Recombinant Human IL-6 Protein (a.a.29-212)

Objective: This study was aimed to investigate the role of Toll-like receptor 4 (TLR4) in advanced glycation end products (AGEs)- induced macrophage polarization toward M1. Methods: Isolated primary macrophages were exposed to prepared AGEs at concentrations of 0, 2.5, 5 and 10 mmol/L. Macrophages were also exposed to hydrogen peroxide (H2O2) which provided exogenous reactive oxygen species (ROS). Receptor for AGEs (RAGE) was over-expressed by a vector. Specific siRNA silencing TLR4 and inhibitor TAK-242 were used to pre-treat the macrophages. Intracellular ROS was determined by DCFH-DA. Immunofluorescence staining was used to evaluate the expression of inducible nitric oxide synthase (iNOS) which is the marker of M1 macrophage phenotype. Real-time PCR was used to assess the mRNA expression level of TLR4 and RAGE. Protein expression levels of cytoplasmic RAGE, TLR4, nuclear signal transducers and activators of transcription 1 (STAT1) and phosphorylation levels of cytoplasmic STAT1 were evaluated by Western blotting. ELISA was used to measure concentrations of interleukin 6 (IL6), IL12 and tumor necrosis factor (TNF)alpha in supernatant of cell culture medium of macrophages. Results: AGEs significantly elevated intracellular ROS generation, expression levels of iNOS, cytoplasmic RAGE, TLR4, nuclear STAT1, phosphorylation levels of cytoplasmic STAT1, as well as IL6, IL12 and TNF alpha contents in a concentration-dependent manner. TLR4 silencing and inhibitor pre-treatment reduced expression levels of cytoplasmic RAGE, TLR4, phosphorylation of STAT1 and nuclear STAT1 in AGEsexposed macrophages without affecting RAGE expression and intracellular ROS production levels. RAGE over-expression elevated both ROS and TLR4 expression levels in macrophages. TLR4 expression elevation was also found in H2O2-treat macrophages. Conclusion: AGEs induced macrophage polarization toward M1 via activating RAGE/ROS/TLR4/STAT1 signaling pathway. (C) 2020 Elsevier Inc. All rights reserved.

Background: Peripheral arterial occlusive disease (PAOD) is a global public health concern that decreases the quality of life of the patients and can lead to disabilities and death. The aim of this study was to identify the genes and pathways associated with PAOD pathogenesis, and the potential therapeutic targets. Methods: Differentially expressed genes (DEGs) and miRNAs related to PAOD were extracted from the GSE57691 dataset and through text mining. Additionally, bioinformatics analysis was applied to explore gene ontology, pathways and protein-protein interaction of those DEGs. The potential miRNAs targeting the DEGs and the transcription factors (TFs) regulating miRNAs were predicted by multiple different databases. Results: A total of 59 DEGs were identified, which were significantly enriched in the inflammatory response, immune response, chemokine-mediated signaling pathway and JAK-STAT signaling pathway. Thirteen genes including IL6, CXCL12, IL1B, and STAT3 were hub genes in protein-protein interaction network. In addition, 513 miRNA-target gene pairs were identified, of which CXCL12 and PTPN11 were the potential targets of miRNA-143, and IL1B of miRNA-21. STAT3 was differentially expressed and regulated 27 potential target miRNAs including miRNA-143 and miRNA-21 in TF-miRNA regulatory network. Conclusion: In summary, inflammation, immune response and STAT3-mediated miRNA-target genes axis play an important role in PAOD development and progression.