Controlled lysosomal rupture was initiated in lysosome-rich, macrophage-like cells by the synthetic lysosomotropic detergent, O-methyl-serine dodecylamide hydrochloride (MSDH). When MSDH was applied at low concentrations, resulting in partial lysosomal rupture, activation of pro-caspase-3-like proteases and apoptosis followed after some hours. Early during apoptosis, but clearly secondary to lysosomal destabilization, the mitochondrial transmembrane potential declined. At high concentrations, MSDH caused extensive lysosomal rupture and necrosis. It is suggested that lysosomal proteases, if released to the cytosol, may cause apoptosis directly by pro-caspase activation and/or indirectly by mitochondrial attack with ensuing discharge of pro-apoptotic factors.z 2000 Federation of European Biochemical Societies.

Historically, consumption of Green tea (Camellia sinensis) has been associated with health benefits against multiple diseases including cancer, atherosclerosis and cardiovascular disorders. Emerging evidence has suggested a pathogenic role for HMGB1, a newly identified "late" mediator of lethal systemic inflammation, in the aforementioned diseases. Here we demonstrated that a major ingredient of Green tea, EGCG, was internalized into HMGB1-containing LC3-positive cytoplasmic vesicles (likely autophagosomes) in macrophages, and induced HMGB1 aggregation in a time-dependent manner. Furthermore, EGCG stimulated LC3-II production and autophagosome formation, and inhibited LPS-induced HMGB1 up-regulation and extracellular release. The EGCG-mediated HMGB1 inhibitory effects were diminished by inhibition of class III phosphatidylinositol-3 kinase (with 3-methyladenine) or knockdown of an essential autophagyregulating protein, beclin-1. Moreover, the EGCG-mediated protection against lethal sepsis was partly impaired by co-administration of an autophagy inhibitor, chloroquine. Taken together, the present study has suggested a possibility that EGCG inhibits HMGB1 release by stimulating its autophagic degradation.

N-acetylserotonin (NAS) is an immediate precursor of melatonin, which we have reported is neuroprotective against ischemic injury.Here we test whether NAS is a potential neuroprotective agent in experimental models of ischemic injury. We demonstrate that NAS inhibits cell death induced by oxygen-glucose deprivation or H 2 O 2 in primary cerebrocortical neurons and primary hippocampal neurons in vitro, and organotypic hippocampal slice cultures ex vivo and reduces hypoxia/ischemia injury in the middle cerebral artery occlusion mouse model of cerebral ischemia in vivo. We find that NAS is neuroprotective by inhibiting the mitochondrial cell death pathway and the autophagic cell death pathway. The neuroprotective effects of NAS may result from the influence of mitochondrial permeability transition pore opening, mitochondrial fragmentation, and inhibition of the subsequent release of apoptogenic factors cytochrome c, Smac, and apoptosis-inducing factor from mitochondria to cytoplasm, and activation of caspase-3, -9, as well as the suppression of the activation of autophagy under stress conditions by increasing LC3-II and Beclin-1 levels and decreasing p62 level. However, NAS, unlike melatonin, does not provide neuroprotection through the activation of melatonin receptor 1A. We demonstrate that NAS reaches the brain subsequent to intraperitoneal injection using liquid chromatography/mass spectrometry analysis. Given that it occurs naturally and has low toxicity, NAS, like melatonin, has potential as a novel therapy for ischemic injury.

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Background: Diabetes mellitus is closely correlated with disc degeneration. Nucleus pulposus (NP) cell apoptosis and senescence are typical cellular features within the degenerative disc. Resveratrol is a newly identified phytoalexin that has protective effects on cartilaginous tissue.Objective: To investigate the whether resveratrol can protect against high glucose-induced NP cell apoptosis and senescence, and the potential mechanism in this process.Methods: Rat NP cells were cultured in either 10% FBS culture medium (control group) or 10% FBS with a high glucose concentration (0.2 M, experiment group) for 3 days. Resveratrol or the combination of resveratrol and LY294002 was added into the culture medium of experiment group to investigate the effects of resveratrol and the PI3K/Akt pathway.Results: High glucose significantly promoted NP cell apoptosis and NP cell senescence compared with the control group. Resveratrol exhibited protective effects against high glucose-induced NP cell apoptosis and senescence. Further analysis showed that resveratrol suppressed reactive oxygen species (ROS) generation and increased the activity of the PI3K/Akt pathway under the high glucose condition. However, the LY294002 had no significant effects on ROS content in the resveratrol-treated high glucose group.Conclusion: Resveratrol can attenuate high glucose-induced NP cell apoptosis and senescence, and the activation of ROS-mediated PI3K/Akt pathway may be the potential mechanism in this process.

The mechanisms involved in the anabolic effect of interferon gamma (IFNc) on bone have not been carefully examined. Using microarray expression analysis, we found that IFNc upregulates a set of genes associated with a tryptophan degradation pathway, known as the kynurenine pathway, in osteogenic differentiating human mesenchymal stem cells (hMSC). We, therefore, hypothesized that activation of the kynurenine pathway plays a role in osteoblastogenesis even in the absence of IFNc. Initially, we observed a strong increase in tryptophan degradation during osteoblastogenesis with and without IFNc in the media. We next blocked indoleamine 2,3-dioxygenase-1 (IDO1), the most important enzyme in the kynurenine pathway, using a siRNA and pharmacological approach and observed a strong inhibition of osteoblastogenesis with a concomitant decrease in osteogenic factors. We next examined the bone phenotype of Ido1 knockout (Ido1 2/2 ) mice. Compared to their wild-type littermates, Ido1 2/2 mice exhibited osteopenia associated with low osteoblast and high osteoclast numbers. Finally, we tested whether the end products of the kynurenine pathway have an osteogenic effect on hMSC. We identified that picolinic acid had a strong and dose-dependent osteogenic effect in vitro. In summary, we demonstrate that the activation of the kynurenine pathway plays an important role during the commitment of hMSC into the osteoblast lineage in vitro, and that this process can be accelerated by exogenous addition of IFNc. In addition, we found that mice lacking IDO1 activity are osteopenic. These data therefore support a new role for the kynurenine pathway and picolinic acid as essential regulators of osteoblastogenesis and as potential new targets of bone-forming cells in vivo. STEM CELLS 2015;33:111-121

It has been reported that genomic DNA methylation decreases gradually during cell culture and an organism's aging. However, less is known about the methylation changes of age-related specific genes in aging. p21(Waf1/Cip1) and p16(INK4a) are cyclin-dependent kinase (Cdk) inhibitors that are critical for the replicative senescence of normal cells. In this study, we show that p21(Waf1/Cip1) and p16(INK4a) have different methylation patterns during the aging process of normal human 2BS and WI-38 fibroblasts. p21(Waf1/Cip1) promoter is gradually methylated up into middle-aged fibroblasts but not with senescent fibroblasts, whereas p16(INK4a) is always unmethylated in the aging process. Correspondently, the protein levels of DNA methyltransferase 1 (DNMT1) and DNMT3a increase from young to middle-aged fibroblasts but decrease in the senescent fibroblasts, while DNMT3b decreases stably from young to senescent fibroblasts. p21(Waf1/Cip1) promoter methylation directly represses its expression and blocks the radiation-induced DNA damage-signaling pathway by p53 in middle-aged fibroblasts. More importantly, demethylation by 5-aza-CdR or DNMT1 RNA interference (RNAi) resulted in an increased p21(Waf1/Cip1) level and premature senescence of middle-aged fibroblasts demonstrated by cell growth arrest and high beta-Galactosidase expression. Our results suggest that p21(Waf1/Cip1) but not p16(INK4a) is involved in the DNA methylation mediated aging process. p21(Waf1/Cip1) promoter methylation may be a critical biological barrier to postpone the aging process.

In vivo, autophagy and the NLRP3 inflammasome were activated in fibrotic patients and positively correlated with oxidation. Treatment with rapamycin promoted autophagy but inhibited oxidation, NLRP3 inflammasome, and lung fibrosis after bleomycin (BLM) infusion. The autophagy inhibitor 3-methyladenine reduced BLM-induced lung fibrosis and concurrently facilitated NLRP3 inflammasome activation and oxidation in fibroblasts. In vitro, AngII promoted intercellular ROS, hydrogen peroxide, and NADPH oxidase 4 (NOX4) protein levels and reduced the glutathione concentration, thereby leading to NLRP3 inflammasome activation and consequent collagen synthesis. AngII induced autophagy, while VAS2870, NOX4, small-interfering RNA (siRNA), and compound C eliminated AngII-induced LC3B augmentation. Moreover, blocking autophagy with bafilomycin A1 or LC3B siRNA resulted in oxidant accumulation, NLRP3 inflammasome hyperactivation, and collagen deposition. Finally, AngII induced P62/SQSTM1, targeting ubiquitinated apoptosis-associated speck-like protein containing a CARD for degradation, thereby contributing to NLRP3 inflammasome inactivation. Innovation and Conclusion: Autophagy attenuates pulmonary fibrosis by regulating NLRP3 inflammasome activation induced by AngII-mediated ROS via redox balance modulation. Antioxid. Redox Signal. 00, 000-000.