基于NLRP3炎症小体介导的细胞焦亡探究黄竹清脑颗粒对脑缺血再灌注损伤大鼠的神经保护作用

Exploring the neuroprotective effect of Huangzhu Qingnao Granules on rats with cerebral ischemia-reperfusion injury based on NLRP3 inflammasome-mediated pyroptosis

关键词:  黄竹清脑颗粒  脑缺血再灌注损伤  CIRI  MCAO/R  NLRP3  细胞焦亡

Key words:  Huangzhu Qingnao Granule  cerebral ischemia-reperfusion injury  CIRI  MCAO/R  NLRP3  pyroptosis

摘要:

[目的] 研究黄竹清脑颗粒对大脑中动脉栓塞再灌注模型（MCAO/R）大鼠的神经保护作用以及对NOD样受体蛋白3（NLRP3）炎症小体介导的细胞焦亡途径的影响。[方法] 40只雄性SD大鼠随机分为假手术组、模型组、丁苯酞组及黄竹清脑颗粒组。采用MCAO/R大鼠模拟脑缺血再灌注损伤（CIRI），造模成功后以相应药物灌胃4周。Zea-longa评分法对大鼠神经功能缺失进行评估；氯化三苯基四氮唑（TTC）染色法测定脑梗死面积；苏木精-伊红（HE）染色观察脑组织病理形态；酶联免疫吸附法（ELISA）检测脑组织中白细胞介素-18（IL-18）、白细胞介素-1β（IL-1β）、血清淀粉样蛋白A（SAA）水平；TUNEL染色观察细胞凋亡水平；免疫荧光双染法检测脑组织中离子钙结合衔接分子-1（IBA-1）、NLRP3阳性表达情况；蛋白免疫印迹法（Western Blot）检测脑组织中NLRP3、Gasdermin D蛋白（GSDMD）、Gasdermin D 蛋白 N 端结构域（GSDMD-N）、半胱天冬酶-1（Caspase-1）、活化半胱天冬酶-1（Cleaved-Caspase-1）、胶质纤维酸性蛋白（GFAP）蛋白相对表达量。[结果] 与模型组比较，黄竹清脑颗粒组大鼠Zea-longa评分及脑梗死面积均不同程度降低（P<0.05或P<0.01），脑组织病理损伤有一定程度减轻，脑组织中IL-18、IL-1β、SAA水平均明显下降（P<0.01）；IBA-1、NLRP3蛋白阳性表达明显下降（P<0.01）；NLRP3、GSDMD-N、Cleaved-Caspase-1、GFAP蛋白相对表达量均明显降低（P<0.01）。[结论] 黄竹清脑颗粒可能通过抑制NLRP3炎症小体介导的细胞焦亡，减少神经促炎因子的释放，减轻MCAO/R大鼠的神经病理损伤及神经运动功能缺损，从而治疗CIRI。

Abstract:

[Objective] To investigate the neuroprotective effect of Huangzhu Qingnao Granules on rats with middle cerebral artery occlusion and reperfusion（MCAO / R）model，and its influence on the NLRP3 inflammasome-mediated pyroptosis pathway. [Methods] Forty male Sprague-Dawley（SD）rats were randomly divided into sham operation group，model group，butylphthalide group， and Huangzhu Qingnao Granules group. The MCAO/R model was used to simulate cerebral ischemia-reperfusion injury（CIRI），and corresponding drugs were administered by gavage for 4 weeks after successful modeling. Neurological deficits were evaluated by Zea-longa scoring；cerebral infarction area was determined by TTC staining；pathological morphology of brain tissue was observed by HE staining；levels of IL- 18，IL-1β，and SAA in brain tissue were detected by ELISA；cell apoptosis was observed by TUNEL staining；positive expression of IBA-1 and NLRP3 in brain tissue was detected by immunofluorescence double staining；relative expressions of NLRP3，GSDMD，GSDMD-N，Caspase-1，Cleaved-Caspase-1，and GFAP in brain tissue were detected by Western Blot. [Results] Compared with the model group，the Zea-longa score and cerebral infarction area in the Huangzhu Qingnao Granules group were significantly reduced（P<0.05 or P<0.01），and pathological damage of brain tissue was alleviated. The levels of IL-18，IL-1β，and SAA in brain tissue were significantly decreased（P<0.01）. The positive expressions of IBA-1 and NLRP3 proteins were significantly reduced（P<0.01），and the relative expressions of NLRP3，GSDMD-N，Cleaved-Caspase-1，and GFAP proteins were significantly decreased（P<0.01）. [Conclusion] Huangzhu Qingnao Granules may treat CIRI by inhibiting NLRP3 inflammasome-mediated pyroptosis，reducing the release of pro-inflammatory neurofactors，and alleviating neuropathological damage and neuromotor function deficits in MCAO/R rats.