Protect Effects And The Underlying Mechanisms Of Idebenone Against Vasculal Endothelial Cells Apoptosis Induced By Oxidized Low Density Lipoprotein

Atherosclerosis is a chronic vascular disorder and results from multifaceted atherogenic mechanisms. The early stages of the atherosclerotic process are initiated by accumulation of oxidized low-density lipoprotein (oxLDL) and damage to the structure or function of the endothelium. Moreover, previous studies have shown that the proapoptotic effects of oxLDL-induced ROS generation in endothelial cells involve the disturbance of mitochondrial membrane permeability followed by cytochrome c release and subsequently the activation of executioner caspases. Increased oxidative stress and reduced ATP synthesis are believed to sensitize endothelial cells to apoptosis, which play an important role in atherosclerosis.endothelial cell damage induced by oxLDL is mediated by the mitochondrial-dependent apoptotic pathway. Furthermore, impaired mitochondrial function in turn enhances reactive oxidative species (ROS) production and oxidative stress in atherogenesis. Recent studies have indicated that the risk factors of cardiovascular and cerebrovascular diseases are closely related to the increase of reactive oxygen species, and can lead to mitochondrial damage and dysfunction of the heart and brain blood vessels. In summary, mitochondria are considered to be an important link in connection with risk factors, oxidative stress and endothelial cell dysfunction, and also the important factors to promote the formation and development of atherosclerotic lesions. As a result, antioxidant supplementation or mitochondria-targeted drug may be a plausible strategy to prevent atherosclerotic diseases by directly quenching excessive ROS and improving mitochondrial function.Coenzyme Q10 is a part of the mitochondrial respiratory chain and can accept the 2 electrons from complex Ⅰ or complex Ⅱof the mitochondria and then transfer electrons to the complex Ⅲ. CoQ10 can act as the potent antioxidant and donate electrons to complex Ⅲ of the ETC. There are the two forms of oxidation and reduction of quinon, the two forms in the cell can be changed to each other, which is the basis of the electron transport.Idebenone, a synthetic quinone with similarities to CoQ10, shares its quinone moiety with CoQ10, but at the same time differs from CoQ10 by the presence of a much shorter, less lipophilic tail. Based on their partial structural relatedness, Idebenone shares the ability to act as potent antioxidants and to donate electrons to complex Ⅲof the ETC. Idebenone is more easily through through biological membranes and blood brain barrier. Idebenone is reduced at the hydrophobic quinone-binding site within complex Ⅰ like CoQ but exhibits very slow dissociation. Consequently it acts as a competitive substrate that impairs endogenous CoQ function without substituting for its electron transfer function to complex Ⅲ.Idebenone binds a second quinone -binding site within complex Ⅰ that overlaps with the NADH binding site and flavin mononucleotide (FMN) moiety. Due to its high lipophilicity, endogenous CoQ is incapable of binding this "non-physiological" hydrophilic quinone-binding site. However, idebenone, which is less lipophilic than CoQ, is reduced by the complex Ⅰ flavin in the hydrophilic site to form an unstable semiquinone that generates superoxide. Idebenone has been investigated for its potential treatment for a variety of diseases like mitochondrial diseases, Friedreich’s ataxia, as well as Alzheimer’s diseases. The antioxidant function of idebenone is critically dependent on two-electron reduction to idebenol without the creation of unstable intermediates. Multiple studies have detected that idebenone has the effect of inhibition of complex Ⅰ, however, idebenone has the ability to act as a potent antioxidant and donate electrons to complex Ⅲ of the electron transport chain (ETC) through complex Ⅱ-Ⅲ and G3PDH shuttle and so on. The additional idebenone-dependent metabolic pathways that transfers energy equivalents from the cytosol directly into the mitochondrial respiratory chain, was reported recently. Previous studies investigated that idebenone was also found to inhibit oxidative stress in isolated brain mitochondria, synaptosomes and cells. More recent studies have showed idebenone is activated predominantly in the cytoplasm and promotes different complex Ⅰ-independent metabolic pathways. Upon entering the cell, idebenone is efficiently reduced by the cytoplasmic enzyme NADH-quinone oxidoreductase 1 (NQO1) as part of the cellular response to detoxify quinones and to prevent the production of ROS. In this pathway, the increase expression of NQO1 plays the improtant role in the potential therapy of idebenone. NQO1 is regulated by the oxidative stress and Nrf2/ARE signalling pathway. Nrf2/ARE signalling pathway can mediate the expression of anti atherosclerosis gene in vitro, and play the important role in the anti-inflammation, anti-apoptosis and anti-atherosclerosis. The occurrence of AS is closely related to oxidative stress, apoptosis, mitochondrial function and Nrf2/ARE signaling pathway. Therefore, the possible of treatment or prevention of atherosclerosis with idebenone is a major concern, particularly in view of the fact that idebenone is already used in clinical trial without reports of serious adverse event. However, few studies have been conducted regarding idebenone’s anti-atherosclerotic effects and the underlying mechanisms.Glycogen synthase kinase 3(3 (GSK3β) is a multifunctional serine/threonine (Ser/Thr) kinase and β-catenin is a key downstream molecule of the GSK3 signaling pathway. GSK3P and β-catenin play the important roles in regulating many physiological and pathological processes such as gene expression, cell cycle, cellular structure, function, apoptosis and survival. GSK3P has two forms of phosphorylation, β-GSK3 (Tyr216) and P-GSK3 (Ser9). When Ser9 sites were phosphorylated, GSK3 activity was inhibited.GSK3β is a pro-apoptotic kinase and the inhibitors of GSK3β, such as LiCl, can attenuate mitochondrial dysfunction and cellular apoptosis Recently studies have showed that pharmacological inhibition of GSK3P, such as lithium chloride (LiCl), is thought to block the opening of the mitochondrial permeability transition pore (mPTP) and attenuate cytochrome c release from mitochondria in cardiomyocytes. At the same time, GSK3P can inhibit the Nrf2/ARE signaling pathway, and promote cell apoptosis. GSK3P inactivation inhibites phosphorylated β-catenin and improves the accumulation of β-catenin in the nuclear, β-catenin in nucleus binds with TCF/LEF transcription factor, and further activates the downstream target genes, this process can improve the cell function and inhibit cell apoptosis. These evidences have suggested that GSK3P and β-catenin proteins are likely to play an important role in atherosclerosis and idebenone may prevent atherosclerosis by modified GSK3β and β-catenin signals.Based on the evidences, we chose the in vitro experimental model of oxLDL-induced human umbilical vein endothelial cells (HUVECs) injury to mimic the oxidative endothelial injury observed during atherogenesis. Our research aimed to investigate the efficacy of idebenone and the underlying mechanisms of its protection against oxLDL-induced mitochondrial dysfunction in vascular endothelial cells. At the same time, we evaluated the effects of GSK3β and β-catenin signaling proteins in the proess of preventing and treating atherosclerosis. This research was divided into two parts.PART IProtect effects of idebenone against vascular endothelial cells apoptosis induced by oxidized low density lipoproteinObjectiveTo investigate the protect effects of idebenone against vascular endothelial cells apoptosis induced by oxidized low density lipoprotein.Methods1. Cell culture and identification.These experiments were approved by the Research Ethics Committee of the Qilu Hospital of Shandong University. After gaining written consent from every volunteer, human umbilical cords were collected from healthy mothers following deliveries. HUVECs were isolated freshly from human umbilical veins, cultured, identifed and were used for the subsequent experiments between passages 3 and 5.2. To study the effect of oxLDL and idebenone on human umbilical vein endothelial cell. HUVECs were randomly divided into control, 100μg/ml oxLDL,0.05μM idebenone+100μg/ml oxLDL,0.1 μM idebenone+100μg/ml oxLDL, 0.2μM idebenone+100μg/ml oxLDL groups. Subsequently, the morphological changes were observed and recorded randomly using an inverted microscope. HUVECs in each group were incubated with CCK-8 and analysised of cellular proliferation with an enzyme-linked immunosorbent assay (ELISA) reader. HUVECs in each treatment group were stained with Hoechst 33258 and investigated by immunofluorescence microscope. With Annexin V-FITC/PI staining, the apoptotic cell rates upon oxLDL in the presence or absence of idebenone were quantified by using flow cytometric analysis. Proteins were extracted from whole cells,which were used to detect the expression of Bcl-2 Bax、cleaved caspase-3 by Western blotting.Results1. Cell identification.HUVECs identities were confirmed by the presence of CD31 and factor VIII related antigen by using immunofluorescence.2. Idebenone protected endothelial cells against oxLDL-induced injury. The morphological changes of HUVECs were observed by the inverted microscopy. Cellular viability of HUVECs was examinated by the CCK-8.24 h incubation of HUVECs with 100μg/ml oxLDL induced significant increase of cell retraction, suspension and cell shrinkage and significant decrease of cellular viability in comparison with that of control group (65.85±4.25%)(P<0.01).2 h pretreatment with idebenone(0.05μM,0.1 μM and 0.2μM) prior to 100μg/ml oxLDL markedly increased cellular viability(75.68±2.59%,78.24±2.12%,81.48±2.39%) and decreased apoptotic morphological changes (P<0.05). OxLDL induced cell retraction, suspension and cell shrinkage. Idebenone can obviously improve the morphological changes and cellular viability of HUVECs induced by oxLDL.3. Idebenone protected endothelial cells against oxLDL-induced apoptosis. The protective effects of idebenone were further confirmed with Hoechst 33258 staining.100μg/ml oxLDL induced chromatin apoptotic, morphological changes including nuclear segmentation, well-distributed deep blue fluorescence and chromatin condensation by immunofluorescence microscope. Idebenone pretreatment significantly improved the chromatin morphological changes of apoptosis induced by oxLDL.With Annexin V-FITC/PI staining, the apoptotic cell rates upon oxLDL in the presence or absence of idebenone were quantified by using flow cytometric analysis. The percentage of apoptotic cells exposed to oxLDL was markedly higher (27.18 ± 2.76%) than that of control HUVECs(5.37±0.69%)(P<0.01). However, the percentage of apoptotic cells was significantly suppressed in HUVECs pretreated with idebenone(18.37±2.32%,16.76±2.65%,13.52±2.43%)(P<0.05).4. Idebenone inhibited oxLDL-mediated HUVECs apoptosis by modulating Bcl-2 family proteins and controlling caspase-3 activation.Compared with control group, the decrease level of Bcl-2 protein and the increase level of Bax protein were markedly investigated in 100μg/ml oxLDL group (P<0.01). Compared with 100μg/ml oxLDL group, pretreatment with idebenone(0.05μM、0.1μM、0.2μM) suppressed the down-regulation of Bcl-2 and up-regulation of Bax in HUVECs exposed to oxLDL(P<0.05). OxLDL significantly increased the protein levels of cleaved caspase-3 in cultured endothelial cells, which was suppressed by preincubation with idebenone.ConclusionsIdebenone at suitable concentrations significantly prevented oxLDL-induced endothelial dysfunction. The underlying mechanisms of idebenone included inhibition of oxidative damage, modulation Bcl-2 family proteins and caspase-3 activation in HUVECs exposed to oxLDL.PART IIThe underlying mechanisms of idebenone against vascular endothelial cells apoptosis induced by oxidized low density lipoproteinObjectiveTo investigate the underlying mechanisms of idebenone against vascular endothelial cells apoptosis induced by oxidized low density lipoprotein and to evaluate the effects of GSK3β and β-catenin signaling proteins in the proess.Methods1. To investigate the effects of idebenone on mitochondrial function. HUVECs were randomly divided into control, 100μg/ml oxLDL,0.05μM idebenone+100μg/ml oxLDL, 0.1μM idebenone+100μg/ml oxLDL,0.2μM idebenone+100μg/ml oxLDL groups. Proteins were extracted from whole cells in each groups and the concentration of MDA and the activities of SOD in the lysates were examined by using thecommerciallymicroscale lipid peroxidation MDA assay kit and SOD assay kit. Mitochondrial membrane potential (MMP, △Ψm) in each groups was assessed with the signal from aggregated and monomeric JC-1 fluorescence by using flow cytometry. Cellular ATP levels of samples were quantified immediately by using luciferase-luciferin assay with the luminometer. Proteins were extracted from cytoplasmic and mitochondrial fractions in each groups. Protein levels of cytochrome c release from mitochondria and endochylema were determined by using western blotting.2. To evaluate the effects of GSK3β and β-catenin signaling proteins in the proess.HUVECs were randomly divided into control, 100μg/ml oxLDL,0.2μM idebenone+100μg/ml oxLDL groups, lOmM LiCL+100μg/ml oxLDL group. Total, cytosolic and nuclear proteins were prepared in each groups, and western blotting were performed with antibodies against GSK3β, phosphorylated GSK3β (P-GSK3β), phosphorylated β-catenin (P-β-catenin) and β-catenin. GAPDH、 β-actin and Histone H2A were used as loading controls.Results1. Idebenone significantly suppressed the oxLDL-induced up-regulation of MDA, and down-regulation of SOD activities.Incubation of HUVECs with 100μg/mL oxLDL for 24 h caused the significant increase of MDA content (3.34±0.23 nmol/mg protein vs 1.48±0.14 nmol/mg protein)and the marked decrease of SOD activity(2.12±0.18 unit/mg protein vs 4.36±0.31 unit/mg protein), compared with those of cells control group (P<0.01). Pre-incubation with idebenone(0.05μM,0.1 μM and 0.2μM) for 2 h markedly attenuated the increase in MDA content(2.72±0.28 nmol/mg protein,2.41±0.15 nmol/mg protein,2.26±0.13 nmol/mg protein) and decreased SOD activities(3.05±0.25 unit/mg protein,3.33±0.23 unit/mg protein,3.59±0.21 unit/mg protein) in response to oxLDL respectively (P<0.01).Therfore, idebenone significantly suppressed the oxLDL-induced up-regulation of MDA, and down-regulation of SOD activities.2. Idebenone inhibited mitochondrial dysfunction and cellular apoptosis induced by oxLDL through the mitochondrial-dependent apoptotic pathway.The fluorescence ratio of aggregated JC-1 and monomeric JC-1 in cells treated with oxLDL decreased when compared with that of control group (P<0.01) and idebenone pretreatment inhibited the disruption of △Ψm induced by oxLDL by using flow cytometry (P<0.05)OxLDL significantly inhibited the basal release of ATP by mitochondria in HUVECs in comparison with that of control group((8.83±1.710μmol/mg protein vs 23.82±3.43μmol/mg protein) (P<0.01). Pretreatment with idebenone markedly attenuated the decrease of ATP release induced by oxLDL(15.16±2.19μmol/mg protein,16.37±3.51μmol/mg protein,17.12±2.06μmol/mg protein) (P<0.05).The proapoptotic effects of oxLDL-induced ROS generation in endothelial cells involve the disturbance of mitochondrial membrane permeability followed by cytochrome c release and subsequently the activation of executioner caspases. Pretreatment with idebenone attenuated oxLDL-induced cytochrome c levels in cytoplasm and reduced the release of cytochrome c from mitochondria into the cytoplasm in HUVECs by using western blotting.In summary, idebenone reduced the effects of oxLDL on mitochondrial membrane potential and increased ATP production in HUVECs. Then, idebenone reduced the release of cytochrome c from mitochondria and attenuated oxLDL-induced cytochrome c levels in cytoplasm. Thus, pro-incubation with idebenone inhibited mitochondrial dysfunction and cellular apoptosis induced by oxLDL through the mitochondrial-dependent apoptotic pathway and can be potential mitochondria-targeted drugs for the treatment of atherosclerosis.3.Idebenone protected against oxLDL-mediated cytotoxicity via GSK3β/β-catenin signaling pathways.100μg/ml oxLDL resulted in a significantly decrease in the levels of phosphorylated GSK3β at Ser9, which was accompanied by a significant increase in the amount of phosphorylated β-catenin and a decrease in the nuclear accumulation of β-catenin(.P<0.05). Furthermore, the levels of GSK3β phosphorylation at Ser9 and the nuclear accumulation of β-catenin of cells, pretreated with 0.2μM idebenone or lOmM LiCl for 2 h, were significantly increased, which was associated with a significant decrease in the amount of phosphorylated β-catenin(P<0.05). The total protein levels of GSK3β and β-catenin were kept unchanged in each group(P>0.05).ConclusionsIdebenone supplementation could reverse the oxidative stress-induced suppression of antioxidant enzyme activity and reduce the effects of oxLDL on mitochondrial membrane potential and increase ATP production in HUVECs. Idebenone inhibited mitochondrial dysfunction induced by oxLDL through the mitochondrial-dependent apoptotic pathway. Idebenone resulted in the phosphorylation of GSK3β at Ser9 and the nuclear accumulation of P-catenin of cells, which was associated with significantly the decrease of phosphorylated β-catenin. In short,pro-incubation with idebenone inhibited mitochondrial dysfunction and cellular apoptosis induced by oxLDL through the mitochondrial-dependent apoptotic pathway and GSK3β/β-catenin signaling pathway s.Idebenone can be potential mitochondria-targeted drugs and a promising agent in the treatment or prevention of atherosclerosis.