The Molecular Mechanism Of Inulicin-Inhibiting Neointimal Formation Induced By Balloon Injury

Percutaneous transluminal coronary angioplasty(PTCA)has now been widely used in the treatment of cardiovascular disease. However the restenosis after angioplasty is about 30%-50% of patients. Although the pathogenic mechanisms of restenosis have not been completely resolved, vascular smooth muscle cell (VSMCs) proliferation and migration is believed to play a key role in the development of the neointima formation after balloon denudation. Mechanical injury can activate VSMCs and leukocytes in vessal. The activated VSMCs and leukocytes are known to release various types of cytokines and growth factors which stimulate migration and proliferation of VSMCs, leading to neointimal hyperplasia. Inhibition of VSMCs proliferation and migration becomes a potentially important therapeutic strategy for the treatment of cardiovascular diseases such as atherosclerosis and restenosis.The inulicin [containing three specific sesquiterpene lactones, britannilactone (BL), 1-O-acetylbritannilactone (1-O-ABL), and 1,6-O,O-diacetylbritannilactone (1,6-O2-ABL)] was isolated from inula britannica-L which is the traditional Chinese medicine for treatment of inflammation diseases. It has been reported that ABL inhibits inflammation in Raw 264.7 macrophage through inhibition of activation of NF-kB. The present study was designed to determine the effect of inulicin on restenosis induced by balloon injury in rats and to investigate the underlying mechanism.RESULTS1 Inulicin inhibits neointimal formation in carotid arteries after balloon injury1.1 Effect of inulicin on neointimal formation in vivo.A deflated balloon catheter was introduced into the aorta of healthy Male Sprague-Dawley (SD) rats weighing 300 to 350g via the left common carotid artery down to the level of the renal arteries. The balloon was then inflated with saline and the catheter withdrawn slowly to the diaphragm level at opposite directions for three times respectively. All procedures were performed by a single operator. In inulicin treatment group, the animal were pretreated with inulicin (contain ABL 2.66mg /ml) dissolved in tween-80 3 days before operation, then inulicin was administered to rat at dose of ABL 26mg/kg/d by gastric gavage orally for 14 days (n=5). An equal volume of tween-80 (3 ml/rat/d) was administrated orally to control animals as model group (n=5). The sham operated animals were used as normal group which were treated by the same operation as control group but without balloon injury (n=5) .At 21 days after balloon injury, the rats were killed by blood-bleeding, and the carotic aortas were quickly isolated for histological and Western blot analysis.H&E-stained for representative sections of carotid arteries of different groups were analyzed by using a computer assisted image analyzer, and the thickness of neointima was measured. We found that intimal thickness was significantly increased in rat carotic aortas at 21 days after balloon injury. Intimal thickness was significantly reduced in rats treated with inulicin (763.96±114.83μm), compared with model animals (1486.89±241.8μm). I/M was significantly reduced in inulicin-treated rats, as compared with model group (0.77±0.16 vs 1.76±0.11). Taken together, these results demonstrate that inulicin inhibits neointima formation induced by balloon injury.1.2 Effect of inulicin on NF-ΚB activation and translocationNF-kB is a family of transcription factors that is usually present as dimmers in the cytoplasm through interaction with inhibitory proteins, the IkBs. Activated NF-κB has been detected in nuclear of VSMC after balloon injury and atherosclerosis lesions. We therefore investigated the effects of inulicin on NF-κB activation .The cytosolic and nuclear NF-κB p65 subunit levels were detected following balloon injury in the presence or absence of inulicin. The positive cells for NF-kB were found in neointima after balloon injury, but not in normal vessels. Western blot analysis showed the same results as morphology. Balloon injury increased p65 translocation to the nucleus in VSMC, and, inulicin attenuated this response. We found that inulicin can strongly inhibit NF-kB activiton.1.3 Inulicin induces degradation of IκB-αIt has been well established that NF-κB activity is regulated by IκB proteins, and the phosphorylation and degradation of IκB allows translocation of NF-kB to the nucleus, where it is active. Translocation of NF-κB is a recognized step leading to its transcriptional activity. To explore whether the inhibitory effect of inulicin on NF-κB activation induced by balloon injury is via inhibition of IκB-αdegradation and NF-κB translocation. IKB-αlevel in neointima was detected. IKB-αexpression was decreased in model group significantly compare with both normal and inulicin treat group. These results further confirmed that inulicin could suppress NF-κB activation and translocation by blockade of degradation of IκB.1.4 Effect of inulicin on the proliferative activity of VSMCsSuppressed neointima formation was accompanied by a reduction in the proliferative activity of VSMCs. In order to elucidate the mechanism of inulicin inhibiting neointima formation, we examined the C-Jun and PCNA protein in neointima after balloon injury. A remarkable decrease in the number of C-Jun and PCNA positive cells were demonstrated in the intima and medial layers of arteries for inulicin treated rats, compared with the model group. These results indicate that inulicin inhibits neointimal formation, which is associated with the down regulation of AP-1 and PCNA.1.5 Effect of inulicin on COX-2 expressionIn order to evaluate the anti-inflammatory activity of inulicin, we examined the COX-2 protein expression in neointima after balloon injury. COX-2 protein was found in VSMC after balloon injury, while little COX-2 protein was detected in normal vessels by immunohistochemical assays. Results of Western blot also showed that the inhibitory effect of inulicin on neointimal hyperplasia is associated with the down-regulation of COX-2. 2 Inulicin inhibits expression of adhensive molecules2.1 Effect of inulicin on OPN and ICAM-1 expressionInitiation of neo-intimal thickening is characterized by migration and accumulation of VSMC within the intima. Both osteopontin (OPN) and ICAM-1, as adhesion molecules, were dramatically decreased in inulicin group, compare with normal group. Immunohistochemical assays and Western bolt suggest that adhensive molecule mediated the neointima formation. Inulicin inhibits neointima formation by decrease in adhensive molecule expression.2.2 Effect of inulicin on ILK expressionILK is an important signal molecule which mediates adhensive signal transduction. Therefore we examined the changes of ILK in order to elucidate inulicin inhibiting adhensive action. Western blot analysis showed that ILK was expressed at high level in model group while less ILK expression was found in inulicin treatment group. The data demonstrate that the decrease in OPN, ICAM-1 and ILK results from the inulicin anti-inflammatory action.2.3 Effect of inulicin on MMP-2/TIMP-2 expressionExtracellular matrix is important in cell growth and shape which is mediated by MMP-2/TIMP-2. Gelatin zymogram analysis for MMP-2 revealed that active MMP-2 was found in model group while less active MMP-2 was found in inulicin group. The changes of TIMP-2 were the same as that of MMP-2, suggesting that the increase in MMP-2 was not due to the decease in TIMP-2. Inhibition of MMP-2 and TIMP-2 may be one of mechanisms for the inhibition of neointima formation by inulicin.3 Inulicin induced apoptosis of VSMC in vivo after balloon injury3.1 Inulicin induced apoptosisIncreasing evidences suggests that cell proliferation and resistance to apoptosis in VSMC are two key mechanisms in neointimal formation. Decreased apoptosis in VSMCs contributes to neointimal hyperplasia and restenosis. In order to determine whether apoptosis is induced by inulicin, TUNEL assay was performed. The results revealed that in normal and model aortas few apoptotic cells were found in the intima while inulicin can increase apoptotic cells significantly. The data suggest that in addition to its antiproliferative effect, inulicin also induces apoptosis of VSMC, which may contribute to its effect in neointimal formation.3.2 Inulicin modulats expression of apoptosis related proteins in vivo.In order to assess the mechanism of inulicin inducing apoptosis some important apoptotic factors in balloon-injured aortic walls were detected by Western blot and immunohistochemical assays. In the inulicin treat group, expression of pro-apoptotic proteins, Bax and caspase-3, was up-regulated while the expression of anti-apoptotic protein Bcl-2 was down-regulated, compare with model group. Taken together these results, inulicin may affect VSMC proliferation and apoptosis through regulation of the expression of growth/apoptosis-related genes.3.3 Inulicin promotes caspase expressionThe expression of caspase-3 was up-regulated significantly in the inulicin treat group, compare with model group, while there is only a little caspase-3 in the normal group. The change of caspase-3 was accordance with Bcl-2.Conclusions1 Inulicin inhibits inflammation by decrease in COX-2 expression inhibiting NF-κB activation and translocation via blockade of IκB-αphosphorylation and degradation in neointimal formation.2. Inulicin significantly inhibits neointima formation after balloon injury by suppressing the expression of proliferative and adhesive gene, such as C-Jun, PCNA, OPN, ICAM-1 and ILK.3 Inulicin significantly inhibits MMP-2/TIMP-2 expression, resulting in decrease in the rate of ECM turnover.4 Inulicin induces the VSMC apoptosis via up-regulating the expression of pro-apoptotic proteins Bax and caspase-3 and down-regulating the expression of anti-apoptotic protein Bcl-2.5 Inulicin inhibits neointima formation by multiple pathways.