AGE-induced Apoptosis Research Articles

Cinnamaldehyde protects SH-SY5Y cells against advanced glycation end-products induced ectopic cell cycle re-entry

Accumulation of advanced glycation end-products (AGEs) in the brain contributes significantly to cognitive impairment in patients with diabetes by disrupting the post-mitotic state of neuronal cells, thereby triggering ectopic cell cycle re-entry (CCR) and subsequent neuronal apoptosis. Cinnamaldehyde (CINA), a potential mitigator of cognitive impairment due to its blood glucose-lowering properties, warrants exploration for its role in counteracting diabetes-related neurological damage. In this study, we examined the neuroprotective effect of CINA on AGE-damaged SH-SY5Y human neuroblastoma cells differentiated in vitro. We investigated the impact of CINA on AGE-induced neuronal CCR and apoptosis, finding that it substantially suppressed aberrant DNA replication, precluded cells from entering the mitotic preparatory phase, and diminished apoptosis. Additionally, CINA inhibited the expression of eIF4E without altering S6K1 phosphorylation. These findings indicate that CINA safeguards neuronal cells from AGE-related damage by preventing abnormal CCR, preserving the post-mitotic state of neuronal cells, and reducing AGE-induced apoptosis, potentially through the inhibition of eIF4E-controlled cell proliferation. Our results highlight the prospective utility of CINA in managing diabetic neuropathy.

Phlorofucofuroeckol-A from Ecklonia Cava attenuates methylglyoxal-induced nephrotoxicity by regulating RAGE and inhibiting the formation of advanced glycosylated end products

Diabetic nephropathy (DN) is the most prevalent and fatal complication of diabetes. The progression of the DN is acceleated by both the formation and accumulation of advanced glycation end products (AGEs). Several synthetic compounds have been proposed to inhibit AGE formation; however, their use is associated with certain side effects. In this study, a natural compound derived from the brown seaweed Ecklonia cava was explored as a natural agent for the prevention and alleviation of DN. Specifically, the protective effect of phlorofucofuroeckol-A (PFFA) was investigated against methylglyoxal-derived AGE-induced apoptosis in mesangial cells. PFFA reduced methylglyoxal glycation and the accumulation of methylglyoxal-derived AGEs. Moreover, PFFA inhibited the interaction between the receptor for AGE (RAGE) and AGEs by forming stable ligands with RAGE. The mechanism underlying the prevention of apoptosis by PFFA was found to involve the inhibition of the phosphorylation of mitogen-activated kinase-related proteins, reversal of the downregulation of intracellular oxidative stress-associated proteins, and normalization of the levels of proteins regulating mitochondria-related intrinsic apoptosis. These findings highlight PFFAs as promising natural therapeutic agents for the prevention or alleviation of DN.

Advanced glycation end products induce nucleus pulposus cell apoptosis by upregulating TXNIP via inhibiting glycolysis pathway in intervertebral disc degeneration.

Accumulation of advanced glycation end products (AGEs) causes apoptosis in human nucleus pulposus cells (NPCs), contributing to intervertebral disc degeneration (IVDD). The purpose of this study was to determine the roles of thioredoxin-interacting protein (TXNIP) in the mechanisms underlying AGE-induced apoptosis of NPCs. TXNIP was silenced or overexpressed in HNPCs exposed to AGEs. Glycolysis was assessed using extracellular acidification rate (ECAR), ATP level, GLUT1, and GLUT4 measurements. AGEs, TXNIP, GLUT1, and GLUT4 levels in IVDD patients were measured as well. In NPCs, AGEs reduced cell viability, induced apoptosis, inhibited glycolysis, and increased TXNIP expression. Silencing TXNIP compromised the effects of AGEs on cell viability, apoptosis, and glycolysis in NPCs. Furthermore, TXNIP overexpression resulted in decreased cell viability, increased apoptotic cells, and glycolysis suppression. Furthermore, co-treatment with a glycolysis inhibitor improved TXNIP silencing's suppressive effects on AGE-induced cell injury in NPCs. In IVDD patients with Pfirrmann Grades II-V, increasing trends in AGEs and TXNIP were observed, while decreasing trends in GLUT1 and GLUT4. AGE levels had positive correlations with TXNIP levels. Both AGE and TXNIP levels correlated negatively with GLUT1 and GLUT4. Our study indicates that TXNIP plays a role in mediating AGE-induced cell injury through suppressing glycolysis. The accumulation of AGEs, the upregulation of TXNIP, and the downregulation of GLUT1 and GLUT4 are all linked to the progression of IVDD.

Long term treadmill exercise affects age-related oxidative stress in the spinal cord of rats

Age-induced apoptosis is believed to be caused by the imbalance between production of reactive oxygen species (ROS) and human body antioxidant defence. Regular aerobic treadmill-exercise has been suggested to enhance the antioxidant defence. This study aimed to investigate the effects of long-term treadmill exercise on age-related oxidative stress and the apoptosis of oligodendrocytes in the spinal cord of the rat. Sixty male rats were divided into six groups: three exercised groups, which underwent 6, 9 and 12 months of mild-to-moderate treadmill exercise and three non-exercised control groups. Spinal cord white or grey matter tissue sampling was done through mid-thoracic laminectomy. The malondialdehyde (MDA; indicator of oxidative stress) levels, the number of apoptotic oligodendrocytes and ultrastructural alterations were also evaluated. Our data showed that treadmill exercise resulted in decreased lipid peroxidation and the number of apoptotic oligodendrocytes in the spinal cord of rats, as compared to non-exercised animals. These results were confirmed by TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labelling) staining and electron microscope. This study suggests that the long-term treadmill exercise can affect oxidative stress and oligodendrocytes apoptosis in the spinal cord of aged rats and further studies are needed to validate these findings in humans.

Panax notoginseng saponins alleviates advanced glycation end product-induced apoptosis by upregulating SIRT1 and antioxidant expression levels in HUVECs.

The present study examined whether Panax notoginseng saponins (PNS) alleviated advanced glycation end product (AGE)-induced apoptosis in human umbilical vein endothelial cells (HUVECs). HUVECs were incubated with 300 µg/ml AGEs alone or AGEs and PNS (0.05, 0.5 or 1 mg/ml) for 48 h. The results of the present study demonstrated that PNS effectively promoted cell viability, inhibited apoptosis and suppressed the activity of caspase-3 in AGE-induced HUVECs. The activities of monocyte chemoattractant protein-1 and malondialdehyde were reduced, and superoxide dismutase activity was increased following treatment with PNS. Furthermore, PNS significantly increased the expression of silent information regulator 1 (SIRT1) and transforming growth factor (TGF)-β1 proteins, and suppressed the expression of inducible nitric oxide synthase and cyclooxyggenase-2 proteins in AGE-induced HUVECs. Therefore, the present study demonstrated that PNS reduced AGE-induced apoptosis by upregulating SIRT1 and antioxidants in HUVECs. The present findings suggest that the PNS may as an important pharmacological agent for AGE-induced cardiovascular injury.

Silencing of Long Non-coding RNA NEAT1 Upregulates miR-195a to Attenuate Intervertebral Disk Degeneration via the BAX/BAK Pathway.

Background/AimsAn increasing body of evidence has demonstrated that long non-coding RNAs (lncRNAs) play a vital regulatory role in intervertebral disk degeneration (IVDD). Nucleus enriched abundant transcript 1 (NEAT1), a novel cancer-related lncRNA, is associated with many malignancies, including ovarian cancer, and esophageal squamous cell carcinoma. Nevertheless, the role of NEAT1 in the progression of IVDD remains to be studied. Here, we explored the effect of NEAT1 on the progression of IVDD and the mechanisms involved.MethodsAn IVDD model was constructed in SD rats in vivo, and degeneration was induced by advanced glycation end product (AGE) in human nucleus pulposus cells (HNPC) in vitro. Quantitative real-time PCR was performed to detect the relative NEAT1 and miR-195a expressions and further confirmed the relationship between NEAT1 and miR-195a. Cell apoptosis was evaluated by TUNEL assay. The related mechanisms were explored by Western blot assay.ResultsThe relative NEAT1 expression was significantly upregulated in the IVDD rat model and the denatured HNPC. Silencing of NEAT1 expression in HNPC significantly promoted the Collagen II and TIMP-1 expression induced by AGE while greatly suppressing the expressions of MMP-3 and cleaved caspase-3. Besides, downregulation of NEAT1 obviously reversed the AGE-induced apoptosis in HNPC. More interestingly, these effects of NEAT1 knockout on HNPC were largely reversed by silencing of miR-195a or overexpression of BAX under the AGE treatment. Mechanically, the direct combination of NEAT1 with miR-195a resulted in upregulation of MMP-3, cleaved caspase-3, BAX, and BAK, as well as downregulation of Collagen II and TIMP-1, which are associated with EMT and apoptosis. We also demonstrated similar results in the in vivo experiments.ConclusionNEAT1 played its role in IVDD progression via partly by mediating the miR-195 expression and might be used as a potential target for IVDD therapy.

Beta carotene protects H9c2 cardiomyocytes from advanced glycation end product-induced endoplasmic reticulum stress, apoptosis, and autophagy via the PI3K/Akt/mTOR signaling pathway.

BackgroundDiabetic cardiomyopathy (DCM), which is associated with many pathological processes, commonly occurs when advanced glycation end products (AGEs) are present. β-carotene (BC) is a well-known vitamin A precursor that is found in many fruits and vegetables. BC can reduce the risk of cancer and cardiovascular disease. This study aimed to investigate the effect of BC on AGE-induced myocardial injury in vitro.MethodsCell viability test was used to select 40 µM concentrations of BC to treat AGE-induced H2c9 cells. The cell apoptosis was detected by flow cytometry. Western blotting was used to measure the protein expression levels of Bcl-2-associated X protein (Bax), B-cell lymphoma-2 (Bcl-2), cleaved caspase-3, activating transcription factor 4 (ATF4), glucose-regulated protein 78 (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP), beclin 1, p62,microtubule-associated protein 1 light chain 3 (LC3), phosphorylated PI3K (p-PI3K), phosphorylated Akt (p-AKT), and phosphorylated mTOR (p-mTOR). Enzyme-linked immunosorbent assay (ELISA) was performed to measure the levels of lactate dehydrogenase (LDH) and cardiac troponin-1 (cTn-I). Reactive oxygen species (ROS) was detected by flow cytometry. The levels of malondialdehyde (MDA), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) were used to determine MDA kits, SOD assay kit and GSH-Px kit, respectively.ResultsBC significantly inhibited AGE-induced cell death and apoptosis in H9c2 cells. BC had a suppressive effect on intracellular ROS production and antioxidative enzyme reduction. Moreover, BC decreased hyperactive endoplasmic reticulum (ER) stress and autophagy in H9c2 cells. Furthermore, BC exerted a cardioprotective effect in AGE-induced H9c2 cells via the activation of the PI3K/Akt/mTOR signaling pathway.ConclusionsBC exhibited a cardioprotective effect AGE-induced apoptosis. Our study provides a foundation for further study into the potential value of BC for treating DCM or other heart diseases.

Diallyl Trisulfide (DATS) Suppresses AGE-Induced Cardiomyocyte Apoptosis by Targeting ROS-Mediated PKCδ Activation.

Chronic high-glucose exposure results in the production of advanced glycation end-products (AGEs) leading to reactive oxygen species (ROS) generation, which contributes to the development of diabetic cardiomyopathy. PKCδ activation leading to ROS production and mitochondrial dysfunction involved in AGE-induced cardiomyocyte apoptosis was reported in our previous study. Diallyl trisulfide (DATS) is a natural cytoprotective compound under various stress conditions. In this study, the cardioprotective effect of DATS against rat streptozotocin (STZ)-induced diabetic mellitus (DM) and AGE-induced H9c2 cardiomyoblast cell/neonatal rat ventricular myocyte (NRVM) damage was assessed. We observed that DATS treatment led to a dose-dependent increase in cell viability and decreased levels of ROS, inhibition of PKCδ activation, and recuded apoptosis-related proteins. Most importantly, DATS reduced PKCδ mitochondrial translocation induced by AGE. However, apoptosis was not inhibited by DATS in cells transfected with PKCδ-wild type (WT). Inhibition of PKCδ by PKCδ-kinase-deficient (KD) or rottlerin not only inhibited cardiac PKCδ activation but also attenuated cardiac cell apoptosis. Interestingly, overexpression of PKCδ-WT plasmids reversed the inhibitory effects of DATS on PKCδ activation and apoptosis in cardiac cells exposed to AGE, indicating that DATS may inhibit AGE-induced apoptosis by downregulating PKCδ activation. Similar results were observed in AGE-induced NRVM cells and STZ-treated DM rats following DATS administration. Taken together, our results suggested that DATS reduced AGE-induced cardiomyocyte apoptosis by eliminating ROS and downstream PKCδ signaling, suggesting that DATS has potential in diabetic cardiomyopathy (DCM) treatment.

1,25-(OH)2D3 protects Schwann cells against advanced glycation end products-induced apoptosis through PKA-NF-κB pathway

AimsTo explore the effect and mechanism of 1, 25-(OH)2D3 on Schwann cell apoptosis induced by advanced glycation end products. Main methodsSchwann cells, isolated from rodent sciatic nerve were incubated with AGE-modified bovine serum albumin(AGE) to mimic diabetic conditions and 1,25-(OH)2D3 was used as protector. Cell apoptosis was detected by PI/Annexin-V staining, caspase 3 activity assay and western blotting for caspase 3 and PARP. The activation of protein kinase A (PKA) and nuclear factor kappa-B (NF-κB) was evaluated by western blot. Immunofluorescent staining was used for intercellular location of NF-κB. Cytokine secretion was evaluated by enzyme-linked immunosorbent assay. Key findingsSchwann cell apoptosis accelerated after incubating with AGE. However, if combining 1,25-(OH)2D3 with AGE, apoptosis decreased significantly. 1,25-(OH)2D3 enhanced PKA activity, but inhibited AGE-induced nuclear translocation of NF-κB. Furthermore, PKA activator (8-bromoadenoside cyclic adenoside monophosphate, 8-Br-cAMP) or NF-κB inhibitor (caffeic acid phenethyl ester, CAPE) could reduce the apoptosis, decreased cleaved caspase 3 and cleaved PARP, suggesting the involvement of PKA and NF-κB pathways in the protection of 1,25-(OH)2D3 on Schwann cells. Moreover, 8-Br-cAMP and CAPE could inhibit AGE-induced secretion of interleukin(IL)-1β, prostaglandin E2(PEG2) and cyclooxygenase 2(COX2). Interestingly, 8-Br-cAMP decreased phospho-NF-κB and inhibited nucleus translocation of NF-κB. It hinted at the regulation of PKA to NF-κB. Finally, a pre-treatment of H-89 (an inhibitor of PKA) could block the protection of 1,25-(OH)2D3 on cell apoptosis. In conclusion, 1,25-(OH)2D3 could protect Schwann cell against AGE-induced apoptosis through PKA/NF-κB pathway. SignificanceThese findings provide experimental rationales for using vitamin D for diabetic neuropathy.

Interleukin-10 Protects Schwann Cells against Advanced Glycation End Products-Induced Apoptosis via NF-κB Suppression.

Demyelination resulting from Schwann cell injury is a main pathological feature of diabetic neuropathy, and a key contributor to this process may be inflammation due to advanced glycation end products (AGEs). Therefore, protection by anti-inflammation agents is anticipated. In this study, we showed that interleukin-10 (IL-10), an anti-inflammatory cytokine, inhibits apoptosis of Schwann cells induced by AGEs in vitro. We isolated and cultured Schwann cells from rat sciatic nerves. As detected by flow cytometry, apoptosis of Schwann cells markedly increased following incubation with AGEs for 48 h. However, pretreatment with IL-10 inhibited AGE-induced apoptosis. The effect of IL-10 on NF-κB, which is a very important regulator of inflammation, was also evaluated, and results showed high levels of phospho-NF-κB and nuclear localization of NF-κB in cells incubated with AGEs but low levels of phospho-NF-κB and cytoplasmic localization in the cells incubated with IL-10, indicating the activation of NF-κB by AGEs and inhibition of NF-κB by IL-10. Moreover, incubating Schwann cells with an NF-κB inhibitor (caffeic acid phenethyl ester) for 30 min before adding AGEs mimicked IL-10, lowering the amount of reactive oxygen species and activity of caspase-3 and also decreasing apoptosis in Schwann cells. These results indicate that IL-10 may protect Schwann cells against AGE-induced apoptosis by attenuating oxidative stress via the inhibition of activation of NF-κB.

Melatonin protects endothelial progenitor cells against AGE-induced apoptosis via autophagy flux stimulation and promotes wound healing in diabetic mice

Wound healing is delayed in diabetic patients. Increased apoptosis and endothelial progenitor cell (EPC) dysfunction are implicated in delayed diabetic wound healing. Melatonin, a major secretory product of the pineal gland, promotes diabetic wound healing; however, its mechanism of action remains unclear. Here, EPCs were isolated from the bone marrow of mice. Treatment of EPCs with melatonin alleviated advanced glycation end product (AGE)-induced apoptosis and cellular dysfunction. We further examined autophagy flux after melatonin treatment and found increased light chain 3 (LC3) and p62 protein levels in AGE-treated EPCs. However, lysosome-associated membrane protein 2 expression was decreased, indicating that autophagy flux was impaired in EPCs treated with AGEs. We then evaluated autophagy flux after melatonin treatment and found that melatonin increased the LC3 levels, but attenuated the accumulation of p62, suggesting a stimulatory effect of melatonin on autophagy flux. Blockage of autophagy flux by chloroquine partially abolished the protective effects of melatonin, indicating that autophagy flux is involved in the protective effects of melatonin. Furthermore, we found that the AMPK/mTOR signaling pathway is involved in autophagy flux stimulation by melatonin. An in vivo study also illustrated that melatonin treatment ameliorated impaired wound healing in a streptozotocin-induced diabetic wound healing model. Thus, our study shows that melatonin protects EPCs against apoptosis and dysfunction via autophagy flux stimulation and ameliorates impaired wound healing in vivo, providing insight into its mechanism of action in diabetic wound healing.

RAGE-dependent mitochondria pathway: a novel target of silibinin against apoptosis of osteoblastic cells induced by advanced glycation end products

Advanced glycation end products (AGEs) can stimulate osteoblast apoptosis and have a critical role in the pathophysiology of diabetic osteoporosis. Mitochondrial abnormalities are closely related to osteoblast dysfunction. However, it remains unclear whether mitochondrial abnormalities are involved in AGE-induced osteoblastic cell apoptosis. Silibinin, a major flavonolignan compound of silimarin, has strong antioxidant and mitochondria-protective properties. In the present study, we explored the possible mitochondrial mechanisms underlying AGE-induced apoptosis of osteoblastic cells and the effect of silibinin on osteoblastic cell apoptosis. We demonstrated that mitochondrial abnormalities largely contributed to AGE-induced apoptosis of osteoblastic cells, as evidenced by enhanced mitochondrial oxidative stress, conspicuous reduction in mitochondrial membrane potential and adenosine triphosphate production, abnormal mitochondrial morphology, and altered mitochondrial dynamics. These AGE-induced mitochondrial abnormalities were mainly mediated by the receptor of AGEs (RAGE). In addition, we found that silibinin directly downregulated the expression of RAGE and modulated RAGE-mediated mitochondrial pathways, thereby preventing AGE-induced apoptosis of osteoblastic cells. This study not only provides a new insight into the mitochondrial mechanisms underlying AGE-induced osteoblastic cell apoptosis, but also lays a foundation for the clinical use of silibinin for the prevention or treatment of diabetic osteoporosis.

Zinc against advanced glycation end products.

Advanced glycation end products (AGEs) are destructive compounds with pathogenic importance in age-related chronic diseases. Zinc has antioxidant, anti-inflammatory and anti-apoptotic potential. This study aimed to summarize effects of zinc on AGE formation and AGE-induced damaging agents. Pubmed, Google scholar, Web of Sciences, and Scopus databases were searched. There was no limitation for publication date. English language original articles (in vitro, experimental and human studies) which examined the effect of external zinc on AGE formation, AGE-induced apoptosis, or oxidative stress in mammals were included. To review the effect of zinc on AGE generation, the search keywords were as follows: "zinc" in title and "AGEs" or "methylglyoxal" or "pentosidine" or "carboxymethyllysine" or "glucosylation" or "carbonyl stress" or "AGEs-induced apoptosis or oxidative stress or inflammation" in title/abstract. In total, 90 titles and abstracts were identified. Fifty-two studies were screened after duplicates were removed. Six articles were chosen for review following analysis of both titles and summaries. Finally, based on intensive critical appraisal, 5 articles were included in the study. The evidence presented indicates that zinc has anti-glycation, anti-oxidative and anti-apoptotic effects. Zinc insufficiency may stimulate AGEs formation. Whereas, zinc supplementation could inhibit formation of AGEs, AGE-induced cell apoptosis and oxidative stress status, and protein carbonyl formation possibly through various signalling pathways.

TRB3 mediates advanced glycation end product-induced apoptosis of pancreatic β-cells through the protein kinase C β pathway.

Advanced glycation end products (AGEs), which accumulate in the body during the development of diabetes, may be one of the factors leading to pancreatic β-cell failure and reduced β-cell mass. However, the mechanisms responsible for AGE-induced apoptosis remain unclear. This study identified the role and mechanisms of action of tribbles homolog 3 (TRB3) in AGE-induced β-cell oxidative damage and apoptosis. Rat insulinoma cells (INS-1) were treated with 200 µg/ml AGEs for 48 h, and cell apoptosis was then detected by TUNEL staining and flow cytometry. The level of intracellular reactive oxygen species (ROS) was measured by a fluorescence assay. The expression levels of receptor of AGEs (RAGE), TRB3, protein kinase C β2 (PKCβ2) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) were evaluated by RT-qPCR and western blot analysis. siRNA was used to knockdown TRB3 expression through lipofection, followed by an analysis of the effects of TRB3 on PKCβ2 and NOX4. Furthermore, the PKCβ2-specific inhibitor, LY333531, was used to analyze the effects of PKCβ2 on ROS levels and apoptosis. We found that AGEs induced the apoptosis of INS-1 cells and upregulated RAGE and TRB3 expression. AGEs also increased ROS levels in β-cells. Following the knockdown of TRB3, the AGE-induced apoptosis and intracellular ROS levels were significantly decreased, suggesting that TRB3 mediated AGE-induced apoptosis. Further experiments demonstrated that the knockdown of TRB3 decreased the PKCβ2 and NOX4 expression levels. When TRB3 was knocked down, the cells expressed decreased levels of PKCβ2 and NOX4. The PKCβ2-specific inhibitor, LY333531, also reduced AGE-induced apoptosis and intracellular ROS levels. Taken together, our data suggest that TRB3 mediates AGE-induced oxidative injury in β-cells through the PKCβ2 pathway.

Far-infrared protects vascular endothelial cells from advanced glycation end products-induced injury via PLZF-mediated autophagy in diabetic mice

The accumulation of advanced glycation end products (AGEs) in diabetic patients induces vascular endothelial injury. Promyelocytic leukemia zinc finger protein (PLZF) is a transcription factor that can be activated by low-temperature far-infrared (FIR) irradiation to exert beneficial effects on the vascular endothelium. In the present study, we investigated the influence of FIR-induced PLZF activation on AGE-induced endothelial injury both in vitro and in vivo. FIR irradiation inhibited AGE-induced apoptosis in human umbilical vein endothelial cells (HUVECs). PLZF activation increased the expression of phosphatidylinositol-3 kinases (PI3K), which are important kinases in the autophagic signaling pathway. FIR-induced PLZF activation led to autophagy in HUVEC, which was mediated through the upregulation of PI3K. Immunofluorescence staining showed that AGEs were engulfed by HUVECs and localized to lysosomes. FIR-induced autophagy promoted AGEs degradation in HUVECs. In nicotinamide/streptozotocin-induced diabetic mice, FIR therapy reduced serum AGEs and AGEs deposition at the vascular endothelium. FIR therapy also reduced diabetes-induced inflammatory markers in the vascular endothelium and improved vascular endothelial function. These protective effects of FIR therapy were not found in PLZF-knockout mice. Our data suggest that FIR-induced PLZF activation in vascular endothelial cells protects the vascular endothelium in diabetic mice from AGE-induced injury.

Autophagy Protects Advanced Glycation End Product-Induced Apoptosis and Expression of MMP-3 and MMP-13 in Rat Chondrocytes.

Aging is one of the most prominent risk factors for the pathological progression of osteoarthritis (OA). One feature of age-related changes in OA is advanced glycation end products (AGEs) accumulation in articular cartilage. Autophagy plays a cellular housekeeping role by removing dysfunctional cellular organelles and proteins. However, the relationship between autophagy and AGE-associated OA is unknown. The aim of this study is to determine whether autophagy participates in the pathology of AGE-treated chondrocytes and to investigate the exact role of autophagy in AGE-induced cell apoptosis and expression of matrix metalloproteinase- (MMP-) 3 and MMP-13. AGEs induced notable apoptosis that was detected by Annexin V/PI double-staining, and the upregulation of MMP-3 and MMP-13 was confirmed by Western blotting. Autophagy-related proteins were also determined by Western blotting, and chondrocytes were transfected with mCherry-GFP-LC3B-adenovirus to monitor autophagic flux. As a result, autophagy significantly increased in chondrocytes and peaked at 6 h. Furthermore, rapamycin (RA) attenuated AGE-induced apoptosis and expression of MMP-3 and MMP-13 by autophagy activation. In contrast, pretreatment with autophagy inhibitor 3-methyladenine (3-MA) enhanced the abovementioned effects of AGEs. We therefore demonstrated that autophagy is linked with AGE-related pathology in rat chondrocytes and plays a protective role in AGE-induced apoptosis and expression of MMP-3 and MMP-13.

Inhibition of autophagy increased AGE/ROS-mediated apoptosis in mesangial cells

The aim of our study was to investigate the role of autophagy, a homeostatic process involved in the lysosomal degradation of damaged cell organelles and proteins, in regulating the survival of mesangial cells treated with advanced glycation end products (AGEs). In the present study, AGEs induced mitochondrial depolarization and led to mitochondrial-dependent apoptosis in mesangial cells, as shown by the loss of the mitochondrial membrane potential; increased Bax processing; increased Caspase-9, Caspase-3 and PARP cleavage; and decreased Bcl-2 expression. Meanwhile, AGEs also triggered autophagy flux in mesangial cells, as confirmed by the presence of autophagic vesicles, the conversion of LC3II/LC3I and the increase/decrease in Beclin-1/p62 expression. Interestingly, this study reported apparent apoptosis and autophagy that were dependent on reactive oxygen species (ROS) production. Scavenging ROS with N-acetyl-l-cysteine could prevent the appearance of the autophagic features and reverse AGE-induced apoptosis. Moreover, AGE-triggered mitophagy, which was confirmed by the colocalization of autophagosomes and mitochondria and Parkin translocation to mitochondria, played a potential role in reducing ROS production in mesangial cells. Additionally, inhibition of autophagy significantly enhanced AGE-induced cell apoptosis. Taken together, our data suggest that ROS were the mediators of AGE-induced mesangial cell apoptosis and that autophagy was likely to be the mechanism that was triggered to repair the ROS-induced damage in the AGE-treated cells and thereby promote cell survival. This study provides new insights into the molecular mechanism of autophagy involved in AGE-induced apoptosis in mesangial cells.

Antioxidants inhibit advanced glycosylation end-product-induced apoptosis by downregulation of miR-223 in human adipose tissue-derived stem cells.

Advanced glycosylation end products (AGEs) are endogenous inflammatory mediators that induce apoptosis of mesenchymal stem cells. A potential mechanism includes increased generation of reactive oxygen species (ROS). MicroRNA-223 (miR-223) is implicated in the regulation of cell growth and apoptosis in several cell types. Here, we tested the hypothesis that antioxidants N-acetylcysteine (NAC) and ascorbic acid 2-phosphate (AAP) inhibit AGE-induced apoptosis via a microRNA-dependent mechanism in human adipose tissue-derived stem cells (ADSCs). Results showed that AGE-HSA enhanced apoptosis and caspase-3 activity in ADSCs. AGE-HSA also increased ROS generation and upregulated the expression of miR-223. Interestingly, reductions in ROS generation and apoptosis, and upregulation of miR-223 were found in ADSCs treated with antioxidants NAC and AAP. Furthermore, miR-223 mimics blocked antioxidant inhibition of AGE-induced apoptosis and ROS generation. Knockdown of miR-223 amplified the protective effects of antioxidants on apoptosis induced by AGE-HSA. miR-223 acted by targeting fibroblast growth factor receptor 2. These results indicate that NAC and AAP suppress AGE-HSA-induced apoptosis of ADSCs, possibly through downregulation of miR-223.

Advanced glycation end-product (AGE) induces apoptosis in human retinal ARPE-19 cells via promoting mitochondrial dysfunction and activating the Fas-FasL signaling.

Advanced glycation end-products (AGEs) are extremely accumulated in the retinal vascular and epithelial cells of diabetes mellitus (DM) patients, particularly with diabetic retinopathy (DR). To elucidate the pathogenesis of the AGE-induced toxicity to retinal epithelial cells, we investigated the role of Fas-Fas ligand (FasL) signaling and mitochondrial dysfunction in the AGE-induced apoptosis. Results demonstrated that the AGE-BSA- induced apoptosis of retinal ARPE-19 cells. And the AGE-BSA treatment caused mitochondrial dysfunction, via deregulating the B-cell lymphoma 2 (Bcl-2) signaling. Moreover, the Fas/FasL and its downstreamer Caspase 8 were promoted by the AGE-BSA treatment, and the exogenous α-Fas exacerbated the activation of Caspase 3/8. On the other side, the siRNA-mediated knockdown of Fas/FasL inhibited the AGE-BSA-induced apoptosis. Taken together, we confirmed the activation of Fas-FasL signaling and of mitochondrial dysfunction in the AGE-BSA-promoted apoptosis in retinal ARPE-19 cells, implying the important role of Fas-FasL signaling in the DR in DM.

The downregulation of thioredoxin accelerated Neuro2a cell apoptosis induced by advanced glycation end product via activating several pathways

Thioredoxin (Trx), a 12 kDa protein, has different functions in different cellular environments, playing important anti-oxidative and anti-apoptotic roles and regulating the expression of transcription factors. Advanced glycation end products (AGEs) are a heterogeneous group of irreversible adducts from glucose–protein condensation reactions and are considered crucial to the development of diabetic nephropathy, retinopathy, neurodegeneration and atherosclerosis. The aim of this study was to use a Trx inhibitor to investigate the effects and mechanism of Trx down-regulation on AGE-induced Neuro2a cell apoptosis. Neuro2a cells were cultured in vitro and treated with different conditions. The apoptosis and proliferation of Neuro2a cells were detected using flow cytometry, DNA-Ladder and CCK8 assays. Rho 123 was used to detect the mitochondrial membrane potential. ROS generation and caspase3 activity were detected using a DCFH-DA probe and micro-plate reader. Western blotting and real-time PCR were used to detect the expression of proteins and genes. We found that the down-regulation of thioredoxin could accelerate AGE-induced apoptosis in Neuro2a cells. A possible underlying mechanism is that the down-regulation of thioredoxin stimulated the up-regulation of ASK1, p-JNK, PTEN, and Txnip, as well as the down-regulation of p-AKT, ultimately increasing ROS levels and caspase3 activity.