脑泰方对脑出血急性期大鼠脑铁代谢的干预作用及神经保护机制

Effect of Naotaifang(脑泰方)on Cerebral Iron Metabolism and Its Neuroprotective Mechanism in Rats with Acute Intracerebral Hemorrhage

目的:研究脑泰方对脑出血急性期大鼠脑组织铁代谢的调节作用,从铁死亡角度探究其神经保护机制。方法:采用自体血注射法构建SD大鼠脑出血模型,分为假手术组、模型组、去铁胺组、脑泰方常规剂量组、脑泰方高剂量组,各组于造模并给药7 d后取材;采用HE染色观察脑组织病理形态改变,采用Zea Longa 5级评分法进行大鼠神经功能缺失评分,采用普鲁士蓝染色观察并检测病灶脑组织铁沉积量,采用免疫组化和Western Blotting技术检测转铁蛋白受体(TfR)、铁调节蛋白-2(IRP-2)、膜铁转运蛋白-1(Fpn-1)的表达,采用生物试剂盒检测病灶脑组织脂质活性氧(lipid-ROS)含量,采用Western Blotting技术检测铁死亡标志物环氧合酶-2(COX-2)的表达。结果:与假手术组比较,模型组大鼠脑组织病理损伤明显加重,神经功能缺失评分明显升高,TfR、IRP-2表达均明显升高,Fpn-1表达明显降低,病灶脑组织铁沉积量、lipid-ROS含量及COX-2表达均明显升高,差异均有统计学意义(P<0.05或P<0.01)。与模型组比较,去铁胺组及脑泰方各剂量组病理损伤明显减轻,神经功能缺失评分明显降低,TfR、IRP-2表达均明显降低,Fpn-1表达明显升高,脑组织铁沉积量、lipid-ROS含量及COX-2表达均明显降低(P<0.05或P<0.01);脑泰方高剂量组铁沉积量高于去铁胺组,而其Lipid-ROS含量及COX-2表达均明显低于去铁胺组与脑泰方常规剂量组,差异均有统计学意义(P<0.05)。结论:脑泰方可通过调节脑出血急性期大鼠神经细胞铁代谢蛋白的表达,减少脑组织铁沉积,从而抑制神经细胞铁死亡而发挥脑保护作用;铁超载并非神经细胞铁死亡的唯一因素,高剂量的脑泰方还可能通过其他途径抑制神经细胞铁死亡而发挥脑保护作用。

Objective:To study the regulatory effect of Naotaifang on iron metabolism in the brain tissue of rats with acute intracerebral hemorrhage,and to explore its neuroprotective mechanism from the perspective of ferroptosis.Methods:The model of intracerebral hemorrhage in SD rats was established by autogenous blood injection.They were divided into sham operation group,model group,deferoxamine group,Naotaifang conventional dose group and Naotaifang high dose group.The rats in each group were taken after 7 days of modeling and administration.The pathological changes of brain tissue were observed by HE staining,and the neurological deficit score was evaluated by Zea longa 5-grade scoring method.Prussian blue staining was used to observe and detect the amount of iron deposition in the brain tissue of the lesion.Immunohistochemistry and Western Blotting te chnology were used to detect the expression of transferrin receptor(TfR),iron regulatory protein-2(IRP-2),and ferroportin-1(Fpn-1).Biological kits were used to detect the content of lipid reactive oxygen species(lipid-ROS)in the brain tissue of the lesions,and Western Blotting technology was used to detect the expression of the ferroptosis marker cyclooxygenase-2(COX-2).Results:Compared with the sham operation group,the pathological damage of brain tissue in model group was significantly aggravated,the score of neurological deficit was significantly increased,the expression of TfR and IRP-2 was significantly increased,the expression of Fpn-1 was significantly decreased,the contents of iron and lipid-ROS and the expression COX-2 were significantly increased(P<0.05 or P<0.01).Compared with model group,the pathological damage of the deferoxamine group and the groups of Naotaifang was obviously alleviated,the score of neurological deficit was obviously decreased,the expression of TfR and IRP-2 was significantly decreased,the expression of fpn-1 was significantly increased,the contents of iron and lipid-ROS and the expression of COX-2 were significantly decreased(P<0.05 or P<0.01).The content of iron in the deferoxamine group was significantly lower than that in the groups of Naotaifang,while the content of lipid-ROS and the expression of COX-2 in the Naotaifang high dose group were significantly lower than that in the deferoxamine group and the Naotaifang conventional dose group(P<0.05).Conclusions:Naotaifang can reduce iron deposition in brain tissue and inhibit ferroptosis by regulating the expression of iron metabolizing proteins in nerve cells of rat models with intracerebral hemorrhage,so as to play a neuroprotective role.Iron overload is not the only factor of ferroptosis,high dose of Naotaifang may also inhibit ferroptosis through other ways.