黄芩苷抑制内皮损伤诱导的血管新生内膜形成及其机制
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摘要
目的:血管平滑肌细胞(vascular smooth muscle cell, VSMC)增殖是血管再狭窄的主要病理基础。黄芩苷(baicalin)是从中草药黄芩中分离出来的一种黄酮类化合物。以往研究发现,黄芩苷对肿瘤细胞的增殖具有明显的抑制作用。但其是否影响血管内皮损伤所诱导的血管内膜增生尚不清楚。本研究从以下几方面探讨黄芩苷对血管新生内膜形成的影响及其作用机制。
方法:
1 VSMC的培养:按贴块法分离培养VSMC,取3～6代细胞进行实验。传代培养的VSMC生长到80%～90%汇合时,给予不同浓度黄芩苷(5、10、20、40μmol/L)预处理。
2 Western blot分析:取等量蛋白的提取液上样,进行SDS-PAGE。电转移至PVDF膜上。随后与相应的一抗及二抗反应,用化学发光法检测抗原抗体结合区带。用数码成像分析系统软件对结果进行定量分析。
3球囊损伤模型的复制及分组
将SD大鼠(250~300g)常规麻醉消毒,沿颈部正中线切口,暴露左侧颈外动脉,在颈外动脉剪一楔形切口,将球囊导管自切口插入至胸腹主动脉远心端,充盈球囊后反复回拉三次,退出,结扎颈外动脉,关闭切口。实验动物分假手术组、球囊损伤组、黄芩苷组。
4标本的处理
于术后14天从股动脉放血处死大鼠,分离颈总动脉,制备病理切片。
结果:
1黄芩苷抑制PDGF诱导的VSMC增殖
MTT分析结果表明,黄芩苷能以浓度(5、10、20、40μmol/L)依赖的方式抑制VSMC增殖,具有明显的量-效关系。
2黄芩苷对VSMC增殖标志基因PCNA表达的影响
免疫细胞化学和Western blot分析显示,正常细胞中PCNA呈低水平表达,PDGF (10 ng/ml)刺激后PCNA水平大幅度升高,而药物预孵育则使PCNA水平下降。提示黄芩苷具有抑制PDGF诱导的VSMC增殖的作用。
3黄芩苷抑制PDGF激活的PDGFR/MEK/ERK1/2信号通路
Western blot结果显示,PDGF刺激细胞15 min,PDGFR磷酸化水平明显升高;而经黄芩苷预处理的VSMC,磷酸化的PDGFR呈浓度依赖性的降低;进而对其下游的MEK、ERK1/2活性进行分析,发现MEK和ERK1/2磷酸化程度也随着药物浓度的增加逐渐降低;但磷酸化的JNK、p38、Akt不受药物处理的影响。提示黄芩苷是通过特异性抑制MEK/ERK1/2通路活化来抑制PDGF诱导的VSMC的增殖。
4黄芩苷抑制球囊损伤后血管内膜的增生
球囊损伤术后14天,黄芩苷组血管内膜的增生程度较球囊损伤组明显减轻,两组的血管内膜/中膜(I/M)厚度比分别为0.43±0.04和2.36±0.15,具有显著性差异(P < 0.01)。表明黄芩苷可以明显抑制球囊损伤诱导的血管内膜增生。
5黄芩苷抑制新生内膜中PCNA表达
形态学分析显示:正常血管内膜中仅有少量PCNA表达,球囊损伤14天后,血管中PCNA阳性的细胞数明显增多;而黄芩苷可明显抑制PCNA的表达,与模型组相比PCNA阳性的细胞数减少了53.4 %,具有显著性差异(P < 0.01)。提示黄芩苷可抑制血管细胞的增殖活性。
6黄芩苷抑制球囊损伤诱导的ICAM-1和VCAM-1的表达
免疫组织化学染色显示:正常血管内膜中可见极少量的ICAM-1和VCAM-1着色颗粒。球囊损伤14天后,两种蛋白阳性染色的细胞数及单细胞染色强度均较正常组显著增加。而黄芩苷能够显著抑制球囊损伤诱导的ICAM-1和VCAM-1的表达,与模型组相比阳性染色的细胞数分别降低了55.8±6.9%和57.2±5.5%,具有显著性差异(P<0.01,P<0.01)。
结论:
1黄芩苷抑制PDGF诱导的VSMC增殖。
2黄芩苷抑制VSMC增殖的机制与阻断PDGFR/MEK/ERK1/2信号通路有关。
3黄芩苷可预防内皮损伤所致的血管内膜增生和管腔狭窄。
4黄芩苷抑制血管内膜增生与其抑制血管细胞增殖和粘附分子表达有关。
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.
Methods
1 VSMC culture
5-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 analysis
Equal 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 model
SD 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 specimen
The 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.
Results
1 The role of baicalin in VSMC proliferation induced by PDGF
The 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 PDGF
The 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 PDGF
Western 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 rat
At 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 rat
Immunohistochemical 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 rat
Immunohistochemistry 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.
Conclusions
1 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.
方法:
1 VSMC的培养:按贴块法分离培养VSMC,取3～6代细胞进行实验。传代培养的VSMC生长到80%～90%汇合时,给予不同浓度黄芩苷(5、10、20、40μmol/L)预处理。
2 Western blot分析:取等量蛋白的提取液上样,进行SDS-PAGE。电转移至PVDF膜上。随后与相应的一抗及二抗反应,用化学发光法检测抗原抗体结合区带。用数码成像分析系统软件对结果进行定量分析。
3球囊损伤模型的复制及分组
将SD大鼠(250~300g)常规麻醉消毒,沿颈部正中线切口,暴露左侧颈外动脉,在颈外动脉剪一楔形切口,将球囊导管自切口插入至胸腹主动脉远心端,充盈球囊后反复回拉三次,退出,结扎颈外动脉,关闭切口。实验动物分假手术组、球囊损伤组、黄芩苷组。
4标本的处理
于术后14天从股动脉放血处死大鼠,分离颈总动脉,制备病理切片。
结果:
1黄芩苷抑制PDGF诱导的VSMC增殖
MTT分析结果表明,黄芩苷能以浓度(5、10、20、40μmol/L)依赖的方式抑制VSMC增殖,具有明显的量-效关系。
2黄芩苷对VSMC增殖标志基因PCNA表达的影响
免疫细胞化学和Western blot分析显示,正常细胞中PCNA呈低水平表达,PDGF (10 ng/ml)刺激后PCNA水平大幅度升高,而药物预孵育则使PCNA水平下降。提示黄芩苷具有抑制PDGF诱导的VSMC增殖的作用。
3黄芩苷抑制PDGF激活的PDGFR/MEK/ERK1/2信号通路
Western blot结果显示,PDGF刺激细胞15 min,PDGFR磷酸化水平明显升高;而经黄芩苷预处理的VSMC,磷酸化的PDGFR呈浓度依赖性的降低;进而对其下游的MEK、ERK1/2活性进行分析,发现MEK和ERK1/2磷酸化程度也随着药物浓度的增加逐渐降低;但磷酸化的JNK、p38、Akt不受药物处理的影响。提示黄芩苷是通过特异性抑制MEK/ERK1/2通路活化来抑制PDGF诱导的VSMC的增殖。
4黄芩苷抑制球囊损伤后血管内膜的增生
球囊损伤术后14天,黄芩苷组血管内膜的增生程度较球囊损伤组明显减轻,两组的血管内膜/中膜(I/M)厚度比分别为0.43±0.04和2.36±0.15,具有显著性差异(P < 0.01)。表明黄芩苷可以明显抑制球囊损伤诱导的血管内膜增生。
5黄芩苷抑制新生内膜中PCNA表达
形态学分析显示:正常血管内膜中仅有少量PCNA表达,球囊损伤14天后,血管中PCNA阳性的细胞数明显增多;而黄芩苷可明显抑制PCNA的表达,与模型组相比PCNA阳性的细胞数减少了53.4 %,具有显著性差异(P < 0.01)。提示黄芩苷可抑制血管细胞的增殖活性。
6黄芩苷抑制球囊损伤诱导的ICAM-1和VCAM-1的表达
免疫组织化学染色显示:正常血管内膜中可见极少量的ICAM-1和VCAM-1着色颗粒。球囊损伤14天后,两种蛋白阳性染色的细胞数及单细胞染色强度均较正常组显著增加。而黄芩苷能够显著抑制球囊损伤诱导的ICAM-1和VCAM-1的表达,与模型组相比阳性染色的细胞数分别降低了55.8±6.9%和57.2±5.5%,具有显著性差异(P<0.01,P<0.01)。
结论:
1黄芩苷抑制PDGF诱导的VSMC增殖。
2黄芩苷抑制VSMC增殖的机制与阻断PDGFR/MEK/ERK1/2信号通路有关。
3黄芩苷可预防内皮损伤所致的血管内膜增生和管腔狭窄。
4黄芩苷抑制血管内膜增生与其抑制血管细胞增殖和粘附分子表达有关。
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.
Methods
1 VSMC culture
5-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 analysis
Equal 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 model
SD 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 specimen
The 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.
Results
1 The role of baicalin in VSMC proliferation induced by PDGF
The 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 PDGF
The 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 PDGF
Western 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 rat
At 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 rat
Immunohistochemical 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 rat
Immunohistochemistry 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.
Conclusions
1 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.
引文
1 Bennett MR, Schwartz SM. Antisense therapy for angioplasty restenosis. Some critical considerations. Circulation, 1995, 92: 1981~1993.
2 Nylande M, Kroess A, Stranden E, et al. Marker of vascular inflammation are association with the extent of atherosclerosis assessed as angiographic score and treadmill walking distances in patients with peripheral arterial occlusive disease. Vasc Medi, 2006, 11: 21~28.
3 Li J, Zhu X, Chen M, et al. Myocardin-related transcription factor B is required in cardiac neural crest for smooth muscle differentiation and cardiovascular development. Proc Natl Acad Sci USA, 2005, 102: 8916~8921.
4 Vindis C, Escargueil-Blanc I, Elbaz MD, et al. Esensitization of platelet-derived growth factor receptor-beta by oxidized lipids in vascular cells and atherosclerotic lesions: prevention by aldehyde scavengers. Circ Res, 2006, 98: 785~792.
5 Boucher P, Gotthardt M, Li W P, et al. LRP: role in vascular wall integrity and protection from atherosclerosis. Science, 2003, 300: 329~332.
6 Okano J, Nagahara T, Matsumoto K, Murawaki Y.Caffeine inhibits the proliferation of liver cancer cells and activates the MEK/ERK/EGFR signalling pathway. Basic Clin Pharmacol Toxicol, 2008, 102: 543~551.
7 Nakata S, Fujita N, Kitagawa Y, et al. Regulation of platelet-derived growth factor receptor activation by afadin through SHP-2: implications for cellular morphology. J Biol Chem, 2007, 282: 37815~37825.
8 Geisterfer AA, Peach MJ, Owens GK. Angiotensin II induces hypertrophy, not hyperplasia, of cultured rat aortic smooth muscle cells. Circ Res, 1988, 62: 749~758.
9 Wang N, Tang LJ, Zhu GQ, et al. Apoptosis induced by baicalin involving up-regulation of P53 and bax in MCF-7 cells. J Asian Nat Prod Res. 2008, 10: 1129~1135.
10 Chou TC, Chang LP, Li CY, et al. The anti-inflammatory and analgesic effects of Baicalin in carrageenan-evoked thermal hyperalgesia. Anesth. Analg. Dec 2003, 97: 1724~1729.
11 Ramos K, Cox LR. Primary cultures of rat aortic endothelial and smooth muscle cells. An in vitro model to study xenobiotic-induced vascular cytotoxicity. In Vitro Cell Dev Biol, 1987, 23: 288~296.
12 Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury, I: smooth muscle growth in the absence of endothelium. Lab Invest, 1983, 49: 327 ~333.
13 Capron L, Jarnet J, Heudes D, et al. Repeated ballooninjury of rat aorta. A model of neointima with attenuated inhibition by heparin. Arterioscler Thromb Vasc Biol, 1997, 17: 1649~1656.
14 Chen KH, Guo X, Ma D, et al. Dysregulation of HSG triggers vascular proliferative disorders, Nat Cell Biol, 2004, 6: 872~883.
15 Liu B, Han M, Wen JK. Acetylbritannilactone inhibits neointimal hyperplasia after balloon injury of rat artery by suppressing nuclear factor-{kappa}B activation. J Pharmacol Exp Ther. 2008, 324: 292~298.
16 Hong M, Mehran R, Mintz G, et al. Restenosis after coronary Angioplasty. Curr Probl cardiol, 1997, 22 : 7.
17 Bauriedel G, Kandoll, Welsch U, et al. Mechanisms of restenosis after angioplasty. Z Kardiol, 1994, 83 (Suppl 4): 31~41.
18 Wang Z, Yang H, Tachado SD, et a1. Phosphatase -mediated crosstalk control of ERK and p38 MAPK signaling in corneal epithelial cells. Invest Ophthalmol Vis Sci, 2006, 47: 5267~5275.
19 Yang GH, Jarvis BB, Chung YJ, et a1. Apoptosis induction by the satratoxins and other trichothecene mycotoxins: relationship to ERK, p38 MAPK, and SAPK/JNK activation. Toxicol Appl Pharmacol, 2000, 164: 149~160.
20 Fecher LA, Amaravadi RK, Flaherty KT. The MAPK pathway in melanoma. Curr Op in Oncol, 2008, 20: 183~189.
21 Lawrence MC, J ivan A, Shao C, et al. The roles of MAPKs in disease. Cell Res, 2008, 18: 436~442.
22 Zhu X, Perry G, Smith MA. Amyotrophic lateral sclerosis: a novel hypothesis involving a gained‘loss of function’in the JNK/SAPK pathway. Redox Rep, 2003, 8: 129~133.
23王玮,吴莹瑶,卢岩,等.野黄芩苷抗炎作用的实验研究.中国医科大学学报, 2003, 32: 503~506.
24袁榴娣,徐红,杜肇宗.黄芩苷对艾氏腹水瘤细胞影响的初步探讨.南京铁道医学院学报, 1997, 16: 231~235.
25 Wu JA, Attele AS, Zhang L, et al. Anti - H IV activity of medicinal herbs: usage and potential development. An J Chin Med, 2001, 29: 69~73.
26熊英,傅颖媛,况南珍,等.黄芩苷抗白色念珠菌作用及机制研究.中国药理学通报, 2004, 20: 1404~1409.
27周智兴,傅颖媛.黄芩苷对白色念珠菌酶活性的影响.时珍国医国药, 2007, 18: 2639~2342.
28 Zhang DY, Wu J, Ye F, et al. Inhibition of cancer cell proliferation and prostaglandin E2 synthesis by Scutellaria baicalensis. Cancer Res. 2003, 63: 4037~4043.
29 Tischer E, Mitchell R, Hartman T, et al. The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing. J Biol Chem, 1991, 266: 11947~11952.
30顾正勤,孙颖浩,许传亮.黄芩苷诱导前列腺癌细胞株DU145凋亡的体外研究.中国中药杂志, 2005, 30:63~66.
31冯正权,沈敏鹤,吴良村.黄芩苷体外调控乳腺癌细胞表达TIMP2的实验研究.中国临床药理学与治疗学, 2005, 10: 705~708.
32宋健辉,孙吉平,贾延劼.黄芩苷对胰岛细胞瘤细胞系增殖的影响.中南大学学报, 2005, 30: 145~148.
33 Britton RS, Bacon BR. Intracellular signaling pathways in stellate cell activation. Alcohol Clin Exp Res 1999, 23: 922~925.
34 Pinzani M. Pdgf and signal transduction in hepatic stellate cells. Front Biosci 2002, 7: D1720~1726.
1 Ross R. The pathogenesis of atherosclerosis: A perspective for the1990s. Nature, 1993, 362: 801.
2 Veno H, Kanellakis P, Agrotis A, et al. Blood flow regulates the development of vascular hypertrophy, smooth muscle cell proliferation, and endothelial cell nitric oxide synthesis in hypertension. Hypertension, 2000, 36: 89~96.
3 Alison M, Devlin, James S, et al. DNA synthesis andapoptosis in smooth muscle cell from a model of genetic hypertension. Hypertension, 2000, 36: 110~115.
4 Rizzoni D, Porteri E, Guefi D, et al. Cellular hypertrophy in subcutaneous small arteries of patients with renovascular hypertension. Hypertension, 2000, 35: 931.
5 Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev. 2004, 84: 767~801.
6 Kong AN, Yu R, Chen C, et a1. Signal transduction events elicited by natural products: role of MAPK an caspase pathways in homeostatic response and induction of apoptosis Arch Pharm Res, 2000, 23: 1~l6.
7 Jo M, Stolz DB, Esplen JE, et a1. Cross-talk between epidermal growth factor receptor C-Met signal pathways in transformed cells. J Biol Chem, 2000, 275: 8866~8881.
8 Eguchi S. Intracellular signaling of angiotensinⅡ-induced p70 S6 kinase phosphorylation at Ser (411) in vascular smooth muscle cells. Possible requirement of epidermal growth factor receptor, Ras, extracellular signal-regulated kinase, and Akt. J Biol Chem, 1999, 274: 36 843~36 851.
9 Liao DF, Duff JL, Daum G, et al. AngiotensionⅡstimulates MAP kinase kinase kinase activity in vascular smooth muscle cell. Role of Raf. Circ Res, 1996, 79: 1007~1 014.
10 Liao DF, Monia B, Dean N, et al. Protein kinase C-ζmediates angiotensinⅡactivation of ERK in vascular smooth muscle cells. J Biol Chem, 1997, 272: 6 146~6 150.
11廖端芳, Berk BC,关永源. P85磷酯肌醇-3激酶/蛋白激酶C-ζ复合物对血管紧张素Ⅱ激活平滑肌细胞p70核蛋白体S60激酶的调节作用.中国动脉硬化杂志, 2000, 8: 193~198.
12 Iton H, Yamamura S, Ware JA, et a1. D/ferential effect of protein kinase C on human vascular smooth muscle cell proliferation and migration. Am J Physiol Heart Cite Physiol, 2001. 28l: H359~H370.
13 Hotio T, Kohno M, Kano H, et a1. AdrenomedullJn¨a novel anti-migration factor of vascular smooth cells. Circ Res, 1995, 77: 660~664.
14 Ali S, Becker MW, Davis MG, et a1. Dissociation of vasoconstrictor-stimulated basic fibroblast growth factor expression from hypertrophic growth in cultured vascular smooth muscle cells. Relevant role of protein kinase c. Circ Res, 1994, 75: 836~843.
15 Marrero MB. Direct stimulation of jak/STAT pathway by the angiotensinⅡAT1 receptor. Nature, 1995, 375: 247~250.
16 Seki Y, Kai H, Shibata R, et al. Role of the JAK/STAT pathway in rat carotid artery remodeling of vascular injury.Circ Res, 2000, 87: 12~18.
17 Rutherford C, Martin W, Salame M, et al. Substantial inhibition of neointimal response to balloon injury in the rat carotid artery using a combinant of antibody to platelet-derived growth factor-BB and basic fibroblast growth factor. Atherosclerosis, 1997, 130: 45~51.
18 Fox JC ,Shanley JR. Antisense inhibition of basic fibroblast growth factor induces apoptosis in vascular smooth muscle cells. J Bib Chem, 1996, 271: 12 578~12 584.
19 Hoch RV, Soriano P. Roles of PDGF in animal development. Development, 2003, 130: 4679~4684.
20 Boucher P, Gotthardt M, Li W P, et al. LRP: role in vascular wall integrity and protection from atherosclerosis. Science, 2003, 300: 329~332.
21 Tallquist M, Kazaluaskas A. PDGF signaling in cells and mice. Cytokine Growth Factor Rev, 2004, 15: 205~213.
22 J echlinger M, Sommer A, Moriggl R et al. Autocrine PDGFR signaling promotes mammary cancer metastasis. J Clin Invest, 2006, 116: 1561.
23 kurland ES, Rosen CJ, Cosman F, et al. Insulin-like growth factor I in men with idiopathic osteporosis. J Clin Endocrinol Metab, 1997, 82: 2799~2805.
24 Coverley JA, Baxter RC. Phosphorylation of IGFBPs. Mol cell Endorinol, 1997, 128: 1~5.
25 Hayry P, Myllarniem M, Aavik E, et al. Stabile D-peptideanalog of insulin-like growth factor-1 inhibits smooth muscle cell proliferation after carotid ballooning injury in rat. FASEB J, 1995, 9: 1336~1344.
26 Massague J. Receptors for the TGF-beta family. Cell. 1992 Jun 26; 69: 1067~1070.
27 Berk BC. Vascular Smooth muscle growth: autocrine growth mechanisms. Physiol Rev, 2001, 81: 999~1030.
28 Nakano N, Morishiea R, Moriguchi A, et al. Negative regulation of local hepatocyte growth factor expression by angiotensinⅡand transforming growth factor-beta in blood vessels: potential role of HGF in cardiovascular disease. Hypertension, 1998, 32: 444~451.
29 Itoh H, Mukoyama M, Pratt RE, et al. Multiple autocrine growth factor modulate vascular smooth muscle cell growth response to angiotensinⅡ. J Clin Invest, 1993, 91: 2268~2274.
30 Fukuda N. Suppression of the exaggerated growth of vascular smooth muscle cells from SHR by antisense oligodeoxynucleotide to TGF beta. Nippon Rinsho, 1993, 51: 1663~1667.
31 Mouszawa K, Kikuchi S, Fukuyama J, et al. Inhibition of PDGF-βand TGF-βinduced collagen synthesis, migration and proliferation by tranilast in vascular smooth muscle cells from spontaneously hypertensive rats. Atherosclerosis, 1995, 118: 213~221.
2 Nylande M, Kroess A, Stranden E, et al. Marker of vascular inflammation are association with the extent of atherosclerosis assessed as angiographic score and treadmill walking distances in patients with peripheral arterial occlusive disease. Vasc Medi, 2006, 11: 21~28.
3 Li J, Zhu X, Chen M, et al. Myocardin-related transcription factor B is required in cardiac neural crest for smooth muscle differentiation and cardiovascular development. Proc Natl Acad Sci USA, 2005, 102: 8916~8921.
4 Vindis C, Escargueil-Blanc I, Elbaz MD, et al. Esensitization of platelet-derived growth factor receptor-beta by oxidized lipids in vascular cells and atherosclerotic lesions: prevention by aldehyde scavengers. Circ Res, 2006, 98: 785~792.
5 Boucher P, Gotthardt M, Li W P, et al. LRP: role in vascular wall integrity and protection from atherosclerosis. Science, 2003, 300: 329~332.
6 Okano J, Nagahara T, Matsumoto K, Murawaki Y.Caffeine inhibits the proliferation of liver cancer cells and activates the MEK/ERK/EGFR signalling pathway. Basic Clin Pharmacol Toxicol, 2008, 102: 543~551.
7 Nakata S, Fujita N, Kitagawa Y, et al. Regulation of platelet-derived growth factor receptor activation by afadin through SHP-2: implications for cellular morphology. J Biol Chem, 2007, 282: 37815~37825.
8 Geisterfer AA, Peach MJ, Owens GK. Angiotensin II induces hypertrophy, not hyperplasia, of cultured rat aortic smooth muscle cells. Circ Res, 1988, 62: 749~758.
9 Wang N, Tang LJ, Zhu GQ, et al. Apoptosis induced by baicalin involving up-regulation of P53 and bax in MCF-7 cells. J Asian Nat Prod Res. 2008, 10: 1129~1135.
10 Chou TC, Chang LP, Li CY, et al. The anti-inflammatory and analgesic effects of Baicalin in carrageenan-evoked thermal hyperalgesia. Anesth. Analg. Dec 2003, 97: 1724~1729.
11 Ramos K, Cox LR. Primary cultures of rat aortic endothelial and smooth muscle cells. An in vitro model to study xenobiotic-induced vascular cytotoxicity. In Vitro Cell Dev Biol, 1987, 23: 288~296.
12 Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury, I: smooth muscle growth in the absence of endothelium. Lab Invest, 1983, 49: 327 ~333.
13 Capron L, Jarnet J, Heudes D, et al. Repeated ballooninjury of rat aorta. A model of neointima with attenuated inhibition by heparin. Arterioscler Thromb Vasc Biol, 1997, 17: 1649~1656.
14 Chen KH, Guo X, Ma D, et al. Dysregulation of HSG triggers vascular proliferative disorders, Nat Cell Biol, 2004, 6: 872~883.
15 Liu B, Han M, Wen JK. Acetylbritannilactone inhibits neointimal hyperplasia after balloon injury of rat artery by suppressing nuclear factor-{kappa}B activation. J Pharmacol Exp Ther. 2008, 324: 292~298.
16 Hong M, Mehran R, Mintz G, et al. Restenosis after coronary Angioplasty. Curr Probl cardiol, 1997, 22 : 7.
17 Bauriedel G, Kandoll, Welsch U, et al. Mechanisms of restenosis after angioplasty. Z Kardiol, 1994, 83 (Suppl 4): 31~41.
18 Wang Z, Yang H, Tachado SD, et a1. Phosphatase -mediated crosstalk control of ERK and p38 MAPK signaling in corneal epithelial cells. Invest Ophthalmol Vis Sci, 2006, 47: 5267~5275.
19 Yang GH, Jarvis BB, Chung YJ, et a1. Apoptosis induction by the satratoxins and other trichothecene mycotoxins: relationship to ERK, p38 MAPK, and SAPK/JNK activation. Toxicol Appl Pharmacol, 2000, 164: 149~160.
20 Fecher LA, Amaravadi RK, Flaherty KT. The MAPK pathway in melanoma. Curr Op in Oncol, 2008, 20: 183~189.
21 Lawrence MC, J ivan A, Shao C, et al. The roles of MAPKs in disease. Cell Res, 2008, 18: 436~442.
22 Zhu X, Perry G, Smith MA. Amyotrophic lateral sclerosis: a novel hypothesis involving a gained‘loss of function’in the JNK/SAPK pathway. Redox Rep, 2003, 8: 129~133.
23王玮,吴莹瑶,卢岩,等.野黄芩苷抗炎作用的实验研究.中国医科大学学报, 2003, 32: 503~506.
24袁榴娣,徐红,杜肇宗.黄芩苷对艾氏腹水瘤细胞影响的初步探讨.南京铁道医学院学报, 1997, 16: 231~235.
25 Wu JA, Attele AS, Zhang L, et al. Anti - H IV activity of medicinal herbs: usage and potential development. An J Chin Med, 2001, 29: 69~73.
26熊英,傅颖媛,况南珍,等.黄芩苷抗白色念珠菌作用及机制研究.中国药理学通报, 2004, 20: 1404~1409.
27周智兴,傅颖媛.黄芩苷对白色念珠菌酶活性的影响.时珍国医国药, 2007, 18: 2639~2342.
28 Zhang DY, Wu J, Ye F, et al. Inhibition of cancer cell proliferation and prostaglandin E2 synthesis by Scutellaria baicalensis. Cancer Res. 2003, 63: 4037~4043.
29 Tischer E, Mitchell R, Hartman T, et al. The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing. J Biol Chem, 1991, 266: 11947~11952.
30顾正勤,孙颖浩,许传亮.黄芩苷诱导前列腺癌细胞株DU145凋亡的体外研究.中国中药杂志, 2005, 30:63~66.
31冯正权,沈敏鹤,吴良村.黄芩苷体外调控乳腺癌细胞表达TIMP2的实验研究.中国临床药理学与治疗学, 2005, 10: 705~708.
32宋健辉,孙吉平,贾延劼.黄芩苷对胰岛细胞瘤细胞系增殖的影响.中南大学学报, 2005, 30: 145~148.
33 Britton RS, Bacon BR. Intracellular signaling pathways in stellate cell activation. Alcohol Clin Exp Res 1999, 23: 922~925.
34 Pinzani M. Pdgf and signal transduction in hepatic stellate cells. Front Biosci 2002, 7: D1720~1726.
1 Ross R. The pathogenesis of atherosclerosis: A perspective for the1990s. Nature, 1993, 362: 801.
2 Veno H, Kanellakis P, Agrotis A, et al. Blood flow regulates the development of vascular hypertrophy, smooth muscle cell proliferation, and endothelial cell nitric oxide synthesis in hypertension. Hypertension, 2000, 36: 89~96.
3 Alison M, Devlin, James S, et al. DNA synthesis andapoptosis in smooth muscle cell from a model of genetic hypertension. Hypertension, 2000, 36: 110~115.
4 Rizzoni D, Porteri E, Guefi D, et al. Cellular hypertrophy in subcutaneous small arteries of patients with renovascular hypertension. Hypertension, 2000, 35: 931.
5 Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev. 2004, 84: 767~801.
6 Kong AN, Yu R, Chen C, et a1. Signal transduction events elicited by natural products: role of MAPK an caspase pathways in homeostatic response and induction of apoptosis Arch Pharm Res, 2000, 23: 1~l6.
7 Jo M, Stolz DB, Esplen JE, et a1. Cross-talk between epidermal growth factor receptor C-Met signal pathways in transformed cells. J Biol Chem, 2000, 275: 8866~8881.
8 Eguchi S. Intracellular signaling of angiotensinⅡ-induced p70 S6 kinase phosphorylation at Ser (411) in vascular smooth muscle cells. Possible requirement of epidermal growth factor receptor, Ras, extracellular signal-regulated kinase, and Akt. J Biol Chem, 1999, 274: 36 843~36 851.
9 Liao DF, Duff JL, Daum G, et al. AngiotensionⅡstimulates MAP kinase kinase kinase activity in vascular smooth muscle cell. Role of Raf. Circ Res, 1996, 79: 1007~1 014.
10 Liao DF, Monia B, Dean N, et al. Protein kinase C-ζmediates angiotensinⅡactivation of ERK in vascular smooth muscle cells. J Biol Chem, 1997, 272: 6 146~6 150.
11廖端芳, Berk BC,关永源. P85磷酯肌醇-3激酶/蛋白激酶C-ζ复合物对血管紧张素Ⅱ激活平滑肌细胞p70核蛋白体S60激酶的调节作用.中国动脉硬化杂志, 2000, 8: 193~198.
12 Iton H, Yamamura S, Ware JA, et a1. D/ferential effect of protein kinase C on human vascular smooth muscle cell proliferation and migration. Am J Physiol Heart Cite Physiol, 2001. 28l: H359~H370.
13 Hotio T, Kohno M, Kano H, et a1. AdrenomedullJn¨a novel anti-migration factor of vascular smooth cells. Circ Res, 1995, 77: 660~664.
14 Ali S, Becker MW, Davis MG, et a1. Dissociation of vasoconstrictor-stimulated basic fibroblast growth factor expression from hypertrophic growth in cultured vascular smooth muscle cells. Relevant role of protein kinase c. Circ Res, 1994, 75: 836~843.
15 Marrero MB. Direct stimulation of jak/STAT pathway by the angiotensinⅡAT1 receptor. Nature, 1995, 375: 247~250.
16 Seki Y, Kai H, Shibata R, et al. Role of the JAK/STAT pathway in rat carotid artery remodeling of vascular injury.Circ Res, 2000, 87: 12~18.
17 Rutherford C, Martin W, Salame M, et al. Substantial inhibition of neointimal response to balloon injury in the rat carotid artery using a combinant of antibody to platelet-derived growth factor-BB and basic fibroblast growth factor. Atherosclerosis, 1997, 130: 45~51.
18 Fox JC ,Shanley JR. Antisense inhibition of basic fibroblast growth factor induces apoptosis in vascular smooth muscle cells. J Bib Chem, 1996, 271: 12 578~12 584.
19 Hoch RV, Soriano P. Roles of PDGF in animal development. Development, 2003, 130: 4679~4684.
20 Boucher P, Gotthardt M, Li W P, et al. LRP: role in vascular wall integrity and protection from atherosclerosis. Science, 2003, 300: 329~332.
21 Tallquist M, Kazaluaskas A. PDGF signaling in cells and mice. Cytokine Growth Factor Rev, 2004, 15: 205~213.
22 J echlinger M, Sommer A, Moriggl R et al. Autocrine PDGFR signaling promotes mammary cancer metastasis. J Clin Invest, 2006, 116: 1561.
23 kurland ES, Rosen CJ, Cosman F, et al. Insulin-like growth factor I in men with idiopathic osteporosis. J Clin Endocrinol Metab, 1997, 82: 2799~2805.
24 Coverley JA, Baxter RC. Phosphorylation of IGFBPs. Mol cell Endorinol, 1997, 128: 1~5.
25 Hayry P, Myllarniem M, Aavik E, et al. Stabile D-peptideanalog of insulin-like growth factor-1 inhibits smooth muscle cell proliferation after carotid ballooning injury in rat. FASEB J, 1995, 9: 1336~1344.
26 Massague J. Receptors for the TGF-beta family. Cell. 1992 Jun 26; 69: 1067~1070.
27 Berk BC. Vascular Smooth muscle growth: autocrine growth mechanisms. Physiol Rev, 2001, 81: 999~1030.
28 Nakano N, Morishiea R, Moriguchi A, et al. Negative regulation of local hepatocyte growth factor expression by angiotensinⅡand transforming growth factor-beta in blood vessels: potential role of HGF in cardiovascular disease. Hypertension, 1998, 32: 444~451.
29 Itoh H, Mukoyama M, Pratt RE, et al. Multiple autocrine growth factor modulate vascular smooth muscle cell growth response to angiotensinⅡ. J Clin Invest, 1993, 91: 2268~2274.
30 Fukuda N. Suppression of the exaggerated growth of vascular smooth muscle cells from SHR by antisense oligodeoxynucleotide to TGF beta. Nippon Rinsho, 1993, 51: 1663~1667.
31 Mouszawa K, Kikuchi S, Fukuyama J, et al. Inhibition of PDGF-βand TGF-βinduced collagen synthesis, migration and proliferation by tranilast in vascular smooth muscle cells from spontaneously hypertensive rats. Atherosclerosis, 1995, 118: 213~221.