| Objective: Percutaneous transluminal balloon angioplasty has now been widely used in the treatment of obstructed atherosclerotic vessels. However, its overall benefits are interrupted by inducing local arterial restenosis. 1-o-acetylbritannilactone (ABL) is a new active extracts from Inula Britannica L, a traditional Chinese medicinal herb. Recently, we reported ABL suppresses NO and PGE2 synthesis in RAW 264.7 macrophages through the inhibition of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) gene expression. The present study was designed to elucidate the effect of ABL on neointimal hyperplasia after balloon injury and its mechanism of action.Methods1 Establishment of animal modelSD rats were randomly divided into three groups, including sham group, injured group and ABL group. Animals were anesthetized with urethane, 600 mg/kg intraperitoneal. The thoracic-abdominal aorta was de-endothelialized. Briefly, the catheter was pushed from left common carotid artery into the aorta down to the level of the renal arteries three times with a 2F Fogarty catheter inserted through the external carotid artery. To attain a constant degree of vessel wall injury for each of the animals, we kept the diameter of the balloon and the resistance during withdrawal constant and the same for each of the animals. All procedures were performed by a single operator.2 Preparation of experimental specimenAll the rats were killed at different time intervals after de-endothelium. The aortas were separated for preparation of sections, and the aortas were separated for preparation of protein extract. Sections were stained with hematoxylin and eosin to detect the suppression of neointimal hyperplasia by ABL. The microscopical pictures were analyzed by using a computer assisted image analyzer, and the thickness of neointima was measured. We identificated the NF-κB /DNA binding activity in nuclear extracts of injured rat aortas by electrophoretic mobility shift assay. The protein extracts from aortas were used to detect the expression of NF-κB p65, p65 nuclear translocation, COX-2 and iNOS by Western blot. In addition, RT-PCR detects the transcription of ICAM-1.Results1 ABL inhibits neointimal hyperplasiaAt 14 days after balloon injury, the injured vessel (injured group) showed abundant neointimal hyperplasia. The ABL-treated animals (ABL group) showed significant suppression of neointimal hyperplasia and reduction of the I/M ratio (I/M ratio, injured versus ABL, 1.87±0.16 versus 0.45±0.03, p < 0.01). ABL treatment significantly reduced neointimal hyperplasia, compared with the injured group.2 ABL suppresses the expression and nuclear translocation of NF-κB p65Western blot analysis showed that the expression of NF-κB p65 increased at 1 day after balloon injury, and reached the peak level at 14 days. To investigate the effect of ABL on p65 expression, Western blot was performed. At 14 days after balloon injury, the activity of p65 expression was reduced in ABL-treated group. Moreover, balloon injury could stimulate the translocation of p65 into the nucleus. However, the amount of p65 translocation into the nucleus was significantly reduced in the injured arteries of ABL-treated rats.3 ABL decreases the level of phosphorylated IκB-αThe results of Western blot analysis showed that there was a high expression of IκB-αin sham group. In addition, balloon injury resulted in a considerable decrease in IκB-αwhich is about 46.7% compared with sham group at 14 days. However, following ABL treatment the level of phosphorylated IκB-αbeing reduced by 89.2%±7.4% (P<0.01). These results suggested that ABL suppresses the phosphorylation and degradation of IκB-α.4 ABL markedly inhibit the DNA binding activity of NF-κB.Double stranded NF-κB consensus oligonucleotide probe was end-labelled withγ-32P-ATP. Nuclear protein-oligonucleotide complexes were resolved by electrophoresis on a 5% non denaturing polyacrylamide gel. The gel was autoradiographed with intensifying screen at -70℃. Subsequently, the specificity of the NF-κB /DNA binding complex was significantly inhibited by ABL.5 ABL inhibits COX-2, iNOS and ICAM-1 expression in vascular tissue induced by balloon injuryWestern blot analysis showed COX-2, iNOS and ICAM-1 expression in vascular tissue. The results showed that balloon injury resulted in significant increase in COX-2 and iNOS expression, compared with sham-operated rats. In ABL-treated rats, the expression of COX-2 and iNOS induced by balloon injury was significantly reduced by 65.7%±7.9% and 47.2%±5.4% (P<0.01,P<0.01). The results of RT-PCR analysis showed that balloon injury at 3 days resulted in a considerable increase in ICAM-1 mRNA. After ABL treatment, increased mRNA of ICAM-1 was reduced by 56.3%±7.3% (P<0.01).6 ABL inhibits the production of PGE2 by suppressing injury-induced COX-2 expressionTo test whether the inhibiting neointimal hyperplasia effect of ABL is related to its anti-inflammatory properties, we examined the effect of ABL on COX-2 gene expression and the production of PGE2 by enzyme immunoassay system. The results showed that the PGE2 levels in sham, injured and ABL group were 1300±129 pg/ml, 2600±195 pg/ml and 945±136 pg/ml respectively. Compared with the injured group, ABL could reduce the production of PGE2. 7 ABL inhibits the expression and phosphorylation of IKK-βAt 14 days after balloon injury, the expression of IKKβand p-IKKβincreased in the tissue extracts. ABL could block the expression of IKK-βtriggered by balloon injury. IKKβand p-IKKβlevels decreased by 46.9%±6.7% and 88.1%±9.2%, compared with injured group (P<0.01,P<0.01).Conclusions1 ABL inhibits neointima formation after balloon injury in rats. Investigation of potential signaling pathways demonstrated that ABL inhibited the NF-κB nuclear translocation and DNA binding activity via the blockade of IKK-βactivation, the suppression of IκB-αphosphorylation and degradation and subsequent NF-κB activation.2 ABL significantly inhibit expression of COX-2, iNOS and ICAM-1 in vascular tissue induced by balloon injury. Furthermore, ABL reduces the serum PGE2 level after balloon injury.
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