Fig 1: Workflow describing steps involved in the lectin microarray. MGCs were prepared from spinal cords isolated by the hydraulic extrusion technique from three‐day‐old post‐natal rats. MGCs were treated with TNF‐α, IL‐1β and IL‐6 (10 ng/ml per cytokine) in combination/s for 1 day (acute) to 21 days (chronic). Lipopolysaccharide (LPS) (10 ng/ml) was used as positive control. Treatments were given every alternate day (n = 3). Membrane proteins were extracted using Mem‐PER™ Plus, a membrane protein extraction kit. Lectin microarray profiling was performed using a panel of 48 lectins to study any alteration in mammalian glycosylation. MGCs were then assessed for glycosylation localisation and association with particular cell types using FITC‐conjugated lectins and glial markers (GFAP and CD11b), respectively, by ICC
Fig 2: Effects of GA on the levels of inflammatory factors in the serum and aortas of DM-AS rats. ELISA was used to detect the concentration of TNF-α, IL-6 and IL-1β in the (A) serum and (B) aortas of rats. ***P<0.001 vs. control group; ##P<0.01 and ###P<0.001 vs. DM-AS group. AS, atherosclerosis; DM, diabetes mellitus; GA, gycyrrhizic acid; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; IL-6, interleukin-6.
Fig 3: Identification of glycan modulations during cytokine combination and LPS treatments. (A) Hierarchical clustering analysis of 48 lectins. Colors define activation as highly expressed (red) and no expression (blue). Only one cytokine combination (i.e., TNF‐α, IL‐1β and IL‐6 combination) was used along with LPS as a positive control. Treatment was given from day‐1 up to day‐21, and at four time points, day‐1, day‐7, day‐14 and day‐21 lectin microarray was performed on membrane proteins. Lectin microarray interaction pattern of control, at day 1, day 7, day 14 and day 21. Alexa‐555‐NHS dye tagged membrane glycoproteins were printed on an NHS‐activated hydrogel‐coated glass surface. Samples at a 3 μg/ml concentration were incubated in the lectin array. Images were obtained by G265BA microarray scanner (Agilent Technologies) and were analyzed with Pro Scan Array Express™ software (PerkinElmer). Labeled‐BSA and control dye samples were incubated at the same concentration conditions as negative lectin binding controls. The cytokine combination group showed an increased signal for the lectins with a higher response. In (B) Narcissus Pseudonarcissus (NPL), (C) Galanthus nivalis agglutinin (GNA), (D) Lycopersicon esculentum (LEL), (E) Datura stramonium (DSL), (F) Solanum tuberosum (STL), (G) Ricinus communis agglutinin‐I (RCA‐I), (H) Sambucus nigra (SNA), (I) Pisum sativum (PSA), (J) Aleuria aurantia lectin (AAL), (K) Jacalin (JAC), (L) Marasmium oreades agglutinin (MOA) and (M) Peanut agglutinin (PNA) (days 1, 7, 14 and 21), indicating higher general glycosylation upon cytokine combination treatment. Data are represented as mean ± SD, n = three independent experiments pulled samples run for six technical replicates, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Man: Mannose, GlcNAc: N‐acetylgalactosamine, Gal: Galactose, LacNAc: N‐acetyl lactosamine, Sial: Sialic acid, Fuc: Fucose, α‐Gal: α‐Galactose
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