*2.6. Effects of Wistin on the Activation of MAPK Pathway in LPS-Induced RAW 264.7 Cells*

The MAPK pathway is known for its role in the modulation of inflammatory responses [41]. We investigated the role of wistin in the phosphorylation of three MAPKs (p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK)). Phosphorylation of p38 was decreased by wistin, but that of ERK and JNK was not affected compared to that in the LPS group (Figure 6). Therefore, these results suggest that wistin could work by negatively regulating the p38 MAPK pathway.

**Figure 6.** The effects of wistin on MAPK (p-p38, p-ERK, and p-JNK) in LPS-induced RAW 264.7cells. 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. Phosphorylation of p38 (**a**), ERK, (**c**) and JNK (**e**) was measured using a Western blot. Quantitative analysis of the p-p38/β-actin (**b**), p-ERK/β-actin (**d**), and p-JNK/β-actin (**f**) using image J. \* *p* < 0.05 compared with the LPS-treated group. The data are presented as the means ± SD; *n* = 2.

#### **3. Discussion**

Inflammation is a protective response to harmful stimuli (PAMP and DAMP) by the immune system [42]. However, chronic inflammation can cause various diseases, such as cardiovascular disease, inflammatory bowel disease, rheumatoid arthritis, and diabetes [5]. Ibuprofen, naproxen, and other NSAIDs are conventional anti-inflammatory drugs used to treat rheumatism, osteoarthritis, arteriosclerosis, neuroinflammatory diseases, and other inflammatory diseases [8,10]. However, these drugs have several side effects, such as an increased risk of gastric mucosal injury, renal injury, and other medical complications [8,10]. Therefore, it is important to identify novel anti-inflammatory agents that can overcome the shortcomings of conventional anti-inflammatory drugs. Several plants have been used as folk medicines for the treatment and prevention of diseases [43]. Currently, sinecatechin extracted from green tea and silymarin extracted from milk thistle seeds (*Silybum marianum*) are used as plant-derived anti-inflammatory drugs for treating HPV and HBV infections, respectively [24–27]. Furthermore, as plant-derived anti-inflammatory drugs, eupatilin and JOINS tablets are used to treat gastritis and knee osteoarthritis [44,45]. Isoflavones have antioxidant, anticancer, antibacterial, and anti-inflammatory properties [32]. Our study highlights the potential of wistin as an anti-inflammatory agent with fewer side effects via modulation of the inflammatory signaling pathway. However, further in vivo and clinical studies of wistin in this regard are warranted.

During inflammation, NO plays an important role in the regulation of immune and inflammatory responses [46]. Additionally, the high production of ROS during inflammation can lead to cell damage through the oxidation of DNA, RNA, and proteins [47]. iNOS and COX-2 are pro-inflammatory mediators that are regulated by pro-inflammatory transcription factors [48]. When we examined the effects of wistin on NO and ROS generation, we found that wistin reduced the levels of both NO and ROS. In addition, wistin significantly decreased the mRNA expression of inflammatory enzymes (iNOS and COX-2) and inflammatory cytokines (IL-1β and IL-6). Furthermore, wistin decreased the protein expression of iNOS and COX-2. Therefore, these results suggest that wistin exerts anti-inflammatory effects by modulating inflammatory enzymes, inflammatory mediators, and cytokines.

NF-κB signaling is a well-known inflammatory pathway that regulates the expression of pro-inflammatory cytokines (IL-1β and IL-6) and pro-inflammatory enzymes (iNOS and COX-2) [49]. p65 (RelA) is a component of NF-κB, and phosphorylation of p65 induces the expression of a variety of genes [50]. In addition, AKT regulates NF-κB by phosphorylating the IκB kinase (IKK) complex, which phosphorylates the p65 subunit [19]. Wistin reduced the phosphorylation of AKT and p65 in LPS-stimulated RAW264.7. In addition, wistin decreased the translocation of p65 from the cytosol to the nucleus. These results suggest that wistin exerts anti-inflammatory effects by inhibiting the AKT/NF-κBsignaling pathway.

The MAPK pathway (ERK, JNK, and p38) regulates pro-inflammatory mediators [41]. In particular, p38 can induce NF-κB activation to induce the expression of pro-inflammatory cytokines [51]. p38 MAPK activates mitogen- and stress-activated protein kinases (MSK), which can activate NF-κB (p65 subunit) signaling [52]. Wistin decreased LPS-induced p38 phosphorylation but has no effect on LPS-induced EKR and JNK phosphorylation. These results suggest that wistin could have anti-inflammatory effects by inhibiting p38 pathways in LPS-stimulated RAW264.7.

In conclusion, we demonstrated that wistin exerts anti-inflammatory effects by downregulating pro-inflammatory mediators in LPS-mediated signaling. Wistin could regulate pro-inflammatory mediators, including NO, ROS, pro-inflammatory cytokines, and enzymes, by inhibiting the NF-κB and p38 signaling pathways (Figure 7). Similarly, genistein and daidzein inhibit inflammatory mediators via the NF-κB and MAPK signaling pathways [30,32]. They belong to the isoflavonoid family and exhibit versatile pharmacological activities [30,32]. Therefore, wistin may be developed as a plant-derived anti-inflammatory agent with fewer side effects than other conventional anti-inflammatory drugs.

**Figure 7.** Wistin suppressed LPS-stimulated inflammation by inhibiting the phosphorylation of the p65 and p38 signaling pathways.

#### **4. Materials and Methods**
