Bcl-xL调控mtDNA/P选择素在糖尿病血瘀证发病中的作用及薯蓣皂苷的干预机制

Mitochondrial dysfunction and damage (MDD) play key roles in blood stasis syndrome of diabetes mellitus(DM).High glucose in DM causes low expression of Bcl-xL,leading to increased expression of p selection (Ps) induced by MDD. Data from clinical research revealed that Diocin(Dic) could reverse blood stasis syndrome of DM, however the underlying mechanism is unknown.Our researh indicated that Dic could alleviate MDD and inhibit platelet aggregation. Meanwhile,we found the high level of mitochondrial DNA (mtDNA) in DM, the increased Ps expression after addition of mtDNA, and the reduced production of mtDNA and Ps expression after Dic treatment.Therefore, we speculate that high glucose causes MDD by supressing Bcl-xL, resulting mtDNA release which trigger platelet activation, while Dio prevents the event induced from high glucose by regulating Bcl-xL to avoid MDD and mtDNA release produced by MDD. It is at the first time proposed that relationship between Bcl-xL and mtDNA release and their roles in blood stasis syndrome of DM, and the interference of Dic and underlying meachanism. Here,we will employ DNA polymerase γ knokout DM mice model，mtDNA knokout platelet model，confocal laser technology，immune coprecipitation techniques to investigate our hypothesis.

血小板线粒体功能障碍和损伤（MDD）在糖尿病（DM）血瘀证发病中起重要作用；高糖导致Bcl-xL表达下调诱发MDD引起P选择素（Ps）表达增高；临床研究显示，薯蓣皂苷（Dic）中成药对DM血瘀证疗效明确，但上述机制不详。我们证实，Dic改善MDD并抑制血小板聚集；预研究发现：DM血小板线粒体DNA（mtDNA）含量增高，mtDNA可增加Ps表达，而Dic能降低DM的mtDNA含量及Ps表达。因此，我们推测“高糖抑制Bcl-xL导致MDD，释放mtDNA引发血小板活化；而Dic通过Bcl-xL介导避免MDD，阻断mtDNA外漏”是DM血瘀证发病及Dic防治DM血瘀证的新机制。我们拟用DNA聚合酶-γ基因敲除鼠的DM动物模型、mtDNA缺失血小板的细胞模型，应用激光共聚焦和免疫共沉淀等技术，从整体-细胞-线粒体-分子层次证明我们的假说。此研究有助探明活血化瘀疗效机制，深化糖尿病瘀血阻滞理论。

糖尿病血瘀证诱发的心脑血管疾病是人类死亡的主要原因，血小板的异常活化是血瘀证发生的关键因素，高糖状态下血小板的活化机制及血小板的异常活化在糖尿病血瘀证的作用尚不明确。薯蓣皂苷类活血化瘀中成药治疗糖尿病性血瘀证疗效明确，作用机制不详。. 课题使用DNA聚合酶-γ基因敲除鼠的DM动物模型、mtDNA缺失血小板的细胞模型，应用激光共聚焦和免疫共沉淀等技术，从整体-细胞-线粒体-分子多个层次展开研究，以期揭示糖尿病血瘀证的病发机制及薯蓣皂苷的活血化瘀作用途径。.依照课题计划，本课题确定了Bcl-xL调控mtDNA/P选择素在糖尿病血瘀证发病中的作用及薯蓣皂苷防治糖尿病血瘀证的功效。. 1) 明确了薯蓣皂苷能够显著降低高糖诱导活化的血小板mtROS水平、胞外mtDNA水平及血小板聚集率。证实了薯蓣皂苷能够阻断mtDNA外漏，改善血小板线粒体功能和结构损伤。. 2）确定了高糖及外源mtDNA均能激活DC-SIGN并导致PLCγ2活化，进而启动血小板活化信号级联反应。. 3）明确了薯蓣皂苷能够显著降低高糖诱导活化的血小板线粒体胞外mtDNA水平，并抑制DC-SIGN及PLCγ2活化，显著降低高糖诱导活化的血小板线粒体功能损伤、mtDNA外漏，并明显阻滞了由DC-SIGN启动的血小板活化信号级联反应，从而探明了薯蓣皂苷“扶正祛瘀”防治糖尿病血瘀证的作用机制。. 4）发现糖尿病患者的血小板活化程度升高，并且Akt通路被上调。当抑制糖尿病患者血小板的Akt通路时，发现糖尿病患者血小板活化程度下降。发现薯蓣皂苷可通过调节血小板Akt通路抑制糖尿病患者的血小板活化。. 本课题研究探明了活血化瘀疗效机制，深化糖尿病瘀血阻滞理论，丰富了中医药的科学内涵。项目的顺利完成为糖尿病血瘀证的诊治与薯蓣皂苷的糖尿病血瘀证治疗研究提供重要的理论依据和数据支撑，同时也为糖尿病血瘀证的诊治提供更多的思路与靶点，极具理论意义和应用价值。