Gambogic acid inhibits LPS-induced macrophage pro-inflammatory cytokine production mainly through suppression of the p38 pathway

Abstract

Objectives: In traditional Chinese medicine, gamboge can detoxify bodies, kill parasites, and act as a hemostatic agent. Recent studies have demonstrated that gambogic acid (GBA) suppressed inflammation in arthritis, and also presented antitumor effect. Thus, this study investigated the new biological properties of GBA on macrophages.

Materials and methods: RAW 264.7 cells were pretreated with GBA at different concentrations (10, 20, 40, 80, 160, 320 nM) for 24 hrs, and then cell viability was measured using Cell Counting Kit (CCK)-8 assays. Pro-inflammatory cytokines such as TNF-α, IL-6 and IL-1β were determined using ELISA kits and qPCR. Then nitrite concentration was calculated according to a standard curve. At last, the effect of GBA on MAPK and NF-κB signaling pathways was assessed by western blot and luciferase reporter gene assay.

Results: GBA (IC50: 260 nM) suppressed the TNF-α, IL-6 and IL-1β expression induced by lipopolysaccharide (LPS) in RAW 264.7 cells. The expression of TNF-α, IL-6 and IL-1β decreased to 30-50% and 70-75% in the high-dose (160 nM) and low-dose (40 and 80 nM) GBA groups, respectively. Furthermore, the nitric oxide (NO) production and the activation of NF-κB, ERK, and JNK pathways were significantly reduced in high-dose (160 nM) GBA only, while p38 pathway was inhibited at both low (40 and 80 nM) and high (160 nM) concentration of GBA.

Conclusion: These data suggested that GBA inhibited LPS-induced production of pro-inflammatory cytokines including TNF-α, IL-6 and IL-1β mainly through the suppression of the p38 pathway.

Keywords: Anti-inflammatory agents; Gambogic acid; MAPK; NF-κB; RAW 264.7 cells; p38.

Figure 1

Effect of gambogic acid (GBA) on lipopolysaccharide (LPS)-induced pro-inflammatory cytokine release in raw 264.7 cells. (A) RAW 264.7 cells were pretreated with the indicated concentrations of GBA for 24 hr, and cell viability was measured using CCK-8 assays. (B) The half-maximal inhibitory concentration (IC50) of GBA was 260 nM. (C, E & G) Cells were pretreated with the indicated concentrations of GBA for 2 hr and then exposed to LPS (100 ng/ml) for 24 hr. The expression levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) in the supernatant were measured by ELISA. (D, F & H) Cells were pretreated with the indicated concentration of GBA for 2 hr and exposed to LPS (100 ng/ml) for 4 hr. The mRNA levels of TNF-α, IL-6 and IL-1β were measured using RT-qPCR. Bars represent the mean±SD of three independent experiments. * P<0.01 vs. normal control group; ** P<0.01 vs. LPS alone

Figure 2

Effect of gambogic acid (GBA) on nitric oxide production, inducible nitric oxide synthase (iNOS) and nuclear factor-κB (NF-κB) pathway activation in lipopolysaccharide (LPS)-stimulated raw 264.7 cells. (A) RAW 264.7 cells were pretreated with the indicated concentration of GBA for 2 hr and stimulated with LPS (100 ng/ml) for 24 hr. Then, the nitrite level in the supernatant was measured using the Griess Reagent System. (B) Cells were pretreated with the indicated concentration of GBA for 2 hr and stimulated with LPS (100 ng/ml) for 24 hr. After cellular proteins were harvested, iNOS protein levels were measured by western blotting. (C) RAW 264.7 cells stably expressing NF-κB luciferase reporter were pretreated with different concentration of GBA for 2 hr and stimulated with LPS (100 ng/ml) for 6 hr. Then, NF-κB activation was measured by monitoring luciferase activity. (D) RAW 264.7 cells were pretreated with the indicated concentration of GBA for 2 hr and stimulated with LPS (100 ng/ml). Cellular proteins were extracted and the IκB levels were measured by western blotting. Bars represent the mean±SD of three independent experiments. * P<0.01 vs. normal control group; ** P<0.01 vs. LPS alone

Figure 3

Effect of gambogic acid (GBA) on lipopolysaccharide (LPS)-induced mitogen-activated protein kinase pathways. (A) RAW 264.7 cells were pretreated with the indicated concentration of GBA for 2 hr and stimulated with LPS (100 ng/ml) for 30 min. The protein level of p-p38 and p38 were measured by western blotting. (B and C) RAW 264.7 cells were pretreated with the indicated concentration of GBA for 2 hr and stimulated with LPS (100 ng/ml) for 30 min. The protein levels of p- extracellular signal-regulated kinases (ERK), ERK, p-c-Jun N-terminal kinase (JNK) and JNK were measured by western blotting. Bars represent the mean±SD of three independent experiments. *P<0.01 vs. normal control group; ** P<0.01 vs. LPS alone

Figure 4

Model of the effects of gambogic acid (GBA) on lipopolysaccharide (LPS)-stimulated raw 264.7 cells. LPS triggers multiple intracellular signaling pathways, including the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. Activation of the NF- κB signaling pathway increases inducible nitric oxide synthase (iNOS), which produces nitric oxide (NO) and promotes pro-inflammatory cytokines. Pro-inflammatory genes, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β), are upregulated upon activation of the MAPK signaling pathway. Low concentrations of GBA suppresses phosphorylation of p38; however, high concentrations of GBA has broader effects on both the MAPK and NF- κB signaling pathways, attenuating the release of pro-inflammatory factors.