Knockout of receptor for advanced glycation end‐products attenuates age‐related renal lesions

Pro‐aging effects of endogenous advanced glycation end‐products (AGEs) have been reported, and there is increasing interest in the pro‐inflammatory and ‐fibrotic effects of their binding to RAGE (the main AGE receptor). The role of dietary AGEs in aging remains ill‐defined, but the predominantly renal accumulation of dietary carboxymethyllysine (CML) suggests the kidneys may be particularly affected. We studied the impact of RAGE invalidation and a CML‐enriched diet on renal aging. Two‐month‐old male, wild‐type (WT) and RAGE^?/? C57Bl/6 mice were fed a control or a CML‐enriched diet (200 μg CML/g_food) for 18 months. Compared to controls, we observed higher CML levels in the kidneys of both CML WT and CML RAGE^?/? mice, with a predominantly tubular localization. The CML‐rich diet had no significant impact on the studied renal parameters, whereby only a trend to worsening glomerular sclerosis was detected. Irrespective of diet, RAGE^?/? mice were significantly protected against nephrosclerosis lesions (hyalinosis, tubular atrophy, fibrosis and glomerular sclerosis) and renal senile apolipoprotein A‐II (ApoA‐II) amyloidosis (p < 0.001). A positive linear correlation between sclerosis score and ApoA‐II amyloidosis score (r = 0.92) was observed. Compared with old WT mice, old RAGE^?/? mice exhibited lower expression of inflammation markers and activation of AKT, and greater expression of Sod2 and SIRT1. Overall, nephrosclerosis lesions and senile amyloidosis were significantly reduced in RAGE^?/? mice, indicating a protective effect of RAGE deletion with respect to renal aging. This could be due to reduced inflammation and oxidative stress in RAGE^?/? mice, suggesting RAGE is an important receptor in so‐called inflamm‐aging.     

Advanced glycation end product ligands for the receptor for advanced glycation end products: biochemical characterization and formation kinetics

Advanced glycation end products (AGEs) accumulate with age and at an accelerated rate in diabetes. AGEs bind cell-surface receptors including the receptor for advanced glycation end products (RAGE). The dependence of RAGE binding on specific biochemical characteristics of AGEs is currently unknown. Using standardized procedures and a variety of AGE measures, the present study aimed to characterize the AGEs that bind to RAGE and their formation kinetics in vitro. To produce AGEs with varying RAGE binding affinity, bovine serum albumin (BSA) AGEs were prepared with 0.5M glucose, fructose, or ribose at times of incubation from 0 to 12 weeks or for up to 3 days with glycolaldehyde or glyoxylic acid. The AGE-BSAs were characterized for RAGE binding affinity, fluorescence, absorbance, carbonyl content, reactive free amine content, molecular weight, pentosidine content, and N-epsilon-carboxymethyl lysine content. Ribose-AGEs bound RAGE with high affinity within 1 week of incubation in contrast to glucose- and fructose-AGE, which required 12 and 6 weeks, respectively, to generate equivalent RAGE ligands (IC50=0.66, 0.93, and 1.7 microM, respectively). Over time, all of the measured AGE characteristics increased. However, only free amine content robustly correlated with RAGE binding affinity. In addition, detailed protocols for the generation of AGEs that reproducibly bind RAGE with high affinity were developed, which will allow for further study of the RAGE-AGE interaction.     

Retracted: Advanced glycation end-products induce oxidative stress through the Sirt1/Nrf2 axis by interacting with the receptor of AGEs under diabetic conditions

Despite the administration of exogenous insulin and other medications used to control many aspects of diabetes mellitus (DM), increased oxidative stress has been increasingly acknowledged in DM development and complications. Therefore, this study aims to investigate the role of advanced glycation end-products (AGEs) in oxidative stress (OS) of thyroid cells in patients with DM. Patients with DM with or without thyroid dysfunction (TD) were enrolled. Thyroid toxic damage was induced by adding AGE-modified bovine serum albumin (AGE-BSA) to normal human thyroid follicular epithelial cells. The cell viability, cell cycle, and cell apoptosis, as well as the content of reactive oxygen species (ROS), catalase (CAT), and malondialdehyde (MDA) in cells were measured. Thyroid hormones, T3, T4, FT3, and FT4 levels were measured by enzyme-linked immunosorbent assay. Receptor for advanced glycation end products (RAGE), sirtuin1 ( Sirt1), and NF-E2-related factor 2 ( Nrf2) expressions were detected, and the mitochondrial membrane potential was measured. We found increased AGEs in the serum of DM patients with TD. By increasing AGE-BSA concentration, cell viability; the thyroid hormones T3, T4, FT3, and FT4 levels; and mitochondrial membrane potential all significantly decreased. However, the increase in AGE-BSA concentration led to an increase in cell apoptosis, RAGE, and nuclear factor-κB expressions but produced the opposite effect on Sirt1, Nrf2, and heme oxygenase-1 expressions, as well as a decrease in antioxidant response element protein levels. The AGE-BSA increased ROS and MDA levels and reduced CAT level in normal human thyroid follicular epithelial cells on a dose independence basis. Our results demonstrated that AGEs-mediated direct increase of RAGE produced OS in thyroid cells of DM by inactivating the Sirt1/Nrf2 axis.     

缬沙坦对人肾小球系膜细胞糖基化终产物受体表达的影响

目的：本实验探讨缬沙坦对糖基化终产物诱导的人肾小球系膜细胞氧化应激水平及糖基化终产物受体（RAGE）表达的影响。方法：体外常规培养人肾小球系膜细胞,运用糖基化修饰的牛血清白蛋白（AGE-BSA）和缬沙坦进行干预,流式细胞术检测细胞内活性氧（ROS）,RT-PCR法检测NADPH氧化酶的亚基p47^phox的mRNA表达,RT-PCR和细胞免疫化学法检测RAGE的表达量。结果：缬沙坦干预组人肾小球系膜细胞的ROS产生量、NADPH氧化酶的亚基p47^phox mRNA表达量、RAGE表达量均低于AGE-BSA组（P〈0．05）,且缬沙坦的抑制作用呈浓度和时间依赖性。结论：缬沙坦可能通过降低氧化应激水平来抑制RAGE的表达。     

Advanced glycation end products regulate anabolic and catabolic activities via NLRP3‐inflammasome activation in human nucleus pulposus cells

Intervertebral disc degeneration is widely recognized as a cause of lower back pain, neurological dysfunction and other musculoskeletal disorders. The major inflammatory cytokine IL‐1β is associated with intervertebral disc degeneration; however, the molecular mechanisms that drive IL‐1β production in the intervertebral disc, especially in nucleus pulposus (NP) cells, are unknown. In some tissues, advanced glycation end products (AGEs), which accumulate in NP tissues and promote its degeneration, increase oxidative stress and IL‐1β secretion, resulting in disorders, such as obesity, diabetes mellitus and ageing. It remains unclear whether AGEs exhibit similar effects in NP cells. In this study, we observed significant activation of the NLRP3 inflammasome in NP tissues obtained from patients with degenerative disc disease compared to that with idiopathic scoliosis according to results detected by Western blot and immunofluorescence. Using NP cells established from healthy tissues, our in vitro study revealed that AGEs induced an inflammatory response in NP cells and a degenerative phenotype in a NLRP3‐inflammasome‐dependent manner related to the receptor for AGEs (RAGE)/NF‐κB pathway and mitochondrial damage induced by mitochondrial reactive oxygen species (mtROS) generation, mitochondrial permeability transition pore (mPTP) activation and calcium mobilization. Among these signals, both RAGE and mitochondrial damage primed NLRP3 and pro‐IL‐1β activation as upstream signals of NF‐κB activity, whereas mitochondrial damage was critical for the assembly of inflammasome components. These results revealed that accumulation of AGEs in NP tissue may initiate inflammation‐related degeneration of the intervertebral disc via activation of the NLRP3 inflammasome.     

艾塞那肽对糖尿病大鼠肾脏损伤的保护作用

目的：探讨GLP-1类似物艾塞那肽（exenatide）对链脲佐菌素诱导的糖尿病大鼠肾脏的保护作用。方法：SD大鼠随机分为正常组（NC组,n=8）和模型组;模型组给予高脂高糖饲料,喂养4周后腹腔注射STZ（30 mg·kg^-1）建模,72 h后以血糖≥ 16.7 mmol·L^-1为糖尿病成模标准,将成模大鼠随机分为糖尿病对照组（DM组,n=10）、3 μg·kg^-1艾塞那肽干预（Ex-1）组和6μg·kg^-1艾塞那肽干预（Ex-2）组;艾塞那肽组连续皮下注射艾塞那肽（bid）12周,NC组和DM组注射等容积溶剂;测定各组大鼠糖脂代谢变化和肾功能指标如血肌酐（Scr）、尿肌酐（Ucr）、尿素氮（BUN）、24 h尿微量白蛋白排出率（24 h UMA）并计算肌酐清除率（Ccr）;测定肾组织氧化应激指标超氧化物歧化酶（SOD）、丙二醛（MDA）和谷胱甘肽过氧化物酶（GSH-Px）;HE染色观察肾组织病理形态及ELISA法测定肾组织糖基化终末产物AGEs水平。结果：与糖尿病组相比,艾塞那肽可明显改善糖尿病大鼠糖脂代谢,血糖、糖基化血红蛋白（HbAlc）、甘油三脂及胆固醇值均下降（P < 0.05）,肾功能指标明显好转（P < 0.05）且肌酐清除率下降（P < 0.05）,提示肾小球高滤过状态;同时改善糖尿病引起的肾组织病理结构改变,AGEs浓度下降（P < 0.05）,氧化应激指标SOD和GSH-Px活力升高,MDA含量降低（P < 0.05）。结论：艾塞那肽具有肾脏保护作用,其机制可能与抑制糖尿病大鼠肾组织的AGEs生成和改善氧化应激有关。     

Caffeic Acid Inhibits the Formation of Advanced Glycation End Products (AGEs) and Mitigates the AGEs‐Induced Oxidative Stress and Inflammation Reaction in Human Umbilical Vein Endothelial Cells (HUVECs)

The advanced glycation end products (AGEs) are the compounds produced by non‐enzymatic glycation reaction of proteins and sugars, which can induce the generation of free radicals and the expression of inflammatory factors, thereby playing an important role in vascular dysfunction in diabetes. To investigate the effects of caffeic acid (CA) on glycation formed by glucose and protein, various spectroscopic techniques and molecular docking methods were carried out. Furthermore, the protective effects of CA on human umbilical vein endothelial cells (HUVECs) damaged by AGEs were detected. The results indicated that CA inhibited AGEs formation in vitro, decreased the expression of IL‐1β, IL‐18, ICAM‐1, VCAM‐1, NLRP3, Caspase‐1 and CRP (C‐reactive protein) and reduced the ROS in HUVECs exposed to AGEs. Our findings suggested that the supplementation with dietary CA could prevent and delay the AGEs‐induced vascular dysfunction in diabetes.     

Pioglitazone attenuates advanced glycation end products‐induced apoptosis and calcification by modulating autophagy in tendon‐derived stem cells

Diabetes mellitus (DM) is one of the prominent risk factors for pathological development and progression of tendinopathy. One feature of DM‐related changes in tendinopathy is accumulation of advanced glycation end products (AGEs) in affected tendons. Pioglitazone (Pio), a peroxisome proliferator‐activated receptor γ agonist, performs a protective effect against AGEs. The present study aimed to investigate the pathogenetic role of AGEs on tendon‐derived stem cells (TDSCs) and to determine the effect of Pio on AGEs‐induced TDSC dysfunctions. Results indicated that AGEs induced TDSC apoptosis as well as compensatory activation of autophagy. Pharmacologic activation/inhibition of autophagy leaded to alleviate/exacerbate apoptosis induced by AGEs. We further confirmed the effect of Pio on autophagy, which ameliorated apoptosis and abnormal calcification caused by AGEs both in vitro and in vivo. Thus, we suggest that Pio ameliorates the dysfunctions of TDSCs against AGEs by promoting autophagy, and we also reveal that Pio is a potential pharmacological choice for tendinopathy.     

Expression and purification of the extracellular domains of human glycoprotein VI (GPVI) and the receptor for advanced glycation end products (RAGE) from Rattus norvegicus in Leishmania tarentolae

Glycosylation is one of the most complex post-translational modifications and may have significant influence on the proper function of the corresponding proteins. Bacteria and yeast are, because of easy handling and cost reasons, the most frequently used systems for recombinant protein expression. Bacteria generally do not glycosylate proteins and yeast might tend to hyperglycosylate. Insect cell- and mammalian cell-based expression systems are able to produce complex N-glycosylation structures but are more complex to handle and more expensive. The nonpathogenic protozoa Leishmania tarentolae is an easy-to-handle alternative expression system for production of proteins requiring the eukaryotic protein folding machinery and post-translational modifications. We used and evaluated the system for the secretory expression of extracellular domains from human glycoprotein VI and the receptor for advanced glycation end products from rat. Both proteins were well expressed and homogeneously glycosylated. Analysis of the glycosylation pattern identified the structure as the conserved core pentasaccharide Man₃GlcNac₂.     

Continuous infusion of substance P into rat striatum alleviates nociceptive behavior via phosphorylation of extracellular signal‐regulated kinase 1/2

Intraplantar injection of 0.4% formalin into the rat hind paw leads to a biphasic nociceptive response; an ‘acute’ phase (0–15 min) and ‘tonic’ phase (16–120 min), which is accompanied by significant phosphorylation of extracellular signal‐regulated kinase (ERK)1/2 in the contralateral striatum at 120 min post‐formalin injection. To uncover a possible relationship between the slow‐onset substance P (SP) release and increased ERK1/2 phosphorylation in the striatum, continuous infusion of SP into the striatum by reverse microdialysis (0.4 μg/mL in microdialysis fiber, 1 μL/min) was performed to mimic volume neurotransmission of SP. Continuous infusion for 3 h of SP reduced the duration of ‘tonic’ phase nociception, and this SP effect was mediated by neurokinin 1 (NK1) receptors since pre‐treatment with NK1 receptor antagonist CP96345 (10 μM) blocked the effect of SP infusion. However, formalin‐induced ‘tonic’ phase nociception was significantly prolonged following acute injection of the MAP/ERK kinase 1/2 inhibitor PD0325901 (100 pmol) by microinjection. The coinfusion of SP and PD0325901 significantly increased the ‘tonic’ phase of nociception. These data demonstrate that volume transmission of striatal SP triggered by peripheral nociceptive stimulation does not lead to pain facilitation but a significant decrease of tonic nociception by the activation of the SP‐NK1 receptor–ERK1/2 system.

     

Anti‐invasion effect of rosmarinic acid via the extracellular signal‐regulated kinase and oxidation–reduction pathway in Ls174‐T cells

Rosmarinic acid is a major phenylpropanoid isolated from Prunella vulgaris L., which is a composition of herbal tea for centuries in China. However, the anti‐invasion activity on Ls174‐T human colon carcinoma cells has not been studied. In this study, we investigated the anti‐metastasis functions according to wound healing assay, adhesion assay, and Transwell assay and found that rosmarinic acid could inhibit migration, adhesion, and invasion dose‐dependently. Rosmarinic acid also could decrease the level of reactive oxygen species by enhancing the level of reduced glutathione hormone. In addition, rosmarinic acid repressed the activity and expression of matrix metalloproteinase‐2,9. According to Western blot and quantitative real‐time PCR assay, rosmarinic acid may inhibit metastasis from colorectal carcinoma mainly via the pathway of extracellular signal‐regulated kinase. In animal experiment, intraperitoneal administration of 2 mg of rosmarinic acid reduced weight of tumors and the number of lung nodules significantly compared with those of control group. Therefore, these results demonstrated that rosmarinic acid can effectively inhibit tumor metastasis in vitro and in vivo. J. Cell. Biochem. 111: 370–379, 2010. © 2010 Wiley‐Liss, Inc.     

Interferon‐γ induces leucine‐rich repeat kinase LRRK2 via extracellular signal‐regulated kinase ERK5 in macrophages

The gene encoding leucine‐rich repeat kinase 2 (LRRK2) comprises a major risk factor for Parkinson's disease. Recently, it has emerged that LRRK2 plays important roles in the immune system. LRRK2 is induced by interferon‐γ (IFN‐γ) in monocytes, but the signaling pathway is not known. Here, we show that IFN‐γ‐mediated induction of LRRK2 was suppressed by pharmacological inhibition and RNA interference of the extracellular signal‐regulated kinase 5 (ERK5). This was confirmed by LRRK2 immunostaining, which also revealed that the morphological responses to IFN‐γ were suppressed by ERK5 inhibitor treatment. Both human acute monocytic leukemia THP‐1 cells and human peripheral blood monocytes stimulated the ERK5‐LRRK2 pathway after differentiation into macrophages. Thus, LRRK2 is induced via a novel, ERK5‐dependent IFN‐γ signal transduction pathway, pointing to new functions of ERK5 and LRRK2 in human macrophages.

     

Advanced oxidation protein products induce pre‐osteoblast apoptosis through a nicotinamide adenine dinucleotide phosphate oxidase‐dependent,mitogen‐activated protein kinases‐mediated intrinsic apoptosis pathway

Osteoblast apoptosis contributes to age‐related bone loss. Advanced oxidation protein products (AOPPs) are recognized as the markers of oxidative stress and potent inducers of apoptosis. We have demonstrated that AOPP accumulation was correlated with age‐related bone loss. However, the effect of AOPPs on the osteoblast apoptosis still remains unknown. Exposure of osteoblastic MC3T3‐E1 cells to AOPPs caused the excessive generation of reactive oxygen species (ROS) by activating nicotinamide adenine dinucleotide phosphate (NADPH) oxidases. Increased ROS induced phosphorylation of mitogen‐activated protein kinases (MAPKs), which subsequently triggered intrinsic apoptosis pathway by inducing mitochondrial dysfunction, endoplasmic reticulum stress, and Ca²⁺ overload and eventually leads to apoptosis. Chronic AOPP loading in aged Sprague‐Dawley rats induced osteoblast apoptosis and activated NADPH oxidase signaling cascade, in combination with accelerated bone loss and deteriorated bone microstructure. Our study suggests that AOPPs induce osteoblast apoptosis by the NADPH oxidase‐dependent, MAPK‐mediated intrinsic apoptosis pathway.     

MRP8/14 mediates macrophage efferocytosis through RAGE and Gas6/MFG‐E8, and induces polarization via TLR4‐dependent pathway

Myeloid‐related protein 8/14 (MRP8/14) participates in various inflammatory responses, however, its effect on macrophage efferocytosis remains unclear. Here, we demonstrate that MRP8/14 significantly inhibits the efferocytosis of apoptotic thymocytes by mouse bone marrow‐derived macrophages (BMDMs), which later proves to be associated with the receptor for advanced glycation end products (RAGE) or for reducing the expression of growth arrest‐specific protein 6 and milk fat globule epidermal growth factor 8, independent of RAGE. Furthermore, MRP8/14 promotes polarization of BMDMs from the M₂‐ to M₁‐like phenotype by upregulating expression of M₁‐related surface receptor proteins and signature M₁‐marker genes and by downregulating signature M₂‐marker gene expression, which depends on Toll‐like receptor 4 and p38 mitogen‐activated protein kinase/nuclear factor κB pathways. Thus, we report a significant inhibitory effect of MRP8/14 on macrophage efferocytosis and MRP8/14‐mediated phenotypic polarization, which may be helpful in developing novel therapeutic strategies leading to inflammation resolution.