Intracellular Reactive Oxygen Species Levels Research Articles

Amauroderma rugosum Extract Improves Brain Function in d‐Galactose‐Induced Aging Mouse Models via the Regulatory Effects of Its Polysaccharides on Oxidation, the mTOR‐Dependent Pathway, and Gut Microbiota

ABSTRACTThe pharmacological effects of Amauroderma rugosum (AR), an edible mushroom found mainly in Southeast Asia, are not well studied, particularly its neuroprotective properties. This study investigated the neuroprotective effects of AR aqueous extract (ARW) in a d‐galactose‐induced accelerated aging mouse model and senescent SH‐SY5Y neuronal cells. Behavioral tests (open field, Morris water maze, Y‐maze, and rotarod) demonstrated that d‐galactose‐induced aging mice exhibited impaired cognitive function, memory loss, anxiety, and reduced locomotor ability, all of which were alleviated by ARW treatment. Histological analysis showed that ARW reduced neuropathological lesions in the hippocampus. In SH‐SY5Y neuronal cells, ARW and AR polysaccharide extract (ARP) enhanced cell viability and decreased intracellular reactive oxygen species (ROS) levels in a concentration‐dependent manner. ARW and ARP also reduced cellular senescence and apoptosis in d‐galactose‐treated cells. Western blot analysis indicated that ARW and ARP upregulated the phosphorylation of mTOR and increased the expression of antioxidant enzymes, including superoxide dismutase 1 and heme‐oxygenase‐1. Additionally, ARW altered the gut microbiota, increasing the relative abundance of beneficial bacteria such as Lactobacillus reuteri and decreasing harmful bacteria like Clostridium scindens. These findings suggest that AR exerts neuroprotective effects primarily through its polysaccharides by modulating oxidative stress, activating the mTOR‐dependent pathway, and influencing the gut microbiota. Consequently, AR could serve as a potential dietary supplement for the prevention and treatment of neurodegenerative diseases.

Bi-targeting of thioredoxin 1 and telomerase by thiotert promotes cell death of myelodysplastic syndromes and lymphoma

Thioredoxin1 (TRX1) and telomerase are both attractive oncology targets that are tightly implicated in tumor initiation and development. Here, we reported that the 6-dithio-2-deoxyguanosine analog thiotert exhibits an effective cytotoxic effect on myelodysplastic syndromes (MDS) cell SKM-1 and lymphoma cell U-937. Further studies confirmed that thiotert effectively disrupts cellular redox homeostasis, as evidenced by elevated intracellular reactive oxygen species (ROS) levels, increased MnSOD, accelerated DNA impairment, and activated apoptosis signal. Mechanistically, our present study revealed that thiotert treatment effectively inhibited the function of the TRX1/TRXR1 system and telomerase reverse transcriptase (TERT), rendering oxidative damage and impairment of telomeres. Meanwhile, pharmacological administration of glutathione (GSH), N-acetylcysteine (NAC), and mitoquinone (MitoQ), or genetic overexpression of TRX1 or TERT in MDS and cells could dampen the toxicity caused by thiotert. Remarkably, the in vivo mouse model of MDS demonstrated that thiotert administration exhibited greater efficacy in tumor reduction compared to the conventional chemotherapy drug cytarabine. Collectively, these results provide experimental insights into the mechanism of thiotert-induced MDS and lymphoma cell death and unveil that thiotert may be an effective and promising new drug for future MDS and lymphoma treatment.Graphical

NPA7: A Dual Receptor Activating Peptide That Inhibits Cardiac Oxidative Stress.

Cardiomyocyte oxidative stress significantly contributes to the progression of hypertension-induced heart failure, highlighting the need for targeted therapies. We developed a novel peptide, NPA7, that coactivates the GC-A (guanylyl cyclase A)/cGMP and MasR (Mas receptor)/cAMP pathway. This study aimed to test NPA7's ability to inhibit oxidative stress by modulating the p62-KEAP1 (Kelch-like ECH-associated protein 1)-NRF2 (nuclear factor erythroid 2-related factor 2) pathway in human cardiomyocytes (HCMs) and a rat model of hypertension. Oxidative stress was induced in HCMs using H2O2 with PBS or NPA7 treatment. Intracellular reactive oxygen species levels were assessed via dihydroethidium staining. Western blotting analysis measured p62, KEAP1, and NRF2 protein levels, while GSH/GSSG ratios and antioxidant gene expression were analyzed. HCMs were transfected with siRNA targeting GC-A, MasR, or p62 before NPA7 and H2O2 treatment. In vivo, spontaneously hypertensive rats received saline or NPA7, with normotensive WKY rats as control and cardiac oxidative stress, KEAP1 protein levels, NOX2, and p67 mRNA levels were measured. NPA7 reduced H2O2-induced reactive oxygen species levels and increased GSH/GSSG ratio in HCMs. Silencing GC-A and MasR reversed NPA7's effects. NPA7 activated the KEAP1-NRF2 pathway, enhancing NRF2's antioxidant target gene expression. In p62 knockdown HCMs, NPA7-induced KEAP1 degradation and NRF2 activation were diminished. Reactive oxygen species levels were elevated in spontaneously hypertensive rat versus WKY hearts however, NPA7 treatment reduced myocardial reactive oxygen species, suppressed KEAP1 protein, and decreased NOX2 and p67 mRNA levels. NPA7 exhibits antioxidant properties in HCMs and spontaneously hypertensive rat hearts by targeting GC-A and MasR through the p62-KEAP1-NRF2 pathway, supporting a novel therapeutic approach against cardiovascular disease-related oxidative stress.

Copper-Based Bio-Coordination Nanoparticle for Enhanced Pyroptosis-Cuproptosis Cancer Immunotherapy through Redox Modulation and Glycolysis Inhibition.

Copper-based nanoparticles have garnered significant interest in cancer therapy due to their ability to induce oxidative stress and cuproptosis in cancer cells. However, their antitumor effectiveness is constrained by the dynamic redox balance and the metabolic shift between oxidative phosphorylation and glycolysis. Here, a polydopamine-coated copper-α-ketoglutaric acid (α-KG) coordination polymer nanoparticle (CKPP) is designed for combined pyroptosis-cuproptosis cancer immunotherapy by amplifying reactive oxygen species (ROS) production and regulating cellular metabolism. The intracellular redox imbalance is achieved through the synergistic effects of α-KG-induced mitochondrial metabolic reprogramming, photothermally enhanced superoxide dismutase-like activity of polydopamine, and glutathione depletion by copper ions. The multifaceted redox modulation results in a substantial increase in intracellular ROS levels, triggering oxidative stress and subsequent pyroptosis in cancer cells. Furthermore, α-KG shifts cellular metabolism from glycolysis to oxidative phosphorylation, thereby enhancing cuproptosis induced by copper ions. The combination of ROS dyshomeostasis and glycolysis inhibition results in a potent enhancement of pyroptosis-cuproptosis-mediated cancer therapy. In a murine model of colorectal cancer, CKPP exhibited a remarkable anticancer effect, achieving a tumor inhibition rate of 96.3% and complete tumor eradication in two out of five cases. Overall, this bio-engineered metal-organic nanocomposite demonstrates significant potential for treating cancer through combined pyroptosis-cuproptosis cancer immunotherapy.

Novel peptide inhibitor of matrix Metalloproteinases-1 from pufferfish skin collagen hydrolysates and its potential Photoprotective activity via the MAPK/AP-1 signaling pathway.

Takifugu bimaculatus, a pufferfish species farmed in Fujian Province, is known for its non-toxic flesh and collagen-rich skin. We identified a novel collagen-derived matrix metalloproteinase 1 (MMP-1) inhibitory peptide, from T. bimaculatus skin with potent anti-photoaging properties. Using multistage membrane and gel filtration chromatography, we purified low-molecular-weight collagen peptides from T. bimaculatus skin (TBSCH-L). Nano-HPLC-MS/MS and virtual molecular docking screening were employed to identify peptides targeting MMP-1. Four anti-photoaging peptide sequences, GDRGFPGE, GPAGPRGA, FPGGPGAK, and RGFPGGDGAA, were identified by assessing the viability of UVB-induced L929 cells. GPAGPRGA (GP8) exhibited the highest MMP-1 inhibitory activity and cellular photoprotection. Surface plasmon resonance confirmed high-affinity binding between MMP-1 and GP8. GP8 significantly reduced intracellular reactive oxygen species (ROS) levels and enhanced superoxide dismutase activity at concentrations of 100-200μM in UVB-exposed L929 cells. At 200μM, GP8 significantly decreased malondialdehyde content. GP8 also accelerated migration of L929 cells, demonstrating its wound-healing potential, markedly reduced intracellular β-galactosidase levels, and downregulated phosphorylation levels of extracellular signal-regulated kinases, c-Jun N-terminal kinases, p38 proteins, and c-Jun protein expression within the MAPK/AP-1 signaling pathway, thereby lowering MMP expression in L929 cells. Exposure of zebrafish to 25-100μM GP8 effectively mitigated UVB-induced damage, restoring up to 31.2% of caudal fin integrity, while significantly reducing ROS levels, lipid peroxidation, and cellular apoptosis. GP8, a novel marine-derived anti-photoaging peptide, holds promise for applications in cosmetic and functional food sectors.

Small but mighty: nanoemulsion particle size dictates bone regeneration potential of FTY720.

The burgeoning field of nano-bone regeneration is yet to establish a definitive optimal particle size for nanocarriers. This study investigated the impacts of nanocarrier's particle size on the bone regeneration efficacy of fingolimod (FTY720)-loaded nanoemulsions. Two distinct particle sizes (60 and 190 nm, designated as NF60 and NF190, respectively) were produced using low-energy and high-energy emulsion techniques, maintaining a consistent surfactant, co-surfactant, and oil. In vitro studies using rat mesenchymal stem cells revealed that both NF60 and NF190 exhibited cell viability and reduced lactate dehydrogenase. Interestingly, NF60 demonstrated superior antioxidant properties, significantly reducing nitric oxide and intracellular reactive oxygen species (ROS) levels compared to NF190. Furthermore, NF60 significantly enhanced ALP activity and calcium deposition during osteogenic differentiation, indicating its potential to promote the early stages of bone formation. In vivo studies using a rat calvarial bone defect model demonstrated that both NF60 and NF190 significantly upregulated the expression of key osteogenic genes, including Runx2, Col, ALP, OCN, and BMP2. Notably, NF60 induced significantly higher expression of Runx2 and BMP2. X-ray and histological investigations revealed significantly improved bone regeneration in the NF60 group, highlighting the superior bone healing potential of smaller FTY720 nanoemulsions, without infiltration of inflammatory cells. The smaller particle size demonstrated superior antioxidant properties, enhanced osteogenic differentiation, and improved bone regeneration, suggesting smaller nanoparticles, with their larger surface area, accelerated drug release rate, and lower viscosity, interact more effectively with cells, leading to increased and effective drug delivery and cellular uptake. Findings highlight the importance of nanocarrier size in optimizing drug delivery for bone tissue engineering applications.

ALOX15-Driven Ferroptosis: The key target in Dihydrotanshinone I's epigenetic Battle in hepatic stellate cells against liver fibrosis.

It is known that ferroptosis promotes hepatic stellate cells (HSCs) inactivation. Arachidonate 15-Lipoxygenase (ALOX15), a ferroptosis driver gene, participates in disease progression. Dihydrotanshinone I (DHI), an active compound from Salvia miltiorrhiza, effectively regulates HSC inactivation. Nonetheless, there still needs to be clear understanding of how DHI affects HSC ferroptosis. This study primarily investigates DHI's protective effects on liver fibrosis in vivo and in vitro. Additionally, we explored the molecular mechanisms by which DHI promotes ferroptosis in HSCs. The relationship between ALOX15 level and methylation was examined. Molecular docking was performed to confirm the targeting between early growth response protein 1 (EGR1) and DHI. DHI exhibited a mitigating effect on liver fibrosis in vivo. DHI-induced inactivation of HSC by promoting ferroptosis, accompanied by an elevation in intracellular iron and reactive oxygen species (ROS) levels. Results of transcriptome sequencing and quantitative real-time PCR (qRT-PCR) confirmed the elevation of ALOX15 (a ferroptosis driver gene) in HSCs with DHI. Loss of ALOX15 inhibited DHI-induced ferroptosis. Interestingly, DNA methyltransferase 1 (DNMT1), an essential DNA methyltransferase, was downregulated by DHI. Overexpression of DNMT1 resulted in decreased ALOX15 expression in cells with DHI. Notably, transcription factor EGR1 was demonstrated to regulate DNMT1 expression. EGR1 deficiency led to an increase in DNMT1 expression, which inhibited DHI-induced ferroptosis. Molecular docking confirmed that EGR1 could serve as a direct pharmacological target of DHI. DHI upregulates EGR1 level, leading to decreased DNMT1 expression and increased ALOX15 demethylation, thereby promoting HSC ferroptosis and inactivation.

A glycosylated AIE-active Fe(III) photosensitizer activated by the tumor microenvironment for synergistic type I photodynamic and chemodynamic therapy.

Photodynamic therapy (PDT) and chemodynamic therapy (CDT) are both promising cancer treatments to inhibit tumor cells by generating highly cytotoxic reactive oxygen species (ROS). Herein, we report a novel tumor microenvironment (TME) stimulus-responsive water-soluble glycosylated photosensitizer (BT-TPE@Fe-Lac), which can serve as a high-efficiency antitumor agent by combining PDT and CDT, based on the coordination of Fe3+ with lactosyl bis(2-pyridylmethyl)amine and an AIE luminogen (benzothiazole-hydroxytetraphenylethene, BT-TPE). BT-TPE@Fe-Lac is stable under physiological conditions and selectively targets HepG2 cells via asialoglycoprotein receptor (ASGPR)-mediated endocytosis. It rapidly dissociates into AIE-active BT-TPE molecules and a lactosyl ferric(III) complex in the acidic lysosomes of cancer cells. Upon exposure to light, BT-TPE produces O2˙- radicals for type I PDT. The ferric(III) complex is reduced to an Fe(II) complex upon depletion of glutathione, which primes the breakdown of endogenous H2O2 within the tumor microenvironment, thus generating highly toxic ˙OH for enhanced CDT. Compared with the monotherapy of PDT or CDT, BT-TPE@Fe-Lac can significantly increase the intracellular ROS levels to induce more tumor cell death under low drug doses and hypoxia-dependent conditions. This strategy leverages the unique properties of the TME to optimize therapeutic outcomes, offering a promising approach for the TME-responsive nanoplatform in advanced cancer therapy.

AZD1390, an Ataxia telangiectasia mutated inhibitor, enhances cisplatin mediated apoptosis in breast cancer cells.

Genomic instability is often caused by deficiencies in DNA damage repair pathways, making therapeutic targeting of DDR beneficial for cancer patients with specific DDR functions. ATM kinase plays a critical role in various cellular processes and its deficiency increases sensitivity to DDR-targeted agents in different cancers. Recent studies highlight ATM inhibition as a potential clinical target, with AZD1390 being a notable ATM inhibitor due to its potent and selective inhibition, ability to accumulate at DNA breaks. The study aimed to evaluate the potential anti-cancer effects of AZD1390, a key component of the DNA damage response, in breast cancer cells. The impact of the combination of AZD1390 and cisplatin on various parameters such as cell viability, proliferation, colony formation capacity, DNA damage, reactive oxygen species (ROS) levels, mitochondrial membrane potential, cell cycle progression, and cell death in breast cancer cells was evaluated using several methodologies, including WST-1 assays, real-time cell analysis, colony formation assays, comet assays, DCF-DA, MMP/JC-1 staining assays, flow cytometry along with Western blot analysis. We found that AZD1390 and cisplatin displayed synergistic antitumor effects in breast cancer cells at low doses. Addinationaly exhibited significant anti-proliferative effects in colony formation and real-time cell proliferation experiments, increasing intracellular ROS levels and mitochondrial membrane potential.The combined treatment also arrested the cell cycle at the G2-M point. Furthermore, combination of AZD1390 with cisplatin enhances its apoptotic effects in MCF-7 and MDA-MB-231cells. These findings could aid in developing new treatments for breast cancer that exploit the genomic instability of cancer cells.

FER-1 inhibits methylglyoxal-induced ferroptosis in mouse alveolar macrophages in vitro

To investigate the inhibitory effect of FER-1 on methylglyoxal-induced ferroptosis in cultured mouse alveolar macrophages. MH-S cells derived from mouse alveolar macrophages treated with 90 μg/mL methylglyoxal, 10 μmol/mL FER-1MG+FER-1, or both were examined for intracellular reactive oxygen species (ROS), malondialdehyde (MDA) and ferrous ion (Fe2+) levels and changes in mitochondrial membrane potential. Western blotting was performed to detect the protein expression levels of glutathione peroxidase 4 (GPX4) and long-chain acyl-CoA synthase 4 (ACSL4). Methylglyoxal treatment of MH-S cells for 24 h significantly decreased the protein expression level of GPX4, upregulated the protein expression of ACSL4, increased intracellular concentrations of ferrous ions, ROS and MDA, caused loss of mitochondrial membrane potential, and decreased cell viability. Treatment of the cells with FER-1 effectively attenuated these detrimental effects of methylglyoxal in MH-S cells by increasing GPX4 expression, reducing ACSL4 expression and intracellular ferrous ions, ROS and MDA levels, and restoring the mitochondrial membrane potential. Methylglyoxal can induce ferroptosis in MH-S cells in a dose-dependent manner, and FER-1 can rescue the cells from methylglyoxal-induced ferroptosis.

Shikonin induces the apoptosis and pyroptosis of EGFR-T790M-mutant drug-resistant non-small cell lung cancer cells via the degradation of cyclooxygenase-2

BackgroundThe T790M mutation in the epidermal growth factor receptor (EGFR) gene is the primary cause of resistance to EGFR-tyrosine kinase inhibitor (TKI) therapy in non-small cell lung cancer (NSCLC) patients. Previous research demonstrated that certain traditional Chinese medicine (TCM) monomers exhibit anti-tumor effects against various malignancies. This study aims to investigate the potentials of shikonin screened from a TCM monomer library containing 1060 monomers in killing EGFR-T790M drug-resistant NSCLC cells and elucidate the underlying mechanisms.MethodsMTT method was used to screen for the TCM monomers with significant killing effects on H1975 cells carrying the EGFR-T790M mutation. The influences of the identified monomer shikonin on cell growth were determined by the colony formation assay. Annexin-V/PI staining and JC-1 staining were applied to detect the effects of shikonin on cell apoptosis. The influences of shikonin on cell membrane integrity were detected by lactate dehydrogenase (LDH) release assay. Reactive oxygen species (ROS) generation was analyzed using DCFH-DA as probe. The mechanisms of shikonin affecting the stability of cyclooxygenase-2 (COX-2) were evaluated by using specific inhibitors for protein degradation pathways. Western blotting was performed to assess the effects of the alteration of COX-2 expression or enzymatic activity on the related signal pathways as well as the apoptotic and pyroptotic markers.ResultsShikonin was identified as a potent cytotoxic compound against EGFR-T790M-mutant NSCLC cells. Shikonin induced cell apoptosis and pyroptosis by triggering the activation of the caspase cascade and cleavage of poly (ADP-ribose) polymerase and gasdermin E by elevating intracellular ROS levels. Further investigations revealed that shikonin induced the degradation of COX-2 via the proteasome pathway, thereby decreasing COX-2 protein level and enzymatic activity and subsequently inhibiting the downstream PDK1/Akt and Erk1/2 signaling pathways through the induction of ROS production. Notably, COX-2 overexpression attenuated shikonin-induced apoptosis and pyroptosis, whereas COX-2 inhibition with celecoxib enhanced the cytotoxic effects of shikonin.ConclusionsCombination treatment with shikonin and COX-2 inhibitor may be a suitable therapeutic strategy for EGFR-T790M-mutant NSCLC treatment.

Luteolin inhibits proliferation of lung cancer A549 cells by increasing ROS production and inhibiting the AKT/mTOR signaling pathway and HO-1 expression

To investigate the mechanism of luteolin for inhibiting proliferation of lung cancer A549 cells. A549 cells treated with different concentrations of luteolin for 48 h were evaluated for changes in cell viability, proliferation, reactive oxygen species (ROS) production and apoptosis using MTT assay, plate cloning assay, EdU staining, DCFH-DA assay and Hoechst33258 staining. The changes in cell autophagy were examined with MDC staining, and the expressions of apoptosis-related proteins (Bax, Bcl-2, and cleaved caspase-9), autophagy-related proteins (LC3B, Beclin 1, and P62), AKT/mTOR pathway proteins, and HO-1 protein were detected using Western blotting. Treatment with luteolin dose-dependently inhibited the viability and proliferation of A549 cells, increased intracellular ROS levels, up-regulated the expressions of Bax, cleaved caspase-9, and Beclin 1, increased the LC3B-II/LC3B-I ratio, down-regulated the expressions of Bcl-2 and P62, and induced cell apoptosis and autophagy. Luteolin also significantly inhibited the phosphorylation of AKT and mTOR and down-regulated the expression of HO-1 protein in the cells. Luteolin induces apoptosis and autophagy to inhibit proliferation of A549 cells by increasing ROS production, inhibiting the AKT/mTOR pathway and down-regulating HO-1 protein expression.

Mutations in AMBRA1 aggravate β-thalassemia by impairing autophagy-mediated clearance of free α-globin.

Accumulation of free α-globin is a critical factor in the pathogenesis of β-thalassemia. Autophagy plays a crucial role in clearing toxic free α-globin, thereby reducing disease severity. However, the impact of natural mutations in autophagy-related genes (ATGs) on the phenotypic variability of β-thalassemia remains unclear. In this study, we systematically investigated the relationship between variants in ATGs and disease phenotypes in a cohort of 1,022 patients with β-thalassemia, identifying four missense mutations in the autophagy and beclin 1 regulator 1 (AMBRA1) gene. Disruption of the Ambra1 gene in β-thalassemic mice was found to reduce autophagic clearance of α-globin in red blood cell precursors, exacerbating disease phenotypes. Functional characterization of the AMBRA1 gene and these mutations in patient-derived CD34+ cells, edited HUDEP-2 cells, and engineered HUDEP-2 β-thalassemic cells confirmed that AMBRA1 facilitates the autophagic clearance of free α-globin in human erythroid cells. Functional studies demonstrated that AMBRA1 missense mutants destabilize ULK1 protein, inhibit LC3 lipidation, and subsequently hinder autophagic flux, leading to increased α-globin deposition. Additionally, these mutations were associated with erythrotoxic effects in vitro, including increased intracellular reactive oxygen species levels, higher apoptosis rates, and impaired erythroid differentiation and maturation. This study sheds light on the molecular association between mutations in ATGs and the exacerbation of β-thalassemia, highlighting the potential role of the AMBRA1 gene as a promising diagnostic and therapeutic target for β-hemoglobinopathies.

Cis-Regulation of an m6A Eraser by an Insertion Variant Associated with Survival of Patients With Non-Small Cell Lung Carcinoma.

N6-methyladenosine (m6A) serves as one of the crucial RNA modifications for genes involved in cancer progression. Here, 7273 expression quantitative trait loci potentially regulating 30 m6A pathway genes are identified from the GTEx database, with 69 single nucleotide polymorphisms significantly associated with survival of non-small cell lung carcinoma (NSCLC) patients (n = 1523) from the ongoing genome-wide association study after false positive probability tests. Notably, the rs151198415 locus, situated in a potential enhancer region, demonstrated a prolonged survival effect with the C>CCACG insertion, which is validated in an independent prospective cohort (n = 237), yielding a pooled hazard ratio of 0.72 (p = 0.007). Mechanistically, the rs151198415 C>CCACG insertion engaged in long-range interaction with the promoter of m6A eraser ALKBH5, promoting ALKBH5 transcription by the creation of an EGR1 binding site. Then, ALKBH5 upregulated FBXL5 expression by m6A demethylation, which is dependent on the ALKBH5 H204 amino acid site and specific m6A sites on FBXL5 mRNA. Finally, the ALKBH5-FBXL5 axis reduces intracellular reactive oxygen species levels, leading to PI3K/AKT and NF-kB pathway inhibition and consequently suppresses NSCLC proliferation and metastasis in vitro and in vivo. Triggered by an insertion variant, this remote cis-regulation of m6A eraser and the downstream molecular events modulate the survival of NSCLC patients.

Flow-cytometric Analysis of Reactive Oxygen Species in Blood Cells: A Potential Tool for Predicting Restenosis - Insights from a Cohort Study.

In-stent restenosis (ISR) is a recurrence of a blockage in a section of the coronary artery that has previously been treated with a stent. Molecular/biochemical pathways underlying ISR are not fully understood, but inflammation and reactive oxygen species (ROS) induced oxidative stress play a significant role in the pathogenesis of restenosis. As blood cells are highly sensitive to oxidative stress and blood is readily accessible compared to other tissues, the current study flow cytometrically investigated intracellular ROS and cytokine profile of blood cells as possible markers of restenosis. Flow cytometry is commonly used for detecting ROS and analyzing oxidative stress but so far, it has not been utilized for prediction of ISR. So, the aim of the study was to explore the potential of flow cytometric assessment of ROS levels in the blood cells as predictor of ISR. The study was carried out in a total of 60 patients who had previously undergone coronary artery stent implantation. They were categorized as Group I - Coronary stent implanted patients without restenosis (n=30) and Group II - Coronary stent implanted patients with restenosis (n=30). Sociodemographics, biochemical and angiographic characteristics were assessed. Intracellular ROS and cytokine estimation in blood cells was done by using flow cytometric analysis. Flow cytometric measurements demonstrated a 1.3-fold increase in ROS levels in red blood cells (RBCs) and 2-fold increase in ROS levels in leucocytes in group II as compared to group I. Mean serum concentrations of pro-inflammatory cytokines: tumor necrosis factor-α (33.54 ± 6.48 vs. 20.10 ± 5.61, p <0.001***), interferon-gamma (21.76 ± 4.46 vs. 20.10 ± 5.61, p <0.001***), interleukin 6 (152.56 ± 30.67 vs. 113.95 ± 23.38, p <0.001***) were found to be higher in restenotic patients as compared to the non-restenotic patients. Correlation analysis showed that intracellular ROS levels of RBCs exhibited a significant positive correlation with late lumen loss in restenotic (r=0.71, p <0.01) as well as non-restenotic patients (r=0.59, p <0.01). Similarly, intracellular ROS levels of WBCs exhibited a significant positive correlation with late lumen loss in restenotic (r=0.72, p <0.01) as well as non-restenotic patients (r=0.61, p <0.01). This study highlights the role of increased levels of intracellular ROS in blood cells in the subsequent development of ISR, which can be detected flow cytometrically. The study suggests that intracellular ROS estimation in blood cells may serve as a potential marker for restenosis and their flow cytometric analysis may facilitate the prediction of ISR.

Allicin attenuates UVB-induced photodamage of keratinocytes by inhibiting NLRP3 inflammasomes and activating the PI3K/Akt pathway

Allicin is a sulfide extracted from garlic bulbs responsible for various physiological and pathophysiological effects, including antioxidant, antibacterial, and anti-parasite activities. However, its efficacy and mechanism of protecting UVB-induced photodamage have not been studied. The research explores Allicin’s protective roles and underlying mechanisms in UVB-induced photodamage of keratinocytes. UVB was employed to generate photodamage in the HaCaT cell line. DCFH-DA fluorescent and Biochemical analyses were carried out to evaluate reactive oxygen species (ROS) and oxidative stress on UVB-induced photodamage to HaCaT cells. RT-qPCR and western blot were performed to measure mRNA and protein expression. Allicin pretreatment (10 and 25 µM) improved cell proliferation and reduced apoptotic rates in UVB-induced HaCaT cells. Allicin (10 and 25 µM) inhibited tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1β) expressions (all, P < 0.001). Allicin reduced the intracellular ROS level and attenuated oxidative stress, with reduced malondialdehyde (MDA) level while increasing the levels of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSh-Px) (all, P < 0.001) in UVB-induced HaCaT cells. Allicin pretreatment inhibited autophagy and reduced the protein expression of Beclin-1 while increasing the p62 protein expression (all, P < 0.001). We also observed that Allicin pretreatment reduced the NLRP3-related protein, such as Caspase-1 (P < 0.001) and increased the protein expressions of the PI3K/Akt pathway molecules, such as PI3K and Akt (all, P < 0.001). Our research data demonstrated that Allicin might inhibit UVB-induced photodamage of keratinocytes via inhibiting NLRP3 inflammasomes and activating the PI3K/Akt pathway.

2-dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione mediates the effect of ROS-enhanced PI3K/Akt/mTOR pathway on autophagy in breast cancer.

Several studies have suggested a potential antitumor effect of 2-dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione (DMDD). To further understand the mechanism of action of this compound, we investigated its effect on the phosphatidylinositol-3-kinase (PI3K)/serine-threonine kinase (Akt)/mammalian target of rapamycin (mTOR) signaling pathway. We show that DMDD application significantly inhibited the proliferation of breast cancer cell lines MDA-MB-231 and ER-α positive MCF-7. Furthermore, DMDD application resulted in increased intracellular reactive oxygen species (ROS) levels, apoptosis and autophagy, whereas it downregulated the expression of PI3K, Akt and mTOR mRNA and proteins, and increased the expression of LC3II/I and p62 proteins. In a mouse breast cancer xenograft model, DMDD inhibited tumor growth. Expression analyses suggest that ROS levels were higher in DMDD treated tumor tissues, whereas immunohistochemical analyses suggest that apoptotic cells were more prevalent in the DMDD treated group compared to the control group. Taken together, our results suggest that the molecular mechanism of action of DMDD may involve the enhancement of breast cancer autophagy through the PI3K/Akt/mTOR signaling pathway by mediating ROS expression.

Enhancement of the Degradation of Phytosterol Side Chains in Mycolicibacterium by Eliminating the Redox Sensitivity of Key Thiolase and Augmenting Cell Activity

Androstenedione (AD) is a vital intermediate in the synthesis of steroid drugs, making its efficient production critical in the steroid drug industry. Acetyl-CoA acetyltransferase (FadA5), a thiolase enzyme, plays an important role in the metabolic process of degrading phytosterol side chains in Mycolicibacterium to produce AD. This work is the first systematic analysis of the role of FadA5 in the transformation of phytosterols by Mycolicibacterium to produce AD. The relationship between redox potential and AD production was examined using resting cells, and it was confirmed that FadA5 is a key enzyme for AD production. Mutating the 87th cysteine of FadA5 to alanine reduced its redox effect, enhancing the substrate tolerance and biotransformation capacity of the strain. Co-expressing Vitreoscilla hemoglobin (VHb) and propionyl-CoA metabolized the transcription activator (PrpR), decreased intracellular reactive oxygen species levels, and improved cell viability. The AD yield of MSP-fA5C87A-VP/ΔfA5 was 2.541 g/L, an increase of 16.83% over the control strain. Using a repeated batch fermentation process, the production efficiency of the MSP-fA5C87A-VP/ΔfA5 strain was 0.658 g/L/d, which was 1.82 times higher than that of the control strain. These findings provide a theoretical basis for understanding and regulating steroid side-chain catabolism in Mycolicibacterium and offer support for the rational modification of industrial strains for steroidal drug precursor production.

Synergistic inhibitory effect of atmospheric pressure plasma and berberine on non‑small cell lung cancer cells via inducing apoptosis.

Non-small cell lung cancer (NSCLC) is a type of lung cancer, the incidence and mortality rate have been high, and the use of monotherapy is easy to make patients develop tolerance. Atmospheric pressure plasma (APP) is an emerging technology for killing cancer cells in recent years, and combination of berberine (BBR) mechanism has not been fully elucidated for NSCLC. The article's primary goal is to investigate the effect of combination on NSCLC and its associated characterization. Antiproliferative effects were detected by cell viability assay and colony formation, and flow cytometry analysis of apoptosis and cycling showed that the combination synergistically induced apoptosis. Then, extracellular reactive oxygen species (ROS)levels and DCFH-DA-based kits examined intracellular ROS levels, and their effects on mitochondrial membrane potential were measured. Study reveals that co-induced apoptosis is associated with ROS accumulation. Subsequently, Western blotting (WB) detected the expression of epidermal growth factor receptor (EGFR), and the important signaling pathway proteins Ras/ERK and AKT/mTOR. Results showed that it could downregulation of EGFR protein expression and inhibit of activation of ERK/AKT signaling pathways. Simultaneous wound healing assay and epithelial-to-mesenchymal transition (EMT) marker detection were performed for the assessment of migration and EMT ability of NSCLC cells. Combination therapy inhibited migration and EMT of NSCLC cells. The results of this study show that the combination can synergistically induce apoptosis of NSCLC by regulating ROS production. EGFR downregulation and AKT/ERK signaling pathway inhibition are linked to the synergistic effect.

The role of FOXM1 in acetylcysteine improving diabetic periodontitis.

Diabetic periodontitis (DP) stems from hyperglycemia-driven oxidative stress amplification and chronic inflammation, leading to periodontal tissue breakdown. Misregulated forkhead box protein M1 (FOXM1) play key roles in this process, exacerbating both inflammation and oxidative stress. In light of N-Acetylcysteine (NAC)'s potent anti-oxidative capacity and anti-inflammatory potential, understanding how it modulates these central pathways to alleviate DP holds high scientific and clinical importance. An animal model of diabetic mice periodontitis was established, and the model mice were injected with FOXM 1 adenovirus to enrich FOXM 1, and the periodontal pathological histology of each group was evaluated by HE staining. Western blotting and RT-PCR evaluated the expression levels of factors involved in bone destruction. ELISA evaluated the amount of inflammatory factors in mice serum. FOXM 1 over-expression and NAC were treated in murine macrophages, and the intracellular reactive oxygen species(ROS) levels in macrophages were measured using a DCFH-DA probe. Receptor activator of NF-κB ligand (RANKL) and lipopolysaccharide (LPS) were used to establish the macrophage osteoclast differentiation model and test the expression level of osteoclast differentiation factors after giving NAC. Hydrogen peroxide was used to establish a peroxidation environment, the plasmid silenced C-JUN, and the DNA binding activity of activating protein-1(AP1) was detected by EMSA. The effect of peroxidation on the osteoclast differentiation level was determined by WB. Mice with DP model had epithelial damage and inflammatory infiltration in periodontal tissues, and in the FOXM1 enriched group, the periodontal epithelial damage was repaired and inflammation was alleviated. FOXM1 enrichment resulted in DP model lower expression of RANKL (P < 0.01), macrophage colony-stimulating factor (M-CSF) (P < 0.01) and elevated expression of osteoprotegerin (OPG) (P < 0.001). Serum levels of pro-inflammatory factors interleukin (IL)-1β, tumor necrosis factor (TNF-α), and inducible nitric oxide synthase (iNOS) were elevated in DP mice (P < 0.001), and anti-inflammatory factor IL-10 was reduced(P < 0.001),, and FOXM1 enrichment significantly reversed inflammatory factor levels (P < 0.01). Overexpression of FOXM1 reduced ROS content in macrophages (P < 0.001), and NAC was performed to further reduce ROS content (P < 0.01). Silencing of FOXM1 elevated the expression of osteoclast-specific genes NFATc1, TRAP and OSCAR (P < 0.01), and the addition of NAC on top of silencing of FOXM1 markedly suppressed the expression level of osteoclast-specific genes (P < 0.01). ROS increased the transcriptional activity of AP1 (P < 0.001), which promoted osteoclast-specific gene expression (P < 0.001), and osteoclast-specific gene expression was decreased after silencing C-JUN (P < 0.01). FOXM1 relieve diabetic periodontitis inflammation and promote bone formation, regulates ROS production and ROS increases the transcriptional activity of AP1 and affects the osteoclastic differentiation of macrophages, which plays a positive role in bone protection in diabetic periodontitis.