Polypharmacological Profiles Underlying the Antitumor Property of Salvia miltiorrhiza Root (Danshen) Interfering with NOX-Dependent Neutrophil Extracellular Traps

Figure 3

Active components derived from Danshen impair NET formation in vitro. (a) Screening natural inhibitors from eight primary components isolated from Danshen to PMA-induced NETosis. NETotic index was used to indicate the extent of DNA release during NETosis. Extracellular DNA release from human neutrophils was quantified by a membrane impermeable DNA-binding dye SYTOX Green. Fluorescence readout obtained from cells lysed with Triton X-100 was considered as 100% DNA release. DNA dissolving drug DNase I served as positive treatment control ( between 0 ng/mL of PMA treatment and 200 ng/mL of PMA treatment, while or between 200 ng/mL of PMA treatment and drug treatment). (b) Neutrophils were pretreated with or without Sal B (200 μM) or DHT I (20 μM) for 2 hours and further incubated with 200 ng/mL of PMA for 4 hours; the structures of NETs were observed by fluorescent microscopy for SYTOX Green staining in formalin-fixed neutrophils at 1000x magnification (scale bar represents 50 μM). (c) NETs were further confirmed by a multicolor fluorescence immunostaining of DNA-histone-MPO complexes at 600x magnification (scale bar represents 20 μM).

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