NGF Attenuates High Glucose-Induced ER Stress,Preventing Schwann Cell Apoptosis by Activating the PI3K/Akt/GSK3β and ERK1/2 Pathways

Diabetic peripheral neuropathy (DPN) is one of the most common and troublesome complications of diabetes mellitus. It has been demonstrated that nerve growth factor (NGF) exerts a pivotal role in the regulation of neuronal growth and the promotion of DPN recovery. However, the exact molecular mechanisms are not well understood. Recent studies have indicated that as a novel therapeutic target, endoplasmic reticulum (ER) stress participates in the onset and progression of DPN. In the present study, it has been demonstrated that NGF prevents the sciatic nerve from degeneration and demyelination in DPN rats. Thus, RSC 96 cells, which retain the characteristic features of Schwann cells (SCs), were cultured in medium containing 30 mM glucose (high glucose, HG) to mimic SCs in DPN mice. The 50-ng/ml dose of NGF was identified to be the optimal concentration for treating an excessive ER stress level under HG conditions for 24 h. We found that NGF treatment significantly inhibits HG-induced ER stress and subsequently suppresses ER-related apoptosis. Further, NGF administration also activates the upstream signaling pathway of ER stress, PI3K/Akt/GSK3β signaling and ERK1/2 signaling. Co-treatment with the PI3K inhibitor LY294002 or ERK1/2 inhibitor U0126 significantly reverses the protective role of NGF on HG-induced excessive ER stress and subsequent apoptosis. These observations suggest that the neuroprotective role of NGF in DPN is mediated by the inhibition of excessive ER stress via the activation of the PI3K/Akt/GSK3β and ERK1/2 signaling pathways.     

Knockdown of FOXO6 inhibits high glucose–induced oxidative stress and apoptosis in retinal pigment epithelial cells

Oxidative stress and apoptosis in retinal pigment epithelium cells are involved in the pathogenesis of diabetic retinopathy (DR). Forkhead box class O 6 (FOXO6) is a member of the FOXO family that can regulate diabetes-induced oxidative stress. However, the role of FOXO6 in DR has not been clarified. The aim of the present study was to investigate the effects of FOXO6 on high glucose (HG)-induced oxidative stress and apoptosis in ARPE-19 cells. The results showed that FOXO6 was overexpressed in clinical vitreous samples from DR patients and in HG-induced ARPE-19 cells. Knockdown of FOXO6 by small interfeing RNA targeting FOXO6 (si-FOXO6) mitigated the HG-induced the production of reactive oxygen species and malondialdehyde, as well as the inhibition of superoxide dismutase activity. Knockdown of FOXO6 reduced the rate of cell apoptosis in HG-induced ARPE-19 cells. The increase in bax expression and decrease in bcl-2 expression caused by HG stimulation were reversed by si-FOXO6 transfection. Furthermore, knockdown of FOXO6 enhanced the activation of Akt/Nrf2 pathway in HG-stimulated ARPE-19 cells. Taken together, suppression of FOXO6 protects ARPE-19 cells from HG-induced oxidative stress and apoptosis, which is in part mediated by the activation of Akt/Nrf2 pathway.     

Pyrroloquinoline quinine protects HK-2 cells against high glucose-induced oxidative stress and apoptosis through Sirt3 and PI3K/Akt/FoxO3a signaling pathway

High glucose(HG)-induced oxidative stress and apoptosis in renal tubular epithelial cells play an important role in the pathogenesis of diabetic nephropathy. Pyrroloquinoline quinine (PQQ), a new B vitamin, has been demonstrated to be important in antioxidant and anti-apoptotic effects. However, its effect on HK-2?cells and the potential mechanism are rarely investigated. In this study, we investigated that PPQ had protective effects against HG-induced oxidative stress damage and apoptosis in vitro model of diabetic nephropathy. PPQ at 10, 100, 500, 1000 and 10000?nM could protect HK-2?cell from HG-induced inhibition. The protective effects of PQQ were associated with increasing the level of antioxidants(SOD2, CAT), inhibition of reactive oxygen species(ROS) production, and dependent modulation of Bcl-2 family proteins. PPQ significantly upregulated the protein and mRNA expression of Sirtuin3(Sirt3) in HG-induced HK-2?cells. PPQ also reduced apoptosis in HG-induced HK-2?cells by the PI3K/Akt/FoxO3a signal pathway. As down-regulated sirt3 or inhibitory the activity of PI3K/Akt/FoxO3a pathway, the protective effects of PPQ were weakened. In conclusion, our data suggest that PPQ achieves the protective effects through PI3K/Akt/FoxO3a pathway and dependent modulation of Sirt3.     

Effects of High Glucose on Cell Viability and Differentiation in Primary Cultured Schwann Cells: Potential Role of ERK Signaling Pathway

Diabetic peripheral neuropathy (DPN) is one of the most common complications of diabetes mellitus and hyperglycemia is considered to be the major factor in the development and progression of DPN. Because of the contribution of Schwann cells (SCs) to the pathology of DPN, we investigated the effects of high glucose on cell proliferation, apoptosis and differentiation in primary cultured SCs. Cell Counting Kit-8 (CCK-8) assay and Hoechst staining showed that high glucose inhibited SCs proliferation and increased apoptosis ratio in time and concentration dependent manner. Western blot and real-time quantitative PCR analysis revealed that the major myelin proteins and genes expressions including P0, MAG and Krox-20, were downregulated time dependently in SCs exposed to high glucose from 48 to 96 h. To further elucidate the underlying pathogenic mechanisms, we also explored the role of ERK signaling pathway in high glucose induced SC injury, which has been proved to drive demyelination of peripheral nerves. The western blot analysis showed that compared with control group phosphorylation level of ERK was increased by 14.3 % in SCs exposed to high glucose for 72 h (P < 0.01). Using immunocytochemistry analysis, we observed that the ERK specific inhibitor U0126 blocked the ERK activation induced by high glucose and reversed the inhibitory effect of high glucose on P0 expression. Taken together, these results suggest that high glucose can cause damage in primary cultured SCs and may exert the inhibitory effect on SC differentiation and myelination through ERK signaling activation.     

Direct activation of mitochondrial apoptosis machinery by c-Jun N-terminal kinase in adult cardiac myocytes.

Although oxidative stress causes activation of c-Jun N-terminal kinase (JNK) and apoptosis in many cell types, how the JNK pathway is connected to the apoptosis pathway is unclear. The molecular mechanism of JNK-mediated apoptosis was investigated in adult rat cardiac myocytes in culture as a model system that is sensitive to oxidative stress. Oxidative stress caused JNK activation, cytochrome c release, and apoptosis without new protein synthesis. Oxidative stress-induced apoptosis was abrogated by dominant negative stress-activated protein kinase/extracellular signal-regulated kinase kinase-1 (SEK1)-mediated inhibition of the JNK pathway, whereas activation of the JNK pathway by constitutively active SEK1 was sufficient to cause apoptosis. Inhibition of caspase-9, an apical caspase in the mitochondrial apoptosis pathway, suppressed oxidative stress-induced apoptosis, whereas inhibition of caspase-8 had no effect, indicating that both the JNK pathway and the mitochondrial apoptosis machinery are central to oxidative stress-induced apoptosis. Both JNK and SEK1 localized on mitochondria where JNK was activated by oxidative stress. Furthermore, active JNK caused the release of apoptogenic factors such as cytochrome c from isolated mitochondria in a cell-free assay. These findings indicate that the JNK pathway is a direct activator of mitochondrial death machinery without other cellular components and provide a molecular linkage from oxidative stress to the mitochondrial apoptosis machinery.     

High glucose-induced repression of RAR/RXR in cardiomyocytes is mediated through oxidative stress/JNK signaling

The biological actions of retinoids are mediated by nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs). We have recently reported that decreased expression of RARα and RXRα has an important role in high glucose (HG)-induced cardiomyocyte apoptosis. However, the regulatory mechanisms of HG effects on RARα and RXRα remain unclear. Using neonatal cardiomyocytes, we found that ligand-induced promoter activity of RAR and RXR was significantly suppressed by HG. HG promoted protein destabilization and serine-phosphorylation of RARα and RXRα. Proteasome inhibitor MG132 blocked the inhibitory effect of HG on RARα and RXRα. Inhibition of intracellular reactive oxidative species (ROS) abolished the HG effect. In contrast, H(2)O(2) stimulation suppressed the expression and ligand-induced promoter activity of RARα and RXRα. HG promoted phosphorylation of ERK1/2, JNK and p38 MAP kinases, which was abrogated by an ROS inhibitor. Inhibition of JNK, but not ERK and p38 activity, reversed HG effects on RARα and RXRα. Activation of JNK by over expressing MKK7 and MEKK1, resulted in significant downregulation of RARα and RXRα. Ligand-induced promoter activity of RARα and RXRα was also suppressed by overexpression of MEKK1. HG-induced cardiomyocyte apoptosis was potentiated by activation of JNK, and prevented by all-trans retinoic acid and inhibition of JNK. Silencing the expression of RARα and RXRα activated the JNK pathway. In conclusion, HG-induced oxidative stress and activation of the JNK pathway negatively regulated expression/activation of RAR and RXR. The impaired RAR/RXR signaling and oxidative stress/JNK pathway forms a vicious circle, which significantly contributes to hyperglycemia induced cardiomyocyte apoptosis.     

MALAT1/miR-185-5p mediated high glucose-induced oxidative stress,mitochondrial injury and cardiomyocyte apoptosis via the RhoA/ROCK pathway

To explore the underlying mechanism of lncRNA MALAT1 in the pathogenesis of diabetic cardiomyopathy (DCM). DCM models were confirmed in db/db mice. MiRNAs in myocardium were detected by miRNA sequencing. The interactions of miR-185-5p with MALAT1 and RhoA were validated by dual-luciferase reporter assays. Primary neonatal cardiomyocytes were cultured with 5.5 or 30 mmol/L D-glucose (HG) in the presence or absence of MALAT1-shRNA and fasudil, a ROCK inhibitor. MALAT1 and miR-185-5p expression were determined by real-time quantitative PCR. The apoptotic cardiomyocytes were evaluated using flow cytometry and TUNEL staining. SOD activity and MDA contents were measured. The ROCK activity, phosphorylation of Drp1^S616, mitofusin 2 and apoptosis-related proteins were analysed by Western blotting. Mitochondrial membrane potential was examined by JC-1. MALAT1 was significantly up-regulated while miR-185-5p was down-regulated in myocardium of db/db mice and HG-induced cardiomyocytes. MALAT1 regulated RhoA/ROCK pathway via sponging miR-185-5p in cardiomyocytes in HG. Knockdown of MALAT1 and fasudil all inhibited HG-induced oxidative stress, and alleviated imbalance of mitochondrial dynamics and mitochondrial dysfunction, accompanied by reduced cardiomyocyte apoptosis. MALAT1 activated the RhoA/ROCK pathway via sponging miR-185-5p and mediated HG-induced oxidative stress, mitochondrial damage and apoptosis of cardiomyocytes in mice.     

NGF通过抑制内质网应激途径保护SCs在高糖环境诱导的凋亡

已知神经生长因子 (nerve growth factor, NGF) 对糖尿病外周神经病变 (diabetic peripheral neuropathy, DPN) 患者具有良好的治疗效果,内质网应激 (endoplasmic reticulum stress, ERS) 在调控DPN 的发生发展方面扮演着重要的角色。然而,二者间的关系未知。本研究以30 mmol/L的高糖处理RSC-96大鼠雪旺细胞 (RSC96 Schwann cells, SCs),模拟DPN患者外周神经的内环境。研究结果证实,在高糖条件下,NGF通过抑制SCs内 ERS的过度激活进而保护SCs的存活且这种抑制作用依靠P13K/AKT/GSK-3β和ERK1/2两条信号通路的调节实现。MTT检测细胞的存活率,结果显示高糖环境培养的SCs在24 h发生最佳程度的抑制,且此时间点加入的NGF浓度为50 ng/mL 时,其存活率最高。Western 印迹检测ERS和相关蛋白质的表达揭示,高糖能够过度激活SCs内ERS蛋白 (GRP-78、ATF-6、ATF-4、XBP-1、CHOP),给予 50 ng/mL的NGF处理后,ERS蛋白的表达水平大幅下调并接近正常,且此时p-AKT、p-ERK1/2、p-GSK3β蛋白的检测水平明显升高。流式细胞术检测细胞凋亡显示,NGF能显著抑制SCs的早期凋亡。上述结果证明,高糖诱导SCs的凋亡增加是由于自身的ERS被过度激活,NGF可通过调节P13K/AKT/GSK-3β和ERK1/2两条信号通路来抑制ERS的过度激活,达到保护SCs存活的目的。此机制为临床治疗 DPN 提供新的理论基础。     

High glucose induces renal mesangial cell proliferation and fibronectin expression through JNK/NF-κB/NADPH oxidase/ROS pathway, which is inhibited by resveratrol

Renal hypertrophy and extracellular matrix accumulation are early features of diabetic nephropathy. Hyperglycemia-induced oxidative stress is implicated in the etiology of diabetic nephropathy. Resveratrol has potent antioxidative and protective effects on diabetic nephropathy. We aimed to examine whether high glucose (HG)-induced NADPH oxidase activation and reactive oxygen species (ROS) production contribute to glomerular mesangial cell proliferation and fibronectin expression and the effect of resveratrol on HG action in mesangial cells. By using rat mesangial cell line and primary mesangial cells, we found that NADPH oxidase inhibitor (apocynin) and ROS inhibitor (N-acetyl cysteine) both inhibited HG-induced mesangial cell proliferation and fibronectin expression. HG-induced elevation of NADPH oxidase activity and production of ROS in mesangial cells was inhibited by apocynin. These results suggest that HG induces mesangial cell proliferation and fibronectin expression through NADPH oxidase-mediated ROS production. Mechanistic studies revealed that HG upregulated NADPH oxidase subunits p22(phox) and p47(phox) expression through JNK/NF-κB pathway, which resulted in elevation of NADPH oxidase activity and consequent ROS production. Resveratrol prevented HG-induced mesangial cell proliferation and fibronectin expression through inhibiting HG-induced JNK and NF-κB activation, NADPH oxidase activity elevation and ROS production. These results demonstrate that HG enhances mesangial cell proliferation and fibronectin expression through JNK/NF-κB/NADPH oxidase/ROS pathway, which was inhibited by resveratrol. Our findings provide novel therapeutic targets for diabetic nephropathy.     

Rosiglitazone attenuates NF-κB-mediated Nox4 upregulation in hyperglycemia-activated endothelial cells

Vascular complications, a major cause of morbidity and mortality in diabetic patients, are related to hyperglycemia-induced oxidative stress. Previously, we reported that rosiglitazone (RSG) attenuated vascular expression and activity of NADPH oxidases in diabetic mice. The mechanisms underlying these effects remain to be elucidated. We hypothesized that RSG acts directly on endothelial cells to modulate vascular responses in diabetes. To test this hypothesis, human aortic endothelial cells (HAECs) were exposed to normal glucose (NG; 5.6 mmol/l) or high glucose (HG; 30 mmol/l) concentrations. Select HAEC monolayers were treated with RSG, caffeic acid phenethyl ester (CAPE), diphenyleneiodonium (DPI), small interfering (si)RNA (to NF-κB/p65 or Nox4), or Tempol. HG increased the expression and activity of the NADPH oxidase catalytic subunit Nox4 but not Nox1 or Nox2. RSG attenuated HG-induced NF-κB/p65 phosphorylation, nuclear translocation, and binding to the Nox4 promoter. Inhibiting NF-κB with CAPE or siNF-κB/p65 also reduced HG-induced Nox4 expression and activity. HG-induced H(2)O(2) production was attenuated by siRNA-mediated knockdown of Nox4, and HG-induced HAEC monocyte adhesion was attenuated by treatment with RSG, DPI, CAPE, or Tempol. These results indicate that HG exposure stimulates HAEC NF-κB activation, Nox4 expression, and H(2)O(2) production and that RSG attenuates HG-induced oxidative stress and subsequent monocyte-endothelial interactions by attenuating NF-κB/p65 activation and Nox4 expression. This study provides novel insights into mechanisms by which the thiazolidinedione peroxisome proliferator-activated receptor-γ ligand RSG favorably modulates endothelial responses in the diabetic vasculature.     

Knockdown of thioredoxin interacting protein attenuates high glucose-induced apoptosis and activation of ASK1 in mouse mesangial cells

Mesangial cell apoptosis contributes to the pathogenesis of diabetic nephropathy. Here we show that thioredoxin interacting protein (TXNIP) is involved in high glucose (HG)-induced mouse mesangial cell (MMC) apoptosis. HG induced activation of apoptosis signal regulating kinase-1 (ASK1) in a time-dependent manner in MMCs. Treatment with antioxidant, tempol, or knockdown of TXNIP in MMCs reduced HG-mediated apoptosis, expression of cleaved caspase-3, Bax/Bcl-2 ratio and activation of ASK1. These data suggest that knockdown of TXNIP prevented HG-induced cell apoptosis and activation of ASK1 may be via reduction of oxidative stress in MMCs.     

Salvianolate ameliorates oxidative stress and podocyte injury through modulation of NOX4 activity in db/db mice

Podocyte injury is associated with albuminuria and the progression of diabetic nephropathy (DN). NADPH oxidase 4 (NOX4) is the main source of reactive oxygen species (ROS) in the kidney and NOX4 is up-regulated in podocytes in response to high glucose. In the present study, the effects of Salvianolate on DN and its underlying mechanisms were investigated in diabetic db/db mice and human podocytes. We confirmed that the Salvianolate administration exhibited similar beneficial effects as the NOX1/NOX4 inhibitor GKT137831 treated diabetic mice, as reflected by attenuated albuminuria, reduced podocyte loss and mesangial matrix accumulation. We further observed that Salvianolate attenuated the increase of Nox4 protein, NOX4-based NADPH oxidase activity and restored podocyte loss in the diabetic kidney. In human podocytes, NOX4 was predominantly localized to mitochondria and Sal B treatment blocked HG-induced mitochondrial NOX4 derived superoxide generation and thereby ameliorating podocyte apoptosis, which can be abrogated by AMPK knockdown. Therefore, our results suggest that Sal B possesses the reno-protective capabilities in part through AMPK-mediated control of NOX4 expression. Taken together, our results identify that Salvianolate could prevent glucose-induced oxidative podocyte injury through modulation of NOX4 activity in DN and have a novel therapeutic potential for DN.     

Eriodictyol inhibits high glucose-induced oxidative stress and inflammation in retinal ganglial cells

Diabetic retinopathy (DR) is one of the most common microvascular complications of diabetes mellitus and is considered as a leading cause of blindness. Oxidative stress and inflammation are significant drivers for the development of DR. Eriodictyol, a flavonoid compound, was proved to possess anti-inflammatory, antioxidative, and antidiabetic activities. However, the role of eriodictyol in DR has not been unveiled. In the current study, we explored the protective effects of eriodictyol on high glucose (HG)-induced rat retinal ganglial cells (RGCs). The results suggested that eriodictyol improved cell viability of HG-induced rat RGC-5 cells in a dose-dependent manner. Eriodictyol reduced the reactive oxygen species production and increased the activities of superoxide dismutase, glutathione peroxidase and catalase in rat RGC-5 cells in response to HG stimulation. The production of proinflammatory cytokines including tumor necrosis factor alpha and interleukin-8 was diminished after eriodictyol treatment. Eriodictyol also suppressed cell apoptosis induced HG in rat RGC-5 cells. Furthermore, eriodictyol enhanced the nuclear translocation of nuclear factor erythroid-2 (E2)-related factor 2 (Nrf2) and elevated the expression of antioxidant enzyme heme-oxygenase-1 (HO-1). These findings suggested that eriodictyol protects the RGC-5 cells from HG-induced oxidative stress, inflammation, and cell apoptosis through regulating the activation of Nrf2/HO-1 pathway.     

SOCS-1 is involved in TNF-α-induced mitochondrial dysfunction and apoptosis in renal tubular epithelial cells

Tumor necrosis factor-α (TNF-α) is suggested to induce mitochondrial dysfunction and apoptosis of renal tubular epithelial cells that possibly exacerbates renal function in chronic kidney disease (CKD). Here we investigated whether suppressor of cytokine signaling-1 (SOCS-1), an inhibitor of cytokine signaling, was involved in TNF-α-induced human renal tubular epithelial cells (HKCs) oxidative stress and apoptosis. TNF-α promoted the protein and mRNA expression of SOCS-1 in a time and dose dependent manner, along with increased cell apoptosis and activation of apoptosis signal regulating kinase-1(ASK1) in HKCs. Furthermore, overexpression of SOCS-1 in HKCs reduced TNF-α-mediated oxidative stress and apoptosis. Meanwhile, We also found that overexpression of SOCS-1 could regulate the activity of JAK/STAT signaling pathway. In addition, a specific JAK2 inhibitor, AG490, that both attenuated TNF-α-induced oxidative stress, also reduced apoptosis. Taken together, overexpression of SOCS-1 prevented TNF-α-mediated cell oxidative stress and apoptosis may be via suppression of JAK/STAT signaling pathway activation in HKCs.     

Mitofusin-2 is a major determinant of oxidative stress-mediated heart muscle cell apoptosis

An inexorable loss of terminally differentiated heart muscle cells is a crucial causal factor for heart failure. Here, we have provided several lines of evidence to demonstrate that mitofusin-2 (Mfn-2; also called hyperplasia suppressor gene), a member of the mitofusin family, is a major determinant of oxidative stress-mediated cardiomyocyte apoptosis. First, oxidative stress with H(2)O(2) led to concurrent increases in Mfn-2 expression and apoptosis in cultured neonatal rat cardiomyocytes. Second, overexpression of Mfn-2 to a level similar to that induced by H(2)O(2) was sufficient to trigger myocyte apoptosis, which is associated with profound inhibition of Akt activation without altering ERK1/2 signaling. Third, Mfn-2 silencing inhibited oxidative stress-induced apoptosis in H9C2 cells, a cardiac muscle cell line. Furthermore, Mfn-2-induced myocyte apoptosis was abrogated by inhibition of caspase-9 (but not caspase-8) and by overexpression of Bcl-x(L) or enhanced activation of phosphatidylinositol 3-kinase-Akt, suggesting that inhibition of Akt signaling and activation of the mitochondrial death pathway are essentially involved in Mfn-2-induced heart muscle cell apoptosis. These results indicate that increased cardiac Mfn-2 expression is both necessary and sufficient for oxidative stress-induced heart muscle cell apoptosis, suggesting that Mfn-2 deregulation may be a crucial pathogenic element and a potential therapeutic target for heart failure.     

Downregulation of BRD4 attenuates high glucose-induced damage of trophoblast cells by inhibiting activation of AKT/mTOR pathway

It was elucidated that bromodomain-containing protein 4 (BRD4) has involvement with diabetic complication. However, the role and molecular mechanism of BRD4 in gestational diabetes mellitus (GDM) are still unclear. In this study, the mRNA and protein contents of BRD4 in placenta tissues of GDM patients and high glucose (HG)-induced HTR8/SVneo cells were detected by qRT-PCR and western blot assay. CCK-8, EdU staining, flow cytometry as well as western blot were applied for the appraisement of cell viability and apoptosis. Wound healing assay and transwell assay were conducted for the assessment of cell migration and invasion. Oxidative stress and inflammatory factors were detected. Additionally, the contents of AKT/mTOR pathway-related proteins were estimated applying western blot. It was discovered that BRD4 expression was ascended in tissues and HG-induced HTR8/SVneo cells. BRD4 downregulation cut down the contents of p-AKT and p-mTOR but had no effects on the total protein levels of AKT or mTOR in HG-induced HTR8/SVneo cells. BRD4 depletion promoted cell viability, enhanced proliferative capability, and reduced cell apoptotic level. Moreover, BRD4 depletion facilitated cell migrative and invasive capabilities, and repressed the oxidative stress as well as inflammatory damage in HG-induced HTR8/SVneo cells. The activation of Akt reversed the protective impacts of BRD4 depletion on HG-induced HTR8/SVneo cells. To sum up, BRD4 silencing may alleviate HG-induced HTR8/SVneo cell damage through the inhibition of the AKT/mTOR pathway.     

Salvianolic acid A protects human SH-SY5Y neuroblastoma cells against H₂O₂-induced injury by increasing stress tolerance ability

Salvianolic acid A (Sal A) is a polyphenol extracted from the root of the Salvia miltiorrhiza bunge. Hydrogen peroxide (H(2)O(2)) is a major reactive oxygen species (ROS), which has been implicated in stroke and other neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. In this study, we investigated the neuroprotective effects of Sal A in human SH-SY5Y neuroblastoma cells against H(2)O(2)-induced injury. Our results showed that cells pretreated with Sal A exhibited enhanced neuronal survival and that this protection was associated with an increase in adenosine triphosphate (ATP) and the stabilization of mitochondrial membrane potential. In addition, Sal A markedly decreased the excessive activation AMP-activated protein kinase (AMPK) and the serine-threonine protein kinase, Akt, in SH-SY5Ycells induced by H(2)O(2). In conclusion, our results demonstrated that Sal A protects SH-SY5Y cells against H(2)O(2)-induced oxidative stress and these protective effects are related to stress tolerance and not energy depletion via inhibition of the AMPK and Akt signaling pathway.     

Role of mitochondrial hOGG1 and aconitase in oxidant-induced lung epithelial cell apoptosis

8-Oxoguanine DNA glycosylase (Ogg1) repairs 8-oxo-7,8-dihydroxyguanine (8-oxoG), one of the most abundant DNA adducts caused by oxidative stress. In the mitochondria, Ogg1 is thought to prevent activation of the intrinsic apoptotic pathway in response to oxidative stress by augmenting DNA repair. However, the predominance of the β-Ogg1 isoform, which lacks 8-oxoG DNA glycosylase activity, suggests that mitochondrial Ogg1 functions in a role independent of DNA repair. We report here that overexpression of mitochondria-targeted human α-hOgg1 (mt-hOgg1) in human lung adenocarcinoma cells with some alveolar epithelial cell characteristics (A549 cells) prevents oxidant-induced mitochondrial dysfunction and apoptosis by preserving mitochondrial aconitase. Importantly, mitochondrial α-hOgg1 mutants lacking 8-oxoG DNA repair activity were as effective as wild-type mt-hOgg1 in preventing oxidant-induced caspase-9 activation, reductions in mitochondrial aconitase, and apoptosis, suggesting that the protective effects of mt-hOgg1 occur independent of DNA repair. Notably, wild-type and mutant mt-hOgg1 coprecipitate with mitochondrial aconitase. Furthermore, overexpression of mitochondrial aconitase abolishes oxidant-induced apoptosis whereas hOgg1 silencing using shRNA reduces mitochondrial aconitase and augments apoptosis. These findings suggest a novel mechanism that mt-hOgg1 acts as a mitochondrial aconitase chaperone protein to prevent oxidant-mediated mitochondrial dysfunction and apoptosis that might be important in the molecular events underlying oxidant-induced toxicity.