Superoxide Accumulation Research Articles

Brevilin A Inhibits VEGF-Induced Angiogenesis through ROS-Dependent Mitochondrial Dysfunction.

Brevilin A (BA), a sesquiterpene lactone isolated from Centipeda minima herb, has been identified to exhibit potent anticancer activity. However, the potential pharmacological effect and mechanism of BA in regulating endothelial cell (EC) angiogenesis, a key event in tumor growth, is poorly understood. In this study, BA was shown to significantly prevent vascular endothelial growth factor (VEGF) induced EC angiogenic capacities in vitro, ex vivo, and in vivo. Subsequent functional assays revealed that BA dose dependently inhibited VEGF-stimulated survival, proliferation, migration, and triggered apoptosis activity in human umbilical vein endothelial cells (HUVECs), as well as suppressed the expression of antiapoptotic protein Bcl-2, increased the expression of proapoptotic protein caspase-3 and Bax, and suppressed PI3K/AKT pathway. Meanwhile, BA was also able to depolarize mitochondrial membranal permeability (MMP), accelerate mitochondrial superoxide accumulation, induce intracellular reactive oxygen species (ROS) production, and decreased intracellular glutathione (GSH) in HUVECs. Furthermore, both mitochondria-specific superoxide scavenger Mito-TEMPOL and broad-spectrum antioxidant N-acetyl-cysteine (NAC) dramatically abolished BA-induced mitochondrial dysfunction and mitochondrial ROS production, causing the reversion of PI3K/AKT pathway and repression of apoptosis, eventually correcting the impaired endothelial behavior in survival, growth, migration, and angiogenesis. Collectively, our data for the first time identified a new mechanism for antiangiogenic effect of BA in vascular EC, one that is based on the regulation of mitochondrial-dependent ROS overproduction.

Inhibitory effect of (E)-2-heptenal on Aspergillus flavus growth revealed by metabolomics and biochemical analyses

The prevention of fungal proliferation in postharvest grains is critical for maintaining grain quality and reducing mycotoxin contamination. Fumigation with natural gaseous fungicides is a promising and sustainable approach to protect grains from fungal spoilage. In this study, the antifungal activities of (E)-2-alkenals (C5-C10) on Aspergillus flavus were tested in the vapor phase, and (E)-2-heptenal showed the highest antifungal activity against A. flavus. (E)-2-Heptenal completely inhibited A. flavus growth at 0.0125 µL/mL and 0.2 µL/mL in the vapor phase and liquid contact, respectively. (E)-2-Heptenal can disrupt the plasma membrane integrity of A. flavus via leakage of intracellular electrolytes. Scanning electron microscopy indicated that the mycelial morphology of A. flavus was remarkably affected by (E)-2-heptenal. Metabolomic analyses indicated that 49 metabolites were significantly differentially expressed in A. flavus mycelia exposed to 0.2 µL/mL (E)-2-heptenal; these metabolites were mainly involved in galactose metabolism, starch and sucrose metabolism, the phosphotransferase system, and ATP-binding cassette transporters. ATP production was reduced in (E)-2-heptenal-treated A. flavus, and Janus Green B staining showed reduced cytochrome c oxidase activity. (E)-2-Heptenal treatment induced oxidative stress in A. flavus mycelia with an accumulation of superoxide anions and hydrogen peroxide and increased activities of superoxide dismutase and catalase. Simulated storage experiments showed that fumigation with 400 µL/L of (E)-2-heptenal vapor could completely inhibit A. flavus growth in wheat grains with 20% moisture; this demonstrates its potential use in preventing grain spoilage. This study provides valuable insights into understanding the antifungal effects of (E)-2-heptenal on A. flavus. KEY POINTS : • (E)-2-Heptenal vapor showed the highest antifungal activity against A. flavus among (C5-C10) (E)-2-alkenals. • The antifungal effects of (E)-2-heptenal against A. flavus were determined. • The antifungal actions of (E)-2-heptenal on A. flavus were revealed by metabolomics and biochemical analyses.

Symbiotic System Establishment between Piriformospora indica and Glycine max and Its Effects on the Antioxidant Activity and Ion-Transporter-Related Gene Expression in Soybean under Salt Stress.

The utilization of symbiosis with beneficial microorganisms has considerable potential for increasing growth and resistance under abiotic stress. The endophytic root fungus Piriformospora indica has been shown to improve plant growth under salt and drought stress in diverse plant species, while there have been few reports of the interaction of P. indica with soybean under salt stress. In this study, the symbiotic system of P. indica and soybean (Glycine max L.) was established, and the effect of P. indica on soybean growth and salt tolerance was investigated. The colonized and non-colonized soybeans were subjected to salt stress (200 mmol/L NaCl), and the impairments in chlorophyll and increasing relative conductivity that can be caused by salt stress were alleviated in the P. indica-colonized plants. The accumulation of malondialdehyde (MDA), hydrogen peroxide (H2O2), and superoxide anion (O2−) were lower than that in non-colonized plants under salt treatment, whereas the activities of antioxidant enzymes were significantly increased by P. indica colonization, including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and glutathione reductase (GR). Importantly, without salt treatment, the Na+ concentration was lower, and the K+ concentration was higher in the roots compared with non-colonized plants. Differential expressions of ion transporter genes were found in soybean roots after P. indica colonization. The P. indica colonization positively regulated the transcription level of PM H+-ATPase, SOS1, and SOS2. The study shows that P. indica enhances the growth and salt tolerance of soybean, providing a strategy for the agricultural production of soybean plants in saline-alkali soils.

Lipophagy As a Mechanism of Resistance in T-ALL Following Energetic Crisis Induced By Oxphos/MCT1 Blockade: Strategies to Eradicate Residual Disease

Acidosis and hypoxia are common hallmarks of cancer associated with poor therapy response and dismal survival. Hypoxia can be due to high demand on oxygen, or due to reduced oxygen supply consequent to pharmacological inhibition of oxidative phosphorylation (OxPhos), causing switch to glycolysis and intracellular acidosis. Both acidosis and hypoxia are known to activate AMPK, stabilize HIF1α, that might orchestrate metabolic switch to alternative sources of energy such as lipophagy, an autophagic degradation of intracellular lipid droplets. We have previously shown that co-inhibition of mitochondrial Complex I and pyruvate-lactate transporter 1 (MCT1) in T-ALL cells is synergistic both in vitro and in vivo (Baran N, et al. ASH 2021). Here we dissect the mechanism of OxPhos/MCT1 blockade (OxPhos/MCT1-i), showing lipophagy as a mechanism of resistance to dual inhibition and provide therapeutic strategy to eradicate the residual disease. In T-ALL cell line models, Complex I/MCT1 blockade led to a significant inhibition of viability and decreased cell proliferation. These effects were accompanied by significant accumulation of reactive oxygen species (ROS) and mitochondrial superoxide, leading to DNA damage and apoptosis (shown by γ-H2AX and cleaved PARP). High level of ROS remained elevated despite co-treatment with ROS-scavengers: ascorbate, α-ketoglutarate, GSH or NAC. To elucidate the mechanisms of cell death, cells were co-treated with inhibitors of autophagy (3-methyladenine, Bafilomycin A, Chloroquine, Wortmannin), apoptosis (QVD-OPH, ZVAD-fmk) and ferroptosis (Ferrostatin1). While all inhibitors improved cell viability, suggesting a mixed contribution of apoptosis, autophagy and ferroptosis, none rescued elevated ROS caused by OxPhos/MCT1-i. Further impairment of mitochondria and Oxphos was evidenced by loss of mitochondrial membrane potential, mitochondria swelling, and reduction of mitochondria number, associated with markers of mitochondria fusion (elevated OPA1, MFN1 and DRP1). Significant reduction of ATP was confirmed by Seahorse and mass spectrometry (MS), indicating TCA cycle blockade and depletion of glutamine, cystathionine and glutathione, likely in response to ROS. ROS and ATP depletion are known to stabilize the hypoxia inducible factor 1 (HIF1α) that controls triglyceride accumulation. Results of GSEA and Western blotting confirmed that dual inhibition induced energetic crisis, activated LKB-AMPK pathway, inhibited mTOR pathway, upregulated fatty acids metabolism, which together with ROS accumulation caused HIF1α stabilization and switch to glycolysis (elevated ECAR by Seahorse and lactate by MS). Glycolysis however, impaired by MCT1 blockade, led to intracellular acidification, reflected by pH Rodo staining. This was associated with significant increase of a key component in lipid turnover acetyl-CoA, and by increased intensity of BODIPY dye, accompanied by increase in level of cellular lysosomes detected by Lysotracker dye, suggesting a possible shift of metabolic dependency toward lipophagy as a remaining source of energy. These results indicate that OxPhos/MCT1-i triggers storage of reduced lipids which subsequently undergo lysosomal degradation during lipophagy, constituting an alternative to OxPhos source of energy. To test if inhibition of lipid metabolism might abrogate the last energetic supply and eradicate residual leukemic cells, T-ALL cells were co-treated with OxPhos/MCT1-i together with Atorvastatin, Etomoxir and Valproate. Concomitant administration of Atorvastatin as well as other inhibitors with both OxPhos/MCT-i significantly reduced cell viability and induced apoptosis, supporting the key role of lipid metabolism in the resistance of T-ALL to dual inhibition. In summary, these results demonstrate that OxPhos/MCT1 inhibition forces switch to lipophagy, sensitizing cells to inhibitors of lipid metabolism, uncovering metabolic checkpoints that can ultimately translate into successful therapies in T-ALL and OxPhos-dependent malignancies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Effects of Glutaminase Inhibition on Venetoclax, Ibrutinib, and AZD-5991 Resistance in Chronic Lymphocytic Leukemia Cell Lines

Introduction: Bruton tyrosine kinase and BCL-2 inhibitors are widely used in CLL, but resistance problem is highly relevant. One of the resistance mechanisms may be metabolic rewiring followed by activation of oxidative phosphorylation. Glutamine plays a major role in cancer metabolism, and glutamine limitation rather than glucose contributes to lower oxygen uptake and mediates apoptosis of cancer cells. It was shown that glutamine metabolism changes contribute to venetoclax (Chen Z. et al, Blood, 2021; Guièze R. et al. Cancer Cell, 2019) and ibrutinib (Galicia-Vázquez G. et al. Frontiers in Oncology, 2018) resistance. We investigated in vitro effects of glutaminase-1 (GLS-1) enzyme inhibition and its potential synergistic effects with venetoclax, ibrutinib, and MCL-1 inhibitor AZD-5991 on resistant cell lines. CB-839 (telaglenastat) is a potent, reversible, and selective GLS-1 inhibitor with 24 nM IC50 for recombinant human mitochondrial glutaminase C. The compound was tested in the clinic for hematological malignancies and solid tumors. Methods: We have tested CB-839 alone or in combination with venetoclax, ibrutinib, and AZD-5991 on resistant HG-3 and MEC-1 CLL cell lines. Cells were incubated with 0.01 to 100 μM of CB-839 for 72 hours. The inhibition of glutamine, glutamate, and glutathione induced by CB-839 alone was measured using a luminescence assay. DCFDA/ H2DCFDA and MitoSOX Red stainings were utilized to evaluate cellular reactive oxygen species (Total ROS) and mitochondrial superoxide (MitoSOX) accumulation, respectively. Intracellular nucleotides including ATP level were measured using high-pressure liquid chromatography. For drug combinations, MTS assay and Annexin V/Propidium Iodide staining were used to assess cell proliferation and apoptosis, respectively. The combination index (CI) for synergy was calculated using Compusyn software. Results: HG3 and MEC1 cell lines were resistant to venetoclax, ibrutinib, and AZD-5991 single agents. The IC50 values for HG3 are 8.1 μM, 1.3 μM, 9.9 μM, and for MEC1 are 2.9 μM, 1.2 μM, and 1.9 μM for venetoclax, ibrutinib, and AZD-5991, respectively. In these cell lines, CB-839 caused a dose-dependent decrease in GLS-1 activity, measured as intracellular glutamate concentration, and inhibited glutathione synthesis (Figure 1A). CB-839 treated cells showed increased MitoSOX, but not Total ROS accumulation. However, as the endpoint effect, the decrease of cell proliferation rather than apoptosis was detected. CB-839 treatment caused a mild decrease in ATP pool in both cell lines. According to MTS assay, venetoclax demonstrated only the particular synergism with CB-839 (CI <1.0 at all effective doses (ED50, ED75, ED90, ED95) for MEC-1, and at ED50, ED75 for HG-3 cell line). In addition, CB-839 increased pro-apoptotic effect of venetoclax in both cell lines. Notably, that CB-839 did not improve any ibrutinib-mediated effects on cell proliferation and apoptosis in CLL cell lines. The most distinct synergism was observed in CB-839 and AZD-5991 combination group (CI <1.0 at all ED for MEC-1 and HG-3 cell lines) with 5- and 19.8-fold shifts in IC50 (Figure 1B). To extend these results, we are performing these combinations in primary CLL cells that are sensitive or resistant to targeted agents such as ibrutinib or venetoclax. Conclusions: Combinations of CB-839 with other agents targeting multiple mechanisms have the potential to overcome metabolic adaptation. Our findings suggest that CB-839 has limited efficacy in overcoming ibrutinib CLL resistance. However, the synergism with apoptosis inducers such as venetoclax and AZD-5991 require further investigations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Cadmium-induced apoptosis of Leydig cells is mediated by excessive mitochondrial fission and inhibition of mitophagy

Cadmium is one of the environmental and occupational pollutants and its potential adverse effects on human health have given rise to substantial concern. Cadmium causes damage to the male reproductive system via induction of germ-cell apoptosis; however, the underlying mechanism of cadmium-induced reproductive toxicity in Leydig cells remains unclear. In this study, twenty mice were divided randomly into four groups and exposed to CdCl2 at concentrations of 0, 0.5, 1.0 and 2.0 mg/kg/day for four consecutive weeks. Testicular injury, abnormal spermatogenesis and apoptosis of Leydig cells were observed in mice. In order to investigate the mechanism of cadmium-induced apoptosis of Leydig cells, a model of mouse Leydig cell line (i.e. TM3 cells) was subjected to treatment with various concentrations of CdCl2. It was found that mitochondrial function was disrupted by cadmium, which also caused a significant elevation in levels of mitochondrial superoxide and cellular ROS. Furthermore, while cadmium increased the expression of mitochondrial fission proteins (DRP1 and FIS1), it reduced the expression of mitochondrial fusion proteins (OPA1 and MFN1). This led to excessive mitochondrial fission, the release of cytochrome c and apoptosis. Conversely, cadmium-induced accumulation of mitochondrial superoxide was decreased by the inhibition of mitochondrial fission through the use of Mdivi-1 (an inhibitor of DRP1). Mdivi-1 also partially prevented the release of cytochrome c from mitochondria to cytosol and attenuated cell apoptosis. Finally, given the accumulation of LC3II and SQSTM1/p62 and the obstruction of Parkin recruitment into damaged mitochondria in TM3 cells, the autophagosome-lysosome fusion was probably inhibited by cadmium. Overall, these findings suggest that cadmium induces apoptosis of mouse Leydig cells via the induction of excessive mitochondrial fission and inhibition of mitophagy.

Endophytic Bacillus subtilis antagonize soil-borne fungal pathogens and suppress wilt complex disease in chickpea plants (Cicer arietinum L.).

The present study aimed to identify potential endophytic bacteria antagonistic against three soil-borne fungal pathogens, Rhizoctonia solani, Sclerotium rolfsii, and Fusarium oxysporum f.sp. ciceri causing root rot, collar rot, and fungal wilt diseases in chickpea plants, respectively. A total of 255 bacterial endophytes were isolated from the leaves, stems, and roots of seven different crop plants (chickpea, tomato, wheat, berseem, mustard, potato, and green pea). The dual culture-based screening for antifungal properties indicated that three endophytic isolates had strong inhibition (>50%) against all three pathogens tested. Based on morphological, biochemical, and molecular characterization, the selected isolates (TRO4, CLO5, and PLO3) were identified as different strains of Bacillus subtilis. The bacterial endophytes (TRO4 and CLO5) were positive for plant growth promoting (PGP) traits viz., ammonia, siderophore, and indole-3-acetic acid (IAA) production. The bio-efficacy of the endophytes (TRO4, CLO5, and PLO3) was tested by an in planta trial in chickpea pre-challenged with R. solani, S. rolfsii, and F. oxysporum f.sp. ciceri. The B. subtilis strains TRO4 and CLO5 were found to be effective in reducing percent disease incidence (p ≤ 0.05) and enhancing plant growth parameters. The different root parameters viz. root length (mm), surface area (cm2), root diameter (mm), and root volume (cm3) were significantly (p ≤ 0.05) increased in TRO4 and CLO5 inoculated chickpea plants. Confocal Scanning Laser Microscopy showed heavy colonization of bacteria in the roots of endophyte-inoculated chickpea plants. The inoculation of endophytic Bacillus subtilis strains TRO4 and CLO5 in chickpea plants through seed biopriming reduced the accumulation of superoxide, enhanced the plant defense enzymes, and induced the expression of Pathogenesis-Related (PR) genes. Semi-quantitative analysis of defense-related genes showed differential activation of PR genes (60srp and IFR) by endophyte inoculation. The results of the present study reveal the antagonistic potential of B. subtilis strains TRO4 and CLO5 against three major soil-borne fungal pathogens and their ability to suppress wilt complex disease in chickpea plants. This is the first report on the simultaneous suppression of three major soil-borne fungal pathogens causing wilt complex in chickpea plants by endophytic B. subtilis strains.

Sirt3 deficiency accelerates ovarian senescence without affecting spermatogenesis in aging mice.

Sirtuin-3 (SIRT3), the main deacetylase in the mitochondria, maintains cellular energy metabolism and redox balance by deacetylating mitochondrial proteins in a NAD+-dependent manner. Growing evidence indicates that decreased Sirt3 expression is involved in various age-related maladies. However, the role of Sirt3 in ovarian and testicular senescence remains unclear. In this study, we observed that sirt3 expression showed age-dependent decreases in the ovary but not the testis. We generated Sirt3 null mice via CRISPR/Cas9-mediated genome editing. We observed that Sirt3 deletion accelerated ovarian aging, as shown by a decrease in offspring sizes, the follicle reserve and oocytes markers (Bmp15 and Gdf9) as well as increased expression of aging and inflammation-related genes (p16, p21, Il-1α, and Il-1β). Sirt3 deficiency led to an accumulation of superoxide and disruption of spindle assembly accompanied by mitochondrial dysfunction (uneven mitochondria distribution, decreased mitochondrial potential as well as reduced mitochondrial DNA content) in aging oocytes. Meanwhile, in ovaries of Sirt3 null mice, the impaired mitochondrial functions were shown by decreases in mitochondrial respiratory complexes, along with lower levels of mitochondrial fusion (OPA1, MFN2) and fission (DRP1, FIS1) proteins. er levels of mitochondrial fusion (OPA1, MFN2) and fission (DRP1, FIS1) proteins. Interestingly, Sirt3-/- male mice exhibited no changes on the testicular histology, serum testosterone levels, germ-cell proliferation, and differentiation of spermatogonia. Meiotic prophase I spermatocytes were also normal. Levels of superoxide, mitochondrial potential as well as expression of mitochondrially-encoded genes were unaltered in Sirt3-/- testes. Collectively, the results indicated that SIRT3 plays a critical role in maintaining the ovarian follicle reserve and oocyte quality in aging mice, suggesting its important role in controlling ovarian senescence.

Kinesin spindle protein inhibitor exacerbates cisplatin-induced hair cell damage

There is emerging evidence indicating that Kinesin family, plays vital roles in influencing the growth of axons, interference with the progression of tumor. However, the function of Kinesin member in the auditory organs remains unknown. SB743921, a kinesin spindle protein (KSP) inhibitor, was applied in mouse organ of Corti and House Ear Institute-Organ of Corti 1 (HEI-OC1) cell line to examine the role of KSP in auditory system with and without cisplatin damage. Cell Counting Kit-8 (CCK-8) and Lactase dehydrogenase (LDH) release assay were conducted to evaluate cell activity and toxicity. Pretreatment with SB743921 increased the sensitivity of HEI-OC1 cells to cisplatin ototoxicity through promoting cell apoptosis and deteriorating superoxide generation mediated damage from cisplatin. SB743921 also enhanced cisplatin induced hair cell damage in explants of mouse cochleae in vitro. Furthermore, the combined N-acetylcysteine (NAC) treatment with cisplatin or with cisplatin and SB743921 both completely rescued the reduced number of hair cells impaired by cisplatin, confirming the strengthening function of superoxide accumulation by SB743921 after cisplatin treatment. Inhibition of kinesin spindle protein enhanced the susceptibility of hair cells to cisplatin induced damage in mouse cochlear explants and HEI-OC1 cells, indicating that kinesin spindle protein might be an unprecedented target to weaken the ototoxicity of platinum medicaments.

The C2H2 zinc finger protein TaZFP13D increases drought stress tolerance in wheat

Wheat is one of the most cultivated crops, and its production is greatly affected by abiotic stresses such as drought. Transcription factors that regulate multiple downstream genes have a good potential to improve stress tolerance due to their ability to modulate various response pathways. Many members of the C1-2i C2H2 zinc finger protein (ZFP) sub-family are up-regulated in response to abiotic stress and specific members were shown to improve oxidative and abiotic stress tolerance in different plant species. Our previous work demonstrated that the most up-regulated TaZFP, TaZFP1B, improves drought tolerance and oxidative stress protection. Based on their strong response to different abiotic stresses, five additional candidate TaZFPs were selected for functional characterization by reverse genetics in three wheat lines, using the four-component Barley Stripe Mosaic Virus system. Of the five candidates studied, TaZFP13D was the only gene that increased drought stress tolerance (resistance to wilting and improved relative water content) in three different wheat lines and significantly improved antioxidant enzymes activities resulting in lower accumulation of superoxide and hydrogen peroxide. Moreover, TaZFP13D down-regulation decreased drought stress tolerance suggesting that this gene does not have a redundant function within the TaZFPs family.

Effect of exogenous melatonin on antioxidant enzyme activities and membrane lipid peroxidation in avocado fruit during ripening

Melatonin functions as a plant growth regulator, has diverse functions and plays an important role in ripening and fruit senescence. In the current study, we investigated the effect of exogenous melatonin (0.1 mM and 0.5 mM) on reactive oxygen species (ROS) metabolism, membrane lipid peroxidation and antioxidant enzyme activity of avocado (Persea americana Mill. cv. 034) during fruit ripening at 22oC ± 1 and 75-80% relative humidity (RH). The results showed that postharvest fruits treated with 0.5 mM melatonin effectively reduced the accumulation of superoxide anion (O2.), hydrogen peroxide (H2O2) and malondialdehyde (MDA) in the mesocarp of the fruit. In addition, melatonin treatment also significantly promoted the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) in avocado. It is suggested that enhanced antioxidant enzyme activity induced by melatonin treatment may contribute to scavenge ROS and alleviating membrane lipid peroxidation in avocado fruit. The results indicate that the MT application might collectively contribute to the delay senescence and maintain postharvest quality of avocado.

Mn-SOD alleviates methotrexate-related hepatocellular injury via GSK-3β affecting anti-oxidative stress of HO-1 and Drp1.

Methotrexate (MTX) is the most common therapeutic agent that may have the risk of drug-induced liver injury. Its pathogenic mechanism is related to oxidative stress caused by mitochondrial dysfunction. Superoxide dismutase (SOD), including manganese-containing SOD (Mn-SOD), can exert its effect of anti-oxidative stress by scavenging superoxide free radicals. Accordingly, this study is performed to explore the underlying molecular mechanism via observing whether Mn-SOD could affect the damage of MTX to hepatocytes. Human hepatocyte cell line L-02 was cultured in vitro and divided into 4 groups, including a blank group with the addition of the same volume of serum-free medium, a MTX group (40 μg/well MTX drug-treatment), a MTX+NC group (40 μg/well MTX drug-treatment+blank plasmid), and a MTX+SOD group (40 μg/well MTX drug-treatment+Mn-SOD plasmid). The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and microRNA-122 (miR-122) in the supernatant of cell culture were respectively detected by automatic biochemical analytical instrument and real-time RT-PCR to evaluate the degree of hepatocyte damage in each group. MitoSOX fluorescent probe was used to label intracellular superoxide in each group, and cell apoptosis was detected by flow cytometry. Meanwhile, the contents of glycogen synthase kinase-3 beta (GSK-3β), hemeoxygenase-1 (HO-1), mitochondrial fission-mediated protein of dynamin-related protein 1 (Drp1), and Mn-SOD were detected by Western blotting. Compared with the blank group, the levels of ALT, AST, and miR-122 in the supernatant of hepatocyte culture of the MTX group and MTX+NC group were significantly elevated (all P <0.05), and that in the MTX+SOD group were significantly decreased ( P <0.05) and equivalent to that in the blank group. MitoSOX staining revealed that the MTX group and MTX+NC had the most abundant superoxide; and the amount was significantly reduced in the MTX+SOD group, without a significant difference when compared with the blank group. Furthermore, the results of flow cytometry indicated that compared with the blank group, the MTX group and MTX+NC group showed significantly increased cell apoptosis ( P <0.05); while there was obviously reduced cell apoptosis in the MTX+SOD group than that in the MTX group and MTX+NC group ( P <0.05). According to the results of Western blotting, the blank group and MTX+SOD group had higher expressions of Mn-SOD, p-GSK-3β, and HO-1; while the MTX group and MTX+NC group exhibited remarkably lower levels of Mn-SOD, p-GSK-3β, and HO-1 than those in the blank group ( P <0.05). Besides, a completely opposite trend was found in the expression of Drp1, which was highly expressed in the MTX group and MTX+NC group, but lowly expressed in the blank group and the MTX+SOD group. MTX may induce hepatocyte damage, and one of the mechanisms may be due to the decrease of intracellular Mn-SOD level, which can cause the accumulation of superoxide, affect the levels of HO-1 and Drp1 through GSK-3β leading to mitochondrial damage and cell apoptosis. High expression of Mn-SOD intracellularly through exogenous introduction can scavenge drug-produced superoxide, affect HO-1 and Drp1 levels through GSK-3β, activate mitochondria, protect cells against damage from oxidative stress, and inhibit hepatocyte apoptosis eventually. So exogenous introduction of SOD may be a potential therapeutic approach to block or reverse MTX-related hepatocyte injury.

1α,25-Dihydroxyvitamin D3 (VD3) Shows a Neuroprotective Action Against Rotenone Toxicity on PC12 Cells: An In Vitro Model of Parkinson's Disease.

Parkinson's disease (PD) is characterized by dopaminergic cell loss in the substantia nigra, and PD brains show neuroinflammation, oxidative stress, and mitochondrial dysfunction. The study evaluated the neuroprotective activity of 1α,25-dihydroxy vitamin D3 (VD3), on the rotenone (ROT)-induced cytotoxicity in PC12 cells. The viability parameters were assessed by the MTT and flow cytometry, on cells treated or not with VD3 and/or ROT. Besides, ROS production, cell death, mitochondrial transmembrane potential, reduced GSH, superoxide accumulation, molecular docking (TH and Keap1-Nrf2), and TH, Nrf2, NF-kB, and VD3 receptor protein contents by western blot were evaluated. VD3 was shown to improve the viability of ROT-exposed cells. Cells exposed to ROT showed increased production of ROS and superoxide, which decreased after VD3. ROT decrease in the mitochondrial transmembrane potential was prevented, after VD3 treatment and, VD3 was shown to interact with tyrosine hydroxylase (TH) and Nrf2. While ROT decreased TH, Nrf2, and NF-kB expressions, these effects were reversed by VD3. In addition, VD3 also increased VD3 receptor protein contents and values went back to those of controls after ROT exposure. VD3 protects PC12 cells against ROT damage, by decreasing oxidative stress and improving mitochondrial function. One target seems to be the TH molecule and possibly an indirect Nrf2 activation could also justify its neuroprotective actions on this PC12 cell model of PD.

Genome-Wide Identification of Polyamine Oxidase (PAO) Family Genes: Roles of CaPAO2 and CaPAO4 in the Cold Tolerance of Pepper (Capsicum annuum L.).

Polyamine oxidases (PAOs), which are flavin adenine dinucleotide-dependent enzymes, catalyze polyamine (PA) catabolism, producing hydrogen peroxide (H2O2). Several PAO family members have been identified in plants, but their expression in pepper plants remains unclear. Here, six PAO genes were identified in the ‘Zunla-1’ pepper genome (named CaPAO1–CaPAO6 according to their chromosomal positions). The PAO proteins were divided into four subfamilies according to phylogenetics: CaPAO1 belongs to subfamily I; CaPAO3 and CaPAO5 belong to subfamily III; and CaPAO2, CaPAO4, and CaPAO6 belong to subfamily IV (none belong to subfamily II). CaPAO2, CaPAO4, and CaPAO6 were ubiquitously and highly expressed in all tissues, CaPAO1 was mainly expressed in flowers, whereas CaPAO3 and CaPAO5 were expressed at very low levels in all tissues. RNA-seq analysis revealed that CaPAO2 and CaPAO4 were notably upregulated by cold stress. CaPAO2 and CaPAO4 were localized in the peroxisome, and spermine was the preferred substrate for PA catabolism. CaPAO2 and CaPAO4 overexpression in Arabidopsis thaliana significantly enhanced freezing-stress tolerance by increasing antioxidant enzyme activity and decreasing malondialdehyde, H2O2, and superoxide accumulation, accompanied by the upregulation of cold-responsive genes (AtCOR15A, AtRD29A, AtCOR47, and AtKIN1). Thus, we identified candidate PAO genes for breeding cold-stress-tolerant transgenic pepper cultivars.

Effect of methyl jasmonate on the quality and antioxidant capacity by modulating ascorbate-glutathione cycle in peach fruit

We investigated the effect of methyl jasmonate (MeJA) on the quality and antioxidant capacity in ‘Xiahui 8’ peach fruit during storage at 20°C and 90–95% relative humidity (RH) for 7 days. Exogenous MeJA maintained better fruit quality during storage, as indicated by reduced weight loss, respiration rate, ethylene production and total soluble solids, as well as maintained appearance and firmness. MeJA treatment significantly improved the activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) in peach fruit, therefore, reducing the accumulation of superoxide anion (O2−), hydrogen peroxide (H2O2) and malondialdehyde (MDA). Additionally, MeJA treatment enhanced the metabolic intensity of AsA-GSH cycle, as key enzyme activities involved in AsA-GSH cycle were markedly increased. These MeJA-promoting effect increased the content of reduced ascorbate (AsA) and glutathione (GSH), and the ratios of reduced to oxidized forms of AsA and GSH, contributing to higher redox status in peach fruit. Collectively, these results suggest that MeJA can maintain better fruit quality and improve antioxidant ability through enhancing AsA-GSH cycle, thus suitable for postharvest application.

CRISPR/Cas9 mediated gene-editing of GmHdz4 transcription factor enhances drought tolerance in soybean (Glycine max [L.] Merr.).

The HD-Zip transcription factors play a crucial role in plant development, secondary metabolism, and abiotic stress responses, but little is known about HD-Zip I genes in soybean. Here, a homeodomain-leucine zipper gene designated GmHdz4 was isolated. Chimeric soybean plants, GmHdz4 overexpressing (GmHdz4-oe), and gene-editing via CRISPR/Cas9 (gmhdz4) in hairy roots, were generated to examine the GmHdz4 gene response to polyethylene glycol (PEG)-simulated drought stress. Bioinformatic analysis showed GmHdz4 belonged to clade δ, and was closely related to other drought tolerance-related HD-Zip I family genes such as AtHB12, Oshox12, and Gshdz4. The GmHdz4 was located in the plant nucleus and showed transcriptional activation activity by yeast hybrid assay. Quantitative real-time PCR analysis revealed that GmHdz4 expression varied in tissues and was induced by PEG-simulated drought stress. The gmhdz4 showed promoted growth of aboveground parts, and its root system architecture, including the total root length, the root superficial area, and the number of root tips were significantly higher than those of GmHdz4-oe even the non-transgenic line (NT) on root tips number. The better maintenance of turgor pressure by osmolyte accumulation, and the higher activity of antioxidant enzymes to scavenge reactive oxygen species, ultimately suppressed the accumulation of hydrogen peroxide (H2O2), superoxide anion (O2−), and malondialdehyde (MDA), conferring higher drought tolerance in gmhdz4 compared with both GmHdz4-oe and NT. Together, our results provide new insights for future research on the mechanisms by which GmHdz4 gene-editing via CRISPR/Cas9 system could promote drought stress and provide a potential target for molecular breeding in soybean.

Tissue Heavy Metals in Liver Diseases

Our objective was to determine the levels of heavy metals, oxidants, and antioxidants in liver tissue of patients with chronic liver disease (CLD) compared with healthy living liver donors (LLDs). We obtained liver specimens from patients undergoing liver transplant for CLD. Samples were also obtained from LLDs. Biochemical analyses were performed on all samples, and the levels of liver tissue, heavy metal, and oxidant-antioxidants biomarker levels in patients with CLD were compared with those measured in LLDs. One hundred and eighteen individuals were included for analyses. Fifty-nine were patients with CLD, and 59 were LLDs. The median levels of liver tissue of superoxide dismutase (P=.009), glutathione peroxidase (P=.042), total oxidant status (P=.006), oxidative stress index (P < .001), and copper (P=.035) were prominently more elevated in CLD than LLDs. On the other hand, the median levels of liver tissue of cadmium (P < .001), selenium (P=.042), and zinc (P < .001) levels were more elevated in the LLDs than patients with CLD. The 2 groups were similar in terms of total antioxidant status, manganese, arsenic, and lead levels. Superoxide accumulation in the liver was higher in patients with CLD. Concerning heavy metals, only the median tissue copper was elevated in patients with CLD with higher Cu/Zn ratio. Cadmium, selenium, and zinc were significantly higher in the healthy LLDs.

Picea wilsonii Transcription Factor PwNAC38 Promotes Drought and Salt Tolerance in Transgenic Arabidopsis and Apple Calli

The mechanism of Picea wilsonii adapting to abiotic stress remains largely unknown. NAC (NAM, ATAF and CUC) transcription factors play significant roles in plant response to adverse environments. In this study, based on our previous RNA-seq, we analyzed the expression patterns of PwNAC38, and revealed its functions in the process of PwNAC38-mediated stress responses. An open-reading frame, encoding PwNAC38 protein with 330 amino acids, was isolated from the cDNA library, a process which can be induced by drought, salt and ABA treatment. Subcellular localization and yeast experiments showed that PwNAC38 was a nuclear-localized transcription factor, and could form homodimers. The full length of PwNAC38 showed transcriptional activity, while the truncated segments, C-PwNAC38 (156–330 aa) and N-PwNAC38 (1–156 aa), did not. The constitutive expression of PwNAC38 (OE lines) in Arabidopsis did not exert influence on the growth of transgenic plants under normal conditions, whereas transgenic seedlings showed higher survival rates, and the seeds had stronger vigor and a higher germination rate under drought and salt stress. The seed germination and root growth of PwNAC38 OE lines were significantly inhibited in the presence of ABA, suggesting the hypersensitivity of PwNAC38 to ABA treatment. Physiological assays showed that the activity of antioxidant enzymes, such as SOD and POD, increased, and that the accumulation of superoxide anion decreased, in OE lines under stress conditions. Moreover, overexpression of PwNAC38 significantly improved drought and salt tolerance in apple calli. A qRT-PCR assay showed that overexpression of PwNAC38 in Arabidopsis promoted the expression of drought or ABA-responsive genes ATHB-7, ANAC019, ERD1, DREB2A, RD29A, ABI5 and NCED3. Taken together, our results revealed that PwNAC38 is positively involved in plants’ response to drought and salt stress by enhancing ROS scavenging efficiency, and is partially dependent on the ABA signaling pathway.

Direct evidence of drought stress memory in mulberry from a physiological perspective: Antioxidative, osmotic and phytohormonal regulations

Drought stress commonly happens more than once during the life cycle of perennial trees. Stress memory endows better capacity to cope with repeated stresses for plants, while the underlying mechanisms are not fully elucidated. In this study, 2-month-old saplings of two mulberry cultivars (Husang32 and 7307 of Morus multicaulis) with or without an early soil water deficit were subjected to subsequent drought for 9 days. The shoot height growth, biomass production, stable carbon isotope discrimination, phytohormones, reactive oxygen species (ROS), osmotic substances and antioxidant enzymes were analyzed after the first and the second drought, respectively. Drought priming saplings sustained comparable or slightly higher biomass accumulation under the second drought than those non-priming. They also exhibited decreased levels of soluble sugars, free proline and soluble proteins, lower accumulation of malonaldehyde (MDA) and superoxide anion (O2•−), reduced activities of superoxide dismutase (SOD) and peroxidase (POD) compared to non-priming plants. Moreover, cultivar Husang32 exhibited elevated abscisic acid (ABA) and jasmonic acid (JA) where 7307 displayed opposite changes. PCA suggests that MDA, H2O2, free proline, SOD and POD in roots, and ROS, soluble sugars and glutamate reductase in leaves are dominant factors influenced by stress memory. ABA and JA in leaves also play important roles in exerting drought imprints. Collectively, stress memory can confer mulberry resistance to recurrent drought via combined regulations of antioxidative protection, osmotic adjustment and phytohormonal responses.

Comprehensive investigations of key mitochondrial metabolic changes in senescent human fibroblasts.

There is a paucity of detailed data related to the effect of senescence on the mitochondrial antioxidant capacity and redox state of senescent human cells. Activities of TCA cycle enzymes, respiratory chain complexes, hydrogen peroxide (H2O2), superoxide anions (SA), lipid peroxides (LPO), protein carbonyl content (PCC), thioredoxin reductase 2 (TrxR2), superoxide dismutase 2 (SOD2), glutathione peroxidase 1 (GPx1), glutathione reductase (GR), reduced glutathione (GSH), and oxidized glutathione (GSSG), along with levels of nicotinamide cofactors and ATP content were measured in young and senescent human foreskin fibroblasts. Primary and senescent cultures were biochemically identified by monitoring the augmented cellular activities of key glycolytic enzymes including phosphofructokinase, lactate dehydrogenase, and glycogen phosphorylase, and accumulation of H2O2, SA, LPO, PCC, and GSSG. Citrate synthase, aconitase, α-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, and complex I-III, II-III, and IV activities were significantly diminished in P25 and P35 cells compared to P5 cells. This was accompanied by significant accumulation of mitochondrial H2O2, SA, LPO, and PCC, along with increased transcriptional and enzymatic activities of TrxR2, SOD2, GPx1, and GR. Notably, the GSH/GSSG ratio was significantly reduced whereas NAD+/NADH and NADP+/NADPH ratios were significantly elevated. Metabolic exhaustion was also evident in senescent cells underscored by the severely diminished ATP/ADP ratio. Profound oxidative stress may contribute, at least in part, to senescence pointing at a potential protective role of antioxidants in aging-associated disease.