Quercetin and Quercitrin Attenuates the Inflammatory Response and Oxidative Stress in LPS-Induced RAW264.7 Cells: In Vitro Assessment and a Theoretical Model

Abstract

Background: Nowadays, atmospheric pollutants, ultraviolet rays, and other factors cause the imbalance of cell redox, resulting in skin oxidative damage. There is an interaction between inflammatory response and oxidative stress, which often involve networks of reactions and serve to amplify each other. Quercetin and quercitrin, with strong antioxidant and anti-inflammatory properties, were widely applied in cardiovascular disease, osteoporsis, pulmonary disease, etc. However, the regulation mechanism of quercetin and quercitrin on various inflammatory skin diseases is still not clear.

Purpose: In this study, quercetin and quercitrin were used to investigate whether they had anti-inflammatory and anti-ROS effects. Besides, theoretical calculation method was also adopted to preliminarily explore the mechanism of the anti-inflammatory and antioxidant effects of these two substances.

Methods: CCK-8 assay was employed to investigate the cytotoxicity. The concentration of NO measured by Griess Reaction System. Moreover, the inflammatory factors (TNF-α, IL-1β, and IL-6) were reduced in LPS-stimulated RAW264.7 cells were tested by ELISA kits. The trend of ROS changes was detected by DCFH-DA method. Finally, the mechanism of the anti-inflammatory and antioxidant effects of these two substances was carried out by DMol3 package in Materials Studio.

Results: CCK-8 assay results guided that the safe concentration of quercetin and quercitrin was lower than 15.0 μg/mL and 22.4 μg/mL, respectively. Also, the concentration of NO could significantly be inhibited by quercetin and quercitrin. Besides, the ELISA results showed that TNF-α, IL-1β, and IL-6 were reduced in LPS-stimulated RAW264.7 cells after interfering with quercetin and quercitrin. The trend of ROS changes was similar to that of inflammatory factors. Finally, the theoretical calculation illustrated that the oxygen atom on B rings may be the main site of electron cloud density changes, which may suggest a possible mechanism for the anti-inflammatory and ROS scavenging effects of quercetin and quercitrin.

Conclusions: This experiment shows that LPS can induce the overactivating of macrophages and the activated macrophages can subsequently induce inflammatory storms and oxidative stress. Both quercetin and quercitrin can inhibit LPS-induced macrophage inflammation and oxidative stress by experiment and theoretical calculations.

Figure 1

The molecular structure formula of quercetin and quercitrin.

Figure 2

The cell survival rate of quercetin (a) and quercitrin (b) on RAW264.7 cells. The results represent the $\overline{x}$ ± s (n = 3). ⁽¹⁾P < 0.01; ⁽²⁾P < 0.05, compared with control group.

Figure 3

The effect of quercetin and quercitrin of NO content on RAW264.7 cells induced by LPS, $\overline{x}$ ± s (n = 3). ⁽¹⁾P < 0.01; ⁽²⁾P < 0.05, compared with control group; ⁽³⁾P < 0.01; ⁽⁴⁾P < 0.05, compared with LPS group.

Figure 4

The effect of quercetin and quercitrin of TNF-α (a), IL-1β (b), and IL-6 (c) contents on RAW264.7 cells induced by LPS, $\overline{x}$ ± s (n = 3). ⁽¹⁾P < 0.01; ⁽²⁾P < 0.05, compared with control group; ⁽³⁾P < 0.01; ⁽⁴⁾P < 0.05, compared with LPS group.

Figure 5

(a): Effects of quercetin and quercitrin on LPS-induced RAW246.7 cells detected by DCFH-DA assay (×100); (b) effects of quercetin and quercitrin on ROS, $\overline{x}$ ± s (n = 3). ⁽¹⁾P < 0.01; ⁽²⁾P < 0.05, compared with control group; ⁽³⁾P < 0.01; ⁽⁴⁾P < 0.05, compared with LPS group.

Figure 6

Interrelation of oxidative stress and inflammation.