Madecassic Acid protects against hypoxia-induced oxidative stress in retinal microvascular endothelial cells via ROS-mediated endoplasmic reticulum stress.

Madecassic acid (MA) is an abundant triterpenoid in Centella asiatica (L.) Urban. (Apiaceae) that has been used as a wound-healing, anti-inflammatory and anti-cancer agent. Up to now, the effects of MA against oxidative stress remain unclear. In this study, we investigated the effect of MA and its mechanisms on hypoxia-induced human Retinal Microvascular Endothelial Cells (hRMECs). hRMECs were pre-treated with different concentrations of MA (0-50μM) for 30min before being incubated under hypoxia condition (37°C, 5% CO2 and 95% N2). Cell apoptosis was evaluated with MTT assay and TUNEL staining, and the expression of apoptosis- and endoplasmic reticulum (ER) stress-related molecules was assessed with western blotting and RT-PCR analysis. Intracellular ROS level was evaluated using DCFH-DA. Intracellular malondialdehyde (MDA), dehydrogenase (LDH), glutathione peroxidase (GSH-PX) and superoxide dismutase (SOD) were evaluated using related Kits. Activating transcription factor 4 (ATF4) nuclear translocation was assessed with western blotting analysis and immunofluorescence staining. MA significantly reduced oxidative stress in hypoxia-induced hRMECs, as shown by increased cell viability, SOD and GSH-PX leakage, decreased TUNEL- and ROS-positive cell ratio, LDH and MDA leakage, caspase-3 and -9 activity, and Bax/Bcl-2 ratio. In addition, MA also attenuated hypoxia-induced ER stress in hRMECs, as shown by reduced mRNA levels of glucose-regulated protein 78 (GRP78), C/EBP homologous transcription factor (CHOP), protein levels of cleaved activating transcription factor 6 (ATF6) and inositol-requiring kinase/endonuclease 1 alpha (IRE1α), phosphorylation of pancreatic ER stress kinase (PERK) and eukaryotic initiation factor 2 alpha (eIF2α), cleaved caspase-12 and ATF4 translocation to nucleus. The current study indicated that the regulation of oxidative stress and ER stress by MA would be a promising therapy to reverse the process and development of hypoxia-induced hRMECs dysfunction.

Yang B ，Xu Y ，Hu Y ，Luo Y ，Lu X ，Tsui CK ，Lu L ，Liang X ... -  《-》

Antioxidative effects of polypyrimidine tract-binding protein-associated splicing factor against pathological retinal angiogenesis through promotion of mitochondrial function.

Dong L ，Lin T ，Li W ，Hong Y ，Ren X ，Ke Y ，Zhang X ，Li X ... -  《-》

Isoquercetin ameliorates tunicamycin-induced apoptosis in rat dorsal root ganglion neurons via suppressing ROS-dependent endoplasmic reticulum stress.

Recent studies showed that Isoquercetin (Iso), a novel extracts of plants and fruits could protect neuronal cells from neurotoxicity and neuro-inflammation. However, its role in acute spinal cord injury (ASCI) have not been elucidated. In the present study, we investigated whether Iso could prevent Tunicamycin (TUN)-induced rat dorsal root ganglion (DRG) neurons from apoptosis and endoplasmic reticulum (ER) stress. DRG neurons were pre-treated with different doses of Iso for 24h (h) and then were stimulated with TUN (0.75μg/ml) for 24h. The cytotoxic effects and apoptosis were detected by MTT assay and TUNEL staining. The protein and mRNA expression levels were detected by Real Time PCR and Western blot, respectively. The localization of cleaved caspase-12 was evaluated by immunofluorescent staining and Western blot. The activation of caspase were measured by colorimetric assays and Western blot. Lactate Dehydrogenase (LDH) and Malondialdehyde (MDA) leakage were detected by the LDH or MDA Detection Kit PLUS. Iso protected TUN-associated DRG neurons from being damaged by cytotoxicity and apoptosis in a dose-dependent manner. Increased LDH and MDA leakage and proportion of TUNEL-positive cells, activation of caspase-3 and -9, increased Bcl-2 Assaciated X protein (Bax)/B cell lymphoma/lewkmia-2 (Bcl-2) ratio and mRNA levels of p53 Upregulated Modulator Of Apoptosis (PUMA) and DP5, and mitochondrial Cytochrome c release. Additionally, Iso down-regulated mRNA levels of ER stress genes, such as glucose-regulated protein 78 (GRP78), GRP94, C/EBP homologous protein (CHOP), and cleaved caspase-12 in TUN-induced DRG neurons. Moreover, Iso blocked the activation of three key branches of unfolded protein response in DRG neurons, including phosphorylation of pancreatic ER stress kinase (PERK), eukaryotic initiation factor 2 alpha (eIF2α), inositol-requiring enzyme 1α (IRE1α), and transcription factor 6 (ATF6). Collectively, Iso prevented TUN-induced DRG neurons apoptosis by inhibiting mitochondrial and ER stress-associated apoptotic signaling cascades, suggesting that Iso possessed neuro-protective ability which could be beneficial in the prevention of ASCI.

Lu T ，Zhang C ，Chai M ，An Y ... -  《-》

Icariin protects vascular endothelial cells from oxidative stress through inhibiting endoplasmic reticulum stress.

To investigate the protective effect and underlying mechanism(s) of icariin (ICA) in preventing hydrogen peroxide (H2O2)-induced vascular endothelial cell injury via endoplasmic reticulum stress (ERS). To study the effects of ICA on H2O2-induced damage, we used the cell counting kit-8 assay to detect cell viability and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay to determine cell adhesion and apoptosis, respectively. Spectrophotometry and enzyme-linked immunosorbent assay were used to measure the expression levels of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Subsequently, glucose-regulated protein 78 (GRP78), activating transcription factor-4 (ATF4) and eukaryotic initiation factor-2α (eIF2α) were detected using Western blotting. In human umbilical vein endothelial cells, different concentrations of ICA exhibited multiple effects, including reduced H2O2 damage, improved cell viability and adhesion, reduced cell apoptosis and increased SOD and GSH-Px activity. Among the ICA concentrations used, only the H2O2 + 100 μmol/L ICA group had significant differences compared to the H2O2 group. ERS activators H2O2 and dl-dithiothreitol (DTT) significantly increased GRP78, ATF4 and eIF2α expressions, decreased cell activity and reduced SOD and GSH-Px activity. In contrast, the H2O2 + 100 μmol/L ICA and H2O2 + 100 μmol/L ICA + DTT groups had significant inhibitory effects on the expressions of GRP78, ATF4 and eIF2α proteins, showing enhanced cell viability and SOD and GSH-Px activity. The results showed the dose-dependent effects of ICA against H2O2-induced injury in vascular endothelial cells. The inhibition of GRP78, ATF4 and eIF2α protein expressions in the ERS, and the subsequent alleviation of oxidative stress damage, might be the molecular mechanism.

Wang FY ，Jia J ，Song HH ，Jia CM ，Chen CB ，Ma J ... -  《-》

Alpha-lipoic acid defends homocysteine-induced endoplasmic reticulum and oxidative stress in HAECs.

Oxidative stress and endoplasmic reticulum (ER) stress play vital roles in the development of atherosclerosis. And hyperhomocysteinemia (HHCY) has been recognized as an independent risk factor for this process. Alpha-lipoic acid, a disulphide-containing compound, can scavenge reactive oxygen species, inhibit the formation of free radicals and chelate metal to maintain the homeostasis in the cells. This study aimed to determine the protecting effects of Alpha-lipoic acid (ALA) on homocysteine (HCY) induced injuries to human aortic endothelial cells (HAECs) and uncover the underlying mechanisms. HAECs were treated with ALA in the presence/absence of HCY. The mechanism of ALA against HCY-induced cell injury was evaluated using Western blotting, real-time RT-PCR. Reactive oxygen Species (ROS) was detected by flow cytometry analysis. Mitochondrial membrane potential in HCY-treated HAECs was measured by Rhodamine 123 staining, and the samples were observed by confocal laser scanning microscopy. ALA suppressed the HCY-stimulated ROS generation, as well as the NF-κB transcriptional activation, and ICAM-1, VCAM-1 expression. ALA also elevated the bcl-2 and reduced caspase3, 9 expressions in the HCY- induced HAECs. Simultaneously, ALA could inhibit activation of ER stress-associated sensors GRP78, IRE1α, ATF6, P-PERK, P-eIF2α, CHOP and ATF4 induced by HCY. In addition, using GSH inhibitor, we proved ALA reduced the expressions of GRP78, ATF4 and IRE1α by generating GSH. ALA ameliorated HCY-induced ER stress and oxidation then reduced cells apoptosis and inflammation. These results suggested ALA played a key role in regulating ER homeostasis in atherosclerosis.

Hu H ，Wang C ，Jin Y ，Meng Q ，Liu Q ，Liu K ，Sun H ... -  《-》