Acetylbritannilactone Inhibits Neointimal Hyperplasia after Balloon Injury of Rat Artery by Suppressing Nuclear Factor- B Activation

Based on our previous observations that 1-O-acetylbritannilactone (R)-4((3aS,4S,7aR)-4-hydroxy-6-methyl-3-methylene-2-oxo2,3,3a,4,7,7a-hexahydrobenzofuran-5-yl)pentyl acetate (ABL) suppresses prostaglandin E2 and nitric oxide synthesis in macrophages, the present study was designed to explore the effect of ABL on neointimal hyperplasia after balloon injury and its mechanism of action. In male Sprague-Dawley rats, 26 mg/kg ABL or polyglycol (control) was administered daily from 3 days before injury to 2 weeks after conventional balloon injury. ABL administration led to a significant reduction in neointimal formation (neointima to media ratio, 1.94 0.43 versus 0.84 0.29, P 0.01) and proliferative activity of vascular smooth muscle cells after balloon injury in rats. Western blot analysis revealed that this is correlated to the inhibition of nuclear factor (NF)B activation and to the reduced expression of cyclooxygenase-2. Investigation of potential signaling pathways demonstrated that ABL inhibited NFB activation via the blockade of the inhibitor of NFB kinaseactivation and the suppression of the degradation of the inhibitors of NFB. These findings suggest that ABL is a potential inhibitor of neointimal formation because it blocks injury-induced NFB activation and may have beneficial effects in reducing the risk of restenosis after angioplasty. Percutaneous transluminal balloon angioplasty is now widely used in the treatment of obstructed atherosclerotic vessels. However, its overall benefits are limited by the fact that it may induce local arterial restenosis in many patients who are initially treated successfully (Bennett and Schwartz, 1995; Boden et al., 2007). Although the mechanisms implicated in the process of restenosis have not been completely resolved, accumulating evidence suggests that inflammation plays a key role in neointimal growth after angioplasty (Danenberg et al., 2002; Welt and Rogers, 2002). The activation of nuclear factor (NF)B has been found in ballooninjured rat carotid arteries and is involved in the process of neointimal hyperplasia, including growth factor-dependent activation of smooth muscle cell (SMC) proliferation, protease-dependent migration of cells to the wounded area, and cytokine-dependent matrix deposition (Tak and Firestein, 2001; Raines et al., 2004; Raines and Ferri, 2005). In particular, it has been demonstrated that activation of NFB is centrally involved in generating the proadhesive phenotype of neointimal smooth muscle cells (Raines et al., 2004; Zeiffer et al., 2004). Because NFB is highly activated at sites of inflammation in diverse diseases, it represents a master switch in many inflammatory diseases (Barnes and Karin, 1997; Chen and Shi, 2002). Thus, interruption of the activation of NFB by anti-inflammatory drugs and agents could suppress the production of inflammatory mediators and in turn block the initiation and progression of inflammationassociated diseases (Karin, 2005). ABL is a new active extract from Inula britannica L., a traditional Chinese medicinal herb (for its structure, see Fig. 1). Sesquiterpene lactones from I. britannica have antitumor and immunomodulatory properties and can inhibit the growth of different types of transformed cells (Song et al., 2002; Rafi et al., 2005). Previous studies showed ABL supThis study was supported by the National Nature Science Foundation of the People’s Republic of China (Grants 30570661 and 30472167) and by the Hebei Province Natural Science Foundation of the People’s Republic of China (Grant C2005000722). J.-K.W. was supported by the Major State Basic Research Development Program of the People’s Republic of China (Grant 2005CCA03100). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.107.127407. ABBREVIATIONS: NF, nuclear factor; SMC, smooth muscle cell; ABL, 1-O-acetylbritannilactone, (R)-4((3aS,4S,7aR)-4-hydroxy-6-methyl-3methylene-2-oxo-2,3,3a,4,7,7a-hexahydrobenzofuran-5-yl)pentyl acetate; PGE2, prostaglandin E2; COX, cyclooxygenase; I, intima; M, media; ELISA, enzyme-linked immunosorbent assay; I B, inhibitor(s) of NFB; IKK, inhibitor of NFB kinase; EMSA, electrophoresis mobility shift assay; PCNA, proliferating cell nuclear antigen. 0022-3565/08/3241-292–298$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 324, No. 1 Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics 127407/3283896 JPET 324:292–298, 2008 Printed in U.S.A. 292 at A PE T Jornals on O cber 5, 2017 jpet.asjournals.org D ow nladed from presses nitric oxide and PGE2 synthesis in macrophages through the inhibition of inducible nitric-oxide synthase and COX-2 gene expression, indicating modulation of inflammation (Han et al., 2004). The present study was designed to elucidate the effect of ABL on neointimal hyperplasia in a model of experimental arterial injury. Materials and Methods Animals and Pretreatments. Male Sprague-Dawley rats weighing 290 to 320 g were fed standard laboratory chow and allowed free access to water in an air-conditioned room with a 12-h light/dark cycle. Before the experiment, animals were housed under these conditions for 7 days to allow acclimatization. All animal experiments were approved by the governmental committee for animal research. ABL (99.5% pure), freshly dissolved in 20% polyglycol, was administered orally by gastric gavage from 3 days before balloon injury to 2 weeks after the injury. Six control rats received an equal volume of 20% polyglycol (3 ml/day) in the same manner. Animal body weights were recorded before and at the end of the 2nd week of the