Andrographolide prevents oxygen radical production by human neutrophils: possible mechanism(s) involved in its anti-inflammatory effect

Abstract

We have reported that andrographolide (ANDRO), an active component of Andrographis paniculata, inhibits inflammatory responses by rat neutrophils. To further elucidate the possible mechanism(s) underlying the ANDRO's effect, N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced adhesion and transmigration of isolated peripheral human neutrophils were studied. Pretreatment with ANDRO (0.1 - 10 microM) concentration-dependently prevented fMLP-induced neutrophil adhesion and transmigration. We further examined the up-expression of surface Mac-1 (CD11b/CD18), an essential integrin mediated in neutrophil adhesion and transmigration. ANDRO pretreatment significantly decreased fMLP-induced up-expression of both CD11b and CD18. Accumulation of reactive oxygen species (ROS) as well as quick intracellular calcium ([Ca(++)](i)) mobilization induced by fMLP displays two important signalling pathways in regulating the up-expression of Mac-1 by neutrophils. That ANDRO pretreatment diminished fMLP-induced production of H(2)O(2) and O(2)*(-), but failed to block that of [Ca(++)](i) mobilization suggested that the ROS but not [Ca(++)](i) signalling could be modulated by ANDRO. To clarify whether ROS production impeded by ANDRO could be an antagonism of fMLP binding, phorbol-12-myristate-13-acetate (PMA), a direct protein kinase C (PKC) activator, was introduced to activate ROS production. PMA triggered remarkable ROS production and adhesion, and were partially reversed by ANDRO. This indicated that a PKC-dependent mechanism might be interfered by ANDRO. We conclude that the prevention of ROS production through, at least in part, modulation of PKC-dependent pathway could confer ANDRO the ability to down-regulate Mac-1 up-expression that is essential for neutrophil adhesion and transmigration.

Figure 1

Mean concentration-response curves for andrographolide (ANDRO) in the inhibition of fMLP (1 μM)-induced neutrophil adhesion and transmigration. BCECF-labeled neutrophils were pretreated with ANDRO (0.1–10 μM) for 10 min at 37°C. For adhesion assay, cells were plated into fibrinogen-coated 24 well plate. After stimulated with fMLP for an additional 30 min at 37°C, non-adherent cells was wash off and adherent cells were quantified by measuring fluorescence intensity. For transmigration assay, cells were plated into upper chamber of fibrinogen-coated inserts. After stimulated with fMLP in the lower chamber for an additional 90 min at 37°C, transmigrated cells in the lower chambers were quantified by measuring fluorescence intensity. Values are mean and vertical lines s.e.m. of six experiments. *, † P<0.05, as compared to samples receiving fMLP alone for transmigration and adhesion, respectively.

Figure 2

Effect of ANDRO on fMLP (1 μM)-induced Mac-1 upregulation. (a) Flow cytometric analysis of total Mac-1 levels on the cell surface of neutrophils. Control neutrophils received neither ANDRO nor fMLP treatment. ANDRO-pretreated sample, designated ‘fMLP+ANDRO', has been stimulated with fMLP. (b) Statistical summary of fMLP-upregulated Mac-1 expression in the presence or absence of ANDRO (1–10 μM). Non-specific IgG₁ was included to contrast the specificity of anti-CD11b or CD18 staining. Values represent the mean of four experiments and horizontal lines show SEM. *, † P<0.05, as compared to samples receiving fMLP alone for CD11b and CD18 staining, respectively.

Figure 3

Effect of ANDRO on fMLP (1 μM)-induced neutrophils ROS (H₂O₂ and O₂^.−) production. (a) Flow cytometric analysis of H₂O₂ (DCF fluorescence, upper panel) and O₂^.− (EB fluorescence, lower panel) production. Control neutrophils received neither ANDRO nor fMLP treatment. ANDRO (0.1–10 μM)-pretreated sample, designated ‘fMLP+ANDRO', has been stimulated with fMLP for 60 min. (b) Statistical summary of fMLP-induced H₂O₂ and O₂^.− production in the presence of ANDRO (10 μM). Values are mean and horizontal lines s.e.m. from six experiments. *, † P<0.05, as compared to sample receiving fMLP alone for DCF and EB fluorescence intensity, respectively.

Figure 4

Effect of ANDRO on fMLP (1 μM) or thapsigargin (0.16 μM)-induced changes in intracellular calcium concentration ([Ca⁺⁺]_i) of neutrophils. [Ca⁺⁺]_i was measured as described in Methods. (a) tracing of changes in [Ca⁺⁺]_i triggered by fMLP (upper panel) or thapsigargin (TSG) (lower panel) in the presence or absence of ANDRO (10 μM). 1 μM verapamil (Verap) and 10 μM BAPTA/AM (BAPTA) were included as calcium channel blocker and intracellular calcium chelator, respectively. (b) Statistical summary of fMLP or TSG-induced changes in [Ca⁺⁺]_i in the presence of ANDRO (10 μM). Net increase in [Ca⁺⁺]_i was calculated by subtracting control values from respective experimental values (control [Ca⁺⁺]_i in resting cell was 76±12 nM). *, † P<0.05, as compared to samples receiving fMLP or TSG alone. Values are mean and vertical lines s.e.m. from five experiments.

Figure 5

Effect of ANDRO on PMA-induced neutrophils ROS (H₂O₂ and O₂^.−) production and adhesion. Flow cytometric analysis of H₂O₂ (DCF fluorescence) and O₂^.− (EB fluorescence) production, and neutrophil adhesion (BCECF fluorescence) were measured as described in Methods. Control neutrophils received neither ANDRO nor PMA treatment. A PKC inhibitor, staurosporine (200 nM)- and ANDRO (1–10 μM)-pretreated samples, designated as ‘PMA+STAU' and ‘PMA+ANDRO', respectively, had been stimulated with PMA (100 ng ml⁻¹). Values are mean and horizontal lines s.e.m. from three experiments. *, †, ‡ P<0.05, as compared to sample receiving PMA alone for BCECF (adhesion), DCF, or EB fluorescence intensity, respectively.