Autophagosome protects proximal tubular cells from aldosterone-induced senescence through improving oxidative stress.
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ABSTRACT:
SUBMITTER: Cao JY
PROVIDER: S-EPMC7993373 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Skin cells are vulnerable to oxidative stress-induced senescence, which may lead to abnormal aging or aging-related disorders. Therefore, strategies that can ameliorate oxidative stress-induced senescence are expected to protect skin from damage, holding the promise of treating skin diseases in the clinic. This study aims to investigate whether caffeine, a well-known purine alkaloid, is able to prevent skin from oxidative stress-induced senescence, and to explore the underlying molecular mechanisms. Methods: A free radical inducer 2,2'-Azobis (2-amidinopropane) dihydrochloride (AAPH) was used to induce oxidative stress and cellular senescence in both transformed skin cells and in normal human epidermal keratinocytes (NHEKs). Ultraviolet (UV) irradiation was established as the in vivo oxidative stress model in mouse skin tissues. Cellular senescence was determined by SA β-galactosidase staining, immunofluorescence and western blotting. Activation of autophagy was confirmed by western blotting, immunofluorescence, and transmission electron microscopy. Reactive oxygen species (ROS) detection by commercial kits, gene knockdown by RNA interference (RNAi) and receptor activation/inactivation by agonist/antagonist treatment were applied in mechanistic experiments. Results: We report that AAPH induced senescence in both transformed skin cells and in NHEKs. Similarly, UV irradiation induced senescence in mouse skin tissues. Remarkably, low dose of caffeine (<10 μM) suppressed cellular senescence and skin damage induced by AAPH or UV. Mechanistically, caffeine facilitated the elimination of ROS by activating autophagy. Using a combination of RNAi and chemical treatment, we demonstrate that caffeine activates autophagy through a series of sequential events, starting from the inhibition of its primary cellular target adenosine A2a receptor (A2AR) to an increase in the protein level of Sirtuin 3 (SIRT3) and to the activation of 5' adenosine monophosphate-activated protein kinase (AMPK). Oral administration of caffeine increased the protein level of SIRT3, induced autophagy, and reduced senescence and tissue damage in UV-irradiated mouse skin. On the other hand, co-administration with autophagy inhibitors attenuated the protective effect of caffeine on UV-induced skin damage in mice. Conclusion: The results reveal that caffeine protects skin from oxidative stress-induced senescence through activating the A2AR/SIRT3/AMPK-mediated autophagy. Our study not only demonstrated the beneficial effect of caffeine using both in vitro and in vivo models, but also systematically investigated the underlying molecular mechanisms. These discoveries implicate the potential of caffeine in the protection of skin disease.
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Project description:Gram-negative sepsis carries high morbidity and mortality, especially when complicated by acute kidney injury (AKI). The mechanisms of AKI in sepsis remain poorly understood. Here we used intravital two-photon fluorescence microscopy to investigate the possibility of direct interactions between filtered endotoxin and tubular cells as a possible mechanism of AKI in sepsis. Using wild-type (WT), TLR4-knockout, and bone marrow chimeric mice, we found that endotoxin is readily filtered and internalized by S1 proximal tubules through local TLR4 receptors and through fluid-phase endocytosis. Only receptor-mediated interactions between endotoxin and S1 caused oxidative stress in neighboring S2 tubules. Despite significant endotoxin uptake, S1 segments showed no oxidative stress, possibly as a result of the upregulation of cytoprotective heme oxygenase-1 and sirtuin-1 (SIRT1). Conversely, S2 segments did not upregulate SIRT1 and exhibited severe structural and functional peroxisomal damage. Taken together, these data suggest that the S1 segment acts as a sensor of filtered endotoxin, which it takes up. Although this may limit the amount of endotoxin in the systemic circulation and the kidney, it results in severe secondary damage to the neighboring S2 segments.
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Project description:Elevated reactive oxygen species (ROS) in type 2 diabetes cause cellular damage in many organs. Recently, the new class of glucose-lowering agents, SGLT-2 inhibitors, have been shown to reduce the risk of developing diabetic complications; however, the mechanisms of such beneficial effect are largely unknown. Here we aimed to investigate the effects of dapagliflozin on cell proliferation and cell death under oxidative stress conditions and explore its underlying mechanisms. Human proximal tubular cells (HK-2) were used. Cell growth and death were monitored by cell counting, water-soluble tetrazolium-1 (WST-1) and lactate dehydrogenase (LDH) assays, and flow cytometry. The cytosolic and mitochondrial (ROS) production was measured using fluorescent probes (H2DCFDA and MitoSOX) under normal and oxidative stress conditions mimicked by addition of H2O2. Intracellular Ca2+ dynamics was monitored by FlexStation 3 using cell-permeable Ca2+ dye Fura-PE3/AM. Dapagliflozin (0.1-10 μM) had no effect on HK-2 cell proliferation under normal conditions, but an inhibitory effect was seen at an extreme high concentration (100 μM). However, dapagliflozin at 0.1 to 5 μM showed remarkable protective effects against H2O2-induced cell injury via increasing the viable cell number at phase G0/G1. The elevated cytosolic and mitochondrial ROS under oxidative stress was significantly decreased by dapagliflozin. Dapagliflozin increased the basal intracellular [Ca2+]i in proximal tubular cells, but did not affect calcium release from endoplasmic reticulum and store-operated Ca2+ entry. The H2O2-sensitive TRPM2 channel seemed to be involved in the Ca2+ dynamics regulated by dapagliflozin. However, dapagliflozin had no direct effects on ORAI1, ORAI3, TRPC4 and TRPC5 channels. Our results suggest that dapagliflozin shows anti-oxidative properties by reducing cytosolic and mitochondrial ROS production and altering Ca2+ dynamics, and thus exerts its protective effects against cell damage under oxidative stress environment.
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