*2.4. Effects of Wistin on the Protein Expression Level of Pro-Inflammatory Enzymes in LPS-Induced RAW 264.7 Cells*

The expression of iNOS and COX-2 regulates key inflammatory mediators [39]. Therefore, we investigated whether wistin exhibits anti-inflammatory effects by inhibiting iNOS and COX-2 protein expression. As shown in Figure 4a,b, wistin significantly reduced the protein expression levels of iNOS and COX-2 compared to those in the LPS group. Therefore, these results suggest that wistin could also inhibit the expression of pro-inflammatory enzymes at the protein level.

**Figure 3.** The effects of wistin on mRNA expression level in LPS-induced RAW 264.7 cells. The cells were treated with the indicated concentrations of wistin for 30 min prior to treatment with LPS (0.1 μg/mL). Then, LPS-induced (**a**) iNOS, (**b**) COX-2, (**c**) IL-1β, and (**d**) IL-6 mRNA level were measured using a quantitative reverse transcription polymerase chain reaction (qRT-PCR). \* *p* < 0.05 compared with the LPS-treated group. ND: not detected. The data are presented as the means ± SD; *n* = 3.

**Figure 4.** The effects of wistin on protein expression of iNOS and COX-2 in LPS-induced RAW 264.7 cells. The cells were treated with the indicated concentrations of wistin for 30 min prior to treatment with LPS (0.1 μg/mL) for 24 h. (**a**) The protein levels of pro-inflammatory enzymes iNOS and COX-2 were determined by Western blot. (**b**) Quantitative analysis of the iNOS/β-actin and COX-2/β-actin using image J. \* *p* < 0.05 compared with the LPS-treated group. The data are presented as the means ± SD; *n* = 2.

#### *2.5. Effects of Wistin on the Activation of AKT/NF-κB Pathway in LPS-Induced RAW 264.7 Cells*

The NF-κB pathway regulates the expression and production of pro-inflammatory enzymes and cytokines [40]. Therefore, we examined the phosphorylation level of LPSinduced AKT and NF-κB (p65 subunit) following wistin (150 μM) treatment for 2 h. Wistin significantly reduced the phosphorylation level of AKT and p65 compared to that in the LPS group (Figure 5a–d). Furthermore, in the absence of LPS stimulation, p65 (red in the merged image) was present in the cytoplasm. Upon LPS stimulation, p65 (pink in merged images) was translocated to the nucleus, whereas wistin treatment reduced the nuclear translocation of p65 (red and pink in merged images) (Figure 5e). These results suggest that wistin suppresses the AKT/NF-κB signaling pathway.

**Figure 5.** The effects of wistin on p-AKT, NF-κB (p-p65 subunit) in LPS-induced RAW 264.7 cells. The cells were treated with the indicated concentrations of wistin for 30 min prior to treatment with LPS (0.1 μg/mL) for 2 h. Then, the phosphorylation of AKT (**a**) and p65 (**c**) were measured using a Western blot. Quantitative analysis of the p-AKT/β-actin (**b**) and p-p65/β-actin (**d**) using image J. (**e**) The effects of wistin on the nuclear translocation of p65 (red) using fluorescence microscopy. The merged images were acquired by overlaying two channels (p65 (red) and DAPI (blue)). \* Indicates translocation of p65 from the cytoplasm to the nucleus. The scale bar represents 10 μm. (**f**) Quantitative analysis of the nuclear translocation of p65 using ImageJ software. \* *p* < 0.05 compared with the LPS-treated group. The data are presented as the means ± SD; *n* = 3.
