牛乳铁蛋白对大鼠脑出血的治疗作用及其与铁代谢和脂质过氧化的关系

Therapeutic effect of bovine lactoferrin on cerebral hemorrhage in rats and its relationship with iron metabolism and lipid peroxidation

目的观察牛乳铁蛋白(bLF)抑制脑出血大鼠铁代谢及脂质过氧化的作用,并探讨其作用机制。方法取成年健康雄性SD大鼠90只,随机分为假手术组、模型组、bLF干预组各30只,采用两步法自体血右侧尾状核注射制作脑出血模型。造模成功后,bLF组给予bLF 85 mg/(kg·d)灌胃,假手术组及模型组给予等量生理盐水灌胃,连续7 d。分别于造模后1、3、7 d,采用改良神经功能缺失评分(mNSS评分)评价大鼠神经缺损情况;处死大鼠,取脑组织,采用HE染色法观察组织病理改变;使用生物试剂盒检测铁离子及活性氧(ROS)含量;Westernblotting法检测IRE/IRP调节系统中的铁蛋白、转铁蛋白受体(TFR)、二价铁金属转运体1(DMT1)、铁调节蛋白2(IRP2)及抗氧化剂谷胱甘肽过氧化物酶4(GPX4)表达;采用Pearson相关分析法分析bLF干预组铁代谢相关蛋白铁蛋白、TFR、DMT1、IRP2与抗氧化酶GPX4的相关性。结果与假手术组相比,模型组脑组织病理损伤明显严重;mNSS评分升高(P均<0.05),在第3天时评分达最高后下降;铁离子及ROS含量增高(P均<0.05),第3天与第7天相比无统计学意义;IRE/IRP调节系统相关蛋白(铁蛋白、TFR、DMT1、IRP2)表达均上升(P均<0.05),在第3天时增加至最高后下降;抗氧化剂GPX4表达减少(P均<0.05),在第3天时表达最低后上升。与模型组相比,bLF组脑组织病理损伤减轻,mNSS评分下降(P均<0.05);铁离子及ROS含量、铁蛋白、TFR、DMT1、IRP2表达均下降(P均<0.05),GPX4表达增多(P均<0.05)。相关性分析显示,bLF组GPX4与铁蛋白、TFR、DMT1、IRP2表达均呈负相关(P均<0.05)。结论牛乳铁蛋白可能下调IRE/IRP调节系统中铁蛋白、TFR、DMT1、IRP2表达,减少铁离子与ROS含量,同时上调抗氧化剂GPX4表达,从而减轻脑出血后脂质过氧化、抑制铁死亡,发挥神经保护作用。

Objective To investigate the effects of bovine lactoferrin(bLF)in inhibiting iron metabolism and lipid peroxidation in rats with intracerebral hemorrhage,and to explore its mechanism of action.Methods Ninety adult healthy male SD rats were randomly divided into sham-operated group,model group and bLF intervention group,with 30 rats in each.The two-step autologous blood right caudate nucleus injection was used to establish the intracerebral hemor⁃rhage models.After successful modeling,the rats in the bLF group were given bLF 85 mg/(kg-d)by gavage,and the rats in the sham-operated group and the model group were given the same amount of normal saline by gavage for 7 d.The neuro⁃logical deficits were evaluated by the modified Neurological Severity Score(mNSS)at 1,3 and 7 d after modeling,respec⁃tively.After executing rats to extract brain tissues,the histopathological changes were observed by HE staining;iron and reactive oxygen species(ROS)content were detected using a bioreagent kit.The expression levels of ferritin,transferrin receptor(TFR),divalent iron metal transporter(DMT1),iron regulatory protein 2(IRP2)and the antioxidant glutathione peroxidase 4(GPX4)in the IRE/IRP regulatory system were detected by Western blotting.The correlation between the iron metabolism-related proteins ferritin,TFR,DMT1,IRP2 and the antioxidant enzyme GPX4 in the bLF intervention group was analyzed by Pearson correlation analysis.Results Compared with the sham-operated group,brain histopatho⁃logical damage was significantly more severe in the model group;mNSS increased(all P<0.05)and decreased after reach⁃ing the highest score on day 3;iron and ROS content levels increased(all P<0.05),but there was no statistically signifi⁃cant difference between the third day and the seventh day;IRE/IRP regulatory system-related proteins(ferritin,TFR,DMT1,IRP2)expression increased(P<0.05),and it increased to the highest level on day 3 and then decreased;the ex⁃pression of antioxidant GPX4 decreased(P<0.05),and it reached the lowest level on day 3 and then increased.Com⁃pared with the model group,the bLF group showed and itreduced brain histopathological damage and decreased mNSS score(both P<0.05),decreased expression of iron and ROS content,IRE/IRP regulatory system-related proteins(ferri⁃tin,TFR,DMT1,IRP2)(all P<0.05),and increased expression of antioxidant GPX4(P<0.05).Correlation analysis showed that the antioxidant GPX4 was negatively correlated with the expression of ferritin,TFR,DMT1,and IRP2 in the bLF group(all P<0.05).Conclusions Bovine lactoferrin may down-regulate the expression of ferritin,TFR,DMT1,and IRP2 in the IRE/IRP regulatory system and reduce iron and ROS content,and meanwhile,it up-regulates the expres⁃sion of the antioxidant GPX4,thereby reducing lipid peroxidation and inhibiting ferroptosis after intracerebral hemorrhage and exerting neuroprotective effects.