OBJECTIVE: To investigate the expression of neutrophil extracellular traps (NETs) and phagocytic function in the peripheral blood of patients with hepatic alveolar echinococcosis (HAE), and to examine their correlations with clinical inflamma tory indicators and liver functions.
METHODS: A total of 50 patients with HAE admitted to Department of Hepatobiliary and Pancreatic Surgery, The Affiliated Hospital of Qinghai University from August 2022 to June 2023 were enrolled, while 50 age- and gender-matched healthy individuals from the Centre for Healthy Examinations of the hospital during the same period served as controls. The levels of NETs markers neutrophil myeloperoxidase (MPO) and neutrophil elastase (NE) were measured using enzyme-linked immunosorbent assay (ELISA). Peripheral blood neutrophils were isolated using density gradient centrifugation, stimulated in vitro using phorbol 12-myristate 13 acetate (PMA), and the levels of MPO and citrullination histone H3 (CitH3) released by neutrophils were quantified using flow cytometry. The phagocytic functions of neutrophils were examined using flow cytometry. In addition, the correlations of MPO and NE levels with clinical inflammatory indicators and liver biochemical indicators were examined using Spearman correlation analysis among HAE patients.
RESULTS: The peripheral blood plasma MPO[(417.15 ± 76.08) ng/mL vs. (255.70 ± 80.84) ng/mL; t = 10.28, P < 0.05], NE[(23.16 ± 6.75) ng/mL vs. (11.92 ± 3.17) ng/mL; t = 10.65, P < 0.05]and CitH3 levels[(33.93 ± 18.93) ng/mL vs. (19.52 ± 13.89) ng/mL; t = 4.34, P < 0.05]were all significantly higher among HAE patients than among healthy controls, and a lower phagocytosis rate of neutrophils was detected among HAE patients than among healthy controls[(70.85 ± 7.32)% vs. (94.04 ± 3.90)%; t = 20.18, P < 0.05], and the ability to produce NETs by neutrophils was higher among HAE patients than among healthy controls following in vitro PMA stimulation. Pearson correlation analysis showed that the phagocytosis rate of neutrophils correlated negatively with platelet-to-lymphocyte ratio (PLR), neutrophil-to-lymphocyte ratio (NLR), interleukin-6 (IL-6) level and C-reactive protein (CRP) level (rs = -0.515 to -0.392, all P values < 0.05), and the MPO and NE levels positively correlated with inflammatory markers NLR, PLR, CRP and IL-6 (rs = 0.333 to 0.445, all P values < 0.05) and clinical liver biochemical indicators aspartic transaminase, alanine aminotransferase, direct bilirubin and total bilirubin among HAE patients (rs = 0.290 to 0.628, all P values < 0.001).
CONCLUSIONS: Excessive formation of NETs is found among HAE patients, which affects the phagocytic ability of neutrophils and results in elevated levels of inflammatory indicators. NETs markers may be promising novel biomarkers for early diagnosis, monitoring, and severity assessment of liver disease.
[摘要] 目的 探索肝泡型棘球蚴病 (hepatic alveolar echinococcosis, HAE) 患者外周血中中性粒细胞胞外诱捕网 (neutrophil extracellular traps, NETs) 表达水平及吞噬功能变化, 以及其与临床炎性指标和肝生化指标间的相关性。方法 选 取2022年8月—2023年6月青海大学附属医院肝胆外科收治的50例HAE患者作为HAE患者组, 选择同期在该院体检 中心体检的年龄、性别匹配的50例健康志愿者作为健康对照组, 采用酶联免疫吸附法 (enzyme-linked immunosorbent assay, ELISA) 检测两组研究对象外周血中NETs标志物髓过氧化物酶 (myeloperoxidase, MPO) 和中性粒细胞弹性酶 (neutrophil elastase, NE) 水平。同时, 采用密度梯度离心法分离两组研究对象外周血中性粒细胞; 经佛波酯 (phorbol 12-myristate 13-acetate, PMA) 体外刺激后, 采用流式细胞术分别定量检测中性粒细胞胞外释放的MPO和瓜氨酸化组蛋白 (citrullinated histone H3, CitH3) 水平。采用流式细胞术比较两组研究对象中性粒细胞吞噬能力差异, 采用Spearman 相关分析评价 HAE患者NETs标志物含量与临床炎性指标及肝生化指标水平间的相关性。结果 HAE患者外周血中NETs标志物 MPO、NE、CitH3 水平分别为 (417.15 ± 76.08)、(23.16 ± 6.75)、(33.93 ± 18.93) ng/mL, 均显著高于健康对照组[ (255.70 ± 80.84)、(11.92 ± 3.17)、(19.52 ± 13.89) ng/mL] (t = 10.28、10.65、4.34, P 均< 0.05); 但HAE组中性粒细胞吞噬率[ (70.85 ± 7.32) %]较健康对照组[ (94.04 ± 3.90) %]下降 (t = 20.18, P < 0.05); 经PMA体外刺激后, HAE患者组中性粒细胞形成 NETs能力较健康对照组显著提高。Spearman相关分析显示, HAE组外周血中性粒细胞吞噬水平和血小板与淋巴细胞比 例 (platelet-to-lymphocyte ratio, PLR)、中性粒细胞与淋巴细胞比例 (neutrophil-to-lymphocyte ratio, NLR)、白细胞介素 (interleukin, IL)-6、C反应蛋白 (C reactive protein, CRP) 水平呈负相关 (rs = −0.515~−0.392, P 均< 0.05), NETs标志物MPO、NE水平与NLR、PLR、CRP、IL-6水平均呈正相关 (rs = 0.333~0.445, P 均< 0.05), 与天冬氨酸氨基转移酶、丙氨酸氨基转 移酶、直接胆红素和总胆红素水平亦呈正相关 (rs = 0.290~0.628, P均< 0.001)。结论 HAE患者体内NETs形成过多, 影 响中性粒细胞吞噬能力, 导致机体炎症水平升高。NETs标志物有望作为肝脏疾病早期诊断、监测和严重程度评估的新 型生物学标志物。.