Cholesterol induces endothelial cells injury by increasing production of reactive oxygen species and activating NF-κB

AIM: To study the increased level of reactive oxygen species in human umbilical vein endothelial cells (HUVECs) and their correlation with the injury caused by cholesterol on HUVECs, and to clarify the original source of intracellular ROS. METHODS: The cells of HUVECs-12 were cultured in F12 medium with 10% FBS and divided into normal control group (without any treatment), solvent group (treated with 0.25% dehydrated alcohol), cholesterol group (treated with 50 mg/L cholesterol) and N-acetyl-L-cysteine(NAC) group(pretreated with 10 mmol/L NAC for 1 h and then treated with 50 mg/L cholesterol for 48 h). The intracellular ROS levels were determined by flow cytometry (FCM) with DCFH-DA as fluorescent probe. Nuclear translocation of NF-κB subunit p65 was detected by immunocytochemistry staining. LDH activity and concentration of nitric oxide in the supernatant of the cell culture were also determined. The concentration of MCP-1 protein in cultured supernatant was measured by ELISA. The intracellular levels of ROS and the changes after adding 4 kinds of enzyme inhibitors (NADPH oxidase inhibitor diphenyl iodide, mitochondrial respiratory chain enzyme complex inhibitor rotenone, NOS inhibitor L-NAME and xanthine oxidase inhibitor oxypurinol) were observed. RESULTS: (1)Compared to the normal control cells, 50 mg/L cholesterol increased intracellular ROS (P<0.01) and activated the nuclear translocation of NF-κB p65. A significant increases in LDH activity and the MCP-l protein were also observed. The NO level decreased in the cells. (2)Compared to the cholesterol control cells, diphenyl iodide decreased intracellular ROS significantly (P<0.01).Retenone also inhibited the generation of ROS partially (P<0.05). The other inhibitors almost did not affect the level of ROS caused by cholesterol (P>0.05). CONCLUSION: Free cholesterol increases ROS generation in endothelial cells, activates intracellular NF-κB, thus leading to endothelial cell injury. NADPH oxidase was the main source of ROS generation in HUVECs cultured with free cholesterol.