Intracellular Superoxide Levels Research Articles

Exosomes from umbilical cord mesenchymal stem cells ameliorate intervertebral disc degeneration via repairing mitochondrial dysfunction

BackgroundReactive oxygen species (ROS), predominantly generated by mitochondria, play a crucial role in the pathogenesis of intervertebral disc degeneration (IVDD). Reduction of ROS levels may be an effective strategy to delay IVDD. In this study, we assessed whether umbilical cord mesenchymal stem cell-exosomes (UCMSC-exos) can be used to treat IVDD by suppressing ROS production caused by mitochondrial dysfunction. Materials and methodsHuman UCMSC-exos were isolated and identified. Nucleus pulposus cells (NPCs) were stimulated with H2O2 in the presence or absence of exosomes. Then, 4D label free quantitative (4D-LFQ) proteomics were used to analyze the differentially expressed (DE) proteins. Mitochondrial membrane potential (MMP), mitochondrial ROS and protein levels were determined via immunofluorescence staining, flow cytometry and western blotting respectively. Additionally, high-throughput sequencing was performed to identify the DE miRNAs in NPCs. Finally, therapeutic effects of UCMSC-exos were investigated in a puncture-induced IVDD rat model. Degenerative grades of rat IVDs were assessed using magnetic resonance imaging and histochemical staining. ResultsUCMSC-exos effectively improved the viability of NPCs and restored the expression of the extracellular matrix (ECM) proteins, collagen type II alpha-1 (COL2A1) and matrix metalloproteinase-13 induced by H2O2. Additionally, UCMSC-exos not only reduced the total intracellular ROS and mitochondrial superoxide levels, but also increased MMP in pathological NPCs. 4D-LFQ proteomics and western blotting further revealed that UCMSC-exos up-regulated the levels of the mitochondrial protein, mitochondrial transcription factor A (TFAM), in H2O2-induced NPCs. High-throughput sequencing and qRT-PCR uncovered that UCMSC-exos down-regulated the levels of miR-194-5p, a potential negative regulator of TFAM, induced by H2O2. Finally, in vivo results showed that UCMSC-exos injection improved the histopathological structure and enhanced the expression levels of COL2A1 and TFAM in the rat IVDD model. ConclusionsOur findings suggest that UCMSC-exos promote ECM synthesis, relieve mitochondrial oxidative stress, and attenuate mitochondrial dysfunction in vitro and in vivo, thereby effectively treating IVDD. The translational potential of this articleThis study provides solid experimental data support for the therapeutic effects of UCMSC-exos on IVDD, suggesting that UCMSC-exos will be a promising nanotherapy for IVDD.

Impact of amino acid substitutions in hepatitis C virus core region on the severe oxidative stress

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease, leading to liver steatosis, fibrosis, and hepatocellular carcinoma (HCC). Despite the accumulation of clinical data showing the impact of amino acid substitutions at positions 70 (R70Q/H) and/or 91 (L91M) in the HCV core protein in progressive liver diseases, including HCC, the underlying mechanisms have not been elucidated.We analyzed 72 liver biopsy specimens from patients with chronic HCV genotype 1b (HCV-1b) infection prior to antiviral treatment. Levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG) and nuclear factor erythroid 2-related factor 2 (NRF2) in the nucleus were quantified using liver tissue immunohistochemistry. The effects of amino acid substitutions in the HCV core region on hepatocellular oxidative stress were investigated using wild-type or double-mutant (R70Q/H+L91M) HCV-1b core transfection and stable expression in human hepatoma HuH-7 cells.Overall, 24, 19, 11, and 18 patients had the wild-type, R70Q/H, L91M, and R70Q/H+L91M genotypes, respectively, in the HCV core. A significantly higher accumulation of hepatocellular 8-OHdG and a lower NRF2/8-OHdG ratio were observed in patients with R70Q/H+L91M than in those with the wild-type disease. Increased levels of intracellular superoxide and hydrogen peroxide in the cytoplasm and mitochondria, mRNA expression of enzymes generating oxidative stress, and nuclear expression of nicotinamide adenine dinucleotide phosphate oxidase 4 were augmented in cells treated with R70Q+L91M.HCV core proteins harboring either or both substitutions of R70Q/H or L91M enhanced hepatocellular oxidative stress in vivo and in vitro. These amino acid substitutions may affect HCC development by enhancing hepatic oxidative stress in patients with chronic HCV-1b infection.

Anti-Necroptotic Effects of Itaconate and its Derivatives.

Itaconate is an unsaturated dicarboxylic acid that is derived from the decarboxylation of the Krebs cycle intermediate cis-aconitate and has been shown to exhibit anti-inflammatory and anti-bacterial/viral properties. But the mechanisms underlying itaconate's anti-inflammatory activities are not fully understood. Necroptosis, a lytic form of regulated cell death (RCD), is mediated by receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL) signaling. It has been involved in the pathogenesis of organ injury in many inflammatory diseases. In this study, we aimed to explore whether itaconate and its derivatives can inhibit necroptosis in murine macrophages, a mouse MPC-5 cell line and a human HT-29 cell line in response to different necroptotic activators. Our results showed that itaconate and its derivatives dose-dependently inhibited necroptosis, among which dimethyl itaconate (DMI) was the most effective one. Mechanistically, itaconate and its derivatives inhibited necroptosis by suppressing the RIPK1/RIPK3/MLKL signaling and the oligomerization of MLKL. Furthermore, DMI promoted the nuclear translocation of Nrf2 that is a critical regulator of intracellular redox homeostasis, and reduced the levels of intracellular reactive oxygen species (ROS) and mitochondrial superoxide (mtROS) that were induced by necroptotic activators. Consistently, DMI prevented the loss of mitochondrial membrane potential induced by the necroptotic activators. In addition, DMI mitigated caerulein-induced acute pancreatitis in mice accompanied by reduced activation of the necroptotic signaling in vivo. Collectively, our study demonstrates that itaconate and its derivatives can inhibit necroptosis by suppressing the RIPK1/RIPK3/MLKL signaling, highlighting their potential applications for treating necroptosis-associated diseases.

Comparative bioactivity analysis of hydro-methanolic extracts of root, leaf, and inflorescence of Tupistra nutans Wall. ex Lindl: An in vitro study on antioxidant and anti-hepatocellular carcinoma activity

Tupistra nutans, native to the Eastern Himalayas, is traditionally used as vegetables and in manaand pressure. Despite being a medicinal agricultural plant, no in-depth antioxidant and anticancer study was done. Therefore, in this study, we investigated antioxidant and anti-hepatocellular carcinoma activities of hydro-methanolic extracts of its root (ETNRt), leaf (ETNLf), and inflorescence (ETNIn). DPPH, ABTS, FRAP, and total antioxidant capacity (TAC) assays revealed that ETNLf had the highest antioxidant potential. Also, extracts showed antioxidant activity in human hepatocellular carcinoma (HepG2) cells by reducing H2O2-induced ROS generation at lower concentrations. However, at higher concentrations, ETNRt, ETNLf, and ETNIn had anticancer effects in HepG2 cells with IC50 (12 h) values of 92.62, 34.60, and 85.55 μg/ml respectively, which was further confirmed by cell migration and clonogenicity reduction. Spectrofluorometric and fluorescence microscopy revealed that ETNRt, ETNLf, and ETNIn increased intracellular ROS, superoxide, and calcium levels and reduced mitochondrial membrane potential and mass. ETNRt, ETNLf, and ETNIn also depleted reduced glutathione levels in HepG2. Acridine orange/propidium iodide and scanning electron microscopy analysis confirmed the membrane damage and apoptotic changes, whereas confocal microscopic analysis with DAPI staining indicated cytoplasmic shrinkage and DNA damage. GC-MS analysis of ETNRt, ETNLf, and ETNIn revealed the differential distribution of anticancer phytochemicals such as beta-citronellol, piperidine, diosgenin, beta-sitosterol, and stigmasterol in Tupistra nutans. Our study suggested that Tupistra nutans had a promising antioxidant and in-vitro anti-hepatocellular carcinoma activity. However, the anticancer potential of inflorescence, used as a vegetable, decreased when it was cooked compared to its hydro-methanolic extract.

Metformin reverses oxidative stress‑induced mitochondrial dysfunction in pre‑osteoblasts via the EGFR/GSK‑3β/calcium pathway.

Oxidative stress is one of the main causes of osteoblast apoptosis induced by post‑menopausal osteoporosis. The authors previously found that metformin can reverse the loss of bone mass in post‑menopausal osteoporosis. The present study aimed to further clarify the effects and mechanisms of action of metformin in post‑menopausal osteoporosis under conditions of oxidative stress. Combined with an in‑depth investigation using the transcriptome database, the association between oxidative stress and mitochondrial dysfunction in post‑menopausal osteoporosis was confirmed. A pre‑osteoblast model of oxidative stress was constructed, and the apoptotic rate following the addition of hydrogen peroxide and metformin was detected using CCK‑8 assay and Annexin V‑FITC/PI staining. Mitochondrial membrane potential was detected using the JC‑1 dye, the intracellular calcium concentration was detected using Fluo‑4 AM, the intracellular reactive oxygen species (ROS) level was observed using DCFH‑DA, and the mitochondrial superoxide level was observed using MitoSOX Red. Bay K8644 was used to increase the level of intracellular calcium. siRNA was used to interfere with the expression of glycogen synthase kinase (GSK)‑3β. Western blot analysis was used to detect the expression of mitochondrial dysfunction‑related proteins. The results revealed that oxidative stress decreased mitochondrial membrane potential and increased intracellular ROS, mitochondrial superoxide and cytoplasmic calcium levels in pre‑osteoblasts; however, metformin improved mitochondrial dysfunction and reversed oxidative stress‑induced injury. Metformin inhibited mitochondrial permeability transition pore opening, suppressed the cytoplasmic calcium influx and reversed pre‑osteoblast apoptosis by promoting GSK‑3β phosphorylation. Moreover, it was found that EGFR was the cell membrane receptor of metformin in pre‑osteoblasts, and the EGFR/GSK‑3β/calcium axis played a key role in metformin reversing the oxidative stress response of pre‑osteoblasts in post‑menopausal osteoporosis. On the whole, these findings provide a pharmacological basis for the use of metformin for the treatment of post‑menopausal osteoporosis.

Mn(III) Porphyrin, MnTnBuOE-2-PyP5+, Commonly Known as a Mimic of Superoxide Dismutase Enzyme, Protects Cardiomyocytes from Hypoxia/Reoxygenation Induced Injury via Reducing Oxidative Stress.

Myocardial ischemia-reperfusion injury (I/R) causes damage to cardiomyocytes through oxidative stress and apoptosis. We investigated the cardioprotective effects of MnTnBuOE-2-PyP5+ (BMX-001), a superoxide dismutase mimic, in an in vitro model of I/R injury in H9c2 cardiomyocytes. We found that BMX-001 protected against hypoxia/reoxygenation (H/R)-induced oxidative stress, as evident by a significant reduction in intracellular and mitochondrial superoxide levels. BMX-001 pre-treatment also reduced H/R-induced cardiomyocyte apoptosis, as marked by a reduction in TUNEL-positive cells. We further demonstrated that BMX-001 pre-treatment significantly improved mitochondrial function, particularly O2 consumption, in mouse adult cardiomyocytes subjected to H/R. BMX-001 treatment also attenuated cardiolipin peroxidation, 4-hydroxynonenal (4-HNE) level, and 4-HNE adducted proteins following H/R injury. Finally, the pre-treatment with BMX-001 improved cell viability and lactate dehydrogenase (LDH) activity in H9c2 cells following H/R injury. Our findings suggest that BMX-001 has therapeutic potential as a cardioprotective agent against oxidative stress-induced H/R damage in H9c2 cardiomyocytes.

Berberine hydrochloride inhibits migration ability via increasing inducible NO synthase and peroxynitrite in HTR-8/SVneo cells

Ethnopharmacological relevanceInadequate trophoblasts migration and invasion is considered as an initial event resulting in preeclampsia, which is closely related to oxidative stress. Berberine hydrochloride (BBR), extracted from the traditional medicinal plant Coptis chinensis Franch., exerts a diversity of pharmacological effects, and the crude drug has been widely taken by most Chinese women to treat nausea and vomit during pregnancy. But there is no research regarding its effects on trophoblast cell function. Aim of the studyThis study aimed to investigate the effect of BBR on human-trophoblast-derived cell line (HTR-8/SVneo) migration ability and its mechanism. Materials and methodsCell viability was detected by CCK-8 assay. The effect of BBR on cells migration function was examined by scratch wound healing assay and transwell migration assay. Intracellular nitric oxide (NO), superoxide (O2−) and peroxynitrite (ONOO−) levels were measured by flow cytometry. The expression levels of inducible NO synthase (iNOS), eNOS, p-eNOS, MnSOD, CuZnSOD, Rac1, NOX1, TLR4, nuclear factor-κB (NF-κB), p-NFκB, pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) in cells were analyzed by Western blotting. Uric acid sodium salt (UA), the scavenger of ONOO−, PEG-SOD (a specific superoxide scavenger), L-NAME (a NOS inhibitor) and antioxidants (Vit E and DFO) were further used to characterize the pathway of BBR action. Results5 μM BBR decreased both the migration distance and the number of migrated cells without affecting cells viability in HTR-8/SVneo cells after 24 h treatment. BBR could increase the level of NO in HTR-8/SVneo cells, and the over-production of NO might be attributable to iNOS, but not eNOS. BBR could increase intracellular O2− levels, and the over-production of O2− is closely related with Rac1 in HTR-8/SVneo cells. The excessive production of NO and O2− further react to form ONOO−, and the increased ONOO− level induced by BBR was blunted by UA. Moreover, UA improved the impaired migration function caused by BBR in HTR-8/SVneo cells. The depressed migration function stimulated by BBR in HTR-8/SVneo cells was diminished by PEG-SOD and L-NAME. Furthermore, BBR increased the expression of IL-6 in HTR-8/SVneo cells, and antioxidants (Vit E and DFO) could decrease the expression of IL-6 and iNOS induced by BBR. ConclusionsBBR inhibits the cell migration ability through increasing inducible NO synthase and peroxynitrite in HTR-8/SVneo cells, indicating that BBR and traditional Chinese medicines containing a high proportion of BBR should be used with caution in pregnant women.

Marine Sponge-Derived Alkaloid Induces Mitochondrial Dysfunction and Inhibits the PI3K/AKT/mTOR Signaling Pathway against Burkitt's Lymphoma.

Burkitt's lymphoma (BL) has a particularly extremely poor prognosis and the fastest growth rate among human tumors, and the development of new drugs for the treatment of BL is urgently needed. In this study, the cytotoxic properties of 3,7-bis(3,5-dimethylphenyl)-aaptamine (AP-51), a new semisynthetic alkaloid derived from the marine natural product aapatamine, were investigated using BL cell lines. Our results showed that AP-51 inhibited the proliferation of Daudi and Raji cells with IC50 values of 3.48 and 2.07 μM, respectively. Flow cytometry and Western blot analyses showed that AP-51 initiated G0/G1 phase arrest by modulating the expression of cyclin-dependent kinases (CDKs). AP-51 also induced apoptosis, as demonstrated by nuclear fragmentation, downregulation of BCL-XL and Mcl-1, and upregulation of cleaved caspase-9, cleaved caspase-3, cleaved-PARP, and cytochrome c, the markers of apoptosis regulated via the mitochondrial pathway. When it comes to mitochondria, AP-51 treatment also significantly increased the levels of intracellular mitochondrial superoxide, decreased ATP content, and reduced the expression of ATP synthase, as well as the expression of the mitochondrial respiratory chain complexes. Finally, AP-51 treatment significantly inhibited the PI3K/AKT/mTOR signaling pathway, which was shown to be associated with the induction of apoptosis. Collectively, these findings indicated that AP-51 initiated cell cycle arrest, induced apoptosis, caused mitochondrial dysfunction, and decreased the phosphorylation of PI3K/AKT/mTOR signaling pathway-related proteins and the protein levels of C-MYC, suggesting that AP-51 has therapeutic potential as a possible treatment for Burkitt's lymphoma.

MS-5, a Naphthalene Derivative, Induces Apoptosis in Human Pancreatic Cancer BxPC-3 Cells by Modulating Reactive Oxygen Species.

Pancreatic cancer is one of the most fatal cancers with a poor prognosis. Standard chemotherapies have proven largely ineffective because of their toxicity and the development of resistance. Therefore, there is an urgent need to develop novel therapies. In this study, we investigated the antitumor activity of MS-5, a naphthalene derivative, on BxPC-3, a human pancreatic cancer cell line. We observed that MS-5 was cytotoxic to BxPC-3 cells, as well as inhibited the growth of cells in a concentration- and time- dependent manner. Flow cytometry analysis revealed that the percentage of annexin V-positive cells increased after MS-5 treatment. We also observed cleavage of caspases and poly (ADP-ribose) polymerase, and downregulation of Bcl-xL protein. Flow cytometry analysis of intracellular levels of reactive oxygen species (ROS) and mitochondrial superoxide suggested that MS-5 induced the generation of mitochondrial superoxide while lowering the overall intracellular ROS levels. Thus, MS-5 may be potential candidate for pancreatic cancer treatment.

The Role of Alpha-Tocotrienol during Development of Primary Hippocampal Neurons

ObjectivesAlpha-tocotrienol (α-TCT), a form of vitamin E, is a lipophilic antioxidant with neuroprotective properties. We recently reported that α-TCT treatment prevents oxidative stress-induced proteolytic cleavage of B-cell lymphoma-extra large (Bcl-xL), a pro-survival mitochondrial protein necessary during neuronal growth. However, it is still unclear if α-TCT exhibits beneficial effects during the physiological development of neurons. In this study, we hypothesized that chronic α-TCT treatment advances the development of primary hippocampal neurons by improving mitochondrial function.MethodsPrimary rat hippocampal neurons were grown in neurobasal media with or without α-TCT for three weeks, and media was replaced with conditioned media containing fresh α-TCT every week. Intracellular α-TCT levels were quantified using HPLC-MS, and intracellular ATP and mitochondrial superoxide levels were determined using luciferase and mitoSOX, respectively. Neurite morphology was examined by Sholl analysis. mRNA and protein levels of Bcl-xL were quantified using qPCR and immunoblotting, respectively.ResultsPrimary hippocampal neurons grown in media containing α-TCT had increased intracellular α-TCT levels and decreased mitochondrial superoxide. Treatment with α-TCT increased mRNA and protein levels of Bcl-xL, neuronal ATP, and the number of neurite branches in primary hippocampal neurons.ConclusionsWe found that primary rat hippocampal neurons treated with α-TCT developed advanced neurite complexity. We suggest that α-TCT treatment improves mitochondrial function via upregulation of Bcl-xL, supporting normal neuron development.Funding SourcesRGC Program (University of Alabama); Crenshaw Research Fund (University of Alabama).

Restricting tumor lactic acid metabolism using dichloroacetate improves T cell functions

BackgroundLactic acid produced by tumors has been shown to overcome immune surveillance, by suppressing the activation and function of T cells in the tumor microenvironment. The strategies employed to impair tumor cell glycolysis could improve immunosurveillance and tumor growth regulation. Dichloroacetate (DCA) limits the tumor-derived lactic acid by altering the cancer cell metabolism.In this study, the effects of lactic acid on the activation and function of T cells, were analyzed by assessing T cell proliferation, cytokine production and the cellular redox state of T cells. We examined the redox system in T cells by analyzing the intracellular level of reactive oxygen species (ROS), superoxide and glutathione and gene expression of some proteins that have a role in the redox system. Then we co-cultured DCA-treated tumor cells with T cells to examine the effect of reduced tumor-derived lactic acid on proliferative response, cytokine secretion and viability of T cells.ResultWe found that lactic acid could dampen T cell function through suppression of T cell proliferation and cytokine production as well as restrain the redox system of T cells by decreasing the production of oxidant and antioxidant molecules. DCA decreased the concentration of tumor lactic acid by manipulating glucose metabolism in tumor cells. This led to increases in T cell proliferation and cytokine production and also rescued the T cells from apoptosis.ConclusionTaken together, our results suggest accumulation of lactic acid in the tumor microenvironment restricts T cell responses and could prevent the success of T cell therapy. DCA supports anti-tumor responses of T cells by metabolic reprogramming of tumor cells.

Addition of Reduced Glutathione (GSH) to Freezing Medium Reduces Intracellular ROS Levels in Donkey Sperm.

In donkeys, the use of frozen-thawed sperm for artificial insemination (AI) leads to low fertility rates. Furthermore, donkey sperm produce a large amount of reactive oxygen species (ROS), and post-AI inflammation induces the formation of neutrophil extracellular traps (NETosis), which further generates many more ROS. These high ROS levels may induce lipid peroxidation in the sperm plasma membrane, thus affecting its integrity. Enzymatic and non-enzymatic antioxidants, mainly found in the seminal plasma (SP), are responsible for maintaining the redox balance. However, this fluid is removed prior to cryopreservation, thereby exposing sperm cells to further oxidative stress. The exogenous addition of antioxidants to the freezing medium can reduce the detrimental effects caused by ROS generation. Therefore, the aim of this study was to evaluate how the addition of different reduced glutathione (GSH) concentrations (control, 2 mM, 4 mM, 6 mM, 8 mM, and 10 mM) to fresh sperm affect their cryotolerance. Total and progressive motility, kinematic parameters and motile sperm subpopulations were significantly (p < 0.05) different from the control in treatments containing 8 mM and 10 mM GSH, but not at lower concentrations. Plasma and acrosome membrane integrity, mitochondrial membrane potential (MMP) and intracellular superoxide levels (O2−) were not affected (p > 0.05) by any GSH concentration. Interestingly, however, the addition of 8 mM or 10 mM GSH reduced (p < 0.05) the percentages of viable sperm with high overall ROS levels compared to the control. In conclusion, frozen-thawed donkey sperm are able to tolerate high GSH concentrations, which differs from what has been observed in other species. This antioxidant capacity suggests that ROS could be important during post-AI and that the impact of using exogenous antioxidants like GSH to improve the sperm resilience to freeze-thawing is limited in this species.

Cementing the relationship between conventional and advanced semen parameters

BackgroundAffordable conventional semen analysis remains a fundamental procedure to be performed routinely during the diagnosis of male infertility. Advanced semen analyses provide valuable clinical insights in treatment-related decision-making, but these are highly expensive and lack universal standardization. This study aimed at determining the relationship between conventional semen parameters, measured with assistance of computer-aided sperm analysis (CASA), and a set of advanced semen tests. Basic semen analysis (n = 124) was performed according to the World Health Organization (WHO) guidelines. Sperm DNA fragmentation and intracellular superoxide (O2−•) levels were assessed by flow cytometry. Seminal plasma thiobarbituric acid reactive substances (TBARS) levels as well as superoxide dismutase (SOD) and catalase (CAT) activity were measured by spectrophotometry. Spearman’s rank correlation coefficient was used, with significance set at p < 0.05.ResultsSemen pH correlated negatively with TBARS (p < 0.01). The proportions of total and progressively motile as well as rapid spermatozoa correlated positively with CAT activity (p < 0.05). Sperm viability correlated negatively with both O2−• (p < 0.05) and DNA fragmentation (p = 0.01), while normal morphology correlated negatively with O2−• levels (p < 0.05) and positively with CAT activity (p < 0.05). Straight-line velocity (VCL) and average-path velocity (VAP) correlated negatively with both O2−• (p < 0.01) and TBARS (p < 0.01). Amplitude of lateral head displacement (ALH) correlated negatively with O2−• (p < 0.01) and DNA fragmentation (p < 0.01), while its correlation with SOD activity was positive (p < 0.05).ConclusionThe results obtained from this study support the validity of some CASA parameters as sensitive indicators of changes in sperm oxidative status and DNA integrity. Predicting advanced from conventional parameters through the building of linear regression models should be considered for future studies.

1,5-Benzodiazepin-2(3H)-ones: In Vitro Evaluation as Antiparkinsonian Agents.

A new series of twenty-three 1,5-benzodiazepin-2(3H)-ones were synthesized and evaluated in the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), ferric reducing antioxidant power (FRAP), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays as a new chemotype with antioxidant and good drug-like properties. All of the derivatives showed low cytotoxicity in comparison to curcumin against the human neuroblastoma SH-SY5Y and the human hepatoma HepG2 cell lines. Experimental solubility in bio-relevant media showed a good relationship with melting points in this series. Five compounds with the best antioxidant properties showed neuroprotectant activity against H2O2-induced oxidative stress in the SH-SY5Y cell line. From them, derivatives 4-phenyl-1H-1,5-benzodiazepin-2(3H)-one (18) and 4-(3,4,5-trimethoxyphenyl)-1H-1,5-benzodiazepin-2(3H)-one (20) yielded good neuroprotection activity in the same neuronal cell line under 6-OHD and MPP+ insults as in vitro models of mitochondrial dysfunction and oxidative stress in Parkinson’s disease (PD). Both compounds also demonstrated a significant reduction of intracellular Reactive Oxygen Species (ROS) and superoxide levels, in parallel with a good improvement of the Mitochondrial Membrane Potential (ΔΨm). Compared with curcumin, compound 18 better reduced lipid peroxidation levels, malondialdehyde (MDA), in SH-SY5Y cells under oxidative stress pressure and recovered intracellular glutathione synthetase (GSH) levels. Apoptosis and caspase-3 levels of SH-SY5Y under H2O2 pressure were also reduced after treatment with 18. Neuroprotection in neuron-like differentiated SH-SY5Y cells was also achieved with 18. In summary, this family of 1,5-benzodiazepin-2-ones with an interesting antioxidant and drug-like profile, with low cytotoxic and good neuroprotectant activity, constitutes a new promising chemical class with high potential for the development of new therapeutic agents against PD.

Superoxide dismutase C affects Dictyostelium contractile vacuole biogenesis and function

Dictyostelium cells cope with hypo-osmotic stress with a contractile vacuole (CV) system, which consists of one or two vacuoles that cyclically charge and discharge. Uniquely, a F-Actin remodeling dependent minimal mixing of the CV membrane components with the target plasmalemma during the fusion and the dischargement warrants the integrity of the CV bladder for an efficient next CV cycle. The effect of hypo-osmotic stress on F-Actin remodeling activity, however, is currently not well understood. Dictyostelium cells increase the level of intracellular superoxide level in response to hypo-osmotic stress, which in turn activates redox-sensitive Ras proteins, but not Akt, which is one of the Ras downstream targets and a major regulator of F-Actin remodeling. However, Akt is not insulated from the active Ras in cells lacking Superoxide dismutase C (SodC). We report here that sodC- cells were compromised in the CV structure and function and the attenuation of Ras/PI3K/Akt signaling in several independent means significantly improved the compromised CV structure but not the function. Interestingly, when sodC- cells were treated with 5-(N,N-Dimethyl) amiloride hydrochloride (EIPA), an inhibitor of sodium proton exchanger (NHE), both the structure and the function of the CV improved. Thus, a proper CV biogenesis in sodC- cells was insufficient to restore their CV function, which in turn indicates the presence of an additional target for SodC and EIPA that modulates CV function.

Suppression of Rotenone-Treated Human Breast Cancer Stem Cell Survival Using Survivin Inhibitor YM155 is Associated to Oxidative Stress Modulation.

Background:Despite recent progress in molecular-targeted therapies, breast cancer remains the primary leading cause of cancer related death among women worldwide. Breast cancer stem cells (BCSCs) are believed to be responsible for therapy resistance and cancer recurrence. We recently demonstrated that human BCSCs (CD24-/CD44+) could survive better than their counterpart non-BCSCs (CD24-/CD44-) when treated with rotenone, possibly due to lower levels of reactive oxygen species (ROS) production, high expression of antioxidant manganese superoxide dismutase (MnSOD), and anti-apoptotic survivin. The aim of this study was to verify the role of survivin on human BCSCs survival under oxidative stress modulation by suppressing its expression using YM155, a survivin inhibitor. Methods:Human BCSCs (ALDH+ cells) were treated with YM155 for 24 h prior to treatment with rotenone for a further 6 h. We determined intracellular superoxide levels were determined using dihydroethidium assay, survivin and MnSOD expression using qRT-PCR, survivin protein level using ELISA, as well as cell viability using trypan blue exclusion and acridine orange/ethidium bromide apoptosis assay. Results:Suppression of survivin expression using YM155 could reduce the survival of rotenone-treated BCSCs, which may be associated with oxidative stress modulation, as shown by increased ROS levels and decreased MnSOD expression. We confirm that survivin is responsible for maintaining BCSCs survival under oxidative stress modulation. Furthermore, YM155 could modulate oxidative stress in BCSCs by reducing MnSOD expression and increasing ROS levels. Conclusion:YM155 treatment could be used to overcome BCSCs resistance to oxidative stress-based anticancer therapies.

Melatonin Rescues the Ti Particle-Impaired Osteogenic Potential of Bone Marrow Mesenchymal Stem Cells via the SIRT1/SOD2 Signaling Pathway.

Wear particles released by joint implants are a major cause of osteolysis around the prosthesis by negatively affecting bone reconstruction. Bone marrow mesenchymal stem cells (BMMSCs) stimulated by wear particles showed an impaired osteogenic potential. Melatonin has been shown beneficial effects on intracellular antioxidant functions and bone formation; however, whether it could restore the osteogenic potential of BMMSCs inhibited by wear particles was unknown. This study aimed to evaluate the protective effect of melatonin on the osteogenic capacity of BMMSCs exposed to titanium (Ti) wear particles and to investigated the underlying mechanisms involving intracellular antioxidant properties. When BMMSCs were exposed to Ti particles in vitro, melatonin treatment successfully improved the matrix mineralization and expression of osteogenic markers in BMMSCs, while decreasing the levels of intracellular reactive oxygen species (ROS) and mitochondrial superoxide. The protective effect of melatonin on osteolysis was validated in a Ti particle-exposed murine calvarial model. Meanwhile, silent information regulator type 1 (SIRT1) and intracellular antioxidant enzymes were significantly up-regulated, particularly superoxide dismutase 2 (SOD2), in melatonin-treated BMMSCs. Furthermore, inhibition of SIRT1 by EX527 completely counteracted the protective effect of melatonin on Ti particle-treated BMMSCs, evidenced by the reduced expression of SOD2, increased ROS and superoxide, and decreased osteogenic differentiation. These results demonstrated that melatonin restored the osteogenic potential and improved the antioxidant properties of BMMSCs through the SIRT1 signaling pathway. Our findings suggest that melatonin is a promising candidate for treating osteolysis induced by wear particles.

Cytotoxic and genotoxic effects on human keratinocytes triggered by sphingomyelinase D from Loxosceles venom.

The spiders of the Loxosceles genus (called brown or violin spiders) are of medical relevance in several countries due to the many human envenomation cases reported. The main component of Loxosceles venom is the enzyme sphingomyelinase D (SMase D), which is responsible for the local and systemic effects induced by the whole venom. Here, we investigated the cytotoxic and genotoxic effects caused by Loxosceles laeta venom and SMase D on human keratinocytes to better understand the dermonecrosis development mechanism. Our findings indicate that whole venom, as well as SMase D, increases intracellular superoxide levels, leading to DNA damage. These effects appear to be dependent on the binding of SMase D to the cell surface, although the complete pathway triggered as a result of the binding still needs to be elucidated. Moreover, after SMase D treatment, we observed the presence of histone γH2AX, suggesting that the cells are undergoing DNA repair. Moreover, when ATR kinase was inhibited, the cell viability of human keratinocytes was decreased. Together, our findings strongly suggest that L. laeta venom, as well as SMase D, increases intracellular superoxide levels, leading to DNA damage in human keratinocytes. Additionally, the induced DNA damage is repaired through the activation of an apparent ATR-mediated DNA-damage response. This knowledge may contribute to a better understanding of the behaviour of human keratinocytes during cutaneous loxoscelism, a condition that affects thousands of people around the world.

Nitroxide Radical-Containing Redox Nanoparticles Protect Neuroblastoma SH-SY5Y Cells against 6-Hydroxydopamine Toxicity

Parkinson's disease (PD) patients can benefit from antioxidant supplementation, and new efficient antioxidants are needed. The aim of this study was to evaluate the protective effect of selected nitroxide-containing redox nanoparticles (NRNPs) in a cellular model of PD. Antioxidant properties of NRNPs were studied in cell-free systems by protection of dihydrorhodamine 123 against oxidation by 3-morpholino-sydnonimine and protection of fluorescein against bleaching by 2,2-azobis(2-amidinopropane) hydrochloride and sodium hypochlorite. Model blood-brain barrier penetration was studied using hCMEC/D3 cells. Human neuroblastoma SH-SY5Y cells, exposed to 6-hydroxydopamine (6-OHDA), were used as an in vitro model of PD. Cells were preexposed to NRNPs or free nitroxides (TEMPO or 4-amino-TEMPO) for 2 h and treated with 6-OHDA for 1 h and 24 h. The reactive oxygen species (ROS) level was estimated with dihydroethidine 123 and Fluorimetric Mitochondrial Superoxide Activity Assay Kit. Glutathione level (GSH) was measured with ortho-phtalaldehyde, ATP by luminometry, changes in mitochondrial membrane potential with JC-1, and mitochondrial mass with 10-Nonyl-Acridine Orange. NRNP1, TEMPO, and 4-amino-TEMPO (25-150 μM) protected SH-SY5Y cells from 6-OHDA-induced viability loss; the protection was much higher for NRNP1 than for free nitroxides. NRNP1 were better antioxidants in vitro and permeated better the model BBB than free nitroxides. Exposure to 6-OHDA decreased the GSH level after 1 h and increased it considerably after 24 h (apparently a compensatory overresponse); NRNPs and free nitroxides prevented this increase. NRNP1 and free nitroxides prevented the decrease in ATP level after 1 h and increased it after 24 h. 6-OHDA increased the intracellular ROS level and mitochondrial superoxide level. Studied antioxidants mostly decreased ROS and superoxide levels. 6-OHDA decreased the mitochondrial potential and mitochondrial mass; both effects were prevented by NRNP1 and nitroxides. These results suggest that the mitochondria are the main site of 6-OHDA-induced cellular damage and demonstrate a protective effect of NRNP1 in a cellular model of PD.

Fluorescein-Containing Superoxide Probes with a Modular Copper-Based Trigger.

Fluorescein-derived superoxide probes featuring a copper(II) complex that can be activated by superoxide to initiate ether bond cleavage and uncage a fluorescein reporter for imaging in live cells are described. Compared to other superoxide sensing moieties, this bond cleavage strategy can be modularly adapted to fluorescent reporters with different properties without compromising the superoxide reactivity and selectivity. A green-emitting probe and its lysosome-targeting analogue have been successfully developed. Both probes are sensitive with more than 30-fold fluorescence enhancement towards superoxide and are highly selective with no significant response towards other reactive oxygen species. A structure-activity relationship study of the copper-based superoxide trigger showed that the secondary coordination environment of the copper(II) center is important for the superoxide reactivity and selectivity. The probes have been applied in imaging changes in intracellular superoxide level in live HeLa and HEK293T cells upon menadione stimulation and also in a cellular inflammation model in RAW 264.7 cells.