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。
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-1βin 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.
作者
褚坦路
章伟
陈静文
潘泽玥
王凌峰
钟晓明
仇凤梅
黄真
CHU Tanlu;ZHANG Wei;CHEN Jingwen;PAN Zeyue;WANG Lingfeng;ZHONG Xiaoming;QIU Fengmei;HUANG Zhen(School of Pharmaceutical Sciences,Zhejiang Chinese Medical University,Hangzhou 310053,China;Jinhua Academy ofZhejiang Chinese Medical University,Jinhua 321053,Zhejiang Province,China)
出处
《浙江大学学报(医学版)》
北大核心
2025年第3期360-371,共12页
Journal of Zhejiang University(Medical Sciences)
基金
浙江省自然科学基金(LY22H280010)
国家自然科学基金(82274330)
浙江中医药大学校级科研项目(2021ZY14)。
