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. 2021 Sep 10;2021:6631929. doi: 10.1155/2021/6631929

Figure 4.

Figure 4

The involvement of the Nrf2/HO-1 pathway in the crocetin-induced inhibitory activity on iNOS expression. (a) Crocetin increased the expressions of Nrf2 and HO-1 and the nuclear translocation of Nrf2. For Nrf2 and HO-1 expressions, RAW264.7 cells were pretreated with the indicated dose of crocetin for 1 h before starvation in serum-free medium for 2.5 h and then exposed to 40 ng/ml LPS for an indicated time. For Nrf2 nuclear translocation, cell culture and crocetin (20 μg/ml) treatment were done as described in Figure 2(c). The expressions of proteins were detected by Western blot analysis using their corresponding antibodies. (b) Nrf2-knockdown (Nrf2-KD) and HO-1-knockdown (HO-1-KD) blocked crocetin-dependent iNOS inhibition. Detection of the knockout efficiency of two monoclonal cells (top). NC and HO-1-KO cell treatment was done as described in (b). (c) HO-1-knockout (HO-1-KO) blocked crocetin-dependent iNOS inhibition. The total and phosphorylated protein kinases were detected with their antibodies, respectively. (d) HO-1-KO blocked crocetin-dependent p-IκB-α inhibition. NC and HO-1-KO cell treatment was done as described in Figure 2(b). Data are presented as the mean ± SD of triplicate tests. ∗p < 0.05 and ∗∗p < 0.01 vs. LPS-treated cells.