Activation of MK-2 by FnIII-1c regulates IL-8 mRNA stability.

<p>Monolayers of dermal fibroblasts were serum-starved overnight and incubated with 10 µM III-1c for 2 h prior to the addition of 10 µM actinomyosin D with or without 20 µM MK-2 inhibitor. After incubation for the indicated times, IL-8 mRNA levels were assessed by RT-PCR. The mRNA level was analyzed relative to the blank control and balanced with the housekeeping gene, β-actin using ΔΔCt (A). The percent (%) change in mRNA expression between 0–30 min and 30–60 min was calculated and analyzed using a two way ANOVA followed by t-test with Bonferroni correction (p<0.05) (B). Error bars indicate mean ± SD of 12 samples. Data are combined from 6 experiments performed in duplicate.</p

Fibronectin Type III domains: Selective induction of cytokines in fibroblasts.

<p>Monolayers of human dermal fibroblasts were serum-starved before treatment with 20 µM FnIII-1c (A), FnEDA (C), FnIII-10n (E), FnIII-13 (F) or 100 µg/ml Tenascin C (G). RNA was extracted and gene expression profiling was performed using a Human Autoimmune and Inflammatory Cytokine PCR array. Dashed lines indicate a 1000 fold change in gene expression (A and C) and a 10 fold increase in gene expression (E, F, G). The 5 genes whose expression was increased ≥1000 fold are shown in tables (B,D).</p

IL-8 expression induced by FnIII domains is NF-κB and TLR4-dependent.

<p>Fibroblasts were serum-starved overnight before treatment with 20 µM FnIII-1c, FnEDA, FnIII-10n, FnIII-13 or PBS in 0.1% BSA-DMEM for the indicated amount of time (A) or for 4 h at the indicated dose (B). Fibroblasts or THP-1 monocytes were incubated with increasing doses of LPS for 4 h (C). Tenascin C (100 µg/ml), LPS (1 µg/ml) or FnEDA (200 µg/ml) were incubated with either dermal fibroblasts or THP-1 monocytes for 4 h (D). Cells were pretreated with the indicated amounts of inhibitor or monoclonal antibody for 1 h prior to the addition of FnIII-1c (E) or FnEDA (F) in 0.1% BSA-DMEM for 4 h. In all experiments, conditioned media was collected and IL-8 protein amounts determined by ELISA. To determine significance, statistical analysis was performed using Student's t test. *p<0.05. Error bars indicate mean ± S.D. of three separate experiments performed in triplicate.</p

FnEDA and FnIII-1c domains work synergistically to regulate IL-8 expression.

<p>Monolayers of human dermal fibroblasts were serum-starved overnight and then incubated with FnIII-1c and FnEDA alone or in combination. After 4 h (A, B, C) or 24 h (D), the amount of IL-8 protein in the conditioned medium was determined by ELISA. Dashed lines and arrows in (C) indicate the approximate Km of FnIII-1c on IL-8 production. Specific amounts of each module are provided in the Figure. Error bars indicate mean ± S.D. of three experiments performed in triplicate.</p

FnIII-1c activates TLR4-dependent p38 MAPK/MK-2 signaling in dermal fibroblasts.

<p>Fibroblasts were serum-starved overnight and treated with the indicated amounts of Fn Type III domains, FnIII-1c, FnIII-10n or FnIII-13 for 1 h. Cell layers were lysed and proteins were electrophoresed and immunoblotted with antibodies to phospho-p38 (A, C) and phospho-MK-2 (B, D). Cells were pretreated with increasing concentrations of antibodies to human TLR4 or 20 µg/ml control antibody, goat IgG (GIgG) for 30 min prior to the addition of 20 µM FnIII-1c for 1 h. Cells were lysed and immunoblotted for phospho-p38 and phospho-MK-2 (E). Immunoblotting for total p38, MK-2 or FAK served as loading controls.</p

FnIII-1c activates NF-κB in parallel with the p38 MAPK signaling pathway.

<p>Monolayers of human fibroblasts were serum-starved overnight and treated with the indicated concentration of p38 inhibitor (SB203580), MK-2 inhibitor (MK-2 inhibitor III), or NFκB inhibitors (PS-1145, Bay11-7082) for 1 h. Cells were then stimulated with 20 µM FnIII-1c for 1vh. Cells were lysed and the nuclear fraction isolated and analyzed by Western blot for the presence of NF-κB protein p65/rel A (A). Blots were quantified by densitometry and normalized to total lamin A/C. Values are mean ± S.D. of 3 separate experiments (B). Cytoplasmic fractions were analyzed by Western blot for p-p38 (C). Membranes were stripped and reprobed with an antibody to nuclear Lamin A/C or FAK, which served as loading control. Proposed signaling pathway (D).</p

The p38 MAPK/MK-2 pathway regulates FnIII-1c induced IL-8 expression.

<p>Fibroblasts were treated with the indicated concentration of inhibitors for p38 (A, B) or MK-2 (C, D) for 1 h prior to stimulation with 20 µM FnIII-1c. The amount of IL-8 protein present in the medium was measured by ELISA at 4 h (A, C). MK-2 phosphorylation in the presence of increasing amounts of p38 inhibitor (SB203580) was assessed using antibodies to phospho-MK-2 (B). HSP27 phosphorylation in the presence of MK-2 inhibitor was assessed by immunoblotting with antibodies to phospho-HSP27. FAK served as loading control (D). Data are expressed as % control where cells which received 20 µM FnIII-1c without inhibitor (0) in A and C are set at 100%. Error bars indicate mean ± SD of 3 separate experiments.</p

Discovery of a Potential Anti-Inflammatory Agent: 3‑Oxo-29-noroleana-1,9(11),12-trien-2,20-dicarbonitrile

Fifteen novel derivatives of glycyrrhetinic acid (GA) were synthesized and evaluated for anti-inflammatory activities. It was found that the introduction of 1-en-3-one and 9(11),12-diene and 2,20-dinitrile functionalities into the scaffold of GA led to the discovery of potent compound <b>19</b> for inhibition of LPS-induced NO production. Furthermore, <b>19</b> effectively inhibited the protein and mRNA expression of inducible NO synthase (iNOS) and the mRNA expression of TNF-α, IL-6, and IL-1β in LPS-stimulated RAW 264.7 macrophages. Mechanistically, <b>19</b> exerted inhibitory effects on the activation of the three main MAPKs and phosphorylation and degradation of IκB-α, as well as the ratio of nuclear/cytosolic content of p65. Importantly, <b>19</b> significantly decreased the mortality rate in the mouse model of LPS-induced sepsis shock. It is noteworthy that inhibitory effect of <b>19</b> on NO production was not blocked by the glucocorticoid receptor antagonist mifepristone, indicating that it does not act through the glucocorticoid receptor

Additional file 1: of Internal limiting membrane peeling and gas tamponade for myopic foveoschisis: a systematic review and meta-analysis

1. Searching strategy in pubmed for the comparison of ILM peeling group & non-ILM peeling group. 2. Searching strategy in pubmed for the comparison of Tamponade group & non-Tamponade group. (DOC 30 kb

Additional file 2: of Internal limiting membrane peeling and gas tamponade for myopic foveoschisis: a systematic review and meta-analysis

1. Outcome indicators of included studies in the comparison of ILM peeling group & non-ILM peeling group. MF, myopic foveoschisis; NR, not reported; Song et al.①, Song et al.②: two sets of data in the study of Song et al. 2. Outcome indicators of included studies in the comparison of Tamponade group & non-Tamponade group. MF, myopic foveoschisis; NR, not reported. (DOC 79 kb