Inhibition Of Reactive Oxygen Species Research Articles

Naringenin-Loaded Bovine Serum Albumin Nanoparticles Inhibit Reactive Oxygen Species (ROS) to Prevent Ulcer and Gastric Cancer

Gastric cancer is a malignance of digestive system and effective treatment measures are key to treatment of gastric cancer. In this experiment, we assessed the effect of bovine serum albumin nanoparticle (BASNP)-coated naringenin (NGN) on reactive oxygen species (ROS) in gastric cancer. After preparation of NGN-BASNP and animal model of gastric cancer, rats were administered with NGN-BASNP, ROS agonists and ROS inhibitors, when the model group was set. After one week of intervention, gastric ulcers in rats were measured and Hematoxylin-eosin (HE) staining was performed. Transwell chamber was used to detect cell invasion ability, while 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was detected proliferation ability of gastric cancer cells. Real Time Quantitative Polymerase Chain Reaction (RT-qPCR) and Western blot determined TNF-α mRNA expression and ROS. With about 100 nm diameter of NGN-BASNP nanoparticles, the nanoparticles presented in good shape. Compared with lesions in the model group, NGN-BASNP treatment greatly improved the condition, relieving the ulcer and decreasing the ulcer area (P < 0.05). After adding ROS agonist, inhibitory effect of NGN-BASNP intervention was amplified (P < 0.01) when the ROS level in the NGN-BASNP+ROS agonist group was decreased and TNF-α expression decreased. Moreover, NGN-BASNP effectively suppressed gastric cancer cell proliferation and migration but induced apoptosis. NGN-BASNP inhibited the expression of TNF-α mRNA and ROS level in gastric cancer cells, thereby alleviating gastric ulcer and delaying gastric cancer cell growth.

N-Acetyl-L-cysteine attenuates titanium dioxide nanoparticle (TiO2 NP)-induced autophagy in male germ cells

Titanium dioxide nanoparticles (TiO2 NPs) are widely used in consumer products, raising concerns about their impact on human health. This study investigates the effects of TiO2 NPs on male germ cells while focusing on cell proliferation inhibition and underlying mechanisms. This was done by utilizing mouse GC-1 spermatogonia cells, an immortalized spermatogonia cell line. TiO2 NPs induced a concentration-dependent proliferation inhibition with increased reactive oxygen species (ROS) generation. Notably, TiO2 NPs induced autophagy and decreased ERK phosphorylation. Treatment with the ROS inhibitor N-Acetyl-l-cysteine (NAC) alleviated TiO2 NPs-induced autophagy, restored ERK phosphorylation, and promoted cell proliferation. These findings call attention to the reproductive risks posed by TiO2 NPs while also highlighting NAC as a possible protective agent against reproductive toxins.

Indoxyl sulfate aggravates podocyte damage through the TGF-β1/Smad/ROS signaling pathway.

Hyperglycaemia induces the production of a large quantity of reactive oxygen species (ROS) and activates the transforming growth factor β1(TGF-β1)/Smad signalling pathway, which is the main initiating factor in the formation of diabetic nephropathy. Indoxyl sulfate (IS) is a protein-binding gut-derived uraemic toxin that localizes to podocytes, induces oxidative stress and inflames podocytes. The involvement of podocyte damage in diabetic nephropathy through the TGF-β1/) signalling pathway is still unclear. In this study, we cultured differentiated rat podocytes in vitro and measured the expression levels of nephrin, synaptopodin, CD2AP, SRGAP2a and α-SMA by quantitative real-time PCR (qRT‒PCR) and western blotting after siRNA-mediated TGF-β1 silencing, TGF-β1 overexpression and the presence of the ROS inhibitor acetylcysteine. We detected the expression levels of nephrin, synaptopodin, CD2AP, SRGAP2a, SRGAP2a in the Smad2/3, phosphorylated-Smad2/3 (p-Smad2/3), Smad7, NADPH oxidase 4 (NOX4), and ROS levels under high glucose (HG) and IS conditions. The results indicated that nephrin, synaptopodin, CD2AP and SRGAP2a expressions were significantly upregulated and α-SMA expression was significantly downregulated in the presence of HG under siRNA-mediated TGF-β1 silencing or after the addition of acetylcysteine. However, in the presence of HG, the expressions of nephrin, synaptopodin, CD2AP and SRGAP2a were significantly downregulated, and the expression of α-SMA was significantly upregulated with the overexpression of TGF-β1. IS supplementation under HG conditions further significantly reduced the expressions of nephrin, synaptopodin, CD2AP and SRGAP2a; altered the expressions of Smad2/3, p-Smad2/3, Smad7 and NOX4; and increased ROS production in podocytes. This study suggests that IS may modulate the expression of nephrin, synaptopodin, CD2AP and SRGAP2a by regulating the ROS and TGF-β1/Smad signalling pathways, providing new theoretical support for the treatment of diabetic nephropathy.

The Power of Synergism: A Novel Drug Combination for Improved Therapy of Glioblastoma

AbstractOne of the biggest obstacles in treating glioblastoma multiforme (GBM) is the plasticity and adaptability of GBM cells, which renders monotherapy ineffective. Novel drug combinations that target multiple pathways are required to overcome this challenge. In the current study, the PI3K/AKT/mTOR pathway is identified as a crucial component in GBM cell survival using the KEGG database and literature search. Hence, it is hypothesized that a potential synergistic therapy can be achieved by targeting different stages of this pathway by combining disulfiram (DSF) and 7‐ethyl‐10‐hydroxycamptothecin (SN‐38). The efficacy of this drug combination is evaluated using 2D and 3D in vitro assays, which exhibited a significant synergism with 5 × 104 times lower IC50 than that of temozolomide (TMZ), the gold‐standard drug. The combination treatment increases intracellular ROS (reactive oxygen species) production, and ROS inhibition by using N‐Acetyl Cysteine (NAC) reduced the cytotoxicity substantially, indicating that ROS played a crucial role in the synergistic cytotoxicity. The combination treatment inhibited GBM cell proliferation, migration, and stem cell marker expression. Mechanistically, the DSF+SN‐38 combination is found to increase p53 expression, inhibit PI3K signaling, and activate caspase 9. Altogether, this study demonstrates that the DSF+SN‐38 combination can present a promising therapeutic strategy for treating GBM.

Reactive Oxygen Species Activate a Ferritin-Linked TRPV4 Channel under a Static Magnetic Field.

Magnetogenetics has shown great potential for cell function and neuromodulation using heat or force effects under different magnetic fields; however, there is still a contradiction between experimental effects and underlying mechanisms by theoretical computation. In this study, we aimed to investigate the role of reactive oxygen species (ROS) in mechanical force-dependent regulation from a physicochemical perspective. The transient receptor potential vanilloid 4 (TRPV4) cation channels fused to ferritin (T4F) were overexpressed in HEK293T cells and exposed to static magnetic fields (sMF, 1.4-5.0 mT; gradient: 1.62 mT/cm). An elevation of ROS levels was found under sMF in T4F-overexpressing cells, which could lead to lipid oxidation. Compared with the overexpression of TRPV4, ferritin in T4F promoted the generation of ROS under the stimulation of sMF, probably related to the release of iron ions from ferritin. Then, the resulting ROS regulated the opening of the TRPV4 channel, which was attenuated by the direct addition of ROS inhibitors or an iron ion chelator, highlighting a close relationship among iron release, ROS production, and TRPV4 channel activation. Taken together, these findings indicate that the produced ROS under sMF act on the TRPV4 channel, regulating the influx of calcium ions. The study would provide a scientific basis for the application of magnetic regulation in cellular or neural regulation and disease treatment and contribute to the development of the more sensitive regulatory technology.

Cellular Imaging and Time-Domain FLIM Studies of Meso-Tetraphenylporphine Disulfonate as a Photosensitising Agent in 2D and 3D Models.

Fluorescence lifetime imaging (FLIM) and confocal fluorescence studies of a porphyrin-based photosensitiser (meso-tetraphenylporphine disulfonate: TPPS2a) were evaluated in 2D monolayer cultures and 3D compressed collagen constructs of a human ovarian cancer cell line (HEY). TPPS2a is known to be an effective model photosensitiser for both Photodynamic Therapy (PDT) and Photochemical Internalisation (PCI). This microspectrofluorimetric study aimed firstly to investigate the uptake and subcellular localisation of TPPS2a, and evaluate the photo-oxidative mechanism using reactive oxygen species (ROS) and lipid peroxidation probes combined with appropriate ROS scavengers. Light-induced intracellular redistribution of TPPS2a was observed, consistent with rupture of endolysosomes where the porphyrin localises. Using the same range of light doses, time-lapse confocal imaging permitted observation of PDT-induced generation of ROS in both 2D and 3D cancer models using fluorescence-based ROS together with specific ROS inhibitors. In addition, the use of red light excitation of the photosensitiser to minimise auto-oxidation of the probes was investigated. In the second part of the study, the photophysical properties of TPPS2a in cells were studied using a time-domain FLIM system with time-correlated single photon counting detection. Owing to the high sensitivity and spatial resolution of this system, we acquired FLIM images that enabled the fluorescence lifetime determination of the porphyrin within the endolysosomal vesicles. Changes in the lifetime dynamics upon prolonged illumination were revealed as the vesicles degraded within the cells.

Erythrocyte-Like Mesoporous PDA@CeO2 Nanozyme with Dual Drugs for Periodontitis Treatment.

Periodontitis is a chronic oral inflammatory disease with the characteristic of excess oxidative stress in the inflammatory site, dramatically decreasing the quality of life. Studies show that nanozymes can be ideal candidates for ROS scavenging in periodontitis. Here, we design a multipath anti-inflammatory mesoporous polydopamine@cerium oxide nanobowl (mPDA@CeO2 NB) with multienzyme mimicking properties, which combines the advantages of both CeO2 NP and mPDA NB for synergistically eliminating reactive oxygen species (ROS), including hydroxyl radical (•OH), hydrogen peroxide (H2O2), and superoxide (O2•-). Besides, the erythrocyte-like structure of mNBs makes them a facility for cell uptake, and the mesopores can load both hydrophobic and hydrophilic drugs for combined anti-inflammatory therapy. In vitro and in vivo experiments prove that the combination of CeO2 and mPDA can synergistically achieve multiple complementary ROS eliminations and suppression of ROS-induced inflammation. Moreover, the ROS regulation plus anti-inflammatory drugs in one mPDA@CeO2 NB prevents the progression of periodontitis in a mouse model. Therefore, the design of mPDA@CeO2 NB with these excellent properties provides a therapeutic strategy for inflammatory diseases.

Deficiency in DNA Damage Repair Proteins Promotes Prostate Cancer Cell Migration through Oxidative Stress

Introduction: DNA damage repair gene deficiency defines a subgroup of prostate cancer patients with early metastatic progression and unfavorable disease outcome. Whether deficiency in DNA damage repair genes directly promotes metastatic dissemination is not completely understood. Methods: The migratory behavior of prostate cancer cells was analyzed after siRNA-mediated knockdown of DNA damage repair and checkpoint proteins, including BRCA2, ATM, and others, using transwell migration assays, scratch assays and staining for F-actin to ascertain cell circularity. Cells deficient in BRCA2 or ATM were tested for oxidative stress by measuring reactive oxygen species (ROS). The effects of ROS inhibition on cell migration were analyzed using the antioxidant N-acetylcysteine (NAC). The correlation between BRCA2 deficiency and oxidative stress was ascertained via immunohistochemistry for methylglyoxal (MG)-modified proteins in 15 genetically defined primary prostate cancers. Results: Prostate cancer cells showed a significantly increased migratory activity after the knockdown of BRCA2 or ATM. There was a significant increase in ROS production in LNCaP cells after BRCA2 knockdown and in PC-3 cells after BRCA2 or ATM knockdown. Remarkably, the ROS scavenger NAC abolished the enhanced motility of prostate cancer cells after the knockdown of BRCA2 or ATM. Primary prostate cancers harboring genetic alterations in BRCA2 showed a significant increase in MG-modified proteins, indicating enhanced oxidative stress in vivo. Conclusions: Our results indicate that DNA damage repair gene deficiency may contribute to the metastatic dissemination of prostate cancer through enhanced tumor cell migration involving oxidative stress.

Diosgenin loaded cellulose nanoonion impedes different stages of protein aggregation induced cell death via alleviating mitochondrial dysfunction and upregulation of autophagy

Protein aggregation is a multifaceted phenomenon prevalent in the progression of neurodegenerative diseases, yielding aggregates of diverse sizes. Recently, increased attention has been directed towards early protein aggregates due to their pronounced toxicity, largely stemming from inflammation mediated by reactive oxygen species (ROS). This study advocates for a therapeutic approach focusing on inflammation control rather than mere ROS inhibition in the context of neurodegenerative disorders. Here, we introduced Camellia sinensis cellulose nanoonion (CS-CNO) as an innovative, biocompatible nanocarrier for encapsulating the phytosteroid diosgenin (DGN@CS-CNO). The resulting nano-assembly, manifesting as spherical entities with dimensions averaging ~180–220 nm, exhibits a remarkable capacity for the gradual and sustained release of approximately 39–44 % of DGN over a 60-hour time frame. DGN@CS-CNO displays a striking ability to inhibit or disassemble various phases of hen egg white lysozyme (HEWL) protein aggregates, including the early (HEWLEA) and late (HEWLLA) stages. In vitro experiments employing HEK293 cells underscore the potential of DGN@CS-CNO in mitigating cell death provoked by protein aggregation. This effect is achieved by ameliorating ROS-mediated inflammation and countering mitochondrial dysfunction, as evidenced by alterations in TNFα, TLR4, and MT-CO1 protein expression. Western blot analyses reveal that the gradual and sustained release of DGN from DGN@CS-CNO induces autophagy, a pivotal process in dismantling intracellular amyloid deposits. In summary, this study not only illuminates a path forward but also presents a compelling case for the utilization of phytosteroid as a formidable strategy against neuroinflammation incited by protein aggregation.

Abstract 2880: Radiation induces myofibroblast differentiation of pericytes

Abstract Radiation not only kills cancer cells directly through DNA damage, but also indirectly by inducing vascular changes. Endothelial cells (ECs) in small blood vessels and capillaries connect with pericytes, a type of mural cells which are important for vascular stability and structure. Effects of radiation on tumor vasculature are critical for the therapeutic efficacy, however, its impacts on pericytes remain largely unknown. In this study, we focused on the effect of radiation on pericytes and sought to elucidate the mechanism by which radiation affects pericytes. We show 20Gy for C3H10T1/2 (10T1/2) mouse embryonic mesenchymal pericyte precursor cells and 10Gy for human pericytes from placenta (hPC-PL) cells induces pericyte differentiation and activation of adhesion molecules. Radiation enhanced lymphocyte adhesion and decreased permeability in co-culture of ECs and pericytes primarily through endothelial cells, not via pericytes. Furthermore, radiation promoted transdifferentiation of pericytes into myofibroblasts. Supernatant from irradiated cells in combination with a TGFβ inhibitor showed that TGFβ signaling is involved in pericyte differentiation by radiation but not all pericyte marker genes are regulated by TGFβ. Therefore, we performed phospho-kinase array analysis and revealed Akt signaling pathway activation 10 minutes following radiation, which was verified by the phosphorylation of GSK3β and PRAS40. Along with this, generation of intracellular reactive oxygen species (ROS) occurs as early as 5 min following radiation. Inhibitors of Akt and ROS suppress the expression of myofibroblast markers increased by radiation. To investigate whether radiation induces pericyte differentiation into myofibroblast in tumors, we employed NG2DsRedBAC (NG2DsRed) transgenic mice where DsRed is expressed under the control of NG2 promoter to trace pericytes. The endogenous expression of DsRed was detected in pericytes in the tumors implanted to NG2DsRed mice. Flow cytometry analysis of tumors harvested 7 days after radiation shows radiation increased vimentin+ cells and decreased DsRed+ cells, which indicates radiation induced pericyte differentiation into myofibroblast. In conclusion, radiation promotes pericyte maturation and transdifferentiation into myofibroblast via Akt signaling. Further study using Akt inhibitor with radiation to tumor-bearing N2DsRed mice will be warranted. Citation Format: Tomoko Yamazaki, Kelley R. Jordan, Marka R. Crittenden, Michael J. Gough, Kristina H. Young. Radiation induces myofibroblast differentiation of pericytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2880.

Neobractatin induces pyroptosis of esophageal cancer cells by TOM20/BAX signaling pathway

BackgroundEmerging evidence suggests that pyroptosis, a form of programmed cell death, has been implicated in cancer progression. The involvement of specific proteins in pyroptosis is an area of growing interest. TOM20, an outer mitochondrial membrane protein, has recently garnered attention for its potential role in pyroptosis. Our previous study found that NBT could induce pyroptosis by ROS/JNK pathway in esophageal cancer cells. PurposeThis study aims to investigate whether NBT induces pyroptosis and verify whether such effects are involved in up-regulation of TOM20 in esophageal cancer cells. MethodsThe University of ALabama at Birmingham CANcer data analysis Portal (UALCAN) was used to analyze the clinical significance of GSDME in esophageal cancer. MTT assay, morphological observation and Western blot were performed to verify the roles of TOM20 and BAX in NBT-induced pyroptosis after CRISPR-Cas9-mediated knockout. Immunofluorescence was used to determine the subcellular locations of BAX and cytochrome c. MitoSOX Red was employed to assess the mitochondrial reactive oxygen species (ROS) level. KYSE450 and TOM20 knockout KYSE450-/- xenograft models were established to elucidate the mechanisms involved in NBT-induced cell death. ResultsIn this study, NBT effectively upregulated the expression of TOM20 and facilitated the translocation of BAX to mitochondria, which promoted the release of cytochrome c from mitochondria to the cytoplasm, leading to the activation of caspase-9 and caspase-3, and finally induced pyroptosis. Knocking out TOM20 by CRISPR-Cas9 significantly inhibited the expression of BAX and the downstream BAX/caspase-3/GSDME pathway, which attenuated NBT-induced pyroptosis. The elevated mitochondrial ROS level was observed after NBT treatment. Remarkably, the inhibition of ROS by N-acetylcysteine (NAC) effectively suppressed the activation of TOM20/BAX pathway. Moreover, in vivo experiments demonstrated that NBT exhibited potent antitumor effects in both KYSE450 and TOM20 knockout KYSE450-/- xenograft models. Notably, the attenuated antitumor effects and reduced cleavage of GSDME were observed in the TOM20 knockout model. ConclusionThese findings reveal that NBT induces pyroptosis through ROS/TOM20/BAX/GSDME pathway, which highlight the therapeutic potential of targeting TOM20 and GSDME, providing promising prospects for the development of innovative and effective treatment approaches for esophageal cancer.

PLEKHM2 deficiency induces impaired mitochondrial clearance and elevated ROS levels in human iPSC-derived cardiomyocytes

Pleckstrin homology domain-containing family M member 2 (PLEKHM2) is an essential adaptor for lysosomal trafficking and its homozygous truncation have been reported to cause early onset dilated cardiomyopathy (DCM). However, the molecular mechanism of PLEKHM2 deficiency in DCM pathogenesis and progression is poorly understood. Here, we generated an in vitro model of PLEKHM2 knockout (KO) induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to elucidate the potential pathogenic mechanism of PLEKHM2-deficient cardiomyopathy. PLEKHM2-KO hiPSC-CMs developed disease phenotypes with reduced contractility and impaired calcium handling. Subsequent RNA sequencing (RNA-seq) analysis revealed altered expression of genes involved in mitochondrial function, autophagy and apoptosis in PLEKHM2-KO hiPSC-CMs. Further molecular experiments confirmed PLEKHM2 deficiency impaired autophagy and resulted in accumulation of damaged mitochondria, which triggered increased reactive oxygen species (ROS) levels and decreased mitochondrial membrane potential (Δψm). Importantly, the elevated ROS levels caused oxidative stress-induced damage to nearby healthy mitochondria, resulting in extensive Δψm destabilization, and ultimately leading to impaired mitochondrial function and myocardial contractility. Moreover, ROS inhibition attenuated oxidative stress-induced mitochondrial damage, thereby partially rescued PLEKHM2 deficiency-induced disease phenotypes. Remarkably, PLEKHM2-WT overexpression restored autophagic flux and rescued mitochondrial function and myocardial contractility in PLEKHM2-KO hiPSC-CMs. Taken together, these results suggested that impaired mitochondrial clearance and increased ROS levels play important roles in PLEKHM2-deficient cardiomyopathy, and PLEKHM2-WT overexpression can improve mitochondrial function and rescue PLEKHM2-deficient cardiomyopathy.

Nortriptyline hydrochloride, a potential candidate for drug repurposing, inhibits gastric cancer by inducing oxidative stress by triggering the Keap1-Nrf2 pathway

Effective drugs for the treatment of gastric cancer (GC) are still lacking. Nortriptyline Hydrochloride (NTP), a commonly used antidepressant medication, has been demonstrated by numerous studies to have antitumor effects. This study first validated the ability of NTP to inhibit GC and preliminarily explored its underlying mechanism. To begin with, NTP inhibits the activity of AGS and HGC27 cells (Human-derived GC cells) in a dose-dependent manner, as well as proliferation, cell cycle, and migration. Moreover, NTP induces cell apoptosis by upregulating BAX, BAD, and c-PARP and downregulating PARP and Bcl-2 expression. Furthermore, the mechanism of cell death caused by NTP is closely related to oxidative stress. NTP increases intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) levels, decreasing the mitochondrial membrane potential (MMP) and inducing glucose (GSH) consumption. While the death of GC cells can be partially rescued by ROS inhibitor N-acetylcysteine (NAC). Mechanistically, NTP activates the Kelch-like ECH-associated protein (Keap1)—NF-E2-related factor 2 (Nrf2) pathway, which is an important pathway involved in oxidative stress. RNA sequencing and proteomics analysis further revealed molecular changes at the mRNA and protein levels and provided potential targets and pathways through differential gene expression analysis. In addition, NTP can inhibited tumor growth in nude mouse subcutaneous tumor models constructed respectively using AGS and MFC (mouse-derived GC cells), providing preliminary evidence of its effectiveness in vivo. In conclusion, our study demonstrated that NTP exhibits significant anti-GC activity and is anticipated to be a candidate for drug repurposing.

DJ-1 protects cell death from a mitochondrial oxidative stress due to GBA1 deficiency.

GBA1 mutations are the most common genetic risk factor for development of Parkinson's disease (PD). The loss of catalytic activity in GBA1, as well as the reduction of the GBA1 protein in certain cellular compartment, may increase disease progression. However, the mechanisms underlying cellular dysfunction caused by GBA1 deficiency are still mostly unknown. In this study, we focus on the genetic interaction between GBA1 deficiency and PD-causing genes, such as DJ-1, in mitochondrial dysfunction. GBA1 knockout (KO) SH-SY5Y cells were used to assess DJ-1 functions against oxidative stress in vitro. The levels of cellular reactive oxygen species were monitored with MitoSOX reagent. The expression of the PARK7 gene was analyzed using the quantitative real-time PCR (qRT-PCR). To understand the mechanism underlying DJ-1 upregulation in GBA1 KO cells, we assess ROS levels, antioxidant protein, and cell viability in GBA1 KO cells with treatment of ROS inhibitor N-acetyl-cysteine or miglustat, which is an inhibitor of glucosylceramide synthase. Dopaminergic degeneration was assessed from Gba1 L444P heterozygous mice mated with Park7 knockout mice. We find that DJ-1 is significantly upregulated in GBA1 KO cells. Elevated levels of DJ-1 are attributed to the transcriptional expression of PARK7 mRNA, but not the inhibition of DJ-1 protein degradation. Because DJ-1 expression is highly linked to oxidative stress, we observe cellular reactive oxygen species (ROS) in GBA1 KO cells. Moreover, several antioxidant gene expressions and protein levels are increased in GBA1 KO cells. To this end, GBA1 KO cells are more susceptible to H2O2-induced cell death. Importantly, there is a significant reduction in dopaminergic neurons in the midbrain from Gba1 L444P heterozygous mice mated with Park7 knockout mice, followed by mild motor dysfunction. Taken together, our results suggest that DJ-1 upregulation due to GBA1 deficiency has a protective role against oxidative stress. It may be supposed that mutations or malfunctions in the DJ-1 protein may have disadvantages in the survival of dopaminergic neurons in the brains of patients harboring GBA1 mutations.

Transcription factor Nrf2 activation regulates NETosis, endothelial injury, and kidney disease in myeloperoxidase-positive antineutrophil cytoplasmic antibody-associated vasculitis

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a systemic autoimmune disease pathologically characterized by vascular necrosis with inflammation. During AAV development, activated neutrophils produce reactive oxygen species (ROS), leading to the aberrant formation of neutrophil extracellular traps (NETs) via NETosis and subsequent fibrinoid vascular necrosis. Nuclear factor-erythroid 2-related factor 2 (Nrf2) functions as an intracellular defense system to counteract oxidative stress by providing antioxidant properties. Herein, we explored the role of Nrf2 in the pathogenesis of AAV. The role and mechanism of Nrf2 in ANCA-stimulated neutrophils and subsequent endothelial injury were evaluated invitro using Nrf2 genetic deletion and Nrf2 activator treatment. In corresponding invivo studies, the role of Nrf2 in ANCA-transfer AAV and spontaneous AAV murine models was examined. Pharmacological activation of Nrf2 invitro suppressed ANCA-induced NET formation via the inhibition of ROS. In contrast, NET formation was enhanced in Nrf2-deficient neutrophils. Furthermore, Nrf2 activation protected endothelial cells from ANC-induced NETs-mediated injury. Invivo, Nrf2 activation ameliorated glomerulonephritis in two AAV models by upregulating antioxidants and inhibiting ROS-mediated NETs. Furthermore, Nrf2 activation restrained the expansion of splenic immune cells, including T lymphocytes and limited the infiltration of Th17 cells into the kidney. In contrast, Nrf2 genetic deficiency exacerbated vasculitis in a spontaneous AAV model. Thus, the pathophysiological process in AAV may be downregulated by Nrf2 activation, potentially leading to a new therapeutic strategy by regulating NETosis.

Diminished NAD+ levels and activation of retrotransposons promote postovulatory aged oocyte (POAO) death

Death is the fate of postovulatory aged or unfertilized oocytes (POAO) in many animals. However, precise molecular mechanisms are yet to be discovered. Here, we demonstrate that increased amounts of reactive oxygen species (ROS), calcium ion (Ca+2) channels, and retrotransposon activity induce apoptosis, which in turn causes POAO death. Notably, suppression of ROS, Ca+2 channels, and retrotransposons delayed POAO death. Further, we found that the histone H4K12 and K16 acetylation increased via downregulation of NAD+ and NAD+ -dependent histone deacetylase SIRT3. Furthermore, adding NMN, sodium pyruvate, or CD38 inhibition delayed the death of postovulatory aged oocytes. Finally, we demonstrate the conservation of retrotransposon-induced DNA damage-dependent POAO death in higher-order vertebrates. Our findings suggest that POAO mortality is caused by cyclic cascade metabolic interactions in which low NAD+ levels increase histone acetylation by inhibiting histone deacetylases, resulting in an increase in retrotransposons, ROS, and Ca+2 channel activity and thus contributing to DNA damage-induced apoptosis.

High glucose intake exacerbates experimental autoimmune prostatitis through mitochondrial reactive oxygen species-dependent TGF-β activation-mediated Th17 differentiation

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a common inflammatory immune disease of the urogenital system. High glucose intake is considered to be a potential promoter of autoimmune diseases. However, the influence of high glucose intake on CP/CPPS is unknown. This research aimed to explore the influences of high glucose intake on experimental autoimmune prostatitis (EAP), a valid animal model of CP/CPPS, and the underlying mechanism. NOD mice received 20% glucose water or normal water treatment during EAP induction. EAP severity and Th17 cell responses were evaluated. Then, we explored the effects of an IL-17A neutralizing antibody, an inhibitor of TGF-β, the reactive oxygen species (ROS) inhibitor NAC, and the mitochondrial ROS (mtROS) antioxidant MitoQ on glucose-fed EAP mice. The results demonstrated that high glucose intake aggravated EAP severity and promoted Th17 cell generation, which could be ameliorated by the neutralization of IL-17A. In vitro experiments showed that high dextrose concentrations promoted Th17 cell differentiation through mtROS-dependent TGF-β activation. Treatment with TGF-β blockade, NAC, or MitoQ suppressed Th17 cell generation both in vivo and in vitro, resulting in the amelioration of EAP manifestations caused by high glucose intake. This study revealed that high glucose intake exacerbates EAP through mtROS-dependent TGF-β activation-mediated Th17 differentiation. Our results may provide insights into the molecular mechanisms underlying the detrimental effects of an environmental factor, such as high glucose intake, on CP/CPPS.

Antibiotic-induced ROS-mediated Fur allosterism contributes to Helicobacter pylori resistance by inhibiting arsR activation of mutS and mutY.

The increasing antibiotic resistance of Helicobacter pylori primarily driven by genetic mutations poses a significant clinical challenge. Although previous research has suggested that antibiotics could induce genetic mutations in H. pylori, the molecular mechanisms regulating the antibiotic induction remain unclear. In this study, we applied various techniques (e.g., fluorescence microscopy, flow cytometry, and multifunctional microplate reader) to discover that three different types of antibiotics could induce the intracellular generation of reactive oxygen species (ROS) in H. pylori. It is well known that ROS, a critical factor contributing to bacterial drug resistance, not only induces damage to bacterial genomic DNA but also inhibits the expression of genes associated with DNA damage repair, thereby increasing the mutation rate of bacterial genes and leading to drug resistance. However, further research is needed to explore the molecular mechanisms underlying the ROS inhibition of the expression of DNA damage repair-related genes in H. pylori. In this work, we validated that ROS could trigger an allosteric change in the iron uptake regulatory protein Fur, causing its transition from apo-Fur to holo-Fur, repressing the expression of the regulatory protein ArsR, ultimately causing the down-regulation of key DNA damage repair genes (e.g., mutS and mutY); this cascade increased the genomic DNA mutation rate in H. pylori. This study unveils a novel mechanism of antibiotic-induced resistance in H. pylori, providing crucial insights for the prevention and control of antibiotic resistance in H. pylori.

Protective effect of FAK inhibitor PF-562271 against human umbilical vein endothelial cell injury induced by aging platelets

To investigate the protective effect of PF-562271, a FAK inhibitor, against aging platelet-induced injury in human umbilical vein endothelial cells (HUVECs). Cultured HUVECs were treated with vehicle, lipopolysaccharide (LPS), LPS+aging platelets, or LPS+aging platelets+PF-562271. The changes in protein expressions of FAK, pFAK and PECAM-1 in the treated cells were detected using Western blotting and immunofluorescence assay, and the level of reactive oxygen species (ROS) was detected with flow cytometry. The changes of barrier function of the cells were assessed with cell permeability test and transendothelial cell resistance test. RT-qPCR was used to analyze mRNA expressions of inflammatory factors, and pro-inflammatory cytokine levels in the culture supernatants was determined with enzyme-linked immunosorbent assay. Immunofluorescence assay was used to examine the effect of the ROS inhibitor vitamin C on PECAM-1 expression in the cells with different treatments. Treatment of HUVECs with LPS and aging platelets significantly increased cellular protein expressions of FAK, pFAK and PECAM-1, which were effectively lowered by addition of PF-562271 (P < 0.05). LPS and aged platelets obviously enhanced ROS production in the cells, which was inhibited by the addition of PF-562271 (P < 0.001). PF-562271 significantly alleviated the damage of endothelial cell barrier function of the cells caused by LPS and aging platelets (P < 0.01). The expressions of TNF-α, IL-6 and IL-8 in HUVECs increased significantly after exposure to LPS and aging platelets, and were obviously lowered after treatment with PF-562271 (P < 0.05). Treatment with vitamin C significantly decreased the expression of PECAM-1 protein in the cells (P < 0.01). The FAK inhibitor PF-562271 alleviates endothelial cell damage induced by LPS and aging platelets by lowering cellular oxidative stress levels and reducing inflammatory responses.

Ototoxicity of polystyrene nanoplastics in mice, HEI-OC1 cells and zebrafish.

Polystyrene nanoplastics are a novel class of pollutants. They are easily absorbed by living organisms, and their potential toxicity has raised concerns. However, the impact of polystyrene nanoplastics on auditory organs remains unknown. Here, our results showed that polystyrene nanoplastics entered the cochlea of mice, HEI-OC1 cells, and lateral line hair cells of zebrafish, causing cellular injury and increasing apoptosis. Additionally, we found that exposure to polystyrene nanoplastics resulted in a significant elevation in the auditory brainstem response thresholds, a loss of auditory sensory hair cells, stereocilia degeneration and a decrease in expression of Claudin-5 and Occludin proteins at the blood-lymphatic barrier in mice. We also observed a significant decrease in the acoustic alarm response of zebrafish after exposure to polystyrene nanoplastics. Mechanistic analysis revealed that polystyrene nanoplastics induced up-regulation of the Nrf2/HO-1 pathway, increased levels of malondialdehyde, and decreased superoxide dismutase and catalase levels in cochlea and HEI-OC1 cells. Furthermore, we observed that the expression of ferroptosis-related indicators GPX4 and SLC7A11 decreased as well as increased expression of ACLS4 in cochlea and HEI-OC1 cells. This study also revealed that polystyrene nanoplastics exposure led to increased expression of the inflammatory factors TNF-α, IL-1β and COX2 in cochlea and HEI-OC1 cells. Further research found that the cell apoptosis, ferroptosis and inflammatory reactions induced by polystyrene nanoplastics in HEI-OC1 cells was reversed through the pretreatment with N-acetylcysteine, a reactive oxygen species inhibitor. Overall, our study first discovered and systematically revealed the ototoxicity of polystyrene nanoplastics and its underlying mechanism.