Intracellular Reactive Oxygen Species Content Research Articles

Targeted tumor killing by pomegranate polyphenols: Pro-oxidant role of a classical antioxidant

One of the key biochemical features that distinguish a cancer cell from normal cells is its persistent pro-oxidative state that leads to intrinsic oxidative stress. Malignant cells have evolved sophisticated adaptation systems that involve high dependency on antioxidant functions and upregulation of pro-survival molecules to counteract the deleterious effects of reactive species and to maintain dynamic redox balance. This situation renders them vulnerable to further oxidative challenges by exogenous agents. In the present study, we advocated that pomegranate polyphenols act as pro-oxidants and trigger ROS-mediated apoptosis in cancer cells. With the help of both in vitro and in vivo models, we have established that pomegranate fruit extract (PFE) can cause a significant reduction in tumor proliferation while leaving normal tissues and cells unharmed. Administration of PFE (0.2% v/v) in Erhlich's ascites carcinoma-bearing mice for 3 weeks, inhibited the nuclear factor (erythroid-derived 2)-like 2-antioxidant response element signaling cascade, increased intracellular reactive oxygen species content, altered glutathione cycle thereby activating reactive oxygen species-induced apoptotic pathway in Erhlich's ascites carcinoma cells. Moreover, PFE mitigated epithelial to mesenchymal transition and migration in triple negative breast cancer cells (MDA-MB 231 cells) by down-regulating nuclear factor kappa light-chain-enhancer of activated B cells. Pre-treatment of tumor cells with N-acetyl cysteine protected these cells from undergoing PFE-induced apoptosis while siRNA-mediated silencing of Nuclear factor (erythroid-derived 2)-like 2 and nuclear factor kappa light-chain-enhancer of activated B cells in tumor cells increased the cytotoxic potential and pro-oxidative activity of PFE, indicating a clear role of these transcription factors in orchestrating the anticancer/pro-oxidative properties of PFE. The seminal findings provided may be exploited to develop potential therapeutic targets for selective killing of malignant cells.

Antibacterial activity of reduced graphene oxide prepared by microbe

In previous work, we successfully bioreduced graphene oxide (B-rGO) utilizing organisms, but the antibacterial properties of B-rGO are still unknown. In this study, Escherichia coli (E. coli) was used as a test target and the antibacterial activity of B-rGO was compared to that of commercial reduced graphene oxide (C-rGO), in order to understand its toxicity. Data from levels of extracellular lactate dehydrogenase (LDH), and intracellular reactive oxygen species (ROS) content demonstrate that cell membrane stress and oxidative stress play a role in this antibacterial process. However, when the concentration of co-existing B-rGO was 50.0 mg/L, E. coli damaged easily, the LDH release concentration was 98.75 U/L, the ROS fluorescence signal was 57.75/106 cells, and the phenomenon known as ‘wrapping’ occurred. Indicated here is that B-rGO does indeed demonstrate very good antibacterial activity as evidenced when using Scanning Electron Microscopy (SEM). Meanwhile, numerous oxygen-containing functional groups existed on the surface of B-rGO as confirmed by Fourier transform infrared spectroscopy (FTIR). The power-time curve of microcalorimetry and flow cytometry analysis confirmed that the antibacterial activity of B-rGO was inadequate when compared to that of C-rGO. Despite E. coli mortality being dosage-dependent for both B-rGO and C-rGO, this implies that B-rGO does in fact achieve good biocompatibility. The outcomes reveal that B-rGO has great potential for environmental remediation.

Ebselen inhibits enterovirus A71-induced apoptosis through reactive oxygen species-mediated signaling pathway.

Enterovirus A71 (EV-A71)is a prevalent infection in severe hand, foot and mouth disease HFMD and can induce acute central nervous system seizures. The three EV-A71 vaccines now circulating in the market are produced for a single subtype. While EV-A71 is constantly evolving and the vaccine's efficacy is gradually reducing, no specialized anti-EV-A71 medication has yet been developed. Therefore, it is crucial to consistently develop new anti-EV-A71 medications. Ebselen, an organoselenium molecule with glutathione oxidase-like activity, is resistant to a range of viruses. In this investigation, we used the Cell counting kit-8 (CCK-8 kit) assay in a Vero cell model to confirm the effectiveness of ebselen against EV-A71 infection. Later, to examine ebselen's anti-EV-A71 mechanism, we measured the apoptosis level of cells in different treatment groups through Annexin V, JC-1, and cell cycle assays, as well as the intracellular reactive oxygen species (ROS) concentration. Ebselen may have an impact on the apoptotic signaling pathway caused by EV-A71 infection, according to the results of a caspase-3 activity experiment. The results showed that Ebselen protected cell damage from ROS generation, decreased the frequency of EV-A71-induced apoptosis, and inhibited caspase-3-mediated apoptosis by lowering caspase-3 activity. To summarize, ebselen is a promising anti-EV-A71 medication.

Effect of reactive oxygen species in cadmium chloride induced apoptosis of mouse Leydig cells

To study the effect of reactive oxygen species(ROS) in cadmium chloride-induced apoptosis of mouse Leydig cells(TM3 cells) and explore the underlying molecular mechanisms.TM3 cells were used as an in vitro model for studying reproductive toxicity induced by cadmium exposure. The cells were treated with different concentrations of CdCl_2(0, 5 and 10 μmol/L) for 24 h. CCK-8 assay was used to detect the effect of CdCl_2 on TM3 cell activity. Hoechst33342 staining was performed to explore the formation of apoptotic bodies. DCFH-DA probe was used to detect the level of ROS in the cells. TM3 cells were pretreated with 1 mmol/L NAC for 1 h and then treated with 10 μmol/L CdCl_2 for 24 h. The protein expression levels of pro-apoptotic proteins Caspase-9 and cleaved Caspase-3 were detected by Western blot; RT-qPCR was used to measure the expression of anti-apoptotic gene Bcl-2 and pro-apoptotic genes Caspase-9 and Caspase-3.After exposure to CdCl_2 for 24 h, viability of TM3 cells decreased and the number of apoptotic bodies increased. Western blot result showed that the protein level of Caspase-9 in the 10 μmol/L CdCl_2 treatment group was increased to 0.86±0.10(P<0.05) compared with the control group(0.56±0.07). Compared with the control group(0.37±0.11), the protein level of cleaved Caspase-3 in the 5 and 10 μmol/L CdCl_2 treatment groups were increased to 0.65±0.03 and 1.05±0.13(P<0.05). Compared with the control group(46.80±1.24), the intracellular ROS content in the 5 and 10 μmol/L treatment groups increased to 60.47±1.39 and 80.63±1.34(P<0.05). Compared with the cadmium-treated group, NAC inhibited Caspase-9(CdCl_2 group: 0.89±0.07; CdCl_2+NAC group: 0.28±0.02)and cleaved Caspase-3(CdCl_2 group: 1.53±0.21; CdCl_2+NAC group: 0.66 ±0.07), the difference was statistically significant(P<0.05). At the same time, NAC decreased the ROS level(62.64±0.93) in the CdCl_2 exposure group(80.13±0.94)(P<0.05). In addition, RT-qPCR result showed that the Caspase-9 mRNA levels in the 5 and 10 μmol/L CdCl_2 treatment groups were 1.40±0.14 and 1.90±0.12(P<0.05), compared with the control group(0.97±0.10). Compared with the control group(0.88±0.08), the cleaved Caspase-3 mRNA levels in the 5 and 10 μmol/L CdCl_2 treatment groups were increased to 1.42±0.11 and 1.59±0.12(P<0.05). While in the 5 and 10 μmol/L CdCl_2-treated group, compared with the control group(0.94±0.02), the Bcl-2 mRNA level were decreased to 0.60±0.02 and 0.50±0.09(P<0.05). Compared with the cadmium treatment group(0.57±0.06), NAC could significantly improve the cadmium-induced Bcl-2 mRNA expression level(0.92±0.03), and Caspase-9(CdCl_2 group: 1.96±0.07; CdCl_2+NAC group: 1.04±0.02) and Caspase-3(CdCl_2 group: 1.65±0.02; CdCl_2+NAC group: 0.66±0.04) were decreased(P<0.05).The Caspase cascade in mouse Leydig cells can be activated by excessive ROS induced by CdCl_2, and inhibition of ROS production can significantly reduce the CdCl_2-induced apoptosis of TM3 cells.

Effect of reactive oxygen species in cadmium chloride induced apoptosis of mouse Leydig cells

To study the effect of reactive oxygen species(ROS) in cadmium chloride-induced apoptosis of mouse Leydig cells(TM3 cells) and explore the underlying molecular mechanisms. TM3 cells were used as an in vitro model for studying reproductive toxicity induced by cadmium exposure. The cells were treated with different concentrations of CdCl_2(0, 5 and 10 μmol/L) for 24 h. CCK-8 assay was used to detect the effect of CdCl_2 on TM3 cell activity. Hoechst33342 staining was performed to explore the formation of apoptotic bodies. DCFH-DA probe was used to detect the level of ROS in the cells. TM3 cells were pretreated with 1 mmol/L NAC for 1 h and then treated with 10 μmol/L CdCl_2 for 24 h. The protein expression levels of pro-apoptotic proteins Caspase-9 and cleaved Caspase-3 were detected by Western blot; RT-qPCR was used to measure the expression of anti-apoptotic gene Bcl-2 and pro-apoptotic genes Caspase-9 and Caspase-3. After exposure to CdCl_2 for 24 h, viability of TM3 cells decreased and the number of apoptotic bodies increased. Western blot result showed that the protein level of Caspase-9 in the 10 μmol/L CdCl_2 treatment group was increased to 0.86±0.10(P<0.05) compared with the control group(0.56±0.07). Compared with the control group(0.37±0.11), the protein level of cleaved Caspase-3 in the 5 and 10 μmol/L CdCl_2 treatment groups were increased to 0.65±0.03 and 1.05±0.13(P<0.05). Compared with the control group(46.80±1.24), the intracellular ROS content in the 5 and 10 μmol/L treatment groups increased to 60.47±1.39 and 80.63±1.34(P<0.05). Compared with the cadmium-treated group, NAC inhibited Caspase-9(CdCl_2 group: 0.89±0.07; CdCl_2+NAC group: 0.28±0.02)and cleaved Caspase-3(CdCl_2 group: 1.53±0.21; CdCl_2+NAC group: 0.66 ±0.07), the difference was statistically significant(P<0.05). At the same time, NAC decreased the ROS level(62.64±0.93) in the CdCl_2 exposure group(80.13±0.94)(P<0.05). In addition, RT-qPCR result showed that the Caspase-9 mRNA levels in the 5 and 10 μmol/L CdCl_2 treatment groups were 1.40±0.14 and 1.90±0.12(P<0.05), compared with the control group(0.97±0.10). Compared with the control group(0.88±0.08), the cleaved Caspase-3 mRNA levels in the 5 and 10 μmol/L CdCl_2 treatment groups were increased to 1.42±0.11 and 1.59±0.12(P<0.05). While in the 5 and 10 μmol/L CdCl_2-treated group, compared with the control group(0.94±0.02), the Bcl-2 mRNA level were decreased to 0.60±0.02 and 0.50±0.09(P<0.05). Compared with the cadmium treatment group(0.57±0.06), NAC could significantly improve the cadmium-induced Bcl-2 mRNA expression level(0.92±0.03), and Caspase-9(CdCl_2 group: 1.96±0.07; CdCl_2+NAC group: 1.04±0.02) and Caspase-3(CdCl_2 group: 1.65±0.02; CdCl_2+NAC group: 0.66±0.04) were decreased(P<0.05). The Caspase cascade in mouse Leydig cells can be activated by excessive ROS induced by CdCl_2, and inhibition of ROS production can significantly reduce the CdCl_2-induced apoptosis of TM3 cells.

Peroxiredoxin 1 alleviates oxygen-glucose deprivation/ reoxygenation injury in N2a cells via suppressing the JNK/caspase-3 pathway.

Cerebral ischemia/reperfusion (I/R) injury inevitably aggravates the initial cerebral tissue damage following a stroke. Peroxiredoxin 1 (Prdx1) is a representative protein of the endogenous antioxidant enzyme family that regulates several reactive oxygen species (ROS)-dependent signaling pathways, whereas the JNK/caspase-3 proapoptotic pathway has a prominent role during cerebral I/R injury. This study aimed to examine the potential mechanism of Prdx1 in Neuro 2A (N2a) cells following oxygen-glucose deprivation and reoxygenation (OGD/R) injury. N2a cells were exposed to OGD/R to simulate cerebral I/R injury. Prdx1 siRNA transfection and the JNK inhibitor (SP600125) were used to interfere with their relative expressions. CCK-8 assay, flow cytometry, and lactate dehydrogenase (LDH) assay were employed to determine the viability and apoptosis of N2a cells. The intracellular ROS content was assessed using ROS Assay Kit. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analyses were conducted to detect the expression levels of Prdx1, JNK, phosphorylated JNK (p-JNK), and cleaved caspase-3. Firstly, Prdx1, p-JNK, and cleaved caspase-3 expression were significantly induced in OGD/R-exposed N2a cells. Secondly, the knockdown of Prdx1 inhibited cell viability and increased apoptosis rate, expression of p-JNK, and cleaved caspase-3 expression. Thirdly, SP600125 inhibited the JNK/caspase-3 signaling pathway and mitigated cell injury following OGD/R. Finally, SP600125 partially reversed Prdx1 down-regulation-mediated cleaved caspase-3 activation and OGD/R damage in N2a cells. Prdx1 alleviates the injury to N2a cells induced by OGD/R via suppressing JNK/caspase-3 pathway, showing promise as a potential therapeutic for cerebral I/R injury.

Synergistic Effect of Bovine Cateslytin-Loaded Nanoparticles Combined With Ultrasound AgainstCandida AlbicansBiofilm

Purpose: To investigate the synergistic effect of bovine cateslytin-loaded nanoparticles (bCAT-NPs) combined with ultrasound against Candida albicans biofilm and uncover the underlying mechanism. Methods: bCAT-NPs were prepared by the double emulsion method, and toxicity was observed by the hemolysis ratio. The metabolic activity and viable cell biomass, morphology and membrane permeability of C. albicans biofilm were observed. The expression of ALS3 mRNA, the content of reactive oxygen species, was detected. Finally, bCAT structure was analyzed. Results & conclusion: The hemolysis ratio of the bCAT-NPs group was significantly lower than that of the bCAT group. bCAT-NPs combined with ultrasound significantly reduced biofilm metabolic activity, inhibited the formation of hyphae, decreased the expression of ALS3 mRNA and increased the intracellular reactive oxygen species content. In the in vivo experiments, the colony-forming units/ml in the ultrasound+bCAT-NPs group decreased, and a few planktonic fungal cells were observed.

Addition of ellagic acid improved the immune ability and delayed the apoptosis of ovarian granulosa cells of Guizhou black goat

Context Follicular development plays an important role in the growth and reproduction of female mammals. Ellagic acid (EA), as a natural antioxidant, has been used in freezing protection of pig semen. However, the effects of EA on immunity and the anti-apoptotic ability of ovarian granulosa cells (GCs) are still unclear. Aims The aim of this study was to analyse the effects of different concentrations of EA on the immune and anti-apoptotic ability of ovarian GCs of Guizhou black goats. Methods In this study, different concentrations of EA (0, 50, 100, 150, 200 μmol/L) were added to the culture of ovarian GCs in vitro, and Cell-Counting Kit 8 (CCK8) assay, cell wound scratch assay, and real-time fluorescence quantitative polymerase chain reaction (RT–qPCR) assay were used to detect the effects of different concentrations of EA on the proliferation, migration, and reproductive marker genes of ovarian GCs. Then the optimal addition concentration of EA was selected and the effects of EA supplementation on immune factors, cytochrome P450 family 19 subfamily A member 1 gene (CYP19A1), estradiol concentrations, intracellular reactive oxygen species concentrations, and apoptosis-related protein expression were detected by RT–qPCR, enzyme-linked immunosorbent assay (ELISA), ROS, and western blotting on the basis of the optimal addition concentration. Key results The CCK8 test and cell scratch test showed that the addition of EA could significantly inhibit the proliferation and migration ability of ovarian GCs compared with the control group, and a dose effect was observed with the increase in concentration. RT–qPCR results showed that different concentrations of EA significantly increased the expression of genes associated with reproduction, including bone morphogenetic protein 15 (BMP15), bone morphogenetic protein receptor 1B (BMPR-1B), growth differentiation fFactor 9 (GDF9), and follicle-stimulating hormone β subunit (FSHβ), and the maximum increase was observed at 150 μmol/L EA. Further analyses using 150 μmol/L EA as the optimal concentration showed significantly increased expressions of CYP19A1, interleukin-10 (IL-10), and superoxide dismutase (SOD2) after EA supplementation, while the expression of IL-8 was significantly decreased compared with those of the control group. ELISA and ROS showed that both intracellular and extracellular estradiol concentrations were higher, while ROS concentrations were significantly lower than those in the control group. Western blotting results showed that 150 μmol/L EA significantly decreased the expression of Caspase-3 and Caspase-9 and the ratio of BCL2-associated X:B-cell lymphoma-2. Conclusions The supplementation of 150 μmol/L EA had significant effects on improving GC immunity and delaying GC apoptosis in goats. The addition of EA also increased the expression of BMP15, BMPR-1B, GDF9, FSHβ, and CYP19A1 and promoted the secretion of estradiol in GCs. Implications These results provided a preliminary lead for further research on the effect of EA on the maturation and development of goat oocytes in vitro.

Construction of Fe-doped ZIF-8/DOX nanocomposites for ferroptosis strategy in the treatment of breast cancer.

Breast cancer has become one of the top five commonest causes of cancer death. The use of ferroptosis to induce the generation of reactive oxygen species (ROS) in cancer cells presents a promising and potential strategy for cancer treatment. Herein, a series of facile bimetallic nanoparticles (x% Fe-doped ZIF-8) were synthesized and tested, and doxorubicin (DOX), a classic drug for breast cancer therapy, was encapsulated. After comparing the ratios of Fe2+/(Fe2+ + Zn2+), 7% Fe-doped ZIF-8 (7FZ) was found to be the most suitable particle for medical application. The drug loading efficiency of DOX@7FZ was 58.01 ± 0.02%. The pH-sensitive DOX@7FZ was degraded and DOX was released in lysosomes once internalized. Both the intracellular content of iron and ROS increased significantly. Meanwhile, the cell viability declined to 13.98% in 24 h at a concentration of 60 μg mL-1 and the IC50 was 42.68 μg mL-1. Moreover, the expression of Bcl-2 and GPX-4 proteins decreased in a time-dependent manner, indicating that DOX@7FZ was able to enhance the ROS level in cancer cells via a synergistic effect between apoptosis and ferroptosis. The mechanism of action of DOX@7FZ was further verified using hematoxylin and eosin staining and immunohistochemical staining of Bcl-2 and GPX-4. These remarkable characteristics of DOX@7FZ may inspire further advancements in the treatment of breast cancer.

Crocetin protects cardiomyocytes against hypoxia/reoxygenation injury by attenuating Drp1-mediated mitochondrial fission via PGC-1α.

Saffron (Crocus sativus L.) has been traditionally used as food, spice, and medicine. Crocetin (CRT), as main bioactive component of saffron, has accumulated pieces of beneficial evidence on myocardial ischemia/reperfusion (I/R) injury. However, the mechanisms are poorly explored. This study aims to investigate the effects of CRT on H9c2 cells under hypoxia/reoxygenation (H/R) and elucidated the possible underlying mechanism. H/R attack was performed on H9c2 cells. Cell counting kit-8 was used to detect the cell viability. Cell samples and culture supernatants were evaluated via commercial kits to measure the superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, and cellular adenosine triphosphate (ATP) content. Various fluorescent probes were used to detect cell apoptosis, intracellular and mitochondrial reactive oxygen species (ROS) content, mitochondrial morphology, mitochondrial membrane potential (MMP), and mitochondrial permeability transition pore (mPTP) opening. Proteins were evaluated via Western Blot. H/R exposure severely reduced cell viability and increased LDH leakage. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) suppression and dynamin-related protein 1 (Drp1) activation were coincided with excessive mitochondrial fission, mitochondrial permeability transition pore (mPTP) opening and mitochondrial membrane potential (MMP) collapse in H9c2 cells treated with H/R. Mitochondria fragmentation under H/R injury induced ROS over-production, oxidative stress, and cell apoptosis. Notably, CRT treatment significantly prevented mitochondrial fission, mPTP opening, MMP loss, and cell apoptosis. Moreover, CRT sufficiently activated PGC-1α and inactivated Drp1. Interestingly, mitochondrial fission inhibition with mdivi-1 similarly suppressed mitochondrial dysfunction, oxidative stress and cell apoptosis. However, silencing PGC-1α with small interfering RNA (siRNA) abolished the beneficial effects of CRT on H9c2 cells under H/R injury, accompanied with increased Drp1 and p-Drp1ser616 levels. Furthermore, over-expression of PGC-1α with adenovirus transfection replicated the beneficial effects of CRT on H9c2 cells. Our study identified PGC-1α as a master regulator in H/R-injured H9c2 cells via Drp1-mediated mitochondrial fission. We also presented the evidence that PGC-1α might be a novel target against cardiomyocyte H/R injury. Our data revealed the role of CRT in regulating PGC-1α/Drp1/mitochondrial fission process in H9c2 cells under the burden of H/R attack, and we suggested that modulation of PGC-1α level may provide a therapeutic target for treating cardiac I/R injury.

Evolutionary and reverse engineering in Saccharomyces cerevisiae reveals a Pdr1p mutation-dependent mechanism for 2-phenylethanol tolerance

Background2-Phenylethanol (2-PE), a higher alcohol with a rose-like odor, inhibits growth of the producer strains. However, the limited knowledge regarding 2-PE tolerance mechanisms renders our current knowledge base insufficient to inform rational design.ResultsTo improve the growth phenotype of Saccharomyces cerevisiae under a high 2-PE concentration, adaptive laboratory evolution (ALE) was used to generate an evolved 19–2 strain. Under 2-PE stress, its OD600 and growth rate increased by 86% and 22% than that of the parental strain, respectively. Through whole genome sequencing and reverse engineering, transcription factor Pdr1p mutation (C862R) was revealed as one of the main causes for increased 2-PE tolerance. Under 2-PE stress condition, Pdr1p mutation increased unsaturated fatty acid/saturated fatty acid ratio by 42%, and decreased cell membrane damage by 81%. Using STRING website, we identified Pdr1p interacted with some proteins, which were associated with intracellular ergosterol content, reactive oxygen species (ROS), and the ATP-binding cassette transporter. Also, the results of transcriptional analysis of genes encoded these proteins confirmed that Pdr1p mutation induced the expression of these genes. Compared with those of the reference strain, the ergosterol content of the PDR1_862 strain increased by 72%–101%, and the intracellular ROS concentration decreased by 38% under 2-PE stress. Furthermore, the Pdr1p mutation also increased the production of 2-PE (11% higher).ConclusionsIn the present work, we have demonstrated the use of ALE as a powerful tool to improve yeast tolerance to 2-PE. Based on the reverse engineering, transcriptional and physiological analysis, we concluded that Pdr1p mutation significantly enhanced the 2-PE tolerance of yeast by regulating the fatty acid proportion, intracellular ergosterol and ROS. It provides new insights on Pdr1p mediated 2-PE tolerance, which could help in the design of more robust yeasts for natural 2-PE synthesis.

Secretoneurin as a Novel Biomarker of Cardiovascular Episodes: Are We There Yet? A Narrative Review

Secretoneurin (SN) is a 33 amino-acid evolutionary conserved neuropeptide from the chromogranin peptide family. SN's main effects may be cardioprotective and are believed to be mediated through its inhibition of calmodulin-dependent kinase II (CaMKII), which influences intracellular calcium handling. SN inhibition of CaMKII suppresses calcium leakage from the sarcoplasmic reticulum through the ryanodine receptor. This action may reduce the risk of ventricular arrhythmias and calcium-dependent remodelling in heart failure. SN is also involved in reducing the intracellular reactive oxygen species concentration, modulating the immune response, and regulating the cell cycle, including apoptosis. SN can predict mortality in different disease states, beyond the classical risk factors and markers of myocardial injury. Plasma SN levels are elevated soon after an arrhythmogenic episode. In summary, SN is a novel biomarker with potential in cardiovascular medicine, and probably beyond.

Biochanin A protects against iron overload associated knee osteoarthritis via regulating iron levels and NRF2/System xc-/GPX4 axis

BackgroundIron homeostasis plays a positive role in articular cartilage health. Excessive iron or iron overload can induce oxidative stress damage in chondrocytes and ferroptosis cell death, advancing knee osteoarthritis (KOA). However, up to date, few effective agents treat iron overload-induced KOA (IOKOA). Chinese herbal medicine (CHM) provides abundant resources for drug selection to manage bone metabolic conditions, including osteoporosis. Biochanin A (BCA) is a novel bioactive multifunctional natural compound isolated from Huangqi, which has protective effects on bone loss. Nevertheless, the function and mechanism of BCA in treating IOKOA are still elusive. PurposeThis study seeks to uncover the potential therapeutic targets and mechanisms of BCA in the management of KOA with iron accumulation. MethodsIron dextrin (500 mg/kg) was intraperitoneally injected into mice to establish the iron overloaded mice model. OA was induced through surgery, and the progression was evaluated eight weeks following surgery. OA severity was evaluated with micro-CT and Safranin-O/Fast green staining in vivo. Iron deposition in the knee joint and synovium was assessed using Perl's Prussian blue staining. Ferric ammonium citrate (FAC) was then administered to primary chondrocytes to evaluate iron regulators mediated iron homeostasis. Toluidine blue staining was utilized to identify chondrocytes in vitro. The vitality of the cells was assessed using the CCK-8 test. The apoptosis rate of cells was measured using Annexin V-FITC/PI assay. The intracellular iron level was detected utilizing the calcein-AM test. Reactive oxygen species (ROS), lipid-ROS, and mitochondrial membrane potentiality were reflected via fluorescence density. Utilizing RT-qPCR and western blotting, the expression level was determined. ResultsMicro-CT and histological staining of knee joints showed greater cartilage degradation and higher iron buildup detected in iron-overloaded mice. BCA can reduce iron deposition and the severity of KOA. Toluidine blue staining and the CCK-8 assay indicated that BCA could rescue chondrocytes killed by iron. Cell apoptosis rates were increased due to iron overload but improved by BCA. Further, the intracellular content of iron, ROS, and lipid-ROS was increased with ferric ammonium citrate (FAC) treatment but restored after treatment with different concentrations of BCA. JC-1 staining revealed that BCA could reduce mitochondrial damage induced by iron overload. ConclusionIron overload was shown to promote chondrocyte ferroptosis in vivo and in vitro. Moreover, iron overload suppressed the expression of collagen II and induced MMP expression by catalyzing ROS generation with mitochondrial dysfunction. Our results showed that BCA could directly reduce intracellular iron concentration by inhibiting TfR1 and promoting FPN but also target the Nrf2/system xc-/GPX4 signaling pathway to scavenge free radicals and prevent lipid peroxidation. The results of this research indicate that BCA regulates iron homeostasis during the progression of osteoarthritis, which can open a new field of treatment for KOA.

Effect of hemoglobin on Nile tilapia (Oreochromis niloticus) kidney (NTK) cell line damage

Bacteria or viral outbreaks can cause tilapia hemorrhage, ensuring considerable volume of hemoglobin (Hb) into the tissue. However, the hemoglobin toxicity on tissue and high doses also effect on tissue this phenomena is still under consideration. Therefore, current study exploited Nile tilapia kidney (NTK) cells to deeply expose the toxic effect of Hb on NTK cells. Toxicity of Hb on NTK cells was determined in terms of cells growth, expression of iron metabolism and inflammation-related genes, consequently examined antioxidant-related enzymes genes expression, intracellular iron and reactive oxygen species (ROS) contents, and apoptosis-related genes expression. The results showed that Hb and heme significantly inhibited NTK cells growth and up-regulated iron metabolism-related genes expression in different degrees. The Hb and heme activated the expression of pro-inflammatory cytokines (TNF-α, tumor necrosis factor-α; IL-1β, interleukin 1β; IL-6, interleukin 6), the anti-inflammatory factor (IL-10, interleukin 10) and the chemotactic factors (IL-4, interleukin 4; IL-8, interleukin 8) through NF-κB pathway, meanwhile activated the expression of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px). Moreover, the Hb significantly increased intracellular iron and ROS contents while the expression of apoptosis-related genes was significantly activated by both Hb and heme. Current investigation suggested that high oxidative activity of Hb could activate iron metabolism- and inflammation-related genes expression, and increase intracellular iron and ROS levels, lead to up-regulated the expression of apoptosis genes in NTK cells.

2D Catalytic, Chemodynamic, and Ferroptotic Vermiculite Nanomedicine

AbstractThe emerging chemodynamic therapy employs an iron‐based catalytic Fenton reaction to transform less‐reactive endogenous hydrogen peroxide within the tumor microenvironment (TME) into a highly toxic hydroxyl radical for killing cancer cells. However, the effective deployment of chemodynamic modality remains challenging, mired by a paucity of Fenton agents and overexpressed antioxidant glutathione (GSH) in cancer cells. Herein, a clay‐based 2D vermiculite nanosheet as a self‐reinforcing chemodynamic nanoagent for efficient lung cancer treatment is engineered. The engineered 2D vermiculite nanosheets are not only biocompatible with normal cells but also capable of regulating the TME through depleting GSH, which ameliorates the antioxidant activity of cancer cells. Meanwhile, GSH consumption results in increased intracellular reactive oxygen species content and enhanced lipid peroxidation level, thus inducing ferroptosis and augmenting chemodynamic cell‐killing efficacy. In particular, the ferrous oxidase hephaestin is the direct therapeutic target for 2D vermiculite nanosheets to fight against lung cancer cells. Systematic in vivo evaluations on a xenografted tumor model verify the favorable biosafety and effective tumor suppression capacity of the engineered 2D vermiculite nanosheets‐mediated chemodynamic tumor therapy by inducing desirable ferroptosis. Therefore, the developed vermiculite nanosheets represent the paradigm of 2D ferroptosis‐inducing nanomedicine for synergistic and efficient cancer treatment.

P53 Promotes Ferroptosis in Macrophages Treated with Fe3O4 Nanoparticles.

Fe3O4 nanoparticles are the most widely used magnetic nanoparticles in the biomedicine field. The biodistribution of most nanoparticles in vivo is determined by the capture of macrophages; however, the effects of nanoparticles on macrophages remain poorly understood. Here, we demonstrated that Fe3O4 nanoparticles could reduce macrophage viability after 48 h of treatment and induce a shift in macrophage polarization toward the M1 phenotype; RNA sequencing revealed the activation of the ferroptosis pathway and p53 upregulation compared to the control group. The expression in p53, xCT, glutathione peroxidase 4 (GPX4), and transferrin receptor (TFR) in macrophages was similar to that in erastin-induced ferroptosis in macrophages, and the ultrastructural morphology of mitochondria was consistent with that of erastin-treated cells. We used DCFH-DA to estimate the intracellular reactive oxygen species content in Fe3O4 nanoparticles treated with Ana-1 and JC-1 fluorescent probes to detect the mitochondrial membrane potential change; both showed to be time-dependent. Fer-1 inhibited the reduction of the glutathione/oxidized glutathione (GSH/GSSG) ratio and inhibited intracellular oxidative stress states; therefore, Fe3O4 nanoparticles induced ferroptosis in macrophages. Finally, we used pifithrin-α hydrobromide (PFT) as a p53 inhibitor to verify whether the high expression of p53 is involved in mediating this process. After PFT treatment, the live/dead cell rate, TFR, p53 expression, and GPX4 consumption were inhibited and mitigated the GSH/GSSG ratio reduction as well. This indicates that p53 may contribute to Fe3O4 nanoparticle-induced ferroptosis of macrophages. We provide a theoretical basis for the molecular mechanisms of ferroptosis in macrophages and the biotoxicity in vivo induced by Fe3O4 nanoparticles.

Effects of Graphene Oxide and Reduced Graphene Oxide Nanostructures on CD4+ Th2 Lymphocytes.

The activation of T helper (Th) lymphocytes is necessary for the adaptive immune response as they contribute to the stimulation of B cells (for the secretion of antibodies) and macrophages (for phagocytosis and destruction of pathogens) and are necessary for cytotoxic T-cell activation to kill infected target cells. For these issues, Th lymphocytes must be converted into Th effector cells after their stimulation through their surface receptors TCR/CD3 (by binding to peptide-major histocompatibility complex localized on antigen-presenting cells) and the CD4 co-receptor. After stimulation, Th cells proliferate and differentiate into subpopulations, like Th1, Th2 or Th17, with different functions during the adaptative immune response. Due to the central role of the activation of Th lymphocytes for an accurate adaptative immune response and considering recent preclinical advances in the use of nanomaterials to enhance T-cell therapy, we evaluated in vitro the effects of graphene oxide (GO) and two types of reduced GO (rGO15 and rGO30) nanostructures on the Th2 lymphocyte cell line SR.D10. This cell line offers the possibility of studying their activation threshold by employing soluble antibodies against TCR/CD3 and against CD4, as well as the simultaneous activation of these two receptors. In the present study, the effects of GO, rGO15 and rGO30 on the activation/proliferation rate of these Th2 lymphocytes have been analyzed by studying cell viability, cell cycle phases, intracellular content of reactive oxygen species (ROS) and cytokine secretion. High lymphocyte viability values were obtained after treatment with these nanostructures, as well as increased proliferation in the presence of rGOs. Moreover, rGO15 treatment decreased the intracellular ROS content of Th2 cells in all stimulated conditions. The analysis of these parameters showed that the presence of these GO and rGO nanostructures did not alter the response of Th2 lymphocytes.

Up-and-coming anti-epileptic effect of aloesone in Aloe vera: Evidenced by integrating network pharmacological analysis, in vitro, and in vivo models.

Background: Aloe vera is a medically valuable plant with anti-epileptic activity; however, its mechanism of action remains unknown. In this study, network pharmacological, in vitro, and in vivo experiments were carried out to explore the potential anti-epileptic components and targets of Aloe vera. Methods: The main active components of Aloe vera were identified by searching the Traditional Chinese Medicine System Pharmacology database. Targets of Aloe vera were predicted using SwissTargetPrediction, whereas information about the epilepsy disease targets was obtained from Gene Cards. The protein–protein interaction network and core targets were screened according to the topological structure and CytoNCA plugin. The glutamate-induced HT22 cell line and pentylenetetrazol-induced seizure rats were used to confirm the effect of aloesone by detecting reactive oxygen species (ROS) and apoptosis, and predicting the targets. Results: A total of 14 core active components were selected based on the screening criteria of oral bioavailability ≥30% and drug-likeness ≥ 0.10. Four compounds, namely linoleic acid, aloesone, isoeleutherol glucosiden qt, and anthranol, demonstrated the potential ability of crossing the blood-brain barrier. A total of 153 targets associated with epilepsy were predicted for the four compounds. Moreover, after network analysis with CytoNCA, 10 targets, namely, MAPK1, SRC, MARK3, EGFR, ESR1, PTGS2, PTPN11, JAK2, PPKCA, and FYN, were selected as the core genes, and SRC, which has been predicted to be the target of aloesone and anthranol, exhibited the highest subgraph centrality value. In vitro experiments confirmed that aloesone treatment significantly inhibited the glutamate-induced neuronal injury by reducing the intracellular ROS content and the early phase of apoptosis. Additionally, treatment with 50 mg/kg aloesone resulted in anti-seizure effects by reducing the seizure score and prolonging the latent period in acute and chronic rats. Furthermore, aloesone treatment increased the phosphorylation of c-SRC at Y418 and reduced the phosphorylation at Y529, simultaneously activating c-SRC. Conclusion: Integrating network pharmacology with in vitro and in vivo experiments demonstrated that aloesone, which inhibited seizure by activating c-SRC, is a potential anti-seizure compound present in Aloe vera.

Engineered nanoceria alleviates thermally induced oxidative stress in free-living Breviolum minutum (Symbiodiniaceae, formerly Clade B)

The breakdown of symbiotic mutualism between cnidarian hosts and dinoflagellate algae partners (i.e., bleaching) has been linked to an immune-like response pathway brought on by a nitro-oxidative burst, a symptom of thermal stress. Stress induced by reactive oxygen species (ROS)/reactive nitrogen species is a problem common to aerobic systems. In this study, we tested the antioxidant effects of engineered poly(acrylic acid)-coated cerium dioxide nanoparticles (CeO2, nanoceria) on free-living Symbiodiniaceae (Breviolum minutum), a dinoflagellate alga that forms symbiotic relationships with reef-building corals and anemones. Results show that poly(acrylic acid)-coated CeO2 with hydrodynamic diameters of ~4 nm are internalized by B. minutum in under 30 min and subsequently localized in the cytosol. Nanoceria exposure does not inhibit cell growth over time, with the treated cultures showing a similar growth trend over the 25-day exposure. Aerobic activity and thermal stress when held at 34°C for 1 h (+6°C above control) led to increased intracellular ROS concentration with time. A clear ROS scavenging effect of the nanoceria was observed, with a 5-fold decrease in intracellular ROS levels during thermal stress. The nitric oxide (NO) concentration decreased by ~17% with thermal stress, suggesting the rapid involvement of NO scavenging enzymes or proteins within 1 h of stress onset. The presence of nanoceria did not appear to influence NO concentration. Furthermore, aposymbiotic anemones (Exaiptasia diaphana, ex Aiptasia pallida) were successfully infected with nanoceria-loaded B. minutum, demonstrating that inoculation could serve as a delivery method. The ability of nanoceria to be taken up by Symbiodiniaceae and reduce ROS production could be leveraged as a potential mitigation strategy to reduce coral bleaching.

Trace mineral mixture supplemented to in vitro maturation medium improves subsequent embryo development and embryo quality in cattle.

Trace minerals participate in reproductive processes and are crucial for oocyte maturation. The objective of the present study was to investigate the effect of combined supplementation with copper (Cu), manganese (Mn), selenium (Se) and zinc (Zn) during bovine in vitro maturation (IVM) on subsequent embryo development and quality. The IVM medium was supplemented as follows: a) Control (no mineral supplementation); b) MScz (6ng/mL Mn + 100ng/mL Se + 200ng/mL Cu + 400ng/mL Zn); c) MScZ (6ng/mL Mn + 100ng/mL Se + 200ng/mL Cu + 1200ng/mL Zn); d) MSCz (6ng/mL Mn + 100ng/mL Se + 600ng/mL Cu + 400ng/mL Zn). Supplementation with MScz and MSCz produced more blastocysts compared with the control. Total blastocyst cell number was higher when minerals were added at any combination. Day-8 blastocysts derived from oocytes treated with minerals had lower intracellular reactive oxygen species concentration and lipid content than the control. In conclusion, combined supplementation with Cu, Mn, Se and Zn during bovine oocyte IVM increased in vitro production performance, improving embryo developmental ability and quality.