Necrotic Death Research Articles

Enhancing KCNQ Channel Activity Improves Neurobehavioral Recovery after Spinal Cord Injury.

Spinal cord injury (SCI) usually leads to acute neuronal death and delayed secondary degeneration, resulting in sensory dysfunction, paralysis, and chronic pain. Excessive excitation is one of the critical factors leading to secondary neural damage initiated by various insults. KCNQ/Kv7 channels are highly expressed in spinal neurons and axons and play an important role in controlling their excitability. Enhancing KCNQ channel activity by using its specific opener retigabine could thus be a plausible treatment strategy to reduce the pathology after SCI. We produced contusive SCI at T10 in adult male rats, which then received 10 consecutive days' treatment with retigabine or vehicle starting 3 hours or 3 days after contusion. Two different concentrations and two different delivery methods were applied. Delivery of retigabine via Alzet osmotic pumps, but not intraperitoneal injections 3 hours after contusion, promoted recovery of locomotor function. Remarkably, retigabine delivery in both methods significantly attenuated the development of mechanical stimuli-induced hyperreflexia and spontaneous pain; however, no significant difference in the thermal threshold was observed. Although retigabine delivered 3 days after contusion significantly attenuated the development of mechanical hypersensitivity and spontaneous pain, the locomotor function is not improved by the delayed treatments. Finally, we found that early application of retigabine attenuates the inflammatory activity in the spinal cord and increases the survival of white matter after SCI. Our results suggest that decreasing neuronal excitability by targeting KCNQ/Kv7 channels at acute stage aids the recovery of locomotor function and attenuates the development of neuropathic pain after SCI. SIGNIFICANCE STATEMENT: Several pharmacological interventions have been proposed for spinal cord injury (SCI) treatment, but none have been shown to be both effective and safe in clinical trials. Necrotic neuronal death and chronic pain are often the cost of pathological neural excitation after SCI. We show that early, brief application of retigabine could aid locomotor and sensory neurobehavioral recovery after SCI, supporting the use of this drug in the clinic to promote motor and sensory function in patients with SCI.

Hydrogen Sulfide Attenuates the Neurotoxicity in the Animal Model of Fetal Alcohol Spectrum Disorders.

Fetal alcohol spectrum disorder (FASD), which is caused by prenatal alcohol exposure, can result in cell death in specific brain regions. Alcohol-induced neurocognitive defects offspring's are included with activation of oxidative-inflammatory cascade followed with wide apoptotic neurodegeneration in many brain's regions such as hippocampus. According to the latest studies, H2S (hydrogen sulfide) can protect neuronal cells via antioxidant, anti-inflammatory, and anti-apoptotic mechanisms in different animal models. Therefore, we aimed to evaluate the protective effects of H2S on ethanol-induced neuroinflammation and neuronal apoptosis in pup hippocampus with postnatal alcohol exposure. Administration of ethanol (5.27g/kg) in milk solution (27.8mL/kg) for each rat pups was performed through intragastric intubation on 2 to 10 postnatal days and NaHS as H2S donor (1mg/kg) was injected on similar time, subcutaneously. For examining the antioxidant and anti-inflammatory effects, ELISA assay was performed to determine the levels of TNF-α, IL1β, and antioxidant enzymes. Immunohistochemical staining was performed to evaluate the expression levels of GFAP and caspase-3 also Nissl staining was done for necrotic cell death evaluation. H2S treatment could significantly increase the activity of total superoxide dismutase, catalase, and glutathione (P < 0.05). It also decreased the levels of TNF-α, IL1β, and malondialdehyde, compared with the ethanol group (P < 0.05). Moreover, the number of hippocampal caspase-3, GFAP-positive cells, and necrotic cells death reduced in the H2S group (P< 0.01). Based on the findings, H2S can inhibit apoptotic signaling that is mediated by the oxidative-inflammatory cascade following ethanol exposure of rat pups on postnatal period.

Anti-cancer therapeutic benefit of red guava extracts as a potential therapy in combination with doxorubicin or targeted therapy for triple-negative breast cancer cells.

Guava extracts purified from leaf and bark have many bio-active molecules with anti-cancer activities. In addition, lycopene-rich extracts obtained from red guava fruit can induce apoptosis in estrogen receptor-positive breast cancers. Triple-negative breast cancer (TNBC) lacks estrogen receptors, progesterone receptors and human epidermal growth factor receptor 2 (HER2) and, therefore, hormone therapy and targeted therapy are not used in the clinic. The purpose of this study was to determine whether red guava fruit extracts can affect the proliferation of TNBC cells. In this study, cell viability was determined by using the MTT assay. Apoptosis and necrosis were analyzed using flow cytometry. Cleaved caspase-3 and PARP were analyzed by western blotting. We found that red guava extracts can, through caspase-3 activation and PARP cleavage signaling, induce apoptotic and necrotic death in TNBC cells. Our results thus show the therapeutic benefit of red guava extracts as a potential cancer treatment for TNBC in combination with doxorubicin or targeted therapy.

Activation of Signal Pathways of Apoptosis under Conditions of Prolonged ER-Stress Caused by Exposure of Mouse Testicular Teratoma Cells to Selenium-Containing Compounds.

Aim to study the molecular mechanisms of apoptotic death of mouse testicular teratocarcinoma cells (line F-9) under exposure to the widely used selenium-containing compounds with antitumor activity, sodium selenite and methylseleninic acid. Methods fluorescence microscopy, MTT assay, Western blotting. Results It was shown that sodium selenite at a concentration of 10 μM and methylseleninic acid at concentrations of 1 and 10 μM cause apoptosis-dependent death of F-9 cells, excluding necrotic death. Western blotting showed an increase in the expression of XBP1s when treating F-9 cells with 1 μM methylseleninic acid. Conclusions 10 μM methylseleninic acid leads to cell apoptosis, most likely by activation of the IRE1 signaling pathway under prolonged stress of the endoplasmic reticulum.

GSK3\u03b2 is a key regulator of the ROS-dependent necrotic death induced by the quinone DMNQ

Signaling pathways controlling necrosis are still mysterious and debated. We applied a shRNA-based viability screen to identify critical elements of the necrotic response. We took advantage from a small molecule (G5) that makes covalent adducts with free thiols by Michael addition and elicits multiple stresses. In cells resistant to apoptosis, G5 triggers necrosis through the induction of protein unfolding, glutathione depletion, ER stress, proteasomal impairments, and cytoskeletal stress. The kinase GSK3β was isolated among the top hits of the screening. Using the quinone DMNQ, a ROS generator, we demonstrate that GSK3β is involved in the regulation of ROS-dependent necrosis. Our results have been validated using siRNA and by knocking-out GSK3β with the CRISPR/Cas9 technology. In response to DMNQ GSK3β is activated by serine 9 dephosphorylation, concomitantly to Akt inactivation. During the quinone-induced pro-necrotic stress, GSK3β gradually accumulates into the nucleus, before the collapse of the mitochondrial membrane potential. Accumulation of ROS in response to DMNQ is impaired by the absence of GSK3β. We provide evidence that the activities of the obligatory two-electrons reducing flavoenzymes, NQO1 (NAD(P)H quinone dehydrogenase 1) and NQO2 are required to suppress DMNQ-induced necrosis. In the absence of GSK3β the expression of NQO1 and NQO2 is dramatically increased, possibly because of an increased transcriptional activity of NRF2. In summary, GSK3β by blunting the anti-oxidant response and particularly NQO1 and NQO2 expression, favors the appearance of necrosis in response to ROS, as generated by the quinone DMNQ.

Suppression of time-dependent decay by controlling the redox balance of human induced pluripotent stem cells suspended in a cryopreservation solution

To scale-up a filling process in a cell manufacturing system, the impact of the processing time on the final product quality should be evaluated. It is known that the suspended cells in a cryopreservation solution are subjected to various types of stress factors, which eventually results in apoptotic or necrotic death. In this study, the aim was to investigate a critical factor of cell decay in human induced pluripotent stem cells suspended in a cryopreservation solution and to propose a methodology to maintain the viability of cells for a longer filling process time. Based on experimental results, it was determined that the accumulation of intracellular reactive oxygen species induces apoptosis of cells suspended in a cryopreservation solution. In addition, treatment with an antioxidant or a low-temperature condition was effective in suppressing the induction of apoptosis and subsequent cell death. In particular, the treatment at low temperature was able to maintain the attachment efficiency of suspended cells at a higher level compared to the treatment with antioxidants. The appropriate control of redox balance in cells suspended in a cryopreservation solution could improve the viability of cells in the filling process and lead to the successful lot size expansion of cell manufacturing systems.

Biopolymer K-carrageenan wrapped ZnO nanoparticles as drug delivery vehicles for anti MRSA therapy

Kappa-Carrageenan wrapped ZnO nanoparticles (KC-ZnO NPs) was synthesized, physico-chemically characterized and evaluated their biocompatibility and antimicrobial therapy against MRSA. XRD showed the highly crystalline and hexagonal phase structure of ZnO NPs. FETEM confirmed the spherical and hexagonal shaped particle with the mean size of 97.03 ± 9.05 nm. The synthesized KC-ZnO NPs exhibited significant antibacterial activity against MRSA. The biofilm growth of MRSA was greatly inhibited at 100 μg/ml as observed through live and dead cell assay. KC-ZnO NPs have shown invitro anti-inflammatory activity (82%) at 500 μg/ml. KC-ZnO NPs was non-toxic to NIH3T3 mouse embryonic fibroblasts cell lines. Further, no apoptotic and necrotic mediated death in NIH3T3 mouse embryonic fibroblasts cells were noticed by flow cytometric analysis. KC-ZnO NPs have good biocompatibility as recorded by the least hemolysis percentage (<3%) up to 100 μg/ml, which is much lesser than the acceptable limit. In addition, ecosafety analysis has shown that KC-ZnO NPs and kappa karrageenan (0–500 μg/ml) caused no mortality of A. salina after 48 h. However, bare zinc acetate has shown 35% mortality of A. salina after 48 h. The results conclude that KC-ZnO NPs could be a novel antibacterial therapy for the treatment of MRSA associated infectious.

Methods for the Measurement of Early Events in Toxoplasma gondii Immunity in Mouse Cells.

Critical steps in resistance of mice against Toxoplasma gondii occur in the first 2 or 3h after the pathogen has entered a cell that has been exposed to interferon γ (IFNγ). The newly formed parasitophorous vacuole is attacked by the IFNγ-inducible IRG proteins and disrupted, resulting in death of the parasite and necrotic death of the cell. Here we describe some techniques that we have used to describe and quantify these events in different combinations of the host and the parasite.

Assays for Inducing and Measuring Cell Death to Detect Macrophage Migration Inhibitory Factor (MIF) Release.

Cell death is a vital process for maintaining tissue homeostasis and removing potentially harmful cells. Cell death can be both programmed and non-programmed and is commonly divided into two main forms, termed apoptotic and necrotic death modes. In this chapter cell death is classified into apoptosis, primary necrosis, pyroptosis, and necroptosis. This chapter outlines the measurement of these different types of cell death and the relationship of measuring MIF release in these assays.

THE IMPACT OF DIABETES MELLITUS ON THE FUNCTIONAL STATE OF THE MITOCHONDRIA IN THE HEART OF MALE AND FEMALE RATS

Type 2 diabetes is one of the main factors of the cardiovascular risk, which leads to a disproportionateincrease in cardiovascular events in women and men. All causes of increased cardiovascular risk in women withtype 2 diabetes mellitus are not established, one of the most likely causes of cardiac activity is considered to begender features of biogenesis and activity of myocardial mitochondria. A comparative analysis of changes in thefunctional state of the heart mitochondria in males and females rats with experimental type 2 diabetes mellituswas conducted. It was established that the activity of succinate dehydrogenase, the part of complex II of theelectron transport chain, reduced more significantly in diabetic males compared with diabetic females. Type 2diabetes mellitus also caused a greater decrease in the activity of the energy metabolism enzyme — aconitase,in heart mitochondria of male compared to female. One of the leading mechanisms of cardiomyocytes’ apoptoticor necrotic death in conditions of mitochondrial dysfunction, in particular induced by diabetes, is the activationof a nonspecific mitochondrial pore opening (mPTP, mitochondrial permeability transition pore). It was founda more pronounced activation of mPTP and more significant decrease in thioredoxin reductase activity inmales with type 2 diabetes mellitus compared with diabetic females. The obtained data may indicate a greatersensitivity of male cardiomyocytes to the damaging effects of proapoptotic factors

Overexpression of Mitochondrial Calcium Uniporter Causes Neuronal Death.

Neurodegenerative diseases are a large and heterogeneous group of disorders characterized by selective and progressive death of specific neuronal subtypes. In most of the cases, the pathophysiology is still poorly understood, although a number of hypotheses have been proposed. Among these, dysregulation of Ca2+ homeostasis and mitochondrial dysfunction represent two broadly recognized early events associated with neurodegeneration. However, a direct link between these two hypotheses can be drawn. Mitochondria actively participate to global Ca2+ signaling, and increases of [Ca2+] inside organelle matrix are known to sustain energy production to modulate apoptosis and remodel cytosolic Ca2+ waves. Most importantly, while mitochondrial Ca2+ overload has been proposed as the no-return signal, triggering apoptotic or necrotic neuronal death, until now direct evidences supporting this hypothesis, especially in vivo, are limited. Here, we took advantage of the identification of the mitochondrial Ca2+ uniporter (MCU) and tested whether mitochondrial Ca2+ signaling controls neuronal cell fate. We overexpressed MCU both in vitro, in mouse primary cortical neurons, and in vivo, through stereotaxic injection of MCU-coding adenoviral particles in the brain cortex. We first measured mitochondrial Ca2+ uptake using quantitative genetically encoded Ca2+ probes, and we observed that the overexpression of MCU causes a dramatic increase of mitochondrial Ca2+ uptake both at resting and after membrane depolarization. MCU-mediated mitochondrial Ca2+ overload causes alteration of organelle morphology and dysregulation of global Ca2+ homeostasis. Most importantly, MCU overexpression in vivo is sufficient to trigger gliosis and neuronal loss. Overall, we demonstrated that mitochondrial Ca2+ overload is per se sufficient to cause neuronal cell death both in vitro and in vivo, thus highlighting a potential key step in neurodegeneration.

Characterization of HemY-type protoporphyrinogen IX oxidase genes from cyanobacteria and their functioning in transgenic Arabidopsis.

We investigated the functions of two cyanobacterial HemY protoporphyrinogen IX oxidase (PPO) genes with in vitro and in vivo assays and evaluated their applicability as resistance traits to PPO-inhibiting herbicides. We isolated HemY-type protoporphyrinogen IX oxidase (PPO) genes from cyanobacteria, OnPPO gene from Oscillatoria nigro-viridis PCC7112 and HaPPO gene from Halothece sp. PCC7418. The alignment of amino acid sequences as well as phylogenetic analyses conducted showed that OnPPO and HaPPO are classified as HemY-type PPO and are more closely related to plastidic PPOs than to mitochondrial PPOs. The PPO-deficient Escherichia coli BT3 strain, which requires heme supplementation, could obtain normal growth in the absence of heme supplementation when complemented with OnPPO and HaPPO. The enzyme assays of OnPPO, HaPPO, and Arabidopsis thaliana PPO1 (AtPPO1) proteins each revealed different kinetic properties in terms of catalytic efficiency, substrate affinity, and the degree of inhibition by PPO inhibitors. In particular, the catalytic efficiencies (kcat/Km) of OnPPO and HaPPO were approximately twofold higher than that of AtPPO1. The elution profiles of all three PPOs, acquired by size-exclusion chromatography, showed only a single peak with a molecular weight of approximately 52-54kDa, which corresponds to a monomeric form. Moreover, functional complementation with OnPPO and HaPPO in AtPPO1-silenced Arabidopsis resulted in restored growth, whereas AtPPO1-silenced wild type Arabidopsis suffered necrotic death. In addition, we observed that overexpression of OnPPO and HaPPO in Arabidopsis conferred resistance to the PPO-inhibiting herbicides tiafenacil and saflufenacil. These results suggest that two HemY-type PPOs of cyanobacteria can functionally substitute for plastidic PPO activity in Arabidopsis and can enhance resistance to tiafenacil and saflufenacil.

Virulence and pathogenicity of a Candida albicans mutant with reduced filamentation.

Deletion of DNA polymerase eta (Rad30/Polη) in pathogenic yeast Candida albicans is known to reduce filamentation induced by serum, ultraviolet, and cisplatin. Because nonfilamentous C. albicans is widely accepted as avirulent form, here we explored the virulence and pathogenicity of a rad30Δ strain of C. albicans in cell-based and animal systems. Flow cytometry of cocultured fungal and differentiated macrophage cells revealed that comparatively higher percentage of macrophages was associated with the wild-type than rad30Δ cells. In contrast, higher number of Polη-deficient C. albicans adhered per macrophage membrane. Imaging flow cytometry showed that the wild-type C. albicans developed hyphae after phagocytosis that caused necrotic death of macrophages to evade their clearance. Conversely, phagosomes kill the fungal cells as estimated by increased metacaspase activity in wild-type C. albicans. Despite the morphological differences, both wild-type and rad30∆ C. albicans were virulent with a varying degree of pathogenicity in mice models. Notably, mice with Th1 immunity were comparatively less susceptible to systemic fungal infection than Th2 type. Thus, our study clearly suggests that the modes of interaction of morphologically different C. albicans strains with the host immune cells are diverged, and host genetic background and several other attributing factors of the fungus could additionally determine their virulence.

In vitro anti-proliferative and anti-bacterial properties of new C7 benzoate derivatives of pinocembrin

In the present work, the in vitro anti-proliferative and anti-bacterial activities of three semi-synthetic benzoate pinocembrin derivatives, isolated from the aerial parts of Glycyrrhiza glabra L., were investigated. As occurs in most natural compounds, the bioavailability of pinocembrin is very poor, therefore it should be improved by chemical strategies aimed to prolong its shelf life and, consequently, its activity. On this basis, three benzoate derivatives of pinocembrin (a1–a3) were synthesised and assayed in order to ascertain their biological value. Among them, compound a1 showed the highest anti-proliferative activity on a wide panel of cancer cell lines, as well as low toxic effects on non-malignant breast cells. The calculated IC50 values in HeLa and SKBR3 cells were 8.5 and 12.7 µM, respectively. Briefly, a1 treatment increased ROS levels, induced mitochondrial membrane damage leading to necrotic death of HeLa cells. Moreover, a1 displayed a promising anti-bacterial activity.

High doses of sodium ascorbate interfere with the expansion of glioblastoma multiforme cells in vitro and in vivo

AimsConstant development of chemotherapeutic strategies has considerably improved the efficiency of tumor treatment. However, adverse effects of chemotherapeutics enforce premature treatment cessation, which leads to the tumor recurrence and accelerated death of oncologic patients. Recently, sodium ascorbate (ASC) has been suggested as a promising drug for the adjunctive chemotherapy of glioblastoma multiforme (GBM) and prostate cancer (PC). To estimate whether ASC can interfere with tumor recurrence between the first and second-line chemotherapy, we analyzed the effect of high ASC doses on the expansion of cells in vitro and in vivo. Main methodsBrightfield microscopy-assisted approaches were used to estimate the effect of ASC (1–14 mM) on the morphology and invasiveness of human GBM, rat PC and normal mouse 3T3 cells, whereas cytostatic/pro-apoptotic activity of ASC was estimated with flow cytometry. These assays were complemented by the in vitro CellROX-assisted analyses of intracellular oxidative stress and in vivo estimation of GBM tumor invasion. Key findingsASC considerably decreased the proliferation and motility of GBM and PC cells. This effect was accompanied by intracellular ROS over-production and necrotic death of tumor cells, apparently resulting from their “autoschizis”. In vivo studies demonstrated the retardation of GBM tumor growth and invasion in the rats undergone intravenous ASC administration, in the absence of detectable systemic adverse effects of ASC. SignificanceOur data support previous notions on anti-tumor activity of high ASC doses. However, autoschizis-related cell responses to ASC indicate that its application in human adjunctive tumor therapy should be considered with caution.

FOLFIRI-Mediated Toxicity in Human Aortic Smooth Muscle Cells and Possible Amelioration with Curcumin and Quercetin

Systemic chemotherapy-mediated cell toxicity is a major risk factor for cardiovascular disease and atherosclerosis. Life-threatening acute events of the FOLFIRI (irinotecan, folinic acid and 5-fluorouracil) regimen are mainly due to DNA damage induced by antimetabolite and topoisomerase inhibition effects. However, the role of human aortic smooth muscle cells (HaVSMCs) in this process and the mechanisms of oxidative stress, DNA and protein damage and apoptosis have not been investigated. Therefore, the effects of curcumin and quercetin on HaVSMC survival in the generation of molecular and cellular toxicity by FOLFIRI treatment and the involvement of vital cellular signalling pathways were investigated. We analysed both FOLFIRI toxicity and the therapeutic potential of quercetin and curcumin in terms of HaVSMC damage using molecular probe and florescence staining, Random Amplified Polymorphic DNA (RAPD), qRT-PCR and Western blot assays. Our study presents two preliminary findings: (a) in HaVSMCs, FOLFIRI treatment significantly induces oxidative damage to both DNA and protein, leading to a dramatic increase in caspase-dependent apoptotic death through P53-mediated Caspase3-dependent mitochondrial apoptosis, and results in TNF-α/Caspase8-mediated necrotic death, and (b) flavonoids not only regulate the expression of genes encoding antioxidant enzymes and increase DNA damage but also limit programmed and necrotic cell death processes in HaVSMCs. Our results clearly indicate the potential for curcumin and, particularly, quercetin as preventative chemotherapeutic interventions for cardiovascular toxicity induced by the FOLFIRI regime in HaVSMCs.

Study of antitumor activity of synthetic peptide ryqlhpyr on the prostate cancer cells

Introduction . The RHAMM (hyaluronan mediated mobility receptor) is overexpressed in many types of human cancer and increased synthesis of the RHAMM usually correlates with a poor prognostic factor. In this paper, we synthesized the peptide-RYQLHPYR modulating the activity of the RHAMM and examined the therapeutic potential of this RHAMM-targeting peptide as an antitumor agent.Objective . Study the effect of the synthetic peptide RYQLHPYR on viability, apoptosis, necrosis, caspase-3 / 7 activity, and invasion of prostate cancer cells.Materials and methods . The peptide RYQLHPYR was prepared by solid phase synthesis. Human prostate cancer cells (PC3 m-LN4), murine embryonic fibroblasts and murine embryonic fibroblasts (RHAMM- / -). To quantify the effect of the peptide on apoptosis and cell necrosis, ELISAPLUS was used. The activity of caspase-3 / 7 was determined by the colorimetric method. Evaluation of the anti-metastatic effect of the peptide in vitro was evaluated by invasion of cells by quantitative analysis of the area of degradation of fluorescent gelatin.Results . It was found that the peptide RYQLHPYR inhibited the growth of tumor cells PC3 m-LN4 at a concentration of 10 μg / ml (2 × 10–7 M) after 24 h by ~80 %. It was shown that the peptide stimulated the level of apoptosis in cancer cells, approximately 10-fold. It was found that the peptide increased the necrotic death of tumor cells by 2.5 times. During the research it was revealed that the peptide increased the caspase-3 / 7 activity in tumor cells by 2 times. At the same time, it was shown that RHAMM-targeting peptide had no significant effect on apoptosis and necrosis of normal cells (fibroblasts) and fibroblasts (RHAMM- / -). It was found that the peptide inhibited invasion of tumor cells by ~99.86 % at a concentration of 10 μg / ml (2 × 10–7 M).Conclusions . The obtained results indicate that the peptide RYQLHPYR has antitumor activity and, therefore, has a therapeutic potential for the treatment of prostate cancer.

Hepatic ferroptosis plays an important role as the trigger for initiating inflammation in nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is a metabolic liver disease that progresses from simple steatosis to the disease state of inflammation and fibrosis. Previous studies suggest that apoptosis and necroptosis may contribute to the pathogenesis of NASH, based on several murine models. However, the mechanisms underlying the transition of simple steatosis to steatohepatitis remain unclear, because it is difficult to identify when and where such cell deaths begin to occur in the pathophysiological process of NASH. In the present study, our aim is to investigate which type of cell death plays a role as the trigger for initiating inflammation in fatty liver. By establishing a simple method of discriminating between apoptosis and necrosis in the liver, we found that necrosis occurred prior to apoptosis at the onset of steatohepatitis in the choline-deficient, ethionine-supplemented (CDE) diet model. To further investigate what type of necrosis is involved in the initial necrotic cell death, we examined the effect of necroptosis and ferroptosis inhibition by administering inhibitors to wild-type mice in the CDE diet model. In addition, necroptosis was evaluated using mixed lineage kinase domain-like protein (MLKL) knockout mice, which is lacking in a terminal executor of necroptosis. Consequently, necroptosis inhibition failed to block the onset of necrotic cell death, while ferroptosis inhibition protected hepatocytes from necrotic death almost completely, and suppressed the subsequent infiltration of immune cells and inflammatory reaction. Furthermore, the amount of oxidized phosphatidylethanolamine, which is involved in ferroptosis pathway, was increased in the liver sample of the CDE diet-fed mice. These findings suggest that hepatic ferroptosis plays an important role as the trigger for initiating inflammation in steatohepatitis and may be a therapeutic target for preventing the onset of steatohepatitis.

361-OR: ER Stress Attenuation Is Involved in PAHSA-Induced Reduction of T1D in Nonobese Diabetic (NOD) Mice

Palmitic Acid esters of Hydroxy Stearic Acids (PAHSAs) are endogenous lipids with antidiabetic and anti-inflammatory effects. We found that PAHSAs protect against T1D by attenuating autoimmune responses and by direct effects on islets. We aimed to determine the mechanisms by which PAHSAs directly promote β-cell survival. Daily oral gavage of PAHSAs in NOD mice starting at 4 or 13 weeks of age delays T1D onset and reduces T1D incidence from 82-90% of mice with vehicle to 35-40% of mice receiving PAHSAs. PAHSA treatment reduces leukocyte infiltration into islets by 50% and reduces T cell activation. PAHSA-treated mice had greater β-cell area relative to total islet area (vehicle 60±7 vs. PAHSA 77±1). PAHSAs enhance β-cell proliferation in NOD mice 5 fold vs. vehicle. PAHSA-treated NOD mice pancreata have lower ER stress markers, CHOP and XBP-1 (vehicle 68±12 vs. PAHSA 42±3), and higher insulin (vehicle 12±2 vs. PAHSA 17±1) immunostaining intensities per β-cell area. PAHSAs attenuate thapsigargin-induced PARP cleavage by 55% in human islets, and JNK1/2 phosphorylation by 40% in MIN6 cells. Also, PAHSAs attenuate apoptotic and necrotic β-cell death under cytokine stress and increase β-cell viability by 34% and proliferation by 31% (p&amp;lt;0.002). Since GLP-1 receptor (GLP-1R) antagonism causes β-cell death and G-protein coupled receptor GPR40 plays a vital role in β-cell function, we assessed if PAHSA beneficial effects on β-cell survival involve these pathways. In vitro, GLP-1R antagonism lowered PAHSA protective effects on β-cell viability by 53% and proliferation by 44% (p&amp;lt;0.005) under cytokine stress but GPR40 antagonism had no effect. Conclusion: In addition to attenuating immune responses, PAHSAs delay the onset and reduce T1D incidence in NOD mice through direct effects. PAHSAs directly reduce cytokine-induced β-cell death and increase cell proliferation in autoimmune diabetes through mechanisms involving ER stress reduction. This appears to be mediated in part by GLP-1R and not by GPR40. Disclosure I. Syed: None. M. Fernandez Rubin de Celis: None. J.F. Mohan: None. P.M. Moraes-Vieria: None. A. Vijayakumar: None. A.T. Nelson: None. D. Siegel: None. A. Saghatelian: None. D. Mathis: None. B. Kahn: Advisory Panel; Self; Alterna Therapeutics, Inc., American Diabetes Association, Harrington Discovery Institute, Janssen Research &amp; Development. Consultant; Self; Ironwood Pharmaceuticals, Inc. Research Support; Self; National Institute of Diabetes and Digestive and Kidney Diseases. Funding National Institutes of Health (R01DK43051, P30DK57521 to B.K.), (R01DK106210 to B.K., A.S.), (K01DK118041 to I.S.); JPB Foundation (to B.K., D.M.); Deutsche Forschungsgemeinschaft (to M.F.R.)

Anticancer potential of zinc oxide nanoparticles against cervical carcinoma cells synthesized via biogenic route using aqueous extract of Gracilaria edulis

Development of novel approach for cancer therapy, sparing healthy normal cells overcoming the limitation of available therapies is of prime importance for cervical cancer treatment. Recently metal oxide based chemotherapeutics has emanated as a promising approach for cancer therapy. Hence, the present study was carried out to assess the anticancer potential of zinc oxide nanoparticles (ZnONPs) synthesized using biogenic source, aqueous extract of Gracilaria edulis. The prepared ZnONPs were characterized using UV–Visible spectroscopy, FTIR, XRD, FESEM, EDX and HRTEM. The anticancer potential of ZnONPs against cervical cancer cell lines (SiHa cells) was evaluated using MTT and the mechanism of apoptosis was evaluated using various staining techniques. UV–Vis spectroscopy exhibited absorption band at 367 nm specific for ZnONPs and the average energy gap was calculated as 3.37 eV. Further characterization by XRD, TEM, and FESEM illustrated the formation of wurtzite structure (hexagonal phase) with size ranging between 20 and 50 nm. EDS of SEM analysis confirmed the presence of Zn and O, which was further substantiated by XPS analysis. PL emission studies showed UV emission peak at 387 nm and broad visible emission peak at 520 nm. Zeta potential value of −28.2 mV depicted the stability of ZnONPs in the dispersion medium. Results of anticancer potential illustrated that ZnONPs exhibited cytotoxic effect against SiHa cells in a dose dependent manner with IC50 value of 35 ± 0.03 μg/ml. AO/EtBr dual staining, JC-1 staining, Hoechst 33258 nuclear staining and comet assay illustrated the ZnONPs induced ROS mediated mitochondrial dependent apoptotic cell death in SiHa cells. Further, flow cytometric analysis using Annexin V/FITC dye demonstrated that ZnONPs induced both apoptotic and necrotic mediated death in SiHa cells. Over all the results conclude that ZnONPs synthesized using algal sources might act as a new medicinal approach for the treatment of cervical carcinoma in conjugation with the current therapy.