Extension of the Lifespan of Caenorhabditis elegans by the Use of Electrolyzed Reduced Water

Electrolyzed reduced water (ERW) has attracted much attention because of its therapeutic effects. In the present study, a new culture medium, which we designated Water medium, was developed to elucidate the effects of ERW on the lifespan of Caenorhabditis elegans. Wild-type C. elegans had a significantly shorter lifespan in Water medium than in conventional S medium. However, worms cultured in ERW-Water medium exhibited a significantly extended lifespan (from 11% to 41%) compared with worms cultured in ultrapure water-Water medium. There was no difference between the lifespans of worms cultured in ERW-S medium and ultrapure water-S medium. Nematodes cultured in ultrapure water-Water medium showed significantly higher levels of reactive oxygen species than those cultured in ultrapure water-S medium. Moreover, ERW-Water medium significantly reduced the ROS accumulation induced in the worms by paraquat, suggesting that ERW-Water medium extends the longevity of nematodes at least partly by scavenging ROS.     

Mechanism of the lifespan extension of Caenorhabditis elegans by electrolyzed reduced water--participation of Pt nanoparticles

Electrolyzed reduced water (ERW) contains a large amount of molecular hydrogen and a small amount of Pt nanoparticles (Pt NPs). We have found that ERW significantly extended the lifespan of Caenorhabditis elegans in a novel culture medium designated Water Medium. In this study, we found that synthetic Pt NPs at ppb levels significantly extended the nematode lifespan and scavenged reactive oxygen species (ROS) in the nematode induced by paraquat treatment. In contrast, a high concentration of dissolved molecular hydrogen had no significant effect on the lifespan of the nematode. These findings suggest that the Pt NPs in ERW, rather than the molecular hydrogen, extend the longevity of the nematode, at least partly by scavenging ROS.     

Alpha-tocopherol ameliorates cypermethrin-induced toxicity and oxidative stress in the nematode Caenorhabdtis elegans

Oxidative stress and other effects induced by cypermethrin (CYP, 15 mM) and their amelioration by alpha-tocopherol (400 microM) was studied in the nematode Caenorhabditis elegans. The worms exposed for 4 h to CYP showed increased levels of reactive oxygen species (46%), H2O2 (37%) and protein carbonyls (29%), accompanied by decreased lifespan and brood size. However, exposure to both CYP and alpha-tocopherol resulted in diminution of above alterations with the worms exhibiting relatively lower levels of ROS (30%), H2O2 (15%), protein carbonyls (14%), altered antioxidant enzyme activities and normal lifespan and brood size. The results suggest that CYP induces oxidative stress in C. elegans and the strategy of intervention with alpha-tocopherol could be exploited to offset this induced oxidative stress.     

Glyoxalase-1 prevents mitochondrial protein modification and enhances lifespan in Caenorhabditis elegans

Studies of mutations affecting lifespan in Caenorhabditis elegans show that mitochondrial generation of reactive oxygen species (ROS) plays a major causative role in organismal aging. Here, we describe a novel mechanism for regulating mitochondrial ROS production and lifespan in C .  elegans: progressive mitochondrial protein modification by the glycolysis-derived dicarbonyl metabolite methylglyoxal (MG). We demonstrate that the activity of glyoxalase-1, an enzyme detoxifying MG, is markedly reduced with age despite unchanged levels of glyoxalase-1 mRNA. The decrease in enzymatic activity promotes accumulation of MG-derived adducts and oxidative stress markers, which cause further inhibition of glyoxalase-1 expression. Over-expression of the C .  elegans glyoxalase-1 orthologue CeGly decreases MG modifications of mitochondrial proteins and mitochondrial ROS production, and prolongs C .  elegans lifespan. In contrast, knock-down of CeGly increases MG modifications of mitochondrial proteins and mitochondrial ROS production, and decreases C .  elegans lifespan.     

rBTI及其突变体对C. elegans寿命的影响

重组荞麦胰蛋白酶抑制剂（recombinant buckwheat trypsin inhibitor,rBTI）是一种来源于荞麦Potato Ⅰ抑制剂家族的丝氨酸蛋白酶抑制剂,具有很好的生物活性及功能。先前的研究表明,rBTI在秀丽隐杆线虫（Caenorhabditis elegans）中具有很好的延长寿命的性质,但其具体的作用机制还不太清楚。本文的研究证明,rBTI能够调节转录因子DAF-16的转录活性,进而影响线虫的寿命,且该性质与其胰蛋白酶抑制活性密切相关。通过定点突变技术,分别对rBTI的45位、53位和44位氨基酸活性位点进行突变,获得了4种不同胰蛋白酶抑制活性的rBTI突变体,分别命名为rBTI-R45A,rBTI-R45F,rBTI-W53R和rBTI-P44T。经典模式生物秀丽隐杆线虫寿命检测实验显示,野生型rBTI可以明显延长C.elegans的寿命,且在0～10 μmol/L 范围内具有浓度依赖性。和未处理对照组相比,10 μmol/L 野生型rBTI延长寿命幅度可达到14.5%,但是突变体rBTI-R45A,rBTI-R45F和rBTI W53R均不同程度失去了延长寿命的功能。利用荧光显微观察及qRT-PCR等方法进一步研究发现,野生型rBTI 可增强寿命调控转录因子DAF-16的转录活性。与寿命检测实验结果一致,4种 rBTI突变体均不能使DAF-16转录活性增强。上述结果表明,在C.elegans中,rBTI可增强长寿因子DAF 16的转录活性,进而延长虫体寿命,且该功能的发挥依赖于其适当的胰蛋白酶抑制活性。本文的结果为进一步研究开发rBTI的功能提供了实验支持和理论基础。     

Induction of stress resistance and extension of lifespan in Chaenorhabditis elegans serotonin-receptor knockout strains by withanolide A

IntroductionApproximately 300 million people worldwide suffer from depression. The COVID-19 crisis may dramatically increase these numbers. Severe side effects and resistance development limit the use of standard antidepressants. The steroidal lactone withanolide A (WA) from Withania somnifera may be a promising alternative. Caenorhabditis elegans was used as model to explore WA's anti-depressive and anti-stress potential.MethodsC. elegans wildtype (N2) and deficient strains (AQ866, DA1814, DA2100, DA2109 and MT9772) were used to assess oxidative, osmotic or heat stress as measured by generation of reactive oxygen species (ROS), determination of lifespan, and mRNA expression of serotonin receptor (ser-1, ser-4, ser-7) and serotonin transporter genes (mod-5). The protective effect of WA was compared to fluoxetine as clinically established antidepressant. Additionally, WA's effect on lifespan was determined. Furthermore, the binding affinities and pKi values of WA, fluoxetine and serotonin as natural ligand to Ser-1, Ser-4, Ser-7, Mod-5 and their human orthologues proteins were calculated by molecular docking.ResultsBaseline oxidative stress was higher in deficient than wildtype worms. WA and fluoxetine reduced ROS levels in all strains except MT9772. WA and fluoxetine prolonged survival times in wildtype and mutants under osmotic stress. WA but not fluoxetine increased lifespan of all heat-stressed C. elegans strains except DA2100. Furthermore, WA but not fluoxetine extended lifespan in all non-stressed C. elegans strains. WA also induced mRNA expression of serotonin receptors and transporters in wildtype and mutants. WA bound with higher affinity and lower pKi values to all C. elegans and human serotonin receptors and transporters than serotonin, indicating that WA may competitively displaced serotonin from the binding pockets of these proteins.ConclusionWA reduced stress and increased lifespan by ROS scavenging and interference with the serotonin system. Hence, WA may serve as promising candidate to treat depression.     

Lonidamine extends lifespan of adult Caenorhabditis elegans by increasing the formation of mitochondrial reactive oxygen species

Compounds that delay aging in model organisms may be of significant interest to antiaging medicine, since these substances potentially provide pharmaceutical approaches to promote healthy lifespan in humans. The aim of the study was to test whether pharmaceutical concentrations of the glycolytic inhibitor lonidamine are capable of extending lifespan in a nematodal model organism for aging processes, the roundworm Caenorhabditis elegans. Several hundreds of adult C. elegans roundworms were maintained on agar plates and fed E. coli strain OP50 bacteria. Lonidamine was applied to test whether it may promote longevity by quantifying survival in the presence and absence of the compound. In addition, several biochemical and metabolic assays were performed with nematodes exposed to lonidamine. Lonidamine significantly extends both median and maximum lifespan of C. elegans when applied at a concentration of 5 micromolar by 8% each. Moreover, the compound increases paraquat stress resistance, and promotes mitochondrial respiration, culminating in increased formation of reactive oxygen species (ROS). Extension of lifespan requires activation of pmk-1, an orthologue of p38 MAP kinase, and is abolished by co-application of an antioxidant, indicating that increased ROS formation is required for the extension of lifespan by lonidamine. Consistent with the concept of mitohormesis, lonidamine is capable of promoting longevity in a pmk-1 sensitive manner by increasing formation of ROS.     

Mitochondrial and Cytoplasmic ROS Have Opposing Effects on Lifespan

Reactive oxygen species (ROS) are highly reactive, oxygen-containing molecules that can cause molecular damage within the cell. While the accumulation of ROS-mediated damage is widely believed to be one of the main causes of aging, ROS also act in signaling pathways. Recent work has demonstrated that increasing levels of superoxide, one form of ROS, through treatment with paraquat, results in increased lifespan. Interestingly, treatment with paraquat robustly increases the already long lifespan of the clk-1 mitochondrial mutant, but not other long-lived mitochondrial mutants such as isp-1 or nuo-6. To genetically dissect the subcellular compartment in which elevated ROS act to increase lifespan, we deleted individual superoxide dismutase (sod) genes in clk-1 mutants, which are sensitized to ROS. We find that only deletion of the primary mitochondrial sod gene, sod-2 results in increased lifespan in clk-1 worms. In contrast, deletion of either of the two cytoplasmic sod genes, sod-1 or sod-5, significantly decreases the lifespan of clk-1 worms. Further, we show that increasing mitochondrial superoxide levels through deletion of sod-2 or treatment with paraquat can still increase lifespan in clk-1;sod-1 double mutants, which live shorter than clk-1 worms. The fact that mitochondrial superoxide can increase lifespan in worms with a detrimental level of cytoplasmic superoxide demonstrates that ROS have a compartment specific effect on lifespan – elevated ROS in the mitochondria acts to increase lifespan, while elevated ROS in the cytoplasm decreases lifespan. This work also suggests that both ROS-dependent and ROS-independent mechanisms contribute to the longevity of clk-1 worms.     

Acacetin promotes healthy aging by altering stress response in Caenorhabditis elegans

The progression in lifespan has been associated with elevated intracellular reactive oxygen species (ROS) and oxidative stress level which contributes to development of age related disorders. The discovery of lifespan modulating phytomolecules may promote development of natural therapies against age related afflictions. Acacetin (5,7-dihydroxy-4-methoxyflavone), is a naturally occurring flavonoid known to possess therapeutic properties. To this end, the present study evaluates effect of acacetin (AC) on lifespan, stress and neurotoxicity for the first time by using well-established free living, multicellular Caenorhabditis elegans model system. The 25?μM dose of AC significantly prolonged the mean lifespan of worms by 27.31% in comparison to untreated control and other tested doses of AC. Additionally, AC enhanced stress resistance against oxidative and thermal stress in worms. Furthermore, AC attenuated age related intracellular ROS level, aggregation of age pigment lipofuscin and increased the mean survival in stress hypersensitive mev-1 mutant by 40.5%. AC supplementation also reduced the alpha synuclein aggregation in transgenic worm model of Parkinson’s disease. The enhanced stress resistance, lifespan and alleviation of age related pathology can be attributed to increment in stress modulatory enzymes like superoxide dismutase (SOD) and catalase (CAT) level. Altogether the results suggest AC exposure maintains stress level, health span and extends mean lifespan of C. elegans. The longevity promoting and neuromodulatory effects of AC are mediated by up regulation of the stress response genes sod-3 and gst-4. The present finding gives new insights of natural remedies and their future prospects in developing therapeutic interventions for managing age related diseases.     

Dietary deprivation extends lifespan in Caenorhabditis elegans

Dietary restriction (DR) is well known as a nongenetic intervention that robustly extends lifespan in a variety of species; however, its underlying mechanisms remain unclear. We have found in Caenorhabditis elegans that dietary deprivation (DD) during adulthood, defined as removal of their food source Escherichia coli after the completion of larval development, increased lifespan and enhanced thermotolerance and resistance to oxidative stress. DD-induced longevity was independent of one C. elegans SIRTUIN, sir-2.1, which is required for the effects of DR, and was independent of the daf-2/insulin-like signaling pathway that independently regulates longevity and larval diapause in C. elegans. DD did not significantly alter lifespan of fem-1(hc17); eat-2(ad465) worms, a genetic model of DR. These findings suggest that DD and DR share some downstream effectors. In addition, DD was detrimental for longevity when imposed on reproductively active young adults, suggesting that DD may only be beneficial in the absence of competing metabolic demands, such as fertility. Adult-onset DD offers a new paradigm for investigating dietary regulation of longevity in C. elegans. This study presents the first evidence that long-term DD, instead of being detrimental, can extend lifespan of a multicellular adult organism.     

Cryopreservation produces limited long-term effects on the nematode Caenorhabditis elegans

Cryopreservation, the freezing and later warming of biological samples with minimal loss of viability, is important in many scientific disciplines. For some applications, particularly those where there is limited available material, it is critical to ensure the maximal survival rates of cryopreserved materials. Most of the challenges encountered with such techniques take place after the warming process where cryodamage affects cell viability and future development. Here we have used the nematode Caenorhabditis elegans to investigate the effects of cryodamage caused by slow-freezing. We find that freezing results in the death of some worms, with an approximately 40% reduction in the number of worms that develop in the frozen populations, but that the effects on worms that survive are limited. For example, there are no differences in the lifetime fecundity or in lifespan between frozen and control worms, although early fecundity and body size was reduced in frozen worms. Similarly, analyses of body wall muscle structure and of pharyngeal function indicates that muscle development and function are not significantly affected by freezing. We do however determine that freezing increases the rates of matricidal hatching, where progeny hatch within the mother. Overall, these results indicate that, for worms that survive, cryopreservation produces limited long-term effects, but do indicate that some phenotypes could be used in further analyses of the cellular damage induced by cryopreservation.     

Differential effects of resveratrol and SRT1720 on lifespan of adult Caenorhabditis elegans

Resveratrol and SRT1720 have been shown to act as sirtuin activators that may ameliorate type 2 diabetes and metabolic diseases in mice. Moreover, resveratrol extends lifespan in model organisms like C. elegans, N. FURZERI, and possibly D. melanogaster. The aim of the study was to test whether pharmacological concentrations of resveratrol and SRT1720 are capable of extending lifespan in a nematodal model organism for aging processes, the roundworm Caenorhabditis elegans. Several hundreds of adult C. ELEGANS roundworms were maintained on agar plates and fed E. COLI strain OP50 bacteria. Resveratrol (5 micromolar, 500 nanomolar) or SRT1720 (1 micromolar, 100 nanomolar) was applied to the agar to test whether they may promote longevity by quantifying survival in the presence and absence of the respective compounds. At a dose of 5 micromolar, which is pharmacologically relevant and 20 times lower than previously published concentrations, resveratrol significantly extends C. elegans lifespan by 3.6% (mean lifespan) and 3.4% (maximum lifespan). By unexpected contrast, SRT1720, which was previously proposed to be several hundred times more active than resveratrol, did not extend lifespan at none of the concentrations tested. Thus, in the model organisms C. elegans, resveratrol is capable of promoting longevity at a concentration that pharmacologically relevant and 20 times lower than previously published doses. The sirtuin activator SRT1720 did not extend lifespan, suggesting that in C. elegans, some relevant effects of resveratrol cannot be mimicked by SRT1720.     

A peroxiredoxin specifically expressed in two types of pharyngeal neurons is required for normal growth and egg production in Caenorhabditis elegans

A family of antioxidant proteins, the peroxiredoxins, serve two purposes, detoxification of reactive oxygen species and cellular signaling. Among the three peroxiredoxins of Caenorhabditis elegans (CePrx1-3), CePrx2 was found to have a very unusual expression pattern, restricted to only two types of pharyngeal neurons; namely, the single pharyngeal interneuron I4 and the sensory interneuron I2. CePrx1 and CePrx3-depleted worms showed no obvious phenotypic alterations, whereas worms devoid of CePrx2 were retarded developmentally and had a significantly reduced brood size. Other features, such as lifespan, pharyngeal activity or defecation rates were indistinguishable from those of wild-type worms. Recombinant CePrx2 revealed antioxidant activity, as it was able to detoxify hydrogen peroxide and butylhydroperoxide (t-BOOH), and to protect glutamine synthetase from inactivation by thiol-dependent metal-catalyzed oxidation. In addition, the molecule was able to act as a terminal peroxidase in the thioredoxin system. Expression of ceprx2 in C.elegans was induced after short-term exposure of worms to t-BOOH but survival of ceprx2 knockout mutants in the presence of reactive oxygen or nitrogen species was not impaired. Thus, CePrx2 may protect specifically the two types of neurons from oxidative damage or, more likely, plays a critical role in peroxide signaling in this nematode.     

Anti-diabetic effects of electrolyzed reduced water in streptozotocin-induced and genetic diabetic mice

Oxidative stress is produced under diabetic conditions and is likely involved in progression of pancreatic beta-cell dysfunction found in diabetes. Both an increase in reactive oxygen free radical species (ROS) and a decrease in the antioxidant defense mechanism lead to the increase in oxidative stress in diabetes. Electrolyzed reduced water (ERW) with ROS scavenging ability may have a potential effect on diabetic animals, a model for high oxidative stress. Therefore, the present study examined the possible anti-diabetic effect of ERW in two different diabetic animal models. The genetically diabetic mouse strain C57BL/6J-db/db (db/db) and streptozotocin (STZ)-induced diabetic mouse were used as insulin deficient type 1 and insulin resistant type 2 animal model, respectively. ERW, provided as a drinking water, significantly reduced the blood glucose concentration and improved glucose tolerance in both animal models. However, ERW fail to affect blood insulin levels in STZ-diabetic mice whereas blood insulin level was markedly increased in genetically diabetic db/db mice. This improved blood glucose control could result from enhanced insulin sensitivity, as well as increased insulin release. The present data suggest that ERW may function as an orally effective anti-diabetic agent and merit further studies on its precise mechanism.     

谷氧还蛋白rbGrx可延缓CL4176秀丽隐杆线虫中β淀粉样蛋白诱导的毒性

阿尔茨海默病（Alzheimer’s disease,AD）是一种与衰老相关的神经退行性疾病,其中β-淀粉样蛋白（β-amyloid,Aβ）诱导的细胞毒性被认为是其发病的主要原因。本文以Aβ转基因秀丽隐杆线虫CL4176为模型,研究了重组荞麦谷氧还蛋白（recombinant buckwheat glutaredoxin, rbGrx）对Aβ诱导的毒性和氧化应激的影响。结果显示,4 μmol/L rbGrx可以延长CL4176线虫平均寿命达20%左右,并增加衰老虫体运动能力约43.6%,延迟产卵高峰期1 d,同时可以有效延缓Aβ毒性诱导的瘫痪表型。进一步研究发现,在正常条件和Aβ诱导毒性时,rbGrx均能降低CL4176线虫体内活性氧（reactive oxygen species,ROS）水平,并上调SOD活性和GSH含量。另外,rbGrx下调Aβ mRNA水平44.1％,减少Aβ沉积量,并且明显上调热激因子1 hsf-1（2.01倍）和hsp-16.2（2.65倍）mRNA表达水平。这表明,rbGrx通过降低CL4176线虫体内的ROS水平和上调热激蛋白质的转录表达水平,降低CL4176秀丽隐杆线虫中Aβ诱导的毒性。结果提示,rbGrx可能具有预防AD的潜力。     

Electrolyzed Reduced Water Supplemented with Platinum Nanoparticles Suppresses Promotion of Two-stage Cell Transformation

In the two-stage cell transformation theory, cancer cells first receive initiation, which is mainly caused by DNA damage, and then promotion, which enhances transformation. Murine Balb/c 3T3 cells are widely used for transformation experiments because they lose contact inhibition ability when transformed. Electrolyzed reduced water (ERW), which is produced near a cathode during electrolysis of water, is an alkaline drinking water that is beneficial to health. ERW contains a high concentration of dissolved hydrogen and scavenge reactive oxygen species (ROS), along with a small amount of platinum (Pt) nanoparticles (Pt nps) derived from Pt-coated titanium electrodes. Pt nps stably disperse in aqueous solution for a long time, and convert hydrogen molecules to active hydrogen (atomic hydrogen) that can scavenge ROS. Therefore, ERW supplemented with synthesized Pt nps is a model strong reduced water. This is the first report that ERW supplemented with synthesized Pt nps strongly prevents transformation of Balb/c 3T3 cells. ERW was prepared by electrolysis of 0.002 M NaOH solution using a batch-type electrolysis device. Balb/c 3T3 cells were treated with 3-methyl cholanthrene (MCA) as an initiation substance, followed by treatment with phorbol-12-myristate-13-acetate (PMA) as a promotion substance. MCA/PMA-induced formation of a transformation focus was strongly suppressed by ERW supplemented with Pt nps but not by ERW or Pt nps individually. ERW supplemented with Pt nps suppressed transformation at the promoter stage, not at initiation, suggesting that ERW supplemented with Pt nps suppressed the PMA-induced augmentation of intracellular ROS. ERW supplemented with Pt nps is a potential new antioxidant against carcinogenesis.