Betulinic acid inhibits endotoxin-stimulated phosphorylation cascade and pro-inflammatory prostaglandin E(2) production in human peripheral blood mononuclear cells

Abstract

Background and purpose: Betulinic acid (BA) is a naturally occurring triterpenoid widely distributed throughout the plant kingdom. We previously reported that BA inhibits lipopolysaccharide (LPS)-induced interleukin-6 production through modulation of nuclear factor κB (NF-κB) in human peripheral blood mononuclear cells (hPBMCs). This study attempted to identify other mechanisms through which BA modulates LPS signalling in mononuclear cells. The effects of BA on signalling pathways downstream were focused on in this study.

Experimental approach: We determined the ability of BA to interfere with p38 and extracellular regulated kinase (ERK) phosphorylation as well as Akt phosphorylation and nuclear factor-κB activation using LPS-activated hPBMCs as an in vitro model. LPS-induced endotoxin shock in mice was the in vivo model employed.

Key results: BA inhibited LPS-induced COX-2 protein expression and prostaglandin E(2) production and also attenuated LPS-induced ERK and Akt phosphorylation, but not p38 in hPBMCs. BA abolished LPS-induced IκBα phosphorylation and thus normalized the levels of IκBα in cytosol. BA also inhibited LPS-induced reactive oxygen species formation and lactate dehydrogenase release. Interestingly, BA improved the life span of mice in endotoxin shock and also inhibited PGE(2) production and myeloperoxidase activity in vivo.

Conclusions and implications: BA modulates LPS-induced COX-2 expression in hPBMCs by inhibiting ERK and Akt pathways as well as by modulating IκBα phosphorylation. At the same time, no cell toxicity was observed. The effect of the drug was confirmed through in vivo experiments. The study gives an insight into the molecular mechanisms of BA.

Figure 1

Chemical structure of betulinic acid [3-beta-hydroxy-20(29)-lupaene-28-oic acid] C₃₀H₄₈O₃ (MW 456.7).

Figure 2

Effect of BA on LPS-induced PGE₂ production and COX-2 protein expression in hPBMCs. (A) BA reduced LPS-induced PGE₂ production in hPBMCs. Cells were pretreated with BA (0.1–10 µg·mL⁻¹ culture medium) 30 min prior to LPS (1 µg·mL⁻¹ culture medium) stimulation. The hPBMCs were cultured for 16 h. After 16 h, PGE₂ was measured in the culture medium as described in the Methods. (B) Cell lysates were subjected to Western blotting with an anti-COX-2 or β-actin antibody. The relative abundance of each band to its own β-actin was quantified, and the LPS control levels (1 µg LPS + 0 µg BA) were set to 100%. Results are expressed as mean ± SEM; n = 6. #Significantly different from basal values (P < 0.05). *Significantly different from LPS control (P < 0.05). BA, betulinic acid; hPBMCs, human peripheral blood mononuclear cells; LPS, lipopolysaccharide; PGE₂, prostaglandin E₂.

Figure 3

Effects of MAPK and Akt inhibitors and BA on LPS-induced PGE₂ production in hPBMCs. Cells were pretreated with pharmacological inhibitors extracellular regulated kinase inhibitor (PD98059; 10 µM), p38 inhibitor (SB203580; 25 µM), JNK inhibitor (SP600125; 10 µM), PI-3 K inhibitor (LY294002; 10 µM) or BA (2 µg·mL⁻¹ culture medium) 30 min prior to stimulation. hPBMCs were stimulated with LPS (1 µg·mL⁻¹ culture medium) for 16 h. After 16 h, PGE₂ was measured in the culture supernatants as described in Methods. Data are presented as mean ± SEM; n = 6. #Significantly different from basal values (P < 0.05). *Significantly different from LPS control (P < 0.05). BA, betulinic acid; hPBMCs, human peripheral blood mononuclear cells; LPS, lipopolysaccharide; PGE₂, prostaglandin E₂.

Figure 4

BA inhibits LPS-induced phosphorylation of extracellular regulated kinase (ERK) 1/2 (A) and Akt (C) but not p38 (B) in hPBMCs. hPBMCs were pretreated with BA (1–2 µg·mL⁻¹ culture medium) for 30 min in RPMI-1640 containing 5% autologous serum. LPS (1 µg·mL⁻¹ culture medium) was then added and incubated for an additional 30 min. Cell lysates were subjected to Western blotting with their relevant antibodies. Photographs of chemiluminescent detection of the blots, which were representative of three independent experiments, are shown. The relative abundance of each band to its own β-actin was quantified. Data are presented as mean ± SEM; n = 6. #Significantly different from basal values (P < 0.05). *Significantly different from LPS control (P < 0.05). BA, betulinic acid; hPBMCs, human peripheral blood mononuclear cells; LPS, lipopolysaccharide.

Figure 5

BA alone does not induce phosphorylation of extracellular regulated kinase (ERK) 1/2 or Akt in normal cells. hPBMCs were pretreated with different concentrations of BA (0.1–2 µg·mL⁻¹ culture medium) for 30 min in RPMI-1640 containing 5% autologous serum. LPS (1 µg·mL⁻¹ culture medium) was then added and incubated for an additional 30 min. Cell lysates were subjected to Western blotting with their relevant antibodies. Photographs of chemiluminescent detection of the blots, which were representative of three independent experiments, are shown. The relative abundance of each band to its own β-actin was quantified. Data are presented as mean ± SEM; n = 6. BA, betulinic acid; hPBMCs, human peripheral blood mononuclear cells; LPS, lipopolysaccharide.

Figure 6

BA inhibits LPS-induced NF-κB signalling in hPBMCs. hPBMCs were pretreated with BA (0.5–2 µg·mL⁻¹ culture medium) for 30 min in RPMI-1640 containing 5% autologous serum. LPS (1 µg·mL⁻¹ culture medium) was then added and incubated for an additional 30 min. (A) Western blots showing that BA inhibits LPS-induced phosphorylation of IκBα. (B) Electrophoretic mobility shift assay showing that BA inhibits LPS-induced NF-κB nuclear translocation. Oct-1 electrophoretic mobility shift assay served as a loading control. (C) Effect of BA (2 µg·mL⁻¹ culture medium), PD98059 (25 µM) and LY294002 (10 µM) on LPS-induced p65 nuclear translocation. Data are presented as mean ± SEM; n = 6. #Significantly different from basal values (P < 0.05). *Significantly different from LPS control (P < 0.05). BA, betulinic acid; hPBMCs, human peripheral blood mononuclear cells; LPS, lipopolysaccharide; NF-κB, nuclear factor κB.

Figure 7

Effect of BA on LPS-induced lactate dehydrogenase release, ROS generation and cell viability (A) BA is not cytotoxic to hPBMCs. (B) BA (2 µg·mL−1 culture medium) inhibited LPS-induced ROS production. ROS production was measured by nitroblue tetrazolium reduction. (C) Normal mononuclear cells. (D) Trypan blue assay showing that BA is non-cytotoxic to normal cells. Data are presented as mean ± SEM; n = 6. #Significantly different from basal values (P < 0.05). *Significantly different from LPS control (P < 0.05). BA, betulinic acid; hPBMCs, human peripheral blood mononuclear cells; LPS, lipopolysaccharide; ROS, reactive oxygen species.

Figure 8

BA protects against LPS-induced toxicity. Mice were pretreated with (i) vehicle [DMSO; control (CON; n = 6]; (ii) LPS (32 mg·kg⁻¹ i.p.; n = 50); and (iii) BA followed by LPS (LPS = 32 mg·kg⁻¹; BA = 20 mg·kg⁻¹ i.p.; n = 60). Mice were observed for 7 days. BA, betulinic acid; hPBMCs, human peripheral blood mononuclear cells; LPS, lipopolysaccharide.

Figure 9

Effect of BA on LPS-induced endotoxic shock and PGE₂ production in mice. Mice were pretreated with BA (20 mg·kg⁻¹ in 0.1% DMSO), injected with LPS and then monitored for a period of 7 days. On day 7, mice (which survived) were killed; liver and lungs were removed as described in Methods. PGE₂ was extracted from tissue homogenates using SPE columns and the elutes obtained were used for PGE₂ quantification. Results are expressed as mean ± SEM (n = 6–8 observations). #Significantly different from normal control (P < 0.05). *Significantly different from LPS control (P < 0.05). BA, betulinic acid; LPS, lipopolysaccharide; PGE₂, prostaglandin E₂.

Figure 10

BA reduces myeloperoxidase activity in liver and lungs of LPS-induced mice. Mice were pretreated with BA (20 mg·kg⁻¹ in 0.1% DMSO), injected with LPS and then monitored for a period of 7 days. On day 7, mice (which survived) were killed; liver and lungs were removed as described in Methods. Results are expressed as mean ± SEM (n = 6–8 observations). #Significantly different from normal control (P < 0.05). *Significantly different from LPS control (P < 0.05). BA, betulinic acid; LPS, lipopolysaccharide.

Figure 11

Betulinic acid abolishes leucocyte infiltration in the lung and liver of LPS-treated animals. Representative photomicrographs of hematoxylin and eosin-stained sections from control (A, E), LPS (B, F), or betulinic acid + LPS (C, G)-treated mice. A, B, C and D are lung sections, and E, F and G are liver sections. LPS, lipopolysaccharide.