Paeonol protects rat vascular endothelial cells from ox-LDL-induced injury in vitro via downregulating microRNA-21 expression and TNF-α release

Aim: Paeonol (2 '-hydroxy-4 '-methoxyacetophenone) from Cortex moutan root is a potential therapeutic agent for atherosclerosis. This study sought to investigate the mechanisms underlying anti-inflammatory effects of paeonol in rat vascular endothelial cells (VECs) in vitro. Methods: VECs were isolated from rat thoracic aortas. The cells were pretreated with paeonol for 24 h, and then stimulated with ox-LDL for another 24 h. The expression of microRNA-21 (miR-21) and PTEN in VECs was analyzed using qRT-PCR. The expression of PTEN protein was detected by Western blotting. TNF-alpha release by VECs was measured by ELISA. Results: Ox-LDL treatment inhibited VEC growth in dose-and time-dependent manners (the value of IC50 was about 20 mg/L at 24 h). Furthermore, ox-LDL (20 mg/L) significantly increased miR-21 expression and inhibited the expression of PTEN, one of downstream target genes of miR-21 in VECs. In addition, ox-LDL (20 mg/L) significantly increased the release of TNF-a from VECs. Pretreatment with paeonol increased the survival rate of ox-LDL-treated VECs in dose-and time-dependent manners. Moreover, paeonol (120 mu mol/ L) prevented ox-LDL-induced increases in miR-21 expression and TNF-a release, and ox-LDL-induced inhibition in PTEN expression. A dual-luciferase reporter assay showed that miR-21 bound directly to PTEN's 3 '-UTR, thus inhibiting PTEN expression. In ox-LDL treated VECs, transfection with a miR-21 mimic significantly increased miR-21 expression and inhibited PTEN expression, and attenuated the protective effects of paeonol pretreatment, whereas transfection with an miR-21 inhibitor significantly decreased miR-21 expression and increased PTEN expression, thus enhanced the protective effects of paeonol pretreatment. Conclusion: miR-21 is an important target of paeonol for its protective effects against ox-LDL-induced VEC injury, which may play critical roles in development of atherosclerosis.