目的 探究芒柄花素磺酸钠（Sul-F）调节中性粒细胞胞外诱捕网（NETs）减轻脑缺血再灌注（I/R）损伤的作用机制。方法 SD大鼠随机分为5组：对照组、模型组、精氨酸阿司匹林组（AAFI，200 mg·kg^-1）和Sul-F低、高剂量组（20、40 mg·kg^-1）。除对照组外，其余各组均采用大脑中动脉栓塞（MCAO）法造模，缺血2 h、再灌注24 h后处死。于再灌注0、12 h后ip给药，对照组和模型组给予等体积0.9%氯化钠溶液。Bederson评分法评估神经功能；TTC染色法测定脑梗死率；Western blotting检测脑匀浆磷脂酶C-β3（PLC β3）和磷酸化PLC β3（p-PLC β3）的表达；酶联免疫法检测脑匀浆血栓素B2（TXB2）及炎症因子白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）含量；免疫荧光检测血小板活化相关分子P选择素（Pselectin）、中性粒细胞活化相关分子中性粒细胞表面黏附分子（CD11b）的表达；三色免疫荧光联合检测组蛋白3B（H3B）、髓过氧化物酶（MPO）和脱氧核糖核酸（DNA），以标识NETs。结果 与对照组相比，模型组大鼠神经功能评分、脑指数、脑梗死率以及脑匀浆IL-1β、IL-6、TXB2蛋白表达显著升高（P＜0.01），p-PLC β3蛋白表达显著降低（P＜0.01），大量NETs形成，P-selectin和CD11b表达均升高（P＜0.01）。与模型组比较，Sul-F显著降低神经功能评分、脑指数、脑梗死率以及脑匀浆IL-1β、IL-6、TXB2蛋白表达（P＜0.05、0.01）；显著升高p-PLC β3蛋白表达（P＜0.05、0.01）；NETs产生明显减少，显著降低P-selectin和CD11b蛋白表达（P＜0.05、0.01）。结论 Sul-F通过促进p-PLC β3蛋白表达，减少血小板活化，抑制血小板介导的NETs形成，减轻炎症反应，进而改善脑I/R损伤。

Objective To study the mechanism of sodium formononetin-3'-sulphonate (Sul-F) in regulating neutrophil extracellular traps (NETs) to alleviate cerebral ischemia/reperfusion (I/R) injury.Methods SD rats were randomly divided into five groups: control group, model group, arginine aspirin group (AAFI, 200 mg·kg^-1), Sul-F low and high dose groups (20, 40 mg·kg^-1). Except for the control group, the other groups were modeled by middle cerebral artery occlusion, and sacrificed after ischemia for 2 h and reperfusion for 24 h. The drugs were given by ip injection at 0 h and 12 h after reperfusion, and the control group and the model group were given the same volume of 0.9% sodium chloride solution. Neurological function was assessed by the Bederson score. TTC staining was used to determine the cerebral infarction volume. Western blotting was used to detect the expression of phospholipase C-β3(PLC β3) and phosphorylated PLC β3(p-PLC β3) in brain homogenate, and to verify its signaling pathway. The contents of thromboxane B2 (TXB2), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in brain homogenate were detected by enzyme-linked immunosorbent assay. The expression of P-selectin and neutrophil surface adhesion molecule (CD11b) on platelet activation and neutrophil activation was detected by immunofluorescence. H3BB (H3B), myeloperoxidase (MPO) and deoxyribonucleic acid (DNA) were detected by three-color immunofluorescence.Results Compared with the control group, the neurological function score, brain index, cerebral infarction rate, and the protein expressions of IL-1β, IL-6, and TXB2 in brain homogenate of rats in the model group were significantly increased (P<0.01), while the protein expression of p-PLC β3 was significantly decreased (P<0.01), and a large number of NETs were formed. The expressions of P-selectin and CD11b were both increased (P<0.01). Compared with the model group, Sul-F significantly reduced the neurological function score, brain index, cerebral infarction rate, and the protein expressions of IL-1β, IL-6, and TXB2 in brain homogenate (P<0.05, 0.01); Significantly increased the protein expression of p-PLC β3 (P<0.05, 0.01); The production of NETs was significantly reduced, and the protein expressions of P-selectin and CD11b were significantly decreased (P<0.05, 0.01).Conclusion Sul-F improves cerebral I/R injury by promoting the expression of p-PLC β3 protein, inhibiting PLC β3 expression, reducing platelet activation, inhibiting NETs formation, and reducing inflammatory response.

R285.5

河北省科学技术厅石家庄平安医院有限公司院士合作重点单位项目