Intracellular ROS Levels Research Articles

Damascenone Inhibits Osteoclastogenesis by Epigenetically Modulating Nrf2-Mediated ROS Scavenge and Counteracts OVX-Induced Osteoporosis

BackgroundBone formation and resorption regulate bone homeostasis. Excessive osteoclastogenesis enhances bone resorption and causes osteoporosis. Although medicines targeting osteoclast have been developed, these drugs have several side effects. Natural compounds have advantages in safety and efficiency, making them potential candidates for osteoporosis treatment. PurposeThis study aims to elucidate the role of damascenone (Dama) in osteoclastogenesis and osteoporosis. Study design and methodsTo demonstrate the effect of Dama on osteoclast differentiation and function, we performed multiple in vitro experiments including TRAP staining, F-actin staining, bone slice resorption assay, real-time PCR, and western bolt. Further, ROS detection, network pharmacology, microscale thermophoresis assay, and ChIP assay were conducted to elucidate the underlying molecular mechanism. Finally, the in vivo effects of Dama were verified using an ovariectomy induced osteoporosis mice model. ResultsDama inhibited RANKL-induced osteoclast differentiation and bone resorptive function in vitro. The expression of osteoclast-related genes and activation of MAPKs and NF-κB signaling in osteoclast were also attenuated by Dama. Meanwhile, Dama reduced intracellular ROS level via up-regulating Nrf2 expression. Network pharmacology demonstrated that HDAC2 is the potential direct target of Dama. Dama inhibited HDAC2 function and increased H3K27ac level of Nrf2, which induced Nrf2 expression and activated ROS scavenging enzymes. Inhibiting NRF2 or activating HDAC2 attenuated the effect of Dama on osteoclastogenesis. Finally, Dama injection suppressed in vivo osteoclastogenesis and ameliorated bone loss induced by OVX. ConclusionDama attenuates osteoclastogenesis by epigenetically modulating Nrf2 expression and ROS scavenge. This study provides evidence for Dama being a potential treatment for osteoporosis.

Evaluation of the bactericidal activity of plasma-activated NaCl solution (PAN) against foodborne pathogens: Inactivation mechanism and application to mackerel

The objective of this study was to assess the bactericidal effect of plasma-activated NaCl solution (PAN) against Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes and apply PAN as a brine salting solution for mackerel. To enhance the bactericidal effect of plasma-activated water (PAW), NaCl solutions (0, 3.5, 7, and 10 %) were treated with plasma for 20 and 40 min to generate PAN. Plasma-activated glycerol solution (PAG) was also included to evaluate the influence of water activity on plasma activation and its effect on microbial activity. Physicochemical analysis revealed that elevating the NaCl concentration of PAN led to a decrease in pH, an increase in oxidation–reduction potential, and higher levels of reactive species such as H2O2 and HOCl. PAN showed greater antibacterial activity compared to PAW and PAG, except for L. monocytogenes, where 40 min activation time and treatment time exceeding 20 min was required for significantly higher reduction to occur. PAN exhibited greater antibacterial activity at higher NaCl concentrations, which was attributed to increased ionic strength and reactive chlorine species. Additionally, we evaluated the microbial mechanisms of PAN by assessing cellular damage and alterations. The common observation across the three pathogens was that PAN resulted in increased cell membrane damage, reduced intermembrane enzyme activity, higher intracellular ROS levels, and changes in zeta potential values, while DNA damage was observed only in PAN-treated L. monocytogenes. Furthermore, when PAW and PAN were stored for up to four weeks, PAN showed higher efficacy compared to PAW. 10 % PAN was also effective against foodborne pathogens on mackerel, achieving log reductions of 3.62 for E. coli O157:H7, 4.62 for S. Typhimurium, and 3.18 for L. monocytogenes after a 20 min treatment without adversely affecting quality. Our results demonstrated the antibacterial activity and action mechanism of PAN, presenting its potential application in the seafood industry.

The Antifungal Effects of Berberine and Its Proposed Mechanism of Action Through CYP51 Inhibition, as Predicted by Molecular Docking and Binding Analysis

Fungal infections present a significant health risk, particularly in immunocompromised individuals. Berberine, a natural isoquinoline alkaloid, has demonstrated broad-spectrum antimicrobial activity, though its antifungal potential and underlying mechanisms against both yeast-like and filamentous fungi are not fully understood. This study investigates the antifungal efficacy of berberine against Candida albicans, Cryptococcus neoformans, Trichophyton rubrum, and Trichophyton mentagrophytes in vitro, as well as its therapeutic potential in a murine model of cryptococcal infection. Berberine showed strong antifungal activity, with MIC values ranging from 64 to 128 µg/mL. SEM and TEM analyses revealed that berberine induced notable disruptions to the cell wall and membrane in C. neoformans. No signs of cell necrosis or apoptosis were observed in fungal cells treated with 2 × MIC berberine, and it did not increase intracellular ROS levels or affect mitochondrial membrane potential. Molecular docking and binding affinity assays demonstrated a strong interaction between berberine and the fungal enzyme CYP51, with a dissociation constant (KD) of less than 1 × 10−12 M, suggesting potent inhibition of ergosterol biosynthesis. In vivo studies further showed that berberine promoted healing in guinea pigs infected with T. mentagrophytes, and in a murine cryptococcal infection model, it prolonged survival and reduced lung inflammation, showing comparable efficacy to fluconazole. These findings indicate that berberine exerts broad-spectrum antifungal effects through membrane disruption and CYP51 inhibition, highlighting its potential as a promising therapeutic option for fungal infections.

Plasma activated solution synergistic with exogenous RONS enhances cancer cell apoptosis for improved tumor therapy

Abstract The role of reactive oxygen and nitrogen species (RONS) in plasma-activated solution (PAS) for inducing cancer cell apoptosis is well-established, but suboptimal concentrations and rapid degradation often limit their efficacy. This study addresses a significant gap in current research by exploring how the incorporation of exogenous RONS into PAS can enhance apoptosis in cancer cells, potentially overcoming the limitations of traditional PAS treatments. In this study, the effect of treating saline with a plasma jet for different durations was investigated to analyze how its chemical composition triggers apoptosis in cancer cells in vitro. It was observed that longer plasma treatment times resulted in lower cell viability, the reactive species of H2O2, NO2- and ONOO- were found to play a crucial role in inducing apoptosis in cancer cells. Based on these findings, the anticancer efficacy of PAS was further evaluated after the addition of specific exogenous RONS, using measures such as cell viability, intracellular ROS levels, and microscopic imaging. Biological experiments demonstrated that the anticancer efficacy followed the order: addition of RONS to saline before plasma discharge treatment > addition of RONS to saline after plasma discharge treatment > exogenous RONS solution without plasma treatment. This suggests that the external introduction of RONS can modify the chemical composition of PAS to effectively enhance its anticancer properties, with the addition of H2O2 before plasma discharge treatment proving to be the most effective strategy. These results provide a novel perspective on harnessing the anticancer potential of PAS and expanding its potential applications in tumor therapy.

Transplantation of human umbilical cord-derived mesenchymal stem cells improves age-related ovarian functional decline via regulating the local renin–angiotensin system on inflammation and oxidative stress

BackgroundAge-related reproductive aging is a natural and irreversible physiological process, and delaying childbearing is increasingly common all over the world. Transplantation of mesenchymal stem cells (MSCs) is considered a new and effective therapy to restore ovarian function, but the relevant mechanisms remain unclear. Recently, it has been found that there is a local Renin–angiotensin system (RAS) in human ovary and it plays a key role.MethodsAfter collecting follicular fluid from women who received oocyte retrieval for pure male factor infertility, the level of RAS components in it were detected, and the correlation analysis by linear regression. Then, the in vivo experiments on female C57BL/6 mice were designed to measure ovarian function, and the transcription and translation levels of RAS pathway were detected by molecular biology methods. Moreover, the role of RAS in regulating inflammation and oxidative stress in the co-culture system were explored in in vitro experiments on KGN cells.ResultsFirst, a total of 139 samples of analyzable follicular fluid were obtained. The local RAS of ovary, which is independent of systemic RAS (P > 0.05), is affected by age (Pearson r < 0, P < 0.05) and related to ovarian function, inflammation, oxidative stress indexes and assisted reproduction laboratory outcomes (P < 0.05). Next, the ovary/body weight of aging mice decreased significantly and serum sex hormones levels changed significantly (P < 0.01). The number of functional follicles decreased, while the atresia follicles increased (P < 0.05). After MSCs transplantation, all the above measures have been partially recovered (P < 0.05). Although several RAS components in aging ovary changed, MSCs only improved the expression level of AT1R (P < 0.05). Furthermore, the secretion ability and mitochondrial membrane potential of aging KGN cells decreased, while the intracellular ROS level and the aging cells ratio increased (P < 0.01). All the above measures have been partially recovered when co-cultured with MSCs (P < 0.05). After Ang(1–7) were added into the co-culture system, the above have been more significantly restored compared with Ang II (P < 0.05). Nevertheless, there was no statistical difference in estradiol level no matter which one was added (P > 0.05).ConclusionsTogether, our findings indicate that a novel possible mechanism to explain how stem cells restore age-related ovarian functional decline.

Integrative Human Genetic and Cellular Analysis of the Pathophysiological Roles of AnxA2 in Alzheimer's Disease.

Alzheimer's disease (AD) is an intractable and progressive neurodegenerative disease. Amyloid beta (Aβ) aggregation is the hallmark of AD. Aβ induces neurotoxicity through a variety of mechanisms, including interacting with membrane receptors to alter downstream signaling, damaging cellular or organelle membranes, interfering with protein degradation and synthesis, and inducing an excessive immune-inflammatory response, all of which lead to neuronal death and other pathological changes associated with AD. In this study, we extracted gene expression profiles from the GSE5281 and GSE97760 microarray datasets in the GEO (Gene Expression Omnibus) database, as well as from the Human Gene Database. We identified differentially expressed genes in the brain tissues of AD patients and healthy persons. Through GO, KEGG, and ROC analyses, annexin A2 (AnxA2) was identified as a putative target gene. Notably, accumulating evidence suggests that intracellular AnxA2 is a key regulator in various biological processes, including endocytosis, transmembrane transport, neuroinflammation, and apoptosis. Thus, we conducted a series of cell biology experiments to explore the biological function of AnxA2 in AD. The results indicate that AnxA2 gene knockdown primarily affects oxidative phosphorylation, cell cycle, AD, protein processing in the endoplasmic reticulum, SNARE interactions in vesicular transport, and autophagy. In SH-SY5Y cells secreting Aβ42, AnxA2 gene knockdown exacerbated Aβ42-induced cytotoxicity, including cell death, intracellular ROS levels, and neuronal senescence, altered cell cycle, and reduced ATP levels, suggesting its critical role in mitochondrial function maintenance. AnxA2 gene knockdown also exacerbated the inhibitory effect of Aβ42 on cell migration. AnxA2 overexpression reduced the inflammatory response induced by Aβ42, while its absence increased pro-inflammatory and decreased anti-inflammatory responses. Furthermore, AnxA2 gene knockdown facilitated apoptosis and decreased autophagy. These results indicated potential pathophysiological roles of AnxA2 in AD.

Elevated circulating LncRNA NORAD fosters endothelial cell growth and averts ferroptosis by modulating the miR-106a/CCND1 axis in CAD patients

Atherosclerosis is a leading cause of cardiovascular diseases, characterized by endothelial dysfunction and lipid accumulation. Long non-coding RNAs (lncRNAs) are emerging as key regulators of endothelial cell behavior. This study aimed to investigate the role of lncRNA NORAD in endothelial cell proliferation and as a potential therapeutic target for atherosclerosis. A total of 75 CAD patients and 76 controls were recruited, and plasma NORAD levels were measured using qRT-PCR. HUVECs were transfected with si-NORAD to evaluate its effects on cell cycle, proliferation, migration, and apoptosis. Plasma NORAD levels were significantly elevated in CAD patients. The NORAD-miRNA-mRNA ceRNA regulatory network was constructed based on GEO database, and G1/S-specific cyclin-D1 (CCND1) was identified as one of the hub factors. NORAD deficiency suppressed cell migration and induced G1 cell cycle arrest in HUVECs by downregulating CCND1 in vitro. NORAD upregulated CCND1 in HUVECs via sponging miR-106a that inhibited cell migration. The dual-luciferase assay confirmed the direct targeting of miR-106a by NORAD, and overexpression of miR-106a inhibited HUVEC proliferation and migration. Si-NORAD transfection resulted in induced early apoptosis, increased intracellular ROS levels, decreased GSH levels, and reduced mitochondrial membrane potential. Additionally, si-NORAD decreased the expression of GPX4, FTH1, KEAP1, NCOA4, and Nrf2, while increasing Xct levels, confirming the involvement of ferroptosis. Our findings reveal that NORAD plays a critical role in endothelial cell proliferation, migration, and apoptosis, and its silencing induces ferroptosis. The regulatory network involving NORAD, miR-106a, and their target genes provides a potential therapeutic avenue for atherosclerosis.

Optimization of the Treatment of Squamous Cell Carcinoma Cells by Combining Photodynamic Therapy with Cold Atmospheric Plasma

Actinic keratosis (AK) is characterized by a reddish or occasionally skin-toned rough patch on sun-damaged skin, and it is regarded as a precursor to squamous cell carcinoma (SCC). Photodynamic therapy (PDT), utilizing 5-aminolevulinic acid (ALA) along with red light, is a recognized treatment option for AK that is limited by the penetration depth of light and the distribution of the photosensitizer into the skin. Cold atmospheric plasma (CAP) is a partially ionized gas with permeability-enhancing and anti-cancer properties. This study analyzed, in vitro, whether a combined treatment of CAP and ALA-PDT may improve the efficacy of the treatment. In addition, the effect of the application sequence of ALA and CAP was investigated using in vitro assays and the molecular characterization of human oral SCC cell lines (SCC-9, SCC-15, SCC-111), human cutaneous SCC cell lines (SCL-1, SCL-2, A431), and normal human epidermal keratinocytes (HEKn). The anti-tumor effect was determined by migration, invasion, and apoptosis assays and supported the improved efficacy of ALA-PDT in combination with CAP. However, the application sequence ALA-CAP–red light seems to be more efficacious than CAP-ALA–red light, which is probably due to increased intracellular ROS levels when ALA is applied first, followed by CAP and red light treatment. Furthermore, the expression of apoptosis- and senescence-related molecules (caspase-3, -6, -9, p16INK4a, p21CIP1) was increased, and different genes of the junctional network (ZO-1, CX31, CLDN1, CTNNB1) were induced after the combined treatment of CAP plus ALA-PDT. HEKn, however, were much less affected than SCC cells. Overall, the results show that CAP may improve the anti-tumor effects of conventional ALA-PDT on SCC cells. Whether this combined application is successful in treating AK in vivo has to be carefully examined in follow-up studies.

Edible thermosensitive chitosan/hydroxypropyl β-cyclodextrin hydrogel with natural licoricidin for enhancing oral health: Biofilm disruption and demineralization prevention

Dental caries, a widespread and significantly detrimental health condition, is characterized by demineralization, pain, compromised tooth functionality, and various other adverse effects. Licoricidin (LC), a natural isoflavonoid, demonstrates potent antimicrobial properties for maintaining oral health. However, its practical application is significantly hindered by its limited water solubility and susceptibility to removal within the oral environment. To tackle this issue, we developed a delivery oral system by an edible thermosensitive chitosan- disodium beta-glycerol phosphate pentahydrate (CS/β-GP) hydrogel to load LC/Hydroxypropyl beta-cyclodextrin (HP-β-CD) inclusion complexes. These hydrogels (LC/HP-β-CD/CS/β-GP) could solidify rapidly at oral temperature and sustainably release LC, thereby preventing its rapid clearance from the oral cavity. We confirmed the significant antibacterial activity of this hydrogel against Streptococcus mutans and Staphylococcus aureus. Additionally, the HP-β-CD combination enhanced LC to penetrate bacterial biofilms and inhibit biofilm growth, leading to leakage of cellular proteins and DNA. Additionally, we studied the effect of LC/HP-β-CD/CS/β-GP on intracellular ROS levels and MMP, comprehensively exploring its antimicrobial mechanism. Furthermore, LC/HP-β-CD/CS/β-GP exhibited the ability to inhibit demineralization and demonstrated excellent biocompatibility. In summary, this study presented a safer approach to oral delivering bioactive substances, offering a promising strategy for enhanced oral health and safety.

Phytochemical Profile and Antioxidant and Protective Activities of Various Types of Extracts from Hyssopus officinalis L. and Grindelia robusta Nutt. Herb Grown in Poland.

The available literature indicates that Hyssopus officinalis and Grindelia robusta are raw materials with great potential for use in prevention and therapy. Therefore, the aims of this study were to assess the phytochemical profile and antioxidant and cytoprotective properties of extracts prepared using various solvents, additionally taking into account different methods of drying the plant material. Hydrodistilled oil was analysed by GC-MS. The chemical composition of the extracts was estimated by spectrophotometry and the HPLC-DAD method. Antioxidant activity was evaluated using DPPH and FRAP and measuring the intracellular level of ROS. Alamar Blue and Neutral Red tests were used to assess the cytotoxicity of the extracts on skin cells - keratinocytes and fibroblasts. The major components of hyssop essential oil were cis- (44.9%) and trans- (18.2%) pinocamphone, while borneol (16.1%), and α-pinene (12.0%) were predominant in grindelia essential oil. Flavonoids were dominant in the extracts (water:ethanol, water:methanol, and water: glycerol) from hot-air dried hyssop herb, while phenolic acids were the predominant compounds in the grindelia herb extracts. The water:ethanol hyssop extract had the highest total content of flavonoids (42.26 mg CE/mL), among which isoquercitrin and rutin were present in the highest quantities (32.61 mg/mL and 21.47 mg/mL, respectively). In the case of grindelia, the highest total phenolic acid content (26.24 mg CAE/mL) was recorded in the water:ethanol extract, and the dominant compounds among them were 1,5-dicaffeoylquinic and chlorogenic acid (10.85 and 6.39 mg/mL, respectively). The water:ethanol extract from both plants also exhibited the highest antioxidant activity in the DPPH and FRAP tests (79.19% and 1.39 mmol/L, respectively, for grindelia and 67.61% and 1.04 mmol/L for hyssop) and was most effective at reducing the level of ROS in cells. In addition, water:ethanol extracts may have a positive impact on the viability of skin cells in vitro. Water:ethanol extracts from H. officinalis and G. robusta herb are promising sources of active compounds and may find application as natural materials with valuable biological properties, which require further in vitro and in vivo testing.

Allergic Potential & Molecular Mechanism of Skin Sensitization of Cinnamaldehyde Under Environmental UVB Exposure

Fragrance, a key ingredient in cosmetics, often triggers skin allergy causes rashes, itching, dryness, and cracked or scaly skin. Cinnamaldehyde (CA), derived from the bark of the cinnamon tree, used as a fragrance and is a moderate skin sensitizer. CA exhibits strong UVB absorption, its allergic potential and the molecular mechanisms underlying skin sensitization under UVB exposure remain largely unexplored. To investigate the allergic potential and molecular mechanisms of CA-induced skin sensitization under ambient UVB radiation, we employed various alternative in-silico, in-chemico and in-vitro tools. CA under ambient UVB isomerizes from trans to cis CA after 1hr of exposure. Furthermore, DPRA assay and docking with simulation studies demonstrated the enhanced allergic potential of cis-CA. Additionally, our study evaluated intracellular ROS levels and the expression of Nrf2, Catalase, and MMP-2, and 9 in KeratinoSens cells, showing significant upregulation under UVB exposure in the presence of CA. Moreover, our findings indicate that CA activates THP-1 cells co-stimulatory surface marker (CD86) via the activation of intracellular ROS, phagocytosis, and genes of the TLR4 pathway. These insights into the mechanisms uncovered by our study are crucial for managing triggers of allergic skin diseases caused by fragrance use and concurrent exposure to environmental UVB/sunlight.

Green synthesis of Au nanoparticles by Scutellaria barbata extract for chemo-photothermal anticancer therapy

IntroductionThe combination of photothermal therapy and chemotherapy has emerged as a promising strategy for cancer treatment. The green synthesis of gold nanoparticles (Au NPs) using extracts from traditional Chinese medicine Scutellaria barbata D. Don (Scu) has revealed an economical, sustainable, and eco-friendly approach for the outstanding anticancer activities. MethodsScu extract was mixed with HAuCl4 solution to fabricate Scu-Au NPs. The synthesis process was optimized by varying the reaction temperature, reaction time, and HAuCl4 concentration. The changes in the components of Scu before and after synthesis were analyzed using the 3,5-dinitrosalicylic acid (DNS) reduction test, the rutin colorimetric method, and the Folin–Ciocalteu method. The morphology, size, and structure of Scu-Au NPs were characterized by TEM, DLS, XRD, and XPS. The photothermal characteristics induced by near-infrared (NIR) irradiation were assessed by continuously monitoring the temperature elevation. The anticancer activity and mechanisms of Scu-Au NPs were investigated using MTT assay, cell uptake studies, ROS level detection, cell apoptosis and necrosis assays, flow cytometry analysis, intracellular Caspase-3 protein activity fluorescence detection, and Western blot analysis on A549, 4T1, and RAW264.7 cells. ResultsScu-Au NPs were successfully fabricated using Scu extract as a reducing agent. The Scu-Au NPs were spherical with a size of 50 nm. During the synthesis process, the levels of reducing sugars, flavonoids, and polyphenols in the Scu extract decreased by 80.8%, 54.3%, and 70.1%, respectively. Scu-Au NPs demonstrated excellent photothermal responsiveness, thermostability, and biocompatibility. For chemo-photothermal anticancer therapy, the cytotoxicity of Scu-Au NPs on 4T1 cells reached approximately 85% upon exposure to 808 nm NIR irradiation. The anticancer activity was attributed to the induction of apoptosis and necrosis, achieved by increasing intracellular ROS levels, causing cell cycle arrest at the S phase, and modulating the expression of apoptosis-related proteins. DiscussionOur synthesized Scu-Au NPs exhibit favorable photothermal conversion characteristics, demonstrating excellent therapeutic efficacy and safety for cancer treatment.

Red blood cell in preeclampsia: attenuated nitric oxide generation and enhanced reactive oxygen species formation and eryptosis

Preeclampsia (PE) pathogenesis is strongly related to diminished nitric oxide (NO) bioavailability and enhanced oxidative stress. Emerging evidence suggests that red blood cells (RBCs) eNOS enzyme contributes to systemic NO bioavailability by its ability of both NO and ROS generation. We aimed to investigate RBC eNOS enzyme activity, NO and ROS generation capacity, eryptosis index and aggregation levels in preeclamptic and uncomplicated pregnant women. Fifty-eight PE patients and 36 healthy pregnant women were included to the investigation. RBC eNOS enzyme activity, intracellular NO, calcium and ROS concentrations and eryptosis levels were determined via flow cytometric methods. RBC deformability and aggregation were measured via LORRCA. Intracellular NO and phosphorylated RBC eNOS levels decreased in PE group compared to healthy pregnant group (p < 0.05, p < 0.001 respectively). Intracellular ROS and calcium levels, eryptosis values and aggregation indexes in the PE group were significantly higher than healthy pregnant group (p < 0.05, p < 0.01, p < 0.05, p < 0.05 respectively). Our results demonstrate for the first time that RBC produce lower NO and higher ROS under PE conditions. Further, RBC of PE patients were more prone to eryptosis and aggregation compared to control group. Our results suggest that, in addition to endothelial cells, RBC also contribute to decreased plasma NO bioavailability via producing less NO and high ROS in PE. Considering increased tendency to eryptosis and aggregation, RBC seem to play role in haemodynamic changes of PE pathogenesis.

Cardiac-targeted and ROS-responsive liposomes containing puerarin for attenuating myocardial ischemia-reperfusion injury.

Aim: This study aimed to construct an ischemic cardiomyocyte-targeted and ROS-responsive drug release system to reduce myocardial ischemia-reperfusion injury (MI/RI).Methods: We constructed thioketal (TK) and cardiac homing peptide (CHP) dual-modified liposomes loaded with puerarin (PUE@TK/CHP-L), which were expected to deliver drugs precisely into ischemic cardiomyocytes and release drugs in response to the presence of high intracellular ROS levels. The advantages of PUE@TK/CHP-L were assessed by cellular pharmacodynamics, in vivo fluorescence imaging and animal pharmacodynamics.Results: PUE@TK/CHP-L significantly inhibited apoptosis and ferroptosis in H/R-injured cardiomyocytes and also actively targeted ischemic myocardium. Based on these advantages, PUE@TK/CHP-L could significantly enhance the drug's ability to attenuate MI/RI.Conclusion: PUE@TK/CHP-L had potential clinical value in the precise treatment of MI/RI.

New insights into evodiamine attenuates IPEC-J2 cells pyroptosis induced by T-2 toxin - Activating Keap1-Nrf2/NF-κB signaling pathway through binding with Keap1

T-2 toxin (T-2) is a highly toxic mycotoxin with a molecular weight of 466.52 g/mol. Evodiamine (EV), an alkaloid component of Evodia, has anti-inflammation and antioxidant properties. As a receptor of oxidative stress, Keap1 with a molecular weight of 70 kDa, is a molecular switch that controls the Nrf2 signaling pathway. In this paper, the effect of EV on Keap1-Nrf2/NF-κB pathway was investigated. Based on our research outcomes, it was observed that T-2 exposure substantially increased IPEC-J2 cells intracellular ROS levels and MDA accumulation, decreased SOD and CAT activities, disrupted intestinal tight junction (ZO-1, occludin, and claudin-1), and up-regulated pyroptosis-related protein (ASC, NLRP3, caspase-1, GSDMD, IL-1β, and IL-18). Additionally, EV could bind well with Keap1, the separating it from Nrf2, promoting Nrf2 into the nucleus, enhanced antioxidant enzyme activities, reduced the production of ROS, down-regulated NF-κB expression, alleviated T-2-induced pyroptosis, and restored tight junction protein expression. However, after treatment with the Nrf2 inhibitor ML385, ML385 reversed the protective effect of EV on IPEC-J2 cells. Collectively, EV can activate the Keap1-Nrf2/NF-κB signaling pathway via binding to Keap1, exert anti-inflammatory and antioxidant effects, inhibit the pyroptosis of IPEC-J2 cells triggered by T-2, and retore intestinal barrier function.

Rosmarinic acid attenuates glioblastoma cells and spheroids’ growth and EMT/stem-like state by PTEN/PI3K/AKT downregulation and ERK-induced apoptosis

BackgroundGlioblastoma (GB) is a highly malignant type of brain cancer with a poor prognosis. Therapeutic strategies for GB are still limited. Rosmarinic acid (RA), a polyphenolic compound, is a promising experimental anticancer agent, but its specific protein targets for GB remain unclear. PurposeThis study aimed to elucidate the anticancer effects of RA in 2D- and 3D-GB cells and the underlying mechanisms. Methods3D-tumor spheroids (mimics in vivo tumors) were obtained by the hanging-drop/agarose method. RA's anti-glioma activity on U-87MG (p53-wt/PTEN-mt) and LN229 (p53-mt/PTEN-wt) cells was evaluated through cell viability, colony-formation, migration/invasion/angiogenesis assays, fluorescence imaging, and spheroid growth analysis. The underlying mechanism of the anticancer effects of RA was investigated by Western blot and immunofluorescence analysis. The MEK inhibitor U0126 was used to block ERK phosphorylation. ResultsRA treatments exerted anti-proliferative and pro-apoptotic effects on human GB cells. RA dose-dependently reduced angiogenesis and intracellular ROS levels, suppressed glioma growth, and migration/invasion in 2D-culture and cancer stem cell (CSC)-like 3D-spheroid culture (SPC). Repeated therapy in SPC was more effective by leading to disrupted structure than a single treatment. Treatments in SPC also suppressed epithelial-mesenchymal transition (EMT) and CSC-like properties. Strikingly, RA downregulated the SIRT1/FOXO1/NF-κB axis independently of p53 or PTEN function in both gliomas. Immunofluorescence labeling revealed decreased SIRT1 and NF-κB-p65 and increased FOXO1 and GAPDH proteins in nuclear location (associated with apoptosis). Surprisingly, RA increased p-ERK1/2 levels, but priming with U0126 abolished RA-mediated p-ERK upregulation; thus, autophagy and apoptosis induction in GB cells were prevented, and the growth of GB spheroids accelerated. Specifically, RA also inhibited the PTEN/PI3K/AKT pathway in U-87MG cells. Due to genetic differences in cells, U-87MG cells were more sensitive to RA treatments than LN229 cells. Meanwhile, our positive control drug trial results with FDA-approved temozolomide (TMZ) used in GB treatment showed that our test compound rosmarinic acid exhibited higher therapeutic effects than TMZ at lower doses. ConclusionSuppression of EMT, downregulation of SIRT1/FOXO1/NF-κB axis, inhibition of PTEN/PI3K/AKT signaling pathway, and ERK-induced apoptosis and autophagy were determined to be involved in stopping glioma progression. Our findings for the first time, revealed that RA may have potential therapeutic use by having multiple targets in human brain cancer with further clinical studies.

A BODIPY-Ferrocene Conjugate for the Combined Photodynamic Therapy and Chemodynamic Therapy with Improved Antitumor Efficiency.

Photodynamic therapy (PDT) can destroy tumor cells by generating singlet oxygen (1O2) under light irradiation, which is limited by the hypoxia of the neoplastic tissue. Chemodynamic therapy (CDT) can produce toxic hydroxyl radical (⋅OH) to eradicate tumor cells by catalytic decomposition of endogenous hydrogen peroxide (H2O2), the therapeutic effect of which is highly dependent on the concentration of H2O2. Herein, we propose a BODIPY-ferrocene conjugate with a balanced 1O2 and ⋅OH generation capacity, which can serve as a high-efficiency antitumor agent by combining PDT and CDT. The ferrocene moieties endow the as-prepared conjugates with the ability of chemodynamic killing of tumor cells. Moreover, combined PDT/CDT therapy with improved antitumor efficiency can be realized after exposure to light irradiation. Compared with the monotherapy by PDT or CDT, the BODIPY-ferrocene conjugates can significantly increase the intracellular ROS levels of the tumor cells after light irradiation, thereby inducing the tumor cell apoptosis at low drug doses. In this way, a synergistic antitumor treatment is achieved by the combination of PDT and CDT.

Knock-out of CD73 delays the onset of HR-negative breast cancer by reprogramming lipid metabolism and is associated with increased tumor mutational burden

ObjectiveCD73 (ecto-5′-nucleotidase, NT5E), a cell-surface enzyme converting 5′-AMP to adenosine, is crucial for cancer progression. However, its role in the tumorigenesis process remains mostly obscure. We aimed to demonstrate CD73's role in breast cancer (BC) tumorigenesis through metabolic rewiring of fatty acid metabolism, a process recently indicated to be regulated by BC major prognostic markers, hormone receptors (HR) for estrogen (ER), and progesterone (PR). MethodsA murine model of chemically induced mammary gland tumorigenesis was applied to analyze CD73 knock-out (KO)-induced changes at the transcriptome (RNA-seq), proteome (IHC, WB), and lipidome (GC-EI-MS) levels. CD73 KO-induced changes were correlated with scRNA-seq and bulk RNA-seq data for human breast tissues and BCs from public collections and confirmed at the proteome level with IHC or WB analysis of BC tissue microarrays and cell lines. ResultsCD73 KO delayed the onset of HR/PR-negative mammary tumors in a murine model. This delay correlated with increased expression of genes related to biosynthesis and β-oxidation of fatty acids (FAs) in the CD73 KO group at the initiation stage. STRING analysis based on RNA-seq data indicated an interplay between CD73 KO, up-regulated expression of PR-coding gene, and DEGs involved in FA metabolism, with PPARγ, a main regulator of FA synthesis, as a main connective node. In epithelial cells of mammary glands, PPARγ expression correlated with CD73 at the RNA level. With cancer progression, CD73 KO increased the levels of PUFAn3/6 (polyunsaturated omega 3/6 FAs), known ligands of PPARγ and target for lipid peroxidation, which may lead to oxidative DNA damage. It correlated with the downregulation of genes involved in cellular stress response (Mlh1, Gsta3), PR–or CD73-dependent changes in the intracellular ROS levels and expression or activation of proteins involved in DNA repair or oxidative stress response in mammary tumor or human BC cell lines, increased tumor mutational burden (TMB) and genomic instability markers in CD73 low HR-negative human BCs, and the prolonged onset of tumors in the CD73 KO HR/PR-negative group. ConclusionsCD73 has a significant role in tumorigenesis driving the reprogramming of lipid metabolism through the regulatory loop with PR and PPARγ in epithelial cells of mammary glands. Low CD73 expression/CD73 KO might enhance mutational burden by disrupting this regulatory loop, delaying the onset of HR-negative tumors. Our results support combining therapy targeting the CD73-adenosine axis and tumor lipidome against HR-negative tumors, especially at their earliest developmental stage.

Design, synthesis, bioactivity and action mechanism of N-substituted N'-phenylpicolinohydrazides against phytopathogenic fungi.

N'-phenylpicolinohydrazide has been proven to be a promising lead compound for research and development of novel fungicides for agriculture in our previous study. As our continuing research, in this study, a series of N-substituted derivatives of N'-phenylpicolinohydrazide were synthesized and explored for the inhibition activity on nine phytopathogenic fungi and action mechanism. The results found that eleven of the compounds had excellent antifungal activity with more than 80% inhibition rates at 50 µg/mL on part or most of the fungi, especially A. solani and P. piricola. Compounds 5i, 5j and 5k showed EC50 values of < 8.0 µg/mL against A. solani superior to positive control carbendazim (EC50 = 36.0 µg/mL) while 5p and 5q exhibited the highest activity with EC50 values of 2.72 and 2.80 µg/mL against P. piricola superior to positive control boscalid (EC50>50.0 µg/mL). Furthermore, 5k also showed significant protective effect against A. solani infection on tomatoes in a concentration-dependent manner. Action mechanism research showed that 5k was able to increase the intracellular ROS level, change both MMP and permeability of cell membrane and damage mycelial morphology. Molecular docking studies showed that 5k could bind into ubiquinone-binding region of succinate dehydrogenase by hydrogen bonds, π-cation, π-π stacked, π-alkyl, and alkyl interactions. Additionally, the antibacterial activity was also investigated. Thus, N-substituted derivatives of N'-phenylpicolinohydrazide were emerged as novel and highly promising antifungal molecular skeletons to develop new fungicides for crop protection.

New insights into mechanism of ellagic acid alleviating arsenic-induced oxidative stress through MAPK/keap1-Nrf2 signaling pathway response, molecular docking and metabolomics analysis in HepG2 cells

The increase of oxidative stress level is one of the vital mechanisms of liver toxicity induced by arsenic (As). Ellagic acid (EA) is widely known due to its excellent antioxidation. Nevertheless, whether EA could alleviate As-induced oxidative stress and the underlying mechanisms remain unknown. Herein, As (2 and 4 μM) and EA (25 and 50 μM) were selected for alone and combined exposure of HepG2 cells to investigate the effects of EA on As-induced oxidative stress. Results indicated that EA could alleviate the oxidative stress caused by As via decreasing intracellular ROS level and MDA content, as well as improving SOD, CAT and GSH-PX activities. qRT-PCR showed that EA might enhance the expression levels of antioxidant enzymes NQO1, CAT and GPX1 by activating MAPK (JNK, p38 and ERK)/keap1-Nrf2 signaling pathway. EA was found to promote dissociation from keap1 and nuclear translocation of Nrf2 by competing with Nrf2 at ARG-380 and ARG-415 sites on keap1 to exert antioxidation using molecular docking. Moreover, metabolomics revealed that EA might maintain the redox balance of HepG2 cells by modulating or reversing disorders of carbon, amino acid, lipid and other metabolisms caused by As. This study provides diversified new insights for the removal of liver toxicity of As and the application of EA.