3'-Sialyllactose protects against LPS-induced endothelial dysfunction by inhibiting superoxide-mediated ERK1/2/STAT1 activation and HMGB1/RAGE axis.

AIM: Endothelial hyperpermeability is an early stage of endothelial dysfunction associated with the progression and development of atherosclerosis. 3'-Sialyllactose (3'-SL) is the most abundant compound in human milk oligosaccharides, and it has the potential to regulate endothelial dysfunction. This study investigated the beneficial effects of 3'-SL on lipopolysaccharide (LPS)-induced endothelial dysfunction in vitro and in vivo. MAIN METHODS: We established LPS-induced endothelial dysfunction models in both cultured bovine aortic endothelial cells (BAECs) and mouse models to determine the effects of 3'-SL. Western blotting, qRT-PCR analysis, immunofluorescence staining, and en face staining were employed to clarify underlying mechanisms. Superoxide production was measured by 2',7'-dichlorofluorescin diacetate, and dihydroethidium staining. KEY FINDINGS: LPS significantly decreased cell viability, whereas 3'-SL treatment mitigated these effects via inhibiting ERK1/2 activation. Mechanistically, 3'-SL ameliorated LPS-induced ROS accumulation leading to ERK1/2 activation-mediated STAT1 phosphorylation and subsequent inhibition of downstream transcriptional target genes, including VCAM-1, TNF-α, IL-1ß, and MCP-1. Interestingly, LPS-induced ERK1/2/STAT1 activation leads to the HMGB1 release from the nucleus into the extracellular space, where it binds to RAGE, while 3'-SL suppressed EC hyperpermeability by suppressing the HMGB1/RAGE axis. This interaction also led to VE-cadherin endothelial junction disassembly and endothelial cell monolayer disruption through ERK1/2/STAT1 modulation. In mouse endothelium, en face staining revealed that 3'-SL abolished LPS-stimulated ROS production and VCAM-1 overexpression. SIGNIFICANCE: Our findings suggest that 3'-SL inhibits LPS-induced endothelial hyperpermeability by suppressing superoxide-mediated ERK1/2/STAT1 activation and HMGB1/RAGE axis. Therefore, 3'-SL may be a potential therapeutic agent for preventing the progression of atherosclerosis.

Ezetimibe attenuates experimental diabetes and renal pathologies via targeting the advanced glycation, oxidative stress and AGE-RAGE signalling in rats.

The current in-vivo study was premeditated to uncover the protective role of ezetimibe (EZ) against advanced glycation endproducts (AGEs)-related pathologies in experimental diabetes. Our results showed that EZ markedly improved the altered biochemical markers of diabetes mellitus (DM) (FBG, HbA1c, insulin, microalbumin, and creatinine) and cardiovascular disease (in-vivo lipid/lipoprotein level and hepatic HMG-CoA reductase activity) along with diminished plasma carboxymethyl-lysine (CML) and renal fluorescent AGEs level. Gene expression study revealed that EZ significantly down-regulated the renal AGEs-receptor (RAGE), nuclear factor-κB (NFκB-2), transforming growth factor-ß (TGF-ß1), and matrix metalloproteinase-2 (MMP-2) mRNA expression, however, the neuropilin-1 (NRP-1) mRNA expression was up-regulated. In addition, EZ also maintained the redox status via decreasing the lipid peroxidation and protein-bound carbonyl content (CC) and increasing the activity of high-density lipoprotein (HDL)-associated-paraoxonase-1 (PON-1) and renal antioxidant enzymes as well as also protected renal histopathological features. We conclude that EZ exhibits antidiabetic and reno-protective properties in diabetic rats.

Verbascoside represses malignant phenotypes of esophageal squamous cell carcinoma cells by inhibiting CDC42 via the HMGB1/RAGE axis.

BACKGROUND: As an aggressive human malignancy, esophageal squamous cell carcinoma (ESCC) is prevalent globally, especially in China. Verbascoside (VE) exerts anti-cancer effects in several human cancers. This work was to investigate the effects of VE on ESCC cells. METHODS: Esophageal squamous cell carcinoma cell proliferation, apoptosis, migration, and invasion were assessed by CCK-8, TUNEL, and Transwell assays. Gene and protein levels were detected by RT-qPCR and western blotting. CDC42 activity was evaluated by G-lisa assay. RESULTS: Verbascoside significantly inhibited cell proliferation, migration, and invasion and induced cell apoptosis in ESCC cells. Furthermore, it was found that VE markedly inhibited HMGB1 and RAGE expression in a dose-dependent manner. Besides, HMGB1/RAGE upregulation partially reversed the anti-cancer effects of VE on ESCC cells. VE repressed HMGB1/RAGE-induced CDC42 activation in ESCC cells. In addition, ML141-mediated CDC42 inactivation further enhanced the effect of VE on ESCC cell proliferation, apoptosis, migration, and invasion. CONCLUSIONS: Our findings indicated that VE has significant anti-tumor potential in ESCC by suppressing HMGB1/RAGE-dependent CDC42 activation.

Endothelial expression of the receptor for advanced glycation end products in experimental diabetes / Expresión endotelial del receptor para productos finales de glicación avanzada en diabetes experimental

The receptor for advanced glycation end products (RAGE) is implicated in the pathogenesis of several chronic diseases including diabetes. The interaction between RAGE and advanced glycation end products (AGEs) promotes gene expression, enhances the release of proinflammatory molecules and causes the generation of oxidative stress in numerous cell types. The aim of this investigation was to evaluate the effect of enalapril and losartan on RAGE expression in abdominal aortic endothelium of rats with experimentally induced diabetes. Male Sprague-Dawley rats, weighing approximately 150 - 200 g, were used. Diabetes was induced in 30 rats by intravenous administration of a single dose of 55 mg/kg body weight of streptozotocin (ETZ). The following groups were studied: control (n=10), diabetic (n=10), losartan-treated diabetic (n=10) and enalapril-treated diabetic (n=10) rats. RAGE expression in aortic endothelium was determined by indirect immunofluorescence. A significant increase in RAGE expression was observed in diabetic animals versus controls (p<0.001), there was a decrease in RAGE expression, in animals treated with losartan versus controls (p<0.01) and in those treated with enalapril (p<0.05) versus control and versus diabetes + vehicle. In conclusion, in the experimental model of ETZ-induced diabetes, there is an increase in RAGE expression at the level of the abdominal aortic endothelium, which can be reversed by treatment with losartan and/or enalapril, two drugs that block the renin-angiotensin system, suggesting its involvement in the molecular events related to vascular damage during diabetes.

El receptor para productos finales de glicación avanzada (RAGE) está implicado en la patogénesis de varias enfermedades crónicas incluyendo la diabetes. La interacción entre RAGE y los productos finales de glicación avanzada (AGEs), promueve la expresión génica, potencia la liberación de moléculas proinflamatorias y provoca la generación de estrés oxidativo en numerosos tipos de células. El objetivo de esta investigación fue evaluar el efecto del enalapril y el losartán sobre la expresión de RAGE en el endotelio de la aorta abdominal de ratas con diabetes inducida experimentalmente. Se utilizaron ratas Sprague-Dawley machos, con un peso aproximado de entre 150 - 200 g. La diabetes se indujo en 30 ratas mediante la administración intravenosa de una sola dosis de 55 mg/Kg de peso corporal de estreptozotocina (ETZ). Se estudiaron los siguientes grupos: ratas control (n=10), diabéticas (n=10), diabéticas tratadas con losartán (n=10) y diabéticas tratadas con enalapril (n=10). La expresión de RAGE en el endotelio aórtico se determinó por inmunofluorescencia indirecta. Se observó un incremento significativo en la expresión de RAGE en los animales diabéticos versus los controles (p<0.001), hubo una disminución en la expresión de RAGE, en los animales tratados con losartán versus los controles (p<0.01) y en los tratados con enalapril (p<0.05) versus control y versus diabetes + vehículo. En conclusión, en el modelo experimental de diabetes inducida por ETZ, existe un incremento en la expresión de RAGE a nivel del endotelio de la aorta abdominal, la cual puede revertirse mediante el tratamiento con losartán y/o enalapril, dos fármacos bloqueadores del sistema renina-angiotensina, lo cual sugiere la participación del mismo en los acontecimientos moleculares relacionados con el daño vascular durante la diabetes.

Involvement of multiple scavenger receptors in advanced glycation end product-induced vessel tube formation in endothelial cells.

Toxic advanced glycation end products (toxic AGEs) derived from glycolaldehyde (AGE3) have been implicated in the development of diabetic vascular complications such as retinopathy characterised by excessive angiogenesis. Different receptor types, such as receptor for AGEs (RAGE), Toll like receptor-4 and scavenger receptors, are expressed in endothelial cells and contribute to AGE-elicited alteration of cell function. In the present study, we examined the involvement of AGE-related receptors on AGE-induced angiogenesis in endothelial cells. The effects of pharmacological inhibitors or receptor neutralizing antibodies on AGE3-induced tube formation were investigated using the in vitro Matrigel tube formation assay in b.End5 cells (mouse endothelial cells). AGE3-induced signalling pathways and receptor expression changes were analysed by Western blot analysis and flow cytometry, respectively. Both FPS-ZM1, a RAGE inhibitor, and fucoidan, a ligand for scavenger receptors, suppressed AGE3-induced tube formation. Cocktails of neutralizing antibodies against the scavenger receptors CD36, CD163 and LOX-1 prevented AGE3-induced tube formation. AGE3 activated mTOR signalling, resulting in facilitation of tube formation. Activation of the AGE-RAGE pathway also led to the upregulation of scavenger receptors. Taken together, our findings suggest that the scavenger receptors CD36, CD163 and LOX-1 in conjunction with the RAGE receptor work together to mediate toxic AGE-induced facilitation of angiogenesis.

Study on the Mechanism of Prunella Vulgaris L on Diabetes Mellitus Complicated with Hypertension Based on Network Pharmacology and Molecular Docking Analyses.

The role of traditional Chinese medicine Prunella vulagaris L in the treatment of tumors and inflammation has been widely confirmed. We found that some signaling pathways of Prunella vulgaris L action can also regulate diabetes and hypertension, so we decided to study the active ingredients, potential targets and signaling pathway of Prunrlla vulgaris L, and explore the "multi-target, multi-pathway" molecular mechanism of Prunella vulgaris L on diabetes mellitus complicated with hypertension(DH). Methods. Based on TCMSP(Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform) and CNKI(China National Knowledge Infrastructure), the components and action targets related to Prunella vulgaris L were screened. The OMIM(Online Mendelian Inheritance in Man) and GeneCards (The human gene database) were used to search for targets related to DH. The "gene - drug - disease" relationship map was drawn by Cytoscape_v3.7.2 plug-in. The target was amplified by the STRING platform, and the "protein - protein" interaction relationship (PPI) network of the interacting target was obtained by the STRING online analysis platform and the Cytoscape_v3.7.2 plug-in. Finally, GO enrichment analysis and KEGG pathway enrichment analysis were conducted on David and Metascape platform to study the co-acting targets. Results. 11 active components, 41 key targets and 16 significant signaling pathways were identified from Prunella vulgaris L. The main active components of Prunella vulgaris L against DH were quercetin and kaumferol, etc, and potential action targets were IL-6 and INS, etc and signaling pathways were AGE-RAGE signaling pathway, TNF signaling pathway, MAPK signaling pathway, PI3K-AKT signaling pathway, etc. It involves in biological processes such as cell proliferation, apoptosis and inflammatory response. Conclusions. The main molecular mechanism of Prunella vulgaris L against DH is that sterols and flavonoids play an active role by affecting TNF signaling pathway, AGE-RAGE signaling pathway, MAPK pathway, PI3K-Akt pathway related targets such as IL-6 and INS.

FBXO10 prevents chronic unpredictable stress-induced behavioral despair and cognitive impairment through promoting RAGE degradation.

AIMS: Depression is one of the leading causes of disability worldwide. The receptor for advanced glycosylation end products (RAGE) is closely related to chronic stress and is a target of F-box protein O10 (FBXO10) which promotes the degradation of RAGE by ubiquitination. Here, we explored the role of FBXO10 and RAGE in chronic unpredictable stress (CUS)-induced behavioral despair, cognitive impairment, neuroinflammation, and the polarization microglia. METHODS: Male C57BL/6 mice with or without infusion of viral in the medial prefrontal cortex (PFC) were subjected to CUS. Then the mice were exposed to forced swim test, sucrose consumption test, novelty-suppressed feeding test, and temporal object recognition task to assess the behavioral despair and cognitive impairment. Inflammatory cytokines and the neurotrophic factor brain-derived neurotrophic factor (BDNF) levels in PFC were assessed by enzyme-linked immunosorbent assay. Immunofluorescence and immunohistochemistry staining were performed to observe the activation and phenotypic transformation of microglia in PFC. LPS-induced cell model was constructed to explore the effect of FBXO10/RAGE axis in the polarization of microglia in vitro. RESULTS: FBXO10 promoted RAGE degradation by ubiquitination in BV2 cells. FBXO10 protein levels were reduced whereas RAGE protein levels were enhanced in CUS mice. FBXO10 overexpression or RAGE knockdown inhibited proinflammatory cytokine release, promoted BDNF expression, mitigated the depressive-like and cognitive impairment behaviors, and affected the polarization of microglia induced by CUS exposure. FBXO10/RAGE axis promoted the polarization of microglia from the M1 to the M2 phenotype in vitro. Moreover, p38 MAPK and NF-κΒ were identified to be the downstream effect factors for FBXO10/RAGE axis. CONCLUSIONS: FBXO10 administration prevents CUS-induced behavioral despair, cognitive impairment, neuroinflammation, and the polarization of microglia through decreasing the accumulation of RAGE, p38 MAPK, and NF-κΒ, suggesting potential therapeutic strategies for the prevention and treatment of depression.

The effects of Danggui-Shaoyao-San on neuronal degeneration and amyloidosis in mouse and its molecular mechanism for the treatment of Alzheimer's disease.

The abnormal deposition of the extracellular amyloid-ß peptide is the typical pathological hallmark of Alzheimer's disease. Strategies to reduce the amyloid-ß deposition effectively alleviate the neuronal degeneration and cognitive deficits of Alzheimer's disease. Danggui-Shaoyao-San has been considered a useful therapeutic agent known for the treatment of Alzheimer's disease. However, the mechanism of Danggui-Shaoyao-San for the treatment of Alzheimer's disease remains unclear. We investigated Danggui-Shaoyao-San's effect on amyloidosis and neuronal degeneration in an APP/PS1 mouse model. We found Danggui-Shaoyao-San alleviated the cognitive deficits in APP/PS1 mice. Additionally, Danggui-Shaoyao-San ameliorated the neuronal degeneration in these mice. Danggui-Shaoyao-San reduced the amyloidosis and amyloid-ß1-42 deposition in APP/PS1 mouse brain and down-regulated the receptor for advanced glycation end products, and up-regulated the level of low-density lipoprotein receptor-related protein-1. However, the protein expression of the ß-amyloid precursor protein, ß-Secretase and presenilin-1 (PS1) in the amyloid-ß production pathway, and the expression of neprilysin and insulin-degrading enzyme in the amyloid-ß degradation pathway were not altered. Our findings collectively suggest that Danggui-Shaoyao-San could ameliorate the amyloidosis and neuronal degeneration of Alzheimer's disease, which may be associated with its up-regulation lipoprotein receptor-related protein-1 and down-regulation of the receptor for advanced glycation end products.

Network Pharmacology and Molecular Docking Study on the Potential Mechanism of Yi-Qi-Huo-Xue-Tong-Luo Formula in Treating Diabetic Peripheral Neuropathy.

OBJECTIVE: To investigate the potential mechanism of action of Yi-Qi-Huo-Xue-Tong-Luo formula (YQHXTLF) in the treatment of diabetic peripheral neuropathy (DPN). METHODS: Network pharmacology and molecular docking techniques were used in this study. Firstly, the active ingredients and the corresponding targets of YQHXTLF were retrieved using the Traditional Chinese Medicine Systems Pharmacology (TCMSP) platform; subsequently, the targets related to DPN were retrieved using GeneCards, Online Mendelian Inheritance in Man (OMIM), Pharmgkb, Therapeutic Target Database (TTD) and Drugbank databases; the common targets of YQHXTLF and DPN were obtained by Venn diagram; afterwards, the "YQHXTLF Pharmacodynamic Component-DPN Target" regulatory network was visualized using Cytoscape 3.6.1 software, and Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed on the potential targets using R 3.6.3 software. Finally, molecular docking of the main chemical components in the PPI network with the core targets was verified by Autodock Vina software. RESULTS: A total of 86 active ingredients and 229 targets in YQHXTLF were screened, and 81 active ingredients and 110 targets were identified to be closely related to diabetic peripheral neuropathy disease. PPI network mapping identified TP53, MAPK1, JUN, and STAT3 as possible core targets. KEGG pathway analysis showed that these targets are mostly involved in AGE-RAGE signaling pathway in diabetic complications, TNF signaling pathway, and MAPK signaling pathway. The molecular docking results showed that the main chemical components of YQHXTLF have a stable binding activity to the core pivotal targets. CONCLUSION: YQHXTLF may act on TP53, MAPK1, JUN, and STAT3 to regulate inflammatory response, apoptosis, or proliferation as a molecular mechanism for the treatment of diabetic peripheral neuropathy, reflecting its multitarget and multipathway action, and providing new ideas to further uncover its pharmacological basis and mechanism of action.

Timely N-Acetyl-Cysteine and Environmental Enrichment Rescue Oxidative Stress-Induced Parvalbumin Interneuron Impairments via MMP9/RAGE Pathway: A Translational Approach for Early Intervention in Psychosis.

Research in schizophrenia (SZ) emphasizes the need for new therapeutic approaches based on antioxidant/anti-inflammatory compounds and psycho-social therapy. A hallmark of SZ is a dysfunction of parvalbumin-expressing fast-spiking interneurons (PVI), which are essential for neuronal synchrony during sensory/cognitive processing. Oxidative stress and inflammation during early brain development, as observed in SZ, affect PVI maturation. We compared the efficacy of N-acetyl-cysteine (NAC) and/or environmental enrichment (EE) provided during juvenile and/or adolescent periods in rescuing PVI impairments induced by an additional oxidative insult during childhood in a transgenic mouse model with gluthation deficit (Gclm KO), relevant for SZ. We tested whether this rescue was promoted by the inhibition of MMP9/RAGE mechanism, both in the mouse model and in early psychosis (EP) patients, enrolled in a double-blind, randomized, placebo-controlled clinical trial of NAC supplementation for 6 months. We show that a sequential combination of NAC+EE applied after an early-life oxidative insult recovers integrity and function of PVI network in adult Gclm KO, via the inhibition of MMP9/RAGE. Six-month NAC treatment in EP patients reduces plasma sRAGE in association with increased prefrontal GABA, improvement of cognition and clinical symptoms, suggesting similar neuroprotective mechanisms. The sequential combination of NAC+EE reverses long-lasting effects of an early oxidative insult on PVI/perineuronal net (PNN) through the inhibition of MMP9/RAGE mechanism. In analogy, patients vulnerable to early-life insults could benefit from a combined pharmacological and psycho-social therapy.

Papaverine provides neuroprotection by suppressing neuroinflammation and apoptosis in the traumatic brain injury via RAGE- NF-B pathway.

The receptor for advanced glycation end products (RAGE)- Nuclear Factor kappa B (NF-κB) signal pathway may represent a new target for the treatment of traumatic brain injury (TBI). The aim of the study is to investigate effects of papaverine on secondary signaling mechanisms through this pathway in mice TBI model.Immunohistochemically, while the number of RAGE and NF- κB positive cells, apoptotic cells increased, the number of NeuN positive cells reduced in TBI.Papaverine reduced the number of RAGE positive cells on glia and the number of NF- κB positive cells on both neuron and glia. At the same time, it decreased the number of microglia labeled with P2RY12 increased due to TBI. It also increased the NeuN positive cells and mitigated the brain edema. Results of this study showed that papaverine reduced TBI- induced neuroinflammation and apoptosis, also provided neuroprotection via the RAGE- NF-κB signal path, which is one of the possible mechanisms in TBI.

Liraglutide improved the cognitive function of diabetic mice via the receptor of advanced glycation end products down-regulation.

Background and aims Advanced glycation end products (AGEs) and receptor of advanced glycation end products (RAGE), are associated with cognition decline. We aim to investigate the effect of liraglutide on cognitive function in diabetic mice. Results Diabetic mice showed decreased cognitive function. Moreover, lower glucagon like peptide-1 (GLP-1) levels in plasma were detected in db/db mice. Additionally, up-regulated RAGE and down-regulated glucagon like peptide-1 (GLP-1R) levels were observed in db/db mice. However, decreased GLP-1R and increased RAGE were reversed by liraglutide. We also found decreased cellular activity in cells with AGEs. Moreover, AGEs up-regulated RAGE in PC12 and HT22 cells. However, liraglutide improved the cell activity damaged by AGEs. Although we did not discover the direct-interaction between RAGE and GLP-1R, elevated RAGE levels induced by AGEs were restored by liraglutide. Conclusion We demonstrated that the cognitive function of diabetic mice was improved by liraglutide via the down-regulation of RAGE. Methods db/db mice and db/m mice were used in this study. Liraglutide was used to remedy diabetic mice. Neurons and RAGE in hippocampus were shown by immunofluorescence. And then, PC12 cells or HT22 cells with AGEs were treated with liraglutide. GLP-1R and RAGE were measured by western blotting.

Morroniside attenuates high glucose-induced BMSC dysfunction by regulating the Glo1/AGE/RAGE axis.

OBJECTIVES: High glucose (HG)-mediated bone marrow mesenchymal stem cell (BMSC) dysfunction plays a key role in impaired bone formation induced by type 1 diabetes mellitus (T1DM). Morroniside is an iridoid glycoside derived from the Chinese herb Cornus officinalis, and it has abundant biological activities associated with cell metabolism and tissue regeneration. However, the effects and underlying mechanisms of morroniside on HG-induced BMSC dysfunction remain poorly understood. MATERIALS AND METHODS: Alkaline phosphatase (ALP) staining, ALP activity and Alizarin Red staining were performed to assess the osteogenesis of BMSCs. Quantitative real-time PCR and Western blot (WB) were used to investigate the osteo-specific markers, receptor for advanced glycation end product (RAGE) signalling and glyoxalase-1 (Glo1). Additionally, a T1DM rat model was used to assess the protective effect of morroniside in vivo. RESULTS: Morroniside treatment reverses the HG-impaired osteogenic differentiation of BMSCs in vitro. Morroniside suppressed advanced glycation end product (AGEs) formation and RAGE expression by triggering Glo1. Moreover, the enhanced osteogenesis due to morroniside treatment was partially blocked by the Glo1 inhibitor, BBGCP2. Furthermore, in vivo, morroniside attenuated bone loss and improved bone microarchitecture accompanied by Glo1 upregulation and RAGE downregulation. CONCLUSIONS: These findings suggest that morroniside attenuates HG-mediated BMSC dysfunction partly through the inhibition of AGE-RAGE signalling and activation of Glo1 and may be a potential treatment for diabetic osteoporosis.

Protective Effect of Buyang Huanwu Decoction on Neurovascular Unit in Alzheimer's Disease Cell Model via Inflammation and RAGE/LRP1 Pathway.

BACKGROUND The aim of this study was to investigate the protective mechanism of neurovascular unit of Buyang Huanwu decoction (BYHWD) in an Alzheimer's disease (AD) cell model via RAGE/LRP1 pathway and find a reliable target for Alzheimer's disease treatment. MATERIAL AND METHODS Rat brain microvessel endothelial cells (BMECs) were cultured in 10% FBS and 1% penicillin/streptomycin. The AD model was established by administration of 24 µmol/L amyloid-ß peptides 25~35. Different concentrations of BYHWD (0.1 mg/mL, 1 mg/mL, and 10 mg/mL) were added as the drug intervention. The morphology of the cells was observed by light microscopy and the ultrastructure of the cells was observed by microscopy. The inflammatory factors IL-1ß, IL-6, TNF-alpha, and Aß25-35 were detected by ELISA. Flow cytometry was used to assess the apoptosis rate. The expressions of RAGE, LRP1, ICAM-1, VCAM-1, Apo J, Apo E, and NF-kappaBp65 were detected by Western blotting. RESULTS The structure of cells in BYHWDM and BYHWDH gradually recovered with increasing dose. BYHWD decreased the apoptotic rate of BMECs induced by Aß25-35. The cells treated with different concentrations of BYHWD had significant difference in terms of anti-apoptotic effect. The therapeutic effect of BYHWD on AD was via the RAGE/LRP1 and NF-kappaBp65 pathways. CONCLUSIONS BYHWD regulates Aß metabolism via the RAGE/LRP1 pathway, inhibits vascular endothelial inflammation induced by ICAM-1 and VCAM-1 via the NF-kappaBP65 pathway, and promotes morphological changes induced by Aß-induced brain microvascular endothelial cell damage.

Fangchinoline Ameliorates Diabetic Retinopathy by Inhibiting Receptor for Advanced Glycation End-Products (RAGE)-Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B Cells (NF-κB) Pathway in Streptozotocin (STZ)-Induced Diabetic Rats.

BACKGROUND Diabetic retinopathy (DR) is a macrovascular complication that occurs in diabetic patients. Conventional treatments for the management of DR have many limitations. Thus, the present investigation evaluated the protective effect of fangchinoline against diabetic retinopathy (DR). MATERIAL AND METHODS DR was induced by streptozotocin (STZ; 60 mg/kg; i.p.) and rats were treated with fangchinoline 1, 3, and 10 mg/kg for 16 weeks. DR was confirmed by determining the concentration of advanced glycation end-products (AGEs) and morphology of retinal tissues. Parameters of oxidative stress and expression of inflammatory cytokines and receptor for advanced glycation end-products (RAGE) in the retinal tissue were determined by Western blot assay and reverse transcription polymerase chain reaction (RT-PCR). Moreover enzyme-linked immunosorbent assay (ELISA) was used to determine the apoptosis index and activity of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the retinal tissues. RESULTS Our study reveals that the concentration of glycosylated hemoglobin (HbA1c) and glucose in the plasma and AGEs in the retinal tissue were significantly reduced in the fangchinoline group compared to the DR group. Moreover, treatment with fangchinoline attenuated the altered retinal morphology and expression of inflammatory mediators and RAGE in the retinal tissues of DR rats. There was a significant (p<0.01) decrease in oxidative stress, activity of NF-κB, and apoptosis index in the fangchinoline group compared to the DR group of rats. CONCLUSIONS Our investigation shows that fangchinoline attenuates the apoptosis of retinal cells in STZ-induced diabetic retinopathy rats by inhibiting the RAGE/NF-κB pathway.

Upregulation of miR-107 expression following hyperbaric oxygen treatment suppresses HMGB1/RAGE signaling in degenerated human nucleus pulposus cells.

BACKGROUND: The expression of both high-mobility group box 1 (HMGB1) and receptor for advanced glycation end-products (RAGE) is upregulated in degenerated discs. HMGB1 is known to function as a coupling factor between hypoxia and inflammation in arthritis, and this inflammatory response is modulated by microRNAs (miRNAs), with miR-107 expression downregulated during hypoxia. In this study, we investigated the regulation of the miR-107/HMGB1/RAGE pathway in degenerated nucleus pulposus cells (NPCs) after hyperbaric oxygen (HBO) treatment. METHODS: NPCs were separated from human degenerated intervertebral disc tissues. The control cells were maintained in 5% CO2/95% air, and the hyperoxic cells were exposed to 100% O2 at 2.5 atmospheres absolute. MiRNA expression profiling was performed via microarray and confirmed by real-time PCR, and miRNA target genes were identified using bioinformatics and luciferase reporter assays. The cellular protein and mRNA levels of HMGB1, RAGE, and inducible nitric oxide synthase (iNOS) were assessed, and the phosphorylation of MAPK (p38MAPK, ERK, and JNK) was evaluated. Additionally, cytosolic and nuclear fractions of the IκBα and NF-κB p65 proteins were analyzed, and secreted HMGB1 and metalloprotease (MMP) levels in the conditioned media were quantified. RESULTS: Using microarray analyses, 96 miRNAs were identified as upregulated and 66 downregulated following HBO treatment. Based on these results, miR-107 was selected for further investigation. Bioinformatics analyses indicated that the 3' untranslated region of the HMGB1 mRNA contained the "seed-matched-sequence" for hsa-miR-107, which was validated via dual-luciferase reporter assays. MiR-107 was markedly induced by HBO, and simultaneous suppression of HMGB1 was observed in NPCs. Knockdown of miR-107 resulted in upregulation of HMGB1 expression in HBO-treated cells, and HBO treatment downregulated the mRNA and protein levels of HMGB1, RAGE, and iNOS and the secretion of HMGB1. In addition, HBO treatment upregulated the protein levels of cytosolic IκBα and decreased the nuclear translocation of NF-κB in NPCs. Moreover, HBO treatment downregulated the phosphorylation of p38MAPK, ERK, and JNK and significantly decreased the secretion of MMP-3, MMP-9, and MMP-13. CONCLUSIONS: HBO inhibits pathways related to HMGB1/RAGE signaling via upregulation of miR-107 expression in degenerated human NPCs.

Protective effects of febuxostat against paraquat-induced lung toxicity in rats: Impact on RAGE/PI3K/Akt pathway and downstream inflammatory cascades.

AIMS: The herbicide paraquat causes fatal lung toxicity by induction of xanthine oxidase, production of free radicals and inflammation. Febuxostat, a xanthine oxidase inhibitor and anti-gout has recently shown anti-inflammatory activity. Accordingly, this study was carried out to investigate whether febuxostat may attenuate paraquat-induced lung toxicity and to explore the possible underlying mechanisms. MAIN METHODS: Rats were administered either vehicle, a single dose of paraquat (30 mg/kg, i.p.), febuxostat (15 mg/kg, oral), or both for 14 successive days. Serum LDH and sRAGE were estimated. Lung tissue xanthine oxidase activity, SOD, TAC, MDA, and RAGE, HMGB1 gene expression, PI3K/Akt and ß-catenin protein expression, MMP-9, IL-8, VEGF and COX-2 gene expression were estimated. KEY FINDINGS: Results showed that paraquat induced lung injury characterized by enhanced oxidative stress and inflammation, upregulated RAGE, HMGB1 gene expression, PI3K/Akt and ß-catenin protein expression. Administration of febuxostat inhibited the deleterious effects of paraquat on lung through inhibition of xanthine oxidase activity and related oxidative stress, downregulation of RAGE/PI3K/Akt pathway, and suppression of ß-catenin protein expression and its downstream inflammatory mediators. SIGNIFICANCE: The present study showed that febuxostat may abrogate paraquat-induced lung toxicity and demonstrated a novel mechanism for its ameliorative effects.

Ethyl pyruvate reduces organic dust-induced airway inflammation by targeting HMGB1-RAGE signaling.

BACKGROUND: Animal production workers are persistently exposed to organic dust and can suffer from a variety of respiratory disease symptoms and annual decline in lung function. The role of high mobility group box-1 (HMGB1) in inflammatory airway diseases is emerging. Hence, we tested a hypothesis that organic dust exposure of airway epithelial cells induces nucleocytoplasmic translocation of HMGB1 and blocking this translocation dampens organic dust-induced lung inflammation. METHODS: Rats were exposed to either ambient air or swine barn (8 h/day for either 1, 5, or 20 days) and lung tissues were processed for immunohistochemistry. Swine barn dust was collected and organic dust extract (ODE) was prepared and sterilized. Human airway epithelial cell line (BEAS-2B) was exposed to either media or organic dust extract followed by treatment with media or ethyl pyruvate (EP) or anti-HMGB1 antibody. Immunoblotting, ELISA and other assays were performed at 0 (control), 6, 24 and 48 h. Data (as mean ± SEM) was analyzed using one or two-way ANOVA followed by Bonferroni's post hoc comparison test. A p value of less than 0.05 was considered significant. RESULTS: Compared to controls, barn exposed rats showed an increase in the expression of HMGB1 in the lungs. Compared to controls, ODE exposed BEAS-2B cells showed nucleocytoplasmic translocation of HMGB1, co-localization of HMGB1 and RAGE, reactive species and pro-inflammatory cytokine production. EP treatment reduced the ODE induced nucleocytoplasmic translocation of HMGB1, HMGB1 expression in the cytoplasmic fraction, GM-CSF and IL-1ß production and augmented the production of TGF-ß1 and IL-10. Anti-HMGB1 treatment reduced ODE-induced NF-κB p65 expression, IL-6, ROS and RNS but augmented TGF-ß1 and IL-10 levels. CONCLUSIONS: HMGB1-RAGE signaling is an attractive target to abrogate OD-induced lung inflammation.