Anti-inflammatory activity of Chios mastic gum is associated with inhibition of TNF-alpha induced oxidative stress

Abstract

Background: Gum of Chios mastic (Pistacia lentiscus var. chia) is a natural antimicrobial agent that has found extensive use in pharmaceutical products and as a nutritional supplement. The molecular mechanisms of its anti-inflammatory activity, however, are not clear. In this work, the potential role of antioxidant activity of Chios mastic gum has been evaluated.

Methods: Scavenging of superoxide radical was investigated by electron spin resonance and spin trapping technique using EMPO spin trap in xanthine oxidase system. Superoxide production in endothelial and smooth muscle cells stimulated with TNF-α or angiotensin II and treated with vehicle (DMSO) or mastic gum (0.1-10 μg/ml) was measured by DHE and HPLC. Cellular H2O2 was measured by Amplex Red. Inhibition of protein kinase C (PKC) with mastic gum was determined by the decrease of purified PKC activity, by inhibition of PKC activity in cellular homogenate and by attenuation of superoxide production in cells treated with PKC activator phorbol 12-myristate 13-acetate (PMA).

Results: Spin trapping study did not show significant scavenging of superoxide by mastic gum itself. However, mastic gum inhibited cellular production of superoxide and H2O2 in dose dependent manner in TNF-α treated rat aortic smooth muscle cells but did not affect unstimulated cells. TNF-α significantly increased the cellular superoxide production by NADPH oxidase, while mastic gum completely abolished this stimulation. Mastic gum inhibited the activity of purified PKC, decreased PKC activity in cell homogenate, and attenuated superoxide production in cells stimulated with PKC activator PMA and PKC-dependent angiotensin II in endothelial cells.

Conclusion: We suggest that mastic gum inhibits PKC which attenuates production of superoxide and H2O2 by NADPH oxidases. This antioxidant property may have direct implication to the anti-inflammatory activity of the Chios mastic gum.

Figures

Figure 1

Effect of mastic gum on cellular production of O2* and H2O2. (A) Production of intracellular O2* was measured by DHE following accumulation of 2-hydroxyethidium using HPLC as described in Materials and Methods [21]. RASMC were stimulated with 20 ng/ml TNF-α for 4-hours. Cells were supplemented with various doses of mastic gum (0-10 μg/ml) for 15-minutes prior to measurements of superoxide. (B) Production of cellular H2O2 was measured by Amplex Red as described in Materials and Methods [21]. RASMC were stimulated with 20 ng/ml TNF-α for 4-hours and then supplemented with various doses of mastic gum (0-10 μg/ml) for 15-minutes prior to measurements of H2O2. *P < 0.01 vs Control, **P < 0.01 vs TNF-α. (C) Superoxide production in bovine aortic endothelial cells (BAEC) stimulated with 200 nM angiotensin II (Ang II) for 4-hours and treated with mastic gum. Control unstimulated BAEC or Ang II-stimulated BAEC were supplemented with 10 μg/ml mastic gum for 15-minutes prior to measurements of superoxide. Data are average from six to eight separate experiments ± Standard Error. *P < 0.01 vs Control, **P < 0.01 vs Ang II.

Figure 2

Spin trapping study of superoxide scavenging by mastic gum. (A) ESR spectrum of EMPO (60 mM) with xanthine (50 μM) and xanthine oxidase (20 mU/ml); (B) ESR spectrum of (A) plus 10 μg/ml mastic gum; (C) ESR spectrum of (A) plus 20 μg/ml mastic gum; (D) ESR spectrum of (A) plus 200 μg/ml mastic gum; (E) ESR spectrum of (A) plus 50 U/ml Cu, Zn-SOD. Computer simulation of ESR spectra (hyperfine coupling constants aN = 13.3 G, aHβ = 10.8 G, aHγ = 1.1 G) [41] and inhibition by SOD (E) confirmed detection of EMPO/*OOH radical adduct. ESR settings were as described in Materials and Methods.

Figure 3

Inhibition of NADPH oxidase in TNF-α stimulated cells treated with mastic gum and attenuation PMA-stimulated superoxide production. (A) Activity of NADPH oxidase was measured as NADPH-dependent O2* production in membrane fractions using ESR as described in Materials and Methods [22]. NADPH oxidase activity was analyzed in membrane fractions of control unstimulated RASMC or RASMC stimulated with 20 ng/ml TNF-α for 4-hours. Mastic gum (10 μg/ml) was applied for 15-minutes prior to isolation of membrane fractions. Direct supplementation of mustic gum to membrane fractions isolated from control or TNF-α stimulated RASMC did not affect NADPH oxidase activity. Data are average from three to six separate experiments ± Standard Error (*P < 0.01 vs Control, **P < 0.01 vs TNF-α). (B) Production of intracellular O2* was measured by DHE following accumulation of 2-hydroxyethidium using HPLC [21] in control or PMA-stimulated RASMC (1 μM PMA, 4-hours) supplemented with various doses of mastic gum (0-10 μg/ml) for 15-minutes prior to measurements of superoxide. Data are average from six separate experiments ± Standard Error (*P < 0.01 vs Control, #P < 0.05 vs TNF-α, **P < 0.01 vs TNF-α).

Figure 4

Inhibition of PKC activity by mastic gum. PKC kinase activity was measured by ELISA-based assay from Enzo Life Sciences in the samples with purified PKC (60 ng) or homogenate of PMA-stimulated RASMC (30 μg). Purified PKC was briefly incubated with 0, 0.1, 1.0 or 10 μg/ml mastic gum prior to PKC activity assay (A). PMA-stimulated RASMC were supplemented with 10 μg/ml mastic gum, 1 μM Go6983 or DMSO as a vehicle for 15-minutes prior to measurements of PKC activity. Data are average from four separate experiments ± Standard Error (*P < 0.01 vs Control, **P < 0.05 vs 0.1 μg/ml Mastic gum).

Figure 5

Proposed mechanism of antioxidant activity of mastic gum via inhibition of PKC-dependent activation of NADPH oxidases.