bims-mideyd 2023-03-19 papers

Issue of 2023‒03‒19
two papers selected by
Rajalekshmy “Raji” Shyam, Indiana University Bloomington

J Ginseng Res. 2023 Mar;47(2): 311-318

Nrf2-mediated activation of HO-1 is required in the blocking effect of compound K, a ginseng saponin metabolite, against oxidative stress damage in ARPE-19 human retinal pigment epithelial cells.

Cheol Park, Hee-Jae Cha, Kyoung-Seob Song, Heui-Soo Kim, EunJin Bang, Hyesook Lee, Cheng-Yun Jin, Gi-Young Kim, Yung Hyun Choi.

Background: The beneficial effects of compound K (CK) on different chronic diseases have been shown to be at least related to antioxidant action. Nevertheless, since its antioxidant activity in human retinal pigment epithelial (RPE) cells is still unknown, here we investigated whether CK alleviates oxidative stress-stimulated damage in RPE ARPE-19 cells.Methods: The cytoprotective consequence of CK in hydrogen peroxide (H2O2)-treated cells was evaluated by cell viability, DNA damage, and apoptosis assays. Fluorescence analysis and immunoblotting were performed to investigate the inhibitory action of CK on reactive oxygen species (ROS) production and mitochondrial dysfunction.
Results: H2O2-promoted cytotoxicity, oxidative stress, DNA damage, mitochondrial impairment, and apoptosis were significantly attenuated by CK in ARPE-19 cells. Furthermore, nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation level and its shuttling to the nucleus were increased, which was correlated with upregulated activation of heme oxygenase-1 (HO-1). However, zinc protoporphyrin, a blocker of HO-1, significantly abrogated the preventive action of CK in H2O2-treated ARPE-19 cells.
Conclusion: This study indicates that activation of Nrf2/HO-1 signaling by CK plays an important role in rescuing ARPE-19 cells from oxidative cellular damage.

Keywords: ARPE-19 cells; Apoptosis; Compound K; Nrf2/HO-1; Oxidative stress

DOI: https://doi.org/10.1016/j.jgr.2022.09.007

bioRxiv. 2023 Mar 02. pii: 2023.03.01.530623. [Epub ahead of print]

Medium depth influences O 2 availability and metabolism in cultured RPE cells.

Daniel T Hass, Qitao Zhang, Gillian Autterson, Richard Bryan, James B Hurley, Jason Ml Miller.

Purpose: RPE oxidative metabolism is critical for normal retinal function and is often studied in cell culture systems. Here, we show that conventional culture media volumes dramatically impact O 2 availability, limiting oxidative metabolism. We suggest optimal conditions to ensure cultured RPE is in a normoxic environment permissive to oxidative metabolism.Methods: We altered the availability of O 2 to human primary RPE cultures directly via a hypoxia chamber or indirectly via the amount of medium over cells. We measured oxygen consumption rates (OCR), glucose consumption, lactate production, 13 C 6 -glucose flux, hypoxia inducible factor (HIF-1α) stability, intracellular lipid droplets after a lipid challenge, trans-epithelial electrical resistance, cell morphology, and pigmentation.
Results: Medium volumes commonly employed during RPE culture limit diffusion of O 2 to cells, triggering hypoxia, activating HIF-1α, limiting OCR, and dramatically altering cell metabolism, with only minor effects on typical markers of RPE health. Media volume effects on O 2 availability decrease acetyl-CoA utilization, increase glycolysis, and alter the size and number of intracellular lipid droplets under lipid-rich conditions.
Conclusions: Despite having little impact on visible and typical markers of RPE culture health, media volume dramatically affects RPE physiology "under the hood". As RPE-centric diseases like age-related macular degeneration (AMD) involve oxidative metabolism, RPE cultures need to be optimized to study such diseases. We provide guidelines for optimal RPE culture volumes that balance ample nutrient availability from larger media volumes with adequate O 2 availability seen with smaller media volumes.

DOI: https://doi.org/10.1101/2023.03.01.530623