Protective effect of Ziziphora clinopodioides flavonoids against H2O2-induced oxidative stress in HUVEC cells.

The present study was designed to study the protective effect of Ziziphora clinopodioides flavonoids (ZCF) against H₂O₂-induced oxidative stress in HUVEC cells. MTT assay was carried out to determine the cell viability of HUVEC cells following pretreatment with ZCF. Fluorescent microscopy measurements were performed to evaluate apoptosis of HUVEC cells. Furthermore, the effects of ZCF on the activities of antioxidants superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), malondialdehyde production (MDA) and lactic dehydrogenase (LDH) levels were analyzed. Apoptosis was observed by Hoechst33258 staining and AO staining. Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) was used to detect the expression of B-cell lymphoma/leukemia-2 (Bcl-2), Bcl-2-associated X protein (Bax) and aspartate proteolytic enzyme-3 (Caspase-3) mRNA. The expression of vascular endothelial growth factor receptor 2 (VEGFR2), protein kinase B (Akt), phosphorylated protein kinase B (p-Akt), Bax, Bcl-2 and Caspase-3 were detected by western blot. ZCF attenuated H₂O₂-induced cell death, as determined by the MTT assay. ZCF decreased malondialdehyde and lactic dehydrogenase levels, increased superoxide dismutase, glutathione peroxidase, catalase activities and inhibited apoptosis. Moreover, pretreatment with ZCF decreased the expression of Bax and Caspase-3 at mRNA level, increased the expression of Bcl-2 mRNA level, decreased the levels of VEGFR2, Bax and Caspase-3 protein, and increased the level of p-Akt / Akt and Bcl-2 protein in HUVEC cells. These results suggested that ZCF protected against H₂O₂-induced injury in HUVEC cells. The mechanism for this effect is related to the enhancement of antioxidant capacity, suppression of angiogenesis and apoptosis.