通阳中药葱白提取物抑制低压低氧诱导促凝血反应的机制研究

Mechanism of activating yang medicine onion extract on inhibiting hypobaric hypoxia-induced procoagulant reaction

目的研究通阳中药葱白提取物(AYMOE)对低压低氧诱导的血小板激活和促凝状态的影响。方法将42只成年雄性SD大鼠随机分为对照组(暴露于常压常氧环境)、低压低氧组(Hyp组,暴露于低压低氧环境)、Hyp+AYMOE-100组(暴露于低压低氧环境并口服AYMOE 100 mg/kg)、Hyp+AYMOE-200组(暴露于低压低氧环境并口服AYMOE 200 mg/kg)、Hyp+AYMOE-300组(暴露于低压低氧环境并口服AYMOE 300 mg/kg)、Hyp+anti-CD42c组(暴露于低压低氧环境并尾静脉注射antiCD42c 5 mg/kg)、Hyp+AYMOE-200+rh Lyn组[暴露于低压低氧环境并口服AYMOE 200 mg/kg联合尾静脉注射重组人酪氨酸蛋白激酶Lyn(rh Lyn) 4.8 mg/kg],每组6只。低压低氧暴露7 d后,ELISA检测各组大鼠血清血栓素A2(TXA2)以及血小板激活因子(PAF)含量,检测大鼠血小板的凝集率,HE染色观察大鼠肺组织病理变化,Western blot检测GPIb-Ⅸ-Ⅴ、Lyn、磷酸化的Lyn(p-Lyn)的表达。结果与对照组比较,暴露于低压低氧的各组大鼠血清TXA2、PAF水平及血小板凝集率升高,GPIb-Ⅸ-Ⅴ、Lyn、p-Lyn蛋白表达上调(P <0.05)。与Hyp组相比,Hyp+AYMOE-100组、Hyp+AYMOE-200组、Hyp+AYMOE-300组的血小板凝集率、血清TXA2、PAF的水平降低,差异均有统计学意义(P <0.05),其中以Hyp+AYMOE-200组抑制效果最明显(P <0.05)。与对照组比较,Hyp组大鼠因低氧而发生血管堵塞,肺静脉血管腔空间狭小;与Hyp组相比,Hyp+AYMOE-200组大鼠肺静脉血管栓塞明显缓解。与Hyp组相比,Hyp+anti-CD42c组和Hyp+AYMOE-200组GPIb-Ⅸ-Ⅴ、Lyn、p-Lyn蛋白表达下调,差异均有统计学意义(P <0.05)。与Hyp+AYMOE-200组比较,Hyp+AYMOE-200+rh Lyn组血小板凝集率、血清TXA2和PAF水平均较高,Lyn和p-Lyn蛋白表达均增加,差异均有统计学意义(P <0.05)。结论 AYMOE可通过抑制GPIb-Ⅸ-Ⅴ和Lyn减少低压低氧诱导的血小板激活并改善促凝状态。

Objective To study the effect of activating yang medicine onion extract( AYMOE) on hypobaric hypoxia-induced platelet activation and procoagulation status. Methods A total of 42 adult male SD rats were randomly divided into the control group( exposed to the normal pressure and normal oxygen environment),the hypobaric hypoxia group( Hyp group,exposed to the hypobaric hypoxia environment),the Hyp + AYMOE-100 group( exposed to the hypobaric hypoxia environment and oral administration of 100 mg/kg of AYMOE),the Hyp +AYMOE-200 group( exposed to the hypobaric hypoxia environment and oral administration of 200 mg/kg of AYMOE),the Hyp + AYMOE-300 group( exposed to hypobaric hypoxia environment and oral administration of 300 mg/kg of the AYMOE),the Hyp + anti-CD42 c group( exposed to the hypobaric hypoxia environment and tail vein injection of 5 mg/kg of anti-CD42 c),and the Hyp + AYMOE-200 + rh Lyn group( exposed to the hypobaric hypoxia environment and oral administration of 200 mg/kg of AYMOE combined with tail vein injection of4. 8 mg/kg of rh Lyn),with 6 rats in each group. After exposed to hypobaric hypoxia environment for 7 days,the serum thromboxane A2( TXA2) and platelet activating factor( PAF) of rats in each group were detected by ELISA and the platelet aggregation rate was detected.The pathological changes of lung tissue of rats were observed by HE staining. The expressions of GPIb-Ⅸ-Ⅴ,Lyn,and phosphorylated-Lyn( p-Lyn) were detected by Western blot. Results Compared with the control group,the levels of serum TXA2 and PAF,platelet aggregation rate of rats in the groups exposed to hypobaric hypoxia environment were higher,and the expressions of GPIb-Ⅸ-Ⅴ,Lyn,p-Lyn protein were up-regulated( P < 0. 05). Compared with the Hyp group,the platelet aggregation rate and the levels of serum TXA2 and PAF decreased in the Hyp + AYMOE-100 group,the Hyp + AYMOE-200 group and the Hyp + AYMOE-300 group,with statistically significant differences( P < 0. 05),and the Hyp + AYMOE-200 group had the most obvious inhibitory effect( P < 0. 05). Compared with the control group,the rats in the Hyp group had vascular blockage due to hypoxia,with narrow pulmonary venous vascular space;the pulmonary vein embolization in the Hyp + AYMOE-200 group was significantly alleviated compared with the Hyp group. The expressions of GPIb-Ⅸ-Ⅴ,Lyn and p-Lyn proteins in the Hyp + anti-CD42 c group and the Hyp + AYMOE-200 group were down-regulated compared with those in the Hyp group,with statistically significant differences( P < 0. 05). Compared with the Hyp + AYMOE-200 group,the platelet aggregation rate and the levels of serum TXA2 and PAF in the Hyp + AYMOE-200 + rh Lyn group were higher,and the expressions of Lyn and p-Lyn proteins were increased,with statistically significant differences( P < 0. 05). Conclusion AYMOE can reduce the hypobaric hypoxia-induced platelet activation and alleviate procoagulant status by inhibiting GPIb-Ⅸ-Ⅴ and Lyn.