Acta Pharmacologica Sinica 2007 August; 28 (8): 1143-1148; doi: 10.1111/j.1745-7254.2007.00595.x

Aim: To investigate the effect of 3,5-dicaffeoylquinic acid (3,5-diCQA) on lipopolysaccharide (LPS)-induced injury in human dermal microvascular endothelial cells (HMEC-1).

Methods: The anti-oxidant effect was detected using the malondialdehyde (MDA) assay in a rat liver microsome model of lipid peroxidation. Cell viability was analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide assay. Cell lipid peroxide injury was measured by lactate dehydrogenase (LDH) release. Apoptotic cells were detected by flow cytometry, and confirmed by DNA fragmentation analysis. Caspase-3 activity was measured using a specific assay kit. The level of intracellular reactive oxygen species (ROS) was determined by flow cytometry with a 2,7-dichlorodihydro-fluorescein diacetate fluorescence probe.

Results: The exposure of microsomes to ascorbate-Fe²⁺ resulted in lipoperoxidation according to an increase in the level of MDA. MDA formation decreased in a dose-dependent manner on treatment with 5, 10, or 50 µmol/L 3,5-diCQA. Treatment with LPS for 16 h resulted in a 60% decrease in cell viability and an increase in LDH release from 47.6% to 61.5%. DNA laddering was observed by agarose gel electrophoresis. The level of apoptotic cells peaked at 27% after treatment with LPS for 12 h. Following treatment with LPS for 12 h, intracellular ROS and caspase-3 activity increased. Pretreatment with 3,5-diCQA at 5, 10, or 50 µmol/L for 1 h attenuated LPS-mediated endothelial cell injury. The anti-apoptotic action of 3,5-diCQA was partially dependent on its capacity for anti-oxidation and the suppression of caspase-3 activity.

Conclusion: 3,5-diCQA displays anti-oxidative and anti-apoptotic activity in HMEC-1 due to scavenging of intracellular ROS induced by LPS, and the suppression of caspase-3 activity.

Keywords: 3,5-dicaffeoylquinic acid; lipopolysaccharide; human dermal microvascular endo-thelial cells; reactive oxygen species; apoptosis; caspase-3