Intracellular NO Research Articles

Macrophage-like THP-1 Cells Derived from High-Density Cell Culture Are Resistant to TRAIL-Induced Cell Death via Down-Regulation of Death-Receptors DR4 and DR5.

Simple SummaryThe mechanisms of leukemic cell resistance to antitumor immunity remains a topical issue. In this work, we found an increase in TRAIL-resistance of human acute myeloid leukemia cells THP-1 in high-density populations in vitro. The results obtained show that a macrophage-like phenotype of the acute myeloid leukemia cells, caused by stressful conditions in high-density culture, can increaser resistance to TRAIL-induced apoptosis, while retaining proliferative potential. The mechanism of the increase in TRAIL-resistance can be related to a decrease in the expression of death receptors DR4 and DR5. The possible realization of these events in vivo may be the reason for tumor progression.Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) is a highly selective and promising anticancer agent due to its specific apoptosis-inducing effect on tumor cells, rather than most normal cells. TRAIL is currently under investigation for use in the treatment of leukemia. However, the resistance of leukemic cells to TRAIL-induced apoptosis may limit its efficacy. The mechanisms of leukemic cell resistance to antitumor immunity remains a topical issue. In this work, we have found an increase in the resistance to TRAIL-induced cell death in human leukemia THP-1 cells, which was caused by differentiation into a macrophage-like phenotype in high-density culture in vitro. Stressful conditions, manifested by the inhibition of cell growth and the activation of cell death in high-density culture of THP-1 cells, induced the appearance of cells adhered to culture dishes. The THP-1ad cell line was derived by selection of these adhered cells. The genetic study, using STR and aCGH assays, has shown that THP-1ad cells were derived from THP-1 cells due to mutagenesis. The THP-1ad cells possessed high proliferative potential and a macrophage-like immunophenotype. The adhesion of THP-1ad cells to the extracellular matrix was mediated by αVβ5 integrin. The cytokine production, as well as the rise of intracellular ROS and NO activities by LPS in THP-1ad cell culture, were characteristic of macrophage-like cells. The THP-1ad cells were found to appear to increase in resistance to TRAIL-induced cell death in comparison with THP-1 cells. The mechanism of the increase in TRAIL-resistance can be related to a decrease in the expression of death receptors DR4 and DR5 on the THP-1ad cells. Thus, the macrophage-like phenotype formation with the maintenance of a high proliferative potential of leukemic cells, caused by stress conditions in high-density cell cultures in vitro, can induce an increase in resistance to TRAIL-induced cell death due to the loss of DR4 and DR5 receptors. The possible realization of these events in vivo may be the reason for tumor progression.

Mechanistic approaches to the light-induced neural cell differentiation: Photobiomodulation vs Low-Dose Photodynamic Therapy

BackgroundNeurodegenerative diseases are the result of irreversible damage in the neuronal cells by effecting vital functions temporarily or even permanently. The use of light for the treatment of these diseases is an emerging promising innovative method. Photobiomodulation (PBM) and Photodynamic Therapy (PDT) are the modalities that have a wide range of use in medicine and have opposite purposes, biostimulation and cell death respectively. MethodsIn this study, we aimed to compare these two modalities (PDT and PBM) at low-level intensities and create a stimulatory effect on the differentiation of PC12 cells. Three different energy densities (1, 3, and 5 J/cm2) were used in PBM and Chlorin e6-mediated PDT applications upon irradiation with 655-nm laser light. The light-induced differentiation profile of PC12 cells was analyzed by morphological examinations, qRT-PCR, cell viability assay, and some mechanistic approaches such as; the analysis of intracellular ROS production, NO release, and mitochondrial membrane potential change. ResultsIt has been observed that both of these modalities were successful at neural cell differentiation. PBM at 1 J/cm2 and low-dose PDT at 3 J/cm2 energy densities provided the best differentiation profiles which were proved by the over-expressions of SYN-1 and GAP43 genes. It was also observed that intracellular ROS production and NO release had pivotal roles in these mechanisms with more cell differentiation obtained especially in low-dose PDT application. ConclusionIt can be concluded that light-induced mechanisms with properly optimized light parameters have the capacity for neural cell regeneration and thus, can be a successful treatment for incurable neurodegenerative diseases.

Low-Dose Piperlongumine Rescues Impaired Function of Endothelial Progenitor Cells and Reduces Cerebral Ischemic Injury in High-Fat Diet-Fed Mice.

It is of great clinical significance to develop potential novel strategies to prevent cardio-cerebrovascular complications in patients with hyperlipidemia. Vascular Endothelial integrity and function play a key role in the prevention of cardio-cerebrovascular diseases. Endothelial progenitor cells (EPCs) can home to sites of ischemic injury and promote endothelial regeneration and neovascularization. Hypercholesterolemia impairs the function of EPC. The present study attempted to identify the effect of piperlongumine on EPCs’ angiogenic potential and cerebral ischemic injury in high-fat diet-fed (HFD-fed) mice. Here, we showed that treatment with low-does piperlongumine (0.25 mg/kg/day) for 8 weeks significantly improved EPCs function and reduced the cerebral ischemic injury (both infarct volumes and neurobehavioral outcomes) in HFD-fed mice. In addition, low-dose piperlongumine administration increased intracellular NO level and reduced intracellular O2 - level in EPCs of HFD-fed mice. Moreover, incubation with piperlongumine (1.0 μM, 24 h) reduced thrombospondin-1/2 (TSP-1/2, a potent angiogenesis inhibitor) expression levels in EPCs from HFD-fed mice, increased the therapeutic effect of EPC from HFD-fed mice on cerebral ischemic injury reduction and angiogenesis promotion in HFD-fed mice, and the donor derived EPCs homed to the recipient ischemic brain. In conclusion, low-dose piperlongumine can enhance EPCs’ angiogenic potential and protect against cerebral ischemic injury in HFD-fed mice. It is implied that treatment with low-dose piperlongumine might be a potential option to prevent ischemic diseases (including stroke) in patients with hyperlipidemia, and priming with piperlongumine might be a feasible way to improve the efficacy of EPC-based therapy for ischemic diseases.

Microcirculatory Function during Endotoxemia-A Functional Citrulline-Arginine-NO Pathway and NOS3 Complex Is Essential to Maintain the Microcirculation.

Competition for the amino acid arginine by endothelial nitric-oxide synthase (NOS3) and (pro-)inflammatory NO-synthase (NOS2) during endotoxemia appears essential in the derangement of the microcirculatory flow. This study investigated the role of NOS2 and NOS3 combined with/without citrulline supplementation on the NO-production and microcirculation during endotoxemia. Wildtype (C57BL6/N background; control; n = 36), Nos2-deficient, (n = 40), Nos3-deficient (n = 39) and Nos2/Nos3-deficient mice (n = 42) received a continuous intravenous LPS infusion alone (200 μg total, 18 h) or combined with L-citrulline (37.5 mg, last 6 h). The intestinal microcirculatory flow was measured by side-stream dark field (SDF)-imaging. The jejunal intracellular NO production was quantified by in vivo NO-spin trapping combined with electron spin-resonance (ESR) spectrometry. Amino-acid concentrations were measured by high-performance liquid chromatography (HPLC). LPS infusion decreased plasma arginine concentration in control and Nos3−/− compared to Nos2−/− mice. Jejunal NO production and the microcirculation were significantly decreased in control and Nos2−/− mice after LPS infusion. No beneficial effects of L-citrulline supplementation on microcirculatory flow were found in Nos3−/− or Nos2−/−/Nos3−/− mice. This study confirms that L-citrulline supplementation enhances de novo arginine synthesis and NO production in mice during endotoxemia with a functional NOS3-enzyme (control and Nos2−/− mice), as this beneficial effect was absent in Nos3−/− or Nos2−/−/Nos3−/− mice.

Preparation, Antioxidant Properties and Ability to Increase Intracellular NO of a New Pyridoxine Derivative B6NO.

In the case of various pathologies, an imbalance between ROS generation and the endogenous AOS can be observed, which leads to excessive ROS accumulation, intensification of LPO processes, and oxidative stress. For the prevention of diseases associated with oxidative stress, drugs with antioxidant activity can be used. The cytotoxic, antioxidant, and NO-donor properties of the new hybrid compound B6NO (di(3-hydroxy-4,5-bis(hydroxymethyl)-2-methylpyridinium) salt of 2-(nitrooxy)butanedioic acid) were studied. It was determined that B6NO chelates iron ions by 94%, which indicates B6NO’s ability to block the Fenton reaction. The hybrid compound B6NO inhibits the process of initiated lipid peroxidation more effectively than pyridoxine. It was shown that B6NO exhibits antioxidant properties by decreasing ROS concentration in normal cells during the oxidative stress induction by tert-Butyl peroxide. At the same time, the B6NO antioxidant activity on tumor cells was significantly lower. B6NO significantly increases the intracellular nitrogen monoxide accumulation and showed low cytotoxicity for normal cells (IC50 > 4 mM). Thus, the results indicate a high potential of the B6NO as an antioxidant compound.

Study on immediate and long-term growth inhibition of Microcystis aeruginosa by non-thermal plasma

There are still few studies on long-term growth inhibition of cyanobacteria though direct algae inactivation by discharge plasma has been widely invetigated. In the work, a home-made DBD system was applied to investigate the immediate and long-term inactivation effect of Microcystis aeruginosa (M. aeruginosa). Owing to discharge plasma in gas–liquid interface or liquid phase, RONS including H2O2, O3 and NO3- and OH radical are produced. The bactericidal effects are time-dependent and a 3.36log reduction (above 99.99%) is achieved after 10-min exposure followed by the loss of membrane integrity resulting in damaged metabolic capacity and partial RS transportation into cells. Plasma discharge also leads to increased intracellular ROS and NO aggravating inactivation efficacy in the interior structure. The concentration changes of MCs indicates a well degradation effect on MCs by DBD plasma. Moreover, growth arrest (10 day for 0.5-min and 15 day for 1-min treated samples) indicates continuous inactivation of short-period DBD. The decreased contents of photosynthetic pigments (phycobilins and Chl-a) and inhibited nutrient absorption efficiency could contribute to residual inactivation effect. The inhibited synthesis of MCs are induced by short-period plasma treatment. The comprehensive regulations of genes associated with physiological processes are potentially responsible for long-term inactivation of M. aeruginosa. This work therefore proposes a type of feasible advanced oxidation process (AOPs) to utilization of DBD plasma that achieves effective short-term and long-term inactivation efficacy of M. aeruginosa.

Crosstalk of Endothelial and Mesenchymal Stromal Cells under Tissue-Related O2

Mesenchymal stromal cells (MSCs) are considered a valuable tool for cell therapy. After systemic administration, the outcome of MSCs and endothelial cells (ECs) interactions strongly depend on the local microenvironment and tissue O2 levels in particular. In vitro analysis of EC effects on MSC regenerative potential in co-culture was performed after short-term interaction at “physiological” hypoxia (5% O2) and acute hypoxic stress (0.1% O2). At 5% O2, MSCs retained stromal phenotype and CFU-f numbers, osteogenic RUNX2 was upregulated. A shift in the expression of adhesion molecules, and an increase in transcription/synthesis of IL-6, IL-8 contributed to facilitation of directed migration of MSCs. In the presence of MSCs, manifestations of oxidative stress in ECs were attenuated, and a decrease in adhesion of PBMCs to TNF-α-activated ECs was observed. Under 0.1% O2, reciprocal effects of ECs and MSCs were similar to those at 5% O2. Meanwhile, upregulation of RUNX2 was canceled, IL-6 decreased, and IL-8 significantly increased. “Protective” effects of MSCs on TNF-α-ECs were less pronounced, manifested as NOS3 downregulation and intracellular NO elevation. Therefore, interaction with ECs at “physiological” hypoxia enhanced pro-regenerative capacities of MSCs including migration and anti-inflammatory modulation of ECs. Under acute hypoxic stress, the stimulating effects of ECs on MSCs and the “protective” potential of MSCs towards TNF-α-ECs were attenuated.

(E)-5-hydroxy-7-methoxy-3-(2′-hydroxybenzyl)-4-chromanone isolated from Portulaca oleracea L. suppresses LPS-induced inflammation in RAW 264.7 macrophages by downregulating inflammatory factors

Context Portulaca oleracea L. is herbaceous succulent annual plant, which belongs to the Portulacaceae family. Many studies have shown its wide spectrum of pharmacological activities such as anti-cancer and anti-diabetic effects. Objectives The objective of this study was to identify the anti-inflammatory effects of HM-chromanone isolated from Portulaca oleracea L. in LPS-stimulated RAW 264.7 macrophages. Materials and methods LPS (1 μg/ml)-stimulated mouse RAW 264.7 macrophages were used to assess the anti-inflammatory effect of HM-chromanone (10–50 μM). Cell viability was evaluated by MTT assay. In addition, the production of intracellular ROS, superoxide anion, lipid peroxide, NO, and PGE2, and activity of antioxidant enzymes were analyzed. The expressions of iNOS, COX-2, IκB, NF-κB, TNF-α, IL-1β and IL-6 were evaluated by western blot analysis. Results HM-chromanone has demonstrated that there is no significant cytotoxic effect on the viability of RAW 264.7 macrophages. In LPS-stimulated RAW 264.7 cells, HM-chromanone treatment was found to significantly inhibit the production of intracellular ROS, superoxide anion and lipid peroxide, while enhancing the activity of antioxidant enzymes such as SOD, catalase, and GSH-px. Additionally, HM-chromanone treatment was observed to inhibit NO and PGE2 production by inhibiting the expression of iNOS and COX-2. Subsequently, HM-chromanone was observed to significantly suppress LPS-induced expression of IκB, NF-κB, TNF-α, IL-1β and IL-6. Discussion and conclusion Overall, our results suggested that HM-chromanone suppresses LPS-induced inflammation in RAW 264.7 macrophages by downregulating the expression of inflammatory factors.

Overview of SARS-CoV-2 Infection and Its Potential Reactions

Context: The immunopathology of SARS-CoV-2 infection in COVID-19 is not well described yet, especially regarding dysregulation of the immune system. In this mini-review, current knowledge about the SARS-CoV-2 infection and immunopathogenesis of COVID-19 disease is described. We also discuss possible induced reactions against SARS-COV-2. Evidence Acquisition: Based on the authors' experience and knowledge, the current review aimed to, firstly, discuss and overview SRAS-CoV-2 infection and reactions in the body, and, secondly, to obtain related subjects from the PubMed database. Results and Conclusion: In most COVID-19 patients, uncontrollable cytokines secretion and mediators are major key points in the pathogenesis of the disease. Of all cytokines and mediators, serum levels of IL-6, IL-1β, TNF-α, IL-8, and soluble TNF-α receptor (sTNFR) have been reported. Lymphopenia and hypoxia, as well as the severity of the disease, can be considered as COVID-19 manifestations. High levels of intracellular NO inside of the red blood cells (RBCs) of patients drive the unexpected silent hypoxia phenotype induced ARDS importantly related to the patient's immune system dysfunction.

Anti-inflammatory activities of amber extract in lipopolysaccharide-induced RAW 264.7 macrophages

Amber is a type of fossil tree resin with several bioactive properties and has been traced in traditional medicines used in Russia and China. However, its anti-inflammatory activities are poorly characterized. Here, the anti-inflammatory effects of the extract of amber mined from Kaliningrad, Russia was investigated in lipopolysaccharide (LPS)-induced RAW 264.7 cells. The effect of the amber extract on cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Further, its effects on the production of intracellular reactive oxygen species (ROS), NO, and inflammatory cytokines were assessed by 2′,7′-dichlorodihydrofluorescein diacetate staining, Griess test, and cytokine enzyme-linked immunosorbent assays, respectively. Western blotting and real-time reverse transcription-polymerase chain reaction analysis were performed to assess the mRNA and protein expression levels of the inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α) interleukin-6 (IL-6), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS). The translocation of the nuclear factor-kappa B (NF-κB) p65 subunit was observed by immunofluorescent staining. Amber extract negatively regulated the LPS-induced differentiation of RAW 264.7 cells to dendritic-like cells and reduced the LPS-induced increase in ROS and NO levels. It also reduced the level of mRNA and protein expressions of TNF-α, IL-6, COX-2, and iNOS in LPS-induced RAW 264.7 macrophages, in a dose-dependent manner. Furthermore, amber extract suppressed the nuclear translocation of the NF-κB p65 subunit. These findings suggest that the potent anti-inflammatory effect of the amber extract is mediated by the inhibition of the NF-κB p65 signaling pathway. Collectively, this study renders amber extract as a potential pharmacological alternative to treat inflammation-related diseases.

Comparison of multi-walled carbon nanotubes and halloysite nanotubes on lipid profiles in human umbilical vein endothelial cells

Tubular nanomaterials (NMs), such as multi-walled carbon nanotubes (MWCNTs) and halloysite nanotubes (HNTs), may be used in biomedicine, but previous studies showed that MWCNTs induced toxicity to endothelial cells (ECs). However, the influence of tubular NMs on EC lipid profiles has gained little attention, probably because ECs are not traditionally considered to be involved in regulating lipid homeostasis. This study compared the different effects of MWCNTs and HNTs on lipid profile changes in human umbilical vein ECs (HUVECs). The results showed that MWCNTs but not HNTs of the same mass concentrations induced cytotoxicity, ultrastuctural changes and intracellular thiol depletion. Meanwhile, only MWCNTs promoted lipid accumulation due to the induction of ER stress leading to up-regulation of fatty acid synthase (FASN). Interestingly, lipidomics results showed that the main lipid classes induced by MWCNTs but not HNTs were ceramide (Cer) and phosphatidylinositol (PI), with most of the lipid classes unaltered or even decreased after NM exposure. Then, extra Cer and PI were added to explore the implications of increase of these lipids. Adding Cer promoted the cytotoxicity of MWCNTs to HUVECs, indicating the lipotoxic role of Cer. Whereas adding PI partially increased intracellular NO and decreased interleukin-6 (IL-6) release due to MWCNT exposure, indicating the signaling role of PI. These results indicated novel roles of lipid dysfunction in NM-induced toxicity to ECs, even though ECs are not the professional cells for controlling lipid homeostasis.

X-ray Dose-Enhancing Impact of Functionalized Au-Fe3O4 Nanoheterodimers on MCF-7 and A549 Multicellular Tumor Spheroids.

The efficiency of nanoparticle-enhanced radiotherapy was studied by loading MCF-7 and A549 multicellular tumor spheroids (MCTSs) with caffeic acid- and nitrosonium-functionalized Au-Fe3O4 nanoheterodimers (Au-Fe3O4 NHDs). Transmission electron microscope images of MCTS cross-sectional sections visualized the invasion and distribution of the nitrosonium- and caffeic acid-functionalized Au-Fe3O4 NHDs (NO- and CA-NHDs) in the A549 and MCF-7 MCTSs, whereas the iron content of the MCTSs were quantified using the ferrozine assay. The synergistic impact of intracellular NO- and CA-NHDs and X-ray irradiation on the growth dynamics of the A549 and MCF-7 MCTSs was surveyed by monitoring their temporal evolution under a light microscope over a period of 14 days. The emergence of hypoxia during the spheroid growth was followed by detecting the lactate efflux of MCTSs without and with NO- and CA-NHDs. The performance of the NO- and CA-NHDs as X-ray dose-enhancing agents in the A549 and MCF-7 MCTSs was clarified by performing clonogenic cell survival assays and determining the respective dose-modifying factors for X-ray doses of 0, 2, 4, and 6 Gy. The NO- and CA-NHDs were shown to perform as potent X-ray dose-enhancing agents in A549 and MCF-7 MCTSs. Moreover, the CA-NHDs boosted their radio-sensitizing efficacy by inhibiting the lactate efflux as impairing metabolic reprogramming. A synergistic effect on the MCTS destruction was observed for the combination of both NHDs since the surfactants differ in their antitumor effect.

Effect of memantine, an anti-Alzheimer\u2019s drug, on rodent microglial cells in vitro

The pathophysiology of Alzheimer’s disease (AD) is related to neuroinflammatory responses mediated by microglia. Memantine, an antagonist of N-methyl-d-aspartate (NMDA) receptors used as an anti-Alzheimer’s drug, protects from neuronal death accompanied by suppression of proliferation and activation of microglial cells in animal models of AD. However, it remains to be tested whether memantine can directly affect microglial cell function. In this study, we examined whether pretreatment with memantine affects intracellular NO and Ca2+ mobilization using DAF-2 and Fura-2 imaging, respectively, and tested the effects of memantine on phagocytic activity by human β-Amyloid (1–42) phagocytosis assay in rodent microglial cells. Pretreatment with memantine did not affect production of NO or intracellular Ca2+ elevation induced by TNF in rodent microglial cells. Pretreatment with memantine also did not affect the mRNA expression of pro-inflammatory (TNF, IL-1β, IL-6 and CD45) or anti-inflammatory (IL-10, TGF-β and arginase) phenotypes in rodent microglial cells. In addition, pretreatment with memantine did not affect the amount of human β-Amyloid (1–42) phagocytosed by rodent microglial cells. Moreover, we observed that pretreatment with memantine did not affect 11 major proteins, which mainly function in the phagocytosis and degradation of β-Amyloid (1–42), including TREM2, DAP12 and neprilysin in rodent microglial cells. To the best of our knowledge, this is the first report to suggest that memantine does not directly modulate intracellular NO and Ca2+ mobilization or phagocytic activity in rodent microglial cells. Considering the neuroinflammation hypothesis of AD, the results might be important to understand the effect of memantine in the brain.

Magnetic Fe3O4 nanoparticles enhance cyanobactericidal effect of allelopathic p-hydroxybenzoic acid on Microcystis aeruginosa by enhancing hydroxyl radical production

Recently, considerable progress has been made in the environmental application of nanotechnology. However, little is known about how nanomaterials might affect the cyanobacterial suppression potential of allelochemicals. In this study, a microcosm was employed to simulate and verify the effect of magnetic Fe3O4 nanoparticles (MFN) on the inhibitory influence of allelopathic hydroxybenzoic acid (p-Ha) on bloom-forming Microcystis aeruginosa. MFN had a hormetic effect on cyanobacterial growth. At a neutral concentration of 182 mg/L, MFN enhanced the algal suppression by p-Ha and decreased the IC50 by half, which was significantly and positively associated with the amount of OH. Furthermore, adding MFN induced a stronger physiological response than treatment with only p-Ha. The cellular integrity was severely disrupted for the cyanobacterium M. aeruginosa. The total protein content decreased rapidly to inactivate the algae by limiting the amounts of extracellular microcystin and polysaccharide released. The modification of the effect of p-Ha by MFN was reflected by the intracellular NO content of M. aeruginosa. In addition, the typical radical scavengers ascorbic acid and 5,5-dimethyl-1-pyrroline N-oxide decreased OH production to weaken algal suppression under the combined treatment with p-Ha and MFN. By contrast, the addition of Fe3+ and increasing the light intensity triggered the generation of OH and strong cyanobacterial suppression. Thus, MFN could enhance the cyanobacterial control efficiency of p-Ha and decrease the input of allelochemicals in the field. These findings suggest a novel mode of allelochemical modification by nanomaterials as a promising cyanobactericide for harmful algal bloom management.

Hot alkali-extracted antioxidative crude polysaccharide from a novel mushroom enhances immune response via TLR-mediated NF-κB activation: A strategy for full utilization of a neglected tribal food.

Mushroom polysaccharides have been revered for decades as incredibly versatile compounds suitable for application in biomedical, pharmaceutical, food, and cosmetic industries. In that essence Russula alatoreticula, a traditionally appraised unique macrofungus, has earlier been explored to contain functional macromolecules elicited by hydrothermal and cold alkali systems. Remnants of that multistep extraction could further be utilized not only to isolate high-value components, but also to reduce waste material. With this in mind, we decided to recycle leftover residue of the tribal delicacy, rather than undervalue, by following heated NaOH reflux. Intriguingly, the effort resulted productive isolation of carbohydrate fraction (RualaHap) enriched in β-glucan that showed excellent antioxidant activity in terms of radical scavenging, chelating ability, and reducing power with EC50 of 207-5000μg/ml. Besides strong immune boosting prowess was also witnessed as exposure of RAW 264.7 cells to the metabolites influenced macrophage viability and phagocytosis in a time-dependent manner. Induction of intracellular NO and ROS generation was also enumerated along with aggrandized production of pseudopods representing hallmark of monocyte stimulation. Moreover, the treatment drastically triggered level of TLR-2, TLR-4, NF-κB, Iκ-Bα, iNOS, COX-2, TNF-α, IFN-γ, and IL-10 maintaining balance between M1 and M2 cytokines via TLR coordinated NF-κB pathway. Conclusively the research offers a roadmap for better utilization of a species boasted in traditional practice aiding future implementation of RualaHap as ingredients of nutraceuticals or functional foods. PRACTICAL APPLICATIONS: At present, macrofungal polysaccharides are regarded as an ideal resource for a range of safe uses encompassing direct contact or ingestion by human. In this context, RualaHap isolated from the wild edible mushroom, R. alatoreticula, could have a broad application prospect, particularly in development of natural therapeutics to ameliorate radical-induced ailment and immune-deficiency condition. The biopolymers could thus be utilized in functional diet or complementary medicine to improve health that in turn might raise local cost of the abandoned taxon boosting rural empowerment.

Tiliacora racemosa leaves induce oxidative stress mediated DNA damage leading to G2/M phase arrest and apoptosis in cervical cancer cells SiHa

Ethnopharmacological relevanceThe Menispermaceae plant Tiliacora racemosa is immensely popular in Indian traditional Ayurvedic medicine as “Krishnavetra” for its remarkable anti-cancerous property, and is commonly used by tribal population for the treatment of skin infections, snake bites and filariasis. Aim of the studyThis present study intends to identify the modus operandi behind the cytotoxic activity of Tiliacora racemosa leaves in cervical cancer cells SiHa. Focus has been instilled in the ability of the plant extract to target multiple signaling pathways leading to cell cycle arrest and cell death in SiHa cells, followed by a pharmacological characterization to identify the bioactive principle. Materials and methodsT. racemosa leaves extracted in methanol, ethyl acetate, hexane and aqueous solvent were screened for cytotoxicity in HeLa, SiHa, C33A (cervical cancer cells) and HEK cells by MTT assay. SiHa cells were treated with the most potent extract (TRM). Cellular morphology, clonogenic and wound healing potential, presence of intracellular ROS and NO, lipid peroxidation, activity of cellular antioxidants (SOD, CAT, GSH), DNA damage detection by comet assay and localisation of γ-H2AX foci, intracellular expression of PARP-1, Bax/Bcl2 and caspase-3, loss in mitochondrial membrane potential by JC1 (flow cytometry) and Rh123 (microscopy), cell cycle analysis, Annexin-FITC assay, AO/EtBr microscopy and apoptotic proteome profiling were undertaken in the treated cells. All the related proteins were studied by immunoblots. Effect of NAC (ROS-scavenger) on cell viability, DNA damage and apoptosis were studied. Phytochemical characterization of all TR extracts was followed by LC-MS analysis of TRM and isolated alkaloid of TR was assessed for cytotoxicity. ResultsThe methanol extract of T. racemosa (TRM) rich in bisbenzylisoquinoline and other alkaloids impeded the proliferation of cervical cancer cells SiHa in vitro through disruption of cellular redox homeostasis caused by increase in cellular ROS and NO with concomitant decrease in the cellular antioxidants. Double-stranded DNA damage was noted from γH2AX foci accumulation and Parp-1 activation leading to ATM-Chk2-p53 pathway arresting the cells at G2/M-phase through cyclin B1 inhibition. The mitochondrial membrane potential was also disturbed leading to caspase-3 dependent apoptotic induction by both extrinsic and intrinsic pathway. Immunoblots show TRM also inhibited PI3K/Akt and NFκB pathway. NAC pre-treatment rescued the cell viability proving DNA damage and apoptosis to be direct consequences of ROS overproduction. Lastly, the therapeutic potential of T. racemosa is was hypothesized to be possibly derived from its alkaloid content. ConclusionThis study proves the age old ethnnopharmacological anticancer role of T. racemosa. The leaf extracts inhibited the anomalous proliferation of SiHa cells by virtue of G2/M-phase cell cycle arrest and apoptotic cell death. Oxidative stress mediated double stranded DNA damage paved the way towards apoptotic cell death through multiple routes, including PI3K/Akt/NFκB pathway. The abundant alkaloid content of T. racemosa was denoted as the probable responsible cytotoxic principle.

A proteoglycan extract from Ganoderma Lucidum protects pancreatic beta-cells against STZ-induced apoptosis.

The pancreatic β-cell death or dysfunction induced by oxidative stress plays an important effect on the development and progression of diabetes mellitus. Based on our previous findings, a natural proteoglycan extracted from Ganoderma Lucidum, named FYGL, could treat T2DM in vivo. In this study, we investigated the effects of FYGL on STZ-induced apoptosis of INS-1 cells and its underlying mechanisms. The results showed that FYGL significantly improved the cell viability and alleviated the apoptosis in STZ-treated INS-1 cells. Moreover, FYGL markedly decreased the intracellular ROS accumulation and NO release, and deactivated NF-κB, JNK, and p38 MAPK signaling pathways in STZ-induced INS-1 cells. Furthermore, FYGL improved the insulin secretion through inhibiting the activation of JNK and improving the expression of Pdx-1 in INS-1 cells damaged by STZ. These results indicated that FYGL could protect pancreatic β-cells against apoptosis and dysfunction, and be used as a promising pharmacological medicine for diabetes management. Abbreviations: T2DM: type 2 diabetes mellitus; FYGL: Fudan-Yueyang G. lucidum; ROS: reactive oxygen species; NO: reactive oxygen species; NF-κB: nuclear factor kappa beta; JNK: c-jun N-terminal kinase; MAPK: mitogen-activated protein kinase; Pdx-1: Pancreatic duodenal homeobox 1.

Titanate nanofibers reduce Kruppel-like factor 2 (KLF2)-eNOS pathway in endothelial monolayer: A transcriptomic study

Although titanate nanofibers (TiNFs) and titanate nanotubes (TiNTs) have been proposed as relatively biocompatible nanomaterials (NMs), there is currently lacking of systemic studies which investigated the toxicity of TiNFs and TiNTs to endothelium. In this study, we developed endothelial monolayer model by using cell culture inserts, and systemically investigated the toxicity of TiNFs and TiNTs by RNA-seq, with a focus on Kruppel-like factor (KLF)-mediated effects, since KLF are transcription factors (TF) involved in the regulation of vascular biology. It was shown that NMs did not significantly induce cytotoxicity despite substantial internalization. However, the expression of many KLF was altered, and Western blot further confirmed that NMs down-regulated KLF2 proteins. Ingenuity pathway analysis (IPA) revealed that NMs altered the expression of KLF2-targed genes, typically the genes involved in inflammatory responses. KLF2-related Gene Ontology (GO) terms and Kyoto Encyclopedia of Gene and Genomes (KEGG) pathways were also altered, and it should be noticed that NMs altered GO terms and KEGG pathways related with endothelial NO synthase (eNOS). This study further verified that NMs decreased intracellular NO and eNOS proteins. All the observed effects were more obvious for TiNFs compared with TiNTs. Combined, this study showed that TiNFs or TiNTs were non-cytotoxic to endothelial monolayer model, but TiNFs and more modestly TiNTs decreased KLF2 leading to decreased eNOS proteins and NO production. Our data may provide novel understanding about the toxicity of TiNFs as well as other Ti-based NMs to endothelium.

Inducible Nitric Oxide Regulates Na-Glucose Co-transport in a Spontaneous SAMP1/YitFc Mouse Model of Chronic Ileitis.

In mammalian small intestine, glucose is primarily absorbed via Na-dependent glucose co-transporter (SGLT1) on the brush border membrane (BBM) of absorptive villus cells. Malabsorption of nutrients (e.g., glucose) leads to malnutrition, a common symptom of inflammatory bowel disease (IBD), where the mucosa is characterized by chronic inflammation. Inducible nitric oxide (iNO) is known to be elevated in IBD mucosa. SAMP1/YitFc (SAMP1) mouse is a spontaneous model of chronic ileitis that develops lesions in its terminal ileum, very similar to human IBD. How SGLT1 may be affected in SAMP1 model of chronic ileitis is unknown. Ten-week-old SAMP1 mice with AKR mice as control were treated with N6-(1-iminoethyl)-L-lysine dihydrochloride (L-NIL) to inhibit iNO production. Intracellular NO levels were found to be increased in villus cells from SAMP1 mice. Moreover, SGLT1 and Na+/K+-ATPase activities and BBM SGLT1 expression were significantly decreased. However, L-NIL treatment reduced the intracellular iNO production, and reversed both downregulated SGLT1 and Na+/K+-ATPase activities in SAMP1 mice. Inhibition of iNO by L-NIL treatment also significantly reversed the BBM SGLT1 protein expression in SAMP1 mice. L-NIL reversed the inflammation mediated downregulation of SGLT1 activity by restoring the BBM SGLT1 expression. Thus, regulation of SGLT1 in chronic ileitis is likely mediated by iNO.

MRP5 nitration by NO-releasing gemcitabine encapsulated in liposomes confers sensitivity in chemoresistant pancreatic adenocarcinoma cells

Pancreatic ductal adenocarcinoma (PDAC) is a therapy recalcitrant disease characterized by the aberrations in multiple genes that drive pathogenesis and drug chemoresistance. In this study, we synthesize a library of seven novel nitric oxide-releasing gemcitabine pro-drugs (NO-GEMs) in order to improve the effectiveness of GEM by exploiting the therapeutic effects of NO. Among these NO-GEM pro-drugs we select 5b as the most effective compound in GEM-resistant PDAC cells. After its encapsulation in liposomes for drug delivery the intracellular NO level increases and nitration associated to activity inhibition of the multidrug resistance associated protein 5 (MRP5; ABCC5) occurs. This results in GEM intracellular accumulation and enhanced apoptotic cell death in GEM-resistant PDAC cells, which express MRP5 at higher levels than GEM-sensitive cells. Our results support the development of a new anti-tumoral strategy to efficiently affect GEM-resistant PDAC cells based on the usage of NO-GEM pro-drugs.