GB was associated with protection of RPEs against H₂O₂-induced oxidative stress and apoptosis, and the findings suggest that AMPK/Nrf2 signaling may contribute to the cytoprotective actions of GB.
Abstract
Objective To investigate whether Ginkgolide B (GB) protects retinal pigment epithelial cells (RPEs) from hydrogen peroxide (H2O2)-induced oxidative stress and apoptosis, and to explore the potential involvement of the adenosine monophosphate-activated protein kinase α (AMPKα)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. Methods Human RPEs were divided into the following groups: control, model (200 μmol/L H₂O₂ for 24 h), GB + H₂O₂, GB + H₂O₂ + Compound C (an AMPK inhibitor), H₂O₂ + Compound C, and GB alone. Cell viability, apoptosis, antioxidant parameters, and AMPKα/Nrf2 expression were assessed using the Cell Counting Kit-8 (CCK-8) assay, flow cytometry, colorimetric biochemical assays, transmission electron microscopy (TEM), quantitative polymerase chain reaction (PCR), and Western blot. Results TEM showed that GB attenuated H₂O₂-induced mitochondrial swelling and cristae disruption, which were reversed by Compound C. Compared with the model group, the GB + H₂O₂ group exhibited increased cell viability, decreased apoptosis, elevated activities of catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD), as well as increased glutathione (GSH) content, and reduced malondialdehyde (MDA) (all p < 0.05), though protein carbonyl (PC) levels showed no significant amelioration. All protective effects were attenuated by Compound C. In the GB-alone group, the percentage of apoptotic cells, Nrf2 mRNA expression, and all other parameters did not differ significantly from those in the control group. Conclusion GB was associated with protection of RPEs against H₂O₂-induced oxidative stress and apoptosis. Because these protective effects were attenuated by pharmacological inhibition of AMPK, the findings suggest that AMPK/Nrf2 signaling may contribute to the cytoprotective actions of GB. However, additional mechanistic studies are required to confirm the involvement of this pathway.
AIM
To investigate the modulator action of resveratrol (RSV) through the suppression of transient receptor potential ankyrin 1 (TRPA1) on the hypoxia (HPO)-caused oxidative degeneration and apoptosis in human retinal pigment epithelial cells (ARPE19).
METHODS
The control (CNT), RSV (50 µmol/L for 24h), HPO (200 µmol/L CoCl2 for 24h), and HPO+RSV groups were induced in the ARPE19. Apoptosis, caspases (caspases 3, 8, and 9), oxidants [lipid peroxidation, oxygen free radical (OFR), and dysfunction of mitochondrial membrane], antioxidants (glutathione and glutathione peroxidase), Ca2+, Zn2+ levels, live cell number and percentage were all measured in the cells of four groups.
RESULTS
The HPO increased Ca2+ and Zn2+ fluorescence intensity in the cells. It also upregulated the apoptosis, caspases, and oxidant indicators while downregulating the antioxidants, and the count of live cells. TRPA1 agonist (cinnamonaldehyde) further upregulated these indicators (all P<0.05). When the HPO-induced increase in TRPA1 activation was treated with RSV and the TRPA1 antagonist (AP18), cell viability and antioxidants rose while the oxidant and apoptotic indicators decreased due to TRPA1 suppression (P<0.05).
CONCLUSION
HPO exposure through TRPA1-mediated Ca2+ signaling, induces mitochondrial oxidative cell cytotoxicity and death, which may offer a treatment option for HPO-induced retinal disorders linked to increased OFR and Ca2+ influx.
Mehmet Argun, Mustafa Nazıroğlu· International Journal of Oph...· 0 citations
AIM
To investigate whether excessive glucocorticoid (GC) exposure disrupts retinal pigment epithelial (RPE) homeostasis through glucocorticoid receptor (GR)-dependent autophagy hyperactivation.
METHODS
Human retinal pigment epithelial cell line (ARPE-19 cells) were exposed to GC (0-100 µmol/L, 48h). GR subcellular localization was analyzed via mitochondrial fractionation and immunofluorescence. Functional assessments included barrier integrity [zonula occludens-1 (ZO-1) immunostaining], phagocytic capacity (FluoSpheres™-labeled photoreceptor outer segment uptake), and ultrastructural analysis (transmission electron microscopy, TEM). Autophagic flux was quantified using mCherry-green fluorescent protein-microtubule-associated protein 1A/1B-light chain 3 (LC3) B reporters. Molecular mechanisms were probed through AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway activity and autophagic flux assessed by LC3-II levels. GR dependency was confirmed using the antagonist RU486, and cell-type specificity was assessed using mouse photoreceptor (661W) cells.
RESULTS
GC induced GR upregulation and mitochondrial translocation in a dose- and time-dependent manner, correlating with pathological autophagy activation. Key findings included: 1) tight junction disruption, evidenced by ZO-1 fragmentation, 2) reduction in phagocytic uptake efficiency, 3) mitochondrial shrinkage and autolysosome accumulation (TEM), 4) altered AMPK/mTOR signaling (p-AMPK up, p-mTOR down) associated with autophagosome formation, 5) elevated LC3-II levels confirming excessive autophagic flux. Critically, all GC-induced effects, including GR upregulation, signaling changes, and increased autophagy, were abolished by GR antagonism with RU486.
CONCLUSION
This study shows that GR-dependent GC exposure induces autophagic flux with ultrastructural evidence suggestive of mitochondria-targeted autophagy and alters AMPK/mTOR signaling, correlating with RPE barrier collapse and phagocytic failure, a possible link to steroid-associated central serous chorioretinopathy (CSC).
Wen-Xue Guan, Yue Guo, Sitong Chen et al.· International Journal of Oph...· 0 citations
BACKGROUND
Ultraviolet B (UVB) irradiation is a primary environmental factor leading to skin oxidative damage and photoaging, primarily by disrupting mitochondrial redox homeostasis in keratinocytes. Dendrobine (DDB), a bioactive sesquiterpene alkaloid derived from the edible and medicinal plant Dendrobium nobile, exhibits diverse pharmacological properties. However, its role and mechanism in protecting against UVB-induced skin damage remain unclear.
MATERIALS AND METHODS
HaCaT human keratinocytes were pretreated with DDB (5-20 μM) prior to UVB irradiation (160 mJ/cm2). Cell viability was assessed using MTT and crystal violet staining assays. Oxidative stress markers, including reactive oxygen species (ROS), 4-hydroxy-2-nonenal (HNE), protein carbonyls, and glutathione (GSH/GSSG) ratio, were measured. Apoptosis was evaluated by flow cytometry, and expression of apoptosis-related proteins (Bax, Bcl-2, cleaved caspase-3, and cleaved caspase-9) and mitochondrial regulators (SIRT3 and SOD2) was analyzed by western blotting. SIRT3 and SOD2 enzymatic activities were determined using commercial kits. Surface plasmon resonance (SPR) and molecular docking were employed to assess direct binding between DDB and SIRT3. Pharmacological inhibition with 3-TYP was used to establish SIRT3 dependency.
RESULTS
DDB significantly rescued HaCaT cells from UVB-induced viability loss in a concentration-dependent manner. DDB attenuated UVB-induced oxidative stress, as evidenced by reduced ROS, HNE, and protein carbonyl levels, and restored the GSH/GSSG ratio. DDB suppressed apoptosis by decreasing Bax/Bcl-2 ratio and cleaved caspase-3/9 levels. Mechanistically, DDB restored UVB-inhibited enzymatic activities of SIRT3 and SOD2 and upregulated their protein expression. SPR analysis revealed that DDB directly binds to SIRT3 with micromolar affinity (KD = 7.603 × 10-6 M). Molecular docking predicted that DDB binds within a distinct pocket of SIRT3 without competing with its substrate peptide. Critically, pharmacological inhibition of SIRT3 with 3-TYP abolished DDB's protective effects against oxidative damage.
CONCLUSION
This study demonstrates that DDB protects keratinocytes from UVB-induced oxidative damage and apoptosis by directly activating the SIRT3-SOD2 axis, thereby restoring mitochondrial redox homeostasis. These findings establish DDB as a promising natural candidate for functional foods or pharmaceuticals aimed at preventing skin photoaging and preserving mitochondrial function.
Jing Xu, Titi Liu, Ziyan Yang et al.· Biochemical and Biophysical...· 0 citations
OBJECTIVE
To investigate the protective effects of dihydroquercetin (DHQ) on mitochondrial function in primary hepatocytes from patients with chronic liver failure (CLF) and discuss the underlying molecular mechanism.
METHODS
Blood samples from 15 CLF patients and 15 healthy subjects were retrospectively analyzed. Primary hepatocytes were isolated using a modified two-step collagenase perfusion method. The optimal concentration of DHQ (20 μmol/L) was determined using the Cell Counting Kit-8 (CCK-8) assay. Cells were divided into a control group, a plasma group, and a DHQ intervention group. The assessed indicators included cell viability (CCK-8), apoptosis rate (flow cytometry), inflammatory factors (IL-1β, IL-6, TNF-α; ELISA), oxidative stress markers (MDA, SOD, GSH-Px), mitochondrial function (JC-1 staining, mtDNA/nDNA ratio) and related protein expression (Western blot).
RESULTS
DHQ adjunctive therapy reduced serum circulating mitochondrial DNA levels in patients with CLF, increased serum SOD2 and Humanin expression, inhibited activation of the NLRP3-mediated pyroptotic pathway, improved remission rates of decompensated complications, and showed favorable safety and tolerability in patients with end-stage liver diseases. In vitro, hepatocyte viability was markedly decreased in the CLF group, with increased apoptosis, elevated IL-1β, IL-6, TNF-α, and MDA levels, as well as declined SOD and GSH-Px activities. DHQ reduced apoptosis, up-regulated mitochondrial functional markers (SOD2, Humanin), restored JC-1 red/green fluorescence ratios, inhibited NLR family pyrin domain-containing 3 (NLRP3) inflammasome activation, and suppressed the caspase-1/IL-1β axis.
CONCLUSION
DHQ exerts multidimensional protective effects on hepatocytes, providing a novel strategy and clinical evidence for mitochondria-targeted CLF therapy.
Ao Shen, Pengxiang Wang, Bing Wu et al.· American journal of translat...· 0 citations
OBJECTIVES
To investigate whether AS-IV alleviates high glucose (HG)-induced podocyte ferroptosis and whether this effect is associated with the GSK3β/Nrf2/GPX4 axis.
METHODS
Differentiated MPC-5 podocytes were exposed to HG (30 mmol/L) with or without AS-IV, the GSK3β inhibitor LY2090314, or the ferroptosis inhibitor Ferrostatin-1 (Fer-1). An osmotic control (mannitol) was included. Cell viability was quantified with the CCK-8 assay. Levels of reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH) and Fe²⁺ were measured. Lipid peroxidation was detected using C11-BODIPY 581/591. Mitochondrial morphology was examined by transmission electron microscopy and protein expression was analyzed by Western blot.
RESULTS
HG exposure induced podocyte injury, characterized by decreased viability, increased oxidative stress (elevated ROS, MDA and lipid ROS), GSH depletion, iron overload and mitochondrial damage. The osmotic control did not reproduce these effects. AS-IV or Fer-1 significantly attenuated the HG-induced damage and lipid peroxidation. At the molecular level, HG downregulated Nephrin, p-GSK3β (Ser9), Nrf2 and GPX4, while upregulating total GSK3β. AS-IV treatment partially reversed these protein expression changes and produced a protective pattern similar to that of LY2090314.
CONCLUSION
AS-IV alleviates HG-induced podocyte injury, possibly by suppressing ferroptosis and this protective effect may involve modulation of the GSK3β/Nrf2/GPX4 axis. The results offer new insights into DN pathogenesis and support AS-IV as a potential therapeutic candidate.
M. Gao, Hong Jiang, Kangya Lei et al.· Pakistan Journal of Pharmace...· 0 citations
BACKGROUND
Oxidative stress-induced retinal ganglion cell (RGC) apoptosis is a common pathological pathway in optic neuropathies. Breviscapine (BVP) possesses antioxidant properties, but its protective effect against RGC apoptosis remains unclear.
OBJECTIVES
To investigate the antagonistic effect of BVP on tert-butyl hydroperoxide (TBHP)-induced apoptosis in primary rat RGCs and evaluate its dose-dependency.
METHODS
Primary RGCs from 10 Sprague-Dawley (SD) rats were exposed to TBHP (100 μmol/L) and treated with BVP (0, 10, 20, 50 μmol/L). Apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and Annexin V/propidium iodide (Annexin V/PI) flow cytometry. Protein expression of synaptophysin, B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax) and cleaved caspase-3 was measured by Western blotting.
RESULTS
BVP treatment significantly increased synaptophysin and Bcl-2 expression, decreased Bax and cleaved caspase-3 expression and reduced the apoptotic rate in a dose-dependent manner (P < 0.05). The apoptotic rates in the blank control, disease control, low-dose, medium-dose and high-dose BVP groups were (2.12 ± 0.73)%, (64.13 ± 10.14)%, (14.13 ± 4.12)%, (10.23 ± 3.03)% and (6.13 ± 3.12)%, respectively. Flow cytometry confirmed that the total apoptotic rate in the high-dose group decreased to (5.79 ± 0.89)%, approaching the level of the blank control group.
CONCLUSION
BVP inhibits TBHP-induced RGC apoptosis in a dose-dependent manner, possibly by regulating the Bcl-2/Bax/caspase-3 pathway. These findings support its potential as a therapeutic candidate for oxidative stress-associated optic neuropathies.
Xiaoyong Huang, Peng Chen· Pakistan Journal of Pharmace...· 0 citations