Paeonol protects rat vascular endothelial cells from ox-LDL-induced injury in vitro via downregulating microRNA-21 expression and TNF-α release
Abstract
Ya-rong LIU1, Jun-jun CHEN2, Min DAI3, *
1Key Laboratory of Chinese Medicine Research and Development, 2Key Laboratory of Xin’an Medicine, Hefei 230038, China; 3Department of Pharmacy, Anhui University of Traditional Chinese Medicine, Hefei 230031, China
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.
Keywords: paeonol; atherosclerosis; microRNA-21; PTEN; vascular endothelial cells; oxidized low density lipoprotein; inflammatory reaction
The studies were mainly supported by research grants from the National Natural Science Foundation of China (81073090 and 81274134). We thank Prof Hou-kai LI (Shanghai University of Traditional Chinese Medicine) for his contribution to the English translation.
* To whom correspondence should be addressed.
E-mail daiminliao@163.com
Received 2013-08-05 Accepted 2013-12-12
Keywords:
1Key Laboratory of Chinese Medicine Research and Development, 2Key Laboratory of Xin’an Medicine, Hefei 230038, China; 3Department of Pharmacy, Anhui University of Traditional Chinese Medicine, Hefei 230031, China
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.
Keywords: paeonol; atherosclerosis; microRNA-21; PTEN; vascular endothelial cells; oxidized low density lipoprotein; inflammatory reaction
The studies were mainly supported by research grants from the National Natural Science Foundation of China (81073090 and 81274134). We thank Prof Hou-kai LI (Shanghai University of Traditional Chinese Medicine) for his contribution to the English translation.
* To whom correspondence should be addressed.
E-mail daiminliao@163.com
Received 2013-08-05 Accepted 2013-12-12