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. 2011 Jan;162(1):147-62.
doi: 10.1111/j.1476-5381.2010.01020.x.

Inhibition of Microsomal Prostaglandin E2 synthase-1 as a Molecular Basis for the Anti-Inflammatory Actions of Boswellic Acids From Frankincense

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Inhibition of Microsomal Prostaglandin E2 synthase-1 as a Molecular Basis for the Anti-Inflammatory Actions of Boswellic Acids From Frankincense

U Siemoneit et al. Br J Pharmacol. .
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Abstract

Background and purpose: Frankincense, the gum resin derived from Boswellia species, showed anti-inflammatory efficacy in animal models and in pilot clinical studies. Boswellic acids (BAs) are assumed to be responsible for these effects but their anti-inflammatory efficacy in vivo and their molecular modes of action are incompletely understood.

Experimental approach: A protein fishing approach using immobilized BA and surface plasmon resonance (SPR) spectroscopy were used to reveal microsomal prostaglandin E(2) synthase-1 (mPGES1) as a BA-interacting protein. Cell-free and cell-based assays were applied to confirm the functional interference of BAs with mPGES1. Carrageenan-induced mouse paw oedema and rat pleurisy models were utilized to demonstrate the efficacy of defined BAs in vivo.

Key results: Human mPGES1 from A549 cells or in vitro-translated human enzyme selectively bound to BA affinity matrices and SPR spectroscopy confirmed these interactions. BAs reversibly suppressed the transformation of prostaglandin (PG)H(2) to PGE(2) mediated by mPGES1 (IC(50) = 3-10 µM). Also, in intact A549 cells, BAs selectively inhibited PGE(2) generation and, in human whole blood, β-BA reduced lipopolysaccharide-induced PGE(2) biosynthesis without affecting formation of the COX-derived metabolites 6-keto PGF(1α) and thromboxane B(2) . Intraperitoneal or oral administration of β-BA (1 mg·kg(-1) ) suppressed rat pleurisy, accompanied by impaired levels of PGE(2) and β-BA (1 mg·kg(-1) , given i.p.) also reduced mouse paw oedema, both induced by carrageenan.

Conclusions and implications: Suppression of PGE(2) formation by BAs via interference with mPGES1 contribute to the anti-inflammatory effectiveness of BAs and of frankincense, and may constitute a biochemical basis for their anti-inflammatory properties.

Figures

Figure 1
Figure 1
Chemical structures of boswellic acids (BAs) and α-amyrin. 3-O-Acetyl-β-boswellic acid (Aβ-BA); 3-O-acetyl-11-keto-β-boswellic acid (AKBA); β-boswellic acid (β-BA); 11-keto-β-boswellic acid (KBA); 3-O-oxaloyl-11-β-keto-boswellic acid (ox-KBA).
Figure 2
Figure 2
Boswellic acids (BAs) bind to microsomal prostaglandin E2 synthase 1 (mPGES1). (A) Supernatants of A549 cell lysates were incubated with β-BA-Seph, KBA-Seph or with Seph, as indicated. (B) Purified, in vitro-translated mPGES1 (200 ng) was incubated with β-BA-Seph, KBA-Seph or Seph beads. Precipitated proteins were separated by SDS-PAGE, and visualized by Western blotting, using specific antibodies against mPGES1 (A,B) or COX-2 (A). An aliquot of the supernatant was used as positive control. Similar results were obtained in three additional experiments. KBA, 11-keto-β-boswellic acid; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis.
Figure 3
Figure 3
Analysis of the binding of boswellic acids to microsomal prostaglandin E2 synthase 1 (mPGES1) by surface plasmon resonance spectroscopy. In vitro-translated mPGES1 was coupled to a CM5 biosensor chip and 3-O-oxaloyl-KBA was used as analyte. Specific binding profiles were obtained after subtracting the signal [response units (RU)] from the untreated control cell. (A) Binding of 3-O-oxaloyl-KBA (ox-KBA) and α-amyrin (10 µM each) to mPGES1. (B) Binding curves for 3-O-oxaloyl-KBA. The equilibrium responses (Req) for 3-O-oxaloyl-KBA at different concentrations were plotted versus the concentration of the compound. (C) Kinetic analysis of 3-O-oxaloyl-KBA-binding to mPGES1. Representative sensograms for the injection of 0.5 µM up to 25 µM 3-O-oxaloyl-KBA are shown. A general analysis was applied to fit the data to a 1:1 binding model (bold lines), and the quality of the fit is displayed by the plots of the residuals. Results are representative for at least three independent experiments. KBA, 11-keto-β-boswellic acid.
Figure 4
Figure 4
Effects of boswellic acids (BAs) on the activity of microsomal prostaglandin E2 synthase 1 (mPGES1) in a cell-free assay. (A) Concentration-response analysis. Microsomal preparations of IL-1β-stimulated A549 cells were pre-incubated with vehicle (DMSO) or BAs for 15 min at 4°C. PGH2 was added and after 1 min, the reaction was stopped and PGE2 was analysed by RP-HPLC as described. The 100% value corresponds to 944 ± 118 pmol PGE2 formed. (B) Reversibility of mPGES1 inhibition. Microsomal fractions of IL-1β-stimulated A549 cells were pre-incubated with 3 µM MK-886 or 10 µM BAs. An aliquot was diluted 10-fold to obtain an inhibitor concentration of 0.3 and 1 µM respectively. For comparison, microsomal preparations were pre-incubated with 0.3 µM MK-886, 1 µM BA or with vehicle (veh, DMSO), and then, 20 µM PGH2 was added (no dilution). After 1 min, PGE2 formation was analysed by RP-HPLC. Data are given as mean + SE, n = 3–4, ***P < 0.001 versus vehicle (DMSO) control. DMSO, dimethyl sulphoxide; RP-HPLC, reversed phase-high performance liquid chromatography.
Figure 5
Figure 5
Effects of boswellic acids (BAs) on PGE2 and 6-keto PGF formation in intact A549 cells. IL-1β-treated A549 cells were pre-incubated (A) with vehicle (veh, DMSO), BAs (30 µM each), α-amyrin (30 µM), MK-886 (30 µM) or celecoxib (cele, 5 µM) (B) with BAs at the indicated concentrations. After 10 min at 37°C, 2.5 µM A23187 plus 1 µM AA and [3H]AA (18.4 kBq) were added (or left untreated = unstim.) and after another 15 min, formed [3H]PGE2 was analysed as described in the Methods. 6-Keto PGF was analysed using High Sensitivity EIA Kits; the 100% value corresponds to 87 ± 11 pg 106 cells. Data are given as mean + SE, n = 3–5. ***P < 0.001 versus vehicle (DMSO) control. AA; arachidonic acid; DMSO, dimethyl sulphoxide.
Figure 6
Figure 6
Effects of boswellic acids (BAs) on prostanoid biosynthesis in human whole blood. Heparinized human whole blood, treated with 1 µM CV4152 and 50 µM aspirin, was pre-incubated with (A) BAs and α-amyrin (10 µM, each) or vehicle (veh, DMSO) for 10 min at RT and then 10 µg·mL−1 LPS was added (or left untreated = unstim.). After 5 h at 37°C, PGE2 was separated by RP-HPLC and quantified by EIA. Controls: MK-886 (30 µM), indomethacin (indo, 50 µM), and celecoxib (cele, 20 µM). (B) Concentration-response of β-BA. (C,D) 6-keto PGF and TXB2 formation. 6-Keto PGF (C) was directly determined in blood plasma from samples above (see A) incubated with β-BA or AKBA (10 µM, each), indo (50 µM), cele (20 µM) or veh (DMSO). Inhibition of TXB2 formation (D) was assessed in heparinized human whole blood without CV4152 and aspirin. Both 6-keto PGF and TXB2 were measured by EIA. The 100% values corresponds to 221.8 ± 19.7 pg·mL−1 PGE2, 382.5 ± 22.3 pg·mL−1 6-keto PGF, and 37.9 ± 4.4 ng·mL−1 TXB2 respectively. (E) 12-HHT formation. Heparinized human blood was pre-incubated with β-BA (50 µM), indo (20 µM) or veh (DMSO) for 10 min, and A23187 (30 µM) was added (or left untreated = unstim.). After 10 min at 37°C, 12-HHT was analysed by HPLC. The 100% value corresponds to 148.8 ± 16.7 ng·mL−1 12-HHT. Data are given as mean + SE, n = 4–5; *P < 0.05; ***P < 0.001 versus vehicle (0.1% DMSO) control. β-BA, β-boswellic acid; AKBA, 3-O-acetyl-11-keto-β-boswellic acid; DMSO, dimethyl sulphoxide; RP-HPLC, reversed phase-high performance liquid chromatography.
Figure 7
Figure 7
Effects of boswellic acids (BAs) in animal models in vivo. (A) Carrageenan-induced pleurisy in rats. Thirty minutes before intrapleural injection of carrageenan, rats (n = 10 for each experimental group) were treated i.p. with BAs (1 mg·kg−1 each), indomethacin (5 mg·kg−1) or vehicle (veh, DMSO 4%). Exudate volume, PGE2 and 6-keto PGF levels as well as inflammatory cell accumulation in pleural cavity were assessed 4 h after carrageenan injection. Data are expressed as mean ± SE, n = 10. *P < 0.05; **P < 0.01; ***P < 0.01 versus vehicle; n.d. = not determined. (B) Carrageenan-induced mouse paw oedema. Animals (n = 10 for each experimental group) were treated i.p. with 0.25 and 1 mg·kg−1β-BA, 5 mg·kg−1 indomethacin (indo) or veh (2% DMSO) 30 min before carrageenan subplantar injection. Data are given as mean + SE, *P < 0.05; **P < 0.01; ***P < 0.001 versus vehicle control. DMSO, dimethyl sulphoxide.

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