Figure 3.

Effects of Alda-1 on HS-induced vascular inflammation, ROS and apoptosis in vitro. Alda-1 (20 μM) was added to HUVECs for 6 h before HS (42°C for 2 h). (A) The ALDH2 activity in cell lysate was determined by measuring NADH production based on the O.D. absorbance at 450 nm in a microplate reader (n = 5). (B) Measurement of ROS production based on DCF fluorescence as determined by using a fluorescence microplate reader with an excitation wavelength of 488 nm and an emission wavelength of 535 nm (n = 5). (C) Measurement of cellular ROS production based on DHE fluorescence as determined using a fluorescence microplate reader with an excitation wavelength of 518 nm and an emission wavelength of 606 nm (n = 5). (D) The viability of HUVECs was measured by the MTT assay based on the O.D. absorbance at 570 nm in a microplate reader (n = 5). (E) The levels of apoptosis were measured by the TUNEL assay as determined by fluorescence microscopy. The percentage of apoptotic cells was determined based on the number of TUNEL-positive cells among the total number of cells. (F) The levels of senescence were measured by β-galactosidase activity detection using bright field microscopy (n = 5). (G) Detection of mitochondrial dysfunction by the JC-1 assay, revealing that the mitochondrial membrane potential (ΔΨm) was decreased in live cells as determined by fluorescence microscopy and a fluorescence microplate reader. The ΔΨm level is expressed as the merge of the red and green channels, and the data were quantified as the ratio of red fluorescence intensity to green fluorescence intensity (n = 5). (H) The protein and 4-HNE levels in lung homogenates were measured by immunoblotting. Densitometric analysis was conducted with imaging processing software. The data were quantified by normalization to GAPDH; phosphorylated proteins were normalized to total proteins (n = 5). The data are expressed as the mean ± SD. Statistical significance is indicated as *p < 0.05.