苯并芘对血小板功能和血栓形成的影响
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摘要
多环芳烃(polycyclic aromatic hydrocarbons,PAHs)是含有两个或两个以上苯环的芳香族化合物。该类化合物性质稳定,污染持久,分布广泛。苯并芘(benzo(a)pyrene, BaP)是多环芳烃的代表物质,存在于汽车尾气、煤、石油、香烟等的不完全燃烧产物中,不适当的烹饪方式也会产生苯并芘。由于其分布广致癌性强,已引起普遍关注。
BaP致癌毒性在过去几年中有了深入的研究。BaP可经过生物体代谢,最终降解为二氢环氧苯并芘(benzo(a)pyrene-7,8-diol-9,10-epoxide, BPDE)。BaP作为强致癌物,可与DNA结合,引起DNA损伤,基因突变,诱发癌症。BaP进入机体后,对神经及免疫系统均有影响。此外,早期的研究显示BaP能引起血栓,对心血管系统也有影响。尽管动物模型和流行病学研究都表明BaP能促进动脉粥样硬化,但确切的机制仍不清楚。
血小板是血液中的有效成分之一,除正常的止血等生理功能外,在血栓形成中也起着重要作用。我们期望通过检测BaP对血小板功能及血栓形成的影响,探讨BaP致心血管疾病的机制。
首先,用血小板聚集仪检测了BaP对血小板聚集的影响。通过聚集实验,我们发现10μmol/L,1μmol/L和0.1μmol/LBaP本身均不能引起血小板聚集。根据文献中细胞实验的浓度和我们的实验结果,确定10μtmol/L为BaP的工作浓度。我们用浓度为10μtmol/L的BaP孵育血小板,并分别用ADP、胶原和凝血酶刺激血小板。结果显示BaP孵育不能增强在胶原和凝血酶的刺激下的血小板聚集。但是,BaP孵育的血小板在ADP的刺激下,聚集率达到0.80±0.10,与未经BaP孵育的血小板相比有极显著差异(P<0.01)。
其次,我们采用流动小室系统观察BaP对血小板粘附的影响。流动小室系统是一套由流动小室、静脉泵、医用硅胶管道和荧光显微镜构建的血液循环模型,能够很好的模拟剪切力作用下的血液状态,研究血小板粘附。我们研究了剪切率为1000s-1时,BaP对血小板粘附的影响。与溶剂对照相比,经BaP孵育的血小板的粘附显著增强(P<0.01)。
流式细胞术以速度快、灵敏度高、准确性好的优点在血液研究中有着广泛的运用。由于P-选择素是反映血小板活化状态的重要指标,因此本研究中我们用流式细胞仪检测BaP对血小板P-选择素表达的影响。结果显示BaP孵育后的血小板P-选择素能在ADP诱导下表达增强(P<0.01),而BaP孵育后的血小板P-选择素不能在凝血酶诱导下表达增强。
动物血栓模型能够模拟人体疾病,有助于更好的评价药物和研究疾病机制。我们建立了FeCl3诱导小鼠颈动脉血栓模型,腹腔注射苯并芘10 mg/kg,观察BaP对小鼠血栓形成时间的影响。结果显示注射BaP的小鼠颈动脉血栓形成时间短于对照组,暗示了BaP对体内血栓形成有促进作用。
通过上述研究,我们得到以下结论：BaP可促进体内血栓形成,引起血小板聚集、粘附和活化。BaP对血小板的影响可能是通过ADP介导的信号通路。
Polycyclic aromatic hydrocarbon (PAHs) are a group of chemicals that consist of two or more benzene rings. PAHs are fairly stable and persistent in the environment. Benzo(a)pyrene (BaP) is a representative PAHs which is widely presented in air pollution, coal tar, and can also be found in cigarette smoke and grilled food.
BaP has been extensively studied due to its wide distribution and strong carcinogenic. The major metabolic of BaP is anti-7,8-dihydrodiol-9,10-epoxide beno(a)pyrene (BPDE). BaP can form DNA adduct, thus inducing DNA damage and gene mutation. BaP can also affect neural and immune systems. Moreover, BaP has been reported to be associated with cardiovascular disease, for example, certain studies have shown that BaP can enhance atherosclerosis. However, few studies have been conducted to investigate its effect on platelet activation.
As one of the active ingredients in blood, platelet plays an important role in thrombosis. Therefore, in the present study, the effects of BaP on platelet aggregation, adhersion, and activation were examined. We also used the FeCl3-induced arterial thrombosis murine model to determine the effect of BaP on thrombosis.
1. The effect of BaP on platelet aggregation. It was found that different concentration of BaP (10μmol/L, 1μmol/L, and 0.1μmol/L) did not induce platelet aggregation. On the other hand, while BaP preincubation failed to enhance platelet aggregation under collagen and thrombin stimulation, BaP preincubation significantly enhanced ADP-induced platelet aggregation.
2. The effect of BaP on platelet adhesion. We used flow chamber system to study platelet adhesion. At a shear rate of 1000s-1, BaP preincubation led to significant platelet adhesion.
3. The effect of BaP on platelet activation. Using whole blood flow cytometry, the expression of P-selectin, an indicator for platlet activation, was evaluated. The results showed that BaP preincubation also increased ADP-induced P-selectin expression but not thrombin-induced P-selectin expression.
4. The effect of BaP on thrombosis. All mice received 10 mg/kg BaP or vehicle only by i.p. injection and anaesthetized to establish thrombus model. We evaluated the effect of BaP on thrombus formation time. Thrombus formation time of BaP-exposed animal is significantly shorter than unexposed controls.
In conclusion, these data indicate that BaP can enhance thrombosis. BaP can stimulate ADP-induced platelet aggregation, platelet adhesion and P-selectin expression, probably through the interaction with ADP-mediated signal pathway.
BaP致癌毒性在过去几年中有了深入的研究。BaP可经过生物体代谢,最终降解为二氢环氧苯并芘(benzo(a)pyrene-7,8-diol-9,10-epoxide, BPDE)。BaP作为强致癌物,可与DNA结合,引起DNA损伤,基因突变,诱发癌症。BaP进入机体后,对神经及免疫系统均有影响。此外,早期的研究显示BaP能引起血栓,对心血管系统也有影响。尽管动物模型和流行病学研究都表明BaP能促进动脉粥样硬化,但确切的机制仍不清楚。
血小板是血液中的有效成分之一,除正常的止血等生理功能外,在血栓形成中也起着重要作用。我们期望通过检测BaP对血小板功能及血栓形成的影响,探讨BaP致心血管疾病的机制。
首先,用血小板聚集仪检测了BaP对血小板聚集的影响。通过聚集实验,我们发现10μmol/L,1μmol/L和0.1μmol/LBaP本身均不能引起血小板聚集。根据文献中细胞实验的浓度和我们的实验结果,确定10μtmol/L为BaP的工作浓度。我们用浓度为10μtmol/L的BaP孵育血小板,并分别用ADP、胶原和凝血酶刺激血小板。结果显示BaP孵育不能增强在胶原和凝血酶的刺激下的血小板聚集。但是,BaP孵育的血小板在ADP的刺激下,聚集率达到0.80±0.10,与未经BaP孵育的血小板相比有极显著差异(P<0.01)。
其次,我们采用流动小室系统观察BaP对血小板粘附的影响。流动小室系统是一套由流动小室、静脉泵、医用硅胶管道和荧光显微镜构建的血液循环模型,能够很好的模拟剪切力作用下的血液状态,研究血小板粘附。我们研究了剪切率为1000s-1时,BaP对血小板粘附的影响。与溶剂对照相比,经BaP孵育的血小板的粘附显著增强(P<0.01)。
流式细胞术以速度快、灵敏度高、准确性好的优点在血液研究中有着广泛的运用。由于P-选择素是反映血小板活化状态的重要指标,因此本研究中我们用流式细胞仪检测BaP对血小板P-选择素表达的影响。结果显示BaP孵育后的血小板P-选择素能在ADP诱导下表达增强(P<0.01),而BaP孵育后的血小板P-选择素不能在凝血酶诱导下表达增强。
动物血栓模型能够模拟人体疾病,有助于更好的评价药物和研究疾病机制。我们建立了FeCl3诱导小鼠颈动脉血栓模型,腹腔注射苯并芘10 mg/kg,观察BaP对小鼠血栓形成时间的影响。结果显示注射BaP的小鼠颈动脉血栓形成时间短于对照组,暗示了BaP对体内血栓形成有促进作用。
通过上述研究,我们得到以下结论：BaP可促进体内血栓形成,引起血小板聚集、粘附和活化。BaP对血小板的影响可能是通过ADP介导的信号通路。
Polycyclic aromatic hydrocarbon (PAHs) are a group of chemicals that consist of two or more benzene rings. PAHs are fairly stable and persistent in the environment. Benzo(a)pyrene (BaP) is a representative PAHs which is widely presented in air pollution, coal tar, and can also be found in cigarette smoke and grilled food.
BaP has been extensively studied due to its wide distribution and strong carcinogenic. The major metabolic of BaP is anti-7,8-dihydrodiol-9,10-epoxide beno(a)pyrene (BPDE). BaP can form DNA adduct, thus inducing DNA damage and gene mutation. BaP can also affect neural and immune systems. Moreover, BaP has been reported to be associated with cardiovascular disease, for example, certain studies have shown that BaP can enhance atherosclerosis. However, few studies have been conducted to investigate its effect on platelet activation.
As one of the active ingredients in blood, platelet plays an important role in thrombosis. Therefore, in the present study, the effects of BaP on platelet aggregation, adhersion, and activation were examined. We also used the FeCl3-induced arterial thrombosis murine model to determine the effect of BaP on thrombosis.
1. The effect of BaP on platelet aggregation. It was found that different concentration of BaP (10μmol/L, 1μmol/L, and 0.1μmol/L) did not induce platelet aggregation. On the other hand, while BaP preincubation failed to enhance platelet aggregation under collagen and thrombin stimulation, BaP preincubation significantly enhanced ADP-induced platelet aggregation.
2. The effect of BaP on platelet adhesion. We used flow chamber system to study platelet adhesion. At a shear rate of 1000s-1, BaP preincubation led to significant platelet adhesion.
3. The effect of BaP on platelet activation. Using whole blood flow cytometry, the expression of P-selectin, an indicator for platlet activation, was evaluated. The results showed that BaP preincubation also increased ADP-induced P-selectin expression but not thrombin-induced P-selectin expression.
4. The effect of BaP on thrombosis. All mice received 10 mg/kg BaP or vehicle only by i.p. injection and anaesthetized to establish thrombus model. We evaluated the effect of BaP on thrombus formation time. Thrombus formation time of BaP-exposed animal is significantly shorter than unexposed controls.
In conclusion, these data indicate that BaP can enhance thrombosis. BaP can stimulate ADP-induced platelet aggregation, platelet adhesion and P-selectin expression, probably through the interaction with ADP-mediated signal pathway.
引文
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