The Galloyl Group Enhances the Inhibitory Activity of Catechins against LPS-Triggered Inflammation in RAW264.7 Cells

Abstract

The galloyl group in catechins was confirmed to be crucial for their health benefits. However, whether the catechins' galloyl group had a contribution to their anti-inflammation remains unclear. This study investigated the anti-inflammation properties and mechanisms of catechins in RAW264.7 cells by using ELISA, fluorometry, flow cytometer, Western blot, and molecular docking. Results showed that the galloyl group enhanced the inhibitory abilities of catechins on inflammatory cytokines (NO, PGE₂, IL-1β, and TNF-α) and ROS release in LPS-induced cells. This suppression was likely mediated by delaying cells from the G0/G1 to the S phase, blocking COX-2 and iNOS via the TLR4/MAPK/NF-κB pathway with PU.1 as an upstream target. The research proved that the existence of galloyl groups in catechins was indispensable for their anti-inflammatory capacities and offered a theoretical basis for the anti-inflammatory mechanism of galloylated catechins. Future research is needed to verify the anti-inflammatory effects of catechins in various sources of macrophages or the Caco-2/RAW264.7 cell co-culture system.

Keywords: TLR4/MAPK/NF-κB pathway; catechins; galloyl group; inflammation; inhibitory.

Figure 1

The chemical structures of four catechins: (A) EC, epicatechin; (B) ECG, epicatechin-3-gallate; (C) EGC, epigallocatechin; and (D) EGCG, epigallocatechin-3-gallate. The group in red is the galloyl group.

Figure 2

Influences of catechins (EC, epicatechin; ECG, epicatechin-3-gallate; EGC, epigallocatechin; EGCG, epigallocatechin-3-gallate) on RAW 264.7 cell viability. Results were displayed as mean ± SD from no less than three experimental replications.

Figure 3

Effects of catechins (EC, epicatechin; ECG, epicatechin-3-gallate; EGC, epigallocatechin; EGCG, epigallocatechin-3-gallate) on the levels of NO (A), PGE₂ (B), IL-1β (C), and TNF-α (D) in RAW264.7 cells. Results were displayed as mean ± SD from no less than three experimental replications. Diverse lowercases in the histogram denote significant differences (p < 0.05).

Figure 4

Impacts of catechins (EC, epicatechin; ECG, epicatechin-3-gallate; EGC, epigallocatechin; EGCG, epigallocatechin-3-gallate) on ROS release in LPS-induced RAW264.7 cells. (A) Fluorescence microscopy observation. (B) Quantitative analysis. The cells were exposed to LPS (1 μg/mL) with or without the addition of catechins (100 µM) for 24 h. Results were displayed as mean ± SD from no less than three experimental replications. Diverse lowercases in the histogram denote significant differences (p < 0.05).

Figure 5

Regulatory influences of catechins (CAs: EC, epicatechin; ECG, epicatechin-3-gallate; EGC, epigallocatechin; EGCG, epigallocatechin-3-gallate) on cell cycle progression. (A) RAW264.7 cells were harvested after 24 h of LPS induction, colored with PI, and subsequently detected using flow cytometry. (B) The modulation of cell cycle by CAs was represented as the relative proportion of cells in three different phases.

Figure 6

Impacts of catechins (CAs: EC, epicatechin; ECG, epicatechin-3-gallate; EGC, epigallocatechin; EGCG, epigallocatechin-3-gallate) on the iNOS and COX-2 expression in macrophages. Cells were exposed to LPS (1 μg/mL) with or without the addition of catechins (100 µM) for 24 h. Results were displayed as mean ± SD from no less than three experimental replications. Diverse lowercases in the histogram denote significant differences (p < 0.05).

Figure 7

Effects of catechins (CAs: EC, epicatechin; ECG, epicatechin-3-gallate; EGC, epigallocatechin; EGCG, epigallocatechin-3-gallate) on MAPK/NF-κB pathway in LPS-triggered macrophages. (A) The NF-κB phosphorylation level in cells. (B) The ERK, JNK, and p38 phosphorylation levels in cells. Results were displayed as mean ± SD from no less than three experimental replications. Diverse lowercases in the histogram denote significant differences (p < 0.05).

Figure 8

Influences of catechins (CAs: EC, epicatechin; ECG, epicatechin-3-gallate; EGC, epigallocatechin; EGCG, epigallocatechin-3-gallate) on TLR4/CD14 pathway in LPS-triggered RAW264.7 cells. The expression levels of CD14, TLR4, MD-2, MyD88, and TRAF6 in cells. Results were displayed as mean ± SD from no less than three experimental replications. Diverse lowercases in the histogram denote significant differences (p < 0.05).

Figure 9

Docking results of the interaction between PU.1 and galloylated catechins (ECG, epicatechin-3-gallate; EGCG, epigallocatechin-3-gallate). (A) The 3D (A) and 2D (B) diagram of ECG-PU.1-DNA interaction. The 3D (C) and 2D (D) diagrams of EGCG-PU.1-DNA interaction.