Inhibition of neutrophil superoxide generation by shikonin is associated with suppression of cellular Ca²⁺ fluxes

Abstract

Shikonin, an anti-inflammatory compound of “Shikon”, inhibits the neutrophil superoxide (O2•−) generation by NADPH oxidase 2 (Nox2); however, the mechanisms of how shikonin affects Nox2 activity remained unclear. We aimed to elucidate the relationship between the inhibition of Nox2 activity and influences on intracellular Ca2+ concentration ([Ca2+]i) by shikonin. For this purpose, we used a simultaneous monitoring system for detecting changes in [Ca2+]i (by fluorescence) and O2•− generation (by chemiluminescence) and evaluated the effects of shikonin on neutrophil-like HL-60 cells stimulated with N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP). Since fMLP activates Nox2 by elevation in [Ca2+]i via fluxes such as inositol 1,4,5-trisphosphate-induced Ca2+ release (IICR) and store-operated Ca2+ entry (SOCE), we also evaluated the effects of shikonin on IICR and SOCE. Shikonin dose-dependently inhibited the fMLP-induced elevation in [Ca2+]i and O2•− generation (IC50 values of 1.45 and 1.12 µM, respectively) in a synchronized manner. Analyses of specific Ca2+ fluxes showed that shikonin inhibits IICR and IICR-linked O2•− generation (IC50 values: 0.28 and 0.31 µM for [Ca2+]i and O2•−, respectively), as well as SOCE and SOCE-linked O2•− generation (IC50 values: 0.39 and 0.25 µM for [Ca2+]i and O2•−, respectively). These results suggested that shikonin inhibits the O2•− generation by Nox2 in fMLP-stimulated neutrophils by targeting Ca2+ fluxes such as IICR and SOCE.

Oral administration of Pantoea agglomerans-derived lipopolysaccharide prevents development of atherosclerosis in high-fat diet-fed apoE-deficient mice via ameliorating hyperlipidemia, pro-inflammatory mediators and oxidative responses

“…To evaluate the oxidative burst activity in the leukocyte fraction obtained from the peripheral blood sample, a real-time dual monitoring device of chemiluminescence/fluorescence developed by Hamamatsu Photonics K.K. [ 23 , 24 ] was applied here. The device was developed to monitor the changes in chemiluminescence intensity by superoxide anion (O 2 ●- ) production and the changes in fluorescence intensity by MPO reaction after PMA stimulation at every 0.5 s. Representative results of the analysis are shown in Fig 6A .…”

“…O 2 ●- production and MPO activity after stimulation with phorbol 12-myristate 13-acetate (PMA; Wako Pure Chemical Industries) in the hemolyzed blood sample was simultaneously determined with a real-time chemiluminescence and fluorescence monitoring system (CFL-P2200; Hamamatsu Photonics K.K., Shizuoka, Japan), comprised of a high speed on/off system with LED excitation light and a chemiluminescence/fluorescence high sensitivity detector system, as previously described [ 23 , 24 ] with some modification. An aliquot of each whole blood sample (30 μl) was mixed with 500 μl of an ammonium chloride-based red blood cells lysis buffer with no fixing reagents (Tonbo Biosciences, San Diego, CA, USA) for 2 min at room temperature (RT).…”

Rapid on-site dual optical system to measure specific reactive oxygen species (O2-• and OCl-) in a tiny droplet of whole blood

“…HL-60 cells, a human acute promyelocytic leukemia cell line, obtained from American Type Culture Collection (Manassas, VA, USA) were maintained and differentiated to neutrophil-like cells with DMSO as previously described [ 10 , 11 , 15 ]. The cells thus differentiated were suspended in RH buffer and kept at 4°C until measurement.…”

“…Data analysis by the dedicated software and Excel. Stimulant enhancements of the signals of CL MCLA and FL APF were determined by calculating the peak areas under the curves (AUC) [ 10 , 11 ].…”

“…We noticed that both O 2 -• and OCl- can be measured simultaneously by modifying the previously developed system that can monitor generated O 2 -• and intracellular Ca 2+ concentrations [ 8 – 11 ], which lead us to develop a new system. The new system uses the same chemiluminescence (CL) probe of MCLA (CL MCLA ) for O 2 -• as before [ 12 , 13 ] while OCl- is monitored as fluorescence (FL) by using APF (FL APF ) [ 14 ].…”