Stress-induced Cell Death Research Articles

Mitochondrial E3 ligase TRIM71 affects mitochondrial complex assembly and sensitizes dopaminergic neuronal cells to apoptosis in Parkinson’s Disease (PD)

Parkinson’s Disease (PD) is a chronic neurodegenerative disorder that impacts the substantia niagra region of the midbrain leading to impaired motor as well as non-motor symptoms of the central nervous system (CNS). Mitochondrial dysfunction has been characterized as the primary cause of dopaminergic neuronal loss, however, the molecular mechanisms leading to mitochondrial dysfunction are not completely understood. PARKIN, E3 ubiquitin ligase, plays a crucial role in maintaining mitochondrial quality control, albeit the role of other E3 ligases in regulating mitochondrial functions is not understood. In the current study, we explored the implication of TRIM71, E3 ubiquitin ligase, in the modulation of mitochondrial functions and neuronal death in PD stress conditions induced by rotenone and 6-OHDA. Ectopic expression of TRIM71 in SH-SY5Y dopaminergic neuronal cells sensitizes to PD stress-induced cell death, while its knock-down rescues neuronal cell death. TRIM71 turnover is enhanced in neurons under PD stress conditions. TRIM71 predominantly localizes on the outer mitochondrial membrane and translocation increases during PD stress conditions. TRIM71 regulates mitochondrial complex I and IV assembly and activity. TRIM71 knock-down decreases mitochondrial ROS and enhances ATP level as well as mitochondrial membrane potential in PD stress conditions. TRIM71-mediated mitochondrial ROS and cell death were rescued by mitoTEMPO, a mitochondrial-targeted antioxidant. Altogether, the evidence strongly suggests TRIM71-mediated modulation of mitochondrial functions and neuronal apoptosis in PD stress conditions.

Curcumin-encapsulated glucan nanoparticles as an oxidative stress modulator against human hepatic cancer cells

In Hepatitis B patients, the virus targets liver cells, leading to inflammation and liver damage, which can result in severe complications such as liver failure, cirrhosis, and liver cancer. Therapeutic options for liver disease are currently limited. Curcumin, a polyphenol with potential protective effects against chronic diseases like cancer, suffers from poor water solubility, restricting its pharmacological applications. This study explores the encapsulation of curcumin in glucan nanoparticles (NPs) and its impact on oxidative stress in liver cancer cells. Two sizes of curcumin-loaded glucan NPs, GC111 (111 nm) and GC398 (398 nm), were produced with nearly 100 % encapsulation efficiency. Cytotoxicity studies revealed that particle size influences the extent of observed effects, with GC111 NPs causing a greater reduction in cell viability. Additionally, the smaller GC111 NPs demonstrated a higher capacity to induce oxidative stress in cancer cells by stimulating the production of ROS, NO, and the chemokine RANTES in a concentration-dependent manner. These findings suggest that the smaller GC111 NPs are promising candidates for future studies aimed at evaluating oxidative stress-induced tumor cell death mechanisms.

Disturbing microtubule-endoplasmic reticulum dynamics by gold nanoclusters for improved triple-negative breast cancer treatment.

Microtubules are highly dynamic structures, and their dynamic instability is indispensable for not only cell growth and movement, but also stress responses, such as endoplasmic reticulum (ER) stress. Docetaxel, a microtubule targeting agent (MTA), is the first-line drug for cancer treatment by simultaneously promoting microtubule dysregulation- and ER stress-induced cell death. However, it also causes adverse effects and drug resistance, especially in triple-negative breast cancer (TNBC) with a poor prognosis and high mortality rate. In this study, we developed a peptide-templated gold nanocluster, namely GA. GA significantly sensitizes TNBC cells to docetaxel, causing severe cell death. This effect is further validated by a 3D tumor spheroid model. Mechanistically, GA disrupted microtubule dynamic instability, meanwhile promoting PERK-mediated ER stress. Interestingly, ER stress inhibitors profoundly suppressed microtubule dysregulation, suggesting a retrograde regulation of ER stress on microtubules. In vivo, the combined administration of docetaxel and GA significantly suppresses tumor growth while docetaxel alone cannot. GA similarly elevated the level of caspases and PERK within tumors as in vitro. Importantly, GA treatment also profoundly promoted the production of anti-tumor inflammatory cytokines. Collectively, we developed an ER-microtubule regulatory nanomaterial that enhanced the therapeutic effect of docetaxel by elevating tumor cell death and anti-tumor cytokine production, providing a potential supplemental strategy for TNBC treatment.

Immunohistochemical examination of immunoreactivity of transient receptor potential melastatin 2, glutathione peroxidase 4 and spexin in lichen planus.

In this study, we aimed to investigate the potential contributions to the disease by examining the immunoreactivities of SPX in LP-affected skin tissue using immunohistochemical methods, in light of its recent prominence as a molecule related to diabetes mellitus, along with apoptosis and ferroptosis mediated by GPX4 and TRPM2 channels facilitating oxidative stress-induced cell death. This research explored the immunohistochemical expressions of TRPM2, GPX4, and SPX in Lichen Planus (LP) patients compared to healthy individuals. Forty skin samples were collected, split equally between LP patients and healthy controls, excluding those with other conditions. Samples underwent immunohistochemical staining for TRPM2, SPX, and GPX4, using secondary antibodies and chromogens AEC or DAB. Histoscores were calculated based on staining diffusiveness and severity. Statistical analyses were performed with SPSS 22.0, using t-tests and ANOVA, with significance set at p < 0.05. There were no demographic differences between groups (p > 0.05). LP patients showed significantly lower TRPM2 and GPX4 histoscores and higher SPX histoscores compared to controls (TRPM2 and GPX4: p < 0.001, SPX: p < 0.001). Gender and age did not affect histoscores significantly. Findings suggest TRPM2, GPX4, and SPX play roles in LP pathogenesis, indicating a need for further molecular studies to clarify their involvement. This contributes to understanding LP beyond the traditional apoptosis perspective.

7936 Type 1 Diabetes Therapeutic Compound MSB-61: Using RNA Sequencing To Identify The Mechanism

Abstract Disclosure: P. Khan: None. K. Corbin: None. N. Ajmal: None. S. Bergmeir: None. G. Gu: None. X. Tong: None. C.S. Nunemaker: None. Abstract Type 1 diabetes (T1D) is a chronic autoimmune disease in which loss of insulin-producing beta-cells leads to hyperglycemia due to which patients eventually require lifelong insulin therapy to maintain normal glycemic control. Insulin remains the gold standard and the only effective treatment for T1D since its discovery 100 years ago. Protecting the pancreatic beta cells from cytokine-induced cell death and restoring insulin secretion are important to treat T1D, two properties that are currently lacking in present therapies. Our lab identified a chemical compound MSB-61 that increases insulin release and protects pancreatic islets against T1D-associated cytokines and endoplasmic reticulum (ER) stress. This study aims to identify the mechanism of action of MSB-61 in protecting pancreatic islets from ER stress-induced cell death. To achieve this aim pancreatic islets obtained from CD-1 strain mice were exposed for one hour to 50uM MSB-61 or vehicle control. After treatment, RNA was collected for RNA sequencing. In addition to RNA extraction for sequencing, MSB-61's ability to secrete insulin was verified by performing ELISA on control,10 and 50uM MSB-61-treated islets, and the activity of protection from cell death was assessed by performing cell death assay on islets treated with ER stressors in the presence and absence of 10 and 50uM MSB-61. Using Genialis, a bioinformatics service, we evaluated 57,010 genes (including non-coding transcripts) in our RNA sequencing data set. The DESeq2 technique of data analysis allowed us to identify 350 differentially expressed genes (DEGs), of which 251 were upregulated and 74 were downregulated. After removing non-coding/undefined transcripts and genes with &amp;lt;5 counts per million, a total of 117 genes remained; 108 up and 9 down. According to the String database (string-db.org), many of the top DEGs formed a common network of genes associated with cell death protection and insulin secretion. A key component of that gene network is a stress-responsive orphan nuclear receptor with known protective effects in beta cells. Testing this orphan nuclear receptor as a possible direct or indirect target of MSB-61 for cell death prevention can aid in identifying the genes and pathways targeted by MSB-61, allowing us to improve our approaches to T1D treatment and develop better medications. Presentation: 6/3/2024

Environmental salinity differentiates responses to acute hypothermal stress in milkfish

Global warming has led to an increase in the frequency of cold extremes, causing significant economic losses in aquaculture, particularly in subtropical regions. Milkfish (Chanos chanos) holds significant importance in aquaculture within subtropical Asian regions. Despite milkfish's ability to tolerate varying salinity levels, frequent cold snaps associated with extreme weather events have caused substantial mortality. Understanding the molecular and cellular mechanisms underlying cold stress-induced cell death is crucial for developing effective strategies to mitigate such losses. Given the pivotal role of the liver in fish physiology, we established a primary milkfish hepatocyte culture demonstrating robust proliferation and expressing a unique marker leptin A. The molecular characterization of cold-treated hepatocytes at 18 °C showed that the mRNA levels of superoxide dismutase (sod1) and catalase (cat), responsible for neutralizing reactive oxygen species (ROS), were downregulated in freshwater (FW) conditions, while cat expression was upregulated in seawater (SW) conditions. This differential modulation of ROS signaling implies distinct responses in FW and SW, leading to higher ROS levels and increased cell death in FW condition compared to those in SW. Transcriptomic analysis of liver tissues from milkfish reared in FW or SW, with or without cold stress, revealed distinct gene expression patterns. Although cold stress affected a common set of genes in both FW and SW conditions, SW-specific cold responsive genes are associated with metabolic pathways while FW-specific genes were linked to cell proliferation pathways. Notably, gene set enrichment analysis highlighted the downregulation of cytochrome-related genes, a major source of ROS production, in response to cold stress in SW. In summary, our study unveils an insightful mechanism whereby the salinity of SW counteracts cold stress-induced ROS signaling, emphasizing the feasibility and practicality of preconditioning fish in SW as a preventive measure against cold stress-induced mortality.

The Effect of In Vitro Digestion on the Anti-allergic, Anti-Inflammatory and Antioxidant Properties of Purple Rice and Purple Barley Phenolic Extracts in Caco-2 and RBL-2H3 Cells

Gastrointestinal diseases are associated with increased oxidative stress and inflammation. Pigmented cereal polyphenols are believed to have therapeutic potential. However, the impact of digestion on their bioactivity has not been fully elucidated. This study investigated the impact of digestion on the ability of purple rice extract (PRx) and purple barley (PBx) to alleviate oxidative stress-induced intestinal epithelial cell death and allergic inflammation. The study used a human colon adenocarcinoma (Caco-2) cell line to simulate intestinal oxidative stress for 4 h using 1 mM hydrogen peroxide. To evaluate the protective effects of digested and undigested cereal extracts, cells were first pre-treated with varying concentrations (50, 100, 200, and 500 μg/ml) of both purple rice (PRx) and purple barley (PBx) extracts. A rat basophilic leukemia (RBL-2H3) cell line was used to investigate the ability of the extracts to prevent calcium ionophore-induced histamine and cytokine release. The results demonstrated that both digested and undigested phenolic extracts prevented oxidative stress-induced cell death in Caco-2 cells and reduced Interleukin (IL)-8 secretion. PRx showed greater cytoprotective effects than PBx. Both digested and undigested extracts showed a time-dependent antihistamine effect, with the greatest inhibitory effects observed after 2 h and 4 h of treatment with PRx. Additionally, the extracts significantly attenuated the release of several cytokines including IL-2, IL-4, IL-13, monocyte chemoattractant protein-1, interferon-gamma, and tumour necrosis factor-alpha. Overall, the study demonstrates that both digested and undigested pigmented rice and barley extracts are potential sources of polyphenols which may promote intestinal health and alleviate allergic inflammation.

Neuroprotective effects of cerebroprotein hydrolysate and its combination with antioxidants against oxidative stress-induced HT22 cell death.

This study aimed to investigate the neuroprotective effects of cerebroprotein hydrolysate (CPH) against oxidative stress-induced HT22 cell death. Additionally, the effect of antioxidants such as quercetin (QC) and N-acetyl-L-cysteine (NAC) on the neuroprotective activity of CPH was evaluated. The mouse-derived hippocampal neuronal cell line HT22 was pretreated with CPH or a mixture of CPH and QC or NAC. HT22 cell death was induced by either 10mM glutamate, 2.5μM amyloid-β (Aβ)25-35, and 300μM cobalt chloride (CoCl2). As results, CPH effectively alleviated HT22 cell death induced by glutamate, Aβ25-35, and CoCl2. In addition, CPH combination with QC augmented cell viability in both glutamate- and Aβ25-35-stressed conditions but had no synergic effect on the CoCl2-stressed condition. The synergic effect of CPH and NAC combination was observed under all cell death conditions. The neuroprotective actions of CPH and its combinations with QC or NAC against various oxidative stress-induced HT22 cell deaths were demonstrated, providing a promising strategy for developing CPH preparations for the prevention and/or treatment of neurodegenerative diseases such as Alzheimer's disease.

Estrogen, via ESR2 receptor, prevents oxidative stress-induced Müller cell death and stimulates FGF2 production independently of NRF2, attenuating retinal degeneration

In this study, we aimed to investigate the effects of the deficient antioxidative gene, nuclear factor-erythroid 2-related factor 2 (Nrf2), on 17β-estradiol (E2)-mediated oxidative stress response, with a specific focus on growth factor production and cell death in Müller cells and retinal tissue. Administration of hydrogen peroxide (H2O2) reduced the viability of Müller cells derived from Nrf2 wild-type (WT) and knockout (KO) mice. However, this effect was more significant in the KO cells than in the WT cells. Pretreatment with E2 inhibited H2O2-induced cell death in both Nrf2 WT and KO Müller cell genotypes. Small interfering RNA-mediated gene silencing of estrogen receptor 2 (Esr2) attenuated the cell survival-promoting activity of E2 in Nrf2 KO Müller cells, while other identified estrogen receptors, Esr1 or G protein-coupled estrogen receptor 1 (Gper1), had no effect. Western blotting revealed higher ESR2 expression levels in Nrf2 KO cells than in WT Müller cells. Conditioned media from E2-and H2O2-treated Nrf2 WT or KO Müller cells enhanced the dissociated retinal cell viability compared with H2O2-treated cells. Both quantitative reverse-transcription polymerase chain reaction assay (qRT-PCR) and enzyme-linked immunosorbent assay exhibited a significant increase in fibroblast growth factor 2 (FGF2) expression levels in E2-and H2O2-treated Nrf2 WT and KO Müller cells compared to those in E2-treated cells. In vivo, administration of N-methyl-N-nitrosourea (MNU) reduced the thickness and cell density of the outer nuclear layer (ONL) in Nrf2 KO mice and enhanced the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive cells in the ONL. However, E2 administration attenuated these defects in MNU-treated mice. Concomitant administration of MNU and E2 enhanced FGF2 protein levels in retinal lysates of Nrf2 KO mice. In conclusion, E2 demonstrated a significant role in preventing oxidative stress-induced retinal cell death by stimulating FGF2 production in Müller cells, independent of the Nrf2 gene. Based on these findings, we anticipate that exogenous administration of estrogens or ESR2-selective agonists could aid in treating patients with oxidative stress-related retinal degenerative diseases such as age-related macular degeneration and retinitis pigmentosa.

Repurposing of Anti-Cancer Drugs Against Moderate and Severe COVID Infection: A Network-Based Systems Biological Approach.

The COVID-19 pandemic caused by SARS-CoV-2 is an unparalleled health risk, needing fast antiviral medication development. One of the most effective strategies for developing therapies against novel and emerging viruses is drug repurposing. Recently, systems biology approaches toward the discovery of repurposing medications are gaining prominence. This study aimed to implement a systems biology approach to identify crucial drug targets as well as potential drug candidates against COVID infection. Our approach utilizes differential gene expression in COVID conditions that enable the construction of a protein-protein interaction (PPI) network. Core clusters were extracted from this network, followed by molecular enrichment analysis. This process identified critical drug targets and potential drug candidates targeting various stages of COVID-19 infection. The network was built using the top 200 differently expressed genes in mild, moderate, and severe COVID-19 infections. Top 3 clusters for each disease condition were identified, representing the core mechanism of the network. Molecular enrichment revealed the majority of the pathways in the mild state were associated with transcription regulation, protein folding, angiogenesis, and cytokine-signaling pathways. Whereas, the enriched pathways in moderate and severe disease states were predominately linked with the immune system and apoptotic processes, which include NF-kappaB signaling, cytokine signaling, TNF-mediated signaling, and oxidative stress-induced cell death. Further analysis identifies 28 potential drugs that can be repurposed to treat moderate and severe COVID-19, most of which are currently used in cancer treatment. Interestingly, some of the proposed drugs have demonstrated inhibitory effects against SARS-CoV-2, as supported by literature evidence. Overall, the drug repurposing method described here will help develop potential antiviral medications to treat emerging COVID strains.

Adenosine Monophosphate as a Metabolic Adjuvant Enhances Antibiotic Efficacy against Drug-Resistant Bacterial Pathogens.

Global bacterial infections are on the rise, and drug resistance to bacteria is gradually rendering existing antibiotics ineffective. Therefore, the discovery of new strategies is urgently needed. Cellular metabolism is a key factor in the regulation of bacterial drug resistance, which cannot be separated from the utilization of energetic substances, suggesting that energetic substances may be associated with bacterial drug resistance. In this study, we found that adenosine monophosphate (AMP) can enhance the bactericidal effect of gentamicin against gentamicin-resistant Staphylococcus aureus. This synergistic effect can be generalized for use with different antibiotics and Gram-positive or Gram-negative bacteria. We also validated that the mechanism of AMP reversal of antibiotic resistance involves enhancing the proton motive force via the tricarboxylic acid cycle to increase antibiotic uptake. Simultaneously, AMP increases oxidative stress-induced cell death. This study presents a strategy for adopting low-dose antibiotics to control drug-resistant bacteria, which is important for future drug development and bacterial control.

Disulfidptosis: disulfide stress-induced novel cell death pathway.

The aberrant accumulation of intracellular disulfides in solute carrier family 7 member 11 (SLC7A11)high cells under glucose starvation induces disulfidptosis. Disulfidptosis has shown potential in tumor diagnosis and treatment.

Resveratrol alleviates endoplasmic reticulum stress-induced cell death and improves functional prognosis after traumatic brain injury in mice.

Resveratrol (RSV) is a polyphenol antioxidant that has been shown to have neuroprotective effects. We sought molecular mechanisms that emphasize the anti-inflammatory activity of RSV in traumatic brain injury (TBI) in mice associated with endoplasmic reticulum stress (ERS). After establishing three experimental groups (sham, TBI, and TBI+RSV), we explored the results of RSV after TBI on ERS and caspase-12 apoptotic pathways. The expression levels of C/EBP homologous protein (CHOP), glucose regulated protein 78kD (GRP78), caspase-3, and caspase-12 in cortical brain tissues were assessed by western blotting. The qPCR analysis was also performed on mRNA expression of tumor necrosis factor (TNF)-α and interleukin (IL)-1β in cortical brain tissue. In addition, the expression of GRP78 in microglia (ionized calcium binding adaptor molecule 1; Iba-1) and neurons (neuronal nuclei; NeuN) was identified by immunofluorescence staining. The neurological function of mice was assessed by modified neurological severity scores (mNSS). After drug treatment, the expression of CHOP, GRP78, caspase-3 and caspase-12 decreased, and qPCR results showed that TNF-α and IL-1β were down-regulated. Immunofluorescence staining showed down-regulation of Iba-1+/GRP78+ and NeuN+/GRP78+ cells after RSV treatment. The mNSS analysis confirmed improvement after RSV treatment. RSV improved apoptosis by downregulating the ERS signaling pathway and improved neurological prognosis in mice with TBI.

Multi-Cohort Transcriptomic Profiling of Medical Gas Plasma-Treated Cancers Reveals the Role of Immunogenic Cell Death.

The therapeutic potential of cold physical gas plasma operated at atmospheric pressure in oncology has been thoroughly demonstrated in numerous preclinical studies. The cytotoxic effect on malignant cells has been attributed mainly to biologically active plasma-generated compounds, namely, reactive oxygen and nitrogen species. The intracellular accumulation of reactive oxygen and nitrogen species interferes strongly with the antioxidant defense system of malignant cells, activating multiple signaling cascades and inevitably leading to oxidative stress-induced cell death. This study aims to determine whether plasma-induced cancer cell death operates through a universal molecular mechanism that is independent of the cancer cell type. Using whole transcriptome data, we sought to investigate the activation mechanism of plasma-treated samples in patient-derived prostate cell cultures, melanoma, breast, lymphoma, and lung cancer cells. The results from the standardized single-cohort gene expression analysis and parallel multi-cohort meta-analysis strongly indicate that plasma treatment globally induces cancer cell death through immune-mediated mechanisms, such as interleukin signaling, Toll-like receptor cascades, and MyD88 activation leading to pro-inflammatory cytokine release and tumor antigen presentation.

WED-105 Nitazoxanide directly protects from stress-induced cell death to alleviate liver damage in preclinical models of acute-on-chronic liver failure

WED-105 Nitazoxanide directly protects from stress-induced cell death to alleviate liver damage in preclinical models of acute-on-chronic liver failure

Mechano-activated connexin hemichannels and glutathione transport protect lens fiber cells against oxidative insults

Long-lived lens fiber cells require a robust cellular protective function against oxidative insults to maintain their hemostasis and viability; however, the underlying mechanism is largely obscure. In this study, we unveiled a new mechanism that protects lens fiber cells against oxidative stress-induced cell death. We found that mechano-activated connexin (Cx) hemichannels (HCs) mediate the transport of glutathione (GSH) into chick embryonic fibroblasts (CEF) and primary lens fiber cells, resulting in a decrease in the accumulation of intracellular reactive oxygen species induced by both H2O2 and ultraviolet B, providing protection to lens fiber cells against cell apoptosis and necrosis. Furthermore, HCs formed by both homomeric Cx50 or Cx46 and heteromeric Cx50/Cx46 were mechanosensitive and could transport GSH into CEF cells. Notably, mechano-activated Cx50 HCs exhibited a greater capacity to transport GSH than Cx46 HCs. Consistently, the deficiency of Cx50 in single lens fiber cells led to a higher level of oxidative stress. Additionally, outer cortical short lens fiber cells expressing full length Cxs demonstrated greater resistance to oxidative injury compared to central core long lens fibers. Taken together, our results suggest that the activation of Cx HCs by interstitial fluid flow in cultured epithelial cells and isolated fiber cells shows that HCs can serve as a pathway for moving GSH across the cell membrane to offer protection against oxidative stress.

Intravascular Laser Blood Irradiation (ILIB) Enhances Antioxidant Activity and Energy Metabolism in Aging Ovaries.

Ovarian aging is characterized by the accumulation of free radicals, leading to tissue damage and affecting reproductive health. Intravascular laser irradiation of blood (ILIB, using a low-energy He-Ne laser) is known for its efficacy in treating vascular-related diseases by reducing free radicals and inflammation. However, its impact on ovarian aging remains unexplored. This study aimed to investigate the effects of ILIB on oxidative stress and energy metabolism in aging ovaries. Genetic analysis was conducted on 75 infertile patients with aging ovaries, divided into ILIB-treated and control (CTRL) groups. Patients underwent two courses of laser treatment, and clinical parameters were evaluated. Cumulus cells were collected for the genetic analysis of oxeiptosis, glycolysis, and the tricarboxylic acid (TCA) cycle. The analysis of gene expression patterns revealed intriguing findings in ILIB-treated patients compared to the untreated group. Notably, ILIB treatment resulted in significant upregulation of oxeiptosis-related genes AIFM1 and NRF2, suggesting a potential protective effect against oxidative stress-induced cell death. Furthermore, ILIB treatment led to a downregulation of glycolysis-associated gene hexokinase 2 (HK2), indicating a shift away from anaerobic metabolism, along with an increase in PDHA levels, indicative of enhanced mitochondrial function. Consistent with these changes, ILIB-treated patients exhibited elevated expression of the key TCA cycle genes citrate synthase (CS), succinate dehydrogenase complex subunit A (SDHA), and fumarate hydratase (FH), signifying improved energy metabolism. The findings from this study underscore the potential of ILIB as a therapeutic strategy for mitigating ovarian aging. By targeting oxidative stress and enhancing energy metabolism, ILIB holds promise for preserving ovarian function and reproductive health in aging individuals. Further research is warranted to elucidate the underlying mechanisms and optimize the application of ILIB in clinical settings, with the ultimate goal of improving fertility outcomes in women experiencing age-related ovarian decline.

High expression of fragile X mental retardation protein inhibits ferroptosis of colorectal tumor cells by activating the RAS/MAPK signaling pathway

To investigate the mechanism by which fragile X mental retardation protein (FMRP) regulates ferroptosis evasion in colorectal cancer (CRC) cells. We examined FMRP expression levels in CRC cell lines using RT-qPCR and Western blotting and analyzed the biological functions and signaling pathways involved in FMRP-mediated regulation of CRC progression using the TCGA database. A lentiviral FMRP overexpression vector (Lv-FMRP) and 3 knockdown vectors (siFMRP-1, siFMRP-2, and siFMRP-3) were constructed, and their effects on proliferation of HCT116 cells were examined using CCK8 assay and plate clone formation assay; the changes in cell ferroptosis level was determined using MDA/ROS/GSH/Fe2+ kits, mitochondrial membrane potential changes were detected using JC-1 fluorescence staining, and the expressions of proteins associated with ferroptosis and the RAS/MAPK signaling pathway were detected using Western blotting. The subcutaneous tumorigenic potential of the transfected cells was evaluated in nude mice. Compared with normal colonic mucosal epithelial NCM460 cells, the CRC cell lines had significantly higher FMRP expression level. Bioinformatics analysis suggested the involvement of FMRP in regulation of reactive oxygen, oxidative stress-induced cell death, mitochondrial respiration, and glutathione metabolism pathways. In the cell experiments, FMRP knockdown significantly inhibited proliferation of HCT116 cells, lowered cellular GSH content, increased MDA and ROS levels, Fe2+ fluorescence intensity, and mitochondrial membrane potential, and decreased SLC7A11/GPX4 protein expressions and the phosphorylation levels of ERK, MEK, MAPK, and RAS proteins; FMRP overexpression resulted in the opposite changes in the cells. In the tumor-bearing nude mice, HCT116 cells with FMRP knockdown showed attenuated tumorigenic potential with lowered xenograft growth rate and reduced SLC7A11 expression in the xenograft. The high expression of FMRP inhibits ferroptosis in CRC cells and promotes progression of CRC by activating the RAS/MAPK signaling pathway.

Prolactin is an Endogenous Antioxidant Factor in Astrocytes That Limits Oxidative Stress-Induced Astrocytic Cell Death via the STAT3/NRF2 Signaling Pathway

Oxidative stress-induced death of neurons and astrocytes contributes to the pathogenesis of numerous neurodegenerative diseases. While significant progress has been made in identifying neuroprotective molecules against neuronal oxidative damage, little is known about their counterparts for astrocytes. Prolactin (PRL), a hormone known to stimulate astroglial proliferation, viability, and cytokine expression, exhibits antioxidant effects in neurons. However, its role in protecting astrocytes from oxidative stress remains unexplored. Here, we investigated the effect of PRL against hydrogen peroxide (H2O2)-induced oxidative insult in primary cortical astrocyte cultures. Incubation of astrocytes with PRL led to increased enzymatic activity of superoxide dismutase (SOD) and glutathione peroxidase (GPX), resulting in higher total antioxidant capacity. Concomitantly, PRL prevented H2O2-induced cell death, reactive oxygen species accumulation, and protein and lipid oxidation. The protective effect of PRL upon H2O2-induced cell death can be explained by the activation of both signal transducer and activator of transcription 3 (STAT3) and NFE2 like bZIP transcription factor 2 (NRF2) transduction cascades. We demonstrated that PRL induced nuclear translocation and transcriptional upregulation of Nrf2, concurrently with the transcriptional upregulation of the NRF2-dependent genes heme oxygenase 1, Sod1, Sod2, and Gpx1. Pharmacological blockade of STAT3 suppressed PRL-induced transcriptional upregulation of Nrf2, Sod1 and Gpx1 mRNA, and SOD and GPX activities. Furthermore, genetic ablation of the PRL receptor increased astroglial susceptibility to H2O2-induced cell death and superoxide accumulation, while diminishing their intrinsic antioxidant capacity. Overall, these findings unveil PRL as a potent antioxidant hormone that protects astrocytes from oxidative insult, which may contribute to brain neuroprotection.

Restraint Stress-Induced Neutrophil Inflammation Contributes to Concurrent Gastrointestinal Injury in Mice.

Psychological stress increases risk of gastrointestinal tract diseases. However, the mechanism behind stress-induced gastrointestinal injury is not well understood. The objective of our study is to elucidate the putative mechanism of stress-induced gastrointestinal injury and develop an intervention strategy. To achieve this, we employed the restraint stress mouse model, a well-established method to study the pathophysiological changes associated with psychological stress in mice. By orally administering gut-nonabsorbable Evans blue dye and monitoring its plasma levels, we were able to track the progression of gastrointestinal injury in live mice. Additionally, flow cytometry was utilized to assess the viability, death, and inflammatory status of splenic leukocytes, providing insights into the stress-induced impact on the innate immune system associated with stress-induced gastrointestinal injury. Our findings reveal that neutrophils represent the primary innate immune leukocyte lineage responsible for stress-induced inflammation. Splenic neutrophils exhibited elevated expression levels of the pro-inflammatory cytokine IL-1, cellular reactive oxygen species, mitochondrial burden, and cell death following stress challenge compared to other innate immune cells such as macrophages, monocytes, and dendritic cells. Regulated cell death analysis indicated that NETosis is the predominant stress-induced cell death response among other analyzed regulated cell death pathways. NETosis culminates in the formation and release of neutrophil extracellular traps, which play a crucial role in modulating inflammation by binding to pathogens. Treatment with the NETosis inhibitor GSK484 rescued stress-induced neutrophil extracellular trap release and gastrointestinal injury, highlighting the involvement of neutrophil extracellular traps in stress-induced gastrointestinal inflammation. Our results suggest that neutrophil NETosis could serve as a promising drug target for managing psychological stress-induced gastrointestinal injuries.