Cell-Free Culture Supernatant of Probiotic Lactobacillus fermentum Protects Against H2O2-Induced Premature Senescence by Suppressing ROS-Akt-mTOR Axis in Murine Preadipocytes

Information regarding cellular anti-senescence attributes of probiotic bacteria vis-à-vis modulation of senescence-associated secretory phenotype (SASP) and mTOR signaling is very limited. The present study assessed anti-senescence potential of secretory metabolites of probiotic Lactobacillus fermentum (Lact. fermentum) using H₂O₂-induced model of senescence in 3T3-L1 preadipocytes. Application of H₂O₂-induced cellular senescence characterized by increased cell size and SA-β-gal activity, activation of SASP and reactive oxygen species (ROS), DNA damage response and induction of cell cycle inhibitors (p53/p21^WAF1/p16^INK4a). Further, a robust stimulation of the PI3K/Akt/mTOR pathway and AMPK signaling was also observed in H₂O₂-treated cells. However, exposure of cells to cell-free supernatant of Lact. fermentum significantly attenuated phosphorylation of PI3K/Akt/mTOR pathway and alleviated senescence markers p53, p21^WAF1, SA-β-gal, p38MAPK, iNOS, cox-2, ROS, NF-κB, and DNA damage response. These results provide evidence that secretory metabolites of Lact. fermentum can mitigate the development as well as severity of stress-induced senescence thereby indicating its utility for use as anti-aging or age-delaying agent.

     

p38 MAPK and Ca2+ contribute to hydrogen peroxide-induced increase of permeability in vascular endothelial cells but ERK does not

To examine the involvement of p38 mitogen-activated protein kinase (p38 MAPK) and extra-cellular signal-regulated kinase (ERK) in the oxidative stress-induced increase of permeability in endothelial cells, the effects of a p38 MAPK inhibitor (SB203580) and ERK inhibitor (PD90859) on the H₂O₂-induced increase of permeability in bovine pulmonary artery endothelial cells (BPAEC) were investigated using a two-compartment system partitioned by a semi-permeable filter. H₂O₂ at 1 mM caused an increase of the permeation rate of fluorescein isothiocyanate (FITC)-labeled dextran 40 through BPAEC monolayers. SB203580 inhibited the H₂O₂-induced increase of permeability but PD98059 did not, though activation (phosphorylation) of both p38 MAPK and ERK was observed in H₂O₂-treated cells in Western blot analysis. An H₂O₂-induced increase of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) was also observed and an intracellular Ca²⁺ chelator (BAPTA-AM) significantly inhibited the H₂O₂-induced increase of permeability. However, it showed no inhibitory effects on the H₂O₂-induced phosphorylation of p38 MAPK and ERK. The H₂O₂-induced increase of [Ca²⁺]_i was not influenced by SB203580 and PD98059. These results indicate that the activation of p38 MAPK and the increase of [Ca²⁺]_i are essential for the H₂O₂-induced increase of endothelial permeability and that ERK is not.     

Novel role of TRPV2 in promoting the cytotoxicity of H2O2-mediated oxidative stress in Human hepatoma cells

Oxidative stress is important for the initiation and progression of cancers, which confers the cells with a survival advantage by inducing oxidative adaption and drug resistance. Therefore, developing strategies to promote oxidative stress-induced cytotoxicity could be important for cancer therapy. Herein, we found that H₂O₂-mediated oxidative stress increases TRPV2 expression in human hepatoma (HepG2 and Huh-7) cells. This occurred at the mRNA and protein levels in a dose-dependent manner. The significance of TRPV2 in promoting H₂O₂-induced cell death was demonstrated in gain and loss of function studies with overexpression and knockdown of TRPV2, respectively. Mechanistically, H₂O₂-induced cell death involves inhibition of pro-survival signaling proteins (Akt, Nrf2) and activation of pro-death signaling proteins (p38, JNK1). Overexpression of TRPV2 in H₂O₂-treated hepatoma cells aggravates the inhibition of Akt and Nrf2, while it enhances the activation of p38 and JNK1 at the early stage of cell death. Interestingly, increased expression of TRPV2 in HepG2 cells improved the efficacy of stress-associated chemicals to induce cell death. Our findings suggest that TRPV2 acts as an important enhancer for H₂O₂-induced cytotoxicity. This process occurred by the inhibition of Akt and Nrf2 as well as the early activation of p38 and JNK1. These findings have important implications for inhibition of oxidative adaption and drug resistance.     

Acute activation of AMP-activated protein kinase prevents H2O2-induced premature senescence in primary human keratinocytes

We investigated the effects of AMPK on H₂O₂-induced premature senescence in primary human keratinocytes. Incubation with 50 µM H₂O₂ for 2 h resulted in premature senescence with characteristic increases in senescence-associated ß-galactosidase (SA-gal) staining 3 days later and no changes in AMPK or p38 MAPK activity. The increase in SA-gal staining was preceded by increases in both p53 phosphorylation (S15) (1 h) and transactivation (6 h) and the abundance of the cyclin inhibitor p21^CIP1 (16 h). Incubation with AICAR or resveratrol, both of which activated AMPK, prevented the H₂O₂-induced increases in both SA-Gal staining and p21 abundance. In addition, AICAR diminished the increase in p53 transactivation. The decreases in SA-Gal expression induced by resveratrol and AICAR were prevented by the pharmacological AMPK inhibitor Compound C, expression of a DN-AMPK or AMPK knock-down with shRNA. Likewise, both knockdown of AMPK and expression of DN-AMPK were sufficient to induce senescence, even in the absence of exogenous H₂O₂. As reported by others, we found that AMPK activation by itself increased p53 phosphorylation at S15 in embryonic fibroblasts (MEF), whereas under the same conditions it decreased p53 phosphorylation in the keratinocytes, human aortic endothelial cells, and human HT1080 fibrosarcoma cells. In conclusion, the results indicate that H₂O₂ at low concentrations causes premature senescence in human keratinocytes by activating p53-p21^CIP1 signaling and that these effects can be prevented by acute AMPK activation and enhanced by AMPK downregulation. They also suggest that this action of AMPK may be cell or context-specific.     

The role of p38 MAP-kinase in stress-induced senescence of human endometrium-derived mesenchymal stem cells

Our recent findings demonstrate that human endometrium-derived mesenchymal stem cells (hMESCs) respond to sublethal oxidative stress by stress-induced premature senescence via the АТМ/Chk2/p53/p21/Rb pathway. Application of SB203580 (SB) inhibitor suggested p38 MAP-kinase involvement in the senescence progression. However, there are several disadvantages concerning this inhibitor: (1) SB is toxic and hardly suitable for in vivo experiments and (2) poor kinase selectivity profile of SB complicates interpretation of the obtained data. Here, to confirm the involvement of p38 in H₂O₂-induced hMESCs senescence, we applied another highly specific p38 inhibitor, BIRB796 (BIRB). In the presence of BIRB, the cell size decreased, the level of reactive oxygen species reduced, proliferation partially resumed, and Rb phosphorylation level increased in comparison to H₂O₂-treated hMESCs. Summarizing these results, we can postulate p38 involvement in H₂O₂-induced senescence of hMESCs and suggest p38 inhibition as a promising approach in prevention of premature senescence.     

The ginsenoside protopanaxatriol protects endothelial cells from hydrogen peroxide-induced cell injury and cell death by modulating intracellular redox status

Ginsenosides, the active components of the famous Chinese herb ginseng, have been suggested to possess cardiovascular-protective effects. The mechanism of ginsenosides is believed to be associated with their ability to prevent cellular oxidative stress. The purpose of this study was to explore the cytoprotective effects of the ginsenoside protopanaxatriol (PPT) on hydrogen peroxide (H₂O₂)-induced endothelial cell injury and cell death. Pretreatment of human umbilical vein endothelial cells (HUVECs) with PPT for 24 h was able to protect the cells against H₂O₂-induced injury. In addition to cell death, pretreatment with PPT could also reduce H₂O₂-induced DNA damage, overactivation of the DNA repair enzyme PARP-1, and concomitant depletion of the intracellular substrate NAD⁺. Furthermore, PPT could reverse the decrease in ATP/ADP ratio caused by H₂O₂. The metabolism of glutathione was also changed. H₂O₂ could induce a significant decrease in GSH level resulting in a decrease in the GSH/GSSG ratio. This could be prevented by pretreatment with PPT. The action was associated with increasing activities of the GSH-metabolizing enzymes glutathione reductase and glutathione peroxidase. These findings suggest that the ginsenoside PPT could protect HUVECs against H₂O₂-induced cell death via its action against oxidative stress, which may be responsible for the cardiovascular-protective action of ginseng.     

Protectin DX prevents H2O2-mediated oxidative stress in vascular endothelial cells via an AMPK-dependent mechanism

Protectin DX (PDX), which is a novel regulator of 5′ adenosine monophosphate-activated protein kinase (AMPK), has recently gained attention for its ability to improve several metabolic diseases. However, the function of PDX in vascular endothelial cells remains unclear. To confirm the protective effects of PDX on endothelial oxidative stress, human umbilical vein endothelial cells (HUVECs) were treated with hydroperoxide (H₂O₂) and PDX. PDX treatment significantly increased the level of AMPK phosphorylation, and this elevation was attenuated after treatment with G-protein coupled receptor 120 (GPR120) antagonist or GPR120 knockdown. Expressions and activities of antioxidant proteins, including catalase and superoxide dismutase 2 (SOD2), were elevated by PDX and were inhibited by treatment with AMPK inhibitor or with GPR120 antagonist. Production of H₂O₂-induced reactive oxygen species (ROS), the Bax/Bcl-2 ratio, and the loss of mitochondrial membrane potential were all reversed by PDX, leading to improved cell viability and reduced release of lactate dehydrogenase (LDH). Using flow cytometry, we also found that PDX significantly reduced the H₂O₂-induced apoptotic population of cells. These protective effects of PDX were all reversed after treatment with AMPK inhibitor or GRP120 antagonist. These results show that the PDX-AMPK axis has a protective role against H₂O₂-induced oxidative stress in vascular endothelial cells.     

Resveratrol inhibits the hydrogen dioxide-induced apoptosis via Sirt 1 activation in osteoblast cells

Sirt 1 plays a critical role in stress responses. We determined the deregulation of Sirt 1 activity, p53 acetylation, Bcl-2 expression, and mitochondria-dependent apoptosis in mouse osteoblast MC3T3-E1 cells which were exposed to H₂O₂. And then we investigated the protective role of Sirt 1 activator, Resveratrol (RSV), against the H₂O₂-induced apoptosis. Results demonstrated that Sirt 1 and Bcl-2 were inhibited, whereas p53 acetylation, Bax, and caspase 9 were promoted by H₂O₂, as was aggravated by the Sirt 1 inhibitor, EX-527. Instead, RSV inhibited the H₂O₂-induced both p53 acetylation and the caspase 9 activation, whereas ameliorated the H₂O₂-induced Bcl-2 inhibition and apoptosis. In conclusion, Sirt 1 was downregulated during the H₂O₂-induced apoptosis in MC3T3-E1 cells. And the chemical activation of Sirt 1 inhibited the H₂O₂-induced apoptosis via the downregulation of p53 acetylation. Our results suggest that Sirt 1 upregulation appears to be an important strategy to inhibit the oxidative stress-induced apoptosis.     

Protective Effect of Selenoprotein X Against Oxidative Stress-Induced Cell Apoptosis in Human Hepatocyte (LO2) Cells via the p38 Pathway

Oxidative stress, as mediated by ROS (reactive oxygen species), is a significant factor in initiating the cells damaged by affecting cellular macromolecules and impairing their biological functions; SelX, a selenoprotein also known as MsrB1 belonging to the methionine sulfoxide reductase (Msr) family, is the redox repairing enzyme and involved in redox-related functions. In order to more precisely analyze the relationship between oxidative stress, cell oxidative damage, and SelX, we stably overexpressed porcine Selx full-length cDNA in human normal hepatocyte (LO2) cells. Cell viability, cell apoptosis rate, intracellular ROS, and the expression levels of mRNA or protein of apoptosis-related genes under H₂O₂-induced oxidative stress were detected. We found that overexpression of SelX can prevent the oxidative damage caused by H₂O₂ and propose that the main mechanism underlying the protective effects of SelX is the inhibition of LO2 cell apoptosis. The results revealed that overexpressed SelX reduced the H₂O₂-induced intracellular ROS generation, inhibited the H₂O₂-induced upregulation of Bax and downregulation of Bcl-2, and increased the mRNA and protein ratio of Bcl-2/Bax. Furthermore, it inhibited H₂O₂-induced p38 MAPK phosphorylation. Taken together, our findings suggested that SelX played important roles in protecting LO2 cells against oxidative damage and that its protective effect is partly via the p38 pathway by acting as a ROS scavenger.     

Anti-apoptotic and anti-senescence effects of Klotho on vascular endothelial cells

Klotho-mutated mice manifest multiple age-related disorders that are observed in humans. A recent study suggested that Klotho protein might function as an anti-aging hormone in mammals. Because it has been reported that apoptosis and senescence in vascular endothelial cells are closely related to the progression of atherosclerosis, we investigated Klotho's ability to interfere with apoptosis and cellular senescence in human umbilical vascular endothelial cells (HUVEC). Klotho overexpression decreased H(2)O(2)-induced apoptosis in COS-1 cells and Jurkat cells. Klotho protein also reduced H(2)O(2)- and etoposide-induced apoptosis in HUVEC. Caspase-3 and caspase-9 activity was lower in Klotho-treated HUVEC than in control cells. Senescence-associated beta-gal staining showed that Klotho protein interferes with H(2)O(2)-induced premature cellular senescence. The expression of p53 and p21 was lower in Klotho-treated cells. Our study suggests that Klotho acts as a humoral factor to reduce H(2)O(2)-induced apoptosis and cellular senescence in vascular cells.     

Parthenolide regulates oxidative stress-induced mitophagy and suppresses apoptosis through p53 signaling pathway in C2C12 myoblasts

Muscle redox disturbances and oxidative stress have emerged as a common pathogenetic mechanism and potential therapeutic intervention in some muscle diseases. Parthenolide (PTL), a sesquiterpene lactone found in large amounts in the leaves of feverfew, possesses anti-inflammatory, anti-migraine, and anticancer properties. Although PTL was reported to alleviate cancer cachexia and improve skeletal muscle characteristics in a cancer cachexia model, its actions on oxidative stress-induced damage in C2C12 myoblasts have not been reported and the regulatory mechanisms have not yet been defined. In our study, PTL attenuated H₂O₂-induced growth inhibition and morphological changes. Furthermore, PTL exhibited scavenging activity against reactive oxygen species and protected C2C12 cells from apoptosis in response to H₂O₂. Meanwhile, PTL suppressed collapse of the mitochondrial membrane potential, thereby contributing to normalizing H₂O₂-induced autophagy flux and mitophagy, correlating with inhibiting degradation of mitochondrial marker protein TIM23, the increase in LC3-II expression and the reduction of mitochondria DNA. Besides its protective effect on mitochondria, PTL also prevented H₂O₂-induced lysosomes damage in C2C12 cells. In addition, the phosphorylation of p53, cathepsin B, and Bax/Bcl-2 protein levels, and the translocation of Bax from the cytosol to mitochondria induced by H₂O₂ in C2C12 cells was significantly reduced by PTL. In conclusion, PTL modulates oxidative stress-induced mitophagy and protects C2C12 myoblasts against apoptosis, suggesting a potential protective effect against oxidative stress-associated skeletal muscle diseases.     

Novel role of silent information regulator 1 in acute endothelial cell oxidative stress injury

Silent information regulator 1 (SIRT1), a class III histone deacetylase, retards aging and plays roles in cellular oxidative stress injury (OSI). However, the biological context in which SIRT1 promotes oxidative injury is not fully understood. Here, we show that SIRT1 essentially mediates hydrogen peroxide (H₂O₂)-induced cytotoxicity in human umbilical vein endothelial cell (HUVEC). In HUVECs, SIRT1 protein expression was significantly increased in a dose-dependent manner after H₂O₂ treatment, whereas the acetylation levels of the NF-κB p65 subunit and p53 were decreased. EX527 (a specific SIRT1 inhibitor) conferred protection to the HUVECs against H₂O₂, as indicated by an improved cell viability, adhesion, an enhanced migratory ability, a decreased apoptotic index, decreased reactive oxygen species (ROS) production and reductions in several biochemical parameters. Immunofluorescence and Western blot analyses demonstrated that H₂O₂ treatment up-regulated SIRT1, phosphorylated-JNK (p-JNK), p-p38MAPK, and p-ERK expression. EX527 pretreatment reversed these effects on SIRT1, p-JNK, and p-p38MAPK but further increased the p-ERK levels. Similar results were confirmed in SIRT1 siRNA experiments. In summary, SIRT1 signaling pathway inhibition imparts protection against acute endothelial OSI, and modulation of MAPKs (JNK, p38MAPK, and ERK) may be involved in the protective effect of SIRT1 inhibition.     

Phytochemical screening of flavonoids with their antioxidant activities from rapeseed (Brassica napus L.)

Ten flavone compounds, including three new flavonoid glycosides, were isolated from defatted rapeseed, and their protective antioxidant effect on H₂O₂-induced oxidative damage in human umbilical vein endothelial cells (ECV-304) was investigated. Three new flavonoid glycosides were identified as kaempferol-3-O-[(6-O-sinapoyl)-β-d-glucopyranosyl-(1 → 2)-β-d-glucopyranoside]-7-O-β-d-glucopyranoside (8), kaempferol-3,7-di-O-β-d-glucopyranoside-4'-O-(6-O-sinapoyl)-β-d-glucopyranoside (9), and kaempferol-3-O-[(3-O-sinapoyl)-β-d-glucopyranosyl-(1 → 2)-β-d-glucopyranoside]-7-O-β-d-glucopyranoside (10). The protective effects of all of the isolated compounds on H₂O₂-induced oxidative damage were assessed, and the activities of superoxide dismutase (SOD) and lactate dehydrogenase (LDH) were measured. All of compounds had a protective effect on H₂O₂-induced oxidative damage in ECV-304 cells and the presence of a substituted sinapoyl group and its position in the structures were used to elucidate the activity differences.     

Mitigation of H2O2-induced autophagic cell death by propofol in H9c2 cardiomyocytes

Autophagy, a self-eating process, is responsible for degradation of long-lived proteins and damaged cellular proteins/organelles. Double-membrane autophagosomes, formed during the process, engulf proteins/organelles and fuse with lysosomes to degrade the contents. It is important to maintain cell homeostasis and many physiological processes including cellular responses to oxidative stress. Oxidative stress induced by myocardial infarction is a major factor of heart failures. In this study, we examined how propofol modulates hydrogen peroxide (H₂O₂)-induced autophagic cell death in H9c2 cardiomyocytes. H₂O₂ dramatically induced cell death, which was similarly reduced in the presence of either propofol or autophagy inhibitors (e.g., wortmannin), suggesting that propofol has a protective effect in H₂O₂-induced autophagic cell death. Acidic autophagic vacuoles were elevated in H₂O₂-treated H9c2 cells, but they were largely decreased in the presence of propofol. Furthermore, many autophagy-related proteins such as LC3-II, ATG proteins, p62, AMPK, and JNK were activated in H₂O₂-treated H9c2 cells and were significantly deactivated in the presence of propofol. These results show that propofol regulates oxidative stress-induced autophagic cell death in cardiomyocytes. We further suggest that propofol can act as a cardioprotectant in heart diseases.     

Selenium inhibits high glucose-induced cyclooxygenase-2 and P-selectin expression in vascular endothelial cells

Selenium as a component of glutathione peroxidase may be beneficial in insulin resistance, hence potentially may modify the risk of diabetes and cardiovascular disease. The aim of our study was to evaluate whether selenium can also alter high glucose (HG), advanced glycation end products (AGE), high insulin (HI) and H₂O₂-induced expression of cyclooxygenase (COX)-2 and P-selectin. Human umbilical vein endothelial cells (HUVECs) were pretreated with selenium and stimulated by HG, AGE, HI and H₂O₂. Selenium significantly inhibited HG, AGE, HI and H₂O₂-induced expression of COX-2 and P-selectin. Moreover, selenium also inhibited HG, AGE, HI and H₂O₂-induced activation of p38 mitogen-activated protein kinase (p38 MAPK), which indicated that the preventive effects of selenium on COX-2 and P-selectin may be associated with p38. Our results indicated that selenium supplementation can reduce HG, AGE, HI and H₂O₂-induced expression of COX-2 and P-selectin by inhibition of the p38 pathway.