The production of superoxide in myocardial tissue was detected using dihydroethidium (DHE) staining under a fluorescence microscope. Briefly, myocardial tissue sections (5 μm) were prepared and then incubated (30 min, 37 °C) in Krebs-HEPES buffer (mM components: NaCl 99, KCl 4.7, MgSO4 1.2, KH2 PO4 1.0, CaCl2 1.9, NaHCO3 25, glucose 11.1, Na HEPES 20; pH 7.4) containing 2 μM DHE in a dark room. The slides were examined with a Nikon TE2000 inverted microscope (Nikon, Tokyo, Japan) at excitation and emission wavelengths of 480 and 610 nm, respectively.
The thiobarbituric acid method (Beyotime) was used to detect the level of malondialdehyde (MDA) in the myocardium. The total antioxidant capacity (T-AOC) of the myocardium was measured using the T-AOC detection kit using the plasma iron reduction capacity method (Beyotime). The activities of total SOD, Cu-Zn/SOD, and Mn-SOD in the myocardium were determined using the WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-)-2 H-tetrazole) method (Beyotime).
The thiobarbituric acid method (Beyotime) was used to detect the level of malondialdehyde (MDA) in the myocardium. The total antioxidant capacity (T-AOC) of the myocardium was measured using the T-AOC detection kit using the plasma iron reduction capacity method (Beyotime). The activities of total SOD, Cu-Zn/SOD, and Mn-SOD in the myocardium were determined using the WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-)-2 H-tetrazole) method (Beyotime).