Figure 1
Flow chart of the study. The study included in vivo animal experiments and in vitro experiments. In experiment 1, mice were divided into seven groups: sham, I/R, I/R + H2, I/R + H2 + LY, I/R + H2 + W, I/R + LY, and I/R + W. A 90-min inhalation of 66.7% H2 and 33.3% O2 gas was initiated immediately after 45 min of ischemia. Cardiac enzymes, infarct area, oxidative parameters, inflammatory parameters, apoptotic parameters, and the phosphorylation of Akt-related proteins were measured 4 h after reperfusion. A hemodynamic test was performed 24 h after reperfusion. In experiment 2, mice were divided into seven groups: sham, I/R, I/R + H2, I/R + A, I/R + H2 + A, I/R + C, and I/R + H2 + C. Cardiac enzymes, apoptotic parameters, and the phosphorylation of Akt-related proteins were measured 4 h after reperfusion. In the in vitro experiments, flow cytometry, TUNEL staining, and measurement of ROS generation were conducted after 4 h of reoxygenation to confirm the optimal dose of H2. Cardiomyocytes were assigned randomly into six groups for investigation of the role of Akt1 and Akt2 in the protective effects of 75% H2 as follows: control, H/R, H/R + HCH, H/R + A + HCH, H/R + C + HCH, and H/R + A + C + HCH. Measurement of ROS generation, X-gal staining, TUNEL staining, MTT assay, flow cytometry, alkaline comet assay, and western blot were performed after 4 h of reoxygenation.
