Mitochondrial Superoxide Research Articles

Agmatine suppresses glycolysis via the PI3K/Akt/mTOR/HIF-1α signaling pathway and improves mitochondrial function in microglia exposed to lipopolysaccharide.

Modulating metabolic pathways in activated microglia can alter their phenotype, which is relevant in uncontrolled neuroinflammation as a component of various neurodegenerative diseases. Here, we investigated how pretreatment with agmatine, an endogenous polyamine, affects metabolic changes in an in vitro model of neuroinflammation, a murine microglial BV-2 cell line exposed to lipopolysaccharide (LPS). Hence, we analyzed gene expression using qPCR and protein levels using Western blot and ELISA. Microglial metabolic status was assessed by measuring lactate release and cellular ATP by enzymatic and luminescence spectrophotometry. Mitochondrial functionality was analyzed by fluorescent probes detecting mitochondrial membrane potential (mtMP) and superoxide production. Our findings suggest that kinase pathways associated with hypoxia-inducible factor-1α (HIF-1α) regulate energy metabolism in pro-inflammatory activated microglia. We have shown that LPS induces HIF-1α and genes for glucose transporter and glycolytic rate, increases lactate production and causes mitochondrial dysfunction, suggesting a metabolic shift towards glycolysis. Agmatine inhibits the PI3K/Akt pathway and negatively regulates mammalian target of rapamycin (mTOR) phosphorylation and HIF-1α levels, reducing lactate and tumor necrosis factor (TNF) production, which is supported by pharmacological blockade of PI3K. Pretreatment with agmatine also rescues mitochondrial function by counteracting the LPS-induced decline in mtMP and increase in mitochondrial superoxide, resulting in an anti-apoptotic effect. Agmatine alone increases intracellular ATP levels and maintains this effect even under pro-inflammatory conditions. Our study emphasizes the ability of agmatine to engage in metabolic reprogramming of pro-inflammatory microglia through increased ATP production and modulation of signaling pathway involved in promoting glycolysis and cytokine release.

Involvement of mitochondrial dysfunction and oxidative stress in the nephrotoxicity induced by high-fat diet in Sprague-Dawley rats.

The prevalence of obesity-associated kidney injury has increased, yet the precise extent of the injury and its underlying mechanisms remain unclear. This study used a Sprague-Dawley (SD) rat model to simulate human exposure scenarios, with the objective of investigating the involvement of mitochondria in obesity-induced renal toxicity. Biochemical analysis revealed significant increases in serum creatinine, cystatin C, urinary protein, urinary microalbumin, and urinary α1-microglobulin levels in rats fed a high-fat diet, indicating a notable decline in glomerular filtration function. Histopathological examination showed mild to moderate degeneration in renal tubular epithelial cells, slight glomerular enlargement, fusion and disappearance of pedunculated cell, and decreased electron density of mitochondrial matrix and cristae, indicating the impaired filtration function of kidney. Furthermore, the study found reduced mitochondrial membrane potential and superoxide dismutase (SOD) levels, along with increased malondialdehyde (MDA) levels, signifying elevated mitochondrial oxidative stress in the kidneys of high-fat diet-fed rats. Additionally, a decrease in the number of mitochondrial proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) and uncoupling protein-2 (UCP-2)-positive cells, as well as reduced protein expression levels in the mitochondria, suggests a reduced renal mitochondrial resistance to oxidative stress. Collectively, these findings indicate that a high-fat diet triggers abnormalities in both renal filtration and structural functionality in SD rats. The observed reduction in renal mitochondrial density and the elevation in oxidative stress levels could potentially serve as underlying mechanisms.

Nepetin limits NLRP3 inflammasome activation and alleviates NLRP3-driven inflammatory diseases via PINK1-dependent mitophagy.

The NLRP3 inflammasome plays a pivotal role in the progression of inflammatory diseases. Mitochondrial damage, oxidative stress and mitochondrial DNA (mtDNA) leak are the key upstream factors for NLRP3 inflammasome activation. Nepetin (Nep), a naturally occurring flavonoid found with anti-inflammatory properties; however, whether it can affect the NLRP3 inflammasome activation and its precise anti-inflammatory mechanism remains unclear. In this study, we demonstrated that Nep enhances PINK1-mediated ubiquitin phosphorylation, which promotes mitophagy and subsequently inhibits NLRP3 inflammasome activation and pyroptosis in macrophages. The administration of Nep to macrophages alleviated of mitochondrial damage, reduced mitochondrial superoxide production, restored mitochondrial membrane potential and prevented the mtDNA leakage. These findings provide compelling evidence for the antioxidant effect of Nep. Furthermore, the pivotal function of mitophagy in the NLRP3 inflammasome inhibitory impact of Nep was substantiated through the utilisation of mitophagy inhibitors and siRNA techniques. Notably, Nep increased survival and reduced organ damage in mice with systemic inflammation by inhibiting NLRP3 inflammasome activation. In addition, Nep suppressed NLRP3 inflammasome activation in obese mice, which led to reduced white adipose and liver inflammation, thereby ameliorating insulin resistance. In conclusion, our findings suggest that Nep is a potent NLRP3 inflammasome inhibitor and a promising candidate for the development of anti-inflammatory therapies.

Hippo-YAP signaling alleviates copper-induced mitochondrial dysfunction and oxidative damage via the ATOX1-PPA2 pathway.

Hippo signaling plays a crucial role in the cellular response to various stressors, such as mechanical stress, metabolic stress, and hypoxic stress. However, its physiological significance in copper (Cu) stress remains poorly understood. Here, we demonstrated aberrant activation of Hippo-YAP signaling in sheep pancreas and pancreatic organoids exposed to excessive Cu, accompanied by significant pathological changes, elevated levels of oxidative stress, and impaired mitochondrial structure and function. The inhibition of Hippo signaling or overexpression of YAP protected against Cu-induced damage by improving mitochondrial function and maintaining cellular Cu homeostasis. YAP interacted with TEAD and upregulated the expression of Cu chaperone ATOX1, a key regulator of intracellular Cu homeostasis. ATOX1 restored mitochondrial function under Cu stress by reducing mitochondrial superoxide levels, increasing ATP production and mitochondrial membrane potential. Additionally, our findings confirmed that ATOX1 indirectly bound to the PPA2 promoter and increased its transcription. Notably, the restoration of ATP production in mitochondria mediated by PPA2 overexpression facilitated efficient intracellular Cu efflux, allowing rapid and precise reestablishment of intracellular Cu homeostasis under Cu stress. Collectively, Hippo-YAP signaling alleviates Cu-induced oxidative damage by restoring mitochondrial function through activation of PPA2 transcription depending on ATOX1, thereby ensuring cellular Cu efflux and enhancing antioxidant capacity.

The Potential Roles of IL-1β, IL-6, and RIPK3 in the Pathogenesis of Stevens–Johnson Syndrome/Toxic Epidermal Necrolysis

Background/Objectives: Stevens–Johnson Syndrome and Toxic Epidermal Necrolysis (SJS/TEN) are rare but severe skin conditions, often triggered by medications, that can be life-threatening. These conditions frequently affect the eyes, causing ocular surface disease, which can result in visual impairment or blindness. Although the exact mechanisms behind SJS/TEN remain unclear, key inflammatory mediators such as IL-1β, IL-6, and RIPK3 are believed to play critical roles in inflammation, necroptosis, and regulatory processes. Investigating these factors offers new insights into the disease’s underlying mechanisms and potential targets for treatment. This study aims to determine the roles of IL-1β, IL-6, and RIPK3 in the pathogenesis of SJS/TEN. Methods: The study examined the expression levels of IL-1β, IL-6, and RIPK3 in skin biopsies from patients with biopsy-confirmed SJS/TEN, using lichen planus as a positive control and normal skin as a baseline control. Immunohistochemistry was employed for this analysis. Additionally, the impact of SJS/TEN patient plasma on mitochondrial function was assessed in platelets and human corneal epithelial (H-CET) cells. Using a fluorescent plate reader, mitochondrial activity and superoxide ion levels were measured, comparing plasma from SJS/TEN patients to normal human plasma. Results: Skin biopsies from SJS/TEN patients showed a significantly higher expression of IL-1β, IL-6, and RIPK3 compared to both lichen planus and normal controls. Furthermore, plasma from SJS/TEN patients significantly reduced platelet viability and increased mitochondrial and total cellular superoxide ions, as demonstrated by elevated levels of MitoSOX Red and CellROX Red. Conclusions: These findings suggest that IL-1β, IL-6, and RIPK3 may contribute to the pathogenesis of SJS/TEN and highlight their potential as targets for therapeutic intervention.

Autophagic flux-lipid droplet biogenesis cascade sustains mitochondrial fitness in colorectal cancer cells adapted to acidosis

Cancer development is associated with adaptation to various stressful conditions, such as extracellular acidosis. The adverse tumor microenvironment also selects for increased malignancy. Mitochondria are integral in stress sensing to allow for tumor cells to adapt to stressful conditions. Here, we show that colorectal cancer cells adapted to acidic microenvironment (CRC-AA) are more reliant on oxidative phosphorylation than their parental cells, and the acetyl-CoA in CRC-AA cells are generated from fatty acids and glutamine, but not from glucose. Consistently, CRC-AA cells exhibit increased mitochondrial mass and fitness that depends on an upregulated autophagic flux-lipid droplet axis. Lipid droplets (LDs) function as a buffering system to store the fatty acids derived from autophagy and to protect mitochondria from lipotoxicity in CRC-AA cells. Blockade of LD biogenesis causes mitochondrial dysfunction that can be rescued by inhibiting carnitine palmitoyltransferase 1 α (CPT1α). High level of mitochondrial superoxide is essential for the AMPK activation, resistance to apoptosis, high autophagic flux and mitochondrial function in CRC-AA cells. Thus, our results demonstrate that the cascade of autophagic flux and LD formation plays an essential role in sustaining mitochondrial fitness to promote cancer cell survival under chronic acidosis. Our findings provide insight into the pro-survival metabolic plasticity in cancer cells under microenvironmental or therapeutic stress and imply that this pro-survival cascade may potentially be targeted in cancer therapy.

Inhibition of acyl-CoA synthetase long-chain isozymes decreases multiple myeloma cell proliferation and causes mitochondrial dysfunction.

Multiple myeloma (MM) is an incurable cancer of plasma cells with a 5-year survival rate of 59%. Dysregulation of fatty acid (FA) metabolism is associated with MM development and progression; however, the underlying mechanisms remain unclear. Herein, we explore the roles of long-chain fatty acid coenzyme A ligase (ACSL) family members in MM. ACSLs convert free long-chain fatty acids into fatty acyl-CoA esters and play key roles in catabolic and anabolic fatty acid metabolism. Analysis of the Multiple Myeloma Research Foundation (MMRF) CoMMpassSM study showed that high ACSL1 and ACSL4 expression in myeloma cells are both associated with worse clinical outcomes for MM patients. Cancer Dependency Map (DepMap) data showed that all five ACSLs have negative Chronos scores, and ACSL3 and ACSL4 were among the top 25% Hallmark Fatty Acid Metabolism genes that support myeloma cell line fitness. Inhibition of ACSLs in myeloma cell lines in vitro, using the pharmacological inhibitor Triacsin C (TriC), increased apoptosis, decreased proliferation, and decreased cell viability, in a dose- and time-dependent manner. RNA-sequencing analysis of MM.1S cells treated with TriC showed a significant enrichment in apoptosis, ferroptosis, and endoplasmic reticulum (ER) stress, and proteomic analysis of these cells revealed enriched pathways for mitochondrial dysfunction and oxidative phosphorylation. TriC also rewired mitochondrial metabolism by decreasing mitochondrial membrane potential, increasing mitochondrial superoxide levels, decreasing mitochondrial ATP production rates, and impairing cellular respiration. Overall, our data support the hypothesis that suppression of ACSLs in myeloma cells is a novel metabolic target in MM that inhibits their viability, implicating this family as a promising therapeutic target in treating myeloma.

Intestine-specific disruption of mitochondrial superoxide dismutase extends longevity.

Reactive oxygen species (ROS) are highly reactive oxygen containing molecules that are generated by normal metabolism. While ROS can cause damage to the building blocks that make up cells, these molecules can also act as intracellular signals that promote longevity. The levels of ROS within the cell can be regulated by antioxidant enzymes, such as superoxide dismutase (SOD), which converts superoxide to hydrogen peroxide. Interestingly, our previous work has shown that disruption of the mitochondrial SOD gene sod-2 results in increased lifespan, suggesting that elevating levels of mitochondrial superoxide can promote longevity. To explore the molecular mechanisms involved, we determined the tissues in which disruption of sod-2 is necessary for lifespan extension and the tissues in which disruption of sod-2 is sufficient to extend lifespan. We found that tissue-specific restoration of SOD-2 expression in worms lacking SOD-2 could partially revert changes in fertility, embryonic lethality and resistance to stress, but did not inhibit the effects of sod-2 deletion on lifespan. Knocking down sod-2 expression using RNA interference specifically in the intestine, but not other tissues, was sufficient to extend longevity. Intestine-specific knockdown of sod-2 also increased resistance to heat stress while decreasing resistance to oxidative stress. Combined, these results indicate that disruption of sod-2 in neurons, intestine, germline, or muscle is not required for lifespan extension, but that decreasing sod-2 expression in just the intestine extends lifespan. This work defines the conditions required for disruption of mitochondrial superoxide dismutase to increase longevity.

Caspase Inhibition Restores Collagen I α1 and Fibronectin Release in Cigarette Smoke Extract-Exposed Human Lung Fibroblasts.

Chronic obstructive pulmonary disease (COPD) is a progressive lung disease characterized by obstructed airflow, airway remodeling, and inflammation, with cigarette smoke (CS) exposure being the main risk factor. While CS extract (CSE) has been shown to activate caspases in various cell types, the role of caspases in human lung fibroblasts (hLFs), in COPD remains poorly understood. Recent studies have linked caspases to extracellular matrix (ECM) remodeling in skin and kidney fibrosis. Caspase activation can be triggered by oxidative stress, with active caspase-3 executing the pore-forming protein gasdermin E (GSDME) in the cleaved N-terminal form GSDME-NT. We investigated whether CSE activates caspases and GSDME in hLFs, and their role in ECM remodeling. MRC-5 lung fibroblasts were treated with CSE with or without the antioxidant N-acetyl cysteine (NAC), and the caspase-8 inhibitor z-IETD-fmk. We measured the effects on caspase-1-8-3/7 activation, GSDME cleavage, ECM remodeling (procollagen Iα1, COLIα1, and fibronectin, FN), and mitochondrial superoxide (mSOX) generation. Key findings were validated in patient-derived hLFs (phLFs). Our results showed that CSE induced caspase-1-8-3/7 activation, mSOX generation, and decreased COLIα1 and FN levels in MRC-5. CSE caused caspase-8-dependent activation of caspase-3, leading to the GSDME cleavage. Treatment with NAC inhibited mSOX and caspase activation. Inhibition of caspase-8 and mSOX restored FN and COLIα1 levels. In phLFs, we confirmed caspase-1 and -8 activation, mSOX increase, COLIα1/FN decrease, and the effects of NAC. Our findings highlight the role of the axis caspase-8-3/7-GSDME and mSOX in regulating ECM protein, suggesting that these pathways may contribute to remodeling in COPD.

Modelling the human Coenzyme Q deficiency in Drosophila melanogaster.

The interference of the expression of each of the genes involved in the synthesis of coenzyme Q (CoQ) in Drosophila melanogaster can help to understand the pathophysiology of CoQ-dependent mitochondrial diseases in humans. We have knocked-down all genes involved in the CoQ biosynthesis pathway at different temperatures to induce depletion of CoQ at different levels throughout the body and in a tissue-specific manner. The efficiency of the knockdowns was quantified by Q-RTPCR and determination of CoQ levels by HPLC-UV+ECD. We performed mitochondria purification and quantified respiratory chain activity, both mitochondrial hydrogen peroxide and superoxide production, resistance to mechanical stress and determination of life expectancy. Finally, we evaluated the effect of CoQ10 supplementation as phenotype rescue therapy. D. melanogaster presents 3 isoforms of CoQ: CoQ8, CoQ9 and CoQ10. The level of depletion depended on the efficiency of the RNAi used and is specific for each gene. The interference of some genes interrupted fly development in embryogenesis (pdss2) or during metamorphosis (pdss1, coq3, coq5, coq8 and coq10), while in other cases viable adults can be obtained (coq2, coq6 and coq7). The knockdown of coq7 accumulated intermediates of the CoQ biosynthesis pathway at all stages of development, altered electron transfer with poor assembly of mitochondrial complexes, and deregulated mitochondrial hydrogen peroxide and superoxide production. Coq7 mutant flies showed partial lethality in metamorphosis, bang sensitivity and reduced life span of surviving animals. CoQ10 supplementation rescued the coq7-mutant phenotypes. Knock-down in the imaginal disc generated gene-specific eye deformities that can be mitigated by CoQ10 supplementation. Our results indicate that interference of the CoQ biosynthesis pathway in D. melanogaster shows a great diversity of phenotypes depending on the target gene, mirroring the heterogeneity of CoQ deficiency syndrome in humans and point to why mutations in certain genes are rarely found in patients.

Unveiling the link between NADPH oxidase 2 activation and mitochondrial superoxide formation in leukemic cell killing induced by arsenic trioxide.

This study focused on the interplay between NADPH oxidase 2 (NOX 2) activation and mitochondrial superoxide (mitoO2.-) formation induced by clinically relevant concentrations of arsenic trioxide (ATO; As2O3) in acute promyelocytic leukemia (APL) cells. Carefully controlled inhibitor studies and small interfering RNA mediated downregulation of p47phox (a component of the NOX 2 complex) expression demonstrated that, in an APL cell line, ATO promotes upstream NOX 2 activation critically connected with the formation of mitoO2.- and with the ensuing mitochondrial permeability transition (MPT)-dependent apoptosis. Instead, acute myeloid leukemia (AML) cell lines respond to ATO with low NOX 2 activation, resulting in a state that is non-permissive for mitoO2.- formation. Consistently, through rescue experiments, we demonstrate that pharmacological stimulation of NOX 2 overcomes resistance in these cells, thereby initiating the same cascade of downstream events observed in APL cells. As a final note, several lines of evidence, including measurement of glutathione, catalase and glutathione peroxidase levels, indicated that the antioxidant machinery was similar in APL and AML cells. The results regarding nuclear factor erythroid 2 p45-related factor 2-dependent antioxidant responses were instead of more complex interpretation as NB4 cells appeared particularly responsive to ATO. Our findings allow a novel interpretation of the interplay between NOX 2 activation and mitoO2.- formation induced by ATO, ultimately steering leukemic cells towards MPT-dependent apoptosis. These mechanistic insights provide a rationale for the disparate responses of APL and AML cells to ATO, offering potential avenues for the development of therapeutic intervention tailored to specific leukemia subtypes.

APP antisense oligonucleotides are effective in rescuing mitochondrial phenotypes in human iPSC-derived trisomy 21 astrocytes.

Antisense oligonucleotides (ASOs) have shown promise in reducing amyloid precursor protein (APP) levels in neurons, but their effects in astrocytes, key contributors to neurodegenerative diseases, remain unclear. This study evaluates the efficacy of APP ASOs in astrocytes derived from an individual with Down syndrome (DS), a population at high risk for Alzheimer's disease (AD). Human induced pluripotent stem cells (hiPSCs) from a healthy individual and an individual with DS were differentiated into astrocytes. Astrocytes were treated with APP ASOs for 10 days, and APP levels were quantified. Mitochondrial morphology and superoxide production in DS astrocytes were analyzed using super-resolution and confocal microscopy. APP ASOs significantly reduced APP levels in astrocytes from both control and DS individuals. In DS astrocytes, treatment restored mitochondrial health, increasing mitochondrial number and size while reducing superoxide production. APP ASOs effectively reduce APP levels and improve mitochondrial health in astrocytes, suggesting their potential as a therapeutic approach for DS and DS-related AD. Further in vivo studies are required to confirm these findings. APP ASOs reduce APP levels in human iPSC-derived astrocytes. APP ASO treatment rescues mitochondrial phenotypes in trisomy 21 astrocytes. This study supports ASOs as a potential therapy for Down syndrome-related Alzheimer's disease.

The Relation Between Mitochondrial Membrane Potential and Reactive Oxygen Species Formation.

Mitochondria are considered one of the main sites of reactive oxygen species (ROS) production in the eukaryotic cells. For this reason, mitochondrial dysfunction associated with increased ROS production underlies various pathological conditions as well as promotes aging. Chronically increased rates of ROS production contribute to oxidative damage to macromolecules, i.e., DNA, proteins, and lipids. Accumulation of unrepaired oxidative damage may result in progressive cell dysfunction, which can finally trigger cell death. The main by-product of mitochondrial oxidative phosphorylation is superoxide, which is generated by the leak of electrons from the mitochondrial respiratory chain complexes leading to one-electron reduction of oxygen. Mitochondrial superoxide dismutase (MnSOD, SOD2) as well as cytosolic superoxide dismutase (Cu/ZnSOD, SOD1), whose smaller pool is localized in the mitochondrial intermembrane space, converts superoxide to H2O2, which can be then degraded by the catalase to harmless H2O.In this chapter, we focus on the relationship between one of the bioenergetic parameters, which is mitochondrial membrane potential, and the rate of ROS formation. We present a set of various methods enabling the characterization of these parameters applicable to isolated mitochondria or intact cells. We also present examples of experimental data demonstrating that the magnitude and direction (increase or decrease) of a change in mitochondrial ROS production depend on the mitochondrial metabolic state.

The mitochondrial and cytoplasmic superoxide anion imbalance trigger the expression of certain cellular aging markers in HaCaT keratinocytes.

In cells, the term "cellular aging" represents a collection of biological changes that can precede the proliferative senescence states. Cells more resistant to proliferative senescence, such as the ones found in the basal layer of the epidermis, may also exhibit these aging patterns. Therefore, cellular aging events could be induced by endogenous signals named here as cellular aging triggers (CATs) components. The superoxide anion (O2⁻) could be a prime candidate for a CATs, as it is continuously produced by eukaryotic cells. To test this hypothesis, mitochondrial and cytoplasmic O2⁻ imbalances were induced in HaCaT keratinocytes using rotenone (ROT, 30µM), which inhibits mitochondrial complex I and paraquat (PQT, 30µM), which increases O2⁻ levels via redox cycling. ROT and PQT reduced cellular proliferation rate and elevated β-Galactosidase and transforming growth factor beta (TGF-β) levels. Furthermore, they increased the frequency of larger cells with nuclear alterations, the levels of oxidative markers, and interleukin 1β, a marker of the Senescence-Associated Secretory Phenotype (SASP). However, the mitochondrial O2⁻ imbalance caused by ROT led to more pronounced alterations compared to PQT. These findings support the hypothesis that the existence of CAT components, such as the O2⁻ anion, plays a significant role in cellular aging.

Succinate Regulates Endothelial Mitochondrial Function and Barrier Integrity.

Endothelial dysfunction is a hallmark of several pathological conditions, including cancer, cardiovascular disease and inflammatory disorders. In these conditions, perturbed TCA cycle and subsequent succinate accumulation have been reported. The role of succinate as a regulator of immunological responses and inflammation is increasingly being recognized. Nevertheless, how endothelial cell function and phenotype are altered by elevated intracellular succinate has not been addressed yet. Thus, we employed numerous in vitro functional assays using primary HUVECs and diethyl succinate (DES), a cell membrane-permeable succinate analogue. An MTS assay 1 h post stimulation with DES suggested reduced metabolic activity in HUVECs. Concurrently, elevated production of ROS, including mitochondrial superoxide, and a reduction in mitochondrial membrane potential were observed. These findings were corroborated by Seahorse mito-stress testing, which revealed that DES acutely lowered the OCR, maximal respiration and ATP production. Given the link between mitochondrial stress and apoptosis, we examined important survival signalling pathways. DES transiently reduced ERK1/2 phosphorylation, a response that was followed by a skewed pro-apoptotic shift in the BAX to BCL2L1 gene expression ratio, which coincided with upregulating VEGF gene expression. This indicated an induction of mixed pro-apoptotic and pro-survival signals in the cell. However, the BAX/BCL-XL protein ratio was unchanged, suggesting that the cells did not commit themselves to apoptosis. An MTS assay, caspase 3/7 activity assay and annexin V/propidium iodide staining confirmed this finding. By contrast, stimulation with DES induced acute endothelial barrier permeability, forming intercellular gaps, altering cell size and associated actin filaments without affecting cell count. Notably, during overnight DES exposure gradual recovery of the endothelial barrier and cell sprouting was observed, alongside mitochondrial membrane potential restoration, albeit with sustained ROS production. COX-2 inhibition and EP4 receptor blockade hindered barrier restoration, implicating a role of COX-2/PGE2/EP4 signalling in this process. Interestingly, ascorbic acid pre-treatment prevented DES-induced acute barrier disruption independently from ROS modulation. In conclusion, succinate acts as a significant regulator of endothelial mitochondrial function and barrier integrity, a response that is counterbalanced by upregulated VEGF and prostaglandin production by the endothelial cells.

Establishment of embryogenic Pinus thunbergii Parl. suspension cultures: growth parameters, dynamic analysis, and plant regenerative capacities

BackgroundPinus thunbergii is an economically important conifer species that plays a fundamental role in forest ecosystems. However, the population has declined dramatically in recent years as a result of the pine wilt disease outbreak. Thus, developing pine wilt-resistant P. thunbergii is an effective strategy for combating this epidemic.ResultsThe somatic embryogenesis of nematode-resistant P. thunbergii was previously reported by our group. The current study looked into the potential commercialization of suspension cultures as a means of large-scale production of nematode-resistant P. thunbergii seedlings. According to our findings, P. thunbergii suspension cultures were suitable for an initial inoculum of embryogenic tissue (2 g) and a subculture inoculum (6.7% (v/v)). Suspension cultures were cultivated for 8–10 days in a 30 mL liquid medium (Gupta and Durzan medium, DCR medium) to facilitate their maturation. The suspension cultures produced a large number of high-quality somatic embryos, which were then used to regenerate the plants and move them into the field. A more accurate assessment of the quality of suspension cultures for somatic embryogenesis could come from the suspension’s dynamics. The results showed that the medium’s phosphate, ammonium, nitrate, and carbohydrates were quickly eaten from day 0 to day 10. In terms of the absorption of nitrogen sources, the ammonium (NH4+) was absorbed prior to nitrate (NO3−). Additionally, the activity of mitochondrial succinate dehydrogenase and superoxide dismutase was directly related to cell growth.ConclusionsThis study presents an approach for selecting appropriate suspension cultures for efficient somatic maturation of P. thunbergii that can also be applied to other conifers. Furthermore, it is possible to commercialize nematode-resistant P. thunbergii seedlings using bioreactors, according to the suspension culture system we describe. To the best of our knowledge, this is the first work to describe a P. thunbergii suspension culture.

M2 Macrophage-Derived Exosomal circ_0088494 Inhibits Ferroptosis via Promoting H3K4me1 Modification of STEAP3 in Cutaneous Squamous Cell Carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is a common type of cutaneous cancer globally. M2 macrophage-derived exosomes (M2 exosomes) facilitate the development of cancer. Ferroptosis, a newly uncovered form of cell death, is linked to cancer progression. The present research planned to study the function and potential mechanism of M2 exosomes on ferroptosis in cSCC. Patients with cSCC were recruited to gather adjacent noncancerous specimens and cSCC tissues. Mononuclear macrophage (THP-1) cells were differentiated into M2 macrophages before exosome extraction, and then the exosomes were added into cSCC cells (A431 and SCL-1). Erastin was applied to induce ferroptosis. Cell viability, mitochondrial superoxide, lipid-ROS, malondialdehyde (MDA), and iron level were detected to validate ferroptosis in cSCC cells. Proteins and RNAs were tested by applying western blot and RT-qPCR. The combination between molecules was validated by ChIP and RIP. Six-transmembrane epithelial antigen of the prostate 3 (STEAP3) was elevated in cSCC specimens, which correlated to reduced ferroptosis. cSCC tissues presented an increase in the number of M2 macrophages. Erastin-elicited ferroptosis was repressed by M2 macrophages, while exosome inhibitor GW4869 neutralized the outcome of M2 macrophages. Furthermore, M2 exosomes repressed ferroptosis of cSCC cells via circ_0088494, which might be related to the upregulation of STEAP3. M2 exosomes-derived circ_0088494 promoted histone 3 lysine 4 monomethylation (H3K4me1) modification of STEAP3 by recruiting histone-lysine N-methyltransferase 2D (KMT2D). The effect of circ_0088494-silenced M2 exosomes on ferroptosis was antagonized by STEAP3 overexpression. M2 exosomes-derived circ_0088494 recruited KMT2D to promote H3K4me1 modification of STEAP3, thereby inhibiting ferroptosis in cSCC. This study might provide a novel target for cSCC treatment.

IL20RA Is the Key Factor Contributing to the Stronger Antioxidant Capacity of Rongchang Pig Sertoli Cells

Variations in disease resistance among pig breeds have been extensively documented, with Sertoli cells (SCs) playing a pivotal role in spermatogenesis. Infections can induce oxidative stress, which can lead to damage to these cells. This study aimed to compare the levels of oxidative stress in SCs from Rongchang and Landrace pig breeds following LPS challenge. SCs were isolated, cultured, and stimulated with LPS to assess cell viability and markers of oxidative stress. Cell viability was evaluated along with oxidative stress markers such as reactive oxygen species (ROS), mitochondrial superoxide, malondialdehyde, and antioxidant enzymes. Mitochondrial function was assessed using JC-1 and Calcein AM probes. Transcriptomic analysis identified differentially expressed genes (DEGs), while ingenuity pathway analysis (IPA) explored enriched pathways. IL20RA, identified through transcriptomics, was validated using the siRNA knockdown technique. The results showed that Rongchang SCs exhibited lower levels of oxidative stress compared to Landrace SCs along with higher activity of antioxidant enzymes. IL20RA emerged as a key regulator since its knockdown affected mitochondrial superoxide production and catalase secretion. The findings suggest that Rongchang SCs possess superior antioxidant capacity, possibly due to the IL20RA-mediated protection of mitochondria, thereby providing insights into breed-specific resistance against oxidative stress and highlighting the role of IL20RA in maintaining stem cell function.

Role of PI3K/AKT/MAOA in glucocorticoid-induced oxidative stress and associated premature senescence of the trabecular meshwork.

The oxidative stress-induced premature senescence of trabecular meshwork (TM) represents a pivotal risk factor for the development of glucocorticoid-induced glaucoma (GIG). This study aimed to elucidate the pathogenesis of TM senescence in GIG. MethodsIntraocular pressure (IOP), transmission electron microscopy and senescence-associated protein expression in TM were evaluated in GIG mice. Protein expression of phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) and monoamine oxidase A (MAOA), phosphorylation of AKT were quantified. ROS and mitochondrial superoxide levels were measured to evaluate cellular oxidative stress. Cell cycle analysis, β-galactosidase staining, senescence-associated protein expression were employed to assess the aging status of primary human trabecular meshwork cells (pHTMs). ResultsmRNA-seq and KEGG analysis indicating PI3K/AKT pathway as a key regulator in TM of GIG. PI3K inhibitor significantly prevented IOP elevation and abnormal mitochondrial morphology of TM in the GIG mouse model. PI3K inhibitor or selective silencing of PIK3R1 alleviated dexamethasone (DEX)-induced oxidative stress, also mitochondrial dysfunction, inhibiting MAOA expression in pHTMs. The same phenomenon was observed in the GIG models with inhibition of MAOA. Further KEGG analysis indicates that cellular senescence is the key factor in the pathogenesis of GIG. TM senescence was observed in both GIG mouse and cell models. Inhibition of the PI3K/AKT/MAOA pathway significantly alleviated DEX-induced premature cellular senescence of TM in GIG models. Glucocorticoids activated the PI3K/AKT/MAOA pathway, leading to mitochondrial dysfunction, oxidative stress, and premature aging in TM, elevating IOP. This mechanism could be associated with the onset and progression of GIG, providing a potential approach for its treatment.

Design and synthesis of novel 3,7-dihydro-1H-purine-2,6-diones as DPP-4 inhibitors: An in silico, in vitro and in vivo approach.

The inhibition of enzyme DPP-4 is pivotal for targeting type 2 diabetes mellitus (DM). The study introduces two series of novel 1,3-dimethyl-3,7-dihydro-1H-purine-2,6-diones derivatives (PB01-PB10) and 3,7-dihydro-1H-purine-2,6-diones compounds (PB11-PB16) were developed using linagliptin scaffold. Sixteen derivatives were synthesized and screened in vitro against DPP-4, revealing IC50 ranging from 15.66±2.546 to 28.45±4.441nM. Compounds PB01 and PB11 demonstrated high potency comparable to reference standard linagliptin (IC50=15.66±2.546, 16.16±1.214, 15.37±2.481nM, respectively). Further studies showed that the compound possesses negligible cytotoxicity up to 100μM concentration. A high glucose-induced DPP-4 upregulation model was further utilized to assess the protective effect of PB01 and PB11, and their efficacy was compared with linagliptin. PB01 and PB11 showed comparable protective effects against high glucose-induced ROS generation and mitochondrial superoxide production, and the compounds also effectively reduced the DPP-4 cellular expression. The in vivo anti-diabetic efficacy, effect on change in body weight, and OGTT due to PB01 and PB11 treatments were evaluated using the STZ-Nicotinamide-induced experimental model of diabetes in mice. Post induction of diabetes, the periodic estimation of blood serum glucose levels reveals that treatment with PB01 and PB11 decreased the high blood serum glucose levels in both acute and chronic studies. The expressions of DPP-4 were observed by IHC, Flowcytometry, and RT-qPCR. The docked complexes of both compounds, along with the standard drug linagliptin, were subjected to molecular dynamics simulation for 140ns to evaluate the complexes' stability and binding affinity.