Abstract
The present study was designed to examine the effects of atorvastatin on vascular inflammatory responses in human coronary artery endothelial cells(HCAECs), when challenged by lipopolysaccharide (LPS), a Toll-like receptor-4 (TLR4) ligand. HCAECs were pretreated with atorvastatin and induced by LPS. The expression of TLR4, interleukin -6(IL-6), monocyte chemoattractant protein 1(MCP-1), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecular-1(ICAM-1), nuclear factor-κB (NF-κB) and p38 mitogen activated protein kinase(p38 MAPK) were evaluated using Real-time polymerase chain reaction, cytokine ELISA assay and Western blotting. The results showed that pretreatment with atorvastatin down-regulated the expression of TLR4 in LPS-activated HCAECs. Atorvastatin also attenuated the LPS-induced expression of interleukin IL-6 and MCP-1, at both the transcription and translation level in HCAECs. LPS-induced endothelial cell adhesion molecules, ICAM-1 and VCAM-1 expression were also reduced by pretreatment with atorvastatin. Furthermore, atorvastatin efficiently suppressed LPS-induced phosphorylation of NF-κB and p38 MAPK in HCAECs. These findings show that atorvastatin suppresses endothelial cell inflammation, suggesting that atorvastatin may be suitable for development as a therapeutic agent for inflammatory cardiovascular disease.
Keywords:
Atorvastatin; TLR4; Lipopolysaccharide; Endothelial cells; Inflammation
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(A) Time-effect TLR4 mRNA responses of LPS stimulation in HCAECs. Statistical analysis was performed on 5min, 15min, 30min and 60min versus 0min group, respectively. (B) Dose-effect TLR4 mRNA response of LPS stimulation in HCAECs. Statistical analysis was performed on 1ng/mL, 10ng/mL, 100ng/mL and 1000ng/mL versus 0ng/mL group, respectively.(C) Time-effect TLR4 protein response of LPS stimulation in HCAECs. (D) Dose-effect TLR4 protein response of LPS stimulation in HCAECs. (E) Time-effect TLR4 protein expression of TLR4 in HCAECs were quantified by using densitometry, and represented by the ratio of β-actin. Statistical analysis was performed on 5min, 15min, 30min and 60min versus 0min group, respectively. (F) Dose-effect TLR4 protein expression of TLR4 in HCAECs were quantified by using densitometry, and represented by the ratio of β-actin. Statistical analysis was performed on 1ng/mL, 10ng/mL, 100ng/mL and 1000ng/mL versus 0ng/mL group, respectively.*P<0.05 indicates significant differences.
(A) Dose effect response showing TLR4 mRNA reduction treatment in HCAECs. Statistical analysis was performed on 6h, 12h, and 24h versus 0h group, respectively. (B) Time-effect response showing TLR4 mRNA reduction after atorvastatin treatment in HCAECs. Statistical analysis was performed on 0.01µmol/L, 0.1µmol/L, 1µmol/L and 10µmol/L versus 0µmol/L group, respectively.*P<0.05 indicates significant differences.
(A) Protein expression of ICAM-1 in HCAECs was assessed by Western blotting after treatment with LPS and atorvastatin. (B) Protein expression of VCAM-1 in HCAECs was assessed by Western blotting after treatment with LPS and atorvastatin. (C) Protein expression of ICAM-1 in HCAECs was quantified using densitometry, and normalized to β-actin. Statistical analysis was performed on Ato, LPS and Ato+ LPS versus Con group, respectively. (D) Protein expression of VCAM-1 in HCAECs was quantified using densitometry, and normalized to β-actin. Statistical analysis was performed on Ato, LPS and Ato+ LPS versus Con group, respectively.Data are expressed as mean values SE of three independent experiments. *P<0.05 indicates significant differences.
(A) The phosphorylation-NF-κB was assessed by Western blotting after treatment with LPS and atorvastatin (B) The phosphorylation of p38 MAPK was assessed by Western blotting after treatment with LPS and atorvastatin. (C) The phosphorylation of NF-κB in HCAECs was quantified using densitometry and represented by the ratio of total NF-κB. Statistical analysis was performed on Ato, LPS and Ato+ LPS versus Con group, respectively. (D) The phosphorylation of p38 MAPK in HCAECs was quantified using densitometry, and represented by the ratio of total p38 MAPK. Statistical analysis was performed on Ato, LPS and Ato+ LPS versus Con group, respectively. Data are expressed as mean values SE of three independent experiments. *P<0.05 indicates significant differences.