Cytoprotective effect of Cissus Quadrangularis extract on human umbilical vein endothelial cells

Abstract

The aims of the present study are to determine whether Cissus quadrangularis (CQ) could protect against hydrogen peroxide (H₂O₂)-induced oxidative injury in human umbilical vein endothelial ECV304 cells and also whether the protective potential may have any relation to the free radical scavenging activities in cell-free system. In cell-free system, superoxide anion, H₂O₂, and hydroxyl radical scavenging activities of the spray drying of compressed fresh CQ (CQWS1) were stronger than ethanolic CQ extract using soxhlet apparatus for 48 h (CQES48). These results were correlated with quercetin and resveratrol, active constituents in CQ, which exhibited the free radical scavenging activities in cell-free system. Based on the MTT assay, no cytotoxicity on ECV304 cells was observed after 24-h exposure to CQWS1 and CQES48 at the concentration not more than 1 mg/mL. Hence, the concentrations up to 1 mg/mL of CQWS1 and CQES48 were chosen for the cytoprotective study from oxidative stress using DCFH-DA assay. Pretreatment of the cells with CQES48 reduced H₂O₂-induced reactive oxygen species (ROS) generation in a concentration-dependent manner, as examined by a decrease of DCF fluorescence intensity induced by H₂O₂ (100 µM). Meanwhile, CQWS1 (1 mg/mL) significantly restored the generation of the intracellular ROS at 10-fold higher concentration than CQES48 (0.1 mg/mL). Additionally, quercetin and resveratrol (5 µM) significantly reduced H₂O₂-induced ROS generation in the cells. The results indicated that the attenuation of intracellular ROS of CQES48 might be due to the presence of quercetin and resveratrol, and might involve in the increase of antioxidant enzymes and endothelial nitric oxide synthase (eNOS) expression. Western blot analysis revealed that treatment of the cells with CQES48 (0.5 mg/mL) as well as quercetin and resveratrol (50 µM) significantly up-regulated the protein expression of antioxidant enzymes, copper/ zinc superoxide dismutase (Cu/Zn-SOD), manganese superoxide dismutase (Mn-SOD) and glutathione peroxidase (GPx), as compared with control. However, treatment with CQES48 for 24 h before exposure to 100 µM H₂O₂ significantly increased the protein expression of Cu/Zn-SOD, Mn-SOD, GPx and eNOS. The results showed that CQES48 attenuated the intracellular ROS level in H₂O₂-induced ECV304 cells injury via the up-regulation of the antioxidant enzymes and eNOS, which was also correlated with H₂O₂ scavenging activity in cell-free system, resulting from its constituents, quercetin and resveratrol.