大鼠脑出血后NF-κB诱导LCN2激活NLRP3炎症小体的研究

NF-κB-induced activation of NLRP3 inflammasome by LCN2 after intracerebral hemorrhage in rats

目的探讨脑出血时脑内运铁蛋白LCN2表达升高的机制及参与出血后炎症反应的机制。方法雄性SD大鼠基底节区注射自体动脉血(100μl)。对照组大鼠术式相同注射等量生理盐水。用NF-κB特异性抑制剂parthenolide和BAY 11-7082脑室注射预处理,用乱序LCN2siRNA或LCN2 siRNA转染后,检测NLRP3、ASC和裂解的Caspase-1蛋白水平作为炎症小体激活状态的指标。Western blot、RT-PCR及免疫组化方法检测LCN2 mRNA和蛋白质的表达水平。结果Western blotting定量发现在出血侧基底节区,出血后1 d、3 d、7 d LCN2蛋白水平显著升高,14 d后下降。脑出血3 d时出血侧LCN2蛋白水平较对侧高71倍(2.70±0.46 vs 0.04±0.01,P<0.001),较对照组高84倍(0.92±0.14 vs 0.01±0.01,P<0.001)。用NF-κB特异性抑制剂BAY117082和parthenolide预处理可抑制LCN2的表达。在乱序siRNA转染后,NLRP3、ASC和裂解的Caspase 1的蛋白质水平显著增加。LCN2 siRNA使这些炎症小体相关蛋白的表达显著减弱。结论本研究提示脑出血后释放的铁离子诱导载铁蛋白LCN2表达。LCN2在出血后的炎症过程中可能起重要作用。

Objective To investigate the mechanism of the elevated expression of LCN2 during intracerebral hemorrhage(ICH)and its involvement in inflammatory response after hemorrhage.Methods Male Sprague-Dawley rats were given an injection of autologous arterial blood(100μl)at the basal ganglia,and the rats in the control group were given an injection of an equal volume of normal saline using the same surgical procedure.Ventricular injection of the NF-κB-specific inhibitors parthenolide and BAY11-7082 was performed for pretreatment,and after transfection with scrambled LCN2RNA or LCN2 siRNA,the levels of NLRP3,ASC,and cleaved caspase-1 were measured as the indicators for the status of inflammasome activation.Western blot,RT-PCR,and immunohistochemistry were used to measure the protein and mRNA expression levels of LCN2.Results Western blotting showed that there was a significant increase in the protein expression level of LCN2 in the ipsilateral basal ganglia on days 1,3,and 7 after hemorrhage and a significant reduction in this level on day 14.On day 3 after intracerebral hemorrhage,the protein expression level of LCN2 in the ipsilateral side was 71 times higher than that in the contralateral side(2.70±0.46 vs 0.04±0.01,P<0.001)and was 84 times higher than that in the control group(0.92±0.14 vs 0.01±0.01,P<0.001).Pretreatment with the NF-κB-specific inhibitors parthenolide and BAY11-7082 could inhibit the expression of LCN2.There were significant increases in the protein expression levels of NLRP3,ASC,and cleaved caspase-1 after transfection with scrambled siRNA,and LCN2 siRNA significantly reduced the expression of these inflammasome-associated proteins.Conclusion This study suggests that iron release after ICH can induce the expression of LCN2,and LCN 2 may play an important role in the inflammatory process after hemorrhage.