血根碱对脂多糖致H9c2细胞氧化损伤的改善作用

Sanguinarine Attenuates H9c2 Cardiomyocytes Oxidative Stress in Vitro Induced by Lipopolysaccharide

关键词:  血根碱  脂多糖  氧化应激  活性氧  H9c2细胞

Key words:  sanguinarine  lipopolysaccharide  oxidative stress  SOD  H9c2 cell

摘要:

目的 观察血根碱（sanguinarine，SAN）对脂多糖（lipopolysaccharide，LPS）诱导的H9c2细胞氧化应激的影响，并对其与HO-1/NOX2途径之间的关系进行探讨。方法 不同因素干预H9c2细胞后，采用细胞活性试剂盒检测不同浓度SAN和/或LPS对H9c2细胞生存的影响；酶标仪及荧光显微镜检测SAN对LPS诱导细胞产生活性氧（reactive oxygen species，ROS）的影响； Real time RT-PCR检测SAN对LPS诱导的NOX2、P47PHOX、HO-1、SOD1、SOD2基因表达的影响； MDA检测试剂盒检测MDA的变化。结果 单用SAN对H9c2细胞活性无影响，LPS可显著降低细胞活性，而SAN与LPS共同干预则可呈浓度依赖性地提高细胞活性，降低ROS产生。LPS处理12 h上调H9c2细胞NOX2、p47phox mRNA表达、下调HO-1 mRNA表达； SAN预处理后，细胞内NOX2、p47phox mRNA下调、HO-1 mRNA上调，而SAN与锌原卟啉IX预处理则可逆转这种现象。SAN上调SOD1及SOD2 mRNA表达，降低MDA产生。结论 SAN可通过活化HO-1/NOX2途径，抑制ROS的产生，从而使H9c2细胞免受LPS介导的损伤，提高细胞活力。

Abstract:

OBJECTIVE To observe the efficiency of sanguinarine (SAN) acting on oxidative stress of H9c2 cell in vitro induced by lipopolysaccharide (LPS), and investigate its relationship with HO-1/NOX2 pathway. METHODS After H9c2 cells received intervention by different factors, cell growth was determined by CCK8 assay, reactive oxygen species (ROS) generation was detected by a Micro plate reader and Fluorescence microscopy, the mRNA expression of NOX2, P47PHOX, SOD1, SOD2 and HO-1 were determined by real time RT-PCR, MDA detection kit was determined to test the change of MDA. RESULTS SAN alone had no effect on cell activity; LPS reduced cell viability significantly, while co-treated the H9c2 cells with SAN and LPS could reverse this condition, improve cell activity and reduce ROS; the mRNA expression of NOX2, p47phox were increased and HO-1 decreased after LPS treated for 12 h, pre-treated with SAN, the mRNA expression of NOX2, p47phox were decreased and HO-1 was increased, while, pre-treated with SnPPIX and SAN could reverse this condition. SAN could increase the mRNA expression of SOD1 and SOD2, decrease the MDA level. CONCLUSION SAN inhibits the ROS generation induced by LPS via activate the HO-1/NOX2 pathway and also enhance ROS scavenging ability of H9c2 cell, and enhance the cell activity in the end.