Transcription Factor Assay Research Articles

Exploring aptamer-aTF sandwich and CRISPR-Cas12a methods for sensitive L-lactate biosensing in human serum and saliva

In this study, a novel transcription factor and aptamer-based sandwich assay is proposed to detect L-lactate in biofluids with excellent selectivity and sensitivity. The assay employs an L-lactate-specific allosteric transcription factor (aTF) fused with a cellulose-binding domain (CBD) and immobilized on cellulose nanocrystals (CNCs). The aTF-CBD/L-lactate complex is coupled with a fluorescently labeled aptamer (fAPTlac), generating a quantifiable fluorescence signal. Remarkably, the proposed assay demonstrated a dynamic detection range from 1 nM to 50 mM, a limit of detection of 28.2 pM, and high selectivity against potentially interfering molecules. Moreover, the proposed method exhibited better performance compared to the CRISPR-Cas12a/aTF-based (CaT) method without compromising the sensitivity or specificity. Furthermore, the assay accurately quantified L-lactate in human serum and saliva samples, with recovery rates between 98 % and 104 %. The proposed method offers a simple, cost-effective, and highly sensitive platform for L-lactate detection in clinical settings, addressing the limitations of existing biosensors for small molecule detection such as matrix interference, low sensitivity, and poor selectivity.

Radiosensitizing effects of CDK4/6 inhibitors in hormone receptor-positive and HER2-negative breast cancer mediated downregulation of DNA repair mechanism and NF-κB-signaling pathway

CDK4/6 inhibitors combined with endocrine therapy prolonged survival in hormone receptor (HR)-positive and HER2-negative advanced breast cancer. We investigated whether CDK4/6 inhibitors enhance radiosensitivity and their underlying mechanisms of this subtype of breast cancer. In vitro and in vivo experiments were conducted using two HR-positive and HER2-negative breast cancer cell lines (MCF-7 and T-47D), CDK4/6 inhibitors (ribociclib and palbociclib) and radiotherapy (RT) to assess the biological functions and mechanisms. The radiation-enhancing effect was assessed using clonogenic assays; γH2AX and 53BP1 levels were assessed by immunofluorescence to evaluate DNA damage. The levels of phospho (p)-ERK, c-Myc, and DNA-double strand break (DSB)-related molecules, p-DNA-PKcs, Rad51, and p-ATM, were assessed by western blotting. We used an NF-κB p65 transcription factor assay kit to evaluate NF-κB activity. We evaluated the antitumor effect of the combination of RT and ribociclib through the MCF-7 orthotopic xenograft model. The synergistic effects of combining RT with ribociclib and palbociclib pretreatment were demonstrated by clonogenic assay. CDK4/6 inhibitors synergistically increased the numbers of RT-induced γH2AX and 53BP1, downregulated the expression of p-DNA-PKcs, Rad51 and p-ATM activated by RT, and reduced RT-triggering p-ERK expression, NF-κB activation, and its down-streaming gene, c-Myc. Combined ribociclib and RT reduced the growth of MCF-7 cell xenograft tumors, and downregulated the immunohistochemical expression of p-ERK, p-NF-κB p65, and c-Myc compared to that in the control group. Combining CDK4/6 inhibitors enhanced radiosensitivity of HR-positive and HER2-negative breast cancer cells at least by reducing DNA-DSB repair and weakening the activation of ERK and NF-κB signaling by RT.

A Novel Analog of the Natural Product Fraxinellone Protects against Endogenous and Exogenous Neurotoxicants.

Numerous insults, both endogenous (e.g., glutamate) and exogenous (e.g., pesticides), compromise the function of the nervous system and pose risk factors for damage or later disease. In previous reports, limonoids such as fraxinellone showed significant neuroprotective activity against glutamate (Glu) excitotoxicity and reactive oxygen species (ROS) production in vitro, albeit with minimal mechanistic information provided. Given these findings, a library of novel fraxinellone analogs (including analogs 1 and 2 described here) was synthesized with the goal of identifying compounds exhibiting neuroprotection against insults. Analog 2 was found to be protective against Glu-mediated excitotoxicity with a measured EC50 of 44 and 39 nM for in vitro assays using PC12 and SH-SY5Y cells, respectively. Pretreatment with analog 2 yielded rapid induction of antioxidant genes, namely, Gpx4, Sod1, and Nqo1, as measured via qPCR. Analog 2 mitigated Glu-mediated ROS. Cytoprotection could be replicated using sulforaphane (SFN), a Nrf2 activator, and inhibited via ML-385, which inhibits Nrf2 binding to regulatory DNA sequences, thereby blocking downstream gene expression. Nrf2 DNA-binding activity was demonstrated using a Nrf2 ELISA-based transcription factor assay. In addition, we found that pretreatment with the thiol N-acetyl Cys completely mitigated SFN-mediated induction of antioxidant genes but had no effect on the activity of analog 2, suggesting thiol modification is not critical for its mechanism of action. In summary, our data demonstrate a fraxinellone analog to be a novel, potent, and rapid activator of the Nrf2-mediated antioxidant defense system, providing robust protection against insults.

Design, synthesis, molecular dynamic simulation, DFT analysis, computational pharmacology and decoding the antidiabetic molecular mechanism of sulphonamide-thiazolidin-4-one hybrids

In search of novel antidiabetic agents, the study was framed to hybridize and synthesize molecules based on scaffolds of benzenesulfonamide and thiazolidine-4-one, efficiently synthesized (6a-6h) and structurally characterized by FTIR, 1H NMR 13C NMR and HRMS spectroscopy. The antidiabetic activity was assessed based on α-amylase inhibition, glucose uptake assay and PPAR-γ transcription factor assay. Compound 6b (4-(2-((E)-5-((Z)-2-hydroxybenzylidene)-4-oxo-2-(phenylimino) thiazolidin-3-yl) ethyl) benzenesulfonamide) demonstrated promising activity toward α-amylase inhibition having IC50 value of 29.51±1.35 µg/ml. A glucose uptake assay was performed for compounds (6a-6h) to assess the capacity of glucose uptake. Among them, Compound 6b exhibits notable glucose uptake with 88.71% in the yeast cell line. To support the glucose uptake findings of compound 6b, expression of PPAR-γ transcription factor was measured of compound 6b as compared to pioglitazone. Furthermore, induced fit docking was carried out to predict the refined pose of synthesized compounds in the α-amylase binding pocket. MD simulation studies confirmed the stability of compound 6b in the α-amylase binding pocket. Molecules were geometrically optimized by DFT theory using B3LYP 6–31 G (d, p**) basis set, and electronic properties were studied. Network pharmacology analysis for compound 6b identified key pathways involved in T2DM and postprandial hyperglycemia like PI3K-Akt pathway, Type II diabetes mellitus pathway metabolic pathway, and digestion of dietary carbohydrates pathway. These findings provide better insights into further developing benzenesulfonamide-based thiazolidin-4-one hybrids as antidiabetic agents.

Anti-psoriasis effect of 18β-glycyrrhetinic acid by breaking CCL20/CCR6 axis through its vital active group targeting GUSB/ATF2 signaling

BackgroundPsoriasis is an immune-mediated chronic inflammatory skin disease. Current research suggests that the long-term persistence and recurrence of psoriasis are closely related to the feedback loop formed between keratinocytes and immune cells, especially in Th 17 or DC cells expressing CCR6. CCL20 is the ligand of CCR6. Therefore, drugs that block the expression of CCL20 or CCR6 may have a certain therapeutic effect on psoriasis. Glycyrrhetinic acid (GA) is the main active ingredient of the plant drug licorice and is often used to treat autoimmune diseases, including psoriasis. However, its mechanism of action is still unclear. MethodsPsoriasis like skin lesion model was established by continuously applying imiquimod on the back skin of normal mice and CCR6-/- mice for 7 days. The therapeutic and preventive effects of glycyrrhetinic acid (GA) on the model were observed and compared. The severity of skin injury is estimated through clinical PASI scores and histopathological examination. qRT-PCR and multiple cytoline assay were explored to detect the expression levels of cytokines in animal dorsal skin lesions and keratinocyte line HaCaT cells, respectively. The dermis and epidermis of the mouse back were separated for the detection of CCL20 expression. Transcription factor assay was applied to screen, and luciferase activity assay to validate transcription factors regulated by GA. Technology of surface plasmon laser resonance with LC-MS (SPR-MS), molecular docking, and enzyme activity assay were used to identified the target proteins for GA. Finally, we synthesized different derivatives of 18beta-GA and compared their effects, as well as glycyrrhetinic acid (GL), on the skin lesion of imiquimod-induced mice to evaluate the active groups of 18beta-GA. Results18β-glycyrrhetinic acid (GA) improved IMQ-induced psoriatic lesions, and could specifically reduce the chemokine CCL20 level of the epidermis in lesion area, especially in therapeutic administration manner. The process was mainly regulated by transcription factor ATF2 in the keratinocytes. In addition, GUSB was identified as the primary target of 18βGA. Our findings indicated that the subject on molecular target research of glycyrrhizin should be glycyrrhetinic acid (GA) instead of glycyrrhizic acid (GL), because GL showed little activity in vitro or in vivo. Apart from that, α, β, -unsaturated carbonyl in C11/12 positions was crucial or unchangeable to its activity of 18βGA, while proper modification of C3 or C30 position of 18βGA may vastly increase its activity. ConclusionOur research indicates that 18βGA exerted its anti-psoriasis effect mainly by suppressing ATF2 and downstream molecule CCL20 predominately through α, β, -unsaturated carbonyl at C11/12 position binding to GUSB in the keratinocytes, and then broke the feedback loop between keratinocytes and CCR6-expressing immune cells. GA has more advantages than GL in the external treatment of psoriasis. A highlight of this study is to investigate the influence of special active groups on the pharmacological action of a natural product, inspired by the molecular docking result.

β-D-Glucan promotes NF-κB activation and ameliorates high-LET carbon-ion irradiation-induced human umbilical vein endothelial cell injury.

Heavy-ion irradiation seriously perturbs cellular homeostasis and thus damages cells. Vascular endothelial cells (ECs) play an important role in the pathological process of radiation damage. Protecting ECs from heavy-ion radiation is of great significance in the radioprotection of normal tissues. In this study, the radioprotective effect of β-D-glucan (BG) derived from Saccharomyces cerevisiae on human umbilical vein endothelial cell (EA.hy926) cytotoxicity produced by carbon-ion irradiation was examined and the probable mechanism was established. EA.hy926 cells were divided into seven groups: a control group; 1, 2, or 4 Gy radiation; and 10 μg/mL BG pretreatment for 24 h before 1, 2, or 4 Gy irradiation. Cell survival was assessed by colony formation assay. Cell cycles, apoptosis, DNA damage, and reactive oxygen species (ROS) levels were measured through flow cytometry. The level of malondialdehyde and antioxidant enzyme activities were analyzed using assay kits. The activation of NF-κB was analyzed using western blotting and a transcription factor assay kit. The expression of downstream target genes was detected by western blotting. BG pretreatment significantly increased the survival of irradiated cells, improved cell cycle progression, and decreased DNA damage and apoptosis. The levels of ROS and malondialdehyde were also decreased by BG. Further study indicated that BG increased the antioxidant enzyme activities, activated Src, and promoted NF-κB activation, especially for the p65, p50, and RelB subunits. The activated NF-κB upregulated the expression of antioxidant protein MnSOD, DNA damage-response and repair-related proteins BRCA2 and Hsp90α, and antiapoptotic protein Bcl-2. Our results demonstrated that BG protects EA.hy926 cells from high linear-energy-transfer carbon-ion irradiation damage through the upregulation of prosurvival signaling triggered by the interaction of BG with its receptor. This confirms that BG is a promising radioprotective agent for heavy-ion exposure.

Radiosensitizing Effects of CDK4/6 Inhibitors in Hormone Receptor-Positive and HER2-Negative Breast Cancer are through Downregulating DNA Repair Mechanism and NF-κB-Signaling Pathway

CDK4/6 inhibitors combined with endocrine therapy are demonstrated to prolong survival for hormone receptor (HR)-positive and HER2-negative advanced breast cancer. We sought to assess whether cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitors can enhance the radiosensitivity and their underlying mechanisms in this subtype of breast cancer through both in vitro and in vivo approaches. We used MCF-7 and T-47D (HR-positive and HER2-negative) breast cancer cell lines, and different doses of CDK4/6 inhibitors (ribociclib and palbociclib) and radiotherapy (RT) to assess the biological functions and mechanisms through in vitro study. The radiation enhancing effect was assessed by clonogenic assay, numbers of the DNA damage response-related molecules, γH2AX and 53BP1, were assessed by immunofluorescence. The p-ERK, c-Myc, and DNA-double strand break (DNA-DSB)-related molecules, p-DNA-PKcs, and p-ATM, were assessed by western blotting. We used NF-κB p65 Transcription Factor Assay Kit to evaluate the transcriptional activity of NF-κB. We evaluated the antitumor effect of combined RT (2 Gy every other day for total 6 Gy) and ribociclib through the MCF-7 orthotopic xenograft model. The synergistic effects of combining RT with different concentrations of ribociclib and palbociclib pretreatment were demonstrated by colonogenic assay. We revealed that CDK4/6 inhibitors synergistically increased the numbers of RT-induced γH2AX and 53BP1, downregulated the expression of p-DNA-PKcs and p-ATM activated by RT, and diminished the expression of RT-triggering p-ERK expression, and NF-κB activation and its down-streaming gene, c-Myc. When compared with control, ribociclib, and RT group, combined ribociclib and RT significantly shrunk the tumor growth of MCF-7 cell xenograft tumors, and downregulated the immunohistochemical expression of p-ERK, p-NF-κB p65, and c-Myc than control group. Our results demonstrated that combining CDK4/6 inhibitors enhanced the radiosensitivity in HR-positive and HER2-negative breast cancer cells, and conceivably radioenhancing mechanisms of CDK4/6 inhibitors are at least through lessening DNA-DSB repairing processes and weakening the activation of ERK signaling and NF-κB signaling by RT.

The MyD88 inhibitor, ST2825, induces cell cycle arrest and apoptosis by suppressing the activation of the NF‑κB/AKT1/p21 pathway in pancreatic cancer.

NF‑κB activation occurs in the majority patients with pancreatic ductal adenocarcinoma (PDAC); however, directly targeting NF‑κB has proven unsuccessful, and recent studies have demonstrated a certain effect of the indirect inhibition of NF‑κB. Myeloid differentiation factor 88 (MyD88) is a common intermediate messenger for NF‑κB activation by inducers. In the present study, the level of MyD88 in PDAC was detected using a public database and a tissue chip. A specific inhibitor (ST2825) of MyD88 was used on PDAC cell lines. Flow cytometry was used to examine apoptosis and cell cycle progression. Transcriptome sequencing was used for ST2825‑treated PANC‑1 cells compared with untreated PANC‑1 cells. The levels of related factors were measured using reverse transcription‑quantitative PCR and western blot analysis. Chromatin immunoprecipitation, co‑immunoprecipitation, transcription factor assay and an NF‑κB phospho‑antibody array were performed to identify the detailed underlying mechanisms. Animal experiments were performed to verify the effects of ST2825 on PDAC, which were found in the invitro experiments. MyD88 was found to be overexpressed in PDAC. ST2825 induced the G2/M phase cell cycle arrest and apoptosis of PDAC cells. ST2825 inhibited MyD88 dimerization to inactivate the NF‑κB pathway. ST2825 inhibited AKT1 expression and induced p21 overexpression to induce G2/M phase cell cycle arrest and apoptosis by inhibiting NF‑κB transcriptional activity. NF‑κB activation, AKT1 overexpression or p21 knockdown partially reversed the effects of ST2825 in PDAC. On the whole, the findings of the present study demonstrate that ST2825 induces G2/M cell cycle arrest and apoptosis via the MyD88/NF‑κB/AKT1/p21 pathway in PDAC. MyD88 may thus serve as a potential therapeutic target in PDAC. ST2825 may serve as a novel agent for the targeted therapy of PDAC in the future.

Fetal hypoxia results in sex- and cell type-specific alterations in neonatal transcription in rat oligodendrocyte precursor cells, microglia, neurons, and oligodendrocytes

BackgroundFetal hypoxia causes vital, systemic, developmental malformations in the fetus, particularly in the brain, and increases the risk of diseases in later life. We previously demonstrated that fetal hypoxia exposure increases the susceptibility of the neonatal brain to hypoxic-ischemic insult. Herein, we investigate the effect of fetal hypoxia on programming of cell-specific transcriptomes in the brain of neonatal rats.ResultsWe obtained RNA sequencing (RNA-seq) data from neurons, microglia, oligodendrocytes, A2B5+ oligodendrocyte precursor cells, and astrocytes from male and female neonatal rats subjected either to fetal hypoxia or control conditions. Substantial transcriptomic responses to fetal hypoxia occurred in neurons, microglia, oligodendrocytes, and A2B5+ cells. Not only were the transcriptomic responses unique to each cell type, but they also occurred with a great deal of sexual dimorphism. We validated differential expression of several genes related to inflammation and cell death by Real-time Quantitative Polymerase Chain Reaction (qRT-PCR). Pathway and transcription factor motif analyses suggested that the NF-kappa B (NFκB) signaling pathway was enriched in the neonatal male brain due to fetal hypoxia, and we verified this result by transcription factor assay of NFκB-p65 in whole brain.ConclusionsOur study reveals a significant impact of fetal hypoxia on the transcriptomes of neonatal brains in a cell-specific and sex-dependent manner, and provides mechanistic insights that may help explain the development of hypoxic-ischemic sensitive phenotypes in the neonatal brain.

Pre-conditioning of gingival epithelial cells with sub-apoptotic concentrations of curcumin prevents pro-inflammatory cytokine release.

Plaque-induced gingival inflammation (gingivitis) is ubiquitous in humans. The epithelial barrier reacts to the presence of oral bacteria and induces inflammatory cascades. The objective of this study was to investigate the mechanism by which the small molecule micronutrient curcumin could decrease inflammatory response in vitro to oral bacterium heat-killed Fusobacterium nucleatum as curcumin could be a useful compound for combatting gingivitis already consumed by humans. H400 oral epithelial cell line was pre-conditioned with curcumin and the production of cytokines was measured by enzyme-linked immunosorbent assay (ELISA) and translocation of transcription factors was used to monitor inflammatory responses. Haem oxygenase (HO-1) expression and molecules that HO-1 releases were evaluated for their potential to reduce the quantity of cytokine production. Immunofluorescence microscopy and Western blotting were used to evaluate changes in transcription factor and enzyme location. Pre-conditioning of H400 cells with a sub-apoptotic concentration of curcumin (20 μM) attenuated secretion of Granulocyte-Macrophage - Colony-Stimulating Factor (GM-CSF) and reduced NFkB nuclear translocation. This pre-conditioning caused an increase in nuclear Nrf2; an initial drop (at 8h) followed by an adaptive increase (at 24 h) in glutathione; and an increase in haem oxygenase (HO-1) expression. Inhibition of HO-1 by SnPPIX prevented the curcumin-induced attenuation of GM-CSF production. HO-1 catalyses the breakdown of haem to carbon monoxide, free iron and biliverdin: the HO-1/CO anti-inflammatory pathway. Elevations in carbon monoxide, achieved using carbon monoxide releasing molecule-2 (CORM2) treatment alone abrogated F. nucleatum-induced cytokine production. Biliverdin is converted to bilirubin by biliverdin reductase (BVR). This pleiotropic protein was found to increase in cell membrane expression upon curcumin treatment. Curcumin decreased inflammatory cytokine production induced by Fusobacterium nucleatum in H400 oral epithelial cells. The mechanism of action appears to be driven by the increase of haem oxygenase and the production of carbon monoxide.

Nitrooleic acid inhibits macrophage activation induced by lipopolysaccharide from Prevotella intermedia

The hypothesis of the present study was that nitro-fatty acids (NO2-FAs) would suppress inflammation associated with periodontal disease. To test this hypothesis, we investigated the influence of nitrooleic acid, a prototypical NO2-FA, on the inflammatory response of murine macrophages activated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen associated the etiology of different types of periodontal diseases. LPS was prepared from P. intermedia cells by using phenol-water protocol. Culture supernatants were assayed for nitric oxide (NO), interleukin-1β (IL-1β), and IL-6. Real-time polymerase chain reaction and immunoblotting analyses were performed to quantify messenger RNA and protein expression, respectively. The secreted embryonic alkaline phosphatase reporter assay was performed to measure NF-κB activation. The transcription factor assay kit was used to measure DNA-binding of NF-κB subunits. Findings obtained from the present study revealed that nitrooleic acid suppresses the generation and messenger RNA expression of inducible NO synthase-derived NO, IL-1β, and IL-6 in RAW264.7 cells activated with P. intermedia LPS and promotes macrophage polarization toward anti-inflammatory M2 phenotype. We also found that nitrooleic acid exerts its effect via heme oxygenase-1 induction and suppression of NF-κB signaling. The inhibition of NO and proinflammatory cytokine production by nitrooleic acid was independent from PPAR-γ, JNK, p38, and STAT1/3. Nitrooleic acid may represent a novel class of agent as a host modulator which has therapeutic benefit in periodontal disease, though more work is needed to confirm this.

Unfolded protein response is involved in the metabolic and apoptotic regulation of oral squamous cell carcinoma

Endoplasmic reticulum (ER) stress and unfolded protein response (UPR) have been shown to be crucial in the pathogenesis and response to treatment in various cancers. However, such response has not been profiled in oral squamous cell carcinoma (OSCC), the most frequent form of cancer in the head and neck region. Cell lines derived from OSCC (SCC4, SCC15 and SCC25) and normal oral mucosa (OKF4, OKF6 and OKP7) were subjected to tunicamycin-induced ER stress (2.5 μg/mL for 24 h) after which the differential regulation of 84 key UPR/ER stress genes were assessed using Quantitative real-time reverse transcription polymerase chain reaction. The expression of the transcription factors SREBP1 and CREB3L3, and the activation of SREBP1, were examined using ELISA and a transcription factor assay. The expression of DDIT3 was immunohistochemically verified in OSCC tissue samples. SREBP1 and CREB3L3 were significantly up-regulated in OSCC with and without tunicamycin-induced ER stress. A significantly higher level of SREBP1 transcriptional activation was observed in OSCC. Apoptosis-associated genes (DDIT3, HTRA4 and HSPA1L) were also significantly up-regulated in OSCC upon ER stress induction. The findings demonstrated the involvement of UPR and ER stress in the pathogenesis of OSCC through the identification of apoptosis-associated genes (DDIT3, HSPA1L and HTRA4) and regulators of metabolism (SREBP1 and CREB3L3) as the key factors differentiating between normal and malignant oral keratinocytes.

The lncARSR/PTEN/Akt/nuclear factor-kappa B feedback regulatory loop contributes to doxorubicin resistance in hepatocellular carcinoma.

Chemoresistance is a major obstacle to hepatocellular carcinoma (HCC) chemotherapy. Our previous study found that long noncoding RNA lncARSR (lncRNA Activated in RCC with Sunitinib Resistance) activated Akt signaling via repressing phosphatase and tensin homolog (PTEN) during doxorubicin resistance in HCC. The purpose of this study is to further explore lncARSR-mediated mechanisms and roles during doxorubicin resistance in HCC. The expression of lncARSR was detected by real-time quantitative polymerase chain reaction (qPCR). Nuclear factor-kappa B (NF-κB) activity was detected by NF-κB luciferase reporter assays, western blot, and NF-κB transcription factor assays. The effects of NF-κB on lncARSR were detected by chromatin immunoprecipitation assay, promoter luciferase reporter assay, and real-time qPCR. The effects of lncARSR/Akt/NF-κB on doxorubicin resistance were detected by Cell Counting Kit-8 assay, capsase-3 activity assay, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelingassay. lncARSR activated NF-κB signaling through activation of Akt. NF-κB transactivated lncARSR through directly binding lncARSR promoter and increasing lncARSR promoter activity. Akt transactivated lncARSR via activating NF-κB signaling. Thus, lncARSR, Akt, and NF-κB formed a positive feedback regulatory loop in HCC. Through this feedback loop, lncARSR auto-regulated its transcription. Drug sensitivity assays showed that the lncARSR/Akt/NF-κB feedback regulatory loop promoted doxorubicin resistance in HCC. These findings identified the lncARSR/Akt/NF-κB feedback regulatory loop in HCC, which represent potential therapeutic targets for improving doxorubicin sensitivity in HCC.

Activity in MCF-7 Estrogen-sensitive Breast Cancer Cells of Capsicodendrin from Cinnamosma fragrans.

Effect of capsicodendrin on the NF-κB pathway was studied in MCF-7 cancer cells. The transcription factor assay was used to screen for NF-κB activity. The effect on IKKβ, ICAM-1, and caspase-7 were studied using western blot. Caspase-1 was studied using Promega Caspase-Glo® assay. Reactive oxygen species (ROS) were detected using the fluorescent probe DCFH-DA. The potentiometric dye JC-1 was used to assess mitochondrial membrane potential (ΔΨm) and the cell cycle was examined using a fluorescence-activated cell sorter. NF-κB p65 inhibitory effect was IC50=8.6 μM and cytotoxic activity was IC50=7.5 μM. The upstream IKK and the downstream ICAM-1 were down-regulated. Sub G1-phase population increased to 81% after 12 h of treatment with capsicodendrin (10 μM) and there was no loss of ΔΨM. Increased levels of intracellular ROS promoted activity of caspase-1 and induced cell death in MCF-7 cells. Capsicodendrin may be a future anticancer agent that prevents the progression of metastatic breast cancer.

In vitro evaluation of the involvement of Nrf2 in maslinic acid-mediated anti-inflammatory effects in atheroma pathogenesis

AimsMaslinic acid (MA) is a naturally occurring pentacyclic triterpene known to exert cardioprotective effects. This study aims to investigate the involvement of nuclear factor erythroid 2-related factor 2 (Nrf2) for MA-mediated anti-inflammatory effects in atheroma pathogenesis in vitro, including evaluation of tumor necrosis factor-alpha (TNF-α)-induced monocyte recruitment, oxidized low-density lipoprotein (oxLDL)-induced scavenger receptors expression, and nuclear factor-kappa B (NF-ĸB) activity in human umbilical vein endothelial cells (HUVECS) and human acute monocytic leukemia cell line (THP-1) macrophages. Materials and methodsAn in vitro monocyte recruitment model utilizing THP-1 and HUVECs was developed to evaluate TNF-α-induced monocyte adhesion and trans-endothelial migration. To study the role of Nrf2 for MA-mediated anti-inflammatory effects, Nrf2 inhibitor ML385 was used as the pharmacological inhibitor. The expression of Nrf2, monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule 1 (VCAM-1), cluster of differentiation 36 (CD36), and scavenger receptor type A (SR-A) in HUVECs and THP-1 macrophages were investigated using RT-qPCR and Western blotting. The NF-κB activity was determined using NF-κB (p65) Transcription Factor Assay Kit. Key findingsThe results showed opposing effects of MA on Nrf2 expression in HUVECs and THP-1 macrophages. MA suppressed TNF-α-induced Nrf2 expression in HUVECs, but enhanced its expression in THP-1 macrophages. Combined effects of MA and ML385 suppressed MCP-1, VCAM-1, and SR-A expressions. Intriguingly, at the protein level, ML385 selectively inhibited SR-A but enhanced CD36 expression. Meanwhile, ML385 further enhanced MA-mediated inhibition of NF-κB activity in HUVECs. This effect, however, was not observed in THP-1 macrophages. SignificanceMA attenuated foam cell formation by suppressing VCAM-1, MCP-1, and SR-A expression, as well as NF-κB activity, possibly through Nrf2 inhibition. The involvement of Nrf2 for MA-mediated anti-inflammatory effects however differs between HUVECs and macrophages. Future investigations are warranted for a detailed evaluation of the contributing roles of Nrf2 in foam cells formation.

Activation of the Keap1-Nrf2 pathway by specioside and the n-butanol extract from the inner bark of Tabebuia rosea (Bertol) DC.

Background: A large number of chemical compounds exert their antioxidant effects by activation of key transcriptional regulatory mechanisms, such as the transcription factor Nrf2. The aim of this study was to evaluate the activation of the Keap1-Nrf2 pathway by both the n-butanol extract obtained from the inner bark of Tabebuia rosea (Bertol) DC and specioside isolated from this extract. Methods: The antioxidant activity of the extract and specioside isolated from the inner bark of T. rosea were evaluated using the oxygen radical absorbance capacity (ORAC) and the 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity (DPPH) techniques, whereas their effects on the viability of HepG2 cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The effects of the compound and the extract on activating the Keap1-Nrf2 pathway were evaluated using a Nrf2 Transcription Factor Assay kit. Induction of the Nrf2-mediated antioxidant response genes HMOX-1 and NQO1 was evaluated by real-time PCR. The protective effects against H 2O 2-induced oxidative stress in HepG2 cells was determined as the percent protection using the MTT method. Results: Both the n-butanol extract and specioside exhibited activity at low concentrations without affecting cellular viability, since the cell viability was greater than 80% after 24 hours of exposure at each tested concentration. In addition, Nrf2 dissociated from Keap1 after treatment with the n-butanol extract at a concentration of 0.25 µg/mL after 4 hours of exposure. An increase in the Nrf2 level in the cytoplasm after 4 hours of exposure to 2 μM specioside was observed. Nrf2 levels stabilized in the nucleus 12 hours after stimulation with both specioside and the extract. After 6 hours of stimulation, both the extract and specioside induced the expression of HMOX-1 and NQO1. Conclusion: The n-butanol extract from the inner bark of T. rosea and specioside produced protective effects against H 2O 2-induced oxidative stress in HepG2 cells.

Activation of the Keap1-Nrf2 pathway by specioside and the n-butanol extract from the inner bark of Tabebuia rosea (Bertol) DC

Background: A large number of chemical compounds exert their antioxidant effects by activation of key transcriptional regulatory mechanisms, such as the transcription factor Nrf2. The aim of this study was to evaluate the activation of the Keap1-Nrf2 pathway by both the n-butanol extract obtained from the inner bark of Tabebuia rosea (Bertol) DC and specioside isolated from this extract. Methods: The antioxidant activity of the extract and specioside isolated from the inner bark of T. rosea were evaluated using the oxygen radical absorbance capacity (ORAC) and the 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity (DPPH) techniques, whereas their effects on the viability of HepG2 cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The effects of the compound and the extract on activating the Keap1-Nrf2 pathway were evaluated using a Nrf2 Transcription Factor Assay kit. Induction of the Nrf2-mediated antioxidant response genes HMOX-1 and NQO1 was evaluated by real-time PCR. The protective effects against H2O2-induced oxidative stress in HepG2 cells was determined as the percent protection using the MTT method. Results: Both the n-butanol extract and specioside exhibited activity at low concentrations without affecting cellular viability, since the cell viability was greater than 80% after 24 hours of exposure at each tested concentration. In addition, Nrf2 dissociated from Keap1 after treatment with the n-butanol extract at a concentration of 0.25 µg/mL after 4 hours of exposure. An increase in the Nrf2 level in the cytoplasm after 4 hours of exposure to 2 μM specioside was observed. Nrf2 levels stabilized in the nucleus 12 hours after stimulation with both specioside and the extract. After 6 hours of stimulation, both the extract and specioside induced the expression of HMOX-1 and NQO1. Conclusion: The n-butanol extract from the inner bark of T. rosea and specioside produced protective effects against H2O2-induced oxidative stress in HepG2 cells.

Activation of the Keap1-Nrf2 pathway by specioside and the n-butanol extract from the inner bark of Tabebuia rosea (Bertol) DC

Background: A large number of chemical compounds exert their antioxidant effects by activation of key transcriptional regulatory mechanisms, such as the transcription factor Nrf2. The aim of this study was to evaluate the activation of the Keap1-Nrf2 pathway by both the n-butanol extract obtained from the inner bark of Tabebuia rosea (Bertol) DC and specioside isolated from this extract. Methods: The antioxidant activity of the extract and specioside isolated from the inner bark of T. rosea were evaluated using the oxygen radical absorbance capacity (ORAC) and the 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity (DPPH) techniques, whereas their effects on the viability of HepG2 cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The effects of the compound and the extract on activating the Keap1-Nrf2 pathway were evaluated using a Nrf2 Transcription Factor Assay kit. Induction of the Nrf2-mediated antioxidant response genes HMOX-1 and NQO1 was evaluated by real-time PCR. The protective effects against H 2O 2-induced oxidative stress in HepG2 cells was determined as the percent protection using the MTT method. Results: Both the n-butanol extract and specioside exhibited activity at low concentrations without affecting cellular viability, since the cell viability was greater than 80% after 24 hours of exposure at each tested concentration. In addition, Nrf2 dissociated from Keap1 after treatment with the n-butanol extract at a concentration of 0.25 µg/mL after 4 hours of exposure. An increase in the Nrf2 level in the cytoplasm after 4 hours of exposure to 2 μM specioside was observed. Nrf2 levels stabilized in the nucleus 12 hours after stimulation with both specioside and the extract. After 6 hours of stimulation, both the extract and specioside induced the expression of HMOX-1 and NQO1. Conclusion: The n-butanol extract from the inner bark of T. rosea and specioside produced protective effects against H 2O 2-induced oxidative stress in HepG2 cells.

The involvement of toll-like receptors 2 and 4 in human platelet signalling pathways

In addition to haemostasis, platelets play an essential role in mechanisms of inflammation and in immunological reactions. Platelets express various toll-like receptors (TLR) on their surface, among them TLR2 and TLR4, which are important for the recognition of bacterial patterns. This study compared TLR2- and TLR4-dependent platelet signalling and their effect on platelet function.Platelet-rich-plasma and washed platelets were prepared from peripheral blood samples of healthy donors. Pam3CSK4 or LPS (lipopolysaccharides from Escherichia coli) were used for stimulation of TLR2 and TLR4. Intracellular signalling pathways were investigated by Western blot. TLR2- and TLR4-mediated specific transcription factor DNA binding activity was measured by the nuclear factor kappa B (NFκB) transcription factor assay kit. Platelet adhesion and glycoprotein Ib function were assessed by immunofluorescence staining and analysis of ristocetin-induced agglutination.Both, Pam3CSK4 and LPS were able to induce NFκB-mediated and classical activating platelet signalling with a higher stimulatory capacity of TLR2. In addition, TLR2 and TLR4 activation led to a similar activation of inhibitory pathways. In contrast to TLR2, stimulation of TLR4 resulted in decreased Akt/protein kinase B phosphorylation conditioned by enhanced protein phosphatase 2A activity. TLR4-mediated signalling induced platelet adhesion and facilitated ristocetin-induced platelet agglutination.In conclusion, Pam3CSK4 directly induces aggregation via classical activation cascades, whereas LPS enhances platelet adhesion and glycoprotein receptor Ib-dependent platelet agglutination.

Activation of the Keap1-Nrf2 pathway by specioside and the n-butanol extract from the inner bark of Tabebuia rosea (Bertol) DC.

Background: A large number of chemical compounds exert their antioxidant effects by activation of key transcriptional regulatory mechanisms, such as the transcription factor Nrf2. The aim of this study was to evaluate the activation of the Keap1-Nrf2 pathway by both the n-butanol extract obtained from the inner bark of Tabebuia rosea (Bertol) DC and specioside isolated from this extract. Methods: The antioxidant activity of the extract and specioside isolated from the inner bark of T. rosea were evaluated using the oxygen radical absorbance capacity (ORAC) and the 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity (DPPH) techniques, whereas their effects on the viability of HepG2 cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The effects of the compound and the extract on activating the Keap1-Nrf2 pathway were evaluated using a Nrf2 Transcription Factor Assay kit. Induction of the Nrf2-mediated antioxidant response genes HMOX-1 and NQO1 was evaluated by real-time PCR. The protective effects against H 2O 2-induced oxidative stress in HepG2 cells was determined as the percent protection using the MTT method. Results: Both the n-butanol extract and specioside exhibited activity at low concentrations without affecting cellular viability, since the cell viability was greater than 80% after 24 hours of exposure at each tested concentration. In addition, Nrf2 dissociated from Keap1 after treatment with the n-butanol extract at a concentration of 0.25 µg/mL after 4 hours of exposure. An increase in the Nrf2 level in the cytoplasm after 4 hours of exposure to 2 μM specioside was observed. Nrf2 levels stabilized in the nucleus 12 hours after stimulation with both specioside and the extract. After 6 hours of stimulation, both the extract and specioside induced the expression of HMOX-1 and NQO1. Conclusion: The n-butanol extract from the inner bark of T. rosea and specioside produced protective effects against H 2O 2-induced oxidative stress in HepG2 cells.