Study of exogenous carbon monoxide-releasing molecules 2 on endotoxin/lipopolysaccharide-induced abnormal activation of platelets of healthy human donors
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摘要: 目的 探讨外源性一氧化碳释放分子2(CORM-2)对LPS所致健康人外周血中血小板过度活化的作用及分子机制。 方法 采集1名健康成年人的静脉血,离心分离出富血小板血浆(PRP),分装于硅化后的试管,按随机数字表法分为正常对照组、LPS组、无活性CORM-2(iCORM-2)组、10 μmol/L CORM-2组、50 μmol/L CORM-2组,每组3管。正常对照组不进行任何处理;LPS组接受20 mL 10 μg/mL的LPS刺激;iCORM-2组、10 μmol/L CORM-2组、50 μmol/L CORM-2组在接受上述LPS刺激的同时分别接受50 μmol/L iCORM-2、10 μmol/L CORM-2、50 μmol/L CORM-2的干预,用量均为20 mL。培养30 min后玻瓶法检测血小板黏附率;免疫荧光法检测血小板侵袭至纤维蛋白原的数量;比浊法检测血小板聚集率;取PRP制备贫血小板血浆(PPP)及血小板,化学荧光素法检测PPP及血小板内ATP含量;流式细胞仪检测血小板膜糖蛋白(GP)Ⅰbα和GPⅥ表达;蛋白质印迹法、免疫沉淀法分别检测血小板糖原合酶激酶3β(GSK-3β)及磷酸化GSK-3β表达。以上指标均重复测定3次。对数据行单因素方差分析、SNK检验。 结果 与正常对照组比较,LPS组、iCORM-2组PRP中血小板黏附率、侵袭至纤维蛋白原上的血小板数量、血小板聚集率、血小板GPⅠbα和GPⅥ表达量及PPP中ATP含量均明显增加,血小板内ATP含量均明显减少,
P 值均小于0.05。与LPS组比较,iCORM-2组上述各指标无明显变化(P 值均大于0.05);10 μmol/L CORM-2组、50 μmol/L CORM-2组血小板内ATP含量明显增加,其余上述各指标均明显减少,P 值均小于0.05。正常对照组、LPS组、iCORM-2组、10 μmol/L CORM-2组、50 μmol/L CORM-2组PRP中血小板GSK-3β表达量分别为0.550±0.060、1.437±0.214、1.210±0.108、0.720±0.010、0.670±0.010,磷酸化GSK-3β表达量分别为0.950±0.070、1.607±0.121、1.420±0.040、1.167±0.015、0.513±0.122。LPS组、iCORM-2组PRP中血小板GSK-3β表达量及磷酸化GSK-3β表达量较正常对照组明显增加(P 值均小于0.05),iCORM-2组PRP中血小板GSK-3β表达量及磷酸化GSK-3β表达量与LPS组相近(P 值均大于0.05),10 μmol/L CORM-2组、50 μmol/L CORM-2组PRP中血小板GSK-3β表达量及磷酸化GSK-3β表达量较LPS组明显减少(P 值均小于0.05)。 结论 LPS刺激可致健康人外周血中血小板异常活化,CORM-2的干预能够有效地抑制血小板的过度活化,其机制可能涉及GP介导的GSK-3β的磷酸化。-
关键词:
- 脓毒症 /
- 血小板 /
- 外源性一氧化碳释放分子2 /
- 糖原合酶激酶3β
Abstract: Objective To explore the effects of exogenous carbon monoxide-releasing molecules 2 (CORM-2) on LPS-induced abnormal activation of platelets in peripheral blood of healthy human donors and its possible molecular mechanism. Methods Venous blood samples were collected from a healthy volunteer, and platelet-rich plasma (PRP) from the blood were isolated by differential centrifugation. The PRP was subpackaged into siliconized test tubes and then divided into control group, LPS group, inactive CORM-2 (iCORM-2) group, 10 μmol/L CORM-2 group, and 50 μmol/L CORM-2 group according to the random number table, with 3 tubes in each group. The PRP in control group did not receive any treatment. The PRP in LPS group received LPS (20 mL, 10 μg/mL) stimulation, and the PRP in iCORM-2 group, 10 μmol/L CORM-2 group, and 50 μmol/L CORM-2 group underwent the same LPS stimulation and treatment of 50 μmol/L iCORM-2, 10 μmol/L CORM-2, and 50 μmol/L CORM-2, respectively, with the dosage of 20 mL. After being cultured for 30 min, the platelet adhesion rate was determined by glass bottle method, the number of platelet spreading on fibrinogen was determined with immunofluorescent method, and the platelet aggregation rate was measured by turbidimetric method. The platelet poor plasma (PPP) was prepared from PRP, the levels of ATP in PPP and platelets were determined by chemical fluorescein method. The expressions of platelet glycoprotein Ⅰ bα (GPⅠbα) and GPⅥ were analyzed by flow cytometer. The expressions of glycogen synthase kinase 3β (GSK-3β) and phosphorylated GSK-3β were determined by Western blotting and immunoprecipitation, respectively. Measurement of the above indices was repeated for 3 times. Data were processed with one-way analysis of variance and SNK test. Results Compared with those in control group, the platelet adhesion rates, numbers of platelets spreading on fibrinogen, platelet aggregation rates, expressions of GPⅠbα and GPⅥ in PRP, levels of ATP in PPP in LPS and iCORM-2 groups were significantly increased, while levels of ATP in platelets were significantly decreased (withP values below 0.05). Compared with those in LPS group, the former 7 indices in iCORM-2 group showed no significant differences (withP values above 0.05), while the levels of ATP in platelets in the 10 μmol/L CORM-2 and 50 μmol/L CORM-2 groups were significantly increased, and the other 6 indices in 10 μmol/L CORM-2 and 50 μmol/L CORM-2 groups were significantly decreased (withP values below 0.05). The expression levels of GSK-3β of the platelets in PRP in control, LPS, iCORM-2, 10 μmol/L CORM-2, and 50 μmol/L CORM-2 groups were 0.550±0.060, 1.437±0.214, 1.210±0.108, 0.720±0.010, and 0.670±0.010, respectively, and the expression levels of the phosphorylated GSK-3β of the platelets in PRP in the above 5 groups were 0.950±0.070, 1.607±0.121, 1.420±0.040, 1.167±0.015, and 0.513±0.122, respectively. Compared with those in control group, both the expression levels of GSK-3β and phosphorylated GSK-3β of the platelets in PRP in LPS and iCORM-2 groups were significantly increased (withP values below 0.05). The expression levels of GSK-3β and phosphorylated GSK-3β of the platelets in PRP between LPS group and iCORM-2 group were similar (withP values above 0.05). The expression levels of GSK-3β and phosphorylated GSK-3β of the platelets in PRP in 10 μmol/L CORM-2 and 50 μmol/L CORM-2 groups were significantly decreased compared with those in LPS group (withP values below 0.05). Conclusions LPS stimulation can abnormally activate the platelets in peripheral blood of healthy human, but the abnormal activation can be inhibited by CORM-2 intervention, and the mechanism of the latter may involve the phosphorylation of GSK-3β mediated by GP.
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