Upregulation Of Oxidative Stress Research Articles

Inflammatory Stimulation Mediates Nucleus Pulposus Cell Necroptosis Through Mitochondrial Function Disfunction and Oxidative Stress Pathway.

The mutual activation between nucleus pulposus (NP) cells death and inflammation is an important pathogenic factor of intervertebral disc degeneration. Whether inflammation mediates NP cells necroptosis, and its relationship with mitochondrial dysfunction and oxidative stress remains unclear. In this study, 50 ng/mL of TNF-α and 20 ng/mL of IL-1β were used to co-treatment with rat NP cells for 0, 24, 48, 72 hours, then Western blot and RT-PCR techniques were utilized to evaluate the expression level of necroptosis-associated target molecules, such as RIPK1, RIPK3 and MLKL. The results established that with prolongation of TNF-α and IL-1β treatment time, the expression level of necroptosis-associated molecules gradually increased. The 48 hours of TNF-α and IL-1β treatment was selected throughout the following experiments. The RIPK1 specific inhibitor necrostatin-1 (Nec-1), RIPK3 inhibitor GSK872, MLKL inhibitor necrosulfonamide (NSA) and small interfering RNA (siRNA) technology were employed. Under the treatment of TNF-α or IL-1β, administration of Nec-1, GSK872 or NSA notably reduced NP cells death and up-regulated NP cells viability. Consistently, SiRNA-mediated knockdown of RIPK3 (SiRIPK3) or MLKL (SiMLKL) promoted the survival of NP cells. However, SiRIPK1 aggravated NP cells death. Furthermore, after 48 hours of TNF-α and IL-1β treatment, the mitochondrial membrane potential decreased, opening of mitochondrial permeability transition pore enhanced, and oxidative stress level notably elevated. The Nec-1, GSK872 or NSA treatment largely restored the normal mitochondrial function and down-regulated oxidative stress. In summary, RIPK1/RIPK3/MLKL-mediated necroptosis play an important role in NP cells death during inflammatory irritation, which might be closely related to mitochondrial dysfunction and up-regulation of oxidative stress.

Up-regulation of oxidative stress and inflammation in the brain of albino wistar rats following sub-acute administration of Synclisia scabrida root extract

Up-regulation of oxidative stress and inflammation in the brain of albino wistar rats following sub-acute administration of Synclisia scabrida root extract

Abstract 12003: TN-C Promotes Cardiac Fibrosis and Endothelial Dysfunction in Duchenne Muscular Dystrophy

Background: Cardiac fibrosis and heart failure are major contributors to mortality in Duchenne muscular dystrophy (DMD) patients. Recently, it has been found that Tenascin C (TN-C) plays a maladaptive role in adverse left ventricular (LV) remodelling and fibrosis. Aims: To 1) elucidate the vascular dysfunction and cardiac fibrosis in link to TN-C in a mouse model of DMD and 2) explore the effect of knocking out TN-C in dystrophic mice. Methods: Male wt, mdx and mdx TN-C KO mice were used. Echocardiography was performed, and fibrosis was assessed on cardiac tissue sections. To test the vascular reactivity wire myography was used and CD31+ endothelial cells (ECs) were isolated from mouse lung tissues and characterized for oxidative stress and inflammatory marker expression. To explore further the signalling pathways contributing to cardiac fibrosis, human cardiac fibroblasts (hCFs) were treated with TN-C or TGF-β, then fibrotic markers and epigenetic regulation of p65 were assessed. Results: Cardiac fibrosis was markedly enhanced in mdx mice compared to age-matched wt animals. This was accompanied by upregulation of TN-C in cardiac tissue and plasma. In addition, vascular endothelial function was impaired in mdx mice. In line with that, endothelial cells isolated from mdx mice also showed a marked upregulation of oxidative stress and inflammatory markers. Interestingly, mdx- TN-C KO mice showed preserved vascular function as well as reduced cardiac fibrosis compared to age-matched mdx mice. hCFs treated with TN-C or TGF-β showed increased collagen production and elevated α-SMA and matrix metalloproteinase 2, 3 and 9 (MMP-2, 3 and 9) expressions. TN-C siRNA reduced the expression of collagen and α-SMA, but not MMPs. In addition, both TN-C and TGF-β promote p65/ NF-κB promoter demethylation and subsequently stimulate pro-inflammatory and pro-fibrotic signalling. These effects were reversed by applying p38 MAPK inhibitor in hCFs. Conclusions: TN-C facilitates oxidative stress and inflammation in ECs and fibroblasts, contributing to severe endothelial dysfunction and cardiac fibrosis. In addition, activation of NF-κB p65 signalling pathway may play a role in TN-C induced fibrosis. Thus, TN-C may be a critical mediator and potential target for therapy in DMD.

A proteomic analysis of the effect of ocean acidification on the haemocyte proteome of the South African abalone Haliotis midae

As a result of increasing CO2 emissions and the prevalence of global climate change, ocean acidification (OA) is becoming more pervasive, affecting many trophic levels, particularly those that rely on succinctly balanced ocean chemistry. This ultimately threatens community structures, as well as the future sustainability of the fishing/aquaculture industry. Understanding the molecular stress response of key organisms will aid in predicting their future survivability under changing environmental conditions. This study sought to elucidate the molecular stress response of the South African abalone, Haliotis midae, an understudied organism with high economic value, utilising a high throughput iTRAQ-based proteomics methodology. Adult abalone were exposed to control (pH 7.9) and experimental (pH 7.5) conditions for 12, 72 and 168 h, following which protein was isolated from sampled haemocytes and subsequently processed. iTRAQ-labelled peptides were analysed using mass spectrometry, while an array of bioinformatics tools was utilised for analysing the proteomic data. COG analysis identified “Cytoskeleton”, “Translation, ribosomal structure and biogenesis”, “Post-translational modification, protein turnover, chaperones”, and “Intracellular trafficking, secretion and vesicular transport” to be the most enriched functional classes, while statistical analysis identified a total of 33 up-regulated and 23 down-regulated effectors of OA stress in abalone. Several of the up-regulated proteins that were identified function in central metabolism (ENO1, PGK, DUOX1, GPD2), the stress/immune response (CAMKI, HSPA5/GRP78, MAPKI), and cytoskeleton, protein sorting and signal transduction (IQGAP1, MYO9B, TLN1, RDX, TCP-1/CCT, SNX6, CHMP1a, VPS13a). Protein-protein interactions were predicted using STRING DB, Cytoscape and Ingenuity Pathway Analysis, providing a model of the effects of OA on the H. midae haemocyte proteome. The data indicated that H. midae underwent a metabolic shift under OA conditions to utilize more energy-efficient mechanisms of ATP generation, while attempts at restoring haemocyte stabilisation and homeostasis were reflected by up-regulation of oxidative stress and cytoskeletal proteins. Our results support other molluscan studies that report a complex array of overlapping functions of both the stress and immune response systems. This interplay of the mounted stress and immune response is maintained and observed through the up-regulation of proteins involved in protein synthesis and turnover, as well as intracellular signalling and transport. The data presented in this study highlight the value of employing sensitive and robust -omics technologies for assessing the effects of changing environmental conditions on marine organisms.

Resveratrol ameliorates type 2 diabetes mellitus-induced alterations to the knee joint articular cartilage ultrastructure in rats

ABSTRACT Diabetes-induced osteoarthritis (OA) is a chronic inflammatory disease that damages the cartilage in the joints and could lead to disability. The protective effect of the antioxidant and anti-inflammatory agent, resveratrol, against alterations to the knee articular cartilage ultrastructure induced by type 2 diabetes mellitus (T2DM) associated with the inhibition of dyslipidemia, oxidative stress, and inflammation has not been investigated before. Therefore, we modeled OA in rats 10 weeks post diabetic induction using a high carbohydrate and fat diet and a single injection of streptozotocin (50 mg/kg body weight), and the protective group of rats started resveratrol (30 mg/kg; orally) treatment 2 weeks before diabetic induction and continued on resveratrol until the end of the experiment at week 12. Blood chemistry analysis confirmed hyperglycemia (elevated glucose and glycated hemoglobin, HbA1c), dyslipidemia (elevated triglyceride, cholesterol, and low-density lipoprotein-cholesterol), and upregulation of oxidative stress (malondialdehyde) and inflammatory (C-reactive protein and tumor necrosis factor-α) biomarkers in the model group. In addition, using light and electron microscopy examinations, we also observed in the model group substantial damage to the articular cartilage and profound chondrocyte and territorial matrix ultrastructural alterations such as chondrocytes with degenerated nucleus and mitochondria, scarce cytoplasmic processes, and absence of the fine fibrillar appearance of territorial matrix. Resveratrol pretreatment significantly (p ≤ 0.0029) but not completely protected from T2DM-induced OA. We conclude that resveratrol protects against alterations to the articular cartilage ultrastructure induced secondary to T2DM in rats, which is associated with the inhibition of glycemia, hyperlipidemia, and biomarkers of oxidative stress and inflammation.

Glucose-6-phosphate dehydrogenase deficiency contributes to metabolic abnormality and pulmonary hypertension

We have previously reported that several patients with idiopathic pulmonary hypertension (PH) had different types of G6PD deficiency. However, the role of G6PD in PH is multifactorial because G6PD is involved in controlling oxidative stress, metabolic switch, and red blood cell fragility. To delineate the contribution of G6PD to PH pathogenesis, we utilized a mouse line with decreased expression of G6PD (10% from wild-type level). We confirmed that mice with G6PD deficiency develop spontaneous pulmonary hypertension with pulmonary artery and right heart remodeling. G6PD deficiency resulted in increased free hemoglobin and activation of the p38 pathway, which we recently reported induces the development of PH in the sugen/hypoxia model via endothelial barrier dysfunction. Metabolomics analysis of G6PD deficient mice indicates the switch to alternative metabolic fluxes that feed into the pentose phosphate pathway (PPP), resulting in the upregulation of oxidative stress, fatty acid pathway, and reduction in pyruvate production. Thus, G6PD deficiency did not reduce PPP flux that is important for proliferation but activated collateral pathways at the cost of increased oxidative stress. Indeed, we found the upregulation of myo-inositol oxidase, reduction in GSH/GSSG ratio, and increased nitration in the lungs of G6PD-deficient mice. Increased oxidative stress also results in the activation of PI3K, ERK1/2, and AMPK that contribute to the proliferation of pulmonary vasculature. Therefore, G6PD deficiency has a multimodal effect, including hemolysis, metabolic reprogramming, and oxidative stress leading to the PH phenotype in mice.

Abstract 17224: Metabolic Switch and Redox Imbalance Contributes to Pulmonary Hypertension in Glucose-6-Phosphate Dehydrogenase Deficiency

Introduction: Pulmonary hypertension (PH) is a fatal disorder with inadequate therapeutic choices and diminished survival rate with later prognosis. We previously reported that several patients with idiopathic pulmonary arterial hypertension had different types of glucose-6 phosphate dehydrogenase (G6PD) deficiency. G6PD is the key regulator enzyme in the pentose phosphate pathway (PPP) and the only source of NADPH in erythrocytes. However, the pathogenic mechanism of how G6PD deficiency contributes to PH development remains elusive. Hypothesis: We hypothesize that G6PD deficiency-induced PH is mediated through a multifactorial mechanism by increased red blood cell fragility, oxidative stress, and a metabolic switch. Methods: To delineate the contribution of G6PD in PH pathogenesis, we utilized a G6PD knockdown mouse line (11-13 week old) with decreased expression of G6PD (10% from wild-type level). Results: Hemodynamic and histological studies confirmed that G6PD deficient mice developed PH phenotype by an increase in right ventricular systolic pressure (30.08±0.91mmHg; p≤0.001), Fulton index (0.358±0.03; p≤0.01) and pulmonary vascular remodeling. G6PD deficiency resulted in increased free hemoglobin and activation of the p38/MAPK pathway, which we recently reported, induces the development of PH in the sugen/hypoxia model via endothelial barrier dysfunction. Metabolomics analysis of G6PD-deficient mice indicates the switch to alternative metabolic fluxes that feed into PPP, resulting in the upregulation of oxidative stress, fatty acid pathway, and reduction in pyruvate production. Thus, G6PD deficiency did not reduce PPP flux that is important for proliferation but activated collateral pathways at the cost of increased oxidative stress. Indeed, we found upregulation of Myo-inositol oxidase (p≤0.05), reduction in GSH/GSSG ratio (p≤0.01), and increased nitration (p≤0.05) in the lungs of G6PD deficient mice. Increased oxidative stress also results in the activation of PI3K, ERK1/2 and AMPK that contributes to the proliferation of pulmonary vasculature. Conclusions: Based on these results we infer that G6PD deficiency has a multi-modal effect, including hemolysis, metabolic reprogramming, and oxidative stress leading to PH.

Carboxyl graphene oxide nanoparticles induce neurodevelopmental defects and locomotor disorders in zebrafish larvae

Graphene-family nanomaterials (GFNs) have been widely used in various fields due to their excellent properties. However, GFNs safety and environmental health have attracted more and more attentions and their potential toxic effects on organisms and the underlying mechanisms are still poorly understood. In this study, we utilized zebrafish to evaluate the toxicity of Carboxyl graphene oxide (GO-COOH). Exposure of zebrafish embryos to 10, 50 and 100 mg/L GO-COOH specifically induced neurodevelopmental abnormalities and altered tendency of locomotor in larval fish. Furthermore, GO-COOH exposure led to increase of AchE and ATPase activities and oxidative stress upregulation, and disrupted the expression of genes involved in neurodevelopment and neurotransmitter pathway. Interestingly, we found that Parkinson’s disease-related genes’ expression were disordered after GO-COOH treatment. Fullerenes and astaxanthin rescued the neurodevelopmental defects, tendency of locomotor and expression of Parkinson’s disease-related genes caused by GO-COOH through downregulating oxidative stress. Therefore, our results suggest that GO-COOH has the potential to induce neurotoxicity and Parkinson’s disease-like symptoms in zebrafish larvae.

Estrogen deficiency accompanied by oxidative stress sustains heme oxygenase 1 expression in cardiomyocytes of ovariectomized rats

Background: Estrogen deficiency is one of the main causes of cardiovascular diseases (CVDs) after menopause, accompanied with the upregulation of oxidative stress. Two isoforms of heme oxygenase (HO), HO-1 and HO-2, have been implicated in the cytoprotective effects via the antioxidant and anti-inflammatory capacities. Aims: This study aimed to investigate the time-course of HO-1 and HO-2 expression in the cardiac tissue of ovariectomized rats and whether oxidative stress is involved in the regulation of HO alteration. Methods: Adult female rats were ovariectomized bilaterally to induce estrogen deficiency and randomly divided into (1) Sham, (2) ovariectomy (Ovx), (3) Ovx + E2 (17β estradiol, 50 μg/kg/day, intramuscular), and (4) Ovx + tempol (1 mM in drinking water, a superoxide dismutase [SOD] mimetic). Rats were sacrificed 12 weeks after Ovx; blood and myocardium samples were collected. Results: Showed that plasma E2 levels of Ovx and Ovx + tempol groups were significantly reduced as compared to Sham group after 4 weeks of Ovx. Superoxide anion in the cardiac tissue was significantly elevated 2 weeks after Ovx, and the increase was drastically reversed by the treatment with E2 and tempol. In addition, Ovx rats showed significantly higher levels of oxidized glutathione (GSSG) than those of Sham group, which were also significantly reduced by E2 and tempol administration. Western blot analysis indicated that HO-1 expression was significantly induced 1 week after Ovx and sustained at high levels until 12 weeks. E2 replacement did not immediately reverse HO-1 until treatment for 4 weeks as well as tempol administration for 5 weeks. Expression of the constitutive enzymes HO-2 did not show significant differences between Sham and Ovx groups, and E2 or tempol administration had no effect on cardiac HO-2 protein expression. Conclusion: E2 deficiency induced upregulation of superoxide anion in the myocardium, which might be the major contributor to the sustained HO-1 expression as adaptive responses to oxidative stress. This study provides new insight into the pathogenesis of CVDs after menopause.

Generation of sphingosine-1-phosphate by sphingosine kinase 1 protects nonalcoholic fatty liver from ischemia/reperfusion injury through alleviating reactive oxygen species production in hepatocytes

BackgroundNonalcoholic fatty liver (NAFL) is emerging as a leading risk factor of hepatic ischemia/reperfusion (I/R) injury lacking of effective therapy. Lipid dyshomeostasis has been implicated in the hepatopathy of NAFL. Herein, we investigate the bioactive lipids that critically regulate I/R injury in NAFL. MethodsLipidomics were performed to identify dysregulated lipids in mouse and human NAFL with I/R injury. The alteration of corresponding lipid-metabolizing genes was examined. The effects of the dysregulated lipid metabolism on I/R injury in NAFL were evaluated in mice and primary hepatocytes. ResultsSphingolipid metabolic pathways responsible for the generation of sphingosine-1-phosphate (S1P) were uncovered to be substantially activated by I/R in mouse NAFL. Sphingosine kinase 1 (Sphk1) was found to be essential for hepatic S1P generation in response to I/R in hepatocytes of NAFL mice. Sphk1 knockdown inhibited the hepatic S1P rise while accumulating ceramides in hepatocytes of NAFL mice, leading to aggressive hepatic I/R injury with upregulation of oxidative stress and increase of reactive oxygen species (ROS). In contrast, administration of exogenous S1P protected hepatocytes of NAFL mice from hepatic I/R injury. Clinical study revealed a significant activation of S1P generation by I/R in liver specimens of NAFL patients. In vitro studies on the L02 human hepatocytes consolidated that inhibiting the generation of S1P by knocking down SPHK1 exaggerated I/R-induced damage and oxidative stress in human hepatocytes of NAFL. ConclusionsGeneration of S1P by SPHK1 is important for protecting NAFL from I/R injury, which may serve as therapeutic targets for hepatic I/R injury in NAFL.

NK cells contribute to hepatic CD8+ T cell failure in hepatitis B virus-carrier mice after alcohol consumption

Despite the fact that both Hepatitis B virus (HBV) infection and excessive alcohol consumption represent health problems worldwide, the mechanism by which alcohol affected the progression of HBV-associated liver disease are not completely understood. Therefore, we studied how alcohol affects the development of HBV infection and the role of T cells and NK cells in the antiviral response. Mononuclear cells (MNCs) derived from HBV-carrier mice and wild type (WT) mice were characterized for phenotype by flow cytometry, HBV antigen and gene expression were detected by Radio Immunoassay (RIA), immunohistochemistry and quantitative real-time (qRT)-PCR. Metabolomics changes were detected in mice liver tissue based on ultra high performance liquid tandem chromatography quadrupole time of flight mass spectrometry (UHPLC-QTOFMS). The mice after ethanol consumption shows higher levels of HBV surface Ag (HBsAg), HBV core antigen (HBcAg) and HBV 3.5 kb RNA expression, and a lower level of CD8+ T cells during HBV persistence, with an increased lymphocyte activation gene-3 (LAG-3) expression on CD8+ T cell. In addition, the energy metabolism was downregulated and the oxidative stress was upregulated in the liver tissue. Furthermore, NK cells depletion results in a lower levels of HBV surface Ag (HBsAg) and HBV 3.5 kb RNA expression, and a higher level of CD8+ T cells with reduced expression of LAG-3. In conclusion, alcohol abuse induces CD8+ T cells failure after acute HBV infection, but depletion of NK cells could retore CD8+ T cell activity. Moreover, downregulation of energy metabolism and upregulation of oxidative stress may also contribute to CD8+ T cell failure.

Regulation of apoptosis and autophagy by luteolin in human hepatocellular cancer Hep3B cells

In several cancer cells, luteolin (3′,4′,5,7-tetrahydroxyflavone) exerts anticancer effects by upregulation of oxidative stress and endoplasmic reticulum (ER) stress, which are shown to activate p53-dependent cell death. Since luteolin-mediated ER stress regulation has not been investigated in hepatocellular carcinoma (HCC) cells, we investigated the role of ER stress in anti-carcinogenic effects using p53-wild type and p53-null HCC cells treated with luteolin. Trypan blue exclusion test was implemented to determine cell viability. Western blot was applied to compare the difference of autophagy, apoptosis, and proliferation event between cell lines. ER stress and p53 activation were determined by RT-PCR. Our results showed that luteolin at 5–10 μmol/L induced higher cytotoxicity in p53-null Hep3B cells than in p53-wild type HepG2 cells. Cytotoxicity was not observed in normal liver cells. Apoptosis activation and proliferation inhibition occurred in only p53-null Hep3B cells in response to luteolin treatment. Also, luteolin induced oxidative stress and ER stress in p53-null Hep3B cells. Although we observed the induction of p21 in Hep3B cells, the concomitant increases in mRNA levels of p53 family members, including TAp63 and TAp73, were not observed. Furthermore, luteolin induced autophagy in Hep3B cells only, which enhanced cell viability. Taken together, this study suggests that luteolin-induced ER stress may exert anticancer effects in a p53-independent manner.

ɑ-MnO2 nanowire induces cytotoxicity of human lung fibroblasts based on a 3D organotypic culture

In this study, we performed a 3D organotypic culture of human lung fibroblast (HLF) in a microfluidic device. This 3D HLF model could partly simulate the biological responses of human lung. ɑ-MnO2 nanowires were successfully synthesized using the hydrothermal approach. Material characterization identified the existence of ɑ-MnO2 nanostructure. Based on this 3D organotypic HLF model, our results demonstrated that ɑ-MnO2 nanowires can induce cytotoxicity in dose-dependently. Because the accumulation of reactive oxygen species (ROS) and depletion of glutathione (GSH) are observed in the nanowire-treated cells. In addition, ROS generation induced intracellular increase of malondialdehyde (MDA), DNA damage and activated alpha-smooth muscle actin expression. Based on these results, we concluded that a mechanism for ɑ-MnO2 nanowire-induced pulmary toxicity, which involves the accumulation of ROS, up-regulation of oxidative stress and inflammatory factors. This investigation may provide a fundamental insight to understand the nanotoxicity of wire-shaped nanomaterials.

Functional human 3D microvascular networks on a chip to study the cytocompatibility of ɑ-MnO2 nanowire

A 3D functional human microvascular network in a microfluidic device was developed in this study. The established model enables endotheilal cells to form vessel-like microtissues and behave physiological functions which are closer to cells in human blood vessels. It provides a possible bridge for how the research outcomes translate to humans. The ɑ-MnO2 nanowires, which were successfully synthesized using the hydrothermal approach. The vascular toxicity was evaluated based on this organotypic 3D microvessel model. Our results demonstrated that ɑ-MnO2 nanowires can induce cytotoxicity in dose-dependently. Because the accumulation of reactive oxygen species (ROS) and depletion of glutathione (GSH) are observed in the nanowire-treated cells. In addition, intravascular accumulation of ɑ-MnO2 nanowires could induce endothelial dysfunction by oxidative stress. This is expressed in disorder of NO expression and IL-6 up-regulation. Based on these results, we concluded that a mechanism for ɑ-MnO2 nanowire-induced vascular toxicity, which involves the accumulation of ROS, up-regulation of oxidative stress and inflammatory factors. This investigation may provide a fundamental insight to understand the nanotoxicity of wire-shaped nanomaterials.

The Up-Regulation of Oxidative Stress as a Potential Mechanism of Novel MAO-B Inhibitors for Glioblastoma Treatment.

Gliomas are malignant brain tumors characterized by rapid spread and growth into neighboring tissues and graded I–IV by the World Health Organization. Glioblastoma is the fastest growing and most devastating IV glioma. The aim of this paper is to evaluate the biological effects of two potent and selective Monoamine Oxidase B (MAO-B) inhibitors, Cmp3 and Cmp5, in C6 glioma cells and in CTX/TNA2 astrocytes in terms of cell proliferation, apoptosis occurrence, inflammatory events and cell migration. These compounds decrease C6 glioma cells viability sparing normal astrocytes. Cell cycle analysis, the Mitochondrial Membrane Potential (MMP) and Reactive Oxygen Species (ROS) production were detected, revealing that Cmp3 and Cmp5 induce a G1 or G2/M cell cycle arrest, as well as a MMP depolarization and an overproduction of ROS; moreover, they inhibit the expression level of inducible nitric oxide synthase 2, thus contributing to fatal drug-induced oxidative stress. Cmp5 notably reduces glioma cell migration via down-regulating Matrix Metalloproteinases 2 and 9. This study demonstrated that our novel MAO-B inhibitors increase the oxidative stress level resulting in a cell cycle arrest and markedly reduces glioma cells migration thus reinforcing the hypothesis of a critical role-played by MAO-B in mediating oncogenesis in high-grade gliomas.

Radiation induces changes in toll-like receptors of the uterine cervix of the rat.

Radiotherapy is an important therapeutic approach against cervical cancer but associated with adverse effects including vaginal fibrosis and dyspareunia. We here assessed the immunological and oxidative responses to cervical irradiation in an animal model for radiation-induced cervicitis. Rats were sedated and either exposed to 20 Gy of ionising radiation given by a linear accelerator or only sedated (controls) and euthanized 1–14 days later. The expressions of toll-like receptors (TLRs) and coupled intracellular pathways in the cervix were assessed with immunohistofluorescence and western blot. Expression of cytokines were analysed with the Bio-Plex Suspension Array System (Bio-Rad). We showed that TLRs 2–9 were expressed in the rat cervix and cervical irradiation induced up-regulation of TLR5, TRIF and NF-κB. In the irradiated cervical epithelium, TLR5 and TRIF were increased in concert with an up-regulation of oxidative stress (8-OHdG) and antioxidant enzymes (SOD-1 and catalase). G-CSF, M-CSF, IL-10, IL- 17A, IL-18 and RANTES expressions in the cervix decreased two weeks after cervical irradiation. In conclusion, the rat uterine cervix expresses the TLRs 2–9. Cervical irradiation induces immunological changes and oxidative stress, which could have importance in the development of adverse effects to radiotherapy.

Oxidative Stress and NO Generation in Cerulein-Induced Acute Pancreatitis in Rats

The interaction between nitric oxide (NO) and superoxides is critical in the development of pancreatitis. Previously, we reported on the up-regulation of oxidative stress and NO-synthase (NOS) in the human chronic pancreatitis and in an animal model of pancreatitis induced by pancreatic duct ligation (PDL) in rats. We have shown that oxidative stress runs ahead of NOS up-regulation, which implies that the NO enhancement in the course of pancreatitis is likely to be an adaptive mechanism aimed at maintaining the homeostatic cellular level of the bioactive NO. Here, we report on the expression of NOS and oxidative stress markers (nitrotyrosine and 8-hydroxyguanosine) in the course of cerulein-induced acute pancreatitis in rats. We found that the pattern of superoxides/NO interaction in this model of acute pancreatitis is similar to that in the PDL-induced rat pancreatitis and in the human chronic pancreatitis. It means that cerulein-induced acute pancreatitis like the PDL-induced pancreatitis is a proper model for further studies of pancreatitis development in humans.

Shikonin inhibits proliferation and induces apoptosis in glioma cells via downregulation of CD147.

Shikonin, a traditional Chinese medicine, has been identified as being capable of inducing apoptosis in various tumors, including glioma, and is thus considered to be a promising therapeutic agent for tumor therapy. However, little is known about the molecular mechanism of shikonin in glioma. The present study investigated the influence of shikonin on the proliferation and apoptosis of glioma cells U251 and U87MG and explored the potential molecular mechanisms. It was identified that shikonin was able to induce apoptosis in human glioma cells in a time- and dose-dependent manner, and a decreased expression level of cluster of differentiation (CD)147 was observed in shikonin-treated U251 and U87MG cells. Knockdown of CD147 inhibited U251 and U87MG cell growth, whereas CD147 overexpression enhanced cell growth and decreased shikonin-induced apoptosis. Additionally, an increased expression level of CD147 suppressed the elevated production of reactive oxygen species and mitochondrial membrane potential levels induced by shikonin. The data indicated that shikonin-induced apoptosis in glioma cells was associated with the downregulation of CD147 and the upregulation of oxidative stress. CD147 may be an optional target of shikonin-induced cell apoptosis in glioma cells.

Natural antioxidants in prevention of accelerated ageing: a departure from conventional paradigms required.

The modern lifestyle is characterised by various factors that cause accelerating ageing by the upregulation of oxidative stress and inflammation-two processes that are inextricably linked in an endless circle of self-propagation. Inflammation in particular is commonly accepted as aetiological factor in many chronic disease states, such as obesity, diabetes and depression. In terms of disease prevention or treatment, interventions aimed at changing dietary and/or exercise habits have had limited success in practise, mostly due to poor long-term compliance. Furthermore, other primary stimuli responsible for eliciting an oxidative stress or inflammatory response-e.g. psychological stress and anxiety-cannot always be easily addressed. Thus, preventive medicine aimed at countering the oxidative stress and/or inflammatory responses has become of interest. Especially in developing countries, such as South Africa, the option of development of effective strategies from plants warrants further investigation. A brief overview of the most relevant and promising South African plants which have been identified in the context of inflammation, oxidative stress and chronic disease is provided here. In addition, and more specifically, our group and others have shown considerable beneficial effects across many models, after treatment with products derived from grapes. Of particular interest, specific cellular mechanisms have been identified as therapeutic targets of grape-derived polyphenols in the context of inflammation and oxidative stress. The depth of these studies afforded some additional insights, related to methodological considerations pertaining to animal vs. human models in natural product research, which may address the current tendency for generally poor translation of positive animal model results into human in vivo models. The importance of considering individual data vs. group averages in this context is highlighted.

Sensitization of neuroendocrine prostate cancer by RRx-001.

280 Background: RRx-001 is a first-in-class dinitroazetidine nontoxic anticancer agent. Its actions include upregulation of oxidative stress, depletion of GSH and NADPH, anti-angiogenesis, epigenetic modulation and induction of viral mimicry. In a phase II clinical study (NCT02489903), RRx-001 reversed resistance to reintroduced platinum doublets in small cell lung cancer (SCLC) and other tumor types. RRx001 demonstrated clinical activity with a complete metabolic response of metastatic castration-resistant neuroendocrine prostate cancer (NEPC) after treatment with RRx-001 and reintroduced platinum doublets. Here, we used a preclinical model to further investigate RRx-001 sensitization of NEPC to platinum therapy. Methods: NCI-H660 spheroids were pre-treated with 1 mM RRx-001 or vehicle prior to treatment with carboplatin or docetaxel and cell proliferation was assessed. Cells were also pre-treated with both 1 mM RRx-001 and 10 mM N-acetyl cysteine (NAC) or 1 mM Buthionine-S,R-sulfoximine (BSO) to investigate the role of reactive oxygen species (ROS) in RRx-001 activity. NCI-H660 xenografts were pre-treated with RRx-001 (mimicking clinical activity) or vehicle prior to treatment with carboplatin, and tumor volume and body weight were monitored. Results: RRx-001 pre-treatment significantly decreased the IC50 of carboplatin from ~86 mM to 36 mM in vitro, whereas no significant effect was observed on the IC50 of docetaxel. The ROS inhibitor NAC reversed > 70% of RRx-001 sensitization to carboplatin, while co-pre-treatment with the ROS generator BSO significantly enhanced RRx-001 activity. In vivo, pre-treatment with RRx-001 prior to carboplatin significantly decreased tumor volume compared to vehicle, with ~50% greater reduction in mean tumor volume at 30 days. Body weight was not affected by RRx-001. Conclusions: This preclinical study supports the concept that “priming” with RRx-001 may be an effective therapeutic strategy to resensitize NEPC to initially highly effective platinum doublets. A phase 3 trial is planned, and studies with additional preclinical NEPC models will identify the mechanism of action of RRx-001.