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目的 研究重组人血小板生成素(rhTPO)对脂多糖(LPS)诱导的血小板减少症小鼠血小板活化和焦亡的影响及机制,为rhTPO的临床使用提供理论依据.方法 将100只C57BL/6小鼠随机(随机数字法)分为5组:空白对照组(Sham组),实验对照组(LPS组),低剂量(L组,1.35×103 U·kg-1·d-1)、中剂量(M组,2.7×103U·kg-1·d-1)、高剂量(H组,5.4×103U·kg-1·d-1)rhTPO治疗组.连续观察72 h.第72 h流式细胞技术检测洗涤血小板CD61/CD62p、Gasdermin D及Caspase-1的阳性表达率;ELISA法检测血浆中IL-1 β、IL-18水平.结果 LPS组与Sham组相比生存率明显降低,差异有统计学意义(P＜0.01);L、M、H组与LPS组相比生存率略有升高,但差异无统计学意义(P＞0.05).Sham组小鼠血小板计数在实验前后无明显变化,LPS组小鼠血小板计数显著下降;第72小时L组小鼠血小板计数明显高于LPS组、M组、H组(P＜0.01),M组、H组较LPS组则无明显升高(P＞0.05).LPS组小鼠洗涤血小板CD61/CD62p、Gasdermin D蛋白表达较Sham组明显升高(P＜0.01),L组较LPS组明显下降(P＜0.05),M、H组较LPS组变化不明显(P＞0.05);Caspase-1表达LPS组较Sham组明显升高(P＜0.01),L组、M组较LPS组明显下降(P＜0.05),H组较LPS组变化不明显(P＞0.05).LPS组小鼠富血小板血浆IL-1 β、IL-18水平较Sham组明显升高(P＜0.01),L组较LPS组下降明显(P＜0.01),M、H组较LPS组变化不明显(P＞0.05).结论 rhTPO可以抑制LPS诱导的血小板减少症小鼠血小板的活化和焦亡,为脓毒症血小板减少症的治疗提供了基础研究依据.

Objective To study the effect and mechanism of recombinant human thrombopoietin (rhTPO) on platelet activation and pyroptosis in mice with lipopolysaccharide (LPS)-induced thrombocytopenia,and provide a theoretical basis for the clinical use of rhTPO.Methods One hundred C57BL/6 mice were randomly(random number) divided into 5 groups:blank control group (sham group),experimental control group (LPS group),low dose (L group,1.35 ×103U · kg-1 · d-1),medium dose (M group,2.7 ×103U · kg-1 · d-1),and high dose (H group,5.4 ×103U · kg-1 · d-1) rhTPO treatment groups.Continuous observation for 72 h.The positive expression rates of CD61/CD62p,Gasdermin D and Caspase-1 in washed platelets were detected by flow cytometry at 72 h,and the levels of IL-1β and IL-18 in plasma were detected by ELISA.Results Compared with the sham group,the survival rate of the LPS group was significantly lower (P＜ 0.01).Compared with the LPS group,the survival rates of the L,M and H groups were slightly increased,but the difference was not statistically significant (P＞0.05).There was no significant change in platelet count of the sham group before and after the experiment.The platelet count in the LPS group decreased significantly.The platelet count at 72 h in the L group was signiftcantly higher than those in the LPS,M and H groups (P＜0.01),and there was no significant difference between the M and H groups and LPS group (P＞0.05).Compared with the sham group,CD61/CD62p and Gasdermin-D protein expressions in the LPS group were significantly increased (P＜0.01),significantly decreased in the L group (P＜0.05),and not significantly changed in the M and H groups (P＞0.05).Caspase-1 expression was significantly increased in the LPS group compared with the sham group (P＜0.01),significantly decreased in the L and M groups compared with the LPS group (P＜0.05),and not significantly changed in the H group compared with the LPS group (P＞0.05).The levels ofplatelet-rich plasma IL-1 beta and IL-18 in the LPS group were significantly higher than those in the sham group (P＜0.01),while those in the L group were significantly lower than those in the LPS group (P＜0.01),and those in the M and H groups were not significantly changed than those in the LPS group (P＞0.05).Conclusions rhTPO can inhibit platelet activation and pyroptosis in LPS-induced thrombocytopenia mice,which provides basic research basis for the treatment of sepsis thrombocytopenia.