Mediators of Inflammation / 2015 / Article / Fig 4

Research Article

Carbon Monoxide Inhibits Tenascin-C Mediated Inflammation via IL-10 Expression in a Septic Mouse Model

Figure 4

CO inhibits TN-C-mediated inflammation via IL-10 induction in RAW 264.7 macrophages and peritoneal macrophages. (a-b) Cells were pretreated with CORM-2 (0, 10, 20, and 40 μM) for 1 h and then stimulated with LPS (100 ng/mL) for 8 h. IL-10 mRNA level was detected by Real Time RT-PCR in RAW 264.7 (a) and peritoneal macrophages (b). After treatment of CO gas (250 ppm) for 2 h and being incubated with LPS (100 ng/mL) stimulated for 8 h, IL-10 mRNA level was detected by Real Time RT-PCR (c). (d-e) Cells with CORM-2 (20 μM) or RuCl3 (20 μM) for 1 h were stimulated with LPS (100 ng/mL) for 8 h. IL-10 mRNA levels increased with CORM-2 which was detected by Real Time RT-PCR in RAW 264.7 (d) and peritoneal macrophages (e). RuCl3 was used as a negative control for CORM-2. (f–i) RAW 264.7 cells were transfected with TN-C siRNA or control siRNA (scRNA). After treatment with CORM-2 (20 μM) for 1 h, cells were stimulated with 100 ng/mL LPS for 8 h to evaluate mRNA levels of TN-C (f) and TNF-α and IL-6 (g) by Real Time RT-PCR. The protein levels of TNF-α and IL-6 were measured by ELISA (h) after 24 h LPS treatment. The mRNA levels of IL-10 were detected by Real Time RT-PCR (i). (j–m) RAW 264.7 macrophages were transfected with IL-10 siRNA or control siRNA (scRNA). After treatment with CORM-2 (20 μM) for 1 h, cells were stimulated with 100 ng/mL LPS for 8 h. The mRNA levels of IL-10 (j), TN-C (k), and TNF-α and IL-6 (l) were detected by Real Time RT-PCR, and protein levels of TNF-α and IL-6 were measured by ELISA (m) after 24 h LPS treatment. RAW 264.7 cells were pretreated with CORM2 (20 μM) or mouse recombinant IL-10 (20 ng/mL) for 1 h followed by the stimulation of LPS (100 ng/mL) for another 8 h. TN-C mRNA levels were determined by Real Time PCR (n). (o-p) RAW 264.7 cells were pretreated with CORM2 or RuCl3 in various concentrations (0, 10, 20, and 40 μM) with or without ZnPP (0, 5, 10, and 20 μM) followed by 8 h LPS treatment. The mRNA levels of HO-1 (o) and TN-C (p) were detected by Real Time PCR. Data represents mean ± SEM, , and as compared with control; , , and ns, nonsignificant, as compared with the cells exposed to LPS alone (normal condition or TN-C/IL-10 siRNA, compared separately).
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