【摘要】 目的:探讨体外培育牛黄（ICCB）减轻脑缺血再灌注损伤（CIRI）的机制。方法:分别构建大脑中动脉阻塞（MCAO）大鼠模型和氧糖剥夺/再灌注（OGD/R）BV2细胞模型。其中，大鼠模型于脑缺血1.5 h再灌注72 h后进行改良的神经损伤严重程度评分（mNSS），并测定脑组织含水量和脑梗死体积；苏木精-伊红染色法检测大脑皮层及海马CA1区组织结构病理变化；免疫荧光法检测皮层小胶质细胞极化情况和NOD样受体热蛋白结构域相关蛋白（NLRP）3炎症小体的表达情况。BV2细胞于氧糖剥夺0.5 h再灌注24 h后，经ICCB和尼日利亚菌素等药物处理并采用MTT法检测细胞存活率。蛋白质印迹法检测OGD/R细胞中NLRP3/含CARD抗体的凋亡相关斑点样蛋白（ASC）/胱天蛋白酶（caspase）1通路蛋白及炎症因子的表达情况。观察NLRP3特异性激动剂尼日利亚菌素预处理24 h对ICCB改善OGD/R细胞活性、炎症因子以及通路相关蛋白的影响。结果:ICCB治疗显著改善大鼠的神经功能，减少脑梗死体积和脑含水量，改善CIRI大鼠皮层和海马CA1区的病理损伤（均P<0.05）。ICCB可刺激大鼠皮层小胶质细胞由M1型向M2型转化，并抑制小胶质细胞中NLRP3的活化（均P<0.01）。ICCB还可以显著降低OGD/R细胞模型中NLRP3、ASC、caspase-1蛋白的表达以及IL-18和IL-1β炎症因子的分泌（均P<0.01）。此外，NLRP3激动剂尼日利亚菌素可逆转ICCB对模型细胞的保护作用及对炎症因子的调控作用（均P<0.05）。结论:ICCB对CIRI具有保护作用，这种保护作用可能与减少小胶质细胞M1型极化、促进其向M2型转化，抑制NLRP3/ASC/caspase-1信号转导从而抑制神经炎症有关。更多还原

【Abstract】 Objective: To investigate the effect of in vitro cultured calculus bovis(ICCB) on cerebral ischemia/reperfusion injury(CIRI) and its mechanism. Methods: A CIRI rat model and a cell model were induced by middle cerebral artery occlusion(MCAO) in Sprague Dawley rats and oxygen glucose deprivation/reperfusion(OGD/R) in BV2 cells, respectively. The CIRI rat model was evaluated using the modified neurological severity score(mNSS), brain water content, and cerebral infarction volume after 1.5 h of ischemia followed by 72 h of reperfusion. Histopathological changes in the cortex and hippocampal CA1 region were observed with hematoxylin and eosin staining. Microglial polarization and NOD-like receptor thermal protein domain associated protein(NLRP) 3 inflammasome expression in the cortex were examined by immunofluorescence. BV2 cell viability was measured via MTT assay after treatment with ICCB and Nigericin. The expressions of NLRP3, ASC, caspase-1 proteins and inflammatory cytokines were detected with Western blotting in OGD/R treated BV2 cells(0.5 h OGD+24 h reperfusion) and in cells pretreated with Nigericin for 24 h. Results: ICCB treatment significantly improved neurological function, reduced cerebral infarct volume and brain water content, and mitigated pathological damage in the cortical and hippocampal CA1 regions of rats subjected to CIRI(all P<0.05). ICCB promoted the transition of cortical microglia from M1 to M2 phenotypes and suppressed NLRP3 activation in microglial cells(all P<0.01). ICCB significantly down-regulated the expression of NLRP3, ASC, and caspase-1 proteins, and reduced the secretion of IL-18 and IL-1β in BV2 cells of OGD/R model(all P<0.01). In addition, Nigericin significantly reversed the salvage effect of ICCB on model cells(both P<0.01) and the modulation of inflammatory cytokines(P<0.05). Conclusion: ICCB exerts a protective effect against CIRI by mitigating neuroinflammation, through the reduction of M1 microglial polarization, promotion of M2 conversion, and suppression of the NLRP3/ASC/caspase-1 signaling pathway.更多还原