前蛋白转化酶枯草溶菌素9对脓毒症相关血小板活化的作用研究

Effect of proprotein convertase subtilisin/kexin type 9 on platelet activation associated with sepsis

目的:探讨前蛋白转化酶枯草溶菌素9(PCSK9)对脓毒症相关血小板活化的作用。方法:①临床试验:采用前瞻性研究方法,选择2021年1月至10月滨州医学院附属医院重症医学科收治的年龄≥18岁且符合脓毒症3.0诊断标准的脓毒症及脓毒性休克患者作为研究对象;以同期健康体检者作为对照。记录患者入院后第1次血常规中血小板计数(PLT);并于确诊1 d取静脉血,采用酶联免疫吸附试验(ELISA)测定血清PCSK9水平。比较两组PCSK9水平及PLT的差异,并针对脓毒症患者根据PLT进行亚组分析;采用Pearson相关法分析脓毒症患者PCSK9水平与PLT的相关性。②动物实验:将80只雄性C57BL/6小鼠按随机数字表法分为对照组、脓毒症模型组〔脂多糖(LPS)组〕、PCSK9抑制剂预处理组(PCSK9 inhibitor+LPS组)及PCSK9抑制剂对照组(PCSK9 inhibitor组),每组20只。腹腔注射LPS 12 mg/kg制备脓毒症小鼠模型;对照组和PCSK9 inhibitor组注射等量无菌生理盐水。PCSK9 inhibitor+LPS组及PCSK9 inhibitor组分别于注射LPS或生理盐水前腹腔注射PCSK9抑制剂5 mg/kg预处理7 d;对照组及LPS组注射等量无菌生理盐水。制模后24 h取肺组织进行病理学及免疫组化观察;取心脏血检测PLT;采用流式细胞仪检测血小板活化情况;采用蛋白质免疫印迹试验(Western blotting)检测血小板活化标志物CD40L的蛋白表达。结果:①临床试验:最终共纳入脓毒症患者57例,同期27例健康体检者作为对照。脓毒症组血清PCSK9水平较健康对照组明显升高(μg/L:232.25±72.21比191.72±54.92,P<0.05),PLT较健康对照组明显降低〔×109/L:146.00(75.50,204.50)比224.00(194.00,247.00),P<0.01〕;进一步亚组分析显示,脓毒症血小板减少者(n=20)血清PCSK9水平较非血小板减少者(n=37)明显升高(μg/L:264.04±60.40比215.06±72.95,P<0.01)。相关分析显示,脓毒症患者血清PCSK9水平与PLT呈显著负相关(r=-0.340,P=0.010)。②动物实验:光镜下显示,对照组与PCSK9 inhibitor组肺组织均无明显病理学改变,且两组PLT、血小板活化情况及血浆CD40L蛋白表达差异均无统计学意义;LPS组小鼠肺间质可见大量炎症细胞浸润,肺泡结构破坏明显,肺泡间隔增厚,肺泡腔内广泛出血,毛细血管扩张并存在出血及血小板聚集,PLT明显降低,血小板活化及血浆CD40L蛋白表达水平显著增加;而给予PCSK9抑制剂预处理后,小鼠肺组织炎症细胞浸润较LPS组有一定程度减少,肺泡间隔增厚减轻,肺组织血小板聚集减少,PLT显著升高(×109/L:515.83±46.60比324.83±46.31,P<0.05),血小板活化及血浆CD40L蛋白表达水平显著降低〔血小板α颗粒膜糖蛋白CD62P阳性表达率:(12.15±1.39)%比(18.33±2.74)%,CD40L蛋白(CD40L/β-actin):0.77±0.08比1.18±0.10,均P<0.05〕。结论:脓毒症中PCSK9水平对促进血小板活化有一定的作用,抑制PCSK9水平对于改善脓毒症血小板减少致不良结局的效果可能具有潜在研究价值。

Objective To investigate the effect of proprotein convertase subtilisin/kexin type 9(PCSK9)on platelet activation in sepsis.Methods①Clinical trial:a prospective study was conducted.Patients with sepsis and septic shock aged≥18 years old who met the diagnostic criteria of Sepsis-3 admitted to the department of intensive care medicine of the Affiliated Hospital of Binzhou Medical College from January to October in 2021 were selected as subjects.Healthy subjects in the same period were taken as healthy control group.Platelet count(PLT)in the first routine blood test after admission was recorded.Venous blood was taken 1 day after diagnosis,and serum PCSK9 level was determined by enzyme-linked immunosorbent assay(ELISA).The differences of PCSK9 level and PLT between the two groups were compared,and subgroup analysis was conducted based on PLT for patients with sepsis.The correlation between PCSK9 level and PLT in septic patients was analyzed by Pearson correlation method.②Animal experiment:80 male C57BL/6 mice were randomly divided into control group,sepsis model group[lipopolysaccharide(LPS)group],PCSK9 inhibitor pretreatment group(PCSK9 inhibitor+LPS group)and PCSK9 inhibitor control group(PCSK9 inhibitor group),with 20 mice in each group.The mouse model of sepsis was reproduced by intraperitoneal injection of LPS 12 mg/kg,and the control group and PCSK9 inhibitor group were intraperitoneally injected with the same amount of sterile normal saline.PCSK9 inhibitor+LPS group and PCSK9 inhibitor group were pretreated with PCSK9 inhibitor 5 mg/kg intraperitoneal injection for 7 days before injection of LPS or normal saline,respectively,and the control group and LPS group were injected with an equal amount of sterile normal saline.The lung tissues were taken for pathological and immunohistochemical observation 24 hours after modeling.Blood was taken from the heart for determining PLT.Platelet activation was detected by flow cytometry.The expression level of platelet-activation marker CD40L was detected by Western blotting.Results①Clinical trial:there were 57 cases in the sepsis group and 27 cases in the healthy control group.Serum PCSK9 level in the sepsis group was significantly higher than that in the healthy control group(μg/L:232.25±72.21 vs.191.72±54.92,P<0.05),and PLT was significantly lower than that in the healthy control group[×109/L:146.00(75.50,204.50)vs.224.00(194.00,247.00),P<0.01].Subgroup analysis showed that the serum PCSK9 level in the thrombocytopenia patients(n=20)was significantly higher than that in the non-thrombocytopenia patients(n=37;μg/L:264.04±60.40 vs.215.06±72.95,P<0.01).Correlation analysis showed a significant negative correlation between serum PCSK9 levels and PLT in septic patients(r=-0.340,P=0.010).②Animal experiment:there were no significant pathological changes in lung tissue in the control group and PCSK9 inhibitor group under light microscope,and no significant differences in PLT,platelet activation and plasma CD40L protein expression was found between the two groups.In the LPS group,a large number of inflammatory cells were infiltrated in the pulmonary interstitium,the alveolar structure was damaged obviously,the alveolar septum was widened,the alveolar cavity was extensively bleeding,the capillary dilatation with bleeding and platelet aggregation were found,the PLT was significantly decreased,the platelet activation and the expression level of CD40L protein in plasma were significantly increased.The infiltration of inflammatory cells in lung tissue of mice in the PCSK9 inhibitor+LPS group was reduced to a certain extent,the thickening of alveolar septa was reduced,the platelet aggregation in lung tissue was decreased as compared with the LPS group,the PLT was significantly increased(×109/L:515.83±46.60 vs.324.83±46.31,P<0.05),the platelet activation and the expression level of CD40L protein in plasma were significantly decreased[positive expression rate of platelet activation dependent granule surface facial mask protein CD62P:(12.15±1.39)%vs.(18.33±2.74)%,CD40L protein(CD40L/β-actin):0.77±0.08 vs.1.18±0.10,both P<0.05].Conclusion PCSK9 level has a certain effect on promoting platelet activation in sepsis,and inhibition of PCSK9 level may have potential research value in improving adverse outcomes caused by sepsis thrombocytopenia.