The Effect of Caffeic Acid Phenethyl Ester (CAPE) on H2O2-Induced Oxidative Stress in Cultured H9c2 Cells Compared to Common Antioxidants

Caffeic Acid Phenethyl Ester (CAPE) is a natural compound that has previously exhibited anti-proliferative, anti-inflammation and antioxidant activities. However, CAPE’s effects have not been fully elucidated in myoblasts under oxidative stress. We compared the effects of 24 hour pretreatment of CAPE to several known antioxidants (caffeic acid, vitamin C, and trolox) in H9c2 cells following oxidative injury by hydrogen peroxide (H2O2). H9c2 cells incubated with H2O2 treatment (100-700 μM, n=4) for 24 hours dose-dependently reduced cell viability (assessed by a cell counting assay). Compared to the reduction in viability from H2O2 500 μM treatment (22 ± 4%), H9c2 cell viability was significantly restored by pretreatment of CAPE (at 10 μM (100 ± 25%); 20 μM (112 ± 15%); 40 μM (109 ± 15%) n=5, p\u3c0.001) and Trolox (at 50 μM (83 ± 10%); 100 μM (89 ± 8%) n=4, p\u3c0.001). In contrast, pretreatment of H9c2 cells with caffeic acid (1-80 μM, n=3) and vitamin C (1000-10,000 μM, n=3) did not restore cell viability following H2O2-induced injury. CAPE’s mechanism was further investigated by measuring reactive oxygen species via a dichlorofluorescin diacetate assay and by evaluating heme oxygenase-1 (HO-1) expression via western blot. Increases in ROS caused by H2O2 500 μM (239 ± 30% of control, n=3) were significantly restored to control by pretreatment of CAPE dose-dependently (n=3, p\u3c0.001). Moreover, CAPE dose-dependently increased HO-1 expression (n=3). These results suggest CAPE can mitigate oxidative stress in H9c2 cells which may involve the induction of HO-1

Synergistic Effects of Methylglyoxal and Hyperglycemia on ROS Generation and the Viability of Cultured H9c2 Myoblast Cells

Heart damage in diabetics may be closely related to the possible synergistic cellular damage from hyperglycemia and increased methylglyoxal levels. This study investigated the effects of glucose and/or methylglyoxal and/or metformin on H9c2 reactive oxygen species (ROS) generation measured by a dichlorofluorescein diacetate (DCFDA) assay and cell viability measured by a cell counting kit-8 assay after various treatments for 24 hours. Glucose treatment (5 mM-40 mM) displayed similar cell viability (n=4) and ROS generation (n=7) when compared to control cells. By contrast, methylglyoxal (5 µM-1400 µM) decreased cell viability at higher concentration (1000 µM (51 ± 8%); 1200 µM (41 ± 5%); 1400 µM (36 ± 8%); all p\u3c0.05, n=5) compared to control cells, which was accompanied by significantly higher ROS generation (1000 µM (167 ± 27%); 1200 µM (204 ± 22%); 1400 µM (201 ± 15%); all p\u3c0.05, n=3). Furthermore, metformin (1 mM-40 mM) reduced methylglyoxal (1200 µM) induced ROS generation and cell death. When H9c2 cells were treated with glucose (25 mM or 40 mM) and different doses of methylglyoxal (600 µM -1400 µM), only higher glucose (40 mM) with different doses of methylglyoxal (600 µM -1400 µM) consistently showed lower cell viability and higher ROS when compared to individual glucose or methylglyoxal. The data suggest that higher concentrations of methylglyoxal, not glucose, induces H9c2 cell damage and metformin can protect cells from the methylglyoxal insult possibly by reduction of ROS production. Moreover, hyperglycemia and methylglyoxal tend to synergistically induce cell damage associated with increased ROS production

Synergistic Effects of Methylglyoxal and High Glucose on Cardiac H9C2 Cells

Methylglyoxal is a precursor of advanced glycation end products which is closely related to vascular complication in diabetes. However, the direct effects of methylglyoxal on cardiac myocytes still need to be elucidated. This study investigated the dose-dependent effects of methylglyoxal on H9C2 myoblastic cell. Furthermore, we determined if metformin would reduce methylglyoxal caused cell damage. Cell viability was evaluated by a cell counting kit-8 assay and intracellular reactive oxygen species (ROS) was evaluated via a dichlorofluorescin diacetate (DCFDA) assay. After incubation of different doses of methylglyoxal (5 µM-1400 µM, n=12-18) for 24 hours, lower dose range of methylglyoxal (5 µM-800 µM) slightly increased the cell viability by 15±4% compared to the control (n=12). By contrast, higher dose methylglyoxal (1000 µM, 1200 µM, 1400 µM) significantly reduced cell viability to 74 ± 6%, 63 ± 5%, and 56 ± 7%, respectively (all