Journal: International journal of obesity (2005)

Article Title: Small fragments of hyaluronan are increased in individuals with obesity and contribute to low-grade inflammation through TLR-mediated activation of innate immune cells.

doi: 10.1038/s41366-022-01187-z

Figure Lengend Snippet: Fig. 4 LMW HA upregulates pro-inflammatory markers through NF-κB-dependent signaling in PBMCs and THP-1 monocytes. A Relative mRNA levels for IL-1β, IL-8, MCP-1, and IL-6 in human monocyte-like THP-1 cells incubated for 6 h in the presence of vehicle (sterile water), LMW HA (100 μg/mL), and Toll-like receptor 2 (TLR2) agonist Pam2CSK4 (100 ng/ml). B Upper panel, Representative immunoblots of IKK α/β, pIKK α/β, pIKB α and β-actin in THP-1 cells incubated with vehicle (0.01% ethanol) or MG132 (10 mM) for 30 min before the addition of vehicle (sterile water), Pam2CSK4 (100 ng/ml) or LMW HA (100 μg/mL) (10 ng/ml) for 2 h. Lower panel, relative mRNA levels for TLR2 in human monocyte-like THP-1 cells incubated for 6 h in the presence of vehicle (sterile water), LMW HA (100 μg/mL), and Toll-like receptor 2 (TLR2) agonist Pam2CSK4 (100 ng/ml). Data are mean ± SEM n = 3 independent experiments performed in duplicate. *p < 0.05, ***p < 0.0005 versus vehicle-treated cells. C Representative immunoblots of IKK α/β, pIKK α/β, pIKB α and ΙΚΒ α in human healthy PBMC incubated with vehicle (0.01% ethanol) or MG132 (10 mM) for 30 min before the addition of vehicle (sterile water), increasing doses of LMW HA (100 pg/ml, and 100 μg/ml), and Pam2CSK4 (100 ng/ml) for 2 h. Results are expressed as mean ± SEM of 3 independent experiments performed in duplicate. *p < 0.05, **p < 0.005, ***p < 0.0005 versus vehicle.

Article Snippet: For the assessment of NFκB intracellular signaling, PBMC, and THP1 cells were incubated in the presence of either vehicle (0.01% EtOH) or 10 μM of the proteasome inhibitor MG132 (Merck Millipore, MA, USA) for 30 min before the addition of either LMW HA (100 μg/ml and 100 pg/ml) or 100 ng/ml of the Toll-like receptor 2 (TLR2) agonist Pam2CSK4 (InvivoGen, CA, USA) for 2 and 6 h. At the end of the incubation periods, cells were washed and collected for further gene expression analysis.

Techniques: Incubation, Sterility, Western Blot

Journal: The Journal of Immunology Author Choice

Article Title: Resolvin D1 Improves the Resolution of Inflammation via Activating NF-κB p50/p50–Mediated Cyclooxygenase-2 Expression in Acute Respiratory Distress Syndrome

doi: 10.4049/jimmunol.1700315

Figure Lengend Snippet: RvD1 facilitates the resolution of inflammation by promoting NF-κB p50–derived COX-2 expression in the ARDS model. Rats were i.v. injected with LPS (3 mg/kg) 12 h before they received tail vein injections with RvD1 (5 μg/kg) or an equivalent volume of ethanol. For treatment, rats were i.v. administered MG-132 at 1 h prior to RvD1 injection, whereas other groups received an equal volume of a DMSO saline solution. Rats were sacrificed 24 h after LPS exposure. Lung tissue samples were collected for morphological evaluation, Western blot, and EMSA supershift assays. (A) Representative photomicrographs of pulmonary histology, as shown by H&E staining. Original magnification, ×100 (inset, ×400). Black arrows indicate LPS-induced thickening of the alveolar walls and red arrows show neutrophil infiltration. (B) Acute lung injury score. (C) COX-2 protein expression was assessed via Western blot and analyzed by densitometry. Values were compared with β-actin expression. (D) NF-κB activation was determined via EMSA supershift assays with the nuclear protein extracts prepared from the lung tissues. EMSAs were used to quantify NF-κB activation in each group. Supershift assays were used to identify the activated NF-κB dimers in the LPS plus RvD1 group. EMSA supershift assays were performed as detailed in Materials and Methods. The NF-κB DNA–protein complexes (open arrow) were separated from the free probe (closed arrow) by electrophoresis in the EMSA. A supershift analysis was conducted with Abs directed against p50 and p65 to characterize specific NF-κB complexes in the LPS plus RvD1 group. Supershifted bands with an asterisk indicate binding of the p50 subunit. CK, negative control; CN, control group; L, LPS group; L+M, LPS plus MG-132 group; L+R, LPS plus RvD1 group; L+R+M, LPS plus RvD1 plus MG-132 group; M, MG-132 group; R, RvD1 group; 100×, competition group. Data are shown as mean ± SEM, n = 5–7 rats per treatment per group, and are representative of at least three independent experiments. *p < 0.05 compared with the control group; &p < 0.05 compared with the LPS group; #p < 0.05 compared with the LPS plus RvD1 group. **p < 0.01, ****p < 0.0001.

Article Snippet: After 24 h of culture with LPS (1 μg/ml) or a control medium, fibroblasts were treated with 100 nM RvD1 or a vehicle solution (0.1% ethanol, as the RvD1 was supplied in ethanol) for an additional 24 h. SP600125 (10 μM), SB603580 (10 μM), MG-132 (10 μM), and BOC-2 (10 μM) were added 30 min prior to RvD1 administration.

Techniques: Derivative Assay, Expressing, Injection, Western Blot, Staining, Activation Assay, Electrophoresis, Binding Assay, Negative Control

Journal: The Journal of Immunology Author Choice

Article Title: Resolvin D1 Improves the Resolution of Inflammation via Activating NF-κB p50/p50–Mediated Cyclooxygenase-2 Expression in Acute Respiratory Distress Syndrome

doi: 10.4049/jimmunol.1700315

Figure Lengend Snippet: NF-κB is responsible for promoting the proresolving COX-2 protein expression in pulmonary fibroblasts. Primary pulmonary fibroblasts were incubated with 1 μg/ml LPS (L) for 24 h followed by administration of 100 nM RvD1 or vehicle (0.1% ethanol) for an additional 24 h, and 10 μM SP600125 (a JNK inhibitor), 10 μM SB203580 (a p38 MAPK inhibitor), and 10 μM MG-132 (an NF-κB inhibitor) were added 30 min prior to RvD1 administration. After incubation, the cells were harvested and sonicated. The expression of COX-2 was determined via Western blot. Cells were differentiated from lung tissues harvested from six rats per condition; n = 8 per treatment per group. Data are shown as mean ± SEM and are representative of at least four independent experiments. **p < 0.01, ***p < 0.001.

Article Snippet: After 24 h of culture with LPS (1 μg/ml) or a control medium, fibroblasts were treated with 100 nM RvD1 or a vehicle solution (0.1% ethanol, as the RvD1 was supplied in ethanol) for an additional 24 h. SP600125 (10 μM), SB603580 (10 μM), MG-132 (10 μM), and BOC-2 (10 μM) were added 30 min prior to RvD1 administration.

Techniques: Expressing, Incubation, Sonication, Western Blot