清通化瘀汤对脑缺血再灌注损伤铁死亡相关基因GPX4、Tf的影响
Effects of Qingtong Huayu Decoction on ferroptosis-related genes GPX4 and Tf in cerebral ischemia-reperfusion injury
胡嘉婧 1左龙 1曲文雨 1李甜 1祝美珍1
作者信息
摘要
目的:研究清通化瘀汤对脑缺血再灌注损伤(CIRI)铁死亡相关基因谷胱甘肽过氧化物酶4(GPX4)、转铁蛋白(Tf)的影响,探究清通化瘀汤防治CIRI的作用机制.方法:240只健康雄性SD大鼠根据体质量随机分为空白组、假手术组、模型组、清通化瘀汤组(2.01 g/kg)和通心络组(0.12 g/kg).按术后再灌注取材时间点分为4个亚组,即3h、24 h、3d和7d,每组12只.各组在制备模型前7 d开始灌胃相应蒸馏水和汤药,直至取材结束,在缺血2 h和取材前对各组大鼠进行神经功能缺损评分,采用DCFH-DA法检测脑组织细胞中活性氧(ROS),根据组织铁含量检测试剂盒测量组织内铁含量的积累,采用免疫组化法检测大鼠缺血侧Tf阳性细胞的表达,采用蛋白免疫印迹法和实时荧光定量PCR法检测大鼠缺血侧GPX4、Tf蛋白及mRNA表达.结果:与空白组和假手术组比较,模型组ROS相对表达水平及Fe24含量明显升高,Tf蛋白及mRNA表达明显升高,GPX4蛋白及mRNA表达明显降低,Tf阳性细胞的表达明显增加.与模型组比较,清通化瘀汤组和通心络组ROS相对表达水平、Fe2+含量、Tf阳性细胞、Tf蛋白及mRNA表达均显著降低(P＜0.05),GPX4蛋白及mRNA表达显著升高(P＜0.05).与通心络组比较,清通化瘀汤对ROS,Fe2+含量,Tf阳性细胞,Tf、GPX4蛋白与mRNA表达的影响更为显著(P＜0.05).结论:清通化瘀汤可以减缓ROS、Fe2+的蓄积,下调Tf的表达和上调GPX4的表达,减轻CIRI,促进脑组织损伤的修复而发挥脑保护的作用.
Abstract
Objective:To study the effects of Qingtong Huayu Decoction on ferroptosis-related genes GPX4 and Tf in cerebral ischemia-reperfusion injury(CIRI),and to explore the mechanism of Qingtong Huayu Decoction in the prevention and treatment of CIRI.Methods:A total of 240 healthy male SD rats were randomly divided into Blank group,Sham group,Ⅰ/R group,QTH group(2.01 g/kg)and TXL group(0.12 g/kg).The rats were divided into 4 subgroups according to the time point of harvesting after reperfusion,namely 3 h,24 h,3 d and 7 d,with 12 rats in each subgroup.The rats in each group were given intragastric administration(distilled water and decoction,respectively)for 7 days before model preparation until the end of sampling.The neurological deficit score was evaluated at 2 hours after ischemia and before sampling.DCFH-DA method was used to detect reactive oxygen species(ROS)in brain tissue cells,and the accumulation of iron content in brain tissue was measured according to the tissue iron content detection kit.Immunohistochemistry was used to detect the expression of Tf positive cells on the ischemic side of rats.The protein and mRNA expressions of GPX4 and Tf on the ischemic side were detected by Western Blot and Real-Time PCR.Results:Compared with the blank group and sham-operation group,the model group had significantly increased ROS and Fe2+content,significantly increased Tf protein and mRNA expression,significantly decreased GPX4 protein and mRNA expression,and significantly increased Tf positive cells expression.Compared with the model group,the contents of ROS and Fe2+,Tf positive cells,Tf protein and mRNA expression in the Qingtong Huayu Decoction group and Tongxinluo group were significantly decreased(P＜0.05),GPX4 protein and mRNA expression were significantly increased(P＜0.05).Compared with Tongxinluo group,Qingtong Huayu Decoction had more significant effects on ROS,Fe2+content,Tf positive cells,Tf protein and mRNA expression,and GPX4 protein and mRNA expression(P＜0.05).Conclusion:Qingtong Huayu Decoction can reduce the accumulation of ROS and Fe2+,down-regulate the expression of Tf and up-regulate the expression of GPX4,alleviate CIRI,and promote the repair of brain tissue damage to play a protective role in brain injury.
关键词
脑缺血再灌注损伤/铁死亡/谷胱甘肽过氧化物酶4/转铁蛋白/清通化瘀汤Key words
Cerebral ischemia-reperfusion injury/Ferroptosis/Glutathione peroxidase 4(GPX4)/Iron-loaded transferrin(Tf)/Qingtong Huayu Decoction引用本文复制引用
基金项目
广西自然科学基金(2021JJA140355)
广西中医药大学研究生教育创新项目基金(YCSW2021222)
广西中医药大学自治区重点实验室课题(20-065-53-04)
出版年
2024
NETL
NSTL
万方数据