Atractylenolide-III suppresses lipopolysaccharide-induced inflammation via downregulation of toll-like receptor 4 in mouse microglia

Abstract

Atractylenolide-III (AIII), a sesquiterpene compound isolated from the rhizome of Atractylodes macrocephala, has been reported to have anti-inflammatory effects in the peripheral organs. However, its effects on brain inflammation remain elusive. The present study investigated the effects of AIII on the response to lipopolysaccharide (LPS) in mouse microglia and clarified the underlying mechanism. In this study, treatment of MG6 cells with AIII (100 μM) significantly decreased the mRNA expression and protein levels of toll-like receptor 4 (TLR4). In addition, pretreatment of MG6 cells and primary cultured microglia cells with AIII (100 μM) significantly decreased the mRNA expression and protein levels of tumor necrosis factor-α, interleukin-1β, interleukin-6, inducible nitric oxide synthase, and cyclooxygenase-2 induced by LPS (5 ng/mL) without cytotoxicity. Subsequently, pretreatment with AIII significantly suppressed the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun NH₂-terminal kinase (JNK) after LPS stimulation in MG6 cells. These results showed that AIII downregulated TLR4 expression, leading to suppression of the p38 MAPK and JNK pathways, which in turn inhibited the production of pro-inflammatory cytokines and enzymes in LPS-stimulated microglia. Our findings, therefore, suggest the potential for AIII as a therapeutic agent for the treatment of brain inflammation, particularly in microglia-associated inflammation.

Keywords: Atractylenolide-III; Cytokine; JNK; Mouse microglia; TLR4; p38 MAPK.

Figure 1

Effects of AIII on TLR4 expression in MG6 cells. Cells were cultured for 1, 3, and 6 h in the absence or presence of AIII (100 μΜ). The mRNA expression of TNF-α (A) was examined by RT-PCR and the protein levels of TLR4 (B) were examined by Western blotting analysis. Non-adjusted images of Western blotting analysis were presented in Supplementary Material Figure 1. (C) Concentration dependence of AIII on TLR4 mRNA expression was examined by RT-PCR after the cells were incubated with AIII (1, 10, and 100 μM) for 3 h. Results are expressed as the mean percentage ±SEM. ∗p < 0.05, ∗∗p < 0.01 compared with the control group, n = 9–15.

Figure 2

Effects of AIII on mRNA expression and protein levels of TNF-α, IL-1β, IL-6, iNOS, and COX-2 induced by LPS in MG6 cells. Cells were cultured for 3 h in the absence or presence of AIII (100 μΜ), followed by the addition of LPS (5 ng/mL) for another 3 h. The mRNA expression of TNF-α (A), IL-1β (B), IL-6 (C), iNOS (D), and COX-2 (E) was examined by RT-PCR. The culture media were collected for analysis of the protein levels of TNF-α (F), IL-1β (G), and IL-6 (H) by ELISA. (I) Effects of AIII on MG6 cell viability was examined by CCK-8 assay. Results are expressed as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, n = 6–9.

Figure 3

Effects of AIII on mRNA expression and protein levels of TNF-α, IL-1β, IL-6, iNOS, and COX-2 induced by LPS in RAW264.7 cells. Cells were cultured for 3 h in the absence or presence of AIII (100 μΜ), followed by the addition of LPS (5 ng/mL) for another 3 h. The mRNA expression of TNF-α (A), IL-1β (B), IL-6 (C), iNOS (D), and COX-2 (E) was examined by RT-PCR. The culture media were collected for analysis of the protein levels of TNF-α (F) and IL-1β (G) by ELISA. Results are expressed as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, n = 9.

Figure 4

Effects of AIII on mRNA expression and protein levels of TNF-α, IL-1β, IL-6, iNOS, and COX-2 induced by LPS in PMC. Cells were cultured for 3 h in the absence or presence of AIII (100 μΜ), followed by the addition of LPS (5 ng/mL) for another 3 h. The mRNA expression of TNF-α (A), IL-1β (B), IL-6 (C), iNOS (D), and COX-2 (E) was examined by RT-PCR. The culture media were collected for analysis of the protein levels of TNF-α (F), IL-1β (G), and IL-6 (H) by ELISA. Results are expressed as mean ± SEM. ∗∗p < 0.01, n = 6–8.

Figure 5

Effects of AIII on the phosphorylation of p38 MAPK, JNK, and NF-κB induced by LPS in MG6 cells. Cells were cultured for 3 h in the absence or presence of AIII (100 μΜ), followed by the addition of LPS (5 ng/mL) for 15, 30, and 60 min. The phosphorylation of p38 MAPK (A), JNK (B), and NF-κB (C) was examined by Western blotting analysis. Non-adjusted images of Western blotting analysis were presented in Supplementary Material Figure 2. Results are expressed as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, n = 3.