Receptor For Advanced Glycation End-products Expression Research Articles

Erjingwan and Alzheimer's disease: research based on network pharmacology and experimental confirmation.

Alzheimer's disease (AD), a challenging neurodegenerative condition, has emerged as a significant global public health concern. The Chinese medicine decoction Erjingwan (EJW) has shown promising efficacy in AD treatment, though its mechanism remains unclear. This study aims to elucidate the mechanism by which EJW treats AD through network pharmacology analysis and in vivo experiments. We identified EJW's components using the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and determined AD-related targets from various databases. A network comprising herbs-compounds-targets was established, and EJW's core targets were ascertained through protein-protein interaction (PPI) analysis. This study assessed the cognitive abilities of APP/PS1 mice using Morris water mazes and Y mazes, in addition to analyzing blood samples for triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) levels. Brain tissues were examined histologically with HE staining, Nissl staining, and immunohistochemistry (IHC) for amyloid β-protein (Aβ) detection. Superoxide dismutase (SOD), reactive oxygen species (ROS), Interleukin-1β (IL-1β), and Interleukin-6 (IL-6) levels in the hippocampal region were measured by ELISA. mRNA expression of apolipoprotein A-I (APOA-I), apolipoprotein B (APOB), apolipoprotein E4 (APOE4), advanced glycation end products (AGE), the receptor for AGE (RAGE), and nuclear factor kappa-B (NF-κB) was evaluated by quantitative PCR (q-PCR). Western blotting was used to detect the expression of AGE, RAGE, NF-κB, and Tau protein. Screening identified 57 chemical components and 222 potential targets of EJW. Ten core targets for AD treatment were identified, with enrichment analysis suggesting EJW's effects are related to lipid metabolism and AGEs/RAGE pathways. EJW enhanced learning and memory in APP/PS1 mice, protected neuronal structure in the hippocampal region, reduced Aβ deposition, and altered levels of TG, TC, LDL, IL-1β, and IL-6, and the expression of APOE4, AGEs, RAGE, NF-κB, and Tau protein, while increasing SOD, APOA-I, and APOB mRNA expression. The study identified four core components of EJW-iosgenin, baicalein, beta-sitosterol, quercetin-and ten core targets including AKT1, IL6, VEGFA, TP53, CASP3, for treating AD. Experimental results demonstrate EJW's capacity to modulate lipid profiles, reduce pathological markers such as Aβ1-42, Tau, IL-6, IL-1β, reactive oxygen species, SOD, and enhance cognitive functions in APP/PS1 mice, potentially through inhibiting the AGEs/RAGE/NF-κB pathway.

Modulating intestinal health: Impact of chitooligosaccharide molecular weight on suppressing RAGE expression and inflammatory response in methylglyoxal-induced advanced glycation end-products

The accumulation of methylglyoxal (MGO) produced in high-temperature processed foods and excessive production in the body contributes to intestinal barrier dysfunction. In this study, we investigated the effects of chitooligosaccharides (COSs) of different molecular weights (<1 kDa, 1–3 kDa, 3–5 kDa, 5–10 kDa, and >10 kDa) on MGO-induced intestinal barrier dysfunction. We investigated the effect of COSs on inhibiting intracellular MGO accumulation/MGO-derived AGEs production and regulating the receptor for AGE (RAGE)-mediated downstream protein expression, including proteins related to apoptosis and inflammation, intestinal barrier integrity, and paracellular permeability. Pretreatment with COSs ameliorated MGO-induced increased RAGE protein expression, activation of apoptotic cascade/inflammatory response, loss of intestinal epithelial barrier integrity, and increased paracellular permeability, ameliorating intestinal dysfunction through MGO scavenging. 1–3 kDa COSs most effectively ameliorated MGO-induced intestinal dysfunction. Our results suggest the potential of COSs in improving intestinal health by ameliorating intestinal barrier dysfunction by acting as an MGO scavenger and highlighting the need for the optimization of the molecular weight of COSs to optimize its protective effects.

Analysis of changes in high-mobility group box 1, receptor for advanced glycation endproducts, and T helper 17/regulatory T balance in severe preeclampsia with acute heart failure.

We measured the levels of High-Mobility Group Box 1 (HMGB1), Receptor for Advanced Glycation Endproducts (RAGE), T Helper 17 cells (Th17), Regulatory T cells (Treg), and related cytokines in the peripheral blood of patients with severe preeclampsia (SPE) complicated with acute heart failure (AHF) to explore the expression changes in these indicators. In total, 96 patients with SPE admitted to Gansu Provincial Maternity and Child-care Hospital between June 2020 and June 2022 were included in the study. The patients were divided into SPE+AHF (40 patients) and SPE (56 patients) groups based on whether they suffered from AHF. Additionally, 56 healthy pregnant women who either received prenatal examinations or were admitted to our hospital for delivery during the same period were selected as the healthy control group. An enzyme-linked immunosorbent assay was performed to detect the expression levels of HMGB1, RAGE, interleukin (IL)-17, IL-6, transforming growth factor β (TGF-β), IL-10, and NT-proBNP in plasma. Flow cytometry was employed to determine the percentages of Th17 and Treg cells. Compared to the healthy control group, the SPE+AHF and SPE groups had higher plasma levels of HMGB1 and RAGE expression, higher Th17 percentage and Th17/Treg ratio, and lower Treg percentage. Compared to the SPE group, the SPE+AHF group had higher plasma levels of HMGB1 and RAGE expression, higher Th17 percentage and Th17/Treg ratio, and lower Treg percentage (P<.05). In patients with SPE with AHF, plasma HMGB1 was positively correlated with RAGE, Th17, Th17/Treg, IL-17, and IL-6 and was negatively correlated with TGF-β and IL-10 (P<.05). Our findings revealed that patients with SPE with AHF had elevated levels of HMGB1 and RAGE while exhibiting Th17/Treg immune imbalance, suggesting that the abnormal expression of these indicators may be involved in the pathogenesis of SPE with AHF.

Comparative Study of the Effects of Dietary-Free and -Bound Nε-Carboxymethyllysine on Gut Microbiota and Intestinal Barrier.

Nε-carboxymethyllysine (CML) is produced by a nonenzymatic reaction between reducing sugar and ε-amino group of lysine in food and exists as free and bound forms with varying digestibility and absorption properties in vivo, causing diverse interactions with gut microbiota. The effects of different forms of dietary CML on the gut microbiota and intestinal barrier of mice were explored. Mice were exposed to free and bound CML for 12 weeks, and colonic morphology, gut microbiota, fecal short-chain fatty acids (SCFAs), intestinal barrier, and receptor for AGE (RAGE) signaling cascades were measured. The results indicated that dietary-free CML increased the relative abundance of SCFA-producing genera including Blautia, Faecalibacterium, Agathobacter, and Roseburia. In contrast, dietary-bound CML mainly increased the relative abundance of Akkermansia. Moreover, dietary-free and -bound CML promoted the gene and protein expression of zonula occludens-1 and claudin-1. Additionally, the intake of free and bound CML caused an upregulation of RAGE expression but did not activate downstream inflammatory pathways due to the upregulation of oligosaccharyl transferase complex protein 48 (AGER1) expression, indicating a delicate balance between protective and proinflammatory effects in vivo. Dietary-free and -bound CML could modulate the gut microbiota community and increase tight-junction expression, and dietary-free CML might exert a higher potential benefit on gut microbiota and SCFAs than dietary-bound CML.

MiR-23a-5p alleviates chronic obstructive pulmonary disease through targeted regulation of RAGE-ROS pathway

BackgroundChronic obstructive pulmonary disease (COPD) is a common respiratory disease and represents the third leading cause of death worldwide. This study aimed to investigate miRNA regulation of Receptor for Advanced Glycation End-products (RAGE), a causal receptor in the pathogenesis of cigarette smoke (CS)-related COPD, to guide development of therapeutic strategies.MethodsRAGE expression was quantified in lung tissue of COPD patients and healthy controls, and in mice with CS-induced COPD. RNA-sequencing of peripheral blood from COPD patients with binding site prediction was used to screen differentially expressed miRNAs that may interact with RAGE. Investigation of miR-23a-5p as a potential regulator of COPD progression was conducted with miR-23a-5p agomir in COPD mice in vivo using histology and SCIREQ functional assays, while miR-23a-5p mimics or RAGE inhibitor were applied in 16-HBE human bronchial epithelial cells in vitro. RNA-sequencing, ELISA, and standard molecular techniques were used to characterize downstream signaling pathways in COPD mice and 16-HBE cells treated with cigarette smoke extract (CSE).ResultsRAGE expression is significantly increased in lung tissue of COPD patients, COPD model mice, and CSE-treated 16-HBE cells, while inhibiting RAGE expression significantly reduces COPD severity in mice. RNA-seq analysis of peripheral blood from COPD patients identified miR-23a-5p as the most significant candidate miRNA interaction partner of RAGE, and miR-23a-5p is significantly downregulated in mice and cells treated with CS or CSE, respectively. Injection of miR-23a-5p agomir leads to significantly reduced airway inflammation and alleviation of symptoms in COPD mice, while overexpressing miR-23a-5p leads to improved lung function. RNA-seq with validation confirmed that reactive oxygen species (ROS) signaling is increased under CSE-induced aberrant upregulation of RAGE, and suppressed in CSE-stimulated cells treated with miR-23a-5p mimics or overexpression. ERK phosphorylation and subsequent cytokine production was also increased under RAGE activation, but inhibited by increasing miR-23a-5p levels, implying that the miR-23a-5p/RAGE/ROS axis mediates COPD pathogenesis via ERK activation.ConclusionsThis study identifies a miR-23a-5p/RAGE/ROS signaling axis required for pathogenesis of COPD. MiR-23a-5p functions as a negative regulator of RAGE and downstream activation of ROS signaling, and can inhibit COPD progression in vitro and in vivo, suggesting therapeutic targets to improve COPD treatment.Graphical

N-acetylserotonin alleviates retinal ischemia-reperfusion injury via HMGB1/RAGE/NF-κB pathway in rats.

To observe the effects of N-acetylserotonin (NAS) administration on retinal ischemia-reperfusion (RIR) injury in rats and explore the underlying mechanisms involving the high mobility group box 1 (HMGB1)/receptor for advanced glycation end-products (RAGE)/nuclear factor-kappa B (NF-κB) signaling pathway. A rat model of RIR was developed by increasing the pressure of the anterior chamber of the eye. Eighty male Sprague Dawley were randomly divided into five groups: sham group (n=8), RIR group (n=28), RIR+NAS group (n=28), RIR+FPS-ZM1 group (n=8) and RIR+NAS+ FPS-ZM1 group (n=8). The therapeutic effects of NAS were examined by hematoxylin-eosin (H&E) staining, and retinal ganglion cells (RGCs) counting. The expression of interleukin 1 beta (IL-1β), HMGB1, RAGE, and nod-like receptor 3 (NLRP3) proteins and the phosphorylation of nuclear factor-kappa B (p-NF-κB) were analyzed by immunohistochemistry staining and Western blot analysis. The expression of HMGB1 protein was also detected by enzyme-linked immunosorbent assay (ELISA). H&E staining results showed that NAS significantly reduced retinal edema and increased the number of RGCs in RIR rats. With NAS therapy, the HMGB1 and RAGE expression decreased significantly, and the activation of the NF-κB/NLRP3 pathway was antagonized along with the inhibition of p-NF-κB and NLRP3 protein expression. Additionally, NAS exhibited an anti-inflammatory effect by reducing IL-1β expression. The inhibitory of RAGE binding to HMGB1 by RAGE inhibitor FPS-ZM1 led to a significant decrease of p-NF-κB and NLRP3 expression, so as to the IL-1β expression and retinal edema, accompanied by an increase of RGCs in RIR rats. NAS may exhibit a neuroprotective effect against RIR via the HMGB1/RAGE/NF-κB signaling pathway, which may be a useful therapeutic target for retinal disease.

Effect of aminoguanidine on plasminogen activator inhibitor-1 and receptor of advanced glycation endproduct in the liver of streptozotocin-induced diabetic rats

Objectives: Advanced glycation end products (AGEs) play an important role in the development and progression of diabetic complications. The receptor for AGE (RAGE) is the ligand-binding site of AGE that initiates and accelerates the atherosclerotic process. Plasminogen activator inhibitor-1 (PAI-1) is a prothrombotic factor that has been proposed as a biological marker for prognostic assessment, monitoring of microvascular and macrovascular complications in diabetes. The purpose of this study is to investigate the effects of aminoguanidine on RAGE and PAI-1 expression levels in the liver of streptozotocin-induced diabetic rats.&#x0D; Methods: Diabetes was induced in rats by intraperitoneal injection of streptozocin (STZ, 50 mg/kg). On day 3, diabetic rats were administered 50, 100, and 200 mg/kg/day of aminoguanidine. The expression of PAI-1 and RAGE in the liver tissue was evaluated using real-time PCR.&#x0D; Results: PAI-1 and RAGE gene expression levels were higher in the liver of the diabetic rats compared to the control group. Aminoguanidine at 50, 100, and 200 mg/kg decreased PAI-1 and RAGE gene expression in the liver (p&lt;0.001 at all doses). However, these genes were downregulated only at a dose of 200 mg/kg in healthy rats (p&lt;0.0001). In addition, hepatic AGE protein levels were significantly decreased following treatment of the diabetic rats with aminoguanidine (p&lt;0.001). There was also a significant correlation between AGE protein concentration and the expression of PAI-1 and RAGE.&#x0D; Conclusion: In summary, the data of the present study suggest that aminoguanidine reduced the expression of PAI-1 and RAGE in the liver of the diabetic rats.

A Potential Role for the Receptor for Advanced Glycation End-Products (RAGE) in the Development of Secondhand Smoke-Induced Chronic Sinusitis.

Chronic sinusitis (CS) is characterized by sinonasal inflammation, mucus overproduction, and edematous mucosal tissue. CS impacts one in seven adults and estimates suggest up to 15% of the general U.S. population may be affected. This research sought to assess a potential role for receptors for advanced glycation end-products (RAGE), an inflammatory receptor expressed in tissues exposed to secondhand smoke (SHS). Human sinus tissue sections were stained for RAGE and S100s, common RAGE ligands. Wild-type mice and mice that over-express RAGE in sinonasal epithelium (RAGE TG) were maintained in room air (RA) or exposed to secondhand smoke (SHS) via a nose-only delivery system five days a week for 6 weeks. Mouse sections were stained for RAGE and tissue lysates were assayed for cleaved caspase 3, cytokines, or matrix metalloproteases. We discovered increased RAGE expression in sinus tissue following SHS exposure and in sinuses from RAGE TG mice in the absence of SHS. Cleaved caspase-3, cytokines (IL-1β, IL-3, and TNF-α), and MMPs (-9 and -13) were induced by SHS and in tissues from RAGE TG mice. These results expand the inflammatory role of RAGE signaling, a key axis in disease progression observed in smokers. In this relatively unexplored area, enhanced understanding of RAGE signaling during voluntary and involuntary smoking may help to elucidate potential therapeutic targets that may attenuate the progression of smoke-related CS.

Biological and clinical significance of the AGE-RAGE axis in the aggressiveness and prognosis of prostate cancer.

Dietary factors and chronic hyperglycemia are linked to the formation of advanced glycation end products (AGEs) and prostate cancer (PCa) risk. The activation of the receptor for AGEs (RAGE) acts as a bridge between various RAGE ligands and certain malignancies. This study showed that the interaction of AGEs and RAGE promoted PCa cell proliferation, invasion, and autophagy-mediated survival in response to chemotherapeutic agents. RAGE-overexpressed PCa cells underwent epithelial-mesenchymal transition and showed increased cancer stem cell-like properties. In mouse xenograft models, RAGE-overexpressed cells showed more substantial tumorigenic capacity than parental cells, whereas RAGE knockdown decreased tumorigenicity. The clinical data validated a positive correlation between high AGE and RAGE expressions with poor clinical outcomes. Our findings suggest that the AGE-RAGE axis facilitates PCa progression and aggressiveness. Prostatic AGEs and RAGE expression levels are associated with PCa prognosis. Adherence to a reduced-AGE diet and targeting RAGE are potential approaches to complement and synergize with the current PCa therapies.

Abstract 14597: Beyond Sex Bias: The Uncharted Role of Rage Signaling in Pulmonary Arterial Hypertension

Background: Pulmonary arterial hypertension (PAH) is a life-threatening disease that initiates in response to pulmonary artery endothelial cells (PAEC) damage followed by inadequate repair. The Receptor for Advanced Glycation Endproducts (RAGE) triggered upon the damage, plays a crucial role in vascular regeneration. However, its specific impact on PAH remains incompletely explored. Hypothesis: In this study, we investigated the sex-specific outcome of RAGE deletion on PAEC homeostasis. Methods: The effect of the global RAGE knockout (KO) on pulmonary hemodynamics, cardiac and endothelial function was studied disaggregated by sex in WT and RAGE KO rats. Endothelial integrity was evaluated via the expression of endothelial junction proteins, the activity of barrier disruptive signaling pathways, and in vivo pulmonary vascular permeability assay. To understand the significance of diminished RAGE signaling in male PAH, we measured membrane RAGE levels in untreated and Sugen/Hypoxia treated rats of both sexes and examined the publicly available single-cell transcriptomic profiles of human PAECs. Results: Male but not female RAGE KO rats developed a spontaneous PAH characterized by a significant increase in the right ventricle (RV) systolic pressure and RV hypertrophy. The PAH phenotype in males corresponded with a male-specific decrease in the expression of CD31 (WT vs. RAGE KO: 1.0 ± 0.07 vs. 0.1 ± 0.02, p &lt;0001, N=6), activation of p38/RhoA signaling axis (p-p38 in WT vs. RAGE KO: 1.0 ± 0.2 vs. 3.2 ± 0.6, p=0.01, N=4), significant PAEC cytoskeletal rearrangement, and increased FITC-dextran extravasation, while female RAGE KOs remained unaffected. We also discovered that Sugen/Hypoxia treatment induced a substantial loss of membrane RAGE, specifically in males. Additionally, human PAEC transcriptomic data revealed that RAGE is expressed primarily in young females but not in males or older females. Conclusions: We conclude that the male sex associates with a reduced RAGE expression and signaling in PAEC, which is further diminished in PAH. The disruption in RAGE signaling, in turn, impairs the pulmonary endothelial barrier, instigates spontaneous PAH, and may contribute to the more severe phenotype observed in male PAH patients.

RAGE as a Novel Biomarker for Prostate Cancer: A Systematic Review and Meta-Analysis.

The receptor for advanced glycation end-products (RAGE) has been implicated in driving prostate cancer (PCa) growth, aggression, and metastasis through the fueling of chronic inflammation in the tumor microenvironment. This systematic review and meta-analysis summarizes and analyzes the current clinical and preclinical data to provide insight into the relationships among RAGE levels and PCa, cancer grade, and molecular effects. A multi-database search was used to identify original clinical and preclinical research articles examining RAGE expression in PCa. After screening and review, nine clinical and six preclinical articles were included. The associations of RAGE differentiating benign prostate hyperplasia (BPH) or normal prostate from PCa and between tumor grades were estimated using odds ratios (ORs) and associated 95% confidence intervals (CI). Pooled estimates were calculated using random-effect models due to study heterogeneity. The clinical meta-analysis found that RAGE expression was highly likely to be increased in PCa when compared to BPH or normal prostate (OR: 11.3; 95% CI: 4.4-29.1) and that RAGE was overexpressed in high-grade PCa when compared to low-grade PCa (OR: 2.5; 95% CI: 1.8-3.4). In addition, meta-analysis estimates of preclinical studies performed by albatross plot generation found robustly positive associations among RAGE expression/activation and PCa growth and metastatic potential. This review demonstrates that RAGE expression is strongly tied to PCa progression and can serve as an effective diagnostic target to differentiate between healthy prostate, low-grade PCa, and high-grade PCa, with potential theragnostic applications.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Spike Protein S1 Induces Methylglyoxal-Derived Hydroimidazolone/Receptor for Advanced Glycation End Products (MG-H1/RAGE) Activation to Promote Inflammation in Human Bronchial BEAS-2B Cells.

The pathogenesis of coronavirus disease 2019 (COVID-19) is associated with a hyperinflammatory response. The mechanisms of SARS-CoV-2-induced inflammation are scantly known. Methylglyoxal (MG) is a glycolysis-derived byproduct endowed with a potent glycating action, leading to the formation of advanced glycation end products (AGEs), the main one being MG-H1. MG-H1 exerts strong pro-inflammatory effects, frequently mediated by the receptor for AGEs (RAGE). Here, we investigated the involvement of the MG-H1/RAGE axis as a potential novel mechanism in SARS-CoV-2-induced inflammation by resorting to human bronchial BEAS-2B and alveolar A549 epithelial cells, expressing different levels of the ACE2 receptor (R), exposed to SARS-CoV-2 spike protein 1 (S1). Interestingly, we found in BEAS-2B cells that do not express ACE2-R that S1 exerted a pro-inflammatory action through a novel MG-H1/RAGE-based pathway. MG-H1 levels, RAGE and IL-1β expression levels in nasopharyngeal swabs from SARS-CoV-2-positive and -negative individuals, as well as glyoxalase 1 expression, the major scavenging enzyme of MG, seem to support the results obtained in vitro. Altogether, our findings reveal a novel mechanism involved in the inflammation triggered by S1, paving the way for the study of the MG-H1/RAGE inflammatory axis in SARS-CoV-2 infection as a potential therapeutic target to mitigate COVID-19-associated pathogenic inflammation.

Soluble RAGE and skeletal muscle tissue RAGE expression profiles in lean and obese young adults across differential aerobic exercise intensities.

Nearly 40% of Americans have obesity and are at increased risk for developing type 2 diabetes. Skeletal muscle is responsible for >80% of insulin-stimulated glucose uptake that is attenuated by the inflammatory milieu of obesity and augmented by aerobic exercise. The receptor for advanced glycation endproducts (RAGE) is an inflammatory receptor directly linking metabolic dysfunction with inflammation. Circulating soluble isoforms of RAGE (sRAGE) formed either by proteolytic cleavage (cRAGE) or alternative splicing (esRAGE) act as decoys for RAGE ligands, thereby counteracting RAGE-mediated inflammation. We aimed to determine if RAGE expression or alternative splicing of RAGE is altered by obesity in muscle, and whether acute aerobic exercise (AE) modifies RAGE and sRAGE. Young (20-34 yr) participants without [n = 17; body mass index (BMI): 22.6 ± 2.6 kg/m2] and with obesity (n = 7; BMI: 32.8 ± 2.9 kg/m2) performed acute aerobic exercise (AE) at 40%, 65%, or 80% of maximal aerobic capacity (V̇o2max; mL/kg/min) on separate visits. Blood was taken before and 30 min after each AE bout. Muscle biopsy samples were taken before, 30 min, and 3 h after the 80% V̇o2max AE bout. Individuals with obesity had higher total RAGE and esRAGE mRNA and RAGE protein (P < 0.0001). In addition, RAGE and esRAGE transcripts correlated to transcripts of the NF-κB subunit P65 (P < 0.05). There was no effect of AE on total RAGE or esRAGE transcripts, or RAGE protein (P > 0.05), and AE tended to decrease circulating sRAGE in particular at lower intensities of exercise. RAGE expression is exacerbated in skeletal muscle with obesity, which may contribute to muscle inflammation via NF-κB. Future work should investigate the consequences of increased skeletal muscle RAGE on the development of obesity-related metabolic dysfunction and potential mitigating strategies.NEW & NOTEWORTHY This study is the first to investigate the effects of aerobic exercise intensity on circulating sRAGE isoforms, muscle RAGE protein, and muscle RAGE splicing. sRAGE isoforms tended to diminish with exercise, although this effect was attenuated with increasing exercise intensity. Muscle RAGE protein and gene expression were unaffected by exercise. However, individuals with obesity displayed nearly twofold higher muscle RAGE protein and gene expression, which positively correlated with expression of the P65 subunit of NF-κB.

The Effect of Free and Protein-Bound Maillard Reaction Products N-ε-Carboxymethyllysine, N-ε-Fructosyllysine, and Pyrraline on Nrf2 and NFκB in HCT 116 Cells.

Maillard reaction products (MRPs) are believed to interact with the receptor for advanced glycation endproducts (RAGE) and lead to a pro-inflammatory cellular response. The structural basis for this interaction is scarcely understood. This study investigates the effect of individual lysine modifications in free form or bound to casein on human colon cancer cells. Selectively glycated casein containing either protein-bound N-ε-carboxymethyllysine (CML), N-ε-fructosyllysine (FL), or pyrraline is prepared and up to 94%, 97%, and 61% of lysine modification could be attributed to CML, FL, or pyrraline, respectively. HCT 116 cells are treated with free CML, pyrraline, FL, or modified casein for 24h. Native casein is used as control. Intracellular MRP content is analyzed by UPLC-MS/MS. Microscopic analysis of the transcription factors shows no activation of NFκB by free or protein-bound FL or CML, whereas casein containing protein-bound pyrraline activates Nrf2. RAGE expression is not influenced by free or casein-bound MRPs. Activation of Nrf2 by pyrraline-modified casein is confirmed by analyzing Nrf2 target proteins NAD(P)H dehydrogenase (quinone 1) (NQO1) and heme oxygenase-1 (HO-1). Studies on the biological effects of glycated proteins require an individual consideration of defined structures. General statements on the effect of "AGEs" in biological systems are scientifically unsound.

#3635 THERAPEUTIC STRATEGIES TARGETING RAGE IMPROVES RENAL OUTCOME IN LUPUS NEPHRITIS

Abstract Background and Aims Lupus nephritis (LN) occurs in up to 60% of patients with systemic lupus erythematosus (SLE). Despite of current development of immunosuppressant agents, LN still impairs the survival and quality of life in SLE patients. Receptor for advanced glycation endproducts (RAGE) is a multi-ligand receptor that belongs to the immunoglobulin superfamily, which is strongly associated with innate immune system. In the present study, we examined whether RAGE is involved in the development of LN. Further, we explored the therapeutic impact of DNA-aptamer directed against RAGE on lupus-related kidney injury. Method [Protocol 1] RAGE expression in kidneys and urinary RAGE excretion (uRAGE) were determined at 8, 12, 16, 18, 20-week-old by real-time PCR and ELISA, respectively in MRL/lpr, SLE-prone mice. [Protocol 2] LN was induced by peritoneally injecting pristane in wild type and RAGE globally knockout mice. [Protocol 3] MRL/lpr mice were subcutaneously administrated with DNA-aptamer raised against RAGE (RAGE-apt) or Control-aptamer (Ctrl-apt) for 10weeks. Results [Protocol 1] uRAGE, but not urinary protein excretion, was increased in 8-week-old MRL/lpr mice when compared to that of the control of MRL/MPJ mice. uRAGE is positively correlated with urinary NAG, a tubular injury marker, suggesting that uRAGE can predict the onset and the progression of LN. Immunofluorescence staining demonstrated that RAGE was upregulated in distal tubules but not in glomerulus in early stage, meanwhile RAGE started to be extensively expressed in nephron at late stage of LN. Not only tubules, but RAGE was also found in infiltrated macrophages in glomerulus. In proximal tubules (PTC), RAGE was co-localized with cathepsine D, a lysosomal aspartyl protease, and Rab7, a marker of late endocytosis, suggesting that PTC RAGE is involved in endosome-related protein degradation. [Protocol 2] Pristane-induced the increase in systolic blood pressure was reduced with improvement of renal dysfunction in RAGE knockouts. RAGE knockouts also showed less fibrosis with the decrease in macrophage infiltration when compared to wild type mice in pristane-induced lupus nephritis. [Protocol 3] Similar to RAGE knockouts, administration of RAGE-apt reduced systolic blood pressure and attenuated the renal dysfunction. RAGE-apt also ameliorated mesangial expansion, crescent formation, and macrophage infiltration into glomerulus in MRL/lpr mice. The level of pro-inflammatory cytokines gene expression including IL-6, TNF-a, and MCP-1 was reduced by RAGE-apt, but not Ctrl-apt, in MRL/lpr mice. Conclusion RAGE could be involved in the pathogenesis of LN, and RAGE-DNA aptamer might be one of the promising therapeutic strategies for preventing the development of LN.

Chemical characterization of the polyphenolic rich fraction of Thunbergia erecta and its therapeutic potential against doxorubicin and cyclophosphamide-induced cognitive impairment in rats.

Thunbergia erecta (Benth.) was traditionally used as anxiolytic, sedative and antidepressant. The study aimed to characterize T. erecta leaf ethyl acetate fraction of alcohol extract (TEAF) and evaluate its neuroprotective effect on doxorubicin and cyclophosphamide-induced chemobrain. Chemical profiling of TEAF was done using (Liquid chromatography coupled with mass (LC-ESI-MS/MS). In vivo chemobrain model was performed by cognitive impairment induced by doxorubicin and cyclophosphamide. Behavioral assessments included moris water maze, y maze, novel object recognition task and passive avoidance tests. Histological examination and oxidative stress markers were investigated. Protein expression of HMDGB1/RAGE/pNF-κB pathway markers was done using western blotting. All results were applied to hippocampus and prefrontal cortex of rats. Molecular docking was done within the active sites of Human Receptor for Advanced Glycation Endproducts (RAGE) using Discovery studio software. Twenty-one phytoconstituents, mostly polyphenolics, were characterized in TEAF of which eleven compounds were tentatively identified for the first time from T. erecta leaves where rosmarinic acid (11) represents the most prevailing compound. TEAF resulted in a marked dose-dependent amelioration of the histopathological changes evidenced by normal histological structure demonstrated in the hypocampal gesture of rats. TEAF demonstrated an enhanced memory and learning functioning in the different behavioral tests assessed especially at 200mg/kg. It showed significant long-term spatial memory enhancement manifested by 50.32% increase in probe trial relative to chemobrain-induced group. It showed pronounced antioxidant activity evidenced by the significant elevation of prefrontal cortical and hippocampal reduced glutathione levels by 2.45 and 2.65 folds, respectively relative to the chemobrain-induced group. The pronounced reduction in hydrogen peroxide (1.24-1.93 folds) and malondialdehyde levels (1.42-2.60 folds) with significant elevation of catalase activity (12.65-31.47%) induced by TEAF supported its potent antioxidant activity. TEAF reversed the inflammatory cytokines release induced by chemotherapy via its interference with HMGB1/RAGE pathway suppressing the expression of HMBG1, RAGE, p65 (NF-kB), and IL-1β. In silico studies showed that rosmarinic acid displayed the best fitting at the active site of RAGE (ΔG=-40.39kcal/mol). Thunbergia erecta can act as a promising remedy for chemobrain that further consolidates its traditional importance.

Flavonoids inhibitory effect and mechanism on laryngeal carcinoma cell cytotoxicity in AGEs/RAGE/NF-κB pathway

Laryngeal carcinoma is a head and neck tumor. Although its survival rate has been improved, some patients die from tumor recurrence or metastasis. This experiment explored the effect and mechanism of flavonoids on laryngeal cancer cell cytotoxicity. Immunohistochemical staining was performed on laryngeal carcinoma tissues. A total of 6 groups were set up in this experiment as follows: the experimental group was added with flavonoids (at 2.5, 5, 10, 20, and 40 mg/L concentrations). Cytotoxicity, proliferation, apoptosis and migration ability were detected respectively. The expressions of Advanced glycation end products (AGEs), receptor for advanced glycation endproducts (RAGE) and Nuclear factor kappa B (NF-κB) proteins were determined. NF-κB/p65 was highly expressed in laryngeal carcinoma tissue cells cytoplasm. The cytotoxicity test proved that, the flavonoids were less toxic to DU4475 and EVC304 cells. After the Hep-2 cells were cultured in vitro for 48 h, as the concentration of flavonoids increased, the cells gradually became round, and their volume and adhesion gradually decreased. The number and density of Hep-2 cells decreased dose and time-dependently. The apoptosis rate and relative wound surface area in experimental group were increased (p &lt;0.05) in a dose-dependent manner. The expressions of AGEs, RAGE and NF-κB in experimental group were decreased (p &lt;0.05). NF-κB/p65 is positively expressed in laryngeal cancer tissues. In conclusion, Flavonoids are less toxic to normal cells and can significantly reduce AGEs, RAGE and NF-κB expressions, also inhibiting Hep-2 cell proliferation. Flavonoids herein significantly inhibited the migration of Hep-2 cells, thus exerting therapeutic effects.

RAGE is a critical factor of sex-based differences in age-induced kidney damage.

Introduction: Advanced glycation end products (AGEs) are a heterogeneous group of molecules with potential pathophysiological effects on the kidneys. Fibrosis together with the accumulation of AGEs has been investigated for its contribution to age-related decline in renal function. AGEs mediate their effects in large parts through their interactions with the receptor for AGEs (RAGE). RAGE is a transmembrane protein that belongs to the immunoglobulin superfamily and has the ability to interact with multiple pro-inflammatory/pro-oxidative ligands. The role of RAGE in aging kidneys has not been fully characterized, especially for sex-based differences. Methods: Therefore, we analyzed constitutive RAGE knockout (KO) mice in an age- and sex-dependent manner. Paraffin-embedded kidney sections were used for histological analysis and protein expression of fibrosis and damage markers. RNA expression analysis from the kidney cortex was done by qPCR for AGE receptors, kidney damage, and early inflammation/fibrosis factors. FACS analysis was used for immune cell profiling of the kidneys. Results: Histological analysis revealed enhanced infiltration of immune cells (positive for B220) in aged (>70weeks old) KO mice in both sexes. FACS analysis revealed a similar pattern of enhanced B-1a cells in aged KO mice. There was an age-based increase in pro-fibrotic and pro-inflammatory markers (IL-6, TNF, TGF-β1, and SNAIL1) in KO male mice that presumably contributed to renal fibrosis and renal damage (glomerular and tubular). In fact, in KO mice, there was an age-dependent increase in renal damage (assessed by NGAL and KIM1) that was accompanied by increased fibrosis (assessed by CTGF). This effect was more pronounced in male KO mice than in the female KO mice. In contrast to the KO animals, no significant increase in damage markers was detectable in wild-type animals at the age examined (>70weeks old). Moreover, there is an age-based increase in AGEs and scavenger receptor MSR-A2 in the kidneys. Discussion: Our data suggest that the loss of the clearance receptor RAGE in male animals further accelerates age-dependent renal damage; this could be in part due to an increase in AGEs load during aging and the absence of protective female hormones. By contrast, in females, RAGE expression seems to play only a minor role when compared to tissue pathology.

Characterization of RAGE and CK2 Expressions in Human Fetal Membranes.

At the feto-maternal interface, fetal membranes (FM) play a crucial role throughout pregnancy. FM rupture at term implicates different sterile inflammation mechanisms including pathways activated by the transmembrane glycoprotein receptor for advanced glycation end-products (RAGE) belonging to the immunoglobulin superfamily. As the protein kinase CK2 is also implicated in the inflammation process, we aimed to characterize the expressions of RAGE and the protein kinase CK2 as a candidate regulator of RAGE expression. The amnion and choriodecidua were collected from FM explants and/or primary amniotic epithelial cells throughout pregnancy and at term in spontaneous labor (TIL) or term without labor (TNL). The mRNA and protein expressions of RAGE and the CK2α, CK2α', and CK2β subunits were investigated using reverse transcription quantitative polymerase chain reaction and Western blot assays. Their cellular localizations were determined with microscopic analyses, and the CK2 activity level was measured. RAGE and the CK2α, CK2α', and CK2β subunits were expressed in both FM layers throughout pregnancy. At term, RAGE was overexpressed in the amnion from the TNL samples, whereas the CK2 subunits were expressed at the same level in the different groups (amnion/choriodecidua/amniocytes, TIL/TNL), without modification of the CK2 activity level and immunolocalization. This work paves the way for future experiments regarding the regulation of RAGE expression by CK2 phosphorylation.

Advanced Glycation End Products and Bone Metabolism in Patients with Chronic Kidney Disease.

Advanced glycation end products (AGEs) accumulation may be involved in the progression of CKD-bone disorders. We sought to determine the relationship between AGEs measured in the blood, skin, and bone with histomorphometry parameters, bone protein, gene expression, and serum biomarkers ofbone metabolism in patients with CKD stages 3 to 5D patients. Serum levels of AGEs were estimated by pentosidine, glycated hemoglobin (A1c), and N-carboxymethyl lysine (CML). The accumulation of AGEs in the skin was estimated from skin autofluorescence (SAF). Bone AGEs accumulation and multiligand receptor for AGEs (RAGEs) expression were evaluated by immunohistochemistry; bone samples were used to evaluate protein and gene expression and histomorphometric analysis. Data are from 86 patients (age: 51 ± 13 years; 60 [70%] on dialysis). Median serum levels of pentosidine, CML, A1c, and SAF were 71.6pmol/mL, 15.2ng/mL, 5.4%, and 3.05 arbitrary units, respectively. AGEs covered 3.92% of trabecular bone and 5.42% of the cortical bone surface, whereas RAGEs were expressed in 0.7% and 0.83% of trabecular and cortical bone surfaces, respectively. AGEs accumulation in bone was inversely related to serum receptor activator of NF-κB ligand/parathyroid hormone (PTH) ratio (R=-0.25; p=0.03), and RAGE expression was negatively related to serum tartrate-resistant acid phosphatase-5b/PTH (R=-0.31; p=0.01). Patients with higher AGEs accumulation presented decreased bone protein expression (sclerostin [1.96 (0.11-40.3) vs. 89.3 (2.88-401) ng/mg; p=0.004]; Dickkopf-related protein 1 [0.064 (0.03-0.46) vs. 1.36 (0.39-5.87) ng/mg; p=0.0001]; FGF-23 [1.07 (0.4-32.6) vs. 44.1 (6-162) ng/mg; p=0.01]; and osteoprotegerin [0.16 (0.08-2.4) vs. 6.5 (1.1-23.7) ng/mg; p=0.001]), upregulation of the p53 gene, and downregulation of Dickkopf-1 gene expression. Patients with high serum A1c levels presented greater cortical porosity and Mlt and reduced osteoblast surface/bone surface, eroded surface/bone surface, osteoclast surface/bone surface, mineral apposition rate, and adjusted area. Cortical thickness was negatively correlated with serum A1c (R=-0.28; p=0.02) and pentosidine levels (R=-0.27; p=0.02). AGEs accumulation in the bone of CKD patients was related to decreased bone protein expression, gene expression changes, and increased skeletal resistance to PTH; A1c and pentosidine levels were related to decreased cortical thickness; and A1c levels were related to increased cortical porosity and Mlt. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.