**1. Introduction**

Inflammation is the body's immune response and defense mechanism against tissue damage caused by exposure to harmful or toxic external agents, infections, and physical injuries [1]. Inflammation resulting from infection or injury is associated with inflammatory responses such as immune cell recruitment and accumulation, release of inflammatory mediators, and changes in blood vessel permeability [2]. Generally, there are two types of inflammation: acute and chronic. Acute inflammation is a rapid process that repairs quickly to minimize damage and restore tissue homeostasis [3]. However, in chronic conditions, the inflammatory response continues, resulting in severe organ damage [4]. Chronic inflammation promotes the progression of several diseases, including cardiovascular disease, inflammatory bowel disease, rheumatoid arthritis and diabetes [5]. Crohn's disease (CD) is the most prevalent IBD syndrome treated using 6-mercaptopurine (6-MP) and its prodrug azathioprine (AZA) [6]. As immunosuppressive drugs, 6-MP and AZA inhibit inflammation by blocking the body's immune response; however, they may cause disease recurrence and side effects, such as hepatotoxicity [6]. Sulfasalazine, a medication for patients with rheumatoid arthritis, has immunomodulatory properties and suppresses pro-inflammatory cytokines; however, it can cause gastrointestinal side effects, such as headache, dizziness,

**Citation:** An, J.; Ryu, G.; Shin, S.-A.; Kim, H.; Ahn, M.-J.; Lee, J.H.; Lee, C.S. Wistin Exerts an Anti-Inflammatory Effect via Nuclear Factor-κB and p38 Signaling Pathways in Lipopolysaccharide-Stimulated RAW264.7 Cells. *Molecules* **2022**, *27*, 5719. https:// doi.org/10.3390/molecules27175719

Academic Editor: Nour Eddine Es-Safi

Received: 10 August 2022 Accepted: 2 September 2022 Published: 5 September 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

rash, and bone marrow suppression [7]. Non-steroidal anti-inflammatory drugs (NSAIDs) are anti-inflammatory drugs used worldwide to treat inflammatory conditions [8]. Among NSAIDs, ibuprofen (IBU) is a non-selective inhibitor of both cyclooxygenase (COX)-1 and -2 isozymes [9]; however, it is associated with the risk of cardiovascular and gastrointestinal complications [10,11]. Naproxen is another NSAID used to treat osteoarthritis, migraine, and rheumatoid arthritis [10]. However, NSAIDs have several side effects, including gastrointestinal toxicity, cardiovascular risk, kidney damage, and hepatotoxicity [8]. Therefore, it is necessary to develop safer anti-inflammatory treatment strategies.

When the human body is infected by pathogens in damaged tissue, immune cells recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) using pattern recognition receptors (PRRs) and promote inflammatory signaling pathways [12]. The Toll-like receptor (TLR) family is mainly expressed in immune cells as major PRRs that play key roles in the induction of innate immune responses and first-line defense [13,14]. Lipopolysaccharide (LPS) is a well-known inflammatory PAMP molecule that exists in the cell walls of gram-negative bacteria [13]. The TLR4–LPS interaction activates the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways [15,16]. TLR4-mediated modulation of myeloid differentiation factor 88 (MyD88) results in activation of NF-κB. Furthermore, NF-κB is regulated by the IκB and IκB kinase (IKK) complex [17], which causes IκB phosphorylation. The phosphorylation of the IκB protein results in the phosphorylation of p65, which translocates to the nucleus [18]. In addition, NF-κB is regulated by phosphatidylinositol 3-kinase (PI3K)-Akt [19]. The MAPK pathway includes extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK), and p38, which result in further activation of the transcription factor activator protein 1 (AP-1) [16]. Activation of the NF-κB and MAPK pathways has been reported to increase the expression of inflammatory enzymes (inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2)) and cytokines (interleukin (IL)-1β and IL-6) in immune cells [16,20]. Therefore, understanding the mechanism by which signaling pathways are controlled at the molecular level might help modulate the inflammatory response and develop new modulators of inflammation.

Phytochemicals extracted from plants have emerged as new agents for the treatment of chronic inflammatory diseases [21]. Current anti-inflammatory disease treatments (nonsteroidal anti-inflammatory drugs and glucocorticoids) have many side effects, including tissue damage, cardiovascular problems, and liver complications. As plant-derived drugs have been reported to have fewer toxic effects, it has been suggested that they could be potential modulators of inflammation [22,23]. Human papillomavirus (HPV) infection can cause chronic inflammation due to the release of pro-inflammatory cytokines [24,25]. Sinecatechin extracted from green tea has been used as a medication to treat HPV infections [24,25]. Silymarin extracted from the seeds of milk thistle (*Silybum marianum*) has anti-inflammatory activity; it inhibits NF-κB activity and improves liver function in patients with hepatitis B (HBV) [26–28]. Eupatilin, a plant-derived drug extracted from *Artemisia asiatica Nakai*, is used to treat gastritis by mediating anti-inflammatory effects and promoting the regeneration of offended mucosa [29]. Among various isoflavones, genistein is known for its antioxidant and anti-inflammatory properties. Genistein prevents endothelial inflammatory damage by inhibiting the NF-κB pathway, which mediates the transcription of proinflammatory cytokines [30]. Although investigations are required to confirm its safety and efficacy, low- (5–15 mg/kg/day) and high-dose (160 mg/kg/day) genistein treatments for patients with mucopolysaccharidosis (MPS) III reportedly have no major side effects [31]. Another isoflavone, daidzein, inhibits the JNK, PARP, and NF-κB signaling pathways to express pro-inflammatory cytokines [32]. In addition, plant polyphenols, particularly flavonoids, have exhibited anti-inflammatory activity both in vitro and in vivo [32]. Compound 4',6-dimethoxyisooflavone-7-*O*-β-d-glucopyranoside (wistin), belonging to the isoflavone family, is an agonist of PPARγ and PPARα in adipocytes and hepatocytes, respectively [33,34]. However, its anti-inflammatory role has not been investigated. Therefore, in

this study, we aimed to identify its anti-inflammatory effects and elucidate the molecular mechanisms underlying its anti-inflammatory effects in LPS-stimulated RAW264.7 cells.
