Effects of Shikonin on the expression of neutrophils extracellular traps induced in diabetic mice
MA Hui-ke,YAO Wen-tao,LIU Xin
Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing Institute of Chinese Medicine, Beijing 100010, China
Abstract:
Objective To investigate whether Shikonin could affect the formation of the bone marrow-derived neutrophil extracellular traps (NETs) induced by phorbol 12-myristate 13- acetate (PMA) in diabetic mice, and provide an experimental basis for its treatment of diabetic foot ulcers. Methods The model of diabetic mice was established by intraperitoneal injection of streptozocin. Bone marrow-derived neutrophils from diabetic mice were extracted by density gradient centrifugation and cultured in vitro. PMA stimulates neutrophils to induce NETs model. CCK-8 method was used to detect the safe dose of Shikonin. Nucleic acid in NETs was quantitatively detected by SYTOX green staining. The content of dsDNA/NETs was quantitatively detected by PicoGreen fluorescent dyes. The level of reactive oxygen species (ROS) in neutrophils was measured by DCFH-DA fluorescence probe. The expression of citrulline histone H3 (Cit-H3) was detected by immunofluorescence. Results Shikonin has no inhibitory effects on the activity of neutrophils from diabetic mice in the concentration range of 0.125-1 μg/mL. Compared with the model group, the fluorescence intensity of NETs in Shikonin group (1, 0.5, 0.25 μg/mL) and the level of dsDNA/NETs were significantly decreased, and the difference was statistically significant (P<0.01). The production of ROS in Shikonin group and the expression of Cit-H3 were significantly reduced, and the difference was statistically significant (P<0.01). Conclusion Shikonin could inhibit the production of NETs by reducing the ROS level of bone marrow-derived neutrophils from diabetic mice, provides a theoretical basis for the treatment of diabetic foot ulcers with Shikonin.
