Fig 1: Aspirin upregulates IL-1Ra in mouse BV-2 microglial cells. A, B) BV-2 cells were treated with 1, 2, 5, and 10μM of aspirin for 2 h under serum-free condition. An increase in IL-1Ra mRNA was significant with p < 0.001 by one-way ANOVA. C, D) An increase in IL-1Ra protein by aspirin was significant at 2μM with p < 0.01 by one-way ANOVA. E, F) Immunostaining and quantification of IL-1Ra showed significance at the p < 0.001 level with 2 and 5μM aspirin by one-way ANOVA. On the other hand, no change in Iba-1 was observed at these doses of aspirin. Results are mean±SEM of three experiments. *p < 0.05, **p < 0.01 and ***p < 0.001.
Fig 2: Aspirin upregulates IL-1Ra in mouse primary astrocytes. A, B) WT mouse astrocytes were treated with 1, 2, 5, and 10μM of aspirin for 2 h under serum-free condition. An increase in IL-1Ra mRNA was significant with p < 0.001. However, increase in either IL-1β or IL-1R was not significant. C, D) Time course of WT astrocytes beginning with 15 min to 240 min using 5μM of aspirin. All treatments after 120 min were significant at the p < 0.001 level by one-way ANOVA. E, F) Time course and related quantification of IL-1Ra protein immunoreactivity showing IL-1Ra with significance at the p < 0.001 level and the p < 0.01 level by one-way ANOVA. G) Representative images of immunostaining of astrocytes showing an increase in IL-1Ra immunoreactivity after aspirin treatment. Results are mean±SEM of three experiments. ***p < 0.001.
Fig 3: HFD alters macrophage polarization during tendon repair.Relative mRNA expression of (A) F4/80; M1 genes (B) TNFa, and (C) CD86; M2 genes (D) Arginase1, (E), Fizz1 (F) CD206, (G) CD163, and (H) IL-1ra was determined by RT-PCR between 3 and 28 days post-repair. Data were normalized to the internal control β-actin. Fold changes are reported relative to LFD day 3 expression. White bars = LFD; black bars = HFD, (*) indicates p<0.05, (**) indicates p<0.001, (***) indicates p<0.0001 between HFD and LFD.
Fig 4: miR-122-5p inhibitor protects A549 cells from LPS-induced injury by targeting IL1RN. (A) MTT assay of cell viability. (B) LDH release assay. (C) Flow cytometry analysis of apoptosis and (D) the apoptotic ratio. (E) Western blotting of cleaved-caspase 3 expression and (F) the cleaved-caspase 3/caspase 3 ratio. **P<0.01 vs. control; ##P<0.01 vs. LPS + inhibitor control; &&P<0.01 vs. LPS + miR-122-5p inhibitor + control-siRNA. miR, microRNA; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; IL1RN, IL-1 receptor antagonist; si, small interfering RNA.
Fig 5: Nec-1 and SB216763 reduce the levels of inflammatory-related cytokines in astrocytes after OGD6 h-Re24 h. (A–D) Representative images from Western blotting (WB) analysis of IL-1Ra, IL-1β, IL-6, and TNF-α in astrocytes. Astrocytes were treated with or without SB216763 (5 μM) during OGD and reoxygenation. β-Actin protein was used as a loading control. Mean ± SD, n = 3. **P < 0.01, vs. non-OGD-Re24 h group; ##P < 0.01 vs. OGD6 h-Re24 h group. (E–H) Representative images from Western blotting (WB) analysis of IL-1Ra, IL-1β, IL-6, and TNF-α. Astrocytes were exposed to OGD for 6 h and reoxygenation for 24 h. Astrocytes were treated with or without Nec-1 (100 μM) during OGD and reoxygenation. β-Actin protein was used as a loading control. Mean ± SD, n = 3. *P < 0.05, **P < 0.01 vs. non-OGD-Re24 h group; #P < 0.05, ##P < 0.01 vs. OGD6 h-Re24 h group.
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