Dichlorodihydrofluorescein Diacetate Research Articles

Extracellular vesicles mirror the beneficial effect of wild olive (acebuche) oil in hypertensive‐like retinal pigment epithelial cells

Aims/Purpose: Arterial hypertension (AH) leads to oxidative and inflammatory imbalance triggering hypertensive organ damage via different pathways. Oil extracted from the wild olive tree (acebuche, ACE) has previously shown antioxidant and anti‐inflammatory properties against hypertensive ocular damage when regularly consumed in the diet1,2. We therefore speculate that circulating extracellular vesicles (EVs) isolated from ACE oil‐fed animals might play a role in modulating pathological mechanisms surrounding AH‐related ocular diseases, including oxidative stress, migration and angiogenesis processes.Methods: Retinal pigment epithelium (ARPE‐19) cells mimicking a hypertensive phenotype induced by incubation with angiotensin II (AngII) were treated with EVs obtained from mice fed ACE oil or a reference extra virgin olive oil (EVOO). The 2′,7′dichlorodihydrofluorescein diacetate (DCFHDA) assay was used to estimate intracellular levels of reactive oxygen species (ROS). Furthermore, kinetic parameters for L‐arginine (L‐Arg) transport and the expression of L‐Arg transporter (CAT‐1) were also analysed. The wound‐healing assay was used to determine the anti‐migratory properties of ACE oil‐derived EVs, and the ability of EVs to modulate angiogenesis was assessed in aortic rings.Results: Both ACE oil‐ and EVOO‐related EVs were able to reduce ROS production in ARPE‐19 cells. The normalization of intracellular redox status by EVs might be exerted through modulation of L‐Arg uptake and CAT‐1 expression. Anti‐migratory and anti‐angiogenesis properties were also displayed by oil‐derived EVs, suggesting their potential benefit in the setting of neovascular ocular diseases.Conclusions: This is the first report to highlight the retinoprotective effects of EVs isolated from the plasma of animals subjected to ACE oil‐ and EVOO‐enriched diets against pathological events related to ocular diseases.

Histone deacetylase 3-specific inhibitor RGFP966 attenuates oxidative stress and inflammation after traumatic brain injury by activating the Nrf2 pathway.

Oxidative stress (OS) and inflammatory reactions play pivotal roles in secondary brain injury after traumatic brain injury (TBI). Histone deacetylase 3 (HDAC3) controls the acetylation of histones and non-histones, which has a significant impact on the central nervous system's reaction to damage. This research determined the implications of RGFP966, a new and specific inhibitor of HDAC3, for the antioxidant (AO) systems mediated by nuclear factor erythroid2-related factor 2 (Nrf2) and the Nod-like receptor protein 3 (NLRP3) inflammasome in TBI. The study also studied the underlying mechanisms of RGFP966's actions. Our objective was to examine the impacts and underlying RGFP966 mechanisms in TBI. In vitro, a rat cortical neuron OS model was induced by H2O2, followed by the addition of RGFP966 to the culture medium. Neurons were collected after 24h for western blot (WB), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and 2'-7'-dichlorodihydrofluorescein diacetate staining. In vivo, RGFP966 (10mg/kg) was administered post-TBI. Brain tissue water content and modified neurological severity scores were assessed 72h post-injury. Cortical tissues surrounding the focal injury were subjected to western blot, TUNEL staining, Nissl staining and immunofluorescence/immunohistochemistry staining, and malondialdehyde level, hindered glutathione content and superoxide dismutase activity were measured. Serum was collected for the enzyme-linked immunosorbent assay. Nrf2-specific shRNA lentivirus was injected into the lateral ventricle of rats for 7days, and cerebral cortex tissue was analyzed by WB and real-time polymerase chain reaction. During in vitro and in vivo experiments, RGFP966 suppressed HDAC3 expression, promoted Nrf2 nuclear translocation, activated downstream AO enzymes, mitigated excessive reactive oxygen species production and alleviated nerve cell apoptosis. RGFP966 effectively reduced brain edema and histological damage and enhanced neurological and cognitive function in rats with TBI. RGFP966 markedly inhibited NLRP3 inflammasome activation mediated by high-mobility group box 1 (HMGB1)/toll-like receptor 4 (TLR4). Nrf2 knockdown in TBI rats attenuated the AO and anti-inflammatory, neuroprotective impacts of RGFP966. Overall, our findings demonstrate that RGFP966 can mitigate the first brain damage and neurological impairments in TBI. The underlying mechanism involves triggering the Nrf2-mediated AO system and negatively regulating the HMGB1/TLR4-mediated NLRP3 inflammasome pathway.

Arzanol, a natural phloroglucinol α-pyrone, protects HaCaT keratinocytes against H2O2-induced oxidative stress, counteracting cytotoxicity, reactive oxygen species generation, apoptosis, and mitochondrial depolarization.

Skin oxidative stress results in structural damage, leading to premature senescence, and pathological conditions such as inflammation and cancer. The plant-derived prenylated pyrone-phloroglucinol heterodimer arzanol, isolated from Helichrysum italicum ssp. microphyllum (Willd.) Nyman aerial parts, exhibits anti-inflammatory, anticancer, antimicrobial, and antioxidant activities. This study explored the arzanol protection against hydrogen peroxide (H2O2) induced oxidative damage in HaCaT human keratinocytes in terms of its ability to counteract cytotoxicity, reactive oxygen species (ROS) generation, apoptosis, and mitochondrial membrane depolarization. Arzanol safety on HaCaT cells was preliminarily examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and microscopic observation. The arzanol pre-incubation (5-100μM, for 24 h) did not induce cytotoxicity and morphological alterations. The phloroglucinol, at 50μM, significantly protected keratinocytes against cytotoxicity induced by 2 h-incubation with 2.5 and 5 mM H2O2, decreased cell ROS production induced by 1 h-exposure to all tested H2O2 concentrations (0.5-5 mM), as determined by the 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) assay, and lipid peroxidation (thiobarbituric acid reactive substances [TBARS] method). The 2-h incubation of keratinocytes with H2O2 determined a significant increase of apoptotic cells versus control cells, evaluated by NucView® 488 assay, from the dose of 2.5 mM. Moreover, an evident mitochondrial membrane potential depolarization, monitored by fluorescent mitochondrial dye MitoView™ 633, was assessed at 5 mM H2O2. Arzanol pre-treatment (50μM) exerted a strong significant protective effect against apoptosis, preserving the mitochondrial membrane potential of HaCaT cells at the highest H2O2 concentrations. Our results validate arzanol as an antioxidant agent for the prevention/treatment of skin oxidative-related disorders, qualifying its potential use for cosmeceutical and pharmaceutical applications.

Synergistic role of thymoquinone and 5-fluorouracil in U-251MG glioblastoma cell line

Abstract Objectives Glioblastoma is a fast-growing and aggressive brain tumor. Despite the current treatment methods, such as chemical and surgical operations, the prognosis is still poor. Therefore, combined therapeutic strategies are proposed to maximize therapeutic efficacy and reduce toxicity. Thymoquinone has been shown to have neuroprotective effects in addition to its anti-cancer effects on different types of cancer. 5-Fluorouracil, on the other hand, is a cytotoxic chemotherapy agent used to treat cancer. As a synergistic combinational approach, this study aimed to examine the antiproliferative effects and production of reactive oxygen species in a glioblastoma cell line. Methods We have tested thymoquinone and 5-fluorouracil alone and in their combination to observe cellular growth with MTT assay. The combinational effects of the agents were determined by the CompuSYN software program. Cell proliferation was assayed with crystal violet assay. Reactive oxygen species production was analyzed by 2′,7′-dichlorodihydrofluorescein diacetate in glioblastoma cells. Results Thymoquinone and 5-fluorouracil inhibited cell growth of glioblastoma cells with half maximal inhibitory concentrations (IC50) of 45.93 and 14.02 µM for 48 h, respectively. At synergistic combinational concentrations, the crystal violet assay demonstrated that there is a positive correlation between combination index values and cell proliferation. Also, an increment in the production of reactive oxygen species was observed upon combinational treatments. Conclusions Our results indicate that the combinational strategy of these two agents reduced cell viability and proliferation in glioblastoma cells and showed strong synergistic anticancer efficiency.

The Role of β-Defensin 1 Against Porphyromonas gingivalis Lipopolysaccharide-Mediated Inflammation in the THP-1 Cell Line.

Introduction Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) is one of the crucial virulence factors of periodontitis. Antimicrobial peptides (AMPs) are emerging as alternatives or adjuncts to antibiotics in the treatment of microbial infections. In this study, cytotoxicity, anti-inflammatory activity, anti-oxidative stress, cell cycle analysis, and apoptosis properties of AMP, β-defensin 1, were studied in Pg-LPS-stimulated THP-1 (Tohoku Hospital Pediatrics - 1) cell line. Methods The cytotoxic nature of Pg-LPS and β-defensin 1 was studied by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. The cytotoxic effect of β-defensin 1 on Pg-LPS-stimulated THP-1 cells was also studied by the same method. The anti-inflammatory role of β-defensin 1 against cyclooxygenase (COX), lipoxygenase (LOX), myeloperoxidase (MPO), and inducible nitric oxide synthase activities were studied. The anti-oxidative nature of β-defensin 1 was analyzed by measuring reactive oxygen species (ROS) generation by dichlorodihydrofluorescein diacetate (DCFDA) assay. Cell cycle distribution and apoptosis were studied by flow cytometry. The hemolytic nature of β-defensin 1 was predicted using the HemoPred web tool. Results The results of the study demonstrated that Pg-LPS showed dose-dependent cytotoxicity to THP-1 cells. β-Defensin 1 had dose-dependent cytotoxicity to THP-1 cells and showed a protective effect on THP-cells up to 1 µg/mL of Pg-LPS, beyond which cell viability decreased. β-Defensin 1 inhibited COX, LOX, MPO, and inducible nitric oxide synthase activities in a concentration-dependent manner. β-Defensin 1 showed anti-oxidative activity by suppressing the generation of ROS measured through fluorescence intensity. From the cell cycle analysis, it was found that β-defensin 1 was able to reduce the Pg-LPS-induced cell cycle arrest at the G0/G1 phase. From the apoptosis profile, β-defensin 1 was found to increase the live cells when compared to THP-1 cells stimulated only with Pg-LPS, indicating that β-defensin 1 provided a protective role to THP-1 cells. β-Defensin 1 was found to be hemolytic in nature by the HemoPred web tool. Conclusion β-Defensin 1 exerted multifunctional activities and can be considered a promising agent for controlling periodontitis.

Ameliorative Effect of Ginsenoside Rg6 in Periodontal Tissue Inflammation and Recovering Damaged Alveolar Bone.

Periodontal disease is a chronic disease with a high prevalence, and in order to secure natural materials to prevent oral diseases, new materials that protect periodontal tissue from inflammation are being sought. Genes were identified using real-time quantitative polymerase chain reaction (RT-qPCR), and proteins were confirmed using Western blot. Dichlorodihydrofluorescein diacetate (DCF-DA) analysis was used, and the antibacterial effects were confirmed through Minimum Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) analysis. To confirm this effect in vivo, Sprague-Dawley rats, in which periodontitis was induced using ligation or Lipopolysaccharide of Porphyromonas gingivalis (PG-LPS), were used. In vitro experiments using human periodontal ligament (HPDL) cells stimulated with PG-LPS showed that Ginsenoside Rg6 (G-Rg6) had anti-inflammatory, antibacterial, antioxidant, and osteoblast differentiation properties. In vivo, G-Rg6 was effective in Sprague-Dawley rats in which periodontitis was induced using ligation or PG-LPS. Therefore, Ginsenoside Rg6 shows potential effectiveness in alleviating periodontitis.

Phytochemically analysed extract of Ageratina adenophora (Sprengel) R.M.King & H. Rob. initiates caspase 3-dependant apoptosis in colorectal cancer cell: A synergistic approach with chemotherapeutic drugs

Ethnopharmacological relevanceAgeratina adenophora (Sprengel) R.M.King & H.Rob. has been used as traditional indigenous medicine all across the globe for its diverse therapeutic applications such as anticancer, analgesic, antipyretic, thermogenic, antiseptic, antimicrobial as well as astringent. The various ethnic groups of India use plant parts to treat cuts and wounds, venomous insect bites, skin lesions, blisters, scabies and other skin irritations, gastritis and indigestion problems, cough, stomach ache and dysentery. The Portuguese traditionally extract the juice from the plant and use it for cancer, diabetes, liver disorder, gallbladder and stomach ailments. Nigerian healers use different parts of the plant to treat diabetes, fever and inflammation. Aim of the studyThe aim of this study is to investigate the cytotoxic potential of A. adenophora hydroalcoholic leaves extract (AHL) on Colorectal cancer (CRC) cell lines (HCT-116, HCT-15 and HT-29), synergistic potential with chemotherapeutic drugs 5FU and Cisplatin as well as reactive oxygen species (ROS) generation, based on the sample collected from Mao district of Manipur, India. Identification of bioactive phytocompounds in AHL was also performed by HRLCMS. MethodsThe AHL was evaluated for its cytotoxic as well as antiproliferative activities by 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide (MTT) assay, clonogenic and cell migration assays. The total phenolic content (TPC) and total flavonoid content (TFC) were quantified by Folin-ciocalteu and Aluminium chloride assays respectively. Caspase 3 activation was evaluated using Caspase-3 Assay Kit. Apoptosis detection by flow cytometry was carried out using annexin V-FITC/PI apoptosis detection kit. The apoptotic cells were also visualized by Giemsa and 4′,6-Diamidino-2-phenylindole (DAPI) staining. The intracellular Reactive oxygen species (ROS) generation was also evaluated using fluorescent probe 2′,7′-dichlorodihydrofluorescein di-acetate (H2DCFDA) in flow cytometry. The combination effects of AHL with chemotherapeutic drugs 5FU and Cisplatin were also evaluated. The identification of phytochemical constituents of AHL were analysed by HR-LCMS. ResultsThe AHL induced cytotoxic activity significantly in HCT-116 with IC50 of 65.65 ± 2.10 μg/mL, but non-cancerous cell HeK-293 was least cytotoxic. Colony formation and cell migration were inhibited in a dose and time dependent manner. The cell morphology upon AHL treatment was significantly altered with apoptotic features. The extract was rich in total phenolic (82.09 ± 0.35mgGAE/g) and total flavonoid (58.31 ± 0.55 mgQAE/g) contents. AHL induced apoptosis as detected by AnnexinV/PI, via activation of caspase 3 and elevated production of Reactive oxygen species (ROS). AHL in combination with 5FU and Cisplatin acts synergistically and potentiates the therapeutic properties of the extract. Sesquiterpenes, phenolic as well as flavonoid derivatives with anticancer properties were detected in AHL by HRLCMS, and these phytoconstituents may be attributed for anticancer property of AHL. ConclusionThe present study evaluates the effectiveness of AHL against Colorectal cancer cell lines. AHL is cytotoxic and induces apoptosis in HCT-116 cells by caspase 3 activation and increased ROS production that can be attributed to sesquiterpenoids. Thus, the plant A. adenophora has therapeutic potential for Colorectal cancer and can be further exploited for developing anticancer drug.

Carnosine supplementation and retinal oxidative parameters in a high-calorie diet rat model

BackgroundTo assess oxidative effects induced by a high-calorie diet on the retina of Wistar rats and test the antioxidative effects of carnosine supplementation.MethodsWistar rats were randomly divided into the following groups: standard diet (SD), high-calorie diet (HcD), standard diet + carnosine (SD + Car), and high-calorie diet + carnosine (HcD + Car). The body weight, adiposity index, plasma glucose, total lipids, high-density lipoprotein (HDL), low-density lipoprotein (LDL), uric acid, creatinine, and triglycerides of the animals were evaluated. The retinas were analyzed for markers of oxidative stress. Hydrogen peroxide production was assessed by 2',7'-dichlorodihydrofluorescein diacetate (DCF) oxidation. The total glutathione (tGSH), total antioxidant capacity (TAC), protein carbonyl, and sulfhydryl groups of the antioxidant system were analyzed.ResultsTAC levels increased in the retinas of the SD + Car group compared to the SD group (p < 0.05) and in the HcD + Car group compared to the HcD group (p < 0.05). The levels of GSH and the GSSH:GSSG ratio were increased in the HcD + Car group compared to the SD + Car group (p < 0.05). An increase in the retinal carbonyl content was observed in the HcD group compared to the SD group (p < 0.05) and in the HcD + Car group compared to the SD + Car group (p < 0.05). A high-calorie diet (HcD) was also associated with a decrease in retinal sulfhydryl-type levels compared to the SD group (p < 0.05).ConclusionThe results suggest that feeding a high-calorie diet to rats can promote an increase in carbonyl content and a reduction in sulfhydryl groups in their retinas. The administration of carnosine was not effective in attenuating these oxidative markers.Trial registrationAnimal Ethics Committee of Botucatu Medical School - Certificate number 1292/2019.

Ajmalicine induces the pyroptosis of hepatoma cells to exert the antitumor effect.

Ajmalicine (AJM) is an alkaloid extracted from the root of Yunan Rauvolfia verticillata. At present, little research has reported the antitumor pharmacological action and mechanism of AJM. Therefore, this work aimed to conduct relevant research. The mouse hepatoma cell line H22 was intervened with a gradient concentration of AJM. Subsequently, the pyroptosis level was detected by flow cytometry. The expression of inflammatory factors and lactate dehydrogenase was measured by enzyme-linked immunosorbent assay. Reactive oxygen species (ROS) expression was detected by dichlorodihydrofluorescein diacetate probe. In addition, the tumor-bearing model mice were also treated with AJM to analyze tumor growth as well as the expression levels of tissue inflammatory factors and proteins. According to our results, AJM promoted the pyroptosis of H22 cells, increased the pyroptosis rate, and upregulated the expression of inflammatory factors tumor necrosis factor α, interleukin-1β, and interleukin-6. At the same time, it enhanced the openness of membrane pores and increased the expression of ROS. Moreover, AJM promoted the expression of Caspase-3 and N-terminal gasdermin E (GSDME). The AJM-induced pyroptosis was suppressed after N-acetylcysteine treatment to inhibit ROS, while Caspase-3 knockdown also inhibited the AJM-induced pyroptosis. In animals, AJM suppressed tumor growth. AJM can activate ROS to induce pyroptosis and exert the antitumor effect via the noncanonical Caspase-3-GSDME pyroptosis pathway.

Assessment of anticancer properties of cumin seed (Cuminum cyminum) against bone cancer.

Early-life osteosarcoma is associated with severe morbidity and mortality, particularly affecting young children and adults. The present cancer treatment regimen is exceedingly costly, and medications like ifosfamide, doxorubicin, and cisplatin have unneeded negative effects on the body. With the introduction of hyphenated technology to create medications based on plant molecules, the application of ayurvedic medicine as a new dimension (formulation, active ingredients, and nanoparticles) in the modern period is rapidly growing. The primary source of lead compounds for the development of medications for avariety of ailments is plants and their products. Traditionally, Cuminum cyminum (cumin) has been used as medication to treat a variety of illnesses and conditions. The cumin seed was successfully extracted with solvents Hexane, Chloroform, Methanol, Ethanol and Acetone. Following the solvent extraction, the extract residue was assayed in MG63 cells for their anti-proliferative properties. First, we used the [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] (MTT) assay to test the extracted residue's cytotoxicity. The results show that hexane extract Half-maximal inhibitory concentration (IC50 86 µG/mL) effciently inhibits cells by causing programmed cell death. Furthermore, using the Acridine orange/ethidium bromide (AO/EB) staining method, the lactate dehydrogenase assay, and the reactive oxygen species assay using the Dichloro-dihydro-fluorescein diacetate (DCHFDA) staining method, we have demonstrated that the hexane extract causes apoptosis in MG63 cells. Furthermore, flow cytometry research revealed that the hexane extract stops the cell cycle in the S phase. In addition, the hexane extract limits colony formation and the migration potential as shown by the scratch wound healing assay. Furthermore, the extract from cumin seeds exhibits remarkable bactericidal properties against infections that are resistant to drugs. Gas chromatography analysis was used to quantitatively determine the hexane and methanolic extract based on the experimental data. The primary chemical components of the extract are revealed by the study, and these help the malignant cells heal. The present study finds that there is scientific validity in using cumin seeds as a novel method of anticancer therapy after undergoing both intrinsic and extrinsic research.

PDCD4 silencing alleviates KA‑induced neurotoxicity of HT22 cells by inhibiting endoplasmic reticulum stress via blocking the MAPK/NF‑κB signaling pathway.

Human programmed cell death 4 (PDCD4) has been reported to participate in multiple neurological diseases. However, the role of PDCD4 in epilepsy, as well as its underlying mechanism, remains unclear. To induce excitotoxicity, 100 µM kainic acid (KA) was applied for the stimulation of HT22 cells for 12 h. Initially, the mRNA and protein expression levels of PDCD4 were evaluated using reverse transcription-quantitative PCR and western blotting. A lactate dehydrogenase assay was performed to detect cell injury. Cell apoptosis was assessed using flow cytometry and western blotting was performed to determine the expression levels of apoptosis-related proteins. Oxidative stress was detected using dichlorodihydrofluorescein diacetate staining, and malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) assay kits. Furthermore, the expression levels of MAPK/NF-κB signaling-related proteins and endoplasmic reticulum (ER) stress-related proteins C/EBP homologous protein, glucose-regulated protein 78, activating transcription factor 4 and phosphorylated-eukaryotic initiation factor-2α were assessed by western blotting. It was revealed that PDCD4 expression was markedly elevated in KA-induced HT22 cells, whereas PDCD4 silencing alleviated KA-induced neurotoxicity of HT22 cells by alleviating cell injury and inhibiting apoptosis. In addition, PDCD4 silencing reduced the levels of reactive oxygen species and MDA, but elevated those of SOD and GSH-Px. PDCD4 silencing also suppressed ER stress by blocking the MAPK/NF-κB signaling pathway. By contrast, the MAPK agonist phorbol myristate acetate reversed the effects of PDCD4 silencing on KA-induced neurotoxicity and oxidative stress in HT22 cells. In conclusion, PDCD4 silencing alleviated KA-induced neurotoxicity and oxidative stress in HT22 cells by suppressing ER stress through the inhibition of the MAPK/NF-κB signaling pathway, which may provide novel insights into the treatment of epilepsy.

Red ginseng polysaccharide promotes ferroptosis in gastric cancer cells by inhibiting PI3K/Akt pathway through down-regulation of AQP3

ABSTRACT Objective This study aims to investigate the effect of red ginseng polysaccharide (RGP) on gastric cancer (GC) development and explore its mechanism. Methods GC cell lines AGS were treated with varying concentrations of RGP (50, 100, and 200 μg/mL). AGS cells treated with 200 μg/mL RGP were transfected with aquaporin 3 (AQP3) overexpression vector. Cell proliferation, viability, and apoptosis were evaluated by MTT, colony formation assay, and flow cytometry, respectively. Real-time quantitative reverse transcription PCR (qRT-PCR) was used to detect the expression of AQP3. The levels of Fe2+, malondialdehyde, and lactate dehydrogenase were measured using their respective detection kits, and the reactive oxygen species levels was determined by probe 2’,7’-dichlorodihydrofluorescein diacetate. The expression of ferroptosis-related protein and PI3K/Akt pathway-related protein were assessed by western blot. In vivo experiments in nude mice were performed and the mice were divided into four groups (n = 5/group) which gavage administrated with 150 mg/kg normal saline, and 75, 150, 300 mg/kg RGP, respectively. Their tumor weight and volume were recorded. Results RGP treatment effectively inhibited the proliferation and viability of AGS cells in a dosage-dependent manner and induced apoptosis. It induced ferroptosis in AGS cells, as well as inhibiting the expression of PI3K/Akt-related proteins. AQP3 overexpression could reversed the effect of RGP treatment on ferroptosis. Confirmatory in vivo experiments showed that RGP could reduce the growth of implanted tumor, with increased RGP concentration resulting in greater tumor inhibitory effects. Conclusion RGP might have therapeutic potential against GC, effectively inhibiting the proliferation and viability of AGS cells.

Stimulation of Hemolysis and Eryptosis by β-Caryophyllene Oxide.

Eryptosis stimulated by anticancer drugs can lead to anemia in patients. β-caryophyllene oxide (CPO) is an anticancer sesquiterpene present in various plants; however, its effect on the structure and function of human red blood cells (RBCs) remains unexplored. The aim of this study was to investigate the hemolytic and eryptotic activities and underlying molecular mechanisms of CPO in human RBCs. Cells were treated with 10-100 μM of CPO for 24 h at 37 °C, and hemolysis, LDH, AST, and AChE activities were photometrically assayed. Flow cytometry was employed to determine changes in cell volume from FSC, phosphatidylserine (PS) externalization by annexin-V-FITC, intracellular calcium by Fluo4/AM, and oxidative stress by 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA). Cells were also cotreated with CPO and specific signaling inhibitors and antihemolytic agents. Furthermore, whole blood was exposed to CPO to assess its toxicity to other peripheral blood cells. CPO induced concentration-responsive hemolysis with LDH and AST leakage, in addition to PS exposure, cell shrinkage, Ca2+ accumulation, oxidative stress, and reduced AChE activity. The toxicity of CPO was ameliorated by D4476, staurosporin, and necrosulfonamide. ATP and PEG 8000 protected the cells from hemolysis, while urea and isotonic sucrose had opposite effects. CPO stimulates hemolysis and eryptosis through energy depletion, Ca2+ buildup, oxidative stress, and the signaling mediators casein kinase 1α, protein kinase C, and mixed lineage kinase domain-like pseudokinase. Development of CPO as an anticancer therapeutic must be approached with prudence to mitigate adverse effects on RBCs using eryptosis inhibitors, Ca2+ channel blockers, and antioxidants.

Evaluating the antioxidant potential of resveratrol-gold nanoparticles in preventing oxidative stress in endothelium on a chip

Vascular endothelial cells play a vital role in the health and maintenance of vascular homeostasis, but hyperglycemia disrupts their function by increasing cellular oxidative stress. Resveratrol, a plant polyphenol, possesses antioxidant properties that can mitigate oxidative stress. Addressing the challenges of its limited solubility and stability, gold nanoparticles (GNps) were utilized as carriers. A microfluidic chip (MFC) with dynamic flow conditions was designed to simulate body vessels and to investigate the antioxidant properties of resveratrol gold nanoparticles (RGNps), citrate gold nanoparticles (CGNps), and free Resveratrol on human umbilical vein endothelial cells (HUVEC). The 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay was employed to measure the extracellular antioxidant potential, and cell viability was determined using the Alamar Blue test. For assessing intracellular oxidative stress, the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) assay was conducted, and results from both the cell culture plate and MFC were compared. Free Resveratrol demonstrated peak DPPH scavenging activity but had a cell viability of about 24–35%. RGNPs, both 3.0 ± 0.5 nm and 20.2 ± 4.7 nm, consistently showed high cell viability (more than about 90%) across tested concentrations. Notably, RGNPs (20 nm) exhibited antioxidative properties through DPPH scavenging activity (%) in the range of approximately 38–86% which was greater than that of CGNps at about 21–32%. In the MFC,the DCFH-DA analysis indicated that RGNPs (20 nm) reduced cellular oxidative stress by 57–82%, surpassing both CGNps and free Resveratrol. Morphologically, cells in the MFC presented superior structure compared to those in traditional cell culture plates, and the induction of hyperglycemia successfully led to the formation of multinucleated variant endothelial cells (MVECs). The MFC provides a distinct advantage in observing cell morphology and inducing endothelial cell dysfunction. RGNps have demonstrated significant potential in alleviating oxidative stress and preventing endothelial cell disorders.

Network pharmacology analysis and experimental validation of Anemarrhenae Rhizoma in treating Alzheimer's disease.

To explore the mechanism of Anemarrhenae Rhizoma in treatment of Alzheimer's Disease (AD). The active ingredients and targets of Anemarrhenae Rhizoma for treatment of AD were screened with network pharmacology methods, the protein-protein interaction (PPI) network was constructed and the core targets were analyzed. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriching analysis was performed. The peripheral blood lymphocytes were extracted and lymphoblastoid cell lines (LCL) were constructed and an in vitro cell model of LCL-SKNMC was established. MTT and CCK-8 methods were used to quantify SKNMC/LCL cells, 2 ´, 7 ´-dichlorodihydrofluorescein diacetate (DCFH-DA) probe was used to detect reactive oxygen species (ROS), and immunofluorescence staining was used to detect the generation of Aβ1-42 in a co-cultured model. Western blotting was used to detect protein expression in the co-culture model. The lifespan of N2 nematodes was observed under oxidative stress, normal state, and heat stress; ROS generated by N2 nematodes was detected by DCFH-DA probes. The paralysis time of CL4176 N2 nematodes was evaluated by paralysis assay, and Aβ deposition in the pharynx was detected by Thioflavin S staining. Through network pharmacology, 15 potential active ingredients and 103 drug-disease targets were identified. PPI analysis showed that the Anemarrhenae Rhizoma might play anti-AD roles through albumin, Akt1, tumor necrosis factor, epidermal growth factor receptor (EGFR), vascular endothelial growth factor A (VEGFA), mammalian target of rapamycin (mTOR), amyloid precursor protein (APP) and other related targets. KEGG analysis showed that the pharmacological effects of Anemarrhenae Rhizoma might involve the biological processes of Alzheimer's disease, endocrine resistance, insulin resistance; and neuroactive ligand-receptor interaction, phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, calcium signaling pathway, AGE-RAGE signaling pathway in diabetes complications, neurotrophic factor signaling pathway and others. The in vitro cell experiments showed that Anemarrhenae Rhizoma was able to reduce the production of ROS and Aβ1-42 (both P<0.01), inhibit the expression of β-secretase 1 (BACE1), APP and Aβ1-42 proteins (all P<0.05), up-regulate the expression of p-PI3K/PI3K, p-AKT/AKT, p-GSK3β/GSK3β in SKNMC cells (all P<0.05). The in vivo studies further confirmed that Anemarrhenae Rhizoma prolonged the lifespan of C. elegans under stress and normal conditions, reduced the accumulation of ROS and the toxicity of Aβ deposition. Anemarrhenae Rhizoma may reduce the production of Aβ in AD and inhibit its induced oxidative stress, which may be achieved by regulating the PI3K/Akt/GSK-3β pathway.

Selection of spermatozoa with high motility and quality from bovine frozen-thawed semen using the centrifuge-free device

This study aimed to assess the potential of the centrifuge-free commercial device (MIGLIS®) in selecting functional frozen-thawed bovine sperm by migration-sedimentation, its effect on embryo development, and compare the potential with that of centrifugation-based techniques, including washing and Percoll density gradient centrifugation (DGC). In experiment 1, different dilutions (1.5×, 2×, and 3×) of frozen-thawed spermatozoa were assessed to identify the adequate one for the MIGLIS method. In experiment 2, the recovery rates, quality, and reactive oxygen species (ROS) concentrations of the spermatozoa selected using MIGLIS, washing, and Percoll DGC were compared. In experiment 3, the resultant in vitro fertilised embryos from spermatozoa selected using the three methods were evaluated for blastocyst formation rates and intracellular ROS concentrations at the 2–4 cell stage. The intracellular ROS concentrations were investigated using 2′, 7′-dichlorodihydrofluorescein diacetate staining. Using the MIGLIS device, significantly more spermatozoa were recovered at 2× dilution compared with the other dilution ratio, but the motility was not affected by the dilution ratio. On the selection of spermatozoa using the three methods, employing MIGLIS decreased the recovery rates. However, the MIGLIS method increased motility, viability, and acrosome integrity rates compared to those in spermatozoa from the other methods. The ROS concentration of spermatozoa in the MIGLIS method was significantly lower than that in the washing method. Nevertheless, blastocyst formation rates were similar among the three methods, but the ROS concentration of early-stage embryos produced using MIGLIS was significantly lower than those produced using Percoll DGC. In conclusion, the MIGLIS method has the potential to select functional, high-quality frozen-thawed bovine spermatozoa.

Resveratrol Reduces ROS by Increasing GSH in Vitrified Sheep Embryos

The in vitro production and cryopreservation of mammalian embryos generates reactive oxygen species (ROS) due to conditions of the system that can overcome their antioxidant protection. Resveratrol is an antioxidant used in in vitro systems to improve blastocyst rates, but its effect on antioxidant enzymes such as glutathione (GSH) in embryos produced by in vitro fertilization (IVF) after vitrification has not been reported. The objective of this study was to evaluate the effects of resveratrol on the in vitro maturation medium (IVM) of sheep oocytes (Ovis aries) on the levels of ROS and GSH in embryos produced by IVF subjected to vitrification. Resveratrol was added at 0 µM, 0.25 µM, 0.5 µM, and 1 µM during oocyte in vitro maturation (IVM). Matured oocytes were fertilized with thawed ram sperm. Embryos were cultured in sequential media until blastocysts, were then vitrified for 24 h, and, after heating, they were stained with DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate) to determine the presence of ROS and with Cell Tracker Blue® for the presence of GSH. The quantitative values of ROS and GSH were obtained through the Image J image processor. The results showed that resveratrol increased GSH and decreased ROS production (p &lt; 0.05) in a dose-dependent manner. It is concluded that its use in sheep oocytes during IVM has a beneficial effect on embryos produced by IVF subjected to vitrification by decreasing reactive oxygen species levels and facilitating the generation of embryo antioxidant enzymes like glutathione.

Echinococcus granulosus cyst fluid inhibits KDM6B-mediated demethylation of trimethylated histone H3 lysine 27 and interleukin-1β production in macrophages

BackgroundEchinococcus granulosus can manipulate its host's immune response to ensure its own survival. However, the effect of histone modifications on the regulation of the NOD-like receptor protein 3 (NLRP3) inflammasome and downstream interleukin-1β (IL-1β) production in response to the parasite is not fully understood.MethodsWe evaluated IL-1β secretion through enzyme-linked immunosorbent assay and assessed reactive oxygen species levels using the dichlorodihydrofluorescein diacetate probe. Western blotting and quantitative real-time polymerase chain reaction were performed to examine the expression of NLRP3 and IL-1β in mouse peritoneal macrophages and Tohoku Hospital Pediatrics-1 cells, a human macrophage cell line. The presence of trimethylated histone H3 lysine 27 (H3K27me3) modification on NLRP3 and IL-1β promoters was studied by chromatin immunoprecipitation.ResultsTreatment with E. granulosus cyst fluid (EgCF) considerably reduced IL-1β secretion in mouse and human macrophages, although reactive oxygen species production increased. EgCF also suppressed the expression of NLRP3 and IL-1β. Mechanistically, EgCF prompted the enrichment of repressive H3K27me3 modification on the promoters of both NLRP3 and IL-1β in macrophages. Notably, the presence of EgCF led to a significant reduction in the expression of the H3K27me3 demethylase KDM6B.ConclusionsOur study revealed that EgCF inhibits KDM6B expression and H3K27me3 demethylation, resulting in the transcriptional inhibition of NLRP3 and IL-1β. These results provide new insights into the immune evasion mechanisms of E. granulosus.Graphical

Hematoporphyrin derivative photodynamic therapy induces apoptosis and suppresses the migration of human esophageal squamous cell carcinoma cells by regulating the PI3K/AKT/mTOR signaling pathway.

Esophageal cancer is one of the most common cancer types in humans worldwide. Photodynamic therapy (PDT) is a promising therapeutic strategy for the treatment of cancer. However, its underlying mechanism needs to be studied thoroughly. The present study focused on the antitumor effect and underlying mechanism of the use of hematoporphyrin derivative (HpD)-PDT against human esophageal squamous cell carcinoma cells via regulation of the PI3K/AKT/mTOR signaling pathway. A Cell Counting Kit-8 assay was used to measure cell viability. Migration was evaluated using a wound healing assay. An annexin V-FITC/PI kit was used to determine cell apoptosis rates. Protein expression levels were analyzed via western blotting. Reverse transcription-quantitative PCR was used to detect gene expression levels. A 2',7'-dichlorodihydrofluorescein diacetate kit was chosen to evaluate intracellular reactive oxygen species levels via flow cytometry. Cell viability and migration were decreased in KYSE-150 cells after HpD-PDT treatment. Cellular apoptosis was induced after HpD-PDT treatment, and the same trend was observed for autophagy. Furthermore, the PI3K/AKT/mTOR signaling pathway was inhibited. The viability and migration of KYSE-150 cells were significantly inhibited, and apoptosis was induced more effectively following treatment with a combination of HpD-PDT and the PI3K inhibitor, a final concentration of 20 µM LY294002. In conclusion, HpD-PDT could suppress esophageal cancer cell viability, induce apoptosis and inhibit migration by downregulating the PI3K/AKT/mTOR signaling pathway. Combination of HpD-PDT with PI3K inhibitor (LY294002) could enhance the therapeutic efficacy compared with that demonstrated by HpD-PDT alone. Further studies on combination therapy are required to achieve improved clinical outcomes.

Vasculoprotective Potential of Baicalein in Angiotensin II-Infused Abdominal Aortic Aneurysms through Inhibiting Inflammation and Oxidative Stress.

Aortic wall inflammation, abnormal oxidative stress and progressive degradation of extracellular matrix proteins are the main characteristics of abdominal aortic aneurysms (AAAs). The nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome dysregulation plays a crucial role in aortic damage and disease progression. The first aim of this study was to examine the effect of baicalein (5,6,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one) on AAA formation in apolipoprotein E-deficient (ApoE-/-) mice. The second aim was to define whether baicalein attenuates aberrant vascular smooth muscle cell (VSMC) proliferation and inflammation in VSMC culture. For male ApoE-/- mice, a clinically relevant AAA model was randomly divided into four groups: saline infusion, baicalein intraperitoneal injection, Angiotensin II (Ang II) infusion and Ang II + baicalein. Twenty-seven days of treatment with baicalein markedly decreased Ang II-infused AAA incidence and aortic diameter, reduced collagen-fiber formation, preserved elastic structure and density and prevented smooth muscle cell contractile protein degradation. Baicalein inhibited rat VSMC proliferation and migration following the stimulation of VSMC cultures with Ang II while blocking the Ang II-inducible cell cycle progression from G0/G1 to the S phase in the synchronized cells. Cal-520 AM staining showed that baicalein decreased cellular calcium in Ang II-induced VSMCs; furthermore, a Western blot assay indicated that baicalein inhibited the expression of PCNA and significantly lowered levels of phospho-Akt and phospho-ERK, along with an increase in baicalein concentration in Ang II-induced VSMCs. Immunofluorescence staining showed that baicalein pretreatment reduced NF-κB nuclear translocation in Ang II-induced VSMCs and furthered the protein expressions of NLRP3 while ASC and caspase-1 were suppressed in a dose-dependent manner. Baicalein pretreatment upregulated Nrf2/HO-1 signaling in Ang II-induced VSMCs. Thus, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining showed that its reactive oxygen species (ROS) production decreased, along with the baicalein pretreatment. Our overall results indicate that baicalein could have therapeutic potential in preventing aneurysm development.