Crocetin Exerts Its Anti-inflammatory Property in LPS-Induced RAW264.7 Cells Potentially via Modulation on the Crosstalk between MEK1/JNK/NF-κB/iNOS Pathway and Nrf2/HO-1 Pathway

Figure 3

The effect of crocetin on the phosphorylation of protein kinases. (a) JNK inhibitor suppressed LPS-induced iNOS expression. RAW264.7 cells were pretreated for 1 h with AZD6244, SB202190, or SP600125 alone and then exposed to 40 ng/ml LPS for 12 h. The expression of iNOS was detected by Western blot analysis. (b) MEK inhibitor reversed the effect on crocetin-mediated iNOS inhibition. RAW264.7 cells were treated with MAPK inhibitors for 1 h, subsequently added crocetin for another 1 h, and then exposed to 40 ng/ml LPS for 12 h. Western blot was applied to detect iNOS expression. (c) Crocetin inhibited LPS-induced phosphorylation of JNK but has no significant effect on MEK and p38. RAW264.7 cells were pretreated with crocetin (10-20 μg/ml) for 1 h and then exposed to 40 ng/ml LPS for 0.5 h. The total and phosphorylated protein kinases were detected with their antibodies, respectively. (d) MEK1-KD and ERK1-KD relieved the crocetin-induced iNOS inhibition. Selecting RAW264.7 cells with scramble shRNA as blank controls (NC), MEK1-KD cell lines or ERK1-KD cell lines were treated with crocetin for 1 h and then exposed to 40 ng/ml LPS for 12 h. The iNOS expression was detected by Western blot analysis. (e) Crocetin increased the phosphorylation of MEK. RAW264.7 cells were pretreated with crocetin (20 μg/ml) at the indicated time or pretreated with crocetin (10-20 μg/ml) for 30 min, and then, total and phosphorylated protein levels of MEK were detected with their antibodies, respectively. (f) JNK is critical for LPS-induced iNOS expression. ERK1-KD (shRNA-2) cells were pretreated with SP600125 (10-20 μM) for 1 h, and the following steps were done as described in (a). The expressions of iNOS were detected by Western blot analysis. Each value represents the of triplicate tests. and vs. LPS-treated cells.

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