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-α 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-α from VECs. Pretreatment with paeonol increased the survival rate of ox-LDL-treated VECs in dose- and time-dependent manners. Moreover, paeonol （120 μmol/L） prevented ox-LDL-induced increases in miR-21 expression and TNF-α 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.