[Calenduloside E inhibits lipopolysaccharide-induced inflammatory response by inhibiting activation of ROS-mediated JAK1-stat3 signaling pathway in RAW264.7 cells]

Abstract

Objective: To investigate the effect of calenduloside E on lipopolysaccharide (LPS)-induced inflammatory response in RAW264.7 cells and explore the underlying molecular mechanism.

Methods: CCK-8 assay was used to examine the effect of different concentrations of calenduloside E (0-30 μg/mL) on the viability of RAW264.7 cells. The release of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in RAW264.7 cells in response to pretreatment with 6, 8, and 10 μg/mL calenduloside E for 2 h followed by stimulation with 100 ng/mL LPS was detected using enzyme-linked immunosorbent assay (ELISA). The expression levels of iNOS and COX-2 and the activation of JAK-stats, MAPKs and NF-кB signaling pathways in the treated cells were determined using Western blotting. A reactive oxygen species (ROS) detection kit was used to detect ROS production in the cells, and the nuclear translocation of the transcription factor stat3 was observed by laser confocal microscopy.

Results: Calenduloside E below 20 μg/mL did not significantly affect the viability of RAW264.7 cells. Calenduloside E dose-dependently decreased the expression levels of iNOS and COX-2 induced by LPS, inhibited LPS-induced release of TNF-α and IL-1β, and suppressed LPS-induced JAK1-stat3 signaling pathway activation and stat3 nuclear translocation. Calenduloside E also significantly reduced ROS production induced by LPS in RAW264.7 cells.

Conclusions: Calenduloside E inhibits LPS-induced inflammatory response by blocking ROS-mediated activation of JAK1-stat3 signaling pathway in RAW264.7 cells.

Keywords: JAK1-stat3 signaling pathway; RAW264.7 cells; calenduloside E; inflammation; lipopolysaccharide.

1

金盏花苷E对RAW264.7细胞活力的影响 Effect of different concentrations of calenduloside E (CE) on RAW264.7 cell viability. **P < 0.01 vs control group

2

金盏花苷抑制LPS诱导炎症蛋白表达(A)及促炎因子释放(B和C) Inhibitory effects of CE on LPS-induced inflammatory proteins (A) and inflammatory cytokines (B, C) in RAW264.7 cells. ^##P < 0.01 vs control group; *P < 0.05, **P < 0.01 vs LPS group.

3

金盏花苷E对LPS诱导的MAPKs及NF-κB信号途径活化的影响 Effects of CE on LPS-induced activation of MAPKs and NF-κB signaling pathways in RAW264.7 cells

4

金盏花苷E对LPS诱导JAK-stats信号通路活化的影响 Effects of CE on LPS-induced activation of JAK-stats signaling pathway in RAW264.7 cells. ^##P < 0.01 vs control group; *P < 0.05, **P < 0.01 vs LPS group

5

金盏花苷E抑制LPS诱导的stat3核转位 Inhibitory effects of CE on LPS-induced nuclear translocation of stat3 in RAW264.7 cells (Scale bar=25 μm)

6

金盏花苷E抑制LPS诱导的ROS产生 Inhibitory effect of CE on LPS-induced ROS production in RAW264.7 cells (Scale bar= 100μm). **P < 0.01 vs LPS group