摘要
目的探讨色胺酮对小鼠血小板聚集、释放功能的影响及机制。方法取昆明(KM)种小鼠10只麻醉采血制备富血小板血浆(PRP)和贫血小板血浆(PPP),再取KM小鼠50只采用多次离心法制备洗涤血小板、制成悬液后分为对照组[1‰二甲基亚砜(DMSO)]、5μmol/L色胺酮组、10μmol/L色胺酮组及0.5 mg/L替罗非班组,除对照组外其余各组小鼠洗涤血小板(或PRP)悬液分别与对应药物孵育,再加3种激动剂[2 mg/L胶原、80 U/L凝血酶及5μmol/L二磷酸腺苷(ADP)],以台式缓冲液(或PPP)作空白对照,采用光比浊法检测3种激动剂刺激下对照组、5μmol/L色胺酮组、10μmol/L色胺酮组及0.5 mg/L替罗非班组小鼠血小板的聚集情况并计算血小板聚集率,采用荧光强度法检测2 mg/L胶原刺激后对照组、10μmol/L色胺酮组及0.5 mg/L替罗非班组小鼠血小板三磷酸腺苷(ATP)的释放功能,采用Western blot检测空白组、对照组、5μmol/L色胺酮组、10μmol/L色胺酮组及0.5 mg/L替罗非班组小鼠血小板中磷酸酰肌醇-3-激酶(PI3K)、蛋白激酶B(PKB或Akt)、糖原合成酶激酶(GSK)3β及蛋白激酶C(PKC)蛋白磷酸化的表达水平。结果胶原刺激血小板聚集时,5、10μmol/L色胺酮组及0.5 mg/L替罗非班组小鼠血小板的聚集率较对照组降低(P<0.05或P<0.01);ADP和凝血酶刺激血小板聚集时,5、10μmol/L色胺酮组小鼠血小板的聚集率与对照组比较,差异无统计学意义(P>0.05);胶原刺激时,10μmol/L色胺酮组和0.5 mg/L替罗非班组小鼠血小板的ATP释放量较对照组降低(P<0.01);胶原诱导的血小板聚集后,5、10μmol/L色胺酮组及0.5 mg/L替罗非班组小鼠血小板中PI3K(p85)、Akt、GSK3β蛋白以及PKC蛋白底物的磷酸化水平较对照组减少(P<0.01)。结论色胺酮可以抑制小鼠血小板的聚集和释放功能,其机制可能与抑制PI3K-Akt-GSK3β和PKC蛋白的磷酸化水平有关。
Objective To explore the effect of tryptanthrin on platelet aggregation and release function and its mechanism.Methods Ten Kunming(KM)mice were anesthetized and their blood samples were extracted to prepare for the platelet-rich plasma(PRP),and platelet-poor plasma(PPP).And other 50 KM mice were collected and their blood samples were extracted to prepare for the washed platelets of mice by multiple centrifugation.After the suspension was made,the subject mice were divided into the control group[1‰dimethyl sulfoxide(DMSO)],5μmol/L tryptanthrin group,10μmol/L tryptanthrin group and 0.5 mg/L tirofiban group.In addition to the control group,the wash platelet(or PRP)suspensions of each group were incubated with the corresponding drugs,and three agonists[2 mg/L collagen,80 U/L thrombin,or 5μmol/L adenosine diphosphate(ADP)]were added respectively,and the Tyrode’s buffer(or PPP)was used as the blank control.The light turbidimetry method was applied to detect the platelet aggregation(PA)in the control group,the 5μmol/L tryptanthrin group,10μmol/L tryptanthrin group,and 0.5 mg/L tirofiban group under the stimulation of three agonists,and PA rate was calculated.Fluorescence intensity method was applied to detect the release function of mouse platelet adenosine triphosphate(ATP)in the control group,10μmol/L tryptanthrin group,and 0.5 mg/L tirofiban group under 2 mg/L collagen stimulation.For the blank group,control group,5 and 10μmol/L tryptanthrin groups,and 0.5 mg/L tirofiban group,western blotting was used to detect the expression levels of phosphoinositide-3-kinase(PI3K),protein kinase B(PKB or Akt),glycogen synthase kinase(GSK)3β,and protein kinase C(PKC)of each group.Results When PA was stimulated by collagend,compared with the control group,the PA rate of the 5 and 10μmol/L tryptanthrin groups,and 0.5 mg/L tirofiban group decreased,and the difference was statistically significant(P<0.05 or P<0.01),but when PA was stimulated by ADP or thrombin,compared with the control group,the PA rate in the 5 and 10μmol/L tryptanthrin groups did not change significantly,and the difference was not statistically significant(P>0.05).When the release of platelets were stimulated by collagen,compared with the control group,the ATP release amount of the 10μmol/L tryptanthrin group and 0.5 mg/L tirofiban group were reduced,and the difference was statistically significant(P<0.01).After collagen-induced PA,compared with the control group,the phosphorylation levels of PI3K(p85),Akt,GSK3β,and PKC protein substrates in the 5 and 10μmol/L tryptanthrin groups,and 0.5 mg/L tirofiban group decreased,and the difference was statistically significant(P<0.01).Conclusion Tryptanthrin could significantly inhibit the aggregation and release of mouse platelets,and its mechanism may be related to the inhibition of the phosphorylation level of PI3K-Akt-GSK3βand PKC protein.
作者
田晓云
袁兆伟
郭芳
熊秀琴
任天和
刘刚
罗俊
TIAN Xiaoyun;YUAN Zhaowei;GUO Fang;XIONG Xiuqin;REN Tianhe;LIU Gang;LUO Jun(Department of Pharmacology,School of Basic Medical Sciences,Guizhou Medical University,Guiyang 550025,Guizhou,China)
出处
《贵州医科大学学报》
CAS
2023年第1期11-16,42,共7页
Journal of Guizhou Medical University
基金
国家自然科学基金(81760653)。
关键词
色胺酮
胶原
血小板
聚集
释放
信号通路
tryptanthrin
bcollagen
lood platelets
aggregation
release
signal pathway
