多沙唑嗪光学异构体对大鼠血管平滑肌细胞增殖与凋亡的影响及其作用机制的实验研究
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摘要
血管平滑肌细胞(vascular smooth muscle cells, VSMCs)位于血管中膜,生理条件下,VSMCs通过收缩和舒张调节血管张力。VSMCs由中膜迁移到内膜下间隙并异常增殖是动脉粥样硬化(atherosclerosis, AS)、高血压和血管再狭窄(restenosis, RS)等疾病共同的细胞病理基础之一。血管内皮细胞损伤导致多种炎症因子和生长调节因子的表达和激活紊乱,诱导VSMCs发生表型改变,刺激VSMCs从血管中膜向内膜下间隙迁移,发生过度增生和凋亡抑制,从而导致血管壁结构改变。因此,血管内皮细胞损伤、VSMCs增殖与迁移及其表型改变是目前研究新生内膜形成和再狭窄的热点。
多沙唑嗪[(±)doxazosin]属长效α1受体阻断药,临床广泛用于治疗良性前列腺增生(benign prostatic hyperplasia,BPH)并发下尿路症状(lower urinary tract symptoms,LUTS);对于BPH/LUTS合并高血压的患者,应用(±)doxazosin可获得双重效果。近年来,国内外研究表明,(±)doxazosin具有抑制主动脉VSMCs增殖的作用,该抑制作用具有浓度依赖性和α1受体非依赖性等特点。
20世纪末,手性药物的研究受到重视。国内外学者利用HPLC技术和高效毛细管电泳技术对(±)doxazosin进行了手性分离,得到其单一光学异构体(+)doxazosin和(-)doxazosin。研究结果表明,在兔离体胸主动脉、颈动脉、肠系膜动脉等血管标本,(-)doxazosin对血管α1受体的阻断作用明显弱于(±)doxazosin和(+)doxazosin;静脉或十二指肠给予(-)doxazosin,其降低麻醉大鼠动脉血压的作用亦明显弱于(±)doxazosin和(+)doxazosin ;但是,(-)doxazosin降低麻醉大鼠和豚鼠的膀胱排尿压的作用与(±)doxazosin相同。这些研究资料说明(-)doxazosin的药理活性,在心血管系统和下尿路系统之间,具有立体结构的选择性。但是,在抑制动脉VSMCs增殖和诱导凋亡方面,(+)doxazosin与(-)doxazosin是否具有立体结构的选择性,与α1受体有无相关性等问题仍不清楚。本研究中,我们采用MTT比色法检测细胞增殖活性,采用流式细胞技术检测细胞增殖周期、凋亡率及凋亡蛋白(Bcl-2和Bax),探讨(-)doxazosin、(+)doxazosin和(±)doxazosin对体外培养大鼠主动脉VSMCs的增殖与凋亡的影响并分析其作用机制。
第一部分多沙唑嗪光学异构体对大鼠主动脉平滑肌细胞增殖的影响及与α1受体相关性的实验研究
观察(±)doxazosin及其光学异构体对大鼠VSMCs增殖的影响。在培养的大鼠主动脉VSMCs,采用MTT比色法检测细胞增殖活性,探讨(-)doxazosin、(+)doxazosin和(±)doxazosin的抗大鼠主动脉VSMCs的增殖作用以及与α1受体的相关性。
1 (±)Doxazosin及其光学异构体对VSMCs增殖的影响(-)Doxazosin和(±)doxazosin在10~30μmol·L-1浓度范围作用于VSMCs 48h或72h,显著抑制大鼠胸主动脉VSMCs增殖;而(+)doxazosin仅在30μmol·L-1具有抑制作用。当延长药物与VSMCs的作用时间至96h时,(-)doxazosin、(+)doxazosin和(±)doxazosin在3～30μmol·L-1浓度均显著抑制主动脉VSMCs增殖。不同浓度的药物作用于大鼠胸主动脉VSMCs 48h、72h及96h时,(-)doxazosin、(+)doxazosin和(±)doxazosin对VSMCs增殖的抑制作用均具有浓度依赖性。双因素方差分析结果显示,药物作用于VSMCs 48h、72h,(-)doxazosin均在浓度为10μmol·L-1时,其抑制率显著高于同浓度(+)doxazosin(P<0.05);作用96h时,(-)doxazosin的抑制率在3～30μmol·L-1浓度范围显著高于同浓度的(+)doxazosin(P<0.01)。以30μmol·L-1浓度的药物处理大鼠胸主动脉VSMCs 48h、72h和96h,(-)doxazosin对主动脉VSMCs增殖的抑制率依次为27.13%、38.05%和58.87%,(+)doxazosin的抑制率依次为24.39%、33.43%和46.10%,(±)doxazosin的抑制率依次为24.29%、36.74%和52.35%。96h时三种药物对主动脉VSMCs增殖的抑制率均显著高于48h(p<0.05和0.01)和72h(p<0.05和0.01)的抑制率。以不同浓度的药物处理大鼠胸主动脉VSMCs 96h,(-)doxazosin、(+)doxazosin和(±)doxazosin抑制VSMCs增殖达40%的浓度(IC40)分别为12.1±2.6、10.2±1.3和20.9±2.2μmol·L-1;(+)doxazosin的IC40值显著大于(±)doxazosin和(-)doxazosin(P<0.01)。
2 (±)Doxazosin及其光学异构体的非α1受体依赖性抗VSMCs增殖作用
与溶媒Ⅰ组中的对照组相比,溶媒Ⅱ组中的对照组(0.001%乙醇自身)以及酚苄明组中的对照组(酚苄明自身)不影响大鼠VSMCs增殖(P>0.05)。在溶媒Ⅰ组中,30μmol·L-1的(-)doxazosin、(+)doxazosin和(±)doxazosin作用于VSMCs 48h,均显著抑制大鼠主动脉VSMCs增殖(P<0.01);其抑制率分别为33.00±2.79%、26.19±5.74%和31.76±4.29%;(+)doxazosin的抑制率显著弱于(-)doxazosin(P<0.05),而与(±)doxazosin的抑制率无显著性差别。在溶媒Ⅱ组与酚苄明组中,(-)doxazosin、(+)doxazosin和(±)doxazosin作用于VSMCs 48h,均显著抑制大鼠主动脉VSMCs增殖(P<0.01);尽管(+)doxazosin对VSMCs增殖的抑制率在绝对值上小于其他两种药物,但是药物的抑制率在三者之间无显著性差异。与溶媒Ⅰ组中(-)doxazosin的抑制作用相比,溶媒Ⅱ组以及酚苄明组中(-)doxazosin的抑制作用无显著性差异( P>0.05 ); (+)Doxazosin和(±)doxazosin的结果同(-)doxazosin。
3 (±)Doxazosin及光学异构体α1受体依赖性抗VSMCs增殖作用
苯肾上腺素(10μmol·L-1)可刺激大鼠主动脉VSMCs增殖,其促增殖作用的强度与含10%小牛血清的DMEM培养液相近。30μmol·L-1的(-)doxazosin、(+)doxazosin和(±)doxazosin作用于VSMCs 48 h,均显著抑制苯肾上腺素刺激的VSMCs增殖(P<0.01);(+)doxazosin的抑制率显著弱于同浓度(-)doxazosin(P<0.05),而与(±)doxazosin组相比无显著性差异。
第二部分多沙唑嗪光学异构体对大鼠主动脉平滑肌细胞增殖周期和凋亡的影响
本研究观察(±)doxazosin及其光学异构体对体外培养的大鼠胸主动脉VSMCs增殖周期和细胞凋亡的影响。应用Giemsa染色观察细胞形态学改变,采用FCM测定细胞周期、细胞凋亡率和凋亡蛋白(Bcl-2和Bax),探讨(±)doxazosin及其光学异构体抗VSMCs增殖的机制。
1 (±)Doxazosin及其光学异构体对VSMCs的形态学影响经30μmol·L-1 (-)doxazosin、(+)doxazosin和(±)doxazosin处理96h后,在普通光学显微镜下的VSMCs,可见细胞体积缩小、染色质浓缩、靠近核膜和核边集现象、细胞核固缩,在细胞质内可见大小不等的颗粒状小体,即为凋亡小体;正常活细胞细胞核染成蓝色或蓝紫色,色泽均一。
2 (±)Doxazosin及其光学异构体对VSMCs细胞增殖周期的影响
VSMCs细胞经25μmol·L-1 (-)doxazosin、(+)doxazosin和(±)doxazosin处理72h后,增殖周期时相分布和增殖指数发生了改变。(±)Doxazosin和(-)doxazosin组G0/G1期细胞均显著增多(P<0.01),S期细胞、G2/M期细胞和增殖指数均显著减少或降低(P<0.01),而(+)doxazosin组仅见S期细胞显著减少(P<0.01)。与(-)doxazosin组比较,(+)doxazosin组G0/G1期细胞显著减少(P<0.01),S期细胞、G2/M期细胞和增殖指数均显著增多或增高(P<0.05和0.01)。
3 (±)Doxazosin及其光学异构体对VSMCs凋亡率的影响
VSMCs经25μmol·L-1 (-)doxazosin、(+)doxazosin和(±)doxazosin处理72h后,(-)doxazosin和(±)doxazosin均可诱导VSMCs凋亡(P<0.01),而(+)doxazosin不具有促进VSMCs凋亡的作用(P>0.05)。(-)Doxazosin和(±)doxazosin诱导VSMCs的凋亡率分别为6.06±0.31%和3.63±0.99%,(-)doxazosin诱导大鼠主动脉VSMCs凋亡的作用显著强于(±)doxazosin(P<0.01)。
4 (±)Doxazosin及其光学异构体对VSMCs中Bcl-2蛋白和Bax蛋白表达的影响
VSMCs经25μmol·L-1的(-)doxazosin、(+)doxazosin和(±)doxazosin处理72h后,实验组细胞Bcl-2蛋白和Bax蛋白含量与溶媒组比较,均无显著性差异(P>0.05)。
VSMCs经(-)doxazosin、(+)doxazosin和(±)doxazosin处理72h后,Bcl-2/Bax蛋白比值分别为0.68±0.01、0.92±0.03和0.82±0.10。与溶媒组(0.99±0.06)相比较,(-)doxazosin和(±)doxazosin均显著降低Bcl-2/Bax的蛋白比值(P<0.05和0.01);而(+)doxazosin不影响VSMCs的Bcl-2/Bax蛋白比值(P>0.05)。(-)Doxazosin组细胞的Bcl-2/Bax蛋白比值亦显著小于(+)doxazosin组(P<0.01)。
第三部分多沙唑嗪光学异构体对多种促增殖因子诱导大鼠主动脉平滑肌细胞增殖的影响
本研究采用MTT比色法测定细胞增殖活性,观察了(±)doxazosin及其光学异构体对血小板源生长因子(PDGF-BB)、血管紧张素II (Ang II)、凝血酶和髙糖诱导的大鼠主动脉VSMCs增殖的影响。
1 (±)Doxazosin及其光学异构体对PDGF-BB诱发VSMCs增殖的影响
PDGF-BB(1nmol·L-1)可刺激大鼠主动脉VSMCs增殖,其促增殖作用的强度显著强于含10%小牛血清的DMEM培养液(P<0.01)。与溶媒组相比,(-)doxazosin、(+)doxazosin和(±)doxazosin(3～30μmol·L-1)作用于VSMCs 48h,均显著抑制PDGF-BB刺激的大鼠主动脉VSMCs增殖(P<0.01)。在抑制率方面,在含10%小牛血清的DMEM培养液中,(±)doxazosin及其光学异构体作用于VSMCs 48h时的最大抑制率为24.29±3.72%～27.13±2.41%;而在含0.4%小牛血清及PDGF-BB的DMEM培养液中,(±)doxazosin及其光学异构体作用于VSMCs 48h时的最大抑制率为95.21±6.05%～99.67±1.99%。双因素方差分析结果显示,在3μmol·L-1浓度时,(+)doxazosin的抑制率显著低于(±)doxazosin(P<0.05)。(-)Doxazosin、(+)doxazosin和(±)doxazosin抑制VSMCs增殖达50%的浓度(IC50)分别为5.44±3.16、7.50±3.40和4.98±4.52μmol·L-1。
2 (±)Doxazosin及其光学异构体对Ang II诱发VSMCs增殖的影响
Ang II(100nmol·L-1)可刺激大鼠主动脉VSMCs增殖,其促增殖作用的强度显著强于含10%小牛血清的DMEM培养液(P<0.01)。与溶媒组相比,(-)doxazosin、(+)doxazosin和(±)doxazosin在3～30μmol·L-1浓度作用于VSMCs 48h,均显著抑制Ang II刺激的大鼠主动脉VSMCs增殖(P<0.01)。在抑制率方面,(-)doxazosin、(+)doxazosin和(±)doxazosin在0.3～30μmol·L-1浓度范围内随着浓度的升高而抑制作用明显增强。双因素方差分析结果显示,(-)doxazosin的抑制率与(+)doxazosin和(±)doxazosin相比,无显著性差异(P>0.05)。在含0.4%小牛血清及Ang II的DMEM培养液中(±)doxazosin及其光学异构体作用于VSMCs 48h时的最大抑制率为93.01±3.23%～94.61±3.11%。(-)Doxazosin、(+)doxazosin和(±)doxazosin抑制VSMCs增殖的IC50值分别为13.38±2.50、14.24±2.47和13.71±2.89μmol·L-1。
3 (±)Doxazosin及其光学异构体对凝血酶诱发VSMCs增殖的影响
凝血酶(1U·ml-1)亦可刺激大鼠主动脉VSMCs增殖,其促增殖作用的强度与含10%小牛血清的DMEM培养液相近。与溶媒组相比,(-)doxazosin、(+)doxazosin和(±)doxazosin在3～30μmol·L-1浓度作用于VSMCs 48h,均显著抑制凝血酶刺激的大鼠主动脉VSMCs增殖(P<0.01)。在抑制率方面,(-)doxazosin、(+)doxazosin和(±)doxazosin在0.3～30μmol·L-1浓度范围内随着浓度的升高而抑制作用明显增强。双因素方差分析结果显示,(-)doxazosin的抑制率与(+)doxazosin和(±)doxazosin相比,无显著性差异(P>0.05)。在含0.4%小牛血清及凝血酶的DMEM培养液中,(±)doxazosin及其光学异构体作用于VSMCs 48h时的最大抑制率为91.87±5.13%～94.63±3.33%。(-)Doxazosin、(+)doxazosin和(±)doxazosin抑制VSMCs增殖的IC50值分别为13.52±2.87、14.60±2.95和14.32±2.75μmol·L-1。
4 (±)Doxazosin及其光学异构体对髙糖诱发VSMCs增殖的影响
高糖(25.6mmol·L-1)可刺激大鼠主动脉VSMCs增殖,其促增殖作用的强度与凝血酶相同。与溶媒组相比,(-)doxazosin、(+)doxazosin和(±)doxazosin在3～30μmol·L-1浓度作用于VSMCs 48h,均显著抑制髙糖刺激的大鼠主动脉VSMCs增殖(P<0.01)。双因素方差分析结果显示,(-)doxazosin在浓度为3μmol·L-1时的抑制率显著高于同浓度(+)doxazosin(P<0.05)。在含0.4%小牛血清及高糖的DMEM培养液中,(±)doxazosin及其光学异构体作用于VSMCs 48h时的最大抑制率为94.17±4.75%～96.16±3.09%。(-)Doxazosin、(+)doxazosin和(±)doxazosin抑制VSMCs增殖的IC50值分别为14.20±6.34、14.10±3.29和13.98±3.53μmol·L-1。
结论
1 (-)Doxazosin、(+)doxazosin和(±)doxazosin均可抑制10%小牛血清DMEM液中大鼠胸主动脉VSMCs的增殖反应;其中,(-)doxazosin的抑制作用明显强于(+)doxazosin,提示doxazosin在该培养条件下的抗VSMCs增殖作用具有手性药理学差异。
2除α1受体非依赖性抗VSMCs增殖作用以外,(-)doxazosin、(+)doxazosin和(±)doxazosin也具有α1受体依赖性抗VSMCs增殖作用。
3 (-)Doxazosin和(±)doxazosin抗大鼠胸主动脉VSMCs增殖的作用与其抑制G0/G1期细胞向S期转化以及降低细胞增殖指数有关;(+)doxazosin的对细胞周期的抑制作用显著弱于(-)doxazosin。
4 (-)Doxazosin和(±)doxazosin具有诱导大鼠胸主动脉VSMCs凋亡的作用,该作用与其调控Bcl-2蛋白/Bax蛋白比值有关;提示(±)doxazosin诱导VSMCs凋亡的物质基础是其光学异构体(-)doxazosin而非(+)doxazosin。
5 (±)Doxazosin及其光学异构体对PDGF-BB、Ang II、凝血酶和高糖诱发大鼠主动脉VSMCs增殖反应具有显著的抑制作用。与小牛血清和苯肾上腺素诱发的VSMCs增殖反应相比,(±)doxazosin及其光学异构体对前四种促增殖剂的抑制作用更强,且未见明显的手性药理学差异。
Vascular smooth muscle cells (VSMCs) located in the middle layer of the arterial wall regulate blood pressure by contraction and relaxation in physiological condition. Migration from the media to the intima space and abnormal proliferation of VSMCs are the common pathological bases of atherosclerosis (AS), hypertension and vascular restenosis (RS). Injury of vascular endothelial cells causes production and activation of a variety of inflammatory cytokines and growth regulation factors, which induce a change in VSMCs phenotype, a migration of VSMCs from the media to the intima, and excessive proliferation as well as apoptosis inhibition. Therefore, protecting vascular endothelial cells and inhibiting proliferation and migration of VSMCs might prevent the diseases such as AS, RS and hypertension.
(±)Doxazosin, a long-acting selectiveα1-adrenergic receptor antagonist, has been extensively used in the treatment of benign prostatic hyperplasia (BPH) complicated with lower urinary tract symptoms (LUTS). BPH/LUTS patients with hypertension treated with (±)doxazosin received two beneficial therapeutic actions. Recent studies demonstrated that (±)doxazosin markedly inhibited proliferation of the aortic VSMCs, and the inhibitory effects had characteristics of concentration-dependent andα1-receptor independent.
In the late 20th century, the study of chiral drug became an important field of the drug development. It was reported that (-)doxazosin and (+)doxazosin were prepared using chiral mobile phase HPLC and high performance capillary electrophoresis. The blocking effect of (-)doxazosin onα1-adrenoceptor was significantly weaker than that of (±)doxazosin and (+)doxazosin in the isolated rabbit thoracic aorta, carotid artery and mesenteric artery. The effect of (-)doxazosin on arterial blood pressure in the anesthetized rats was also significantly weaker than that of (±)doxazosin and (+)doxazosin administered intravenously or intraduodenally. However, the effect of decreasing urinary bladder pressure by (-)doxazosin in the anesthetized rats and guinea-pig was the same as that of (±)doxazosin. These findings suggested that the pharmacological activity of (-)doxazosin had chiral selective effect between the cardiovascular system and lower urinary tract system. However, it remains to be clarified whether (+)doxazosin and (-)doxazosin have chiral selective effect on the cell proliferation and apoptosis of VSMCs and whether their effects are related toα1-adrenoceptor blockade. In the present study, MTT assay was used to determine the cell proliferation; and cell cycle distribution, cell apoptosis and apoptotic proteins (Bcl-2 and Bax) were analyzed using flow cytometry (FCM). The purpose of the study is to observe the effects of (±)doxazosin and its enantiomers on the cell proliferation and apoptosis in cultured VSMCs of the rat aorta and to analyze the related mechanisms.
Part 1 Effects of doxazosin and its enantiomers on cell proliferation in VSMCs of the rat aorta and a relationship of the effects withα1-adrenoceptor
VSMCs of the rat aorta were cultured, and MTT assay was used to determine the cell proliferation. The effects of (-)doxazosin, (+)doxazosin and (±)doxazosin on the cell proliferation of VSMCs and a relationship of the effects induced by (-)doxazosin and (+)doxazosin withα1-adrenoceptor were investigated.
1 Effects of (±)doxazosin and its enantiomers on cell proliferation in VSMCs of the rat aorta
Treatment with (-)doxazosin and (±)doxazosin at 10~30μmol·L-1 for 48h or 72h significantly inhibited cell proliferation in VSMCs of the rat aorta, but (+)doxazosin had an inhibitory effect only at 30μmol·L-1. When the VSMCs were incubated with the three agents for 96h, (-)doxazosin, (+) doxazosin and (±)doxazosin at 3~30μmol·L-1 inhibited the cell proliferation significantly. Treatments with (-)doxazosin, (+)doxazosin and (±)doxazosin at used concentrations for 48h, 72h or 96h inhibited cell proliferation of the VSMCs in a concentration-dependent manner. Statistic results with two-way ANOVA showed that the inhibition rate of cell proliferation by (-)doxazosin at 10μmol·L-1 was significantly higher than that by (+)doxazosin in VSMCs exposed to the drugs for 48h or 72h (P <0.05 ), and the inhibition rate of cell proliferation by (-)doxazosin at 3~30μmol·L-1 was significantly higher than that by (+)doxazosin in VSMCs exposed to the drugs for 96h (P<0.01). when the VSMCs were treated with the three agents at 30μmol·L-1 for 48h, 72h and 96h, the inhibition rates of cell proliferation in VSMCs were 27.13%, 38.05% and 58.87% by (-)doxazosin, 24.39%, 33.43% and 46.10% by (+)doxazosin, and 24.29%, 36.74% and 52.35% by (±)doxazosin, respectively. The inhibition rates of cell proliferation in VSMCs incubated with the three agents for 96h were significantly higher than those for 48h (p<0.05 and 0.01) and 72h (p<0.05 and 0.01). When the VSMCs were exposed to (-)doxazosin, (+)doxazosin and (±)doxazosin at different concentrations for 96h, the concentrations required for 40-percent inhibition (IC40) of the cell proliferation were 10.2±1.3μmol·L-1, 20.9±2.2μmol·L-1 and 12.1±2.6μmol·L-1, respectively. The IC40 value of (+)doxazosin was significantly higher than that of (±)doxazosin or (-)doxazosin (P<0.01).
2α1-Adrenoceptor-independent effects of (±)doxazosin and its enantiomers on cell proliferation in VSMCs of the rat aorta
The value of cell proliferation in sub-control group (0.001% alcohol) of solventⅡgroup or in sub-control group (phenoxybenzamine) of phenoxybenzamine group was not significantly different from that in sub-control group (distilled water) of solventⅠgroup (P>0.05). In solventⅠgroup, the proliferation of VSMCs was significantly inhibited after 48h-incubation with (-)doxazosin, (+)doxazosin and (±)doxazosin at 30μmol·L-1 (P<0.01), and the inhibition rates were 33.00±2.79%, 26.19±5.74% and 31.76±4.29%, respectively. The inhibition rate of (+)doxazosin was significantly lower than that of (-)doxazosin (P<0.05), but was not significantly different from that of (±)doxazosin. In solventⅡgroup and phenoxybenzamine group, the proliferation of VSMCs was significantly inhibited after 48h-incubation with (-)doxazosin, (+)doxazosin and (±)doxazosin as well (P<0.01). Although the inhibition rate of VSMCs proliferation by (+)doxazosin was lower than that by (-)doxazosin or (±)doxazosin, there was no statistical differences among the three agents. In comparison with the sub-group [(-)doxazosin] of solventⅠgroup, the inhibition of VSMCs proliferation by (-)doxazosin in solventⅡgroup and in phenoxybenzamine group was not significantly different (P>0.05); and the same results were obtained in the experiments treated with (+)doxazosin and (±)doxazosin.
3α1-Adrenoceptor-dependent effects of (±)doxazosin and its enantiomers on cell proliferation in VSMCs of the rat aorta
Phenylephrine (10μmol·L-1) was able to stimulate VSMCs proliferation of the rat thoracic aorta, and the extent of cell proliferation promoted by phenylephrine was similar to that by 10% fetal bovine serum contained in DMEM. The VSMCs proliferation stimulated by phenylephrine was significantly inhibited after 48h-incubation with (-)doxazosin, (+)doxazosin and (±)doxazosin at 30μmol·L-1 (P<0.01). The inhibition rate of VSMCs proliferation by (+)doxazosin was significantly lower than that by (-)doxazosin (P<0.05), but was to the same extent as (±)doxazosin.
Part 2 Effects of doxazosin and its enantiomers on cell cycle and apoptosis in VSMCs of the rat aorta
Effects of doxazosin and its enantiomers on cell cycle and apoptosis in cultured VSMCs of the rat thoracic aorta were investigated. Morphological changes in VSMCs were analyzed using Giemsa staining method, and the cell cycle, cell apoptosis and apoptotic proteins (Bcl-2 and Bax) were detected by FCM in order to investigate mechanisms of antiproliferation and induction of apoptosis b (±)doxazosin and its enantiomers in VSMCs of the rat thoracic aorta.
1 Morphological changes in VSMCs of the rat aorta induced by (±)doxazosin and its enantiomers
Morphological changes in cultured VSMCs of the rat thoracic aorta incubated with (-)doxazosin, (+)doxazosin and (±)doxazosin at 30μmol.L-1 for 96h were seen under an ordinary optical microscope. The overall cell shrinkage, chromatin condensation, nuclear margination toward the nuclear membrane, nuclear shrinkage, and apoptotic bodies of various sizes in the cytoplasm were observed,and the nucleus of alive normal cells uniformly stained blue or violet-blue.
2 Effects of (±)doxazosin and its enantiomers on cell cycle in VSMCs of the rat aorta
Cell cycle distribution and proliferation index (PI) of the rat aortic VSMCs changed significantly after incubation with (-)doxazosin, (+)doxazosin and (±)doxazosin at 25μmol·L-1 for 72h. In the VSMCs treated with (±)doxazosin or (-)doxazosin, the proportion of cells in G0/G1-phase was significantly increased; and the proportions of cells in S-phase and G2/M-phase, and PI were significantly decreased (P<0.01). In the (+)doxazosin group, however, only the proportion of cells in S-phase was decreased significantly (P<0.01). The proportions of cells in G0/G1-phase, S-phase and G2/M-phase, and PI value in the VSMCs treated with (-)doxazosin were significantly different from that treated with (+)doxazosin (P<0.05 and 0.01).
3 Effects of (±)doxazosin and its enantiomers on the cell apoptosis in VSMCs of the rat aorta
In the VSMCs incubated with (-)doxazosin, (+)doxazosin and (±)doxazosin at 25μmol·L-1 for 72h, the cell apoptosis was induced by (-)doxazosin and (±)doxazosin (P<0.01), but not by (+)doxazosin (P>0.05). The apoptotic rates of the cultured VSMCs treated with (-)doxazosin and (±)doxazosin were 6.06±0.31% and 3.63±0.99%, respectively. The induction of VSMCs apoptosis induced by (-)doxazosin was significantly stronger than that by (±)doxazosin (P<0.01).
4 Effects of (±)doxazosin and its enantiomers on the expression of apoptosis protein Bcl-2 and Bax in VSMCs of the rat aorta
After incubation of VSMCs with (-)doxazosin, (+)doxazosin and (±)doxazosin at 25μmol·L-1 for 72h, the expression of antiapoptotic protein Bcl-2 and proapoptotic protein Bax did not change significantly in comparison with solvent-treated VSMCs (P>0.05). On the other hand, the ratios of Bcl-2 protein/Bax protein in the cultured VSMCs treated with (-)doxazosin, (+)doxazosin and (±)doxazosin were 0.68±0.01, 0.92±0.03 and 0.82±0.10, respectively. The ratios of Bcl-2 protein/Bax protein in the cultured VSMCs were significantly reduced by (-)doxazosin and (±)doxazosin in comparison with the VSMCs treated with solvent (P<0.05 and 0.01), but (+)doxazosin did not significantly affect the ratio of Bcl-2 protein/Bax protein (P>0.05). The ratio of Bcl-2 protein/Bax protein in VSMCs treated by (-)doxazosin was significantly lesser than that by (+)doxazosin (P<0.01).
Part 3 Effects of doxazosin and its enantiomers on cell proliferation of the rat aortic VSMCs induced by different stimulators
Effects of (±)doxazosin and its enantiomers on cell proliferation determined by MTT assay in the rat aortic VSMCs stimulated by platelet-derived growth factor-BB (PDGF-BB), angiotensin II, high concentration glucose and thrombin.
1 Effects of (±)doxazosin and its enantiomers cell proliferation of the rat aortic VSMCs induced by PDGF-BB
PDGF-BB (1nmol·L-1) induced cell proliferation of the rat aortic VSMCs, and the extent of cell proliferation promoted by PDGF-BB (1nmol·L-1) was significantly stronger than that by 10% fetal bovine serum contained in DMEM (P<0.01). Incubation of the rat aortic VSMCs with (-)doxazosin, (+)doxazosin and (±)doxazosin (3～30μmol·L-1) for 48h inhibited the cell proliferation stimulated by PDGF-BB significantly (P<0.01). In the culture medium of DMEM containing 10% fetal bovine serum, the maximal inhibition rates of VSMCs proliferation by 48h-incubation with (-)doxazosin, (+)doxazosin and (±)doxazosin were from 24.29±3.72% to 27.13±2.41%, in the culture medium of DMEM containing 0.4% fetal bovine serum and 1nmol·L-1PDGF-BB, however, the maximal inhibition rates of cell proliferation by 48h-incubation with (-)doxazosin, (+)doxazosin and (±)doxazosin were from 95.21±6.05% to 99.67±1.99%. Statistic results with two-way ANOVA showed that the inhibition rate of cell proliferation by (+)doxazosin at 3μmol·L-1 was significantly lower than that by (±)doxazosin at the same concentration (P<0.05). A concentration required for 50-percent inhibition (IC50) of the VSMCs proliferation by (-)doxazosin, (+)doxazosin or (±)doxazosin was 5.44±3.16, 7.50±3.40 or 4.98±4.52μmol·L-1.
2 Effects of (±)doxazosin and its enantiomers cell proliferation of the rat aortic VSMCs induced by angiotensin II
Angiotensin II (100 nmol·L-1) induced cell proliferation of the rat aortic VSMCs, and the extent of cell proliferation promoted by angiotensin II (100 nmol·L-1) was significantly stronger than that by 10% fetal bovine serum contained in DMEM (P<0.01). Incubation of the rat aortic VSMCs with (-)doxazosin, (+)doxazosin and (±)doxazosin (3～30μmol·L-1) for 48h inhibited the cell proliferation stimulated by angiotensin II significantly (P<0.01). The inhibition rates of the VSMCs proliferation by (-)doxazosin, (+)doxazosin and (±)doxazosin were increased with the increase in concentrations used (0.3～30μmol·L-1). Statistic results with two-way ANOVA showed that the inhibition rate of VSMCs proliferation by 48h-incubation with (-)doxazosin was not significantly different from that with (+)doxazosin or (±)doxazosin (P>0.05). In the culture medium of DMEM containing 0.4% fetal bovine serum and 100 nmol·L-1 angiotensin II, the maximal inhibition rates of cell proliferation by 48h-incubation with (-)doxazosin, (+)doxazosin and (±)doxazosin were from 93.01±3.23% to 94.61±3.11%. A concentration required for 50-percent inhibition (IC50) of the VSMCs proliferation by (-)doxazosin, (+)doxazosin or (±)doxazosin was 13.38±2.50, 14.24±2.47 or 13.71±2.89μmol·L-1.
3 Effects of (±)doxazosin and its enantiomers cell proliferation of the rat aortic VSMCs induced by thrombin
Thrombin (1μmol·L-1) induced cell proliferation of the rat aortic VSMCs, and the extent of cell proliferation promoted by thrombin (1μmol·L-1) was similar to that by 10% fetal bovine serum contained in DMEM. Incubation of the rat aortic VSMCs with (-)doxazosin, (+)doxazosin and (±)doxazosin (3～30μmol·L-1) for 48h inhibited the cell proliferation stimulated by angiotensin II significantly (P<0.01). The inhibition rates of the VSMCs proliferation by (-)doxazosin, (+)doxazosin and (±)doxazosin were increased with the increase in concentrations used (0.3～30μmol·L-1). Statistic results with two-way ANOVA showed that the inhibition rate of VSMCs proliferation by 48h-incubation with (-)doxazosin was not significantly different from that with (+)doxazosin or (±)doxazosin (P>0.05). In the culture medium of DMEM containing 0.4% fetal bovine serum and 1μmol·L-1 thrombin, the maximal inhibition rates of cell proliferation by 48h-incubation with (-)doxazosin, (+)doxazosin and (±)doxazosin were from from 91.87±5.13% to 94.63±3.33%. A concentration required for 50-percent inhibition (IC50) of the VSMCs proliferation by (-)doxazosin, (+)doxazosin or (±)doxazosin was 13.52±2.87, 14.60±2.95 or 14.32±2.75μmol·L-1.
4 Effects of (±)doxazosin and its enantiomers cell proliferation of the rat aortic VSMCs induced by a high concentration of glucose
A high concentration of glucose (25.6mmol·L-1) induced cell proliferation of the rat aortic VSMCs, and the extent of cell proliferation promoted by the high concentration of glucose (25.6mmol·L-1) was similar to that by 1μmol·L-1 thrombin contained in DMEM. Incubation of the rat aortic VSMCs with (-)doxazosin, (+)doxazosin and (±)doxazosin (3～30μmol·L-1) for 48h inhibited the cell proliferation stimulated by the high concentration of glucose significantly (P<0.01). Statistic results with two-way ANOVA showed that the inhibition rate of VSMCs proliferation by 48h-incubation with 3μmol·L-1 (-)doxazosin was significantly higher than that with (+)doxazosin at the same concentration (P<0.05). In the culture medium of DMEM containing 0.4% fetal bovine serum and 25.6mmol·L-1 glucose, the maximal inhibition rates of cell proliferation by 48h-incubation with (-)doxazosin, (+)doxazosin and (±)doxazosin were from 94.17±4.75% to 96.16±3.09%. A concentration required for 50-percent inhibition (IC50) of the VSMCs proliferation by (-)doxazosin, (+)doxazosin or (±)doxazosin was 14.20±6.34, 14.10±3.29 or 13.98±3.53μmol·L-1.
Conclusion
1 (-)Doxazosin, (+)doxazosin and (±)doxazosin inhibit the rat aortic VSMCs proliferation induced by 10% fetal bovine serum, and the extent of inhibition of cell proliferation by (-)doxazosin is stronger than that by (+)doxazosin, suggesting that (±)doxazosin and its enantiomers have chirally selective effect on cell proliferation in the used experimental condition.
2Αnα1-adrenoceptor-dependent mechanism is involved in the anti-proliferation of the rat aortic VSMCs by (±)doxazosin and its enantiomers besides a knownα1-adrenoceptor-independent mechanism.
3 (-)Doxazosin and (±)doxazosin are able to inhibit cell proliferation by a cell-cycle arrest in G0/G1 phase and a decrease in PI. The effect on cell cycle by (-)doxazosin is significantly stronger than that by of (+)doxazosin.
4 (-)Doxazosin and (±)doxazosin are able to induce cell apoptosis in the rat aortic VSMCs, and the proapoptotic effects are partially related to a reduced ratio of Bcl-2 protein/Bax protein, indicating that (-)doxazosin might be the main component of (±)doxazosin to induce cell apoptosis in the rat aortic VSMCs.
5 (±)Doxazosin and its enantiomers significantly inhibit cell proliferation in the rat aortic VSMCs stimulated by PDGF-BB, angiotensin II, thrombin and high concentration of glucose without chirally selective effects. The inhibition extent of cell proliferation by (-)doxazosin is much stronger in the VSMCs stimulated with PDGF-BB, angiotensin II, thrombin or high concentration of glucose than that in the VSMCs stimulated with fetal bovine serum or phenylephrine.
多沙唑嗪[(±)doxazosin]属长效α1受体阻断药,临床广泛用于治疗良性前列腺增生(benign prostatic hyperplasia,BPH)并发下尿路症状(lower urinary tract symptoms,LUTS);对于BPH/LUTS合并高血压的患者,应用(±)doxazosin可获得双重效果。近年来,国内外研究表明,(±)doxazosin具有抑制主动脉VSMCs增殖的作用,该抑制作用具有浓度依赖性和α1受体非依赖性等特点。
20世纪末,手性药物的研究受到重视。国内外学者利用HPLC技术和高效毛细管电泳技术对(±)doxazosin进行了手性分离,得到其单一光学异构体(+)doxazosin和(-)doxazosin。研究结果表明,在兔离体胸主动脉、颈动脉、肠系膜动脉等血管标本,(-)doxazosin对血管α1受体的阻断作用明显弱于(±)doxazosin和(+)doxazosin;静脉或十二指肠给予(-)doxazosin,其降低麻醉大鼠动脉血压的作用亦明显弱于(±)doxazosin和(+)doxazosin ;但是,(-)doxazosin降低麻醉大鼠和豚鼠的膀胱排尿压的作用与(±)doxazosin相同。这些研究资料说明(-)doxazosin的药理活性,在心血管系统和下尿路系统之间,具有立体结构的选择性。但是,在抑制动脉VSMCs增殖和诱导凋亡方面,(+)doxazosin与(-)doxazosin是否具有立体结构的选择性,与α1受体有无相关性等问题仍不清楚。本研究中,我们采用MTT比色法检测细胞增殖活性,采用流式细胞技术检测细胞增殖周期、凋亡率及凋亡蛋白(Bcl-2和Bax),探讨(-)doxazosin、(+)doxazosin和(±)doxazosin对体外培养大鼠主动脉VSMCs的增殖与凋亡的影响并分析其作用机制。
第一部分多沙唑嗪光学异构体对大鼠主动脉平滑肌细胞增殖的影响及与α1受体相关性的实验研究
观察(±)doxazosin及其光学异构体对大鼠VSMCs增殖的影响。在培养的大鼠主动脉VSMCs,采用MTT比色法检测细胞增殖活性,探讨(-)doxazosin、(+)doxazosin和(±)doxazosin的抗大鼠主动脉VSMCs的增殖作用以及与α1受体的相关性。
1 (±)Doxazosin及其光学异构体对VSMCs增殖的影响(-)Doxazosin和(±)doxazosin在10~30μmol·L-1浓度范围作用于VSMCs 48h或72h,显著抑制大鼠胸主动脉VSMCs增殖;而(+)doxazosin仅在30μmol·L-1具有抑制作用。当延长药物与VSMCs的作用时间至96h时,(-)doxazosin、(+)doxazosin和(±)doxazosin在3～30μmol·L-1浓度均显著抑制主动脉VSMCs增殖。不同浓度的药物作用于大鼠胸主动脉VSMCs 48h、72h及96h时,(-)doxazosin、(+)doxazosin和(±)doxazosin对VSMCs增殖的抑制作用均具有浓度依赖性。双因素方差分析结果显示,药物作用于VSMCs 48h、72h,(-)doxazosin均在浓度为10μmol·L-1时,其抑制率显著高于同浓度(+)doxazosin(P<0.05);作用96h时,(-)doxazosin的抑制率在3～30μmol·L-1浓度范围显著高于同浓度的(+)doxazosin(P<0.01)。以30μmol·L-1浓度的药物处理大鼠胸主动脉VSMCs 48h、72h和96h,(-)doxazosin对主动脉VSMCs增殖的抑制率依次为27.13%、38.05%和58.87%,(+)doxazosin的抑制率依次为24.39%、33.43%和46.10%,(±)doxazosin的抑制率依次为24.29%、36.74%和52.35%。96h时三种药物对主动脉VSMCs增殖的抑制率均显著高于48h(p<0.05和0.01)和72h(p<0.05和0.01)的抑制率。以不同浓度的药物处理大鼠胸主动脉VSMCs 96h,(-)doxazosin、(+)doxazosin和(±)doxazosin抑制VSMCs增殖达40%的浓度(IC40)分别为12.1±2.6、10.2±1.3和20.9±2.2μmol·L-1;(+)doxazosin的IC40值显著大于(±)doxazosin和(-)doxazosin(P<0.01)。
2 (±)Doxazosin及其光学异构体的非α1受体依赖性抗VSMCs增殖作用
与溶媒Ⅰ组中的对照组相比,溶媒Ⅱ组中的对照组(0.001%乙醇自身)以及酚苄明组中的对照组(酚苄明自身)不影响大鼠VSMCs增殖(P>0.05)。在溶媒Ⅰ组中,30μmol·L-1的(-)doxazosin、(+)doxazosin和(±)doxazosin作用于VSMCs 48h,均显著抑制大鼠主动脉VSMCs增殖(P<0.01);其抑制率分别为33.00±2.79%、26.19±5.74%和31.76±4.29%;(+)doxazosin的抑制率显著弱于(-)doxazosin(P<0.05),而与(±)doxazosin的抑制率无显著性差别。在溶媒Ⅱ组与酚苄明组中,(-)doxazosin、(+)doxazosin和(±)doxazosin作用于VSMCs 48h,均显著抑制大鼠主动脉VSMCs增殖(P<0.01);尽管(+)doxazosin对VSMCs增殖的抑制率在绝对值上小于其他两种药物,但是药物的抑制率在三者之间无显著性差异。与溶媒Ⅰ组中(-)doxazosin的抑制作用相比,溶媒Ⅱ组以及酚苄明组中(-)doxazosin的抑制作用无显著性差异( P>0.05 ); (+)Doxazosin和(±)doxazosin的结果同(-)doxazosin。
3 (±)Doxazosin及光学异构体α1受体依赖性抗VSMCs增殖作用
苯肾上腺素(10μmol·L-1)可刺激大鼠主动脉VSMCs增殖,其促增殖作用的强度与含10%小牛血清的DMEM培养液相近。30μmol·L-1的(-)doxazosin、(+)doxazosin和(±)doxazosin作用于VSMCs 48 h,均显著抑制苯肾上腺素刺激的VSMCs增殖(P<0.01);(+)doxazosin的抑制率显著弱于同浓度(-)doxazosin(P<0.05),而与(±)doxazosin组相比无显著性差异。
第二部分多沙唑嗪光学异构体对大鼠主动脉平滑肌细胞增殖周期和凋亡的影响
本研究观察(±)doxazosin及其光学异构体对体外培养的大鼠胸主动脉VSMCs增殖周期和细胞凋亡的影响。应用Giemsa染色观察细胞形态学改变,采用FCM测定细胞周期、细胞凋亡率和凋亡蛋白(Bcl-2和Bax),探讨(±)doxazosin及其光学异构体抗VSMCs增殖的机制。
1 (±)Doxazosin及其光学异构体对VSMCs的形态学影响经30μmol·L-1 (-)doxazosin、(+)doxazosin和(±)doxazosin处理96h后,在普通光学显微镜下的VSMCs,可见细胞体积缩小、染色质浓缩、靠近核膜和核边集现象、细胞核固缩,在细胞质内可见大小不等的颗粒状小体,即为凋亡小体;正常活细胞细胞核染成蓝色或蓝紫色,色泽均一。
2 (±)Doxazosin及其光学异构体对VSMCs细胞增殖周期的影响
VSMCs细胞经25μmol·L-1 (-)doxazosin、(+)doxazosin和(±)doxazosin处理72h后,增殖周期时相分布和增殖指数发生了改变。(±)Doxazosin和(-)doxazosin组G0/G1期细胞均显著增多(P<0.01),S期细胞、G2/M期细胞和增殖指数均显著减少或降低(P<0.01),而(+)doxazosin组仅见S期细胞显著减少(P<0.01)。与(-)doxazosin组比较,(+)doxazosin组G0/G1期细胞显著减少(P<0.01),S期细胞、G2/M期细胞和增殖指数均显著增多或增高(P<0.05和0.01)。
3 (±)Doxazosin及其光学异构体对VSMCs凋亡率的影响
VSMCs经25μmol·L-1 (-)doxazosin、(+)doxazosin和(±)doxazosin处理72h后,(-)doxazosin和(±)doxazosin均可诱导VSMCs凋亡(P<0.01),而(+)doxazosin不具有促进VSMCs凋亡的作用(P>0.05)。(-)Doxazosin和(±)doxazosin诱导VSMCs的凋亡率分别为6.06±0.31%和3.63±0.99%,(-)doxazosin诱导大鼠主动脉VSMCs凋亡的作用显著强于(±)doxazosin(P<0.01)。
4 (±)Doxazosin及其光学异构体对VSMCs中Bcl-2蛋白和Bax蛋白表达的影响
VSMCs经25μmol·L-1的(-)doxazosin、(+)doxazosin和(±)doxazosin处理72h后,实验组细胞Bcl-2蛋白和Bax蛋白含量与溶媒组比较,均无显著性差异(P>0.05)。
VSMCs经(-)doxazosin、(+)doxazosin和(±)doxazosin处理72h后,Bcl-2/Bax蛋白比值分别为0.68±0.01、0.92±0.03和0.82±0.10。与溶媒组(0.99±0.06)相比较,(-)doxazosin和(±)doxazosin均显著降低Bcl-2/Bax的蛋白比值(P<0.05和0.01);而(+)doxazosin不影响VSMCs的Bcl-2/Bax蛋白比值(P>0.05)。(-)Doxazosin组细胞的Bcl-2/Bax蛋白比值亦显著小于(+)doxazosin组(P<0.01)。
第三部分多沙唑嗪光学异构体对多种促增殖因子诱导大鼠主动脉平滑肌细胞增殖的影响
本研究采用MTT比色法测定细胞增殖活性,观察了(±)doxazosin及其光学异构体对血小板源生长因子(PDGF-BB)、血管紧张素II (Ang II)、凝血酶和髙糖诱导的大鼠主动脉VSMCs增殖的影响。
1 (±)Doxazosin及其光学异构体对PDGF-BB诱发VSMCs增殖的影响
PDGF-BB(1nmol·L-1)可刺激大鼠主动脉VSMCs增殖,其促增殖作用的强度显著强于含10%小牛血清的DMEM培养液(P<0.01)。与溶媒组相比,(-)doxazosin、(+)doxazosin和(±)doxazosin(3～30μmol·L-1)作用于VSMCs 48h,均显著抑制PDGF-BB刺激的大鼠主动脉VSMCs增殖(P<0.01)。在抑制率方面,在含10%小牛血清的DMEM培养液中,(±)doxazosin及其光学异构体作用于VSMCs 48h时的最大抑制率为24.29±3.72%～27.13±2.41%;而在含0.4%小牛血清及PDGF-BB的DMEM培养液中,(±)doxazosin及其光学异构体作用于VSMCs 48h时的最大抑制率为95.21±6.05%～99.67±1.99%。双因素方差分析结果显示,在3μmol·L-1浓度时,(+)doxazosin的抑制率显著低于(±)doxazosin(P<0.05)。(-)Doxazosin、(+)doxazosin和(±)doxazosin抑制VSMCs增殖达50%的浓度(IC50)分别为5.44±3.16、7.50±3.40和4.98±4.52μmol·L-1。
2 (±)Doxazosin及其光学异构体对Ang II诱发VSMCs增殖的影响
Ang II(100nmol·L-1)可刺激大鼠主动脉VSMCs增殖,其促增殖作用的强度显著强于含10%小牛血清的DMEM培养液(P<0.01)。与溶媒组相比,(-)doxazosin、(+)doxazosin和(±)doxazosin在3～30μmol·L-1浓度作用于VSMCs 48h,均显著抑制Ang II刺激的大鼠主动脉VSMCs增殖(P<0.01)。在抑制率方面,(-)doxazosin、(+)doxazosin和(±)doxazosin在0.3～30μmol·L-1浓度范围内随着浓度的升高而抑制作用明显增强。双因素方差分析结果显示,(-)doxazosin的抑制率与(+)doxazosin和(±)doxazosin相比,无显著性差异(P>0.05)。在含0.4%小牛血清及Ang II的DMEM培养液中(±)doxazosin及其光学异构体作用于VSMCs 48h时的最大抑制率为93.01±3.23%～94.61±3.11%。(-)Doxazosin、(+)doxazosin和(±)doxazosin抑制VSMCs增殖的IC50值分别为13.38±2.50、14.24±2.47和13.71±2.89μmol·L-1。
3 (±)Doxazosin及其光学异构体对凝血酶诱发VSMCs增殖的影响
凝血酶(1U·ml-1)亦可刺激大鼠主动脉VSMCs增殖,其促增殖作用的强度与含10%小牛血清的DMEM培养液相近。与溶媒组相比,(-)doxazosin、(+)doxazosin和(±)doxazosin在3～30μmol·L-1浓度作用于VSMCs 48h,均显著抑制凝血酶刺激的大鼠主动脉VSMCs增殖(P<0.01)。在抑制率方面,(-)doxazosin、(+)doxazosin和(±)doxazosin在0.3～30μmol·L-1浓度范围内随着浓度的升高而抑制作用明显增强。双因素方差分析结果显示,(-)doxazosin的抑制率与(+)doxazosin和(±)doxazosin相比,无显著性差异(P>0.05)。在含0.4%小牛血清及凝血酶的DMEM培养液中,(±)doxazosin及其光学异构体作用于VSMCs 48h时的最大抑制率为91.87±5.13%～94.63±3.33%。(-)Doxazosin、(+)doxazosin和(±)doxazosin抑制VSMCs增殖的IC50值分别为13.52±2.87、14.60±2.95和14.32±2.75μmol·L-1。
4 (±)Doxazosin及其光学异构体对髙糖诱发VSMCs增殖的影响
高糖(25.6mmol·L-1)可刺激大鼠主动脉VSMCs增殖,其促增殖作用的强度与凝血酶相同。与溶媒组相比,(-)doxazosin、(+)doxazosin和(±)doxazosin在3～30μmol·L-1浓度作用于VSMCs 48h,均显著抑制髙糖刺激的大鼠主动脉VSMCs增殖(P<0.01)。双因素方差分析结果显示,(-)doxazosin在浓度为3μmol·L-1时的抑制率显著高于同浓度(+)doxazosin(P<0.05)。在含0.4%小牛血清及高糖的DMEM培养液中,(±)doxazosin及其光学异构体作用于VSMCs 48h时的最大抑制率为94.17±4.75%～96.16±3.09%。(-)Doxazosin、(+)doxazosin和(±)doxazosin抑制VSMCs增殖的IC50值分别为14.20±6.34、14.10±3.29和13.98±3.53μmol·L-1。
结论
1 (-)Doxazosin、(+)doxazosin和(±)doxazosin均可抑制10%小牛血清DMEM液中大鼠胸主动脉VSMCs的增殖反应;其中,(-)doxazosin的抑制作用明显强于(+)doxazosin,提示doxazosin在该培养条件下的抗VSMCs增殖作用具有手性药理学差异。
2除α1受体非依赖性抗VSMCs增殖作用以外,(-)doxazosin、(+)doxazosin和(±)doxazosin也具有α1受体依赖性抗VSMCs增殖作用。
3 (-)Doxazosin和(±)doxazosin抗大鼠胸主动脉VSMCs增殖的作用与其抑制G0/G1期细胞向S期转化以及降低细胞增殖指数有关;(+)doxazosin的对细胞周期的抑制作用显著弱于(-)doxazosin。
4 (-)Doxazosin和(±)doxazosin具有诱导大鼠胸主动脉VSMCs凋亡的作用,该作用与其调控Bcl-2蛋白/Bax蛋白比值有关;提示(±)doxazosin诱导VSMCs凋亡的物质基础是其光学异构体(-)doxazosin而非(+)doxazosin。
5 (±)Doxazosin及其光学异构体对PDGF-BB、Ang II、凝血酶和高糖诱发大鼠主动脉VSMCs增殖反应具有显著的抑制作用。与小牛血清和苯肾上腺素诱发的VSMCs增殖反应相比,(±)doxazosin及其光学异构体对前四种促增殖剂的抑制作用更强,且未见明显的手性药理学差异。
Vascular smooth muscle cells (VSMCs) located in the middle layer of the arterial wall regulate blood pressure by contraction and relaxation in physiological condition. Migration from the media to the intima space and abnormal proliferation of VSMCs are the common pathological bases of atherosclerosis (AS), hypertension and vascular restenosis (RS). Injury of vascular endothelial cells causes production and activation of a variety of inflammatory cytokines and growth regulation factors, which induce a change in VSMCs phenotype, a migration of VSMCs from the media to the intima, and excessive proliferation as well as apoptosis inhibition. Therefore, protecting vascular endothelial cells and inhibiting proliferation and migration of VSMCs might prevent the diseases such as AS, RS and hypertension.
(±)Doxazosin, a long-acting selectiveα1-adrenergic receptor antagonist, has been extensively used in the treatment of benign prostatic hyperplasia (BPH) complicated with lower urinary tract symptoms (LUTS). BPH/LUTS patients with hypertension treated with (±)doxazosin received two beneficial therapeutic actions. Recent studies demonstrated that (±)doxazosin markedly inhibited proliferation of the aortic VSMCs, and the inhibitory effects had characteristics of concentration-dependent andα1-receptor independent.
In the late 20th century, the study of chiral drug became an important field of the drug development. It was reported that (-)doxazosin and (+)doxazosin were prepared using chiral mobile phase HPLC and high performance capillary electrophoresis. The blocking effect of (-)doxazosin onα1-adrenoceptor was significantly weaker than that of (±)doxazosin and (+)doxazosin in the isolated rabbit thoracic aorta, carotid artery and mesenteric artery. The effect of (-)doxazosin on arterial blood pressure in the anesthetized rats was also significantly weaker than that of (±)doxazosin and (+)doxazosin administered intravenously or intraduodenally. However, the effect of decreasing urinary bladder pressure by (-)doxazosin in the anesthetized rats and guinea-pig was the same as that of (±)doxazosin. These findings suggested that the pharmacological activity of (-)doxazosin had chiral selective effect between the cardiovascular system and lower urinary tract system. However, it remains to be clarified whether (+)doxazosin and (-)doxazosin have chiral selective effect on the cell proliferation and apoptosis of VSMCs and whether their effects are related toα1-adrenoceptor blockade. In the present study, MTT assay was used to determine the cell proliferation; and cell cycle distribution, cell apoptosis and apoptotic proteins (Bcl-2 and Bax) were analyzed using flow cytometry (FCM). The purpose of the study is to observe the effects of (±)doxazosin and its enantiomers on the cell proliferation and apoptosis in cultured VSMCs of the rat aorta and to analyze the related mechanisms.
Part 1 Effects of doxazosin and its enantiomers on cell proliferation in VSMCs of the rat aorta and a relationship of the effects withα1-adrenoceptor
VSMCs of the rat aorta were cultured, and MTT assay was used to determine the cell proliferation. The effects of (-)doxazosin, (+)doxazosin and (±)doxazosin on the cell proliferation of VSMCs and a relationship of the effects induced by (-)doxazosin and (+)doxazosin withα1-adrenoceptor were investigated.
1 Effects of (±)doxazosin and its enantiomers on cell proliferation in VSMCs of the rat aorta
Treatment with (-)doxazosin and (±)doxazosin at 10~30μmol·L-1 for 48h or 72h significantly inhibited cell proliferation in VSMCs of the rat aorta, but (+)doxazosin had an inhibitory effect only at 30μmol·L-1. When the VSMCs were incubated with the three agents for 96h, (-)doxazosin, (+) doxazosin and (±)doxazosin at 3~30μmol·L-1 inhibited the cell proliferation significantly. Treatments with (-)doxazosin, (+)doxazosin and (±)doxazosin at used concentrations for 48h, 72h or 96h inhibited cell proliferation of the VSMCs in a concentration-dependent manner. Statistic results with two-way ANOVA showed that the inhibition rate of cell proliferation by (-)doxazosin at 10μmol·L-1 was significantly higher than that by (+)doxazosin in VSMCs exposed to the drugs for 48h or 72h (P <0.05 ), and the inhibition rate of cell proliferation by (-)doxazosin at 3~30μmol·L-1 was significantly higher than that by (+)doxazosin in VSMCs exposed to the drugs for 96h (P<0.01). when the VSMCs were treated with the three agents at 30μmol·L-1 for 48h, 72h and 96h, the inhibition rates of cell proliferation in VSMCs were 27.13%, 38.05% and 58.87% by (-)doxazosin, 24.39%, 33.43% and 46.10% by (+)doxazosin, and 24.29%, 36.74% and 52.35% by (±)doxazosin, respectively. The inhibition rates of cell proliferation in VSMCs incubated with the three agents for 96h were significantly higher than those for 48h (p<0.05 and 0.01) and 72h (p<0.05 and 0.01). When the VSMCs were exposed to (-)doxazosin, (+)doxazosin and (±)doxazosin at different concentrations for 96h, the concentrations required for 40-percent inhibition (IC40) of the cell proliferation were 10.2±1.3μmol·L-1, 20.9±2.2μmol·L-1 and 12.1±2.6μmol·L-1, respectively. The IC40 value of (+)doxazosin was significantly higher than that of (±)doxazosin or (-)doxazosin (P<0.01).
2α1-Adrenoceptor-independent effects of (±)doxazosin and its enantiomers on cell proliferation in VSMCs of the rat aorta
The value of cell proliferation in sub-control group (0.001% alcohol) of solventⅡgroup or in sub-control group (phenoxybenzamine) of phenoxybenzamine group was not significantly different from that in sub-control group (distilled water) of solventⅠgroup (P>0.05). In solventⅠgroup, the proliferation of VSMCs was significantly inhibited after 48h-incubation with (-)doxazosin, (+)doxazosin and (±)doxazosin at 30μmol·L-1 (P<0.01), and the inhibition rates were 33.00±2.79%, 26.19±5.74% and 31.76±4.29%, respectively. The inhibition rate of (+)doxazosin was significantly lower than that of (-)doxazosin (P<0.05), but was not significantly different from that of (±)doxazosin. In solventⅡgroup and phenoxybenzamine group, the proliferation of VSMCs was significantly inhibited after 48h-incubation with (-)doxazosin, (+)doxazosin and (±)doxazosin as well (P<0.01). Although the inhibition rate of VSMCs proliferation by (+)doxazosin was lower than that by (-)doxazosin or (±)doxazosin, there was no statistical differences among the three agents. In comparison with the sub-group [(-)doxazosin] of solventⅠgroup, the inhibition of VSMCs proliferation by (-)doxazosin in solventⅡgroup and in phenoxybenzamine group was not significantly different (P>0.05); and the same results were obtained in the experiments treated with (+)doxazosin and (±)doxazosin.
3α1-Adrenoceptor-dependent effects of (±)doxazosin and its enantiomers on cell proliferation in VSMCs of the rat aorta
Phenylephrine (10μmol·L-1) was able to stimulate VSMCs proliferation of the rat thoracic aorta, and the extent of cell proliferation promoted by phenylephrine was similar to that by 10% fetal bovine serum contained in DMEM. The VSMCs proliferation stimulated by phenylephrine was significantly inhibited after 48h-incubation with (-)doxazosin, (+)doxazosin and (±)doxazosin at 30μmol·L-1 (P<0.01). The inhibition rate of VSMCs proliferation by (+)doxazosin was significantly lower than that by (-)doxazosin (P<0.05), but was to the same extent as (±)doxazosin.
Part 2 Effects of doxazosin and its enantiomers on cell cycle and apoptosis in VSMCs of the rat aorta
Effects of doxazosin and its enantiomers on cell cycle and apoptosis in cultured VSMCs of the rat thoracic aorta were investigated. Morphological changes in VSMCs were analyzed using Giemsa staining method, and the cell cycle, cell apoptosis and apoptotic proteins (Bcl-2 and Bax) were detected by FCM in order to investigate mechanisms of antiproliferation and induction of apoptosis b (±)doxazosin and its enantiomers in VSMCs of the rat thoracic aorta.
1 Morphological changes in VSMCs of the rat aorta induced by (±)doxazosin and its enantiomers
Morphological changes in cultured VSMCs of the rat thoracic aorta incubated with (-)doxazosin, (+)doxazosin and (±)doxazosin at 30μmol.L-1 for 96h were seen under an ordinary optical microscope. The overall cell shrinkage, chromatin condensation, nuclear margination toward the nuclear membrane, nuclear shrinkage, and apoptotic bodies of various sizes in the cytoplasm were observed,and the nucleus of alive normal cells uniformly stained blue or violet-blue.
2 Effects of (±)doxazosin and its enantiomers on cell cycle in VSMCs of the rat aorta
Cell cycle distribution and proliferation index (PI) of the rat aortic VSMCs changed significantly after incubation with (-)doxazosin, (+)doxazosin and (±)doxazosin at 25μmol·L-1 for 72h. In the VSMCs treated with (±)doxazosin or (-)doxazosin, the proportion of cells in G0/G1-phase was significantly increased; and the proportions of cells in S-phase and G2/M-phase, and PI were significantly decreased (P<0.01). In the (+)doxazosin group, however, only the proportion of cells in S-phase was decreased significantly (P<0.01). The proportions of cells in G0/G1-phase, S-phase and G2/M-phase, and PI value in the VSMCs treated with (-)doxazosin were significantly different from that treated with (+)doxazosin (P<0.05 and 0.01).
3 Effects of (±)doxazosin and its enantiomers on the cell apoptosis in VSMCs of the rat aorta
In the VSMCs incubated with (-)doxazosin, (+)doxazosin and (±)doxazosin at 25μmol·L-1 for 72h, the cell apoptosis was induced by (-)doxazosin and (±)doxazosin (P<0.01), but not by (+)doxazosin (P>0.05). The apoptotic rates of the cultured VSMCs treated with (-)doxazosin and (±)doxazosin were 6.06±0.31% and 3.63±0.99%, respectively. The induction of VSMCs apoptosis induced by (-)doxazosin was significantly stronger than that by (±)doxazosin (P<0.01).
4 Effects of (±)doxazosin and its enantiomers on the expression of apoptosis protein Bcl-2 and Bax in VSMCs of the rat aorta
After incubation of VSMCs with (-)doxazosin, (+)doxazosin and (±)doxazosin at 25μmol·L-1 for 72h, the expression of antiapoptotic protein Bcl-2 and proapoptotic protein Bax did not change significantly in comparison with solvent-treated VSMCs (P>0.05). On the other hand, the ratios of Bcl-2 protein/Bax protein in the cultured VSMCs treated with (-)doxazosin, (+)doxazosin and (±)doxazosin were 0.68±0.01, 0.92±0.03 and 0.82±0.10, respectively. The ratios of Bcl-2 protein/Bax protein in the cultured VSMCs were significantly reduced by (-)doxazosin and (±)doxazosin in comparison with the VSMCs treated with solvent (P<0.05 and 0.01), but (+)doxazosin did not significantly affect the ratio of Bcl-2 protein/Bax protein (P>0.05). The ratio of Bcl-2 protein/Bax protein in VSMCs treated by (-)doxazosin was significantly lesser than that by (+)doxazosin (P<0.01).
Part 3 Effects of doxazosin and its enantiomers on cell proliferation of the rat aortic VSMCs induced by different stimulators
Effects of (±)doxazosin and its enantiomers on cell proliferation determined by MTT assay in the rat aortic VSMCs stimulated by platelet-derived growth factor-BB (PDGF-BB), angiotensin II, high concentration glucose and thrombin.
1 Effects of (±)doxazosin and its enantiomers cell proliferation of the rat aortic VSMCs induced by PDGF-BB
PDGF-BB (1nmol·L-1) induced cell proliferation of the rat aortic VSMCs, and the extent of cell proliferation promoted by PDGF-BB (1nmol·L-1) was significantly stronger than that by 10% fetal bovine serum contained in DMEM (P<0.01). Incubation of the rat aortic VSMCs with (-)doxazosin, (+)doxazosin and (±)doxazosin (3～30μmol·L-1) for 48h inhibited the cell proliferation stimulated by PDGF-BB significantly (P<0.01). In the culture medium of DMEM containing 10% fetal bovine serum, the maximal inhibition rates of VSMCs proliferation by 48h-incubation with (-)doxazosin, (+)doxazosin and (±)doxazosin were from 24.29±3.72% to 27.13±2.41%, in the culture medium of DMEM containing 0.4% fetal bovine serum and 1nmol·L-1PDGF-BB, however, the maximal inhibition rates of cell proliferation by 48h-incubation with (-)doxazosin, (+)doxazosin and (±)doxazosin were from 95.21±6.05% to 99.67±1.99%. Statistic results with two-way ANOVA showed that the inhibition rate of cell proliferation by (+)doxazosin at 3μmol·L-1 was significantly lower than that by (±)doxazosin at the same concentration (P<0.05). A concentration required for 50-percent inhibition (IC50) of the VSMCs proliferation by (-)doxazosin, (+)doxazosin or (±)doxazosin was 5.44±3.16, 7.50±3.40 or 4.98±4.52μmol·L-1.
2 Effects of (±)doxazosin and its enantiomers cell proliferation of the rat aortic VSMCs induced by angiotensin II
Angiotensin II (100 nmol·L-1) induced cell proliferation of the rat aortic VSMCs, and the extent of cell proliferation promoted by angiotensin II (100 nmol·L-1) was significantly stronger than that by 10% fetal bovine serum contained in DMEM (P<0.01). Incubation of the rat aortic VSMCs with (-)doxazosin, (+)doxazosin and (±)doxazosin (3～30μmol·L-1) for 48h inhibited the cell proliferation stimulated by angiotensin II significantly (P<0.01). The inhibition rates of the VSMCs proliferation by (-)doxazosin, (+)doxazosin and (±)doxazosin were increased with the increase in concentrations used (0.3～30μmol·L-1). Statistic results with two-way ANOVA showed that the inhibition rate of VSMCs proliferation by 48h-incubation with (-)doxazosin was not significantly different from that with (+)doxazosin or (±)doxazosin (P>0.05). In the culture medium of DMEM containing 0.4% fetal bovine serum and 100 nmol·L-1 angiotensin II, the maximal inhibition rates of cell proliferation by 48h-incubation with (-)doxazosin, (+)doxazosin and (±)doxazosin were from 93.01±3.23% to 94.61±3.11%. A concentration required for 50-percent inhibition (IC50) of the VSMCs proliferation by (-)doxazosin, (+)doxazosin or (±)doxazosin was 13.38±2.50, 14.24±2.47 or 13.71±2.89μmol·L-1.
3 Effects of (±)doxazosin and its enantiomers cell proliferation of the rat aortic VSMCs induced by thrombin
Thrombin (1μmol·L-1) induced cell proliferation of the rat aortic VSMCs, and the extent of cell proliferation promoted by thrombin (1μmol·L-1) was similar to that by 10% fetal bovine serum contained in DMEM. Incubation of the rat aortic VSMCs with (-)doxazosin, (+)doxazosin and (±)doxazosin (3～30μmol·L-1) for 48h inhibited the cell proliferation stimulated by angiotensin II significantly (P<0.01). The inhibition rates of the VSMCs proliferation by (-)doxazosin, (+)doxazosin and (±)doxazosin were increased with the increase in concentrations used (0.3～30μmol·L-1). Statistic results with two-way ANOVA showed that the inhibition rate of VSMCs proliferation by 48h-incubation with (-)doxazosin was not significantly different from that with (+)doxazosin or (±)doxazosin (P>0.05). In the culture medium of DMEM containing 0.4% fetal bovine serum and 1μmol·L-1 thrombin, the maximal inhibition rates of cell proliferation by 48h-incubation with (-)doxazosin, (+)doxazosin and (±)doxazosin were from from 91.87±5.13% to 94.63±3.33%. A concentration required for 50-percent inhibition (IC50) of the VSMCs proliferation by (-)doxazosin, (+)doxazosin or (±)doxazosin was 13.52±2.87, 14.60±2.95 or 14.32±2.75μmol·L-1.
4 Effects of (±)doxazosin and its enantiomers cell proliferation of the rat aortic VSMCs induced by a high concentration of glucose
A high concentration of glucose (25.6mmol·L-1) induced cell proliferation of the rat aortic VSMCs, and the extent of cell proliferation promoted by the high concentration of glucose (25.6mmol·L-1) was similar to that by 1μmol·L-1 thrombin contained in DMEM. Incubation of the rat aortic VSMCs with (-)doxazosin, (+)doxazosin and (±)doxazosin (3～30μmol·L-1) for 48h inhibited the cell proliferation stimulated by the high concentration of glucose significantly (P<0.01). Statistic results with two-way ANOVA showed that the inhibition rate of VSMCs proliferation by 48h-incubation with 3μmol·L-1 (-)doxazosin was significantly higher than that with (+)doxazosin at the same concentration (P<0.05). In the culture medium of DMEM containing 0.4% fetal bovine serum and 25.6mmol·L-1 glucose, the maximal inhibition rates of cell proliferation by 48h-incubation with (-)doxazosin, (+)doxazosin and (±)doxazosin were from 94.17±4.75% to 96.16±3.09%. A concentration required for 50-percent inhibition (IC50) of the VSMCs proliferation by (-)doxazosin, (+)doxazosin or (±)doxazosin was 14.20±6.34, 14.10±3.29 or 13.98±3.53μmol·L-1.
Conclusion
1 (-)Doxazosin, (+)doxazosin and (±)doxazosin inhibit the rat aortic VSMCs proliferation induced by 10% fetal bovine serum, and the extent of inhibition of cell proliferation by (-)doxazosin is stronger than that by (+)doxazosin, suggesting that (±)doxazosin and its enantiomers have chirally selective effect on cell proliferation in the used experimental condition.
2Αnα1-adrenoceptor-dependent mechanism is involved in the anti-proliferation of the rat aortic VSMCs by (±)doxazosin and its enantiomers besides a knownα1-adrenoceptor-independent mechanism.
3 (-)Doxazosin and (±)doxazosin are able to inhibit cell proliferation by a cell-cycle arrest in G0/G1 phase and a decrease in PI. The effect on cell cycle by (-)doxazosin is significantly stronger than that by of (+)doxazosin.
4 (-)Doxazosin and (±)doxazosin are able to induce cell apoptosis in the rat aortic VSMCs, and the proapoptotic effects are partially related to a reduced ratio of Bcl-2 protein/Bax protein, indicating that (-)doxazosin might be the main component of (±)doxazosin to induce cell apoptosis in the rat aortic VSMCs.
5 (±)Doxazosin and its enantiomers significantly inhibit cell proliferation in the rat aortic VSMCs stimulated by PDGF-BB, angiotensin II, thrombin and high concentration of glucose without chirally selective effects. The inhibition extent of cell proliferation by (-)doxazosin is much stronger in the VSMCs stimulated with PDGF-BB, angiotensin II, thrombin or high concentration of glucose than that in the VSMCs stimulated with fetal bovine serum or phenylephrine.
引文
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