Abstract: Background　Oxidative stress（OS）is closely related to the occurrence and development of ischemic heart disease（IHD）．Antioxidant is an important prevention and treatment strategy in IHD.The results showed that Taxifolin（Tax）has anti-inflammatory，anti-tumor and anti-OS effects，but its mechanism of anti-OS is not fully understood.Objective　To investigate the protective mechanism of Tax on oxidative stress damage induced by hydrogen peroxide（H2O2）in myocardial cell H9C2 and provide reference for research and development of new drugs for OS damage diseases.Methods　H9C2 cells were randomly divided into control group〔0.1% dimethyl sulfoxide（DMSO）〕，H2O2 treatment group（treated with 200 μmol/ml H2O2 for 12 h），Tax+H2O2 treatment group（pretreated with Tax 100 μmol/ml for 6 h，and then exposed to H2O2 200 μmol/ml for next 12 h），and Tax treatment group（treated with Tax 100 μmol/ml for 6 h）from March 2017 to March 2018.For the control group，H2O2 treatment group and Tax+H2O2 treatment group，we observed the cell morphology by optical microscope，and intracellular reactive oxygen species（ROS）by fluorescence inverted microscope.We detected the expression levels of inducible nitric oxide synthase（iNOS），osteopontin（OPN），and high mobility group protein B1（HMGB1）mRNA through reverse transcription-polymerase chain reaction（RT-PCR）method.For the control group and Tax treatment group，we detected the expression levels of p38，phosphorylated p38（p-p38），racellular signal-regulated kinase（ERK）1/2，phosphorylated ERK（p-ERK）1/2，c-Jun N-terminal kinases（JNK），phosphorylated JNK（p-JNK）of mitogen-activated protein kinase（MAPK）signal transduction pathway by Western blotting.Results　Compared with the control group，the cell shape of the H2O2 treatment group showed hypertrophy，rounder and irregular，while in the Tax+H2O2 treatment group，the shape of the cells was close to fusiform and the degree of cell roundness and hypertrophy was lighter than that in the H2O2 treatment group.The quantity of irregular morphological cells decreased.The green fluorescence of ROS in H2O2 treatment group was significantly higher and brighter than that in the control group，while in the Tax+H2O2 treatment group，the green fluorescence of ROS was less and dimmer.In Tax+H2O2 treatment group，the mRNA expression of iNOS and OPN was less，and the mRNA expression of HMGB1 was more than that in the control group and H2O2 treatment group（P<0.05）．The protein expression of p38 in the Tax treatment group was less，and the protein expression of p-p38，ERK1/2，p-ERK1/2，JNK，p-JNK was more than those in the control group in MAPK signal pathway（P<0.05）．Conclusion　Tax can improve the morphological changes caused by H2O2 and reduce OS level in cells.Tax may play its anti-OS role via stimulating MAPK signaling pathway，activating the mRNA expression of HMGB1，and inhibiting the mRNA expression of oxidative stress related to gene iNOS，OPN.

Key words: Oxidative stress, MAP kinase signaling system, Nitric oxide synthase typeⅡ, Osteopontin, High mobility group proteins, Taxifolin, H9C2 cells

摘要： 背景　氧化应激（OS）与缺血性心脏病（IHD）的发生发展密切相关，抗OS损伤在IHD防治中至关重要。花旗松素（Tax）具有抗炎、抗肿瘤、抗OS作用，但其抗OS机制尚未完全明了。目的　探讨Tax对过氧化氢（H2O2）诱导的H9C2心肌细胞OS损伤的保护机制，为OS损伤性疾病的新药研发提供基础依据。方法　2017年3月—2018年3月，将H9C2细胞随机分为对照组〔仅加入0.1%二甲基亚砜（DMSO）〕、H2O2处理组（加入200 μmol/ml H2O2处理12 h）、Tax+H2O2处理组（加入100 μmol/ml Tax预处理6 h，换液后加入200 μmol/ml H2O2处理12 h）、Tax处理组（加入100 μmol/ml Tax处理6 h）。对于对照组、H2O2处理组、Tax+H2O2处理组，采用光学显微镜观察细胞形态，荧光倒置显微镜观察细胞染色情况，RT-PCR法检测诱导型一氧化氮合酶（iNOS）、骨桥蛋白（OPN）、高迁移率族蛋白1（HMGB1）mRNA表达水平。采用Western blotting法检测对照组、Tax处理组丝裂原活化蛋白激酶（MAPK）信号通路p38、磷酸化p38（p-p38）、细胞外调节蛋白激酶（ERK）1/2、磷酸化ERK（p-ERK）1/2、c-Jun氨基末端激酶（JNK）、磷酸化JNK（p-JNK）表达水平。结果　与对照组相比，H2O2处理组细胞出现肥大，呈圆形及不规则形状，而Tax+H2O2处理组细胞形态接近梭形，细胞变圆及肥大程度较H2O2处理组轻，不规则形态细胞数量减少。H2O2处理组绿色荧光量较对照组明显增多且亮度增强，而Tax+H2O2处理组较H2O2处理组绿色荧光量及亮度减弱。Tax+H2O2处理组iNOS mRNA、OPN mRNA表达水平低于对照组、H2O2处理组（P<0.05）；Tax+H2O2处理组HMGB1 mRNA表达水平高于对照组、H2O2处理组（P<0.05）。Tax处理组MAPK信号通路p38表达水平低于对照组，p-p38、ERK1/2、p-ERK1/2、JNK、p-JNK表达水平高于对照组（P<0.05）。结论　Tax可以改善H2O2对细胞造成的形态学改变并减少细胞内OS水平，其机制可能通过激活MAPK信号通路，激活HMGB1表达，抑制OS相关基因iNOS、OPN的表达来发挥抗OS作用。

关键词: 氧化性应激, MAP激酶信号系统, 一氧化氮合酶Ⅱ型, 骨桥蛋白质, 高迁移率族蛋白质类, 花旗松素, H9C2细胞