Baicalin Inhibits Neointimal Hyperplasia After Rat Artery Balloon Injury

It is known that the proliferation of vascular smooth muscle cells (VSMC) plays an important role in the pathogenesis of restenosis. Baicalin is a major flavonoid extracted from the traditional Chinese medicinal herb Scutellaria baicalensis Georgi. Baicalin has been used in the treatment of breast, hepatocellular, pancreatic, and prostatic cancers. However, its effects on the neointimal hyperplasia remain unclear. Therefore, the present study was designed to elucidate the effect of baicalin on the neointimal hyperplasia after balloon injury and the related mechanism.Methods1 VSMC culture5-week-old male SD rats were selected. The VSMCs from rat aorta were isolated and were cultured in DMEM medium containing 10%FBS. The cells used for the experiment were passage 3 ~ 6. The cells grown into 80%~90% confluences were serum-starved for 24 hours. Then the VSMC was treated with baicalin using different doses (5, 10, 20, 40μmol/l) for 24 h.2 Western blot analysisEqual amounts of protein extracts from VSMC were separated on 10% SDS-PAGE, and then blotted onto PVDF membrane. The membrane was immunologically stained with specific antibodies. The results were analyzed by digital imaging system.3 Establishment of the animal modelSD rats were randomly divided into three groups, including sham group, injured group, baicalin group. Rats were anesthetized with urethane. The aorta and carotid artery were de-endothelialized with balloon catheter. Briefly, the catheter was pushed from left carotid artery into the aorta down to the level of the renal arteries three times with a 2F Fogarty catheter, and then recovered the blood stream.4 Preparation of experimental specimenThe rats were killed at 14 days after de-endothelium. The carotid arteries were separated for preparation of sections. Sections were stained with hematoxylin and eosin to detect the neointimal hyperplasia. The microscopical pictures were analyzed using a computer assisted image analyzer, and the thickness of neointima was measured. Furthermore, the effects of baicalin on the expression of PCNA, ICAM-1 and VCAM-1 were analyzed by immunohistochemistry.Results1 The role of baicalin in VSMC proliferation induced by PDGFThe results of MTT assays showed that baicalin (5, 10, 20, 40μmol/L) treatment resulted in a significant reduction of VSMC proliferation in a dose-dependent manner.2 The effect of baicalin on PCNA expression induced by PDGFThe results of immunocytochemistry and Western blot analysis showed that expression of PCNA was rarely detected in control group. PDGF (10 ng/ml) stimulation resulted in significant increase in PCNA expression. However, the amount of PCNA was significantly reduced in the baicalin-treated group, suggesting that baicalin inhibits the proliferation of VSMC induced by PDGF.3 Baicalin markedly inhibits the PDGFR/MEK/ERK1/2 signaling pathway activated by PDGFWestern blot analysis showed that PDGF stimulation resulted in significant increase in the phosphorylation of PDGFR, MEK and ERK1/2, compared with control group. However, the phosphorylation of PDGFR, MEK and ERK1/2 induced by PDGF was significantly reduced in baicalin-treated group.4 Baicalin inhibits neointimal hyperplasia induced by balloon injury in carotid arteries of ratAt 14 days after balloon injury, the injured group showed neointimal formation in carotid arteries of rat. However, baicalin significantly reduced neointimal hyperplasia and I/M ratio (I/M ratio, baicalin versus injured, 0.43±0.04 versus 2.36±0.15, p < 0.05) compared with the injured group.5 Baicalin inhibits the expression of PCNA induced by balloon injury in carotid arteries of ratImmunohistochemical staining showed that the expression of PCNA was rarely detected in in carotid arteries of sham group. However, the number of PCNA positive cells in injured group was significantly increased. However, the expression of PCNA in carotid arteries injured by balloon was significantly reduced by 53.4 % (P<0.01) at 14 day in baicalin-treated group.6 Baicalin inhibits the expression of ICAM-1 and VCAM-1 induced by balloon injury in carotid arteries of ratImmunohistochemistry results showed that the expression of ICAM-1 and VCAM-1 in carotid arteries was not detected in sham group. Balloon injury resulted in significant increase in ICAM-1 and VCAM-1 expression, compared with sham-operated rats. Staining results also showed that the expression of ICAM-1 and VCAM-1 induced by balloon injury was significantly reduced by 55.8% and 57.2% (P<0.01) in baicalin-treated group.Conclusions1 Baicalin inhibits the proliferation of VSMC induced by PDGF.2 Baicalin blocks PDGFR-MEK-ERK1/2 signaling pathway activated by PDGF in VSMCs.3 Baicalin inhibits neointimal hyperplasia via inhibiting vascular cell proliferation and adhesion molecules expression induced by balloon injury.