收稿日期: 2025-04-30
网络出版日期: 2025-08-11
基金资助
国家自然科学基金项目(82272200);国家自然科学基金项目(82241062);国家自然科学基金项目(82130062);国家资助博士后研究人员计划(GZC20242288)
The programmed death of megakaryocytes and its impact on platelet-production copacity and coagulation function in mice with sepsis
Received date: 2025-04-30
Online published: 2025-08-11
目的 研究脓毒症时小鼠骨髓巨核细胞发生程序性死亡（坏死性凋亡、凋亡、自噬、铁死亡和焦亡）的情况及其对产血小板能力、小鼠凝血功能的影响。 方法 将C57BL/6J小鼠随机分为假手术组（Sham组）和脓毒症模型组（CLP组）。Sham组与CLP组小鼠于术后24 h进行腹主动脉采血测定外周血血小板及凝血功能，小鼠处死后取双下肢长骨，经巨核细胞分离液和免疫磁珠分选法提取骨髓巨核细胞。激光共聚焦显微镜观察程序性死亡相关标志分子在小鼠骨髓巨核细胞中的活化情况，流式细胞仪检测巨核细胞程序性死亡率、产血小板表型及血小板表面标志物（CD41、CD42b、CD61），Western blot检测巨核细胞中程序性死亡相关蛋白的表达情况。 结果 与Sham组相比，CLP组小鼠血小板数量在急性脓毒症期（24 h）较假手术组明显下降（P < 0.01），血小板分布宽度（PDW）和平均血小板体积（MPV）则显著上升（P < 0.01）；凝血酶时间（TT）、凝血酶原时间（PT）、 活化部分凝血活酶时间（APTT）显著延长（P < 0.01），而纤维蛋白原（FIB）显著降低（P < 0.01）。与Con/Sham组相比，LPS/CLP组巨核细胞产血小板表型、上清液中PLP数量及血小板表面标志物（CD41、CD42b、CD61）表达水平均显著上升。巨核细胞坏死性凋亡、凋亡、焦亡、铁死亡率在CLP术后24 h显著升高。激光共聚焦显微镜观察显示，CLP术后小鼠巨核细胞LC3、P-MLKL、Caspase-1、Fe2+活化明显。Western blot检测结果显示，CLP组巨核细胞坏死性凋亡相关蛋白P-MLKL的活化率较Sham组显著上升（P < 0.01）；焦亡相关蛋白GSDMD及GSDMD-N剪切显著增加（P < 0.01）；铁死亡相关蛋白ACSL4表达显著上升（P < 0.01），GPX4表达显著降低（P < 0.01）；凋亡相关蛋白Bax表达显著上升（P < 0.05）；自噬相关蛋白LC3B-II剪切显著增加（P < 0.01），P62表达显著降低（P < 0.01）。而使用细胞程序性死亡抑制剂抑制凋亡后巨核细胞产血小板功能下降，抑制坏死性凋亡和焦亡对巨核细胞产血小板功能的影响有限，抑制铁死亡及自噬能增强巨核细胞的产血小板功能。 结论 在脓毒症急性期（24 h）可见明显的巨核细胞程序性死亡。其中，凋亡是巨核细胞产血小板表型分化及产血小板能力升高的重要机制；而巨核细胞过度自噬反应及铁死亡可能导致巨核细胞功能障碍，是脓毒症凝血异常的重要机制。
华天桢 , 汪海涛 , 魏淑婷 , 童森 , 董宁 , 祝筱梅 , 姚咏明 , 刘伟 . 脓毒症小鼠巨核细胞程序性死亡及对产血小板能力、凝血功能的影响[J]. 实用医学杂志, 2025 , 41(15) : 2325 -2335 . DOI: 10.3969/j.issn.1006-5725.2025.15.006
Objective To investigate programmed death including necroptosis， apoptosis， autophagy， ferroptosis， and pyroptosis in bone marrow megakaryocytes of mice during sepsis and its impact on platelet production capacity and coagulation function in mice. Methods C57BL/6J mice were randomly divided into a sham operation group （sham group） and a sepsis model group （CLP group）. Peripheral blood platelets and coagulation function were measured by abdominal aortic blood sampling at 24 h postoperatively in both sham and CLP groups. After the mice were sacrificed， long bones of both lower limbs were taken， and bone marrow megakaryocytes were extracted using megakaryocyte separation solution and immunomagnetic bead separation. Laser confocal microscopy was used to observe the activation of programmed death-related marker molecules in mouse bone marrow megakaryocytes. Flow cytometry was used to detect programmed death rate， platelet production phenotype， and platelet surface markers （CD41， CD42b， CD61） of megakaryocytes. Western blotting was used to detect the expression of programmed death-related proteins in megakaryocytes. Results Compared with the sham group， the CLP group showed significant decreases in the number of platelets during acute sepsis （24 h） （P < 0.000 1）， significant increases in platelet distribution width （PDW） and mean platelet volume （MPV） （P < 0.01）， significant prolonging of thrombin time （TT）， prothrombin time （PT）， and activated partial thromboplastin time （APTT） （P < 0.000 1， P < 0.001， P < 0.01）， and significant reduction in fibrinogen （Fib） （P < 0.000 1）. Compared with the Con/sham group， the LPS/CLP group exhibited significant increases in the platelet production phenotype of megakaryocyte， the number of PLP in the supernatant， and the expression levels of platelet surface markers （CD41， CD42b， CD61）. The rates of megakaryocyte necroptosis/apoptosis， pyroptosis， and ferroptosis were significantly elevated at 24 h post-CLP surgery. Laser confocal microscopy showed significant activation of LC3， P-MLKL， Caspase-1， and Fe2+ in megakaryocytes of mice after CLP surgery. Western blotting results revealed that the CLP group exhibited a significant increase in the activation rate of necroptosis-related protein P-MLKL （P < 0.001）， a significant increase in the cleavage of pyroptosis-related proteins GSDMD and GSDMD-N （P < 0.01， P < 0.001， respectively）， a significant increase in the expression of ferroptosis-related protein ACSL4 （P < 0.01）， and a significant decrease in the expression of GPX4 （P < 0.01） compared to the sham group. Additionally， the CLP group demonstrated significant increases in the expression of apoptosis-related protein Bax， the cleavage of autophagy-related protein LC3B-Ⅱ， and the expression of P62 （P < 0.05， P < 0.001， P < 0.001， respectively）. Inhibition of apoptosis with programmed cell death inhibitors decreased platelet production function of megakaryocyte， while inhibition of necroptosis and pyroptosis had limited effects on platelet production function of megakaryocyte. Inhibition of ferroptosis and autophagy enhanced platelet production function of megakaryocyte. Conclusion Significant programmed death of megakaryocytes was observed during the acute phase of sepsis （24 h）. Among those megakaryocytes， apoptosis is an important mechanism for the differentiation of platelet production phenotype and increased platelet production capacity of megakaryocyte. Overactive autophagy and ferroptosis in megakaryocytes lead to megakaryocyte dysfunction， which is an important mechanism for coagulation abnormalities in sepsis.
Key words： sepsis; coagulation; megakaryocyte; platelet; programmed cell death
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