Antioxidants

Careful balance of the redox status of the embryo and reduction of oxidative stress is crucial in early development. Here we show that the culture of preimplantation mouse embryos in the conditionally non-essential amino acid L-proline (Pro) increases the intracellular concentration of the potent antioxidant glutathione as shown by staining of 2-cell, 4-cell and 8-cell embryos with tetrafluoroterephthalonitrile (4F-2CN). Further, liquid-chromatography/mass spectrometry showed increased GSH levels in all Pro-treated preimplantation stages of development compared to controls. The GSH:GSSG ratio also showed a Pro-dependent increase. Overall, our results indicate that the beneficial effect of Pro in preimplantation embryo culture is due to the reduction in oxidative stress mediated through an increase in cellular GSH concentration.

Oxidative post-translational modifications on the sulfhydryl group of cysteines can occur spontaneously or enzymatically. The dioxygenation of N-terminal cysteines has emerged as a new oxygen sensing paradigm, catalysed by 2-aminoethanethiol dioxygenase (ADO) in mammals. Conflicting evidence has been reported in recent years on whether this reaction can occur in the absence of ADO. Here we sought to address whether physiological oxidative stress can interfere with ADO-catalysed N-terminal dioxygenation. Using a system to produce titratable intracellular levels of H2O2, we demonstrate that the stability of RGS4 and 5 is not affected by oxidative stress, whether ADO is present or not. However, cytotoxic levels of oxidative stress did induce an increase in RGS4/5 protein levels that occurred independently of the Cys N-degron pathway. This effect of tBHP was reduced by Fe2+ chelation and perturbations of lysosomal function, suggesting the possible involvement of ferroptosis. We conclude that N-terminal cysteine dependent proteolysis of RGS4/5 is not sensitive to physiological oxidative stress, but these proteins can be stabilised during the process of oxidative stress-induced cell death through an N-terminal cysteine independent mechanism.

Mitochondrial morphology and function are critical determinants of neuronal function and survival, with disruptions in mitochondrial dynamics often preceding the overt neuronal dysfunction seen in neurodegenerative diseases such as Alzheimers disease, Huntingtons disease and Parkinsons disease. The kynurenine pathway accounts for 95% of dietary tryptophan catabolism and many of the metabolites are neuroactive, including redox-active 3-hydroxykynurenine (3-HK). 3-HK is present under normal physiological conditions in the central nervous system (CNS) and is elevated during inflammation. While supraphysiological levels of 3-HK have been associated with neurotoxicity, the effects of physiological concentrations on neuronal cells, and specifically their mitochondria, remain poorly understood. Here we assessed viability, ATP levels and redox status to determine cellular health and function in neuronal cells exposed to physiological levels of 3-HK, alongside confocal imaging and transcriptomic profiling, finding significant alterations in mitochondrial function and morphology. Interestingly, a biphasic influence of 3-HK on mitochondrial morphology was observed, with an elongated network as well as decreased surface area and volume being observed only at the lowest concentration of 3-HK, reflecting normal physiological levels. At the highest 3-HK concentration tested, reflecting an inflammatory situation, an increased number of mitochondria were present, accompanied by increased activation of caspase-3/7 and enhanced production of mitochondrial superoxide. These results highlight a previously unknown role for 3-HK in regulating mitochondrial function and structure, possibly through altered fission and fusion events, suggesting that subtle changes in kynurenine pathway metabolism may contribute to early mitochondrial dysfunction in neurological disease.

Rhodiola rosea is a traditional medicinal plant often classified as an adaptogen, with reported effects in supporting the bodys response to physical, environmental, and emotional stressors. The present study investigated the antioxidant properties of Rhodiola rosea extract and its major chemical constituents to provide insight into their potential mechanisms of action. Through in vitro biochemical assays, we demonstrated that Rhodiola rosea extract has the capacity to reduce hydrogen peroxide (H2O2) levels. Among its primary chemical components, rosavin significantly decreased H2O2, whereas salidroside had no effect. Neither compound affected superoxide levels. Structural analysis revealed that the intact phenylpropanoid glycoside architecture of rosavin is required for activity, as its individual components, arabinose and rosin, showed no inhibitory effect. Further investigation demonstrated that rosavin attenuates H2O2-mediated oxidation of thiol groups, supporting a role in cellular redox regulation. In cultured human cells, rosavin mitigated reductions in cell viability induced by exposure to H2O2, indicating cytoprotective effects under oxidative stress conditions. Finally, in an in vivo model, administration of SARS-CoV-2 spike protein increased circulating levels of H2O2, which were subsequently reduced following rosavin treatment. Collectively, these findings identify rosavin as a structurally dependent antioxidant component of Rhodiola rosea that modulates H2O2-associated oxidative stress and supports further investigation of phenylpropanoid glycosides as adaptogens.

The production of reactive oxygen species (ROS) in response to cadmium (Cd) has been extensively studied, demonstrating that they play a key role in the plants response to this heavy metal. While the role of enzymes like RBOHs has been thoroughly studied, the function of other ROS-producing enzymes, such as peroxisomal glycolate oxidase (GOX), remains largely overlooked. Peroxisomal GOX is a core metabolic enzyme of the photorespiratory pathway occurring in chloroplasts, mitochondria and peroxisomes. Using Arabidopsis (Arabidopsis thaliana) mutants lacking the main peroxisomal GOX genes, GOX1 (gox1-1) and GOX2 (gox2-1) we explored their function in plant response to Cd. Although photosynthetic capacity appears to be affected to the same extent in both mutants under control and Cd stress conditions, GOX2 seems to play a greater role in ROS production in response to the metal. Transcriptomic analyses on WT and gox2-1 pointed to the mitochondrial electron transport chain (mETC) as a target of Cd stress. We further investigated the individual GOX1 and GOX2 functions in mETC regulation and redox state. Although oxidative ratio of mitochondria was higher in both mutants, it was more pronounced in the absence of GOX1. Furthermore, the mETC is affected in both mutants but the regulation of its components differs in each mutant. These results point out the different functions of the two photorespiratory GOX isoforms in Arabidopsis, leading to a better understanding of the photorespiratory pathway.

BackgroundFerroptosis has emerged as a promising therapeutic target in IRI. However, it remains largely unclear how and when this iron-dependent regulated cell death manifests during IRI. Therefore, we explored malondialdehyde (MDA), a byproduct of lipid peroxidation, and glutathione peroxidase 4 (GPX4), as a marker of redox capacity, in multiple IRI models. With this explorative study, we aimed to uncover MDA dynamics in renal and hepatic IRI, which could provide valuable insights for future internal studies. MethodsHistorical plasma and tissue samples from rat and porcine models of renal and hepatic IRI were selected based on varying conditions of ischemic injury, reperfusion and perfusion. MDA was measured using a colorimetric assay with N-methyl-2-phenylindole, methanol, acetonitrile and hydrochloric acid and quantified at 595 nm. GPX4 protein concentrations were investigated using standard western blotting. ResultsIn rat clamping models, plasma MDA concentrations revealed no difference between control and IRI settings. However, an increasing trend could be observed in tissue samples after IRI. Similarly, a decrease in tissue GPX4 concentrations was observed after IRI. In porcine studies, MDA concentrations were increased during reperfusion of kidneys exposed to prolonged warm ischemia and livers exposed to short periods of cold ischemia. Dynamic preservation could attenuate MDA concentrations. ConclusionWe found that MDA and GPX4 are affected within the first hours after reperfusion, stressing the need for early sampling in studies focusing on characterizing ferroptosis. Moreover, MDA dynamics during organ perfusion revealed an increased vulnerability of ischemic organs to lipid peroxidation and a potential protective effect of dynamic preservation. These preliminary results should be confirmed in studies focusing on ferroptosis characterization, as notable observations regarding sample age and storage conditions and experimental design limit the validity of this study.

Pregnancy complications such as preeclampsia are associated with circulating cell-free mitochondrial DNA (mtDNA), a damage-associated molecular pattern capable of activating Toll-like receptor 9 (TLR9). We hypothesized that acute mtDNA exposure induces maternal inflammation and endothelial dysfunction during pregnancy via TLR9 activation. Non-pregnant and pregnant rats (gestational days 14-15) were treated intravenously with saline or purified mtDNA and euthanized 4 h after treatment. mtDNA increased cytokine mRNA expression in lung and liver of non-pregnant and pregnant rats, with magnitude varying by pregnancy status and organ. Aortas from pregnant, but not non-pregnant, rats exhibited reduced acetylcholine (ACh)-induced relaxation following mtDNA treatment (Emax, saline: 90.1 {+/-} 3.9 % vs. mtDNA: 62.1 {+/-} 20.7 % KClmax, p<0.05), while uterine artery function was preserved, indicating vascular bed-specific effects. Ex vivo incubation of aortic rings with mtDNA {+/-} white blood cells did not replicate in vivo findings, implicating systemic rather than direct vascular mechanisms. Nuclear DNA did not affect ACh-induced relaxation (p>0.05), confirming that the vascular effects were mtDNA-specific. Pharmacological antagonism of TLR9 with ODN2088 partially attenuated mtDNA-induced maternal endothelial dysfunction. Although overt vascular ROS increases were not detected, aortas from pregnant rats had reduced sod-1 expression (p<0.05) and increased eNOS protein abundance (p<0.05). Acute mtDNA exposure during pregnancy induces maternal organ inflammation and impairs endothelium-dependent vasodilation, with partial TLR9 involvement. In conclusion, aortic transcriptional changes in antioxidant pathways and increased eNOS abundance were also observed, though their functional significance remains to be determined. New & NoteworthyTo our knowledge, this is the first study to demonstrate that acute exposure to circulating mtDNA induces pregnancy-specific maternal endothelial dysfunction and organ-selective inflammatory responses. Our findings reveal pregnancy- and vascular-bed specific responses of the maternal vasculature to mitochondrial danger signals, with partial TLR9 involvement. Aortic transcriptional changes in antioxidant pathways and increased nitric oxide synthase abundance were identified as molecular correlates of this dysfunction.

Photosynthetic electron transport is mediated by several protein supercomplexes that are spatially arranged in the thylakoid membranes of chloroplasts. The chloroplast NADH dehydrogenase-like (NDH) complex is part of the photosynthetic alternative electron transport (AET) chain, which reduces the plastoquinone (PQ) pool using reduced ferredoxin as a substrate. This NDH complex is associated with photosystem I (PSI) and mediates a portion of AET in stroma lamellae, whereas photosystem II (PSII) is concentrated in grana stacks. This study presents the findings regarding post-illumination chlorophyll fluorescence increase (PIFI), a protein crucial for regulating AET via the NDH pathway. A marked increase in NDH activity and a reduction in the PQ pool in the dark were observed in PIFI-deficient mutant strains (g-pifi) generated by genome editing. Blue native PAGE analysis indicated that PIFI was associated with the NDH-PSI supercomplex in the wild type, and the NDH complex was dissociated from PSI in the g-pifi mutants. Additionally, the g-pifi mutants exhibited a decrease in the maximum quantum yield of PSII (Fv/Fm). Notably, Fv/Fm was restored in a double mutant harboring both g-pifi and NDH-deficient pnsl1 mutations, demonstrating that deregulated NDH activity in g-pifi causes downregulation of PSII efficiency. However, the lower Fv/Fm was not observed in a mutant lacking thioredoxin m4 (trxm4), which showed deregulated NDH activity but maintained the NDH-PSI supercomplex. These data suggest that PIFI stabilizes the NDH-PSI supercomplex and maintains the spatial localization of PQ reduction via AET in thylakoid membranes, which is essential for the proper functioning of PSII.

Age-related macular degeneration (AMD) is a progressive complex eye disease and one of the leading causes of blindness. AMD progression is marked by molecular changes in the retinal pigmented epithelium (RPE) which include increased reactive oxygen species (ROS) accumulation, mitochondrial dysfunction - eventually leading to dysfunctional RPE. Mitophagy regulator, Pink1, is reduced in the RPE of AMD patients and Pink1 loss leads to a shift from mitochondrial respiration to glycolysis. Serine is a non-essential amino acid which is de novo synthesized from glycolytic intermediate 3-PG via the rate limiting enzyme PHGDH. Serine is tightly integrated into anabolic processes like glutathione (GSH) cycling, maintaining NADH/NADPH pools leading to changes in AMPK signaling. Here, we show that Pink1 loss leads to a reduction in PHGDH and serine levels in the RPE leading to impaired mitochondrial structure and function, increased ROS mediated damage, increased inflammation, and hampered retinal function. Serine supplementation rescued ROS accumulation, balanced GSH abundance, and increased retinal function. Overall, our study highlights the potential of dietary serine in ROS management in AMD.

RAP2.6, an AP2/ERF transcription factor (TF), regulates plant stress responses; however, its role in floral transition remains unexplored. Here, we evaluated RAP2.6s role in flowering and the associated transcriptional changes in Arabidopsis thaliana under long-day conditions. RAP2.6-overexpressing line showed early flowering with fewer rosette leaves, whereas rap2.6-1 mutant flowered later, had more rosette leaves, and higher expression of the floral repressor FLOWERING LOCUS C (FLC). Early flowering in the overexpressing line was accompanied by transcriptional activation of the floral integrators GIGANTEA (GI), FLOWERING LOCUS T (FT), and COSTANS (CO), potentially through RAP2.6 interaction with GCC/DRE cis-regulatory elements. RAP2.6-mediated floral transition depended on nitric oxide (NO), with flowering time largely varying based on NO bioactivity. RAP2.6 was found to be a downstream regulator of Arabidopsis S-NITROSOGLUTATHIONE REDUCTASE 1 (GSNOR1) in controlling S-nitrosothiol (SNO) levels, flowering time, and silique formation. The NITRIC OXIDE-ASSOCIATED 1 (NOA1)-dependent reduction in NO levels abolished early flowering in 35S::RAP2.6 plants without affecting silique formation. Furthermore, enhanced cytokinin sensitivity and upregulation of cytokinin biosynthetic genes suggest cytokinin involvement in RAP2.6-mediated flowering. Together, these findings highlight the crucial role of RAP2.6 in regulating flowering time by integrating redox and hormonal signaling to coordinate reproductive development in A. thaliana.

Organic germanium, particularly carboxyethyl germanium sesquioxide (Ge-132), has been investigated for decades in relation to diverse biological effects, with a strong emphasis on its antioxidant properties. However, the available literature remains dispersed, encompassing heterogeneous experimental models and endpoints that limit mechanistic interpretation. While antiglycative activity has been described at the biochemical level, its downstream gene regulatory consequences under glycative stress remain inconsistently characterized. Here, we combined systematic review of the literature of experimental studies with targeted molecular analysis in a standardized cellular model. The literature mapping was used to guide pathway selection rather than to establish quantitative associations. Based on patterns emerging from literature, we focused on pathways associated with glycative stress responses, including carbonyl stress, inflammatory signaling, and autophagy regulation. Gene expression analysis revealed a limited and selective modulation of regulatory pathways under glycative stress conditions, consistent with a context-dependent effect rather than broad transcriptional reprogramming. In parallel, protein analysis showed reduced intracellular accumulation of advanced glycation end products (AGEs) in Ge-132-treated cells under glycative stress conditions. Importantly, these findings support a dissociation between glycative damage reduction and cellular stress-response pathways. This combined approach helps interpretation of previously fragmented observations across the literature and highlights gene regulation under glycative stress as a relevant but still unresolved aspect of organogermanium biology.

O_LIJuglone is the phytotoxic 1,4-naphthoquinone responsible for the allelopathic effects of black walnut (Juglans nigra), yet how plants perceive and respond to juglone remain poorly understood. C_LIO_LIWe conducted transcriptome profiling of rosettes and roots of Arabidopsis thaliana exposed to juglone from 30 min to 5 d, along with targeted metabolic profiling, biochemical assays, and untargeted proteomics to gain a systems-level understanding of how plants respond to juglone and to test hypotheses underlying its phytotoxicity. C_LIO_LIJuglone exposure induced expression of genes involved in glutathione, cysteine, and sulfur metabolism pathways, and in protein homeostasis. We found that juglone depletes the pool of reduced glutathione (GSH) in roots, in part, through conjugation. We demonstrate that via upregulation of transcription factors (NAC53 and NAC78), the response to juglone activates components of the proteasome stress regulon and triggers extensive proteome remodeling with engagement of the autophagy pathway when proteasome capacity is limited. C_LIO_LIOur findings (i) indicate that thiol depletion and disruption of proteostasis through juglones dual redox cycling and alkylation activities are central to its phytotoxicity, (ii) cast doubt on previous reports that juglone targets a specific enzyme in plants or other organisms, and (iii) provide insight into how the chemical properties of allelopathic quinones shape their ecological roles. C_LI

Seed longevity is a key determinant of crop establishment, productivity, and germplasm conservation. During storage and germination, reactive oxygen species accumulate and contribute to seed aging through oxidative damage and loss of viability. The maintenance of redox homeostasis therefore relies on NADPH-dependent antioxidant systems, which require a continuous supply of reducing power. NADP-dependent malic enzyme 1 (NADP-ME1), represents a source of NADPH supporting antioxidant defense during seed aging. Here, we show that enhanced expression of NADP-ME1 positively contributes to seed vigor and longevity in Arabidopsis thaliana. NADP-ME1 overexpression lines exhibited faster germination and higher overall germination after accelerated aging, whereas knockout mutants showed markedly reduced germination performance. Enhanced post-aging vigor in the overexpression lines was associated with reduced oxidative damage as indicated by lower malondialdehyde and hydrogen peroxide accumulation, along with preservation of specific polyunsaturated fatty acids, and increased {gamma}-tocopherol levels in aged dry seeds. Enhanced expression of NADP-ME1 reshapes the transcriptome of germinated seeds under fresh conditions compared with the wild type, while only minimal differences between genotypes are detected in aged seeds. These results suggest that NADP-ME1 contributes to the establishment of a transcriptional state associated with enhanced seed vigor and improved post-aging germination. Finally, co-immunoprecipitation coupled to mass spectrometry and bimolecular fluorescence complementation identified aspartate aminotransferase 2 as a NADP-ME1 interactor, pointing to a link between malate metabolism and amino acid-related metabolic adjustment. Together, these results identify NADP-ME1 as a determinant of seed resilience to aging and a potential target for improving seed quality.

The tight regulation of iron homeostasis is of great importance for cellular health. An increase in intracellular iron levels results in the formation of free radicals, which damages macromolecules and membranes, eventually resulting in cell death by Ferroptosis. Recently, we showed that patients with mutations in VPS41 display a severe neurodegenerative phenotype with iron deposition in the brain. VPS41 is well known as subunit of the HOPS complex required for fusion of late endosomes and autophagosomes with lysosomes. However, VPS41 has also been identified as inhibitor of Ferroptosis and regulator of redox homeostasis. How VPS41 exerts these functions and if these are dependent on the HOPS complex is unknown. Here we show that depletion of VPS41 results in increased intracellular iron levels, ROS formation and mitochondrial fission. Our findings indicate an important role for VPS41 in the regulation of iron homeostasis and mitochondrial fission and suggest Ferroptosis as a possible cause for neurodegeneration in VPS41 patients.

C4 photosynthesis is a CO2-concentration mechanism that separates CO2 fixation between two cell types, thereby reducing photorespiration and making C4 plants more efficient than their C3 counterparts. While the C4 cycle has evolved multiple times across different genera, this study evaluates very closely related C3 and C4 species within the genus Flaveria. Apart from their carbon metabolism, C4 plants also possess adaptations in their mineral nutrition. One key nutrient which is also directly involved in photosynthesis is phosphorus. It is absorbed by the plant in the form of inorganic phosphate and is an essential component of DNA, ATP, lipids, and carbohydrates. In the Flaveria C4 species, but not in the C3 species, phosphate limitation was shown to affect the dark reactions of photosynthesis. This study investigates how phosphate deficiency impacts the light reactions in C3 and C4 Flaveria plants. We observed a differential response in the functionality of photosynthetic energy conversion between the two species. When exposed to a limited phosphate supply, the C3 species reduced its linear electron transport rate while dissipating excess energy through high-energy quenching, which was regulated by a higher pH gradient across the thylakoid membrane. In contrast, the C4 species did not regulate its photosynthetic light reaction under phosphate limitation. Instead, it exhibited increased stress levels, evidenced by a stronger biomass reduction and the induction of stress markers in the leaves. Additionally, this study uncovered an acceleration in NPQ relaxation during phosphate limitation, regardless of the photosynthesis type. HighlightPhosphate deficiency reduced linear electron transport rates and induced dissipation of excess energy through non-photochemical quenching in the C3 Flaveria species, while in the C4 species, despite elevated stress levels, the photosynthetic light reactions were unaffected.

PurposeAlanine transaminases (ALT), encoded by the GPT gene, catalyzes the reversible conversion of pyruvate and glutamate to alanine and alpha-ketoglutarate, thereby correlating carbohydrate and amino acid metabolism. However, its role in the human neural retina remains unclear. This study aimed to explore the expression, localization, and metabolic function of ALT in the human neural retina and its potential involvement in retinal diseases. MethodsALT1 and ALT2 expression and localization were examined in the retinas of healthy and diabetic retinopathy (DR) donors via immunoblotting and immunofluorescence. ALT function was assessed in ex vivo human retinal explants using pharmacological inhibition with beta-chloro-L-alanine (BCLA), followed by the analyses of enzyme activity, tissue injury, and transcriptomic responses. Stable-isotope tracing with 13C-and 15N-labelled substrates combined with GC-MS was used to define ALT-dependent carbon and nitrogen fluxes in macular and peripheral retinas. Redox level (NADPH/NADP+) was also evaluated under tert-butyl hydroperoxide-induced oxidative stress. ResultsALT1 and ALT2 were both expressed in the human neural retina, with prominent localization in Muller glia and photoreceptor inner segments. ALT1 displayed a diffuse cytoplasmic distribution, whereas ALT2 demonstrated a punctate pattern consistent with mitochondrial localization. In DR retinas, ALT1 expression was spatially disorganized and heterogeneous, while ALT2 remained comparatively preserved. Inhibition of ALT with BCLA markedly reduced ALT activity without causing overt cytotoxicity or major transcriptional changes. Isotope tracing demonstrated that retinal ALT predominantly channels pyruvate-derived carbon into alanine, whereas alanine was minimally contributed to pyruvate production under basal conditions. ALT inhibition suppressed alanine synthesis and release, redirected nitrogen flux towards glutamate, glutamine, and aspartate, and uncovered distinct metabolic adaptations in macular but not peripheral retinas. Under oxidative stress, ALT inhibition induced the decrease of NADP+/NADPH ratio and LDH release, indicating improved redox balance and reduced tissue injury. ConclusionsALT is previously unrecognized as a regulator of carbon and nitrogen partitioner in the human neural retina, contributing to redox homeostasis under stress. The altered distribution of ALT1 in DR retina and the protective metabolic effects of ALT inhibition suggest ALT as a potential contributor to retinal metabolic vulnerability and a candidate therapeutic target in retinal diseases.

BackgroundCarnitine plays an obligatory role in energetics owing to its role in the translocation of long-chain fatty acids into the mitochondrion for oxidation. Here, we determined the metabolic and behavioral consequences of systemic carnitine deficiency (SCD) in mice. MethodsFemale C57BL/6J mice were randomized to receive normal drinking water (control, n = 8) or drinking water supplemented with mildronate 4g.L-1 (mildronate, n = 8) for 21 days. Body composition was assessed at baseline and post treatment. Metabolic and behavioral phenotyping was performed continuously over 72 hours following 14 days of control or mildronate treatment. Stable isotope were used to assess whole-body substrate oxidation. Carnitine subfractions were quantified in skeletal muscle and liver, as was mitochondrial respiratory function. Liver and muscle samples also underwent proteomic analysis. ResultsMildronate treatment depleted total carnitine in muscle and liver by [~]97% (P < 0.001) and [~]90% (P < 0.001), respectively. Carnitine depletion was accompanied by lower total energy expenditure (P = 0.01), attributable to lower voluntary wheel running (P = 0.01). Oxidation rates of palmitate (P < 0.01) but not octanoate were lower whereas rates of glucose oxidation were greater in carnitine depleted mice (P < 0.01). Mitochondrial respiratory capacity was unaltered by carnitine deficiency. Carnitine deficiency remodeled muscle and liver proteomes to support lipid oxidation and energy production. SummaryIn mice, carnitine deficiency is characterized by decreased long-chain fatty acid oxidation despite preserved mitochondrial respiratory capacity. Carnitine deficiency resulted in lower voluntary exercise and a concomitant reduction in energy expenditure.

Background/ObjectivesNeuroinflammation-driven iron dysregulation and neurotoxic astrocyte polarization are increasingly recognized as interconnected pathological mechanisms in neurodegenerative diseases. Systemic inflammation triggered by strenuous exercise or infection can engage the central nervous system and astrocytic inflammatory responses and perturb iron homeostasis; however, targeted nutritional strategies to counteract these processes remain limited. Inflamate(R) is a multi-component botanical supplement comprising boswellic acids, astilbin, xanthohumol, and cinnamaldehyde, each with documented anti-inflammatory properties. However, whether this combined formulation can modulate the inflammatory-iron metabolic axis and astrocyte phenotypic polarization remains unexplored. This study aimed to investigate the effects of Inflamate(R) on LPS-induced pro-inflammatory gene expression, iron metabolism-related gene regulation, and A1/A2 astrocyte phenotypic polarization in mouse astrocytes. MethodsMouse astrocytes (AWT) were pre-treated with Inflamate(R) (0.0375 g/mL) or DMSO vehicle for 24 h, followed by lipopolysaccharide (LPS; 1 g/mL) stimulation for an additional 24 h. The non-cytotoxic working concentration was determined by morphological assessment, CCK-8 cell viability, and LDH cytotoxicity assays. Expression of 14 target genes spanning pro-inflammatory mediators (NOS2, IL6, C3, COX2, PLA2g15, SOCS3), iron metabolism regulators (FTH1, Hepcidin, TFRC, SLC40A1, RGMa, RGMb), and astrocyte polarization markers (S100A10, GFAP) was quantified by qRT-PCR. ResultsUnder normal culture conditions, Inflamate(R) did not significantly alter the expression of any target gene except S100A10, confirming the absence of baseline cytotoxicity or transcriptional homeostatic perturbation. Upon LPS stimulation, Inflamate(R) selectively suppressed NOS2 (approximately 64% reduction, p < 0.0001), IL6 (approximately 37% reduction, p < 0.0001), and C3 (approximately 47% reduction, p < 0.0001), while COX2, PLA2g15, and SOCS3 remained unaffected. Concurrently, Inflamate(R) significantly reduced LPS-induced Hepcidin expression to approximately 17% of the control level (p < 0.05) and attenuated FTH1 upregulation (p < 0.01), without altering the expression of iron transporters (TFRC, SLC40A1) or BMP-SMAD pathway components (RGMa, RGMb). Furthermore, Inflamate(R) upregulated the neuroprotective A2 marker S100A10 under both basal (p < 0.05) and LPS-stimulated conditions (p < 0.01), while the general reactivity marker GFAP remained unchanged. ConclusionsInflamate(R) exerts a selective, multi-target modulatory effect at the transcriptional level in LPS-stimulated astrocytes, encompassing suppression of the iNOS-NO and IL-6 signaling axes, attenuation of inflammation-driven hepcidin-ferritin iron dysregulation via the IL-6-STAT3 pathway, and promotion of a phenotypic shift from neurotoxic A1 toward neuroprotective A2 astrocyte polarization. Given that the IL-6-JAK-STAT3-hepcidin axis is also activated during exercise-induced systemic inflammation, these findings suggest that Inflamate(R) may represent a targeted nutritional strategy for preserving CNS iron homeostasis and supporting neuroprotective astrocyte function in both neurodegenerative and exercise-related neuroinflammatory contexts. Further validation in in vivo neurodegenerative and exercise models, including protein-level analyses, is warranted to confirm these transcriptional findings.

Parkinsons disease (PD) is the second most progressive degenerative disorder of the brain due to dopaminergic (DA) neuron degenerations and alpha-synuclein (-Syn) accumulations. At present, the disease has no effective treatment. Therefore, the current study objective is to identify a novel anti-PD formula (Zhi-Shi-Huang-Wu Formula, F-2) computed at 8:4:2:1 ratio from HSP 70 promoter activators Valeriana jatamansi (V), Acori talarinowii (A), Scutellaria baicalensis (S), Fructus Schisandrae (F). Traditionally, V is used to cure memory impairments, A treats mental disorders, and chronic mild stress, S for neuroprotection, and F showed multiple therapeutic actions to treat insomnia. This study investigated the neuroprotective potential of the V, A, S, F, formula F-2 and its underlying molecular mechanisms in transgenic Caenorhabditis elegans models. A, S, F, and F-2 successfully restored 6-hydroxydopamine intoxicated DA neuron degenerations, reduced food-sensing behavior disabilities, and attenuated -Syn aggregations. Moreover, activates the lipid deposition and proteasome expressions to confirm -Syn degradations at the cellular level. Reactive oxygen species (ROS) cause oxidative stress, and A, S, F, and F-2 repressed ROS and raised SOD-3 expressions. Overall, these data indicate that V, A, S, F combined into F-2 (22.3%) are more effective against PD progression-like symptom than individual drugs V (0.7%), A (11.4%), S (9.6%), and F (12.6%). These improved neuroprotective actions of F-2 possibly due to following the antioxidative pathway. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/709540v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1a6f1f7org.highwire.dtl.DTLVardef@157a270org.highwire.dtl.DTLVardef@69a238org.highwire.dtl.DTLVardef@1194b5e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Inflammation and oxidative stress (OS) are key to Parkinsons disease (PD). We performed a cross-dataset integrative transcriptomic analysis to identify OS- and inflammation-related hub genes persistently dysregulated in PD and to evaluate their response to nutrigenomic interventions using publicly available datasets. Four GEO datasets (GSE7621, GSE20141, GSE20146, GSE49036) were analysed to identify differentially expressed genes (DEGs), which were intersected with GeneCards OS-inflammation gene sets. Functional enrichment analyses, including gene ontology (GO), pathway over-representation analysis (ORA), and protein-protein interaction (PPI) analysis, were used to identify key pathways and hub genes. Gene-food bioactive compound (FBC) association was explored by integrating PD signatures with nutrigenomic profiles from NutriGenomeDB. We identified 183 DEGs in PD, enriched in synaptic, dopaminergic, OS, and inflammatory pathways. Intersection analysis yielded 26 OS-inflammation-related genes and 10 central regulators, including TH, DDC, SNCA, LRRK2, HSPB1, and HSPA1B. revealed opposing transcriptional patterns, with several FBCs suppressing stress-related genes and upregulating dopaminergic markers such as TH, GCH1, and DDC. Overall, this integrative analysis highlights OS-inflammation gene networks in PD and identifies candidate diet-gene interactions that warrant further experimental validation