摘要
目的 研究核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎性小体抑制剂MCC950通过抑制小胶质细胞焦亡对大鼠脑出血预后的影响。方法 54只成年雄性SD(Sprague Dawley)大鼠,按简单随机法将其分为对照组、脑出血组和MCC950治疗组,每组18只。对照组仅做钻孔处理;脑出血组经胶原酶诱导脑出血;MCC950治疗组在使用胶原酶诱导脑出血之前,先通过侧脑室注射MCC950。Longa五级评分1~3分为造模成功,分别于造模成功后24 h和72 h对3组大鼠相关指标进行检测及组间比较,即利用蛋白免疫印迹(WB)分析和免疫荧光测定血肿周围组织中小胶质细胞NLRP3炎性小体、含半胱氨酸的天冬氨酸蛋白水解酶1(Caspase-1)和成熟白细胞介素1β(IL-1β)的蛋白表达量,同时行苏木素-伊红(HE)染色观察炎性细胞浸润程度。用干湿重法测定脑水肿,用改良神经功能损伤严重程度评分(mNSS)评估大鼠脑出血恢复程度。取27只新生SD乳鼠(0~24 h),提取原代小胶质细胞,随机分为对照组、凝血酶激活组、MCC950治疗组,每组细胞数为5×10~5个/ml,共2 ml。对照组为不进行任何干预措施;凝血酶激活组应用20 U凝血酶进行激活;MCC950治疗组使用0.2 nmol MCC950干预1 h后加入20 U凝血酶激活。于干预后24 h分别对3组原代小胶质细胞相关指标进行检测及组间比较。透射电镜下观察小胶质细胞形态,检测细胞焦亡的发生,并测定乳酸脱氢酶(LDH)释放率。应用WB法分别测定3组小胶质细胞中NLRP3、活化Caspase-1、成熟IL-1β和凋亡相关斑点样蛋白(ASC)的蛋白表达量。结果 (1)体外实验结果显示,对照组、凝血酶激活组、MCC950治疗组小胶质细胞LDH释放率分别为(4.0±0.5)%、(21.0±0.7)%、(14.0±0.8)%,3组间差异有统计学意义(F=1 178.172,P<0.01);凝血酶激活组LDH释放率高于对照组,MCC950治疗组LDH释放率低于凝血酶激活组,差异均有统计学意义(均P<0.01)。(2)体外实验结果显示,�
Objective To investigate whether nucleotide-binding oligomerization domain-like receptor protein(NLRP)3 inflammasome inhibitor MCC950 can inhibit microglia pyroptosis and improve the prognosis of intracerebral hemorrhage in rats. Methods Fifty-four adult male Sprague Dawley(SD) rats were randomly divided into control group, intracerebral hemorrhage group and MCC950 treatment group, with 18 rats in each group. The control group was treated with drilling. Intracerebral hemorrhage was induced by collagenase in intracerebral hemorrhage group. In the MCC950 treatment group, MCC950 was injected through the lateral ventricle before collagenase was used to induce intracerebral hemorrhage. The Longa score of 1-3 was considered as successful modeling. The relevant indexes of the three groups were detected and compared between groups at 24 h and 72 h after successful modeling. The protein expressions of microglia NLRP3 inflammasome, activated cysteinyl aspartate specific proteinase 1(Caspase-1) and mature interleukin-1β(IL-1β) in microglial cells around hematoma were determined by western blotting(WB) and immunofluorescence. Hematoxylin-eosin(HE) staining was used to observe the degree of inflammatory cell infiltration. Brain edema was measured by dry wet weight method, and the recovery degree of intracerebral hemorrhage was evaluated by modified neurological severity score(mNSS). Primary microglia were extracted from 27 neonatal SD Suckling rats(0-24 h) and randomly divided into control group, thrombin activation group and MCC950 treatment group, with 5×10~5 cells/ml in each group(total 2 ml). The control group received no intervention. The thrombin activation group was activated with 20 U thrombin. MCC950 treatment group was treated with 0.2 nmol MCC950 for 1 h, and then 20 U thrombin was added for activation. At 24 h after intervention, the related indexes of primary microglia were detected and compared among the three groups. The morphology of microglia, pyroptosis and lactate dehydrogenase(LDH) release rate were obs
作者
梁洪生
程兴博
方晓丰
桂斌斌
荣毅伟
马威
姜振锋
蔺铁
张相彤
Liang Hongsheng;Cheng Xingbo;Fang Xiaofeng;Gui Binbin;Rong Yiwei;Ma Wei;Jiang Zhenfeng;Lin Tie;Zhang Xiangtong(Department of Neurosurgery,First Affiliated Hospital of Harbin Medical University,Harbin 150001,China)
出处
《中国脑血管病杂志》
CAS
CSCD
北大核心
2022年第9期618-628,共11页
Chinese Journal of Cerebrovascular Diseases
基金
国家自然科学基金(81571108)。
