Zhankuic acid A isolated from Taiwanofungus camphoratus is a novel selective TLR4/MD-2 antagonist with anti-inflammatory properties

Figures

FIGURE 1.

ZAA inhibits the production of…

FIGURE 1.

ZAA inhibits the production of inflammation-related molecules in LPS- and IFN-γ–stimulated murine macrophages.…

FIGURE 1. ZAA inhibits the production of inflammation-related molecules in LPS- and IFN-γ–stimulated murine macrophages. (A) Murine peritoneal macrophages were pretreated with or without ZAA for 1 h, followed by incubation with LPS (0.5 μg/ml) for 24 h. Total cell lysates were subjected to immunoblotting for detecting COX2 and iNOS. Relative expression levels of COX2 and iNOS protein were quantified by densitometric analysis with ImageJ software and normalized according to the β-actin reference band. (B) Murine peritoneal macrophages were pretreated with ZAA for 1 h, followed by incubation with LPS (0.5 μg/ml) or IFN-γ (50 ng/ml) for 24 h. The presence of nitrite in the culture medium was analyzed with the Griess assay and used as an indication of NO levels (n = 4. *p < 0.05, ***p < 0.001 versus LPS- or IFN-γ–induced cells). (C) Raw264.7 cells were cotransfected with pNFκB-Luc and pβ-actin-LacZ plasmids. After 48 h, the cells were treated with or without ZAA for 1 h and then treated with LPS (0.5 μg/ml) for 24 h. Total cell lysates were harvested, and their luciferase activities were determined and normalized on the basis of β-galactosidase activities. Values are means ± SD (n = 4. *p < 0.05, **p < 0.01 versus LPS-stimulated cells). (D and E) ZAA inhibits NF-κB, MAPK, and Akt signaling pathways in LPS-stimulated RAW264.7 cells. Cells were treated with ZAA for 1 h, followed by stimulation with LPS (0.5 μg/ml) for 30 min. Total cell lysates were examined for the indicated proteins by immunoblotting. Numbers below the blots in (D) and those shown in the table below the blots in (E) represent the relative expression levels quantified by densitometric analysis with ImageJ software and normalized according to the β-actin reference bands. Similar results were obtained in three independent experiments. N.D., not detectable.

FIGURE 2.

ZAA interacts with the hydrophobic…

FIGURE 2.

ZAA interacts with the hydrophobic binding pocket of MD-2. ( A ) ZAA…

FIGURE 2. ZAA interacts with the hydrophobic binding pocket of MD-2. (A) ZAA assumes a matched configuration to fit into the binding pocket otherwise occupied by LPS. The clipped surface model of MD-2 (PDB entry: 3FXI) is represented along with the ribbon model to depict the LPS binding pocket buried inside the MD-2 protein. The amino acids Gly110–Asn158 shown in magenta, which reside within the immunogenic peptide fragment of MD-2 aas 110–160, are around the ZAA-binding site in the ribbon model of MD-2. Gly110 and Asn158 are indicated by arrows. (B) Interactions involved in the binding of ZAA to the hydrophobic amino acid residues within the MD-2 binding pocket are shown. (C) The docked ZAA conformer (depicted in black) is shown superimposed over the terminal carbon chains of LPS within the complex structure of the 3FXI MD-2 model. (D) Recombinant human MD-2 protein (0.15 μg) was incubated with LPS (1 or 10 μg) or ZAA for 3 h, subjected to native PAGE, and immunoblotted with Abs against different antigenic determinants of MD-2 (aas 110–160 and 2–160). (E) Recombinant human TLR4/MD-2 complex (1 μg) was incubated with LPS (10 μg) or ZAA for 3 h, subjected to native PAGE, and immunoblotted with an Ab against MD-2 (amino acids 110–160). Similar results were obtained in three independent experiments.

FIGURE 3.

ZAA inhibits TNF-α and IL-6…

FIGURE 3.

ZAA inhibits TNF-α and IL-6 production in LPS- or S. choleraesuis –treated RAW264.7…

FIGURE 3. ZAA inhibits TNF-α and IL-6 production in LPS- or S. choleraesuis–treated RAW264.7 cells and mice. RAW264.7 cells in a 96-well plate (2 × 104 cells/well) were incubated with ZAA for 1 h, followed by treatment with (A) LPS (0.25 or 0.5 μg/ml) or (C) S. choleraesuis (2 × 103 CFU/well). The supernatants collected after 4 and 6 h were assessed for TNF-α and IL-6 levels with ELISA, respectively. (B) C3H/HeJ and C3H/HeN mice were pretreated i.p. with 2 mg/kg ZAA for 30 min, followed by i.p. injection of 4 mg/kg LPS. (D) C57BL/6 mice were pretreated with 10 mg/kg ZAA for 30 min, followed by oral administration of S. choleraesuis (2 × 109 CFU/mouse). Levels of TNF-α and IL-6 were measured in the plasma after 6 h with ELISA. Values are means ± SD (n = 6–8). Similar results were obtained in at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. S.C., S. choleraesuis.

FIGURE 4.

ZAA reduces LPS-induced pathological changes…

FIGURE 4.

ZAA reduces LPS-induced pathological changes in mice. ( A–D ) C3H/HeN and C3H/HeJ…

FIGURE 4. ZAA reduces LPS-induced pathological changes in mice. (A–D) C3H/HeN and C3H/HeJ mice were pretreated i.p. with ZAA (20 mg/kg) or the vehicle for 30 min and then injected i.p. with LPS (4 mg/kg). After 10 h, mice were sacrificed, and their lung and kidney tissues were removed. (A) Representative microscopic images of hematoxylin-and-eosin-stained sections of lung and kidney tissues are shown. Scale bars, 20 μm. Original magnification ×200. (B) The number of infiltrated PMNs in each alveolus was observed by light microscopy. The number of PMNs was counted in four randomly chosen fields per slide for each mouse and normalized to the number of alveoli. (C and D) ZAA decreases the levels of BUN (C) and serum creatinine (D). Values shown in (B)–(D) are means ± SD (n = 10. **p < 0.01, ***p < 0.001). (E) C3H/HeN and C3H/HeJ mice that had been pretreated i.p. with ZAA (2 or 10 mg/kg) or the vehicle for 30 min were injected i.p. with a lethal dose of LPS (20 mg/kg). Survival time was monitored, and Kaplan-Meier survival curves were shown in four groups (n = 10. ***p < 0.001 versus LPS-treated C3H/HeN mice). Similar results were obtained in at least three independent experiments.

FIGURE 5.

ZAA ameliorates S. choleraesuis –induced…

FIGURE 5.

ZAA ameliorates S. choleraesuis –induced diarrhea, body weight loss, and infection in the…

FIGURE 5. ZAA ameliorates S. choleraesuis–induced diarrhea, body weight loss, and infection in the gastrointestinal tract. (A and B) C57BL/6 mice that had been pretreated i.p. with ZAA (2 or 10 mg/kg) or the vehicle for 30 min were orally administered with kanamycin-resistant S. choleraesuis (2 × 109 CFU/mouse). (A) Diarrhea was scored after 2 d on a 0–3 scale (0 = normal pellets, 1 = slightly loose feces, 2 = loose feces, and 3 = watery diarrhea). (B) Body weight was recorded every 2 d for 2 wk (n = 9–12; **p < 0.01, ***p < 0.001). (C) Fecal samples were collected at 24-h intervals until 96 h after S. choleraesuis infection and assessed for viable bacterial CFU counts (n = 10; **p < 0.01, ***p < 0.001). (D and E) C57BL/6 mice that were treated orally with ZAA (2 mg/kg) or vehicle for 30 min were orally administered pCMV-Luc-transformed S. choleraesuis (2 × 108 CFU/mouse). After 48 h, bioluminescence imaging of the mice was conducted after injection with d-luciferin. (D) Whole-body images are shown. The photon flux scale is shown on the right. (E) Quantification of bioluminescent imaging data. Radiance values are expressed as means ± SD (***p < 0.001). S.C., S. choleraesuis.