these compounds for lipids and membrane domains may facilitate their accumulation in the cells of living organisms.
Lipoxygenases (LOX) are found in many organisms and oxidize polyunsaturated fatty acids to hydroperoxides. The first tertiary structures predicted already 30 years ago a dedicated oxygen channel to the non-heme iron metal center, which has been a topic of discussion ever since. Analysis of more than fifteen lipoxygenases revealed (mostly) conserved channels from the iron metal center to the surface. They were lined by residues of α-helices 9 and 11 and characteristically passed through a loop between two β-strands below the surface. The channel orifices were generally open and the other ends were opposite to the metal center and linked to substrate cavities by residues at mainly conserved positions of α-helices 9 and 11, which included the Gly/Ala switch and nearby residues. Two observations suggest that they mediate oxygen perfusion. Investigations of 15S-LOX1 and soybean LOX1 by molecular dynamic analysis and replacements in the middle of the channels with bulky residues reduced the perfusion of oxygen and altered substrate turnover and the products. This report revealed a common channel design but with heterogeneity between animal, plant and a subfamily of fungal lipoxygenases with iron replaced by manganese. Their tertiary structures can now be compared and investigated for perfusion of O2.
Cationic polymers (polycations) represent a promising class of antimicrobial compounds whose physicochemical and biological properties can be tailored through appropriate structural design. The positive charge of these macromolecules indicates possible interactions with biological membranes. In this work, polycations differing in chemical structure (poly(2-(dimethylamino)ethyl methacrylate (PDMAEMA), poly(3-methacrylamido propyl trimethyl ammonium chloride) (PMAPTAC)) and molecular weight were investigated for their antifungal activity. The studies focused on the interactions of the polycations with model Candida albicans membranes that is the monolayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and ergosterol. Model systems were employed to verify the mechanisms underlying polycation-membrane interactions. The antifungal activity of these compounds against various fungi was also tested. The results showed that the tested compounds exhibit different modes and strengths of interaction with ergosterol-containing membranes. Among the analyzed polymers, PDMAEMA exhibited significant effects on model systems causing changes in lipid packing and an increase in membrane fluidity, whereas PMAPTAC was less effective. Moreover, the polymer with a lower molecular weight modifies membrane properties more strongly compared to its higher-molecular-weight counterpart and additionally exhibits a greater affinity for membranes with a higher ergosterol content. PDMAEMA and PMAPTAC polymers exhibit selective antifungal activity against several opportunistic molds, particularly against Fusarium spp., Trichophyton spp., Candida parapsilosis, and Cryptococcus neoformans. The coexistence of selective antifungal activity and overall comparable antimycotic efficacy among the tested polymers highlights the prominent role of fungal cell properties in determining their susceptibility to polymers tested. Moreover, the selective toxicity of these compounds may be determined by the content of ergosterol in membranes.
Surface lipids in lipoproteins interact with proteins and receptors at the surface of cell membranes, mediating lipoprotein-cell signaling and trafficking events. Despite this, little is known about lipoproteins' surface lipid composition and any changes introduced by type-2 diabetes mellitus (T2DM). Herein, we investigate the surface lipid composition in purified VLDL, LDL and HDL populations isolated from patients with poor (PC, HbA1c>8.5%) and good glycemic control (GC, HbA1c<6.5%) using high-performance thin-layer chromatography (HPTLC) and targeted liquid chromatography-mass spectrometry (LC-MRM-MS) approaches, which were complemented with biophysical measurements. Composed largely of free cholesterol (Chol), phosphatidylcholine (PC), and sphingomyelin (SM), the surface of lipoproteins contains minor quantities of phosphatidylethanolamine (PE), phosphatidylinositol (PI), monoglycerides (MG), ceramides (Cer) and glucosyl-ceramides (GlcCer). Our findings show that PE levels are altered in VLDL and LDL in T2DM. Screening of predominant Chol and PC lipids derivatives, cholesterol sulfate (CholS) and oxidized phosphatidylcholines (oxPC), revealed pronounced changes in LDL. At the biophysical level, T2DM impacted VLDL and HDL surface properties but not those of LDL. The distinct behavior of lipoproteins' composition and biophysical parameters in T2DM patients and in response to medication, diet, and physical exercise, may contribute to particle-membrane interactions.
This study presents a biochemical and isoenzyme-level characterization of cellulase from Coptotermes gestroi, with emphasis on its regulation by metal ions and surfactants using an integrated spectrophotometric-zymographic approach. The enzyme exhibited optimal activity at pH 5.0 and 45 °C and remained stable after 24 h, retaining 88 % and 112 % of its activity at 45 °C and 27 °C, respectively. Ca2+ significantly enhanced enzymatic activity, whereas Cu2+, Fe2+, Mn2+, and SDS markedly inhibited enzyme function. Electrophoretic analyses revealed multiple cellulase isoforms, with several activity bands detected by native PAGE and at least six active isoenzymes (∼37 to >116 kDa) resolved by SDS-PAGE. Zymographic analysis confirmed ion-dependent modulation of cellulase activity and demonstrated isoenzyme-specific regulatory responses not evident from ensemble spectrophotometric assays. Quantitative image analysis showed strong agreement with spectrophotometric measurements, supporting the reliability of band-based activity profiling. Collectively, these findings demonstrate that the cellulolytic system of Coptotermes gestroi comprises functionally distinct isoenzymes with differential regulatory properties and highlight the value of integrating spectrophotometric and zymographic analyses to resolve enzymatic heterogeneity in complex systems.
Ionic liquids (ILs) have been the focus of significant amounts of study regarding their interaction with biomolecules for a variety of applications including stabilization and destabilization of biomolecular structure, enhanced activities for biotechnology applications, and as designer solvents to promote specific interactions related to biomolecular function. Here we evaluated the impact of four different fatty acid ionic liquids (FAILs) on the stability of the model protein myoglobin and how the effects of these ILs compared to their individual components. The ILs were composed of a biocompatible cation, either choline or tetramethylguanidine, and an anionic fatty acid, either octanoic acid or decanoic acid. Using a combination of absorbance, fluorescence, and circular dichroism spectroscopy, we evaluated the impact of varying FAIL concentrations on the thermodynamic stability of myoglobin when denatured by either temperature or the chaotrope guanidinium hydrochloride. Both series of FAILs destabilized the myoglobin compared to controls, however the FAILs generally were equally or less destabilizing than the fatty acid component alone. Destabilization by the FAILs was determined to affect both the ΔHunfolding and ΔSunfolding values for myoglobin, with larger impacts on both terms from the octanoate-based series of molecules. Interestingly, the octanoate-containing series of molecules exhibited stronger disruption of the myoglobin compared to the decanoates. The highest concentrations of choline- and TMG-octanoate resulted in a change in ΔHunfolding between 75 and 100 kJ, while the octanoate alone caused a change of >120 kJ. The ΔSunfolding followed a similar trend, although to a lesser extent, ranging between 0.08 and 0.35 JK-1 for the octanoate series.
Myocardial ischemia-reperfusion (I/R) injury remains a major clinical challenge that undermines the benefits of reperfusion therapy and contributes to adverse cardiac remodeling. Despite their regenerative potential, the therapeutic efficacy of mesenchymal stem cells (MSCs) is limited by poor engraftment and low survival under ischemic conditions. To address these limitations, we developed a combinatorial strategy utilizing low-density lipoprotein receptor-related protein 6 (LRP6)-targeted chimeric antigen receptor-engineered MSCs (CAR-MSCs) to enhance site-specific homing to injured myocardium, coupled with overexpression of microsomal glutathione S-transferase 1 (MGST1) to strengthen cellular antioxidant defense. We evaluated this approach in both cellular and animal models of I/R injury. In vitro, under oxygen-glucose deprivation/reoxygenation (OGD/R) conditions, treatment with MSCs, CAR-MSCs, or MGST1 overexpression alone each attenuated OGD/R-induced injury. The combination of unmodified MSCs with MGST1 overexpression (MSCs + MGST1) further improved cell viability and reduced apoptosis compared to MSCs alone. Notably, the combination of CAR-MSCs and MGST1 overexpression (CAR-MSCs + MGST1) exhibited the most pronounced protective effects, significantly outperforming all other groups, including MSCs + MGST1, in enhancing cell viability, reducing apoptosis and intracellular reactive oxygen species (ROS) levels, modulating oxidative stress markers (MDA, SOD, CAT), and promoting the secretion of reparative growth factors (VEGF, IGF-1, HGF). In a rat I/R model, combined treatment significantly reduced infarct size, ameliorated histological damage, decreased collagen deposition and apoptosis, and consistently modulated serum oxidative and regenerative biomarkers. Mechanistically, the combined intervention activated the Nrf2/Keap1 signaling pathway, upregulating downstream effectors NQO1 and HO-1. The cardioprotective effects were partially abolished by Nrf2 inhibition. In summary, LRP6-targeted CAR-MSCs coupled with MGST1 overexpression deliver synergistic protection against I/R injury by activating the Nrf2/Keap1 antioxidant program, offering a clinically translatable strategy to enhance precision MSC therapy and mitigate reperfusion-driven cardiac damage.
Acute kidney injury (AKI) is a critical clinical condition characterized by a sudden loss of kidney function, accompanied by complex metabolic and structural changes. In this study, the early molecular and metabolic effects of AKI were investigated using an ischemia-reperfusion (IR) model. The study aims to evaluate the levels of total autofluorescent molecules, analyze diffuse reflectance spectroscopy (DRS) findings, and understand the roles of oxidative stress biomarkers and functional parameters in the kidney IR. The IR model was applied in rats, DRS data were measured in vivo from the kidney and from isolated mitochondria using a fiber optic probe. Additionally, NADH and FAD levels were assessed in serum and mitochondrial isolates using fluorescence spectrometer. Kidney function/injury parameters (creatinine, KIM-1, NGAL and L-FABP) and oxidative stress biomarkers (MDA, L-OOH, dityrosine, kynurenine, and AOPP) were analyzed. In the IR group, significant increases in NADH and FAD levels were observed in both serum and mitochondrial isolates. Oxidative stress biomarkers such as kynurenine, dityrosine, and lipid hydroperoxides also increased in the IR group. DRS analyses of mitochondrial isolates revealed a marked reduction in optical signals in the IR group, but no significant changes were detected in in vivo DRS analyses. Significant correlations were identified between functional and oxidative stress parameters in serum and mitochondria. Although not significant at the tissue, the alterations observed at the mitochondrial level demonstrate the efficacy of the DRS. This study highlights the importance of integrating optical and biochemical approaches to elucidate the effects of IR on mitochondrial-related metabolic processes.
Nonheme iron enzymes encompass one of the most chemically versatile families of metalloenzymes, catalyzing an extraordinary range of oxidative transformations essential to metabolism, natural product biosynthesis, and environmental biodegradation. Despite their mechanistic diversity, these enzymes share a unifying principle: the controlled activation of molecular oxygen to drive selective substrate oxidation. Many mononuclear nonheme iron enzymes employ a conserved 2-His-1-carboxylate facial triad and frequently utilize 2-oxoglutarate (2OG) as a co-substrate to generate Fe(IV)O intermediates that mediate diverse C-H and CC bond oxidations. Organic cofactor-independent nonheme iron enzymes have been recognized for decades, with early examples including catechol dioxygenases and intradiol-cleaving enzymes. Structural and mechanistic advances over the past two decades have revealed that many of these cofactor-independent enzymes feature alternative 3-His or 4-His metal-binding motifs, which confer distinct electronic properties and enable oxygen activation pathways beyond those accessible to 2-His-1-carboxylate systems. Parallel advances in the study of dinuclear nonheme iron enzymes have illuminated equally rich mechanistic diversity, particularly within the ferritin-like (FDO) and heme-oxygenase-like (HDO) families. These diiron systems employ cooperative metal-metal interactions to form diiron-oxygen intermediates that enable multielectron oxidation chemistry beyond the reach of mononuclear centers. Structural and mechanistic studies of HDO-type enzymes involved in natural product biosynthetic pathways have revealed noncanonical coordination environments and substrate selectivity, underscoring the evolutionary and functional plasticity of nonheme iron catalysis. This review summarizes emerging insights into His-rich mononuclear enzymes and HDO-type diiron systems, highlighting their structural innovations, mechanistic principles, and collective contributions to expanding the frontiers of oxygen activation chemistry.
Diabetic encephalopathy (DE), a severe neurological complication of diabetes, is characterized by cognitive decline and neuronal damage. While gut microbiota dysbiosis has been implicated in diabetes pathogenesis, its specific role and molecular mechanisms in DE remain unclear. A multi-omics approach integrating 16S rRNA sequencing and untargeted metabolomics was performed on fecal samples from 29 DE patients and 31 diabetic controls (DM). An in vitro DE model was established using high glucose (HG)-treated HT22 cells, which were further incubated with sterile fecal microbiota supernatant (FMS) from DE patients. Neuronal viability, apoptosis, oxidative stress markers (SOD, MDA, ROS), and miR-493-3p expression were assessed. The miR-493-3p/RAF1 interaction was validated using dual-luciferase reporter assays and Western blot. No significant differences in overall microbial diversity were identified in DE and DM cohorts. However, DE patients exhibited distinct gut microbiota composition, with elevated Verrucomicrobiotaand Bacteroidota, and reduced Proteobacteriaand Firmicutes. Metabolomic analysis revealed 160 differentially abundant metabolites enriched in amino acid and lipid metabolism pathways. In vitro, DE-derived FMS dose-dependently exacerbated HG-induced neuronal oxidative damage and apoptosis, concomitant with miR-493-3p upregulation. Inhibition of miR-493-3p attenuated these damaging effects and restored RAF1 expression. RAF1 was confirmed as a direct target of miR-493-3p, and its downregulation was critical in mediating FMS-induced neuronal injury. This study identified a novel gut-brain axis pathway in DE, whereby gut microbiota dysbiosis and metabolic alterations promote neuronal damage via the miR-493-3p/RAF1 signaling axis. These findings provide new insights into DE pathogenesis and suggest potential therapeutic targets for this debilitating complication.
AlphaB-crystallin is a small heat shock protein that is found in the eye lens, retina, heart, brain, skin, and skeletal muscle. Mutations of αB-crystallin lead to the development of cataracts, human muscle and heart disorders. The role of non-enzymatic glycation of αB-crystallin in the development of pathological processes has been little studied. The aim of this study was to evaluate the influence of enhanced methylglyoxal (MGO) level on the functions of αB-crystallin. We modified recombinant αB-crystallin (aB-Cr) by MGO and investigated the effect of this modification on different functions of aB-Cr: ability to suppress protein thermo-aggregation, promote protein refolding, and suppress the amyloid-like aggregation of α-synuclein A53T (αSynA53T, a mutation associated with early-onset Parkinson's disease). It was shown that MGO modifies 8 Arg residues per αB-Cr subunit yielding methylglyoxal-derived hydroimidazolone (MG-H) and results in cross-links between the protein subunits. The modification reduces the ability of αB-Cr to suppress thermo-aggregation of catalase and to promote refolding glyceraldehyde-3-phosphate dehydrogenase. At the same time, modification by MGO enhances the ability of αB-Cr to suppress fibrillization of αSynA53T: while the addition of native αB-Cr results in shorter fibrils, MGO-modified αB-Cr completely prevents fibrillization of αSynA53T, yielding amorphous aggregates that are less toxic to the cells compared to αSynA53T fibrils. Therefore, the effect of MGO on various αB-crystallin functions may be different, resulting in a decrease or increase in its activity.
The global obesity epidemic is worsening in many parts of the world, leading to a gradual increase in the occurrence of metabolic illnesses such as type 2 diabetes mellitus (T2DM) and cardiovascular disorders. Emerging evidence highlights protein S-nitrosylation as a critical regulatory mechanism in diabetes-associated disease. S-nitrosylation, a post-translational redox-based modification that refers to the formation of a covalent bond between nitric oxide and a cysteine residue's reduced thiol, adds an S-nitrosothiol group to substrate proteins. Several recent investigations have shown that dysregulated protein S-nitrosylation contributes significantly to the development of insulin resistance, mitochondrial dysfunction, oxidative stress, inflammation, and ER stress in a wide range of metabolic disorders. In this review, we will describe the dynamic change between protein S-nitrosylation and denitrosylation in the etiology of diabetes and diabetes-associated disease, as well as the levels of S-nitrosylation enzyme activity and modification proteins in these diseases. This will highlight the therapeutic potential of focusing on the enzyme's activity to modify the levels of protein S-nitrosylation, thereby improving our understanding of nitric oxide's function in redox signaling and metabolic diseases.