Effects of sCD40L on O2− production in HCAECs and porcine coronary arteries. (A) DHE staining in HCAECs. Cells were treated with sCD40L for 24 hours, and intracellular O2− levels were determined by DHE staining and flow cytometric analysis. U test. n = 3. (B) Porcine coronary artery rings. The vessel rings were treated with sCD40L for 24 hours and O2− levels in the endothelial layer of porcine coronary arteries were tested with lucigenin-enhanced chemiluminescence assay. *P < .05 compared with the control. t test. n = 6. (C) Mitochondrial membrane potential (representative histograms of flow cytometric analysis). HCAECs were treated with sCD40L for 24 hours, and mitochondrial membrane potential was determined by JC-1 staining and flow cytometric analysis. (D) Quantitative data of JC-1 staining showed a significant decrease in mitochondrial membrane potential after sCD40L treatment. Antioxidant SeMet effectively reversed these changes. U test. n = 3. (E) ATP content. HCAECs were treated with sCD40L for 24 hours, and ATP production was determined by a commercial ATPLite kit. Antioxidant SeMet was included. t test. n = 3. (F) Effect of mitochondrial complex II inhibitor TTFA on eNOS protein levels. HCAECs were treated with sCD40L in the presence or absence of mitochondrial complex II inhibitor TTFA (10 μM) for 24 hours, and eNOS protein levels were determined by Western blot analysis. *P < .05 and **P < .01, compared with the control. #P < .05 compared with sCD40L treatment. U test. n = 3. Data are means and SE of multiple experiments (n).
