Scutellarein Inhibits LPS-Induced Inflammation through NF-κB/MAPKs Signaling Pathway in RAW264.7 Cells

Abstract

Inflammation is a severe topic in the immune system and play a role as pro-inflammatory mediators. In response to such inflammatory substances, immune cells release cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). Lipopolysaccharide (LPS) is known as an endotoxin in the outer membrane of Gram-negative bacteria, and it catalyzes inflammation by stimulating the secretion of inflammatory-mediated cytokines such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) by stimulated immune cells. Among the pathways involved in inflammation, nuclear factor kappa (NF-кB) and mitogen-activated protein kinases (MAPKs) are important. NF-kB is a diploid composed of p65 and IkBα and stimulates the pro- gene. MAPKs is a family consisting of the extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38, JNK and p38 play a role as proinflammatory mediators. Thus, we aim to determine the scutellarein (SCU) effect on LPS stimulated RAW264.7 cells. Furthermore, since scutellarein has been shown to inhibit the SARS coronavirus helicase and has been used in Chinese medicine to treat inflammatory disorders like COVID-19, it would be required to examine scutellarein's anti-inflammatory mechanism. We identified inflammation-inducing substances using western blot with RAW264.7 cells and SCU. And we discovered that was reduced by treatment with SCU in p-p65 and p-IκBα. Also, we found that p-JNK and p-ERK were also decreased but there was no effect in p-p38. In addition, we have confirmed that the iNOS was also decreased after treatment but there is no change in the expression of COX-2. Therefore, this study shows that SCU can be used as a compound to treat inflammation.

Keywords: LPS-induced inflammation; MAPK; NF-кB; scutellarein (SCU).

Figure 1

Cytotoxic effect of SCU on RAW264.7 cells treated with or without LPS (1 μg/mL) and treated with SCU(0, 25, 50, 75, 100, and 125 μM) at 37 °C for 24 h and 48 h. (A) Chemical structure of SCU. (B) SCU (0, 25, 50, 75, 100, and 125 μM) was treated by concentration, and then the toxicity of SCU to cells for 24 h and 48 h were measured. (C) Cell viability when LPS and SCU were treated together for 24 h and 48 h. Comparison with SCU and LPS treated group * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2

Effect of SCU on LPS-induced protein expression of iNOS and COX-2. (A) Relatibve density of iNOS expression, despite (B) Relative density of COX-2 expression. Comparison with only LPS treated ### p < 0.001. Comparison with SCU and LPS treated group * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Effect of SCU on LPS-induced protein expression of p-p65 and p-IкBα. RAW264.7 cells were treated with LPS (1 μg/mL) and treated with SCU (0, 25, 50, and 75 μM) for at 37 °C 48 h. (A) Relative density of p-p65 (B) Relative density of p-IkBα. Comparison with only LPS treated group # p < 0.05, ## p < 0.01.** p < 0.01, *** p < 0.001. Comparison with SCU and LPS treated group (C) Molecular docking with NF-κB and SCU.

Figure 4

Effect of SCU on LPS-induced MAPKs protein expression. (A) Expression of p-JNK affected by SCU. (B) Expression of p-ERK affected by SCU. Comparison with only LPS treated group ## p < 0.01, ### p < 0.001. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

Summary of pathways in which the SCU effect.