摘要
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(PLCβ3)和磷酸化PLCβ3(p-PLCβ3)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-PLCβ3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-PLCβ3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-PLCβ3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(PLCβ3)and phosphorylated PLCβ3(p-PLCβ3)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-PLCβ3 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-PLCβ3(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-PLCβ3 protein,inhibiting PLCβ3 expression,reducing platelet activation,inhibiting NETs formation,and reducing inflammatory response.
作者
李思博
万彦军
陈娟
庄佳慧
李雅楠
黄怀鹏
LI Sibo;WAN Yanjun;CHEN Juan;ZHUANG Jiahui;LI Yanan;HUANG Huaipeng(College of Pharmacy,Hebei University of Traditional Chinese Medicine,Shijiazhuang 050091,China;State Key Laboratory of Bioactive Substance and Function of Natural Medicines,Institute of Materia Medica,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100050,China;Department of Traditional Chinese Medicine,Shijiazhuang Ping’an Hospital,Shijiazhuang 050051,China;College of Integrated Traditional Chinese and Western Medicine,Hebei University of Traditional Chinese Medicine,Shijiazhuang 050091,China;Department of Clinical Laboratory,Shijiazhuang Ping’an Hospital,Shijiazhuang 050051,China)
出处
《药物评价研究》
北大核心
2025年第7期1798-1806,共9页
Drug Evaluation Research
基金
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。
作者简介
李思博(1998-),女,硕士研究生,研究方向为中药生产与质量控制。E-mail:1399378938@qq.com;通信作者:黄怀鹏(1971-),男,医学博士,研究员,研究方向为中西医结合临床。E-mail:huang71526@163.com;通信作者:李雅楠(1989-),女,医学硕士,副主任检验技师,研究方向为临床检验诊断学。E-mail:niwawa941@163.com。
