硝石雄黄散对血管内皮细胞缺氧损伤的保护机制

Protective mechanism of Xiaoshi Xionghuang San on hypoxia injury of vascular endothelial cells

目的:探索硝石雄黄散对血管内皮细胞缺氧损伤的保护机制。方法:采用Griess法检测中药硝石(XS)、雄黄(XH)、硝石雄黄散(XSXH)中硝酸盐(NO3-)和亚硝酸盐(NO2-)含量及各组小鼠血清一氧化氮(NO)水平;制备硝石雄黄散各组分药的小鼠血清,将人冠状动脉内皮细胞(HCAEC)随机分为空白(Blank)组、缺氧(Hypoxia)组、Hypoxia+XS组、Hypoxia+XH组和Hypoxia+XSXH组,Blank常氧处理,其余组别予缺氧24 h制备细胞缺氧模型;利用小鼠血清干预HCAEC,采用四甲基罗丹明甲酯(TMRM)和线粒体超氧化物指示剂(MitoSox)检测各组HCAEC线粒体膜电位及活性氧(ROS)水平;Griess法检测各组HCAEC中NO水平及ELISA法检测ET-1、IL-6和TNF-α水平。结果:XSXH组NO2-含量高于XS组而NO3-含量较XS组低(P<0.001),XH组NO3-和NO2-含量最低(P<0.001,P<0.01);与Blank组相比,XS组、XH组和XSXH组均能提高小鼠血清NO水平(P<0.05,P<0.01,P<0.001);细胞结果显示,Hypoxia组能显著降低细胞NO生物活性、升高ET-1和炎症因子(IL-6、TNF-α)水平、线粒体膜电位降低及生成大量ROS(P<0.001);与Hypoxia组相比,Hypoxia+XS组、Hypoxia+XH组和Hypoxia+XSXH组对上述指标均有明显改善(P<0.05,P<0.01,P<0.001),其中Hypoxia+XSXH组作用最强,Hypoxia+XS组强于Hypoxia+XH组(P<0.05)。结论:XS、XH和XSXH均能不同程度地提高NO生物活性,通过降低ET-1水平和炎症因子水平,恢复细胞线粒体膜电位以及减少ROS生成来保护血管内皮细胞缺氧损伤,其中XSXH作用最强。

Objective:To explore the protective mechanism of Xiaoshi Xionghuang San on vascular endothelia cell hypoxia injury.Methods:Griess method was used to detect the content of nitrate (NO3-) and nitrite (NO2-) in Chinese medicine Xiaoshi (XS),Xionghuang (XH),and Xiaoshi Xionghuang San (XSXH),and the level of serum nitric oxide (NO) in mice of each group.Mouse serum of each component of Xiaoshi Xionghuang San was prepared and human coronary crtery endothelial cells (HCAEC) were divided into Blank,Hypoxia,Hypoxia+XS Hypoxia+XH and Hypoxia+XSXH groups.Blank group was treated with normoxia,and the other groups were given hypoxia for 24 h to prepare cell hypoxia models.Serum of mice was used to intervene HCAEC;tetramethylrhodamine (TMRM) and mitochondrial superoxide indicator (MitoSOX) were used to detect mitochondrial membrane potential and reactive oxygen species (ROS) levels of HCAEC in each group.The levels of nitric oxide (NO) in HCAEC of each group were detected by Griess and the levels of ET-1 and IL-6and TNF-α were detected by ELISA.Results:The content of NO2-in XSXH group was higher than that in XS group and the content of NO3-was lower than that in XS group (P<0.001),and the content of NO3-and NO2-was the lowest in XH group(P<0.001,P<0.01).Compared with Blank group,levels of serum NO of mice in XS group,XH group and XSXH group increased (P<0.05,P<0.01,P<0.001).Cell results showed that Hypoxia group could significantly reduce the biological activity of NO in cells,increase the levels of ET-1 and inflammatory factors (IL-6,TNF-α),decrease mitochondrial membrane potential and generate a large number of ROS (P<0.001).Compared with Hypoxia group,the above indicators in the Hypoxia+XS,Hypoxia+XH and Hypoxia+XSXH groups significantly improved (P<0.05,P<0.01,P<0.001),of which the Hypoxia+XSXH group had the strongest effect and the Hypoxia+XS group was stronger than the Hypoxia+XH group (P<0.05).Conclusion:XS,XH and XSXH can all increase the biological activity of NO to varying degrees and protect the hypoxia injury of vascular endothelial cells by reducing the levels of ET-1 and inflammatory factors,restoring mitochondrial membrane potential and reducing the generation of ROS,among which XSXH has the strongest effect.