Mediators of Inflammation / 2015 / Article / Fig 2

Research Article

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

Figure 2

CO inhibits LPS-induced TN-C expression and proinflammatory cytokines in RAW 264.7 macrophages. (a–d) To detect the TN-C expression and proinflammatory cytokine production, macrophages 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. The protein levels (upper) and mRNA levels (lower) of TN-C were measured by Western blotting and Real Time RT-PCR. The mRNA levels (b, c) and protein levels (d) of TNF-α and IL-6 were reduced by CORM-2. The amount of mRNA with Real Time RT-PCR was detected and protein expression was measured with ELISA after 24 h of LPS treatment. (e–h) Cells were pretreated with CORM-2 (20 μM) or RuCl3 (20 μM) for 1 h followed by stimulation in the presence or absence of LPS (100 ng/mL) for 8 h. RuCl3 was used as a negative control for CORM-2. The levels of mRNA in TN-C (e), TNF-α (f), and IL-6 (g) after treatment of CORM-2 were measured by Real Time RT-PCR and the protein levels of TNF-α and IL-6 were detected by ELISA after 24 h of LPS treatment (h). (i–k) Cells were pretreated with CO gas (250 ppm) for 2 h and incubated with LPS (100 ng/mL) stimulated for 8 h. TN-C (i), TNF-α (j), and IL-6 (k) mRNA levels were detected by Real Time RT-PCR. Data represents mean ± SEM, as compared with control; and ns, nonsignificant, as compared with the cells exposed to LPS alone.

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