Improved Cell Survival Research Articles

The Role of Beta-Hydroxybutyrate in Mitigating the Inflammatory and Metabolic Consequences of Uric Acid

Background: Uric acid (UA), a metabolite of purine and fructose metabolism, is linked to inflammation and metabolic disorders, including gout and cardiovascular disease. Its pro-inflammatory effects are largely driven by the activation of the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, leading to increased cytokine production. Beta-hydroxybutyrate (BHB), a ketone produced during fasting or carbohydrate restriction, has been shown to reduce inflammation. This study explores the role of BHB in mitigating the inflammatory and metabolic effects of elevated uric acid levels. Methods: We utilized a murine muscle cell culture treated with UA and BHB. Results: Muscle cells treated with UA had increased production of pro-inflammatory cytokines and reduced cell viability. Co-treatment with BHB reversed these effects, improving cell survival and reducing cytokine levels. Additionally, uric acid impaired mitochondrial function and increased oxidative stress, which were mitigated by BHB. Furthermore, uric acid disrupted insulin signaling, but BHB co-treatment restored insulin sensitivity. Conclusions: These findings suggest that BHB holds therapeutic potential by counteracting the inflammatory and metabolic disruptions caused by elevated uric acid, making it a promising target for conditions such as hyperuricemia and metabolic syndrome.

Human adenovirus type 7 (HAdV-7) infection induces pulmonary vascular endothelial injury through the activation of endothelial autophagy

BackgroundHAdV-7 is a prevalent pathogen that can cause severe pneumonia in children. Previous studies have shown a significant increase in serum levels of vascular permeability factor (VPF/VEGF) and viral load in pediatric patients with fatal HAdV-7 infection, suggesting potential damage to the pulmonary vascular endothelium. Further research is necessary to elucidate the underlying mechanism.MethodsThe human lung microvascular endothelial cell line-5a and human CD46 mice were used for in vitro and in vivo experiments, respectively. RNA-seq was employed for correlative omics analysis. Viral infection and copy status were examined using transmission electron microscopy to observe virus particles, immunofluorescence to detect the viral protein Hexon, and qPCR to assess HAdV-7 fiber gene copies. Various methods, including ELISAs for VEGF and other injury markers, the CCK8 assay for cell viability, and flow cytometry for endothelium numbers, were employed to evaluate endothelial damage. Acute lung injury severity was evaluated by scoring pathological inflammation and measuring pulmonary vascular permeability. Autophagy activation was assessed by observing autophagosomes and validating marker proteins.ResultsGSEA analysis showed significant enrichment of gene sets related to endothelial functions (barrier, defense, and regeneration) and ALI in the HAdV-7-infected group. GO analysis indicated an enrichment of autophagy-related pathways linked to cell death. Subsequently, successful signs of HAdV-7 infection and replication were observed in the endothelium, including cytopathic effects, intracellular virions, and increased HAdV-7 fiber gene copies. Endothelial injury, including mitochondrial damage, decreased endothelium, and elevated levels of endothelial injury markers such as VEGF, sICAM-1, sVCAM-1, E-selectin, ESM1, MCP1, and IL1β were observed after HAdV-7 infection. Additionally, evidence of leaky lung blood vessels and ALI was observed, including progressive weight loss, elevated pulmonary vascular permeability, and severe lung consolidation. Furthermore, HAdV-7 infection induced autophagosome formation in the endothelium and triggered complete cell autophagy. Importantly, inhibiting autophagic flux reduced VEGF levels and other endothelial injury markers, decreased viral load, improved cell survival rate, alleviated pulmonary vessel leakage, and mitigated lung inflammation.ConclusionsHAdV-7 successfully infects pulmonary vascular endothelium and replicates effectively, causing injury to the endothelium, high VEGF expression and viral load in the serum, as well as ALI/ARDS. Autophagy inhibitors can alleviate endothelial injury, inhibit viral replication, relieve leakage from the vasculature, and reduce lung inflammation.

Exosomal microRNA-21-5p from gastric cancer cells promotes angiogenesis by targeting LEMD3 in human endothelial cells

Abstract Objectives The effect of exosome-derived miR-21-5p from gastric cancer (GC) on angiogenesis remains unclear. This study aims to examine the angiogenic impact of GC exosome-derived miR-21-5p. Methods Exosomes were isolated from GC cells and co-cultured with human umbilical vein endothelial cells (HUVECs). miR-21-5p levels in HUVECs were measured by qPCR. Flow cytometry was used to assess apoptosis, and the Cell Counting Kit-8 was used to assess cell growth. Bioinformatics analysis was used to identify the miR-21-5p target genes, which were then verified by dual-luciferase gene reporter experiments. qPCR and western blotting were employed to assess the expression of genes and proteins, respectively. Functional rescue assays were conducted to verify that miR-21-5p regulates endothelial cell function by targeting the LEM domain containing 3 (LEMD3). Additionally, cell migration was assessed using a scratch assay. Results Co-cultivation with GC-derived exosomes improved cell survival, decreased apoptosis, and raised miR-21-5p levels in HUVECs. Increases in vascular endothelial growth factor A (VEGFA) and the TGF-β/Smad signaling pathway were seen. It was shown that miR-21-5p targets LEMD3. The biological effects of miR-21-5p were lessened when miR-21-5p was inhibited, or LEMD3 was overexpressed. Conclusions By targeting LEMD3, miR-21-5p in GC cell exosomes stimulates angiogenesis by triggering the TGF-β/Smad signaling cascade and upregulating VEGFA expression. This leads to increased proliferation, survival, and migration of HUVECs, underscoring the potential of targeting this pathway in cancer therapy.

Raloxifene Protects Oxygen-Glucose-Deprived Astrocyte Cells Used to Mimic Hypoxic-Ischemic Brain Injury.

Perinatal asphyxia (PA) is a clinical condition characterized by oxygen supply suspension before, during, or immediately after birth, and it is an important risk factor for neurodevelopmental damage. Its estimated 1/1000 live births incidence in developed countries rises to 5-10-fold in developing countries. Schizophrenia, cerebral palsy, mental retardation, epilepsy, blindness, and others are among the highly disabling chronic pathologies associated with PA. However, so far, there is no effective therapy to neutralize or reduce PA-induced harm. Selective regulators of estrogen activity in tissues and selective estrogen receptor modulators like raloxifene have shown neuroprotective activity in different pathological scenarios. Their effect on PA is yet unknown. The purpose of this paper is to examine whether raloxifene showed neuroprotection in an oxygen-glucose deprivation/reoxygenation astrocyte cell model. To study this issue, T98G cells in culture were treated with a glucose-free DMEM medium and incubated at 37 °C in a hypoxia chamber with 1% O2 for 3, 6, 12, and 24 h. Cultures were supplemented with raloxifene 10, and 100 nM during both glucose and oxygen deprivation and reoxygenation periods. Raloxifene 100 nM and 10 nM improved cell survival-65.34% and 70.56%, respectively, compared with the control cell groups. Mitochondrial membrane potential was preserved by 58.9% 10 nM raloxifene and 81.57% 100 nM raloxifene cotreatment. Raloxifene co-treatment reduced superoxide production by 72.72% and peroxide production by 57%. Mitochondrial mass was preserved by 47.4%, 75.5%, and 89% in T98G cells exposed to 6-h oxygen-glucose deprivation followed by 3, 6, and 9 h of reoxygenation, respectively. Therefore, raloxifene improved cell survival and mitochondrial membrane potential and reduced lipid peroxidation and reactive oxygen species (ROS) production, suggesting a direct effect on mitochondria. In this study, raloxifene protected oxygen-glucose-deprived astrocyte cells, used to mimic hypoxic-ischemic brain injury. Two examiners performed the qualitative assessment in a double-blind fashion.

Prussian blue nanocages as efficient radical scavengers and photothermal agents for reducing amyloid-beta induced neurotoxicity

The unusual accumulation of amyloid-beta 1–42 (Aβ42) is an essential pathological feature of Alzheimer’s disease (AD), and development of Aβ42 nanomodulators offers a potentially therapeutic approach to AD. Here, we report facile synthesis of the hollow mesocrystalline Prussian blue nanocages (HMPBs), which serve as versatile Aβ42 modulators. Due to the hollow nanostructures and large specific surface area, they can effectively inhibit Aβ42 aggregation by adsorption. They also exhibit robust near-infrared (NIR) photothermal effect for light-to-heat transition, which promotes the depolymerization of Aβ42 fibers. Besides, they display ROS quenching ability to scavenge hydroxyl radicals (•OH) caused by Aβ42 fibers, alleviate cellular oxidative stress, and improve cell survival. This work provides a new kind of Prussian blue nanomaterial for multimodal Aβ modulation.

CRISPRi/a screens in human iPSC-cardiomyocytes identify glycolytic activation as a druggable target for doxorubicin-induced cardiotoxicity

Doxorubicin is limited in its therapeutic utility due to its life-threatening cardiovascular side effects. Here, we present an integrated drug discovery pipeline combining human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iCMs), CRISPR interference and activation (CRISPRi/a) bidirectional pooled screens, and a small-molecule screening to identify therapeutic targets mitigating doxorubicin-induced cardiotoxicity (DIC) without compromising its oncological effects. The screens revealed several previously unreported candidate genes contributing to DIC, including carbonic anhydrase 12 (CA12). Genetic inhibition of CA12 protected iCMs against DIC by improving cell survival, sarcomere structural integrity, contractile function, and calcium handling. Indisulam, a CA12 antagonist, can effectively attenuate DIC in iCMs, engineered heart tissue, and animal models. Mechanistically, doxorubicin-induced CA12 potentiated a glycolytic activation in cardiomyocytes, contributing to DIC by interfering with cellular metabolism and functions. Collectively, our study provides a roadmap for future drug discovery efforts, potentially leading to more targeted therapies with minimal off-target toxicity.

Tetramethylpyrazine Analogue T-006 Protects Neuronal and Endothelial Cells Against Oxidative Stress via PI3K/AKT/mTOR and Nrf2 Signaling.

T-006, a novel neuroprotective derivative of tetramethylpyrazine (TMP), exhibits multifunctional neuroprotective properties. T-006 has been shown to improve neurological and behavioral functions in animal models of ischemic stroke and neurodegenerative diseases. The present study aims to further elucidate the mechanisms underlying the protective effects of T-006 against oxidative injuries induced by glutamate or hypoxia. Mouse hippocampal HT22 cells were used to evaluate the neuroprotective effects of T-006 against glutamate-induced injuries, while mouse brain endothelial bEnd.3 cells were used to evaluate the cerebrovascular protective effects of T-006 against oxygen-glucose deprivation followed by reperfusion (OGD/R)-induced injuries. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry were used to measure cell viability and oxidative stress. Western blot and immunofluorescence analyses of protein expression were used to study cell signaling pathways. T-006 exhibited significant protective effects in both oxidative injury models. In HT22 cells, T-006 reduced cell death and enhanced antioxidant capacity by upregulating mTOR and nuclear factor erythroid 2-related factor 2/Heme oxygenase-1 (Nrf2/HO-1) signaling. Similarly, in bEnd.3 cells, T-006 reduced oxidative injuries and preserved tight junction integrity through Nrf2/HO-1 upregulation. These effects were inhibited by LY294002, a Phosphoinositide 3-kinase (PI3K) inhibitor. T-006 may exert its neuroprotective and cerebrovascular protective effects via the regulation of PI3K/AKT-mediated pathways, which facilitate downstream mTOR and Nrf2 signaling, leading to improved cell survival and antioxidant defenses.

Comprehensive analysis of Hibisci mutabilis Folium extract's mechanisms in alleviating UV-induced skin photoaging through enhanced network pharmacology and experimental validation.

Skin photoaging induced by ultraviolet A (UVA) and ultraviolet B (UVB) radiation manifests as skin roughness, desquamation, pigmentation, and wrinkle formation. Current treatments, such as sunscreen, hormones, and antioxidants, have limitations and side effects. Traditional Chinese Medicine Hibisci Mutabilis Folium (HMF), or Mu-Fu-Rong-Ye in Chinese name, refers to the dried leaves of the plant Hibiscus mutabilis L., which belongs to the Malvaceae family. It has been used traditionally to treat acute mastitis, parotitis, neurodermatitis, burns. The reported activities of HMF include anti-inflammatory and anti-oxidant effects. However, the therapeutic potential of HMF in preventing and treating UV-induced skin photoaging remains unexplored. This study aimed to investigate the protective effects of HMF extract (EHMF) against UV-induced skin photoaging and the underlying mechanisms of action, by using network pharmacology and experimental verification. Network pharmacology was employed to identify the effective chemical components of EHMF. Potential targets were identified via PPI network analysis. Representative compounds were characterized using UPLC-MS/MS. In vitro validation involved assessing HaCaT cell viability, observing live/dead cell staining through fluorescence microscopy, and measuring inflammatory factors using ELISA. For in vivo validation, a UV-induced skin photoaging mice model was treated transdermally with EHMF or Methotrexate daily for 7 days. Dermatitis severity, skin morphology, and collagen fiber pathology were evaluated. Inflammatory cytokine and protein expression in dorsal skin lesions was confirmed using Elisa Kits, Western blot and immunohistochemistry. A total of 22 active ingredients of EHMF were identified. GO enrichment and KEGG pathway analyses revealed a focus on inflammatory signaling pathways. In vitro experiments showed that EHMF significantly reduced UV-induced inflammatory factors in HaCaT cells and improved cell survival rates. In vivo, EHMF alleviated back skin lesions in UV-exposed mice, reducing epidermal and dermal thickening and pathological inflammatory cell infiltration. It also decreased abnormal MMP-9 expression and collagen fiber proliferation, along with levels of inflammatory factors like TNF-α, IL-6, IL-17, and EGFR. Western blot and immunohistochemistry results indicated that the over-activation of the AKT-STAT3 signaling pathway was inhibited. EHMF effectively reduced UV-induced skin damage, inflammation, and wrinkles, providing strong support for its clinical application as a dermatological agent.

Curcumin‐Loaded Co‐Axial Electrospun Chitosan/Polyvinyl Alcohol/Calcium Chloride Nanofibrous Membranes for Wound Healing Enhancement

AbstractNanofibrous membranes were developed by combining chitosan (CS), polyvinyl alcohol (PVA), and calcium chloride (CC) to incorporate curcumin (CCM) through a co‐axial electro‐spinning method to improve CCM bioavailability. The optimal parameters were PVA/CS ratio of 7:3 v/v, PVA/CCM ratio of 8:2 v/v, CC amount of 0.5 g, fiber collection distance of 15 cm, voltage value of 18 kV, and injection flow of 0.1 mL/h (both core and shell layer). The prepared PVA/CCM@CS/PVA/CC nanofiber showed smooth surfaces and uniform bead‐free morphology with core‐shell structure. The average fiber diameter was 301.55 ± 76.77 nm with a narrow distribution. Using the Korsmeyer‐Peppas kinetic release model, the nanofiber membrane effectively released ∼85% CCM at pH 7.4 over 96 h via Fickian diffusion transport mechanism. In vitro antibacterial tests demonstrated that the membrane was efficient against both Gram‐positive (Staphylococcus aureus) and Gram‐negative (Pseudomonas aeruginosa) bacteria. When compared to free CCM, the membrane improved cell survival due to its nontoxic and cytobiocompatible features. In vivo tests found that the membrane significantly improved healing in incised‐wound rats, reducing inflammation, fibroblast proliferation, and collagen deposition. These findings indicate that the novel PVA/CCM@CS/PVA/CC nanofiber membrane, which has numerous biological functions, could be a promising candidate for wound dressing therapeutic applications.

Revolutionizing pressure ulcer regeneration: Unleashing the potential of extracellular matrix-derived temperature-sensitive injectable antioxidant hydrogel for superior stem cell therapy

Pressure ulcers are a common issue in elderly and medically compromised individuals, posing significant challenges in healthcare. Human umbilical cord mesenchymal stem cells (HUMSCs) offer therapeutic benefits like inflammation modulation and tissue regeneration, yet challenges in cell survival, retention, and implantation rates limit their clinical application. Hydrogels in three-dimensional (3D) stem cell culture mimic the microenvironment, improving cell survival and therapeutic efficacy. A thermosensitive injectable hydrogel (adEHG) combining gallic acid-modified hydroxybutyl chitosan (HBC-GA) with soluble extracellular matrix (adECM) has been developed to address these challenges. The hybrid hydrogel, with favorable physical and chemical properties, shields stem cells from oxidative stress and boosts their therapeutic potential by clearing ROS. The adEHG hydrogel promotes angiogenesis, cell proliferation, and collagen deposition, further enhancing inflammation modulation and wound healing through the sustained release of therapeutic factors and cells. Additionally, the adEHG@HUMSC composite induces macrophage polarization towards an M2 phenotype, which is crucial for wound inflammation inhibition and successful healing. Our research significantly propels the field of stem cell-based therapies for pressure ulcer treatment and underscores the potential of the adEHG hydrogel as a valuable tool in advancing regenerative medicine.

DDB2 expression lights the way for precision radiotherapy response in PDAC cells, with or without olaparib

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers. Therapeutic options for PDAC are primarily restricted to surgery in the early stages of the disease or chemotherapy in advanced disease. Only a subset of patients with germline defects in BRCA1/2 genes can potentially benefit from personalized therapy, with the PARP inhibitor olaparib serving as a maintenance treatment for metastatic disease. Although the role of radiotherapy in PDAC remains controversial, the use of radiosensitizers offers hope for improving cancer management. Previously, we have shown that damage-specific DNA binding protein 2 (DDB2) is a potential prognostic and predictive biomarker for chemotherapy response in PDAC. In this study, we investigated the function of DDB2 in radiotherapy response, with and without radiosensitization by olaparib in PDAC cells. Our findings demonstrated DDB2 resistance to radiation effects, thereby improving cell survival and enhancing the repair of ionizing radiation-induced DNA double-strand breaks. We observed that DDB2 expression enhances the cell cycle arrest in the G2 phase by phosphorylating Chk1 and Chk2 cell cycle checkpoints. Additionally, we identified a novel link between DDB2 and PARP1 in the context of radiotherapy, which enhances the expression and activity of PARP1. Our findings highlight the potential of low-DDB2 expression to potentiate the radiosensitization effect of olaparib in PDAC cells. Collectively, this study provides novel insights into the impacts of DDB2 in the radiotherapy response in PDAC, enabling its employment as a potential biomarker to predict resistance to radiation. Furthermore, DDB2 represents a significant step forward in precision radiotherapy by widening the scope of patients who can be benefiting from olaparib as a radiosensitizer. Hence, this research has the potential to enrich the limited use of radiotherapy in the care of patients with PDAC.

Icariin alleviates cellular injury induced by cardiac ischemia-reperfusion injury by inhibiting IRE1/JNK-induced ferroptosis

BackgroundIschemia-induced cellular damage and stress responses significantly impact cellular viability and function. Icariin (ICA), known for its protective effects, has been studied to understand its role in mitigating oxygen-glucose deprivation/reperfusion (OGD/R)-induced endoplasmic reticulum (ER) stress and ferroptosis in H9C2 cardiomyoblast cells. MethodsWe employed an in vitro OGD/R model using H9C2 cells. ICA's effects were analyzed across multiple concentrations. Key indicators of ER stress, autophagy, and ferroptosis—including markers like Bip, PERK, IRE1, ATF6, P62, FTH1, LC3II/LC3I, and NCOA4—were assessed using Western blotting, electron microscopy, and biochemical assays. Additionally, the role of the IRE1/JNK pathway in mitochondrial dynamics and its influence on mitochondrial dynamics protein was explored through specific inhibition and activation experiments. ResultsICA significantly reduced the activation of UPR pathways, decreased autophagic vacuole formation, and maintained cell viability in response to OGD/R and Erastin-induced ferroptosis. These protective effects were associated with modulated autophagic processes, reduced lipid peroxidation, and decreased ferrous ion accumulation. Inhibition of the IRE1/JNK pathway and subsequent Drp1 activity demonstrated reduced mitochondrial recruitment and mitophagy, correlating with decreased ferroptosis markers and improved cell survival. ConclusionOur findings highlight ICA's potential in modulating IRE1/JNK pathway, autophagy, providing a therapeutic avenue for mitigating ferroptosis in myocardial ischemia-reperfusion injury (MIRI).

M6A-activated BACH1 exacerbates ferroptosis by epigenetic suppression HSPB1 in severe acute pancreatitis.

Severe acute pancreatitis (SAP) is characterized by acute inflammation of the pancreas. The transcription factor BTB and CNC homology 1 (BACH1) has been implicated in various biological processes, including oxidative stress, apoptosis, and cell cycle regulation. However, its involvement in the pathogenesis of SAP remains relatively understudied. In the present work, our data demonstrated that BACH1 level was significantly increased in SAP patients, cellular, and animal models, while heat shock protein B1 (HSPB1) expression was weakened. Mechanistic assays validated that BACH1 acted as a transcriptional inhibitor of HSPB1. Moreover, HPDE6-C7 cells were stimulated with cerulein (Cer) and LPS to mimic the pathological stages of SAP in vitro. Depletion of BACH1 remarkably improved cell survival and alleviated the oxidative stress, ferroptosis, and inflammatory responses in SAP cell models. However, these changes were dramatically reversed upon co-inhibition of HSPB1. Animal findings confirmed that loss of BACH1 decreased pancreatic injury, inflammatory responses, and ferroptosis, but these effects were weakened by HSPB1 silence. Overall, these findings elucidate that the overexpression of BACH1 favors the ferroptosis and inflammation by transcriptionally inhibiting HSBP1, thereby exacerbating SAP progression.

Calcineurin activation improves cell survival during amino acid starvation in lipid droplet-deficient yeasts

Lipid droplets (LD) are storage sites for neutral lipids that can be used as a source of energy during nutrient starvation, but also function as hubs for fatty acid (FA) trafficking between organelles. In the yeast Saccharomyces cerevisiae, the absence of LD causes a severe disorganization of the endomembrane network during starvation. Here we show that cells devoid of LD respond to amino acid (AA) starvation by activating the serine/threonine phosphatase calcineurin and the nuclear translocation of its target protein Crz1. This activation was inhibited by treatments that restore a normal endomembrane organization, i.e. inhibition of FA synthesis with cerulenin or deletion of the inhibitory transcription factor Opi1. Activation of calcineurin increased the lifespan of LD-deficient cells during AA starvation. Indeed, deletion of its regulatory or catalytic subunits accelerated cell death. Surprisingly, calcineurin activation appeared to be calcium-independent. An increase in intracellular calcium was observed in LD-deficient cells during AA starvation, but its inhibition by genetic deletion of MID1 or YVC1 did not affect calcineurin activity. In contrast, calcineurin activation required the direct regulator of calcineurin Rcn1 and its activating (GSK-3)-related protein kinase Mck1.

GALNT9 enrichment attenuates MPP+-induced cytotoxicity by ameliorating protein aggregations containing α-synuclein and mitochondrial dysfunction

BackgroundGALNTs (UDP-GalNAc; polypeptide N-acetylgalactosaminyltransferases) initiate mucin-type O-GalNAc glycosylation by adding N-GalNAc to protein serine/threonine residues. Abnormalities in O-GalNAc glycosylation are involved in various disorders such as Parkinson’s disease (PD), a neurodegenerative disorder. GALNT9 is potentially downregulated in PD patients.MethodsTo determine whether GALNT9 enrichment ameliorates cytotoxicity related to PD-like variations, a pcDNA3.1-GALNT9 plasmid was constructed and transfected into SH-SY5Y cells to establish a GALNT9-overexpressing cell model.ResultsDownregulation of GALNT9 and O-GalNAc glycosylation was confirmed in our animal and cellular models of PD-like variations. GALNT9 supplementation greatly attenuated cytotoxicity induced by MPP+ (1-Methyl-4-phenylpyridinium iodide) since it led to increased levels of tyrosine hydroxylase and dopamine, reduced rates of apoptosis, and significantly ameliorated MPP+-induced mitochondrial dysfunction by alleviating abnormal levels of mitochondrial membrane potential and reactive oxygen species. A long-lasting mPTP (mitochondrial permeability transition pores) opening and calcium efflux resulted in significantly lower activity in the cytochrome C-associated apoptotic pathway and mitophagy process, signifying that GALNT9 supplementation maintained neuronal cell health under MPP+ exposure. Additionally, it was found that glycans linked to proteins influenced the formation of protein aggregates containing α-synuclein, and GALNT9 supplement dramatically reduced such insoluble protein aggregations under MPP+ treatment. Glial GALNT9 predominantly appears under pathological conditions like PD-like variations.ConclusionsGALNT9 enrichment improved cell survival, and glial GALNT9 potentially represents a pathogenic index for PD patients. This study provides insights into the development of therapeutic strategies for the treatment of PD.

Gelsemium low doses protect against serum deprivation-induced stress on mitochondria in neuronal cells

Gelsemium dynamized dilutions (GDD) are known as a remedy for a wide range of behavioral and psychological symptoms of depression and anxiety at ultra-low doses, yet the underlying mechanisms of the mode of action of G. sempervirens itself are not well understood. The present study was designed to examine the neuroprotective effects of Gelsemium preparations in counteracting stress-related mitochondrial dysfunctions in neuronal cells. We started by studying how serum deprivation affects the mitochondrial functions of human neuroblastoma (SH-SY5Y) cells. Next, we looked into the potential of various Gelsemium dilutions to improve cell survival and ATP levels. After identifying the most effective dilutions, 3C and 5C, we tested their ability to protect SH-SY5Y cells from stress-induced mitochondrial deficits. We measured total and mitochondrial superoxide anion radicals using fluorescent dyes dihydroethidium (DHE) and the red mitochondrial superoxide indicator (MitoSOX). Additionally, we assessed total nitric oxide levels with 4,5-diaminofluorescein diacetate (DAF-2DA), examined the redox state using pRA305cells stably transfected with a plasmid encoding a redox-sensitive green fluorescent protein, and analyzed mitochondrial network morphology using an automated high-content analysis device, Cytation3. Furthermore, we investigated bioenergetics by measuring ATP production with a bioluminescence assay (ViaLighTM HT) and evaluated mitochondrial respiration (OCR) and glycolysis (ECAR) using the Seahorse Bioscience XF24 Analyzer. Finally, we determined cell survival using an MTT reduction assay. Our research indicates that Gelsemium dilutions (3C and 5C) exhibited neuroprotective effects by: - Normalizing total and mitochondrial superoxide anion radicals and total nitric oxide levels. - Regulating the mitochondrial redox environment and mitochondrial networks morphology. - Increasing ATP generation as well as OCR and ECAR levels, thereby reducing the viability loss induced by serum withdrawal stress. These findings highlight that dynamized Gelsemium preparations may have neuroprotective effects against stress-induced cellular changes in the brain by regulating mitochondrial functions, essential for the survival, plasticity, and function of neurons in depression.

A reciprocal relationship between mitochondria and lipid peroxidation determines the chondrocyte intracellular redox environment

In orthopedic research, many studies have applied vitamin E as a protective antioxidant or used tert-butyl hydroperoxide to induce oxidative injury to chondrocytes. These studies often support the hypothesis that joint pathology causes oxidative stress and increased lipid peroxidation that might be prevented with lipid antioxidants to improve cell survival or function and joint health; however, lipid antioxidant supplementation was ineffective against osteoarthritis in clinical trials and animal data have been equivocal. Moreover, increased circulating vitamin E is associated with increased rates of osteoarthritis. This disconnect between benchtop and clinical results led us to hypothesize that oxidative stress-driven paradigms of chondrocyte redox function do not capture the metabolic and physiologic effects of lipid antioxidants and prooxidants on articular chondrocytes. We used ex vivo and in vivo cartilage models to investigate the effect of lipid antioxidants on healthy, primary, articular chondrocytes and applied immuno-spin trapping techniques to provide a broad indicator of high levels of oxidative stress independent of specific reactive oxygen species. Key findings demonstrate lipid antioxidants were pro-mitochondrial while lipid prooxidants decreased mitochondrial measures. In the absence of injury, radical formation was increased by lipid antioxidants; however, in the presence of injury, radical formation was decreased. In unstressed conditions, this relationship between chondrocyte mitochondria and redox regulation was reproduced in vivo with overexpression of glutathione peroxidase 4. In mice aged 18 months or more, overexpression of glutathione peroxidase 4 significantly decreased the presence of pro-mitochondrial peroxisome proliferation activated receptor gamma and deranged the relationship between mitochondria and the redox environment. This complex interaction suggests strategies targeting articular cartilage may benefit from adopting more nuanced paradigms of articular chondrocyte redox metabolism.

MTORC1 Signaling Inhibition Modulates Mitochondrial Function in Frataxin Deficiency.

Lysosomes regulate mitochondrial function through multiple mechanisms including the master regulator, mechanistic Target of Rapamycin Complex 1 (mTORC1) protein kinase, which is activated at the lysosomal membrane by nutrient, growth factor and energy signals. mTORC1 promotes mitochondrial protein composition changes, respiratory capacity, and dynamics, though the full range of mitochondrial-regulating functions of this protein kinase remain undetermined. We find that acute chemical modulation of mTORC1 signaling decreased mitochondrial oxygen consumption, increased mitochondrial membrane potential and reduced susceptibility to stress-induced mitophagy. In cellular models of Friedreich's Ataxia (FA), where loss of the Frataxin (FXN) protein suppresses Fe-S cluster synthesis and mitochondrial respiration, the changes induced by mTORC1 inhibitors lead to improved cell survival. Proteomic-based profiling uncover compositional changes that could underlie mTORC1-dependent modulation of FXN-deficient mitochondria. These studies highlight mTORC1 signaling as a regulator of mitochondrial composition and function, prompting further evaluation of this pathway in the context of mitochondrial disease.

Abstract Tu136: HIF-1α a novel therapeutic target to reduce Cardiac Ischemia Reperfusion Injury during AMI

Acute myocardial infarction (AMI) is a major cause of mortality worldwide. In preclinical models of AMI, using a trans-valvular pump (TVP) to reduce left ventricular (LV) workload for 30 minutes before reperfusion reduces infarct size by 50%. The mechanisms underlying the cardioprotective effect of LV unloading remain poorly understood. Hypoxia inducible factor 1-alpha (HIF-1α) is an oxygen-sensitive transcription factor that activates cardioprotective signaling pathways, improves cell survival and mitochondrial function during myocardial ischemia. We hypothesized that LV unloading limits myocardial injury by activating hypoxia inducible factor 1-a (HIF-1α). To explore the effect of LV unloading on HIF-1α stabilization during ischemia, we performed left anterior descending artery (LAD) occlusion for a total of 120 minutes in adult swine in the presence and absence of LV unloading and further 180 minutes of reperfusion was performed following 120 minutes of LAD occlusion. Trans-valvular unloading activation significantly decreased infarct size both during ischemia (IS/AAR: 4.78±4 % vs 1.5±3%) and post reperfusion when compared ischemia alone and IR alone groups (36.04±8.39 % vs 16.02±3.52 %, p<0.0001) respectively. Interestingly, HIF-1α stabilization is significantly reduced with LV unloading during ischemia and significantly increased post reperfusion in the infarct zone of the swine model of AMI. To test whether HIF1a is necessary for cardioprotection with LV unloading, adult swine were treated with intracoronary delivery of the HIF-1α inhibitor (2-methoxyestradiol; 2ME) before reperfusion. Compared to TVP activation for 30 minutes before reperfusion, addition of 2ME abolished the cardioprotective effect of TVP resulting in increased infarct size (16.03% vs 35.86% p<0.03) and decreased HIF-1a stabilization. Stabilizing HIF-1α post-reperfusion by using a well know HIF-1a stabilizing agent Roxadustat, immediately before reperfusion significantly decreased infarct size in both with and without TVP groups compared to IRI alone. Treatment with Roxadustat also preserved mitochondrial function and increased RISK signaling. We report the novel finding that stabilizing HIF-1a activity before reperfusion plays a critical role in reducing Infarct size in preclinical model of AMI.

Notch Signaling Hydrogels Enable Rapid Vascularization and Promote Dental Pulp Tissue Regeneration.

Successful dental pulp regeneration is closely associated with rapid revascularization and angiogenesis, processes driven by the Jagged1(JAG1)/Notch signaling pathway. However, soluble Notch ligands have proven ineffective in activating this pathway. To overcome this limitation, a Notch signaling hydrogel is developed by indirectly immobilizing JAG1, aimed at precisely directing the regeneration of vascularized pulp tissue. This hydrogel displays favorable mechanical properties and biocompatibility. Cultivating dental pulp stem cells (DPSCs) and endothelial cells (ECs) on this hydrogel significantly upregulate Notch target genes and key proangiogenic markers expression. Three-dimensional (3D) culture assays demonstrate Notch signaling hydrogels improve effectiveness by facilitating encapsulated cell differentiation, enhancing their paracrine functions, and promoting capillary lumen formation. Furthermore, it effectively communicates with the Wnt signaling pathway, creating an odontoinductive microenvironment for pulp-dentin complex formation. In vivo studies show that short-term transplantation of the Notch signaling hydrogel accelerates angiogenesis, stabilizes capillary-like structures, and improves cell survival. Long-term transplantation further confirms its capability to promote the formation of pulp-like tissues rich in blood vessels and peripheral nerve-like structures. In conclusion, this study introduces a feasible and effective hydrogel tailored to specifically regulate the JAG1/Notch signaling pathway, showing potential in advancing regenerative strategies for dental pulp tissue.