脓毒症大鼠血清假性血友病因子、血栓调节蛋白、血小板活化因子、抗凝血酶-Ⅲ变化及辛伐他汀的干预作用

Endothelial protective effect of simvastatin on coagulation system in septic rats

目的探讨脓毒症大鼠血清假性血友病因子(vWF)、血栓调节蛋白(TM)、血小板活化因子(PAF)、抗凝血酶-Ⅲ(AT-Ⅲ)的变化及辛伐他汀对腹主动脉内皮的保护作用。方法雄性SD大鼠54只,分为3组,(1)健康对照组:6只,大鼠腹腔注射生理盐水5 ml,3 h后再次注射生理盐水5 ml。(2)脓毒症模型组:24只,大鼠腹腔注射生理盐水5 ml,3 h后腹腔注射脂多糖2.5 mg制作脓毒症模型。(3)辛伐他汀干预组:24只,大鼠腹腔注射辛伐他汀5 ml,3 h后腹腔注射脂多糖2.5 mg制作脓毒症模型。3组大鼠于1 h、3 h、6 h、12 h时取血测vWF、TM、PAF、AT-Ⅲ。采用扫描电镜和透射电镜分别观察大鼠腹主动脉内皮细胞的形态及凋亡情况。结果与健康对照组比,脓毒症模型组1 h、3 h、6 h、12 h大鼠血清vWF[1 h:(68.3±4.8)ng/ml;3 h:(59.2±5.1)ng/ml;6 h:(74.2±20.1)ng/ml;12 h:(53.5±4.0)ng/ml]、TM[1 h:(1.4±0.3)ng/ml;3 h:(1.6±0.4)ng/ml;6 h:(2.8±0.9)ng/ml;12 h:(1.4±0.5)ng/ml]、PAF[(29.1±6.5)pg/ml;3 h:(28.6±1.5)pg/ml;6 h:(28.7±2.7)pg/ml;12 h:(18.2±4.1)pg/ml]、AT-Ⅲ[(262.2±38.1)mg/L;3 h:(233.0±70.4)μg/ml;6 h:(218.7±54.7)μg/ml;12 h:(162.2±37.2)μg/ml]水平明显升高(P<0.05)。与同时间点的脓毒症模型组比,辛伐他汀干预组1 h、3 h、6 h、12 h的PAF水平[1 h:(15.6±2.5)pg/ml;3 h:(10.4±5.3)pg/ml;6 h:(9.3±1.4)pg/ml;12 h:(11.0±2.7)pg/ml],6 h的TM水平[(1.6±0.9)ng/ml],1 h、6 h、12 h的AT-III水平[1 h:(190.3±29.2)μg/ml;6 h:(104.4±33.6)μg/ml;12 h:(73.6±39.0)μg/ml]均明显降低(P<0.05)。电镜下可见脓毒症模型组大鼠腹主动脉内皮细胞损伤,细胞内空泡化,细胞膜模糊甚至破裂、微绒毛化,核染色质边聚。辛伐他汀干预组大鼠腹主动脉内皮细胞损伤明显减轻,细胞膜清晰,细胞器空泡化减少,少量细胞器。结论脓毒症时毒素及过度激活的炎性因子损伤血管内皮,大量vWF、TM、PAF、AT-Ⅲ释放入血进而引起早期的高凝状态;辛伐他汀对脓毒症大鼠血管内皮具有保护作用,明显降低脓毒症时血vWF、TM、PAF、AT-Ⅲ水平,可改善脓毒症的血管内皮损伤及凝血功能紊乱。

Objective To investigate the endothelial protective effects of simvastatin on the coagulation system in septic rats.Methods A total of 54 SD male rats were divided into 3 groups.Six healthy rats were intraperitoneally injected with normal salineas control group.Twenty-four rats in septic group were intraperitoneally injected with normal saline followed by lipopolysaccharide 2.5 mg.Study group had 24 rats intraperitoneally injected with simvastatin followed by lipopolysaccharide.Plasma von Willebrand factor(vWF),thrombomodulin(TM),platelet activating factor(PAF)and antithrombin-Ⅲ(AT-Ⅲ)were tested at 1 h,3 h,6 h and 12 h after treatment.Scanning electron microscopy and transmission electron microscopy were used to observe the morphology and apoptosis of rat aorta endothelial cells.Results Compared with healthy control group,vWF[(68.3±4.8)ng/ml,(59.2±5.1)ng/ml,(74.2±20.1)ng/ml,(53.5±4.0)ng/ml,respectively],TM[(1.4±0.3)ng/ml,(1.6±0.4)ng/ml,(2.8±0.9)ng/ml,(1.4±0.5)ng/ml,respectively],PAF[(29.1±6.5)pg/ml,(28.6±1.5)pg/ml,(28.7±2.7)pg/ml,(18.2±4.1)pg/ml,respectively]and AT-Ⅲ[(262.2±38.1)μg/ml,(233.0±70.4)μg/ml,(218.7±54.7)μg/ml,(162.2±37.2)μg/ml,respectively]were significantly increased in the sepsis group at 1 h,3 h,6 h and 12 h(P<0.05).Compared with the sepsis group,the plasma levels of PAF in simvastatin intervention group at 1 h[(15.6±2.5)pg/ml,3 h(10.4±5.3)pg/ml,6 h(9.3±1.4)pg/ml,12 h(11.0±2.7)pg/ml]were significantly decreased,so were the TM level at 6 h(1.6±0.9)ng/ml,and the AT-Ⅲlevels at 1 h[(190.3±29.2)μg/ml],6 h[(104.4±33.6)μg/ml]and 12 h[(73.6±39.0)μg/ml,P<0.05].Conclusion In the condition of sepsis,toxins and over-activated inflammatory factors damage the vascular endothelium.A large amount of circulating vWF,TM,PAF,and AT-Ⅲcause early hypercoagulability.Simvastatin significantly reduces plasma amount of these procoagulants,suggesting it smodification of coagulopathy and vascular protective effectsin a septic rat model.