Oxygen Species Production Research Articles

P0089KEAP1/NRF2 PATHWAY REGULATES GFR BY INCREASING THE GLOMERULAR EFFECTIVE AREA WITHOUT AFFECTING THE AFFERENT/EFFERENT ARTERIOLE RATIO

Abstract Background and Aims The Keap1/Nrf2 pathway regulates the expression of a series of cytoprotective, anti-inflammatory and antioxidant genes. The Nrf2 activator, bardoxolone methyl (BARD), has consistently increased estimated GFR (eGFR) in clinical studies in patients with chronic kidney disease. BARD demonstrated improvement of renal function assessed by inulin clearance, the clinical gold standard for measuring GFR, in diabetic kidney disease patients. These findings suggest the Keap1/Nrf2 system is deeply involved in the regulatory mechanisms of GFR. However, the precise mechanisms are not fully elucidated. We pharmacologically and genetically investigated the mechanisms of GFR regulation by Keap1/Nrf2 system using in vivo multiphoton microscope (MPM) imaging techniques. Method C57BL/6 (Cont), Nrf2 knockout (Nrf2-KO), and Nrf2-activated Keap1-knockdown mice (Keap1-KD) were used. The mice were treated the synthetic triterpenoid RTA dh404 (10 mg/kg/day by gavage) which is a Nrf2 activator for rodents, for a week. We successfully developed the technique to evaluate single-nephron GFR (SNGFR) using MPM (Circulation 2019). The glomerular hemodynamics, diameter of the afferent/efferent arterioles and glomerular permeability were also evaluated. The calcium influx into cells in response to ATP and angiotensin II stimulation and the effect on [Ca2+]i by RTAdh404 were evaluated using Fluo 4 and Fura red in cultured mesangial cells and podocytes. Production of reactive oxygen species and nitric oxide (NO) availability were assessed by fluorescent method using CellROX® Deep Red and diaminofluorescein-FM diacetate (DAF-FM DA) upon the exposure to these stimuli. Results SNGFR in Keap1-KD mice was significantly higher than in the control (9.13±0.55 vs 4.40±0.39 nl/min, Figure 1). RTA dh404 increased SNGFR in the control but not in the Nrf2-KO mice (6.00±0.40 vs 4.66±0.35 nl/min, Figure 1). There was no significant change in the ratio of the glomerular afferent/efferent arteriole diameter in all groups. RTA dh404 treatment increased glomerular volume but did not affect the glomerular permeability of albumin and 40kd-dextran. RTA dh404-treatment inhibited calcium influx into cultured podocytes and mesangial cells induced by angiotensin II or ATP, thereby affecting contractile responses. Oxidative stress and NO-bioavailablity were also ameliorated with RTA dh404. Conclusion The Keap1/Nrf2 pathway plays a pivotal role in controlling GFR and presumably underlies the effect of BARD on GFR in patients.

Sargassum duplicatum Extract Reduced Artritis Severity Score and Periarticular Tissue Matrix Metalloproteinase-1 (Mmp-1) Expression in Ajuvan Artritis Exposed to Cold Stressor

In rheumatoid arthritis, significant increase of matrix metalloproteinase-1 (MMP-1) expression results in destruction of cartilage’s collagen, joint’s bone and tendon. Cold stressor increase reactive oxygen species (ROS) production caused by increased oxidative phosphorylation and oxidant compounds can increase MMP-1 production. Sargassum duplicatum contains antioxidant compounds, including polyphenols (flavonoids and phlorotannin) and fucoxanthin. The purpose of this study was to show the effect of Sargassum duplicatum extract (ESD) in reducing arthritis severity score and inhibiting periarticular tissue MMP-1 expression in adjuvant arthritic rat exposed to cold stressor. In this study 30 male adjuvant arthritis (AA) rats divided into three groups: 1) without treatment; 2) exposed to cold stressor of 5o C for 15 minutes on day 8 - 14; 3) exposed to cold stressor and given oral ESD extract 400 mg / kg BW / day on day 1 - 21. At the end of the study, subjective arthritis severity score were assessed and periarticular tissue MMP-1 expression were analysed using immunohistochistry methods. Mann Whitney statistical analysis showed exposure to cold stressor increase arthritis severity score significantly (p=0.002). Treatment with ESD reduce arthritis severity score significantly in AA rats exposed to cold stressors (p=0.001). Anova test results showed that MMP-1 expression were significantly increase in AA rats exposed to cold stressors compared to the group unexposed to cold stressors (p=0.001). ESD treatment significantly reduce the MMP-1 expression in AA rats exposed to cold stressors (p=0.001). In conclusion this study showed that cold stressor increased arthritis severity score and MMP-1 expression in AA, while antioxidant content of ESD decreased arthritis severity score and MMP-1 expression.

18-LB: Increased Acidosis and Free-Radical Production Causes Recurrent Hypoglycemia–Induced Increase in Post-ischemic ER Stress in Treated Diabetic Rats

Cerebral ischemia is a serious complication of diabetes. Treatment of diabetes causes recurrent hypoglycemia (RH). Prior RH exposure increases ischemic brain damage in insulin-treated diabetic (ITD) rats. However, the mechanism of this injury is not well understood. We showed that ischemic acidosis increases reactive oxygen species (ROS) production in RH-exposed ITD (ITD + RH) rats. As enhanced acidosis and ROS production in normal subjects activate PERK, we hypothesized that increased ischemic acidosis and ROS production in RH-exposed ITD rats activate PERK. Therefore, we determined if the administration of tris-(hydroxymethyl)-aminomethane (THAM; an alkalizing agent) and N-acetylcysteine (NAC; an antioxidant) decrease RH-induced post-ischemic PERK activation in ITD + RH rats. Wistar rats were treated with insulin 2-3 weeks after streptozotocin induced diabetes. After 2 weeks, hyperinsulinemic hypoglycemia was induced for 3 hours for 5 days. Following groups were employed: (I) ITD + vehicle (Veh) (n = 6); (II) ITD + RH + Veh (n = 7); (III) ITD + RH + THAM (0.3 M, 3 ml/kg/hr, i.v.) (n = 6); and (IV) ITD + RH + NAC (150 mg/kg, i.v.) (n = 6). Transient global cerebral ischemia was induced by bilateral carotid artery occlusion with hypotension a day after RH. THAM/Veh was infused from 15 min before to 88 minutes after ischemia. NAC/Veh was administered 5 min before and 12 h after ischemia. Based on Western blot analysis, pPERK to total PERK ratio in the ITD + RH + THAM and ITD + RH + NAC groups were significantly lower by 32% and 26%, respectively, when compared with ITD + RH + Veh group. CHOP levels in the ITD + RH + THAM and ITD + RH + NAC group were lower by 38% and 63%, respectively, when compared with ITD + RH + Veh group. Therefore, ischemic acidosis and ROS production may mediate RH-induced ischemic activation of PERK. Understanding the ischemic damage in RH-exposed ITD rats may help identify new treatments for diabetes induced increase in ischemic injury. Disclosure A.K. Rehni: None. S. Cho: None. K.R. Dave: None. Funding National Institutes of Health (NS073779); American Heart Association (18POST34070061)

P1146ENHANCED EXPRESSION OF THIOREDOXIN-INTERACTING-PROTEIN (TXNIP) MAY REGULATE OXIDATIVE DAMAGE IN PERITONEAL DIALYSIS PATIENTS

Abstract Background and Aims Glucose-based solutions used as peritoneal dialysis fluids influence the peritoneal membrane. Exposure to high glucose-based peritoneal dialysis solutions induces reactive oxygen species (ROS) production due to upregulation of TXNIP as shown by several studies in rats as well as in human primary cells. Enhanced expression of TXINP also regulates oxidative damage. Therefore the aim of this study was to investigate the expression of TXNIP and the extent of oxidative damage in human peritoneal biopsies. Method Human peritoneal biopsies of healthy controls, PD patients and patients with EPS were collected. TXNIP and thioredoxin-1 (TRX) mRNA expression was determined by qPCR. Protein expression and the extent of oxidative damage were examined by immunohistochemistry. Results Biopsies from the peritoneum of 7 healthy controls, 36 patients on PD without signs of EPS as well as of 12 patients with EPS were collected. The age of the healthy controls was higher (median 64.00 years, IQR: 53.00-70.00) than in the other subgroups (PD: median 60.50 years, IQR: 46.00-69.00 and EPS: median 51.00 years, IQR: 38.00-58.75). Furthermore, compared to the PD- (39 %) and EPS-group (33 %), there were more female participants in the control-group (86 %). Time on PD was longer in EPS patients (median 70.00 months) than in PD patients (median 33.50 months). In a preliminary study, exposure to high glucose-based peritoneal dialysis solutions did not markedly influence the mRNA expression of TXNIP and TRX. However, on protein level a significant glucose-related upregulation of TXNIP could be observed especially in PD patients. Interestingly, there was no glucose-related change in protein expression of its interacting partner and cellular anti-oxidant TRX. To study the effect of TXNIP expression on the generation of oxidative damage, pH2AX positive nuclei were counted on peritoneal membrane sections. A slight increase of pH2AX positive nuclei upon exposure to dialysis solutions could be observed in the cohort analysed. Conclusion Here, we show for the first time a clear tendency for upregulation TXNIP in human peritoneal tissue after exposure to high glucose-based peritoneal dialysis solutions especially in PD patients. This increase in TXNIP expression may correlate with the accumulation of oxidative damage of macromolecules. Therefore, manipulation of TXNIP expression may be a promising therapeutic target to improve peritoneal membrane function.

1739-P: Insulin Rapidly Increases Skeletal Muscle Mitochondrial ADP Sensitivity, Mitigating HFD-Induced Mitochondrial Dysfunction

Introduction: Causality of the association between impairments in mitochondrial function and insulin resistance remains debatable. We have previously demonstrated that mitochondrial ADP sensitivity is impaired in high-fat diet (HFD)-induced insulin resistance, which contributes to bioenergetic imbalance and higher reactive oxygen species (ROS) production. However, insulin regulates several metabolic processes within skeletal muscle, and we tested the hypothesis that insulin could rapidly improve mitochondrial ADP sensitivity, a phenomenon possibly lost in HFD-induced insulin resistance. Methods: Male and female C57Bl/6 mice were fed a control diet (10% fat) or HFD (60% fat) for 8 weeks. At the end of treatment, insulin was injected intraperitoneally (3 U/kg) twice (0 and 30 minutes). At 60 minutes red gastrocnemius muscle was excised to evaluate mitochondrial bioenergetics, ADP sensitivity, and ROS production. Results: In control mice, insulin increased respiration by ∼2-fold in the presence of submaximal (100-1000 μM ADP) but not maximal ADP-supported respiration. As a result, insulin decreased the apparent ADP Km from 700 to 380 μM ADP. Despite these respiratory changes, insulin had no effect on mitochondrial maximal succinate-supported ROS emission or the ability of ADP to suppress ROS emission. HFD consumption impaired whole-body glucose intolerance, skeletal muscle insulin resistance (decreased insulin-stimulated Akt Thr-308 and Ser-473 phosphorylation) and impaired mitochondrial submaximal ADP-supported respiration by ∼50% (ADP Km ∼1100 μM). However, despite the reduction in insulin signaling following HFD, insulin increased submaximal ADP-supported respiration by ∼2-fold, decreased the apparent ADP Km to ∼400 μM and improved the ability of ADP to suppress mitochondrial ROS emission. Conclusion: Insulin rapidly improves mitochondrial ADP sensitivity, mitigating the apparent HFD-induced mitochondrial dysfunction. Disclosure H. Brunetta: None. H.L. Petrick: None. E.A. Nunes: None. G. Holloway: None. Funding Natural Sciences and Engineering Research Council of Canada (400360); Coordination for the Improvement of Higher Education Personnel

THE EFFECT OF NIMODIPINE ON CALCIUM INTRACELLULAR AND ROS EXPRESSION IN NEURON CULTURE CELL LINE SH-SY5Y INDUCE CHRONIC HIPERGLYCEMIA

Introduction: A frequent microvascular complication of diabetes mellitus were peripheral neuropathies with a prevalence of around 30-50%. Generally, oxidative stress is the key to the pathological process that induces damage to peripheral nerves in sensory neurons thereby increasing the production of reactive oxygen species (ROS) and mitochondrial dysfunction which causes deregulation of calcium ion homeostasis and disrupted calcium signaling. Nimodipine is an L-type Ca2+ inhibitor that reactivates the ATPase pathway, has a neuroprotective effect and can improve the regulation of blood and nerve flow in restoring the adrenergic function of the vasa nervorum. Aim: The purpose of this study was to determine the effect of 0.5µm nimodipine on intracellular calcium and ROS expression in SH-SY5Y cell cultures induces chronic hyperglycemia.
 Method: Normoglycemic glucose-induced cell culture of neurons 25mM, 50mM hyperglycemia for 6 days and then given nimodipine and without 0.5µM nimodipine.
 Results: Based on the results of the One-Way ANOVA test, there was a significant difference (p<0.05) in intracellular calcium expression and ROS in cell cultures exposed to chronic hyperglycemia. Nimodipine 0.5µM significantly reduced intracellular calcium expression but did not reduce ROS expression. The Spearman rank correlation test (r=0.5; p>0.05) given nimodipine 0.5µM therapy found a positive correlation, but not significant.
 Conclusion: Based on this study it can be concluded that the administration of 0.5µM nimodipine can reduce intracellular calcium expression but has not been able to reduce ROS expression in 50mM glucose-induced cell cultures.

β-Naphthoflavone and Ethanol Reverse Mitochondrial Dysfunction in A Parkinsonian Model of Neurodegeneration.

The 1-methyl-4-phenylpyridinium (MPP+) is a parkinsonian-inducing toxin that promotes neurodegeneration of dopaminergic cells by directly targeting complex I of mitochondria. Recently, it was reported that some Cytochrome P450 (CYP) isoforms, such as CYP 2D6 or 2E1, may be involved in the development of this neurodegenerative disease. In order to study a possible role for CYP induction in neurorepair, we designed an in vitro model where undifferentiated neuroblastoma SH-SY5Y cells were treated with the CYP inducers β-naphthoflavone (βNF) and ethanol (EtOH) before and during exposure to the parkinsonian neurotoxin, MPP+. The toxic effect of MPP+ in cell viability was rescued with both βNF and EtOH treatments. We also report that this was due to a decrease in reactive oxygen species (ROS) production, restoration of mitochondrial fusion kinetics, and mitochondrial membrane potential. These treatments also protected complex I activity against the inhibitory effects caused by MPP+, suggesting a possible neuroprotective role for CYP inducers. These results bring new insights into the possible role of CYP isoenzymes in xenobiotic clearance and central nervous system homeostasis.

Oxidative Stress Level in Onco-Surgical Treatment Dynamics at Patients with Malignant Colo-Rectal Tumors

Colorectal cancer (CRC) is one of the most common human malignancies, affecting one of 20 persons in areas with high socio-economic standard. In Romania, the frequency of colorectal cancer is growing rapidly placing the country among countries with an average incidence of the disease. There are some etiologic factors involved and treatment of disease is carried out after proper staging. Biochemical mechanisms underlying malignant transformation in colorectal cancer are not all fully understood, therefore our work trying to enter in the path of oxygen metabolism at patients surgically treated. The aim of the study is to follow the production of active metabolites of oxygen, in the dynamics of the surgical procedure, and how the endogenous natural protection systems are activated, following the invasive procedure. Oxidative stress biochemistry assays, realized before and after surgical excision showed a direct relationship between the production of reactive oxygen species and the presence of tumor, without being able to distinguish exactly if malignant tissue is able to induce oxidative stress, or the latter occurs due to neoplastic changes. Based on the results we can say with certainty that the reactive oxygen species ROS primary attack occurs in the lipids, and then the proteins, following activation of endogenous antioxidant defence.

Preventive Effects of Neuroprotective Agents in a Neonatal Rat of Photothrombotic Stroke Model.

Neonatal ischemic stroke has a higher incidence than childhood stroke. Seizures are the first sign for the need for clinical assessment in neonates, but many questions remain regarding treatments and follow-up modalities. In the absence of a known pathophysiological mechanism, only supportive care is currently provided. Stroke-induced microglia activation and neuroinflammation are believed to play a central role in the pathological progression of neonatal ischemic stroke. We induced a photothrombotic infarction with Rose Bengal in neonatal rats to investigate the effects of pre- and post-treatment with Aspirin (ASA), Clopidogrel (Clop), and Coenzyme Q10 (CoQ10), which are known for their neuroprotective effects in adult stroke. Pre-stroke medication ameliorates cerebral ischemic injury and reduces infarct volume by reducing microglia activation, cellular reactive oxygen species (ROS) production, and cytokine release. Post-stroke administration of ASA, Clop, and CoQ10 increased motor function and reduced the volume of infarction, and the statistical evidence was stronger than that seen in the pre-stroke treatment. In this study, we demonstrated that ASA, Clop, and CoQ10 treatment before and after the stroke reduced the scope of stroke lesions and increased behavioral activity. It suggests that ASA, Clop, and CoQ10 medication could significantly have neuroprotective effects in the neonates who have suffered strokes.

Role of mutant KRAS as a biomarker in FL118-induced apoptosis, reactive oxygen species (ROS) production and inhibition of survivin, Mcl-1 and XIAP in human bladder cancer.

e15651 Background: Effective treatment of bladder cancer patients has not been realized over the past two decades due to heterogeneity with key oncogenic and tumor suppressor gene mutations, which is a major hurdle for effective treatment of the disease. Methods: Cancer cell growth/viability was determined using MTT assays; protein gene expression profiles were determined by both Western blots and proteomics technologies; PARP cleavage were determined by Western blots; genetic modulation of protein gene expression was realized by shRNA silencing or forced expression of the gene; reactive oxygen species (ROS) production was measured using a ROS assay kit; and human bladder xenograft tumor growth was determined using animal models. Results: Our study indicated that FL118, an innovative small molecule, exhibits differential anticancer efficacy in high-grade bladder cancer cell lines (HT1376, T24, UMUC-3). FL118 is highly effective at inhibiting the viability of T24 and UMUC-3 bladder cancer cells, which have Hras and Kras mutations, respectively. In contrast, HT1376 with wild-type Ras exhibits insensitivity to FL118-mediated inhibition of cell viability. Consistently, FL118 strongly induced PARP cleavage (hallmark of apoptosis) and inhibited the expression of survivin, XIAP and/or Mcl-1 in both T24 and UMUC-3 cells but not in the HT1376 cells. Our studies further indicated that the constitutive AKT and ERK1/2 signaling does not play a major role in these FL118-mediated effects. Instead, mutant Kras appeared to be involved in FL118 efficacy and played a positive role in mediating FL118 effects. Silencing of mutant Kras via shRNA reduced both FL118-induced PARP cleavage and downregulation of survivin, XIAP and Mcl-1 in UMUC-3 cells, suggesting mutant Kras is required for FL118 to exhibit high efficacy. FL118 increases ROS production in T24 and UMUC-3 cells but not in HT1376 cells. Silencing of mutant Kras with shRNA in UMUC-3 cells significantly reduced FL118-mediated ROS generation. Additionally, proteomics analyses of UMUC3 and HT1376 cell protein profiles after FL118 treatment support the obtained results described above. Consistent with these in vitro results, in vivo studies indicated that UMUC3 is highly sensitive and HT1376 is highly resistant to FL118 treatment. Conclusions: Our studies suggest that mutant Kras is a favorable biomarker for FL118 targeted treatment in human bladder cancer.

FRET mechanism between a fluorescent breast-cancer drug and photodynamic therapy sensitizers

Tamoxifen is one of the most frequently used drugs for the treatment of estrogen receptor positive breast cancer, which is the most prevalent form of hormone dependent breast cancer. A few years ago, we developed a fluorescent derivative of tamoxifen formed by the covalent binding of tamoxifen to a common dye biomarker. The new compound, known as FLTX1, showed the pharmacological activity of the tamoxifen moiety and efficient fluorescence properties, which could be used synergistically to improve the effect of the drug. In this paper, we demonstrate that irradiation at the absorption band of FLTX1 can result in fluorescence resonance energy transfer to photosensitizers such as Rose Bengal and Merocyanine 540, activating the production of reactive oxygen species (ROS). Indeed, the generation of ROS was demonstrated using a colorimetric assay. Since FLTX1 mostly binds estrogen-receptor overexpressing cancer cells, the results obtained are very promising and suggest a new therapeutic strategy combining chemo- and photodynamic therapies.

Interferon-regulating activity of the CelAgrip drug and its influence on the formation of reactive oxygen species and expression of innate immunity genes in Burkitt’s lymphome cell cultures

Interferons (IFN) and IFN inducers are effective in suppressing viral reproduction and correcting of the innate immunity mechanisms. The aim of the study was to test the hypothesis of the possible involvement of the IFN inducer CelAgrip (CA) as an activator or suppressor of antiviral effects in Burkitt's lymphoma (LB) cell cultures with different ability to produce Epstein-Barr virus antigens (EBV). The kinetic analysis of the dynamics of reactive oxygen species (ROS) production and determination of gene group expression by real-time PCR in response to CA treatment were done in human cell lines LB P3HR-1 and Namalva, spontaneously producing and not producing EBV antigens. When treating CA in Namalva cells, a decrease in the ROS activation index was found; in P3HR-1 cells, an increase was observed. After treatment with CA, there was no reliable activation of the IFN-α, IFN-β and IFN-λ genes in Namalva cells, but the expression of the ISG15 and P53(TP53) genes was increased more than 1200 times and 4.5 times, respectively. When processing the CA of P3HR-1 cells, the expression of IFN-α genes increased by more than 200 times, IFN-λ - 100 times, and the ISG15 gene - 2.2 times. The relationship between IFN-inducing action of CA and the activity of ISG15 and ROS in LB cell cultures producing and not producing EBV antigens is supposed. In Namalva cells that do not produce EBV antigens the treatment of CA results in suppression of ROS generation and activation of the expression of genes ISG15 and P53 (TP53); in P3HR-1 cells producing EBV antigens, the opposite picture is observed - the formation of ROS and the expression of the IFN-α and IFN-λ genes are activated and the activity of the ISG15 and P53 (TP53) genes is suppressed.

Production of Reactive Oxygen Species, Unbalanced by Endogenous Antioxidant Capture Mechanisms Monitored in Surgically Treated Breast Cancer

Oxidative stress is defined as an imbalance between the production of oxygen reactive species (ROS) and their endogenous capacity of capturing them. Oxidative stress activates multiple effects generated by the non-specific growth of the ROS and by the decrease of antioxidant concentrations and neutralization mechanisms. The most accurate understanding of the mechanisms underlying oxidative stress and the deepening of its implications in different cancers has become very important in recent decades. Cancer cells are usually subjected to high levels of ROS, which continually activates the malignant phenotype by stimulating cell proliferation � by increasing the mitotic rate, angiogenesis, invasiveness and metastasis rates. The role of the ROS in the etiology and progression of breast cancer is in continuous processing. However, less attention has been paid to the development of redox-targeted breast cancer therapy strategies. Excess ROS production is detrimental for the survival and proliferation of neoplastic cells, through the oxidative-destructive mechanisms of biomolecules essential for life. This study departs from the hypothesis that the tumor is an inductive factor of oxidative stress, and an antioxidant treatment prior to surgery may influence the response to surgical treatment in breast cancer patients. This response is highlighted by determining certain essential compounds of the redox balance, after initiating the antioxidant therapy. Moreover, we have tried to assess the possible biochemical changes given by age, tumor size and asymmetry/laterality of the neoplasm in the investigated patients, and to determine the prognostic or predictive factors of these changes.

Anti-inflammatory effect of hispidin on LPS induced macrophage inflammation through MAPK and JAK1/STAT3 signaling pathways

Severe inflammatory reactions caused by macrophage activation can trigger a systemic immune response. In the present study, we observed the anti-inflammatory properties of hispidin on LPS induced RAW264.7 macrophage cells. Our results showed that hispidin treatment significantly reduced the production of cellular NO, IL-6 and reactive oxygen species (ROS) while has not inhibitory effect on TNF-α productions. Excitingly, hispidin treatment retains the phagocytosis ability of macrophages which enabling them to perform the function of removing foreign invaders. Signaling studies showed, hispidin treatment dramatic suppressed the LPS induced mitogen activated protein kinases (MAPK) and JAK/STAT activations. In conclusion, our findings suggest that hispidin may be a new therapeutic target for clinical treatment of macrophages-mediated inflammatory responses.

Citron C-05 inhibits both the penetration and colonization of Xanthomonas citri subsp. citri to achieve resistance to citrus canker disease

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is a serious bacterial disease that affects citrus production worldwide. Citron C-05 (Citrus medica) is the only germplasm in the Citrus genus that has been identified to exhibit strong resistance to Xcc. However, it has not been determined when, where, and how Xcc is restricted in the tissues of Citron C-05 during the infection process. In the present study, we investigated the spatiotemporal growth dynamics of an eGFP-labeled virulent Xcc (eGFP-Xcc) strain in Citron C-05 along with five susceptible biotypes (i.e., lemon, pummelo, sour orange, sweet orange, and ponkan mandarin) upon inoculation via the spraying or leaf infiltration of a bacterial suspension. The results from extensive confocal laser scanning microscopy analyses showed that while Xcc grew rapidly in plants of all five susceptible genotypes, Xcc was severely restricted in the epidermal and mesophyll cell layers of the leaves of Citron C-05 in the early stage of infection. Not surprisingly, resistance against Xcc in Citron C-05 was found to be associated with the production of reactive oxygen species and hypersensitive response-like cell death, as well as greater upregulation of several defense-related genes, including a pathogenesis-related gene (PR1) and a glutathione S-transferase gene (GST1), compared with sweet orange as a susceptible control. Taken together, our results not only provide further valuable details of the spatiotemporal dynamics of the host entry, propagation, and spread of Xcc in both resistant and susceptible citrus plants but also suggest that resistance to Xcc in Citron C-05 may be attributed to the activation of multiple defense mechanisms.

The therapeutic value of SC66 in human renal cell carcinoma cells

The PI3K-AKT-mTOR cascade is required for renal cell carcinoma (RCC) progression. SC66 is novel AKT inhibitor. We found that SC66 inhibited viability, proliferation, migration and invasion of RCC cell lines (786-O and A498) and patient-derived primary RCC cells. Although SC66blocked AKT-mTORC1/2 activation in RCC cells, it remained cytotoxic in AKT-inhibited/-silenced RCC cells. In RCC cells, SC66 cytotoxicity appears to occur via reactive oxygen species (ROS) production, sphingosine kinase 1inhibition, ceramide accumulation and JNK activation, independent of AKT inhibition. The ROS scavenger N-acetylcysteine, the JNK inhibitor (JNKi) and the anti-ceramide sphingolipid sphingosine-1-phosphate all attenuated SC66-induced cytotoxicity in 786-O cells. In vivo, oral administration of SC66 potently inhibited subcutaneous 786-O xenograft growth in SCID mice. AKT-mTOR inhibition, SphK1 inhibition, ceramide accumulation and JNK activation were detected in SC66-treated 786-O xenograft tumors, indicating that SC66 inhibits RCC cell progression through AKT-dependent and AKT-independent mechanisms.

SiRNA Interference with Notch1 Affects LPS-Induced Apoptosis of Rat Intestinal Epithelial Cells

Notch signaling is associated with the pathogenesis of ulcerative colitis (UC). This study investigated whether the Notch regulates intestinal epithelial cell apoptosis and reactive oxygen species (ROS) production. Rats were separated into UC model group and control group followed by analysis of the expression of Notch1, Hes1 and OLFM4 by real-time PCR and western blot, content of malondialdehyde (MDA), superoxide dismutase (SOD) and caspase-3 as well as cell apoptosis by flow cytometry. Under LPS stimulation conditions, IEC-6 cells were divided into LPS + siRNA-NC group and LPS + siRNA-Notch1 group and Notch1, Hes1 and OLFM4 level as well as ROS was measured. Compared with control group, the activity of caspase-3, MDA and apoptosis in colon tissue of UC model were significantly increased with decreased SOD activity and increased Notch1, Hes1 and OLFM4 expression. LPS treatment can significantly up-regulate the expression of Notch1, Hes1 and OLFM4 in IEC-6 cells, increase cell apoptosis and ROS production. Notch1 interference by siRNA significantly downregulated Notch1, Hes1, and OLFM4 in IEC cells, and increased cell apoptosis and ROS production. UC lesions can activate Notch signaling pathway in colon tissue, which may play a role in repair. Interference with Notch1 signaling may aggravate intestinal epithelial cell apoptosis and ROS production in an inflammatory environment.

The potential protective roles of zinc, selenium and glutathione on hypoxia-induced TRPM2 channel activation in transfected HEK293 cells

Hypoxia induces cell death through excessive production of reactive oxygen species (ROS) and calcium (Ca2+) influx in cells and TRPM2 cation channel is activated by oxidative stress. Zinc (Zn), selenium (Se), and glutathione (GSH) have antioxidant properties in several cells and hypoxia-induced TRPM2 channel activity, ROS and cell death may be inhibited by the Zn, Se, and GSH treatments. We investigated effects of Zn, Se, and GSH on lipid peroxidation (LPO), cell cytotoxicity and death through inhibition of TRPM2 channel activity in transfected HEK293 cells exposed to hypoxia defined as oxygen deficiency.We induced four groups as normoxia 30 and 60 min evaluated as control groups, hypoxia 30 and 60 min in the HEK293 cells. The cells were separately pre-incubated with extracellular Zn (100 µM), Se (150 nM) and GSH (5 mM). Cytotoxicity was evaluated by lactate dehydrogenase (LDH) release and the LDH and LPO levels were significantly higher in the hypoxia-30 and 60 min-exposed cells according to normoxia 30 and 60 min groups. Furthermore, we found that the LPO and LDH were decreased in the hypoxia-exposed cells after being treated with Zn, Se, and GSH according to the hypoxia groups. Compared to the normoxia groups, the current densities of TRPM2 channel were increased in the hypoxia-exposed cells by the hypoxia applications, while the same values were decreased in the treatment of Zn, Se, and GSH according to hypoxia group. In conclusion, hypoxia-induced TRPM2 channel activity, ROS and cell death were recovered by the Se, Zn and GSH treatments.

Defense Responses of Wheat Seedling under Biotic Stress Mimicked by the Linear β-(1,3)-Glucan

Reactive oxygen species (ROS) production causes damage, and to better deal with the toxic effects of ROS, the seeds have developed detoxification mechanisms, among which the enzymes of the antioxidant system (catalase, superoxide dismutase, ascorbate peroxidase). Another result supports the link between ROS and redox regulation catalyzed by redoxin family in the seed. Among which, thioredoxins (Trxs) and peroxiredoxins (Prxs), particularly 1-Cys-Prx, expressed during maturation and germination steps.

High-throughput, Label-Free Quantitative Proteomic Studies of the Anticancer Effects of Electrical Pulses with Turmeric Silver Nanoparticles: an in vitro Model Study

Triple negative breast cancer (TNBC) represents 15–20% of the over one million new breast cancer cases occurring each year. TNBC is an aggressive cancer phenotype, with low 5-year survival rates, high 3-year recurrence rates, and increased risk of metastasis. A lack of three commonly exploited hormone receptors renders TNBC resistant to endocrine therapies and lends to its critical absence of viable therapeutic targets. This necessitates the development of alternate and effective novel therapeutic strategies for TNBC. Towards this, our current work seeks to develop the technique of Electrical pulse (EP)-mediated Turmeric silver nanoparticles (TurNP) therapy, known as Electrochemotherapy (ECT), to effectively target TNBC cells. This technique involves the efficient delivery of natural bioactive molecules with anti-cancer effects via a biophysical means. In these experiments, the bioactive molecules are turmeric, a dried rhizome of Curcuma longa that has been used for centuries, both as a dietary supplement and as a medicine in Ayurveda (science of life) in the Indian subcontinent and in traditional Chinese medicine. Our results reveal the combined effect of TurNP + EP treatment in reducing MDA-MB-231 cell viability to as low as 9% at 12 h. Showing biological selectivity, this combination treatment has a substantially lower effect on non-tumorigenic mammary epithelial MCF10A cells (67% viability). To gain mechanistic insights into the actions of TurNP-based ECT treatment, we performed high-throughput, label-free quantitative proteomics studies. Proteomics results indicate that TurNP + EP treatment significantly influenced expression of a diverse list of proteins, including receptors, transcription factors, structural proteins, kinases, and metabolic enzymes. This include the downregulation of 25 proteins in PI3K-Akt signaling pathway (such as GRB2, EGFR, EPHA2, GNB1, GNB2, 14–3–3 family, and Integrin family proteins), and 12 proteins (AKR1A1, ALDOA, ALDOC, PGK1, PGM1, PGAM1, ENO1, ENO2, GAPDH, TPI1, LDHA, and LDHB) in the glycolytic pathway with concomitant reduction in metabolite levels (glucose uptake, and intracellular- lactate, glutamine, and glutamate). Compared to TurNP alone, TurNP + EP treatment upregulated 66 endoplasmic reticulum and 193 mitochondrial proteins, enhancing several processes and pathways, including Pyruvate Metabolism, Tricarboxylic acid (TCA) cycle, and Oxidative Phosphorylation (OXPHOS), which redirected the TNBC metabolism to mitochondria. This switch in the metabolism caused excessive production of H2O2 reactive oxygen species (ROS) to inflict cell death in MDA-MB-231 cells, demonstrating the potency of this treatment.