Reactive Oxygen Species Assay Research Articles

Abrusamide H Impairs the Secretion of the Cytokines in RAW264.7 Cells and the Inflammatory Infiltration in Tail Transection-Induced Zebrafish.

Abrus mollis Hance (Leguminosae) has a variety of biological activities, including anti-inflammatory, antioxidant, antibacterial, antiviral, and antitumor activities. However, the specific substances responsible for the anti-inflammatory effects are unknown. Abrusamide H (BJBS) is a truxillic acid derivative obtained from the leaves of Abrus mollis Hance and has potential anti-inflammatory effects. In this study, we aimed to estimate the potential effect and mechanism of BJBS in inflammation by establishing lipopolysaccharide (LPS)-stimulated RAW264.7 cells in vitro and an injured zebrafish tail fin in vivo. The RAW264.7 cells were treated with different concentrations of BJBS after LPS stimulation. The production of nitric oxide (NO) was detected by Griess reaction, and reactive oxygen species (ROS) were detected by an ROS assay kit. The levels of proinflammatory cytokines, including interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and interleukin 18 (IL-18) were measured by ELISA. Results showed that BJBS at all concentrations inhibited the proliferation of RAW264.7 macrophages after LPS stimulation by cell counting kit-8 and the production of NO and ROS. In the BJBS treatment group, the levels of IL-6, TNF-α, IL-1β, and IL-18 decreased in a concentration-dependent manner. The results in vivo showed that no significant difference in the survival of zebrafish between the BJBS and blank groups and BJBS inhibited the migration and aggregation of zebrafish neutrophils in a dose-dependent manner in inflammation induced by tail transection-induced inflammation. In conclusion, BJBS inhibited the production of NO and ROS, decreased the levels of secreted IL-6, TNF-α, IL-1β, and IL-18, and reduced the migration and aggregation of zebrafish neutrophils.

DETERMINATION OF INTRACELLULAR REACTIVE OXYGEN SPECIES IN T-CELL SUBSETS OF HIV+ PATIENTS ON CONTINUOUS cART

Background: Reactive oxygen species (ROS) are generated at physiological levels as a result of cellular metabolism and contribute to cellular interaction and immune response. Elevated ROS may cause cell stress, damage, and apoptosis, and have been detected in different pathological states of infectious and non-infectious etiology. Aim: To evaluate the association between intracellular ROS in T-cell subsets and HIV VL in chronic HIV infection. Material and methods: Whole blood samples (Li-heparin, n=33) were analyzed during routine immune monitoring in two groups of HIV+ patients: A (n=21), on continuous cART for at least 2y, with sustained viral suppression (HIV VL<40 copies/ml) and group B (n=12) on cART for less than 2y, average HIV VL 92330 c/ml. Percentage and absolute counts (AC) of CD4+ and CD8+T cells were determined by flow cytometry (Multitest, BD Trucount™ tubes, FACS Canto II). Fluorometric ROS assay kit (Sigma-Aldrich) was adapted for flow cytometry analysis to detect intracellular ROS in CD4+ and CD8+ T-cells (FACSDiva 6.1.2). Results: The average CD4AC did not differ significantly between group A and B (714 vs. 568, p>0.05), unlike the CD4/CD8 ratio (1.2 vs. 0.6, p<0.01). The mean fluorescence intensity (MFI) of CD4+T intracellular ROS was significantly lower in group A (mean MFI 1744 vs. 2492, p<0.05), unlike the CD8+T cell ROS content (1753 vs. 2129, p>0.05). Noteworthy, CD4+T intracellular ROS correlated positively with HIV VL (R=0.5, p<0.05), unlike CD8+T ROS. On the other hand, positive correlations between CD8+T ROS and cART duration, as well as age (R=0.5, p<0.05 for both) were observed in group A. Conclusions: CD4+T ROS production may be an indicator of residual HIV activity in the settings of undetectable HIV VL. The combined effects of ageing and long-term cART affect mostly the CD8+T cell compartment.

Reversal of multidrug resistance by Fissistigma latifolium–derived chalconoid 2-hydroxy-4,5,6-trimethoxydihydrochalcone in cancer cell lines overexpressing human P-glycoprotein

Cancer treatment is an evolving field with various challenges to clinical practice. One unresolved problem in this field is multidrug resistance (MDR) mediated by ABC efflux transporters, particularly P-glycoprotein (P-gp). In this study, by prescreening compounds, we identified the potential of a dihydrochalcone compound, 2-hydroxy-4,5,6-trimethoxydihydrochalcone, for P-gp inhibition. Therefore, we investigated its ability to inhibit P-gp and reverse P-gp-mediated MDR, as well as the underlying mechanisms. The P-gp-inhibitory effects of 2-hydroxy-4,5,6-trimethoxydihydrochalcone were investigated as follows. A P-gp efflux assay and an ATPase assay were used to understand the modulatory mechanisms in the drug-binding and ATP-binding areas, respectively. Prominent reversal effects observed in MDR cancer cell lines; thus, reversal, cytotoxicity, cell cycle, apoptosis, and reactive oxygen species assays were conducted to investigate the underlying mechanism. The results indicated that 2-hydroxy-4,5,6-trimethoxydihydrochalcone functionally inhibited P-gp in a noncompetitive manner, and this inhibition was unrelated to expression. In addition, 2-hydroxy-4,5,6-trimethoxydihydrochalcone served as an ATPase stimulator but not as a P-gp substrate. Moreover, a low binding energy of − 6.85 kcal/mol and one hydrogen bond were obtained, indicating that 2-hydroxy-4,5,6-trimethoxydihydrochalcone has a high affinity for P-gp. P-gp-mediated MDR was reversed by 31.6 μM 2-hydroxy-4,5,6-trimethoxydihydrochalcone in combination with paclitaxel, with a reversal fold value of 379.42. In conclusion, this study provides evidence of the ability of 2-hydroxy-4,5,6-trimethoxydihydrochalcone to inhibit P-gp and reverse MDR.

Ethanolic extract of Origanum syriacum L. leaves exhibits potent anti-breast cancer potential and robust antioxidant properties.

Background: Breast cancer (BC) is the second most common cancer overall. In women, BC is the most prevalent cancer and the leading cause of cancer-related mortality. Triple-negative BC (TNBC) is the most aggressive BC, being resistant to hormonal and targeted therapies. Hypothesis/Purpose: The medicinal plant Origanum syriacum L. is a shrubby plant rich in bioactive compounds and widely used in traditional medicine to treat various diseases. However, its therapeutic potential against BC remains poorly investigated. In the present study, we screened the phytochemical content of an ethanolic extract of O. syriacum (OSEE) and investigated its anticancer effects and possible underlying mechanisms of action against the aggressive and highly metastatic human TNBC cell line MDA-MB-231. Methods: MTT, trans-well migration, and scratch assays were used to assess cell viability, invasion, or migration, respectively. Antioxidant potential was evaluated in vitro using the DPPH radical-scavenging assay and levels of reactive oxygen species (ROS) were assessed in cells in culture using DHE staining. Aggregation assays were used to determine cell-cell adhesion. Flow cytometry was used to analyze cell cycle progression. Protein levels of markers of apoptosis (BCL-2, pro-Caspase3, p53), proliferation (p21, Ki67), cell migration, invasion, or adhesion (FAK, E-cadherin), angiogenesis (iNOS), and cell signaling (STAT3, p38) were determined by immunoblotting. A chorioallantoic Membrane (CAM) assay evaluated in ovo angiogenesis. Results: We demonstrated that OSEE had potent radical scavenging activity in vitro and induced the generation of ROS in MDA-MB-231 cells, especially at higher OSEE concentrations. Non-cytotoxic concentrations of OSEE attenuated cell proliferation and induced G0/G1 cell cycle arrest, which was associated with phosphorylation of p38 MAPK, an increase in the levels of tumor suppressor protein p21, and a decrease of proliferation marker protein Ki67. Additionally, only higher concentrations of OSEE were able to attenuate inhibition of proliferation induced by the ROS scavenger N-acetyl cysteine (NAC), indicating that the anti-proliferative effects of OSEE could be ROS-dependent. OSEE stimulated apoptosis and its effector Caspase-3 in MDA-MB-231 cells, in correlation with activation of the STAT3/p53 pathway. Furthermore, the extract reduced the migration and invasive properties of MDA-MB-231 cells through the deactivation of focal adhesion kinase (FAK). OSEE also reduced the production of inducible nitric oxide synthase (iNOS) and inhibited in ovo angiogenesis. Conclusion: Our findings reveal that OSEE is a rich source of phytochemicals and has robust anti-breast cancer properties that significantly attenuate the malignant phenotype of MD-MB-231 cells, suggesting that O. syriacum may not only act as a rich source of potential TNBC therapeutics but may also provide new avenues for the design of novel TNBC drugs.

5-Bromo-3,4-dihydroxybenzaldehyde attenuates endothelial cells injury from high glucose-induced damage

Hyperglycemia triggers metabolic and inflammatory responses, which lead to vascular inflammation and consequently induce microvascular and/or macrovascular diabetic complications. 5-bromo-3,4-dihydroxybenzaldehyde (BDB), a marine red algae-derived bromophenol compound, is found to have diverse bioactivities, including the effect of anti-inflammation and anti-diabetes, though the mechanism of which is still unclear. To evaluate the anti-vasculopathy of BDB and explore the possible mechanism involved. Firstly, MTT assay was used to optimize the treatment concentration of glucose and BDB with HUVECs. Subsequently, we adopted two concentrations of BDB (50 µM and 100 µM) to verify the protective effect of BDB on vascular model, which was established by HUVECs from high glucose (30 mM)-induced damage. The cell migration and tube formation were used to evaluate the function of HUVECS. Moreover, the related mechanisms were analyzed by assays for flow cytometry, ELISA, qPCR, intracellular ROS and western blot. The present study demonstrated that BDB could protect endothelial cells from apoptosis caused by high glucose treatment. BDB also significantly reduced the secretion of inflammatory cytokines, such as TNF-α, IL-1β and IL-6, induced by high glucose, which was also in agreement to the decrease of p65 protein expression and activities of NF-ĸB regulated by BDB. The reactive oxygen species (ROS) production and phosphorylation of 38 protein expression were also down-regulated by BDB compared to high glucose alone treatment. Furthermore, BDB reserved the endothelial cells functions of migration and tube formation under high glucose condition, which suggested that BDB could be a potential candidate in treating vascular inflammation induced by hyperglycemia.

Identification and functional characterization of ORF19.5274, a novel gene involved in both azoles susceptibility and hypha development in Candida albicans.

Azole resistance is becoming increasingly serious due to the frequent recurrence of fungal infections and the need for long-term clinical prevention. In our previous study, we discovered ORF19.5274 with an unknown function by TMT™ quantitative proteomics technology after fluconazole (FLC) treatment of Candida albicans. In this study, we created the target gene deletion strain using CRISPR-Cas9 editing technology to see if ORF19.5274 regulates azole sensitivity. The data showed that ORF19.5274 was involved in hyphal development and susceptibility to antifungal azoles. Deleting this gene resulted in defective hyphal growth in solid medium, while only a weak lag in the initiation of hyphal development and restoring hyphal growth during the hyphal maintenance phase under liquid conditions. Moreover, intracellular reactive oxygen species (ROS) assay and propidium iodide staining assays showed increased endogenous ROS levels and membrane permeability, but decreased metabolic activity of biofilm in orf19.5274Δ/Δ after treatment with FLC in comparison with either SC5314 or orf19.5274Δ/Δ::ORF19.5274 strains. More importantly, orf19.5274Δ/Δ significantly enhanced the FLC efficacy against C. albicans in infected Galleria mellonella larvae. The above characteristics were fully or partially restored in the complemented strain indicating that the changes caused by ORF19.5274 deletion were specific. In summary, the ORF19.5274 gene is required for hyphal development of C. albicans, and is correlated with the response to antifungal azoles in vitro and in vivo. The identification of ORF19.5275 is promising to expand the potential candidate targets for azoles.

Antiproliferative Effects of Artabotrys odoratissimus Fruit Extract and its Bioactive Fraction through Upregulation of p53/γH2AX Signals and G2/M Phase Arrest in MIA PaCa-2 Cells.

Artabotrys odoratissimus (Annonaceae) is a medicinal and ornamental plant widely cultivated in Southeast Asia for its famous ylang ylang essential oil. The fruits of this plant are used for health benefits, but very little is studied about the bioactive principles, their role in regulating oxidative stress and tumour progression. The study aimed to evaluate the antiproliferative effects of fruit extract of Artabotrys odoratissimus and its bioactive fraction using cell-based assays. The free radical scavenging and antiproliferative effects of Artabotrys odoratissimus fruit ethyl acetate (FEA) extract and its bioactive fraction were evaluated using cell viability assays, colony formation assay, double staining assay, reactive oxygen species (ROS) assay, comet assay, cell cycle analysis, and western blotting. The extract showed phenolic content of 149.8±0.11μg/mg Gallic acid equivalents and flavonoid content of 214.47±4.18 μg/mg Quercetin. FEA showed an IC50 value of 76.35 μg/ml in the ABTS assay and an IC50 value of 134.3±7.8 μg/ml on MIA PaCa-2 cells. The cells treated with 125 μg/ml and 250 μg/ml FEA showed increased apoptotic cells in Double staining assay, DNA damage during comet assay, enhanced ROS, and cell cycle arrest at G2M phase at 125 μg/ml and 250 μg/ml. The active fraction AF5 showed an IC50 value of 67±1.26 μg/ml on MIA PaCa-2 cells during MTT assay, displayed potential antiproliferative effects, and showed a marked increase in the expression of γH2AX and p53. These results prove that the fruit extract and the bioactive fraction demonstrate oxidative stress-mediated DNA damage, leading to apoptosis in the MIA PaCa-2 cell line.

Mechanism of acteoside-activated let-7g-5P attenuating Aβ-induced increased permeability and apoptosis of brain microvascular endothelial cells based on experimental and network pharmacology.

Amyloid β-protein (Aβ)-induced apoptosis and oxidative stress of human brain microvascular endothelial cells(BMECs) are contributors to the development of Alzheimer's disease (AD). Acteoside has shown its therapeutic potential for AD treatment. Therefore, this study investigated the effect of acteoside on Aβ-induced blood-brain barrier damage, oxidative stress and apoptosis as well as to explore the underlying mechanisms through network pharmacology. The study used Aβ to induce human BMECs to construct an in-vitro injury model. Following treatment with acteoside, transendothelial electrical resistance (TEER), RT-qPCR and Western blot were used to evaluate the permeability of BMECs. The apoptosis level was detected by TUNEL and Western blot, ROS assay kit was used for the detection of reactive oxygen species (ROS) expression. The let-7g-5p expression level was detected by RT-qPCR. After additional treatment with let-7g-5p inhibitor, corresponding assays were performed again. Finally, network pharmacology was used to verify the mechanism. Acteoside decreased the permeability, oxidative stress and cell apoptosis of Aβ-stimulated cells. More importantly, acteoside-activated let-7g-5p and additional treatment with let-7g-5p inhibitor abated the effects of acteoside on Aβ-induced permeability, oxidative stress and apoptosis of Aβ-stimulated BMECs. According to network pharmacology, 233 targeted genes of acteoside and 122 potential targets of let-7g-5p were determined by screening several databases, and two targets called Casp-3 and ITGB3 were obtained after taking the intersection. In conclusion, these results reveal that acteoside-activated let-7g-5p attenuating Aβ-induced increased permeability and apoptosis of human BMECs.

Mechanistic efficacy assessment of selected novel methanimine derivatives against vincristine induced Neuropathy: In-vivo, Ex-vivo and In-silico correlates

Vincristine induced peripheral neuropathy (VIPN) is a serious untoward side effect suffered by cancer patients, which still lacks an adequate therapeutic approach. This study examined the alleviating potential of novel methanimine derivatives i.e. (E)-N-(4-nitrobenzylidene)-4-chloro-2-iodobenzamine (KB 9) and (E)-N-(2-methylbenzylidene)-4-chloro-2-iodobenzamine (KB 10) in VIPN. Vincristine was injected in BALB/c mice for 10 days to instigate nociceptive neuropathy. Dynamic and static allodynia, thermal (hot and cold) hyperalgesia were evaluated at 0, 5, 10 and 14 days using cotton brush, Von Frey filament application, hot plate test, acetone drop and cold water respectively. Tumour necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), lipid peroxide (LPO), glutathione-S-transferase (GST), catalase (CAT), superoxide dismutase (SOD) and reactive oxygen species (ROS) assays were performed to assess the efficacy of KB9 and KB10 against neuroinflammation and oxidative stress utilizing ELISA, immunohistochemistry and western blot analysis in brain and sciatic nerve tissues. Computational studies were executed to determine the stable binding conformation of both compounds with respect to COX-2 and NF-κB. Interestingly, both compounds substantially reduced protein expression related to neuroinflammation, oxidative stress (LPO, GST, SOD, CAT) and pain (NF-κB, COX-2, IL-1β and TNF-α). This molecular analysis suggested that the neuroprotective effect of KB9 and KB10 was mediated via regulation of inflammatory signaling pathways. Overall, this study demonstrated that KB9 and KB10 ameliorated vincristine induced neuropathy, through anti-inflammatory, anti-nociceptive and antioxidant mechanisms.

Synergistic antibacterial activity of compact silver/magnetite core-shell nanoparticles core shell against Gram-negative foodborne pathogens.

The development of innovative antibacterial drugs against foodborne pathogens has led to an interest in novel materials such as nanomaterials. The unique features of nanomaterial qualify it for use as an antibacterial treatment. Noble metals and metal oxide nanoparticles, such as silver and magnetite nanoparticles, have been shown to be effective antibacterial medications against a range of microorganisms. In this work, Ag@Fe3O4 -NPs were fabricated by using a wet chemical reduction and modified co-precipitation techniques. The antibacterial efficiency of the Ag/Fe3O4 core shell nanoparticles was investigated by applying various techniques, such as the Kirby–Bauer Disk Diffusion test, minimum inhibitory concentration (MIC) and bactericidal concentration (MBC), Colony Forming Unit (CFU), and kill time assay. The toxicity mechanism of Ag@Fe3O4 -NPs against Salmonella typhimurium and Escherichia coli was studied by apoptosis and reactive oxygen species (ROS) assays. The data revealed that a cubic core was surrounded by a silver shell, which indicated the regular morphology of silver magnetite core shell nanoparticles without any aggregation. Furthermore, Ag@Fe3O4 -NPs is more toxic against S. typhimurium and E. coli than Ag-NPs and Fe3O4 NPs. The MIC values for Ag/Fe3O4 NPs against S. typhimurium and E. coli were 3.1 and 5.4 μg/ml, respectively, whereas the MIC values for Ag-NPs and MNPs against S. typhimurium and E. coli were 4.1 and 8.2 μg/ml for Ag-NPs and 6.9 and 10.3 μg/ml for MNPs. The results showed the ability of Ag@Fe3O4 -NPs to induce apoptosis by generating ROS. Also, the ability of Ag@Fe3O4 -NPs to liberate free Ag+ and generate ROS via the Haber-Weiss cycle may be a plausible mechanism to explain the toxicity of Ag@Fe3O4 -NPs - NPs.

Antioxidant activity and protective effect of wheat germ peptides in an in vitro celiac disease model via Keap1/Nrf2 signaling pathway

Celiac disease (CD) is an allergic intestinal disease caused primarily by gliadin and is widespread in the population. Alpha gliadin peptide causes cellular damage by substantially increasing cellular reactive oxygen species (ROS) levels. In this study, we examined the protective effect of 25 wheat germ peptides (WGPs) on the ɑ-gliadin peptide (P31-43)-treated Caco-2 cells. The experimental results showed that three peptides, WGP2, WGP7, and WGP11, significantly promoted cell viability and greatly alleviated the damage of Caco-2 cells by P31-43. According the assay of ROS, The three WGPS significantly reduced ROS to normal levels, which were elevated by P31-43 peptide. The results in terms of antioxidant-related enzymes showed that WGPs significantly increased catalase (CAT), Glutathione Reductases (GR), Glutathione peroxidase (GPx), and Glutathione (GSH)/ oxidized Glutathione (GSSG) levels, thus significantly enhancing the antioxidant level of cells. By studying the key protein expression levels of the Kelch-like ECH-associated protein 1 (Keap1)/NF-E2-related factor 2 (Nrf2) pathway, the results show that WGPs could activate Nrf2 and glutamate-cysteine ligase catalytic subunit (GCLC) up-regulation. For glutamate-cysteine ligase modifier (GCLM), WGP2 and WGP7 lead to its down-regulation, while WGP11 leads to its significant up-regulation.The present study found that peptides from wheat germ can effectively mitigate the cellular damage induced by the ɑ-gliadin peptide, which provides a new perspective for the prevention and treatment of CD.

Resveratrol pretreatment improves mitochondrial function and alleviates myocardial ischemia-reperfusion injury by up-regulating mi R-20b-5p to inhibit STIM2

This study aimed to explore the mechanism of resveratrol(RES) pretreatment in improving mitochondrial function and alleviating myocardial ischemia-reperfusion(IR) injury by inhibiting stromal interaction molecule 2(STIM2) through microRNA-20 b-5 p(miR-20 b-5 p). Ninety rats were randomly assigned into sham group, IR group, IR+RES(50 mg·kg~(-1) RES) group, IR+RES+antagomir NC(50 mg·kg~(-1) RES+80 mg·kg~(-1) antagomir NC) group, and IR+RES+miR-20 b-5 p antagomir(50 mg·kg~(-1) RES+80 mg·kg~(-1) miR-20 b-5 p antagomir) group, with 18 rats/group. The IR rat model was established by ligation of the left anterior descending coronary artery. Two weeks before the operation, rats in the IR+RES group were intraperitoneally injected with 50 mg·kg~(-1) RES, and those in the sham and IR groups were injected with the same dose of normal saline, once a day. Ultrasonic instrument was used to detect the left ventricular internal diameter at end-diastole(LVIDd) and left ventricular internal diameter at end-systole(LVIDs) of rats in each group. The 2,3,5-triphenyte-trazoliumchloride(TTC) method and hematoxylin-eosin(HE) staining were employed to detect the myocardial infarction area and histopathology, respectively. Real-time quantitative PCR(qRT-PCR) was carried out to detect the expression of miR-20 b-5 p in myocardial tissue. Oxygen glucose deprivation/reoxygenation(OGD/R) was performed to establish an OGD/R model of H9 c2 cardiomyocytes. CCK-8 assay was employed to detect H9 c2 cell viability. H9 c2 cells were assigned into the control group, OGD/R group, OGD/R+RES group(25 μmol·L~(-1)), OGD/R+RES+inhibitor NC group, OGD/R+RES+miR-20 b-5 p inhibitor group, mimic NC group, miR-20 b-5 p mimic group, inhibitor NC group, and miR-20 b-5 p inhibitor group. Flow cytometry was employed to detect cell apoptosis. Western blot was employed to detect the expression of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), cleaved-cysteine proteinase 3(cleaved-caspase-3), and STIM2 in cells. The mitochondrial membrane potential(MMP) assay kit, reactive oxygen species(ROS) assay kit, and adenosine triphosphate(ATP) assay kit were used to detect the MMP, ROS, and ATP levels, respectively. Dual luciferase reporter gene assay was adopted to verify the targeting relationship between miR-20 b-5 p and STIM2. Compared with the sham group, the modeling of IR increased the myocardial infarction area, LVIDd, LVIDs, and myocardial pathology and down-regulated the expression of miR-20 b-5 p(P<0.05). These changes were alleviated in the IR+RES group(P<0.05). The IR+RES+miR-20 b-5 p antagomir group had higher myocardial infarction area, LVIDd, LVIDs, and myocardial pathology and lower expression of miR-20 b-5 p than the IR+RES group(P<0.05). The OGD/R group had lower viability of H9 c2 cells than the control group(P<0.05) and the OGD/R+RES groups(25, 50, and 100 μmol·L~(-1))(P<0.05). Additionally, the OGD/R group had higher H9 c2 cell apoptosis rate, protein levels of Bax and cleaved caspase-3, and ROS level and lower Bcl-2 protein, MMP, and ATP levels than the control group(P<0.05) and the OGD/R+RES group(P<0.05). The OGD/R+RES+miR-20 b-5 p inhibitor group had higher H9 c2 cell apoptosis rate, protein levels of Bax and cleaved-caspase 3, and ROS level and lower Bcl-2 protein, MMP, and ATP levels than the OGD/R+RES group(P<0.05). miR-20 b-5 p had a targeting relationship with STIM2. The expression of STIM2 was lower in the miR-20 b-5 p mimic group than in the mimic NC group(P<0.05) and lower in the inhibitor NC group than in the miR-20 b-5 p inhibitor group(P<0.05). RES pretreatment can inhibit the expression of STIM2 by promoting the expression of miR-20 b-5 p, thereby improving the function of mitochondria and alleviating myocardial IR damage.

Cell-line and culture model specific responses to organic contaminants in house dust: Cell bioenergetics, oxidative stress, and inflammation endpoints

Exposure to organic contaminants in house dust is linked to the development or exacerbation of many allergic and immune disorders. In this work, we evaluate the effects of organic contaminants on different cell bioenergetics endpoints using five different cell lines (16HBE14o-, NuLi-1, A549, THP-1 and HepG2), and examine its effects on lung epithelial cells using conventional 2D and 3D (air-liquid interface/ALI) models. Proposed rapid bioenergetic assays relies on a quick, 40min, exposure protocol that provides equivalent dose-response curves for ATP production, spare respiratory capacity, and cell respiration. Although cell-line differences play an important role in assay performance, established EC50 concentrations for immortalized lung epithelial cells ranged from 0.11 to 0.15mg/mL (∼2 µg of dust in a 96-well microplate format). Bioenergetic response of distinct cell types (i.e., monocytes and hepatocytes) was significantly different from epithelial cells; with HepG2 showing metabolic activity that might adversely affect results in 24h exposure experiments. Like in cell bioenergetics, cell barrier function assay in ALI showed a dose dependent response. Although this is a physiologically relevant model, measurements are not as sensitivity as cytokine profiling and reactive oxygen species (ROS) assays. Observed effects are not solely explained by exposure to individual contaminants, this suggests that many causal agents responsible for adverse effects are still unknown. While 16HBE14o- cells show batter barrier formation characteristics, NuLi-1 cells are more sensitivity to oxidative stress induction even at low house dust extract concentrations, (NuLi-1 2.11-fold-change vs. 16HBE14o- 1.36-fold change) at 0.06µg/mL. Results show that immortalized cell lines can be a suitable alternative to primary cells or other testing models, especially in the development of high-throughput assays. Observed cell line specific responses with different biomarker also highlights the importance of careful in-vitro model selection and potential drawbacks in risk assessment studies.

Ferula gummosa gum exerts cytotoxic effects against human malignant glioblastoma multiforme in vitro.

Ferula gummosa (F. gummosa), a potent medicinal herb, has been shown to possess anticancer activities in vitro. The present examination evaluated the cytotoxic and apoptogenic impacts of F. gummosa gum on the U87 glioblastoma cells. MTT assay to determine the cell viability, flow cytometry by annexin V/FITC-PI to apoptosis evaluation, reactive oxygen species (ROS) assay, and quantitative RT-PCR were performed. The results revealed that F. gummosa inhibited the growth of U87 cells in a concentration- and time-dependent manner with IC50 values of 115, 82, and 52 μg/mL obtained for 24, 48, and 72 h post-treatment, respectively. It was also identified that ROS levels significantly decreased following 4, 12, and 24 h after treatment. The outcomes of flow cytometry analysis suggested that F. gummosa induced a sub-G1 peak which translated to apoptosis in a concentration-dependent manner. Further examination revealed that F. gummosa upregulated Bax/Bcl-2 ratio and p53 genes at mRNA levels. Collectively, these findings indicate that sub-G1 apoptosis and its related genes may participate in the cytotoxicity of F. gummosa gum in U87 cells.

Preparation and in vitro biocompatibility of PBAT and chitosan composites for novel biodegradable cardiac occluders

Abstract The biodegradable composites were prepared by melt blending of chitosan (CS) and poly(butyleneadipate-co-terephthalate) (PBAT). By utilizing Fourier transformed infrared spectroscopy, scanning electron microscopy-energy dispersive spectroscopy, mechanical properties analysis, water contact angle measuring, differential scanning calorimetry, and thermogravimetric analysis, it was demonstrated that the CS of the PBAT-CS10 composite was relatively evenly dispersed in the PBAT matrix, the mechanical properties were significantly improved, the hydrophilicity was increased, the cold crystallization temperature was increased, and a good range of melt working temperature was obtained. The PBAT-CS10 composite was used to fabricate a cardiac occluder by fused deposition modeling of three-dimensional printing, and finite element analysis, and in vitro implantation testing proved the occluder’s mechanical support and sealing function under extreme boundary conditions. In vitro degradation experiments, neutral red uptake cytotoxicity assay, CCK-8 cell proliferation detection, immunofluorescence staining of the cytoskeleton, cell apoptosis detection, and reactive oxygen species assay were all performed on the composite, confirming that it and the occluder made of it could be hydrolyzed under physiological conditions and had no adverse effects on the cell membrane, lysosome membrane, cell proliferation, cell morphology, cell apoptosis, or ROS level, and had good biocompatibility.

Study on the biological mechanism of urolithin a on nasopharyngeal carcinoma in vitro

Context Urolithin A (UroA) can inhibit the growth of many human cancer cells, but it has not be reported if UroA inhibits nasopharyngeal carcinoma (NPC) cells. Objective To explore the inhibitory effect of UroA on NPC and potential mechanism in vitro. Materials and methods RNA-sequencing-based mechanistic prediction was conducted by comparing KEGG enrichment of 40 μM UroA-treated for 24 h with untreated CNE2 cells. The untreated cells were selected as control. After NPC cells were treated with 20–60 μM UroA, proliferation, migration and invasion of were measured by colony formation, wound healing and transwell experiments. Apoptosis, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) were measured by flow cytometry, Hoechst 33342, Rhodamine 123, JC-1 staining and ROS assay methods, respectively. Gene and protein expression were measured by RT-qPCR and Western blotting assay. Results RNA-sequencing and KEGG enrichment revealed UroA mainly altered the ECM receptor interaction pathway. UroA inhibited cells proliferation, epithelial–mesenchymal-transition pathway, migration and invasion with IC50 values of 34.72 μM and 44.91 μM, induced apoptosis, MMP depolarization and increase ROS content at a concentration of 40 μM. UroA up-regulated E-cadherin, Bax/Bcl-2, c-caspase-3 and PARP proteins, while inhibiting COL4A1, MMP2, MMP9, N-cadherin, Vimentin and Snail proteins at 20–60 μM. Moreover, co-treatment of UroA (40 μM) and NAC (5 mM) could reverse the effect of UroA on apoptosis-related proteins. Discussion and conclusions RNA-sequencing technology based on bioinformatic analyses may be applicable for studiying the mechanism of drugs for tumour treatment.

Abstract P3024: Mitochondrial Dysfunction Underlies Sinoatrial Node Dysfunction In Heart Failure With Preserved Ejection Fraction

Background: Metabolic comorbidities are frequent in patients with heart failure with preserved ejection fraction (HFpEF). We recently demonstrated that chronotropic incompetence and exercise intolerance are associated with intrinsic sinoatrial (SAN) dysfunction in animal models of HFpEF. However, there are no studies investigating whether metabolic dysfunction in HFpEF can lead to mitochondrial dysfunction in the SAN. Hypothesis: Our recent findings uncovering SAN pacemaker dysfunction in HFpEF upon metabolic stress led us to test the hypothesis that mitochondrial dysfunction may underlie this condition. Methods: Male C57Bl6 mice fed high fat diet (HFD) plus nitric oxide inhibitor (L-NAME) or regular chow served as HFpEF and control, respectively. Optical mapping, transcriptomics, targeted quantitative proteomics of mitochondrial proteins, mitochondrial respiration and reactive oxygen species (ROS) assays were conducted using explanted mouse SAN tissue or isolated pacemaker cells. Results: SAN from HFpEF-verified animals after 20 weeks of HFD+LNAME exhibited prolonged SAN recovery time after pacing (100%; P<0.05). RNA sequencing revealed augmentation of gene clusters related to metabolism (i.e. ucp and pdk4 ) and extracellular matrix expansion (i.e. col, postn and Cilp1 ). Targeted proteomics validated SAN RNA-sequencing findings, showing upregulation of proteins related to ROS scavenging, fatty acid transport and TCA/OXPHOS (i.e. SOD, CPT2 and DHSA/B). Depressed maximal mitochondrial respiration was seen in isolated mitochondria from SAN of HFpEF animals compared to controls (-42%; P<0.05). This was supported by increased generation of mitochondrial ROS in single pacemaker cells from HFpEF SAN (70%; P<0.05). Acute mitochondrial dysfunction induced by a mitochondrial uncoupler (FCCP) elicited a pronounced prolongation of SAN recovery time in HFpEF compared to controls (165%; P<0.05). Conclusion: Our results indicate that mitochondrial function is depressed in SAN from HFpEF animals, and is associated with SAN pacemaker dysfunction. Further studies are required to test cause-and-effect and to evaluate potential therapeutic strategies targeting mitochondrial pathways for HFpEF associated abnormalities of sinus rhythm.

LTBP2 Knockdown Promotes Ferroptosis in Gastric Cancer Cells through p62-Keap1-Nrf2 Pathway.

Gastric cancer (GC) is one of the most common gastrointestinal malignancies. Ferroptosis is a new type of peroxidation-driven and iron-dependent cell death. However, the biological functions and exact regulatory mechanisms of ferroptosis in GC remain elusive. Here, we performed RNAi and gene transfection, cell viability assay, lipid peroxidation assay, reactive oxygen species (ROS) assay, glutathione assay, qRT-PCR, Western blotting, and transmission electron microscopy (TEM) to study ferroptosis in gastric cancer. The results revealed that silencing latent transforming growth factor β binding proteins (LTBP2) can significantly inhibit GC cell proliferation and decrease cellular GSH levels, reduce GPX4 activity, and increase ROS generation and malondialdehyde (MDA) levels, leading to ferroptosis in GC cells. In addition, we demonstrate that suppression of LTBP2 could regulate the p62-Keap1-Nrf2 pathway, thereby downregulating the GPX4 and xCT expression and upregulating the PTGS2 and 4HNE expression. Our findings described a new role of LTBP2 in regulating ferroptosis, which heralds the prospect of ferroptosis-mediated cancer therapy.

Preparation and Evaluation of the Cytoprotective Activity of Micelles with DSPE-PEG-C60 as a Carrier Against Doxorubicin-Induced Cytotoxicity.

To deliver doxorubicin (DOX) with enhanced efficacy and safety in vivo, fullerenol-modified micelles were prepared with the amphiphilic polymer DSPE-PEG-C60 as a carrier, which was synthesized by linking C60(OH)22 with DSPE-PEG-NH2. Studies of its particle size, PDI, zeta potential, and encapsulation efficiency were performed. DOX was successfully loaded into the micelles, exhibiting a suitable particle size [97 nm, 211 nm, 260 nm, vector: DOX = 5:1, 10:1; 15:1 (W/W)], a negative zeta potential of around −30 mv, and an acceptable encapsulation efficiency [86.1, 95.4, 97.5%, vector: DOX = 5:1, 10:1; 15:1 (W/W)]. The release behaviors of DOX from DSPE-PEG-C60 micelles were consistent with the DSPE-PEG micelles, and it showed sustained release. There was lower cytotoxicity of DSPE-PEG-C60 micelles on normal cell lines (L02, H9c2, GES-1) than free DOX and DSPE-PEG micelles. We explored the protective role of DSPE-PEG-C60 on doxorubicin-induced cardiomyocyte damage in H9c2 cells, which were evaluated with a reactive oxygen species (ROS) assay kit, JC-1, and an FITC annexin V apoptosis detection kit for cellular oxidative stress, mitochondrial membrane potential, and apoptosis. The results showed that H9c2 cells exposed to DSPE-PEG-C60 micelles displayed decreased intracellular ROS, an increased ratio of red fluorescence (JC-1 aggregates) to green fluorescence (JC-1 monomers), and a lower apoptotic ratio than the control and DSPE-PEG micelle cells. In conclusion, the prepared DOX-loaded DSPE-PEG-C60 micelles have great promise for safe, effective tumor therapy.

CD147 protein expression and Temozolomide resistance in glioma cells: an Ex vivo and In vivo study

It has been noted that temozolomide resistance occurs in a number of malignancies, including glioma, although the underlying cause of this is unknown. The goal of the studyin vivo investigation to show that increased CD147 expression in glioma cells is a factor in their resistance to the chemotherapy drug temozolomide. Proliferation assays, TUNEL assays, reactive oxygen species assays, protein degradation assays, immunohistochemistry, Western blotting, quantitative polymerase chain reactions, and tumorigenicity assays were all carried out. Using the human protein atlas databases, the expression levels of CD147 in different kinds of malignancies were examined. For immunohistochemistry, a total of 7, 12, 19, 15, and 16 glioma samples were taken from para-carcinoma tissue, representing stage I, stage II, stage III, and stage IV gliomas, respectively. The expression of CD147 proteins is correlated with the tumor's aggressiveness. Cell development was slowed by suppressing the expression of the CD147 protein. The expression of the CD147 protein contributed to the emergence of temozolomide resistance. Expression of the CD147 protein reduced mRNA expression. The growth-inhibitory impact of temozolomide on glioma cells was enhanced by the suppression of CD147 protein.Nuclear factor E2-related factor 2 expression and CD147 protein expression showed a significant reciprocal connection with each other (p 0.0001, r2 = 0.3254). In glioma, resistance to temozolomide is due to overexpression of CD147 protein and induction of nuclear factor E2-related factor 2.