普伐他汀对C反应蛋白刺激下大鼠颈动脉血栓形成的影响

The effect of Pravastatin on carotid artery thrombosis in rats under the stimulus of C reactive protein

目的探讨普伐他汀对C反应蛋白(CRP)刺激下大鼠颈动脉血栓形成的影响。方法将32只大鼠分为4组,普通对照组:常规饲料喂养;普伐他汀组:常规饲料喂养的基础上加用普伐他汀干预;CRP组:常规饲料喂养的基础上加用CRP干预;普伐他汀+CRP组:常规饲料喂养的基础上加用普伐他汀和CRP干预。采用20%三氯化铁浸润的滤纸包裹大鼠颈动脉建立血栓模型,记录形成闭塞血栓的时间并称量所形成血栓的重量。分离富血小板血浆,通过流式细胞仪检测体外给予PAR-4激动剂刺激的血小板P选择素的表达。采用酶联免疫法检测循环中可溶性P选择素和凝血酶的水平。结果普伐他汀治疗后的大鼠颈动脉形成闭塞血栓时间有明显延长,CRP静脉注射对该模型的血栓形成时间无明显影响,而各组形成血栓的质量无明显差别。流式细胞检测各组血小板P选择素的表达水平均较低且无明显差异。体外给予PAR-4激动剂AYPGKF-NH2(100μmol/L)刺激后血小板表面P选择素表达显著增加,普通对照组P选择素的阳性百分率从3.4%增加至46%。而与普通对照组比较,普伐他汀或CRP均使得GYPGKF-NH2刺激的血小板P选择素表达明显下降。在普伐他汀治疗的基础上给予CRP注射的大鼠血小板P选择素表达更进一步下降至25%。大鼠血浆中基础可溶性P选择素的表达水平较低,CRP刺激后血清可溶性P选择素水平增加约22%,普伐他汀治疗能明显降低CRP刺激的循环中可溶性P选择素水平[(9.64±0.81)pg/mL vs(11.34±0.74)pg/mL,P<0.05]。静脉给予CRP注射4 h后血清F1+2水平较基础状态明显增高[(2.57±0.4)pg/mL vs(0.72±0.11)pg/mL,P<0.01];而预先给予普伐他汀治疗能明显降低CRP刺激的血清F1+2水平(1.93±0.25)pg/mL。结论普伐他汀能延长CRP刺激下氧化损伤所致大鼠颈动脉血栓形成时间,在炎症状态下他汀的抗血栓作用可能带来更多临床益处。

Objective To investigate the effect of Pravastatin on carotid artery thrombosis in rats under the stimulus of C reactive protein （CRP）. Methods Thirty - two Sprague - Dawley rats were divided into four groups, normal control group （normal diet and placebo） ; pravastatin group （fed with normal diet plus pravastatin （5 mg/kg · d） intervention） ; CRP group （fed with normal diet and were also given CRP （3 mg/kg） intravenously four hours prior to the induction of thrombus） ; pravastatin ＋ CRP group （Based on the normal diet feed plus pravastatin and CRP intervention）. An occlusive thrombus was created in the carotid artery by application of Whatman paper soaked in 20% FeCl3. The time when occlusive thrombus formatted and weight of thrombus were recorded. The expression of platelet P - selectin was detected by flow cytometry with or without stimulus of PAR - 4 agonist in vitro. The levels of soluble P and prothrombin fragments were detected by ELISA. Results The time when occlusive tbrombus formatted was not altered by intravenous of CRP in the rats fed with or without Pravastatin. However, the duration of thrombosis was significantly increased in the group given Pravastatin （17.5 ±1. 5 minutes vs. 13 ±1.41 minutes, P 〈0. 01）. There was no significant difference in the weight of the thrombosis among all groups. The expression of P - selectin on platelets was low in all groups without significant difference among them, but it was significantly increased by PAR-4 agonist AYPGKF- Nh2 （100μmol/L） in vitro, as the positive percentage of P - selectin was increased to 46% from 3.4% at baseline. The P - selectin expression on platelets stimulated by AYPGKF - NH2 was reduced by either Pravastatin or CRP intervention, as P - seleetin was reduced by about 25% in the combination of Pravastatin and CRP. Serum soluble P - selectin was enhanced by about 22% by CRP, which was significantly inhibited by Pravastatin （9. 64 ±0. 81 vs. 11.34 ±0. 74 pg/mL, P 〈0. 05）. Serum F1 ＋ 2 level was significantly increased 4 hours after intravenous CRP intervention （2. 57 ± 0. 4 pg/mL vs. 0. 72 ± 0. 11 pg/ mL, P 〈 0. 01 ）, while pretreatment with Pravastatin could significantly reduce serum levels of F1 ＋ 2 （ 1.93 ± 0. 25 pg./ mL） stimulated by CRP. Conclusion This study shows that Pravastatin delays thrombus formation in rat carotid arterial exposed to oxidant stress under the stimulus of CRP. The antithrombotic effect of statins in inflammatory state may bring more clinical benefits.