Micheliolide inhibits LPS-induced inflammatory response and protects mice from LPS challenge

Abstract

Sepsis is the principal cause of fatality in the intensive care units worldwide. It involves uncontrolled inflammatory response resulting in multi-organ failure and even death. Micheliolide (MCL), a sesquiterpene lactone, was reported to inhibit dextran sodium sulphate (DSS)-induced inflammatory intestinal disease, colitis-associated cancer and rheumatic arthritis. Nevertheless, the role of MCL in microbial infection and sepsis is unclear. We demonstrated that MCL decreased lipopolysaccharide (LPS, the main cell wall component of Gram-negative bacteria)-mediated production of cytokines (IL-6, TNF-α, MCP-1, etc) in Raw264.7 cells, primary macrophages, dendritic cells and human monocytes. MCL plays an anti-inflammatory role by inhibiting LPS-induced activation of NF-κB and PI3K/Akt/p70S6K pathways. It has negligible impact on the activation of mitogen-activated protein kinase (MAPK) pathways. In the acute peritonitis mouse model, MCL reduced the secretion of IL-6, TNF-α, IL-1β, MCP-1, IFN-β and IL-10 in sera, and ameliorated lung and liver damage. MCL down-regulated the high mortality rate caused by lethal LPS challenge. Collectively, our data illustrated that MCL enabled maintenance of immune equilibrium may represent a potentially new anti-inflammatory and immunosuppressive drug candidate in the treatment of sepsis and septic shock.

Figure 1. MCL does not induce cellular apoptosis in Raw264.7.

(a) Chemical structure of MCL; (b) Raw264.7 cells (1.2 × 10⁵) were plated overnight, and stimulated by different concentrations of MCL with or without LPS (100 ng/mL) for 18 h. Cells were harvested and labeled with Annexin V and 7-AAD, and analyzed by FACS. Similar results were obtained in three independent experiments. (c) Cell counting kit (CCK) cell proliferation assay. Raw264.7 cells (2 × 10⁴/well) were seeded in 96-well plates. The culture medium was replaced with a medium containing MCL (0 μM, 5 μM or 10 μM). After 4 h of assay using the CCK, the optical density of each well was determined at 24 h, 48 h or 72 h (450/650 nm). Data are shown as mean ± SD of three independent experiments.

Figure 2. MCL inhibits LPS-triggered cytokine production in Raw264.7.

Plate seeding (2 × 10⁵/well) was carried out in 24-well plates overnight. Cells were stimulated by different concentrations of MCL with or without LPS (100 ng/mL) for the indicated time periods. IL-6 (a), TNF-α (b), MCP-1 (d), IFN-β (e) and IL-10 (f) in the supernatants were measured by ELISA. The IL-1β (c) mRNA expression was examined by qRT-PCR. Data are shown as mean ± SD of three independent experiments; *p < 0.05.

Figure 3. MCL inhibits the production of LPS-induced proinflammatory cytokines, chemokines, type I interferon and anti-inflammatory cytokines in mouse peritoneal macrophages.

Mouse peritoneal macrophages (3.5 × 10⁵/300 μL) were generated as indicated, and stimulated by MCL with or without LPS (100 ng/mL) for 6 h or 18 h. The concentrations of IL-6 (a), TNF-α (b), MCP-1 (c), IFN-β (d) and IL-10 (e) in the supernatants were measured by ELISA. Data are shown as mean ± SD of three independent experiments; *p < 0.05.

Figure 4. MCL also inhibits LPS-triggered inflammatory responses in human monocytic cell line THP-1 and human CD14⁺ monocytes.

THP-1 cells (1.8 × 10⁵/500 μL) were seeded in a 24-well plate with phorbol-12-myristate-13-acetate (PMA, 10 ng/mL) overnight. Cells were stimulated by MCL with or without LPS (100 ng/mL) for 6 h or 18 h. The concentrations of IL-6 (a), TNF-α (b), MCP-1 (c), IFN-β (d) and IL-10 (e) in the supernatants were measured by ELISA. Peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation from healthy donors and the monocytes were sorted by magnetic beads conjugated with CD14⁺ antibodies. (f) Human CD14⁺ monocytes were plated (3.5 × 10⁵/300 μL) overnight and stimulated with MCL and LPS as indicated. The concentration of TNF-α in the supernatants was measured by ELISA. Data are shown as mean ± SD of three independent experiments; *p < 0.05.

Figure 5. MCL inhibits the activation of NF-κB signaling pathway after LPS stimulation.

Raw264.7 cells (1 × 10⁶/well) was plated in 6-well plates overnight, and stimulated with LPS (100 ng/mL) with or without MCL (10 μM) for the indicated time periods. (a) Phospho-ERK, p-JNK, p-p38, and the corresponding total ERK, JNK and p38 were detected by Western blot. Similar results were obtained in three independent experiments. (b) Phospho-IκBα, total IκBα and β-Actin were detected in Raw264.7 cells by Western blot. Similar results were obtained in three independent experiments. (c) Human monocytic cell line THP-1 (1 × 10⁶/well) was plated in 6-well plates overnight with PMA (10 ng/mL), and stimulated with LPS and MCL as indicated. Phospho-IκBα, total IκBα and β-Actin were detected by Western blot. Similar results were obtained in three independent experiments. (d) Raw264.7 cell line was cotransfected with NF-κB luciferase reporter plasmid and pRL-TK-Renilla-luciferase plasmid. After 30 h, cells were stimulated with LPS (100 ng/mL) for 24 h, and luciferase activities were measured. The NF-κB luciferase activities were presented as fold increase.

Figure 6. MCL inhibits PI3K/Akt/p70S6K activation after LPS stimulation, accounting for the decreased IL-10 expression.

Raw264.7 cells (1 × 10⁶/well) were plated in 6-well plates overnight, and stimulated with LPS (100 ng/mL) with or without MCL (10 μM) for the indicated time periods. (a) Phospho-Akt at Ser473, total Akt, phospho-p70S6K and β-Actin were detected by Western blot. (b) Raw264.7 cells (1 × 10⁶/well) were seeded overnight, and pretreated with MCL (10 μM) or LY294002 (10 μM) for 30 min. Subsequently, cells were stimulated with LPS (100 ng/mL) or MCL (10 μM) as indicated for 15 min or 30 min. Western blot analyses of Akt (Ser473) phosphorylation, total Akt and β-Actin were carried out. (c,d) Raw264.7 cells were transfected with Akt-expressing plasmid (Myr-Akt-HA) and empty plasmid (mock), respectively. Phosphorylation of Akt (Ser473) was confirmed by Western blot. Forty-eight hours after transfection, cells were stimulated as indicated and IL-10 secretion was tested using ELISA. Similar results were obtained in three independent experiments. Data are shown as mean ± SD of three independent experiments; *p < 0.05.

Figure 7. MCL attenuates serum cytokine secretion, organ damage and mortality in LPS-challenged mice.

C57BL/6J mice were injected intraperitoneally with PBS (0.2 mL/mouse), MCL (20 mg/kg), LPS (10 mg/kg), LPS + MCL (20 mg/kg), LPS + DXM (7 mg/kg) and LPS + DXM (7 mg/kg) + MCL (10 mg/kg). (a–f) Two hours later, mice were sacrificed and plasma samples were clotted for 3 h at 4 °C and centrifuged at 3500 rpm for 25 min. The concentrations of various cytokines in the sera were examined by ELISA. Data are shown as mean ± SE of 9 mice per group; *p < 0.05. (g) H&E-staining of lung or liver tissue sections from the indicated group (×200). N = 5 mice per group. DXM, dexamethasone. (h) Septic shock mouse model was created by intraperitoneal LPS injection (25 mg/kg; Sigma). Survival experiments were carried out by several groups as indicated. The protective roles of the low, medium and high doses of MCL (5, 10, 20 mg/kg) were assessed. The survival status of all the groups was recorded at different intervals up to 6 days, and data were analyzed using Log-Rank test. The survival curve was generated by Sigmaplot software. N = 10 mice per group; **p < 0.01.

Figure 8. MCL regulates LPS-induced inflammatory response.

MCL attenuated the activation of NF-κB and PI3K/Akt signaling pathways, decreasing the downstream cytokine expression.