Tetramethylpyrazine inhibits NETs formation induced by high glucose by regulating NRF2/HO-1 pathway

Abstract

Objective To investigate the effect and mechanism of tetramethylpyrazine (TMP) on the formation of neutrophil extracellular traps (NETs) induced by high glucose. Methods The viability of neutrophils treated with TMP of 0, 5, 10, 20, 40, and 80 μmol/L for 4 h was measured by CCK-8 assay. Neutrophils were divided into Control group (1.5 g/L glucose), TMP group (40 μmol/L TMP), HG group (4.5 g/L glucose), HG+TMP group (40 μmol/L TMP+4.5 g/L glucose), HG+TMP+ML385 group (4.5 g/L glucose+40 μmol/L TMP+10 μmol/L ML385), HG+ML385 group (4.5 g/L glucose+10 μmol/L ML385) and HG+DPI group (4.5 g/L glucose+10 μmol/L DPI). The structure of NETs was observed with immunofluorescence assay. The contents of reactive oxygen species (ROS) and superoxide anions were detected using dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay and dihydroethidine (DHE) method. MPO-DNA and dsDNA in the culture medium was measured with ELISA and Pico-Green kit, respectively. Western blotting was used to detect the intracellular expression of H3-cit, NRF2, HO-1, and NOX2 proteins. Results TMP of 80 μmol/L had significant effect on neutrophil (P < 0.01), but no such effects were seen in TMP at other doses viability. Compared with the Control group, the expression levels of NRF2 and HO-1 were increased significantly in TMP group (P < 0.01), while that of H3-cit was obviously decreased after TMP treatment (P < 0.05), and the contents of dsDNA and MPO-DNA in the medium were decreased notably (P < 0.01). NETs formation was not observed by immunofluorescence assay in the HG+TMP group. The productions of ROS and superoxide anions in the HG+TMP group were significantly lower than those produced in the HG group (P < 0.01). The effects of TMP on the expression of H3-cit, NRF2, and HO-1 were reversed after treatment with NRF2 inhibitor ML385. Compared with the HG+TMP group, the contents of dsDNA and MPO-DNA were increased significantly (P < 0.01), and the contents of intracellular ROS and superoxide anions were elevated remarkably in HG+TMP+ML385 group (P < 0.01). Conclusion TMP can activate the NRF2/HO-1 pathway and then suppress the production of ROS, and thus inhibit the NETs formation induced by high glucose.

Keywords

• tetramethylpyrazine
• neutrophils
• neutrophil extracellular traps
• reactive oxygen species
• diabetic retinopathy
• high glucose
• nrf2/ho-1