Oxidative Stress Protection Mechanism of Taxifolin in H9C2 Cells

doi:10.12114/j.issn.1007-9572.2019.00.006

Abstract

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

ZENG Zhihui,WANG Xiaoli,YE Yanqiong, et al. Oxidative Stress Protection Mechanism of Taxifolin in H9C2 Cells　[J]. Chinese General Practice, 2019, 22(15): 1794-1799. DOI: 10.12114/j.issn.1007-9572.2019.00.006.
曾志辉,王晓莉,叶艳琼等. 花旗松素对H9C2细胞氧化应激保护作用机制研究[J]. 中国全科医学, 2019, 22(15): 1794-1799. DOI: 10.12114/j.issn.1007-9572.2019.00.006.

References

［1］BARQUERA S，PEDROZA-TOBíAS A，MEDINA C，et al．Global overview of the epidemiology of atherosclerotic cardiovascular disease［J］．Arch Med Res，2015，46（5）：328-338．DOI：10.1016/j.arcmed.2015.06.006.
［2］LI H，SONG F，DUAN L R，et al．Paeonol and danshensu combination attenuates apoptosis in myocardial infarcted rats by inhibiting oxidative stress：roles of Nrf2/HO-1 and PI3K/Akt pathway［J］．Sci Rep，2016，6：23693．DOI：10.1038/srep23693.
［3］韩晓燕.NADPH氧化酶NOX家族与疾病的关系［J］．国际病理科学与临床杂志，2010，30（6）：513-517．DOI：10.3969/j.issn.1673-2588.2010.06.012.
HAN X Y.NOX family of NADPH oxidase and diseases［J］．Journal of International Pathology and Clinical Medicine，2010，30（6）：513-517．DOI：10.3969/j.issn.1673-2588.2010.06.012.
［4］陈丽云，吴艳青，张正红，等．NADPH氧化还原反应平台及其在AngⅡ介导ROS信号通路中的调节作用［J］．生理科学进展，2012，43（6）：439-444．DOI：10.3969/j.issn.0559-7765.2012.06.008.
CHEN L Y，WU Y Q，ZHANG Z H，et al．NADPH redox reaction platform and its regulatory role in AngⅡ-mediated ROS signaling pathway［J］．Progress in Physiological Sciences，2012，43（6）：439-444．DOI：10.3969/j.issn.0559-7765.2012.06.008.
［5］BRAUNERSREUTHER V，MONTECUCCO F，ASRIH M，et al．
Role of NADPH oxidase isoforms NOX1，NOX2 and NOX4 in myocardial ischemia/reperfusion injury［J］．J Mol Cell Cardiol，2013，64：99-107．DOI：10.1016/j.yjmcc.2013.09.007.
［6］仝瑶瑶，刘晖.花旗松素的研究进展［J］．中西医结合心血管病电子杂志，2014，2（3）：139-140.
TONG Y Y，LIU H.Advances in research of Taxifolin［J］．Cardiovascular Disease Journal of Integrated Traditional Chinese and Western Medicine，2014，2（3）：139-140.
［7］张昊，陈芳，申杰.P38MAPK信号通路及其与动物氧化应激反应关系的研究进展［J］．湖北畜牧兽医，2015，36（7）：13-15．DOI：10.3969/j.issn.1007-273X.2015.07.005.
ZHANG H，CHEN F，SHEN J.P38MAPK signaling pathway and its relationship with animal oxidative stress in animals［J］．Hubei Journal of Animal and Veterinary Sciences，2015，36（7）：13-15．DOI：10.3969/j.issn.1007-273X.2015.07.005.
［8］SONG J，LIU Q，TANG H，et al．Activation of PI3Kγ/Akt pathway increases cardiomyocyte HMGB1 expression in diabetic environment［J］．Oncotarget，2016，7（49）：80803-80810．DOI：10.18632/oncotarget.13096.
［9］师智勇，孙宗全，王国华.花旗松素对大鼠心肌缺血再灌注损伤的保护作用［J］.华中医学杂志，2008，32（4）：287-289.
SHI Z Y，SUN Z Q，WANG G H.The study of Taxifolin's effect on myocardial ischemia-reperfusion injury in rats［J］．Central China Medical Journal，2008，32（4）：287-289.
［10］苏其利，朱平，庄建，等．氧化应激与缺血性心肌病的研究进展［J］．岭南心血管病杂志，2017，23（5）：628-632．DOI：10.3969/j.issn.1007-9688.2017.05.36.
SU Q L，ZHU P，ZHUANG J，et al．Advances in oxidative stress and ischemic cardiomyopathy［J］．South China Journal of Cardiovascular Diseases，2017，23（5）：628-632．DOI：10.3969/j.issn.1007-9688.2017.05.36.
［11］马丽雅，张辉锋.一氧化氮合酶（NOS）与心血管疾病［J］．临床医学研究，2010，27（2）：349-352．DOI：10.3969/j.issn.1671-7171.2010.02.063.
MA L Y，ZHANG H F.Nitric oxide synthase（NOS）and cardiovascular disease［J］．Journal of Clinical Research，2010，27（2）：349-352．DOI：10.3969/j.issn.1671-7171.2010.02.063.
［12］DENHARDT D T，GIACHELLI C M，RITTLING S R.Role of osteopontin in cellular signaling and toxicant injury［J］．Annu Rev Pharmacol Toxicol，2001，41：723-749．DOI：10.1146/annurev.pharmtox.41.1.723.
［13］魏露观.影响细胞毒性T淋巴细胞在病毒性心肌炎中分布的相关研究［D］．大连：大连医科大学，2013.
WEI L G.Study on the distribution of cytotoxic T lymphocyte in viral myocarditis［D］．Dalian：Dalian Medical University，2013.
［14］罗俊，苏威，朱腾腾，等．特发性肺动脉高压患者血清高迁移率族蛋白B1水平及其临床意义［J］．中山大学学报（医学科学版），2017，38（6）：860-865.
LUO J，SU W，ZHU T T，et al．Serum HMGB1 levels and its clinical significance in patients with idiopathic pulmonary arterial hypertension［J］．Journal of Sun Yat-sen University（Medical Sciences），2017，38（6）：860-865.
［15］NAVARRO-GONZáLEZ J F，MORA-FERNáNDEZ C，MUROS DE FUENTES M，et al．Inflammatory molecules and pathways in the pathogenesis of diabetic nephropathy［J］．Nat Rev Nephrol，2011，7（6）：327-340．DOI：10.1038/nrneph.2011.51.
［16］KLUNE J R，DHUPAR R，CARDINAL J，et al．HMGB1：endogenous danger signaling［J］．Mol Med，2008，14（7/8）：476-484．DOI：10.2119/2008-00034.Klune.
［17］MAGNA M，PISETSKY D S.The role of HMGB1 in the pathogenesis of inflammatory and autoimmune diseases［J］．Mol Med，2014，20：138-146．DOI：10.2119/molmed.2013.00164.
［18］PORTO A，PALUMBO R，PIERONI M，et al．Smooth muscle cells in human atherosclerotic plaques secrete and proliferate in response to high mobility group box 1 protein［J］．FASEB J，2006，20（14）：2565-2566．DOI：10.1096/fj.06-5867fje.
［19］INOUE K，KAWAHARA K，BISWAS K K，et al．HMGB1 expression by activated vascular smooth muscle cells in advanced human atherosclerosis plaques［J］．Cardiovasc Pathol，2007，16（3）：136-143．DOI：10.1016/j.carpath.2006.11.006.
［20］宋琦，王振杰.MAPK信号转导通路与失血性休克相关性的研究进展［J］．中国急救医学，2018，38（8）：684-689．DOI：10.3969/j.issn.1002-1949.2018.08.009.
SONG Q，WANG Z J.Progress of relationship between MAPK signal transduction pathway and hemorrhagic shock［J］．Chinese Journal of Critical Care Medicine，2018，38（8）：684-689．DOI：10.3969/j.issn.1002-1949.2018.08.009.
［21］LIU F C，LEE H C，LIAO C C，et al．Tropisetron protects against acetaminophen-induced liver injury via suppressing hepatic oxidative stress and modulating the activation of JNK/ERK MAPK pathways［J］．Biomed Res Int，2016，2016：1952947．DOI：10.1155/2016/1952947.

[1]	LIU Rong, LI Guoxin, LI Yunze, LIANG Lizhe, LU Fei, DANG Shijing, WU Hongjiang, LIU Xiaoyi. Study on the Correlation and Diagnostic Value of Multiple Inflammatory and Oxidative Stress Indices in Lower Extremity Arteriosclerotic Obliteration [J]. Chinese General Practice, 2025, 28(18): 2234-2240.
[2]	LIU Qingfang, WEI Youshi, XIAO Fei, ZHOU Chunxiang, LIU Qun, YUAN Hui, CAO Tianran. Ergothionein Improves Cognitive Dysfunction in Vascular Dementia Rats by Activating NRF2/HO-1 Signaling Pathway [J]. Chinese General Practice, 2025, 28(17): 2156-2162.
[3]	ZHANG Ning, FAN Shiming, REN Ming, SHEN Youlu, MA Yanmei, DOU Xiaohong, PU Cuo, LU Xingqin, HUANG Qiang, XUE Rui, WANG Fuyan, LIN Ying, ZHANG Jihui, TIAN Huizhen, HAN Ping, HAN Yujuan, HAN Shuping, LIU Qingling, LU Tianlong, LU Yongliang, E Weijian. Study on the Relationship between Homocysteine Levels, Polymorphisms of Homocysteine Metabolizing Enzyme Genes, Blood Pressure, and Oxidative Stress Levels in Hypertensive Patients at Different Altitudes [J]. Chinese General Practice, 2025, 28(15): 1862-1870.
[4]	ZHU Jing, XIE Ji, HU Xiaoxia. Study on Anti-inflammatory and Anti-oxidative Stress Mechanism of Sitagliptin: a Dipeptidyl Peptidase-4 Inhibitor [J]. Chinese General Practice, 2025, 28(12): 1543-1548.
[5]	LIU Xinyi, LIU Zhandong. Research Progress of Anxiety and Depression Related to Chronic Fatigue Syndrome [J]. Chinese General Practice, 2023, 26(35): 4477-4482.
[6]	ZHU Li, XING Jiajia, WEI Juanfang, WANG Wenchun, ZHANG Anren. Research Advances in the Mechanism of Short-chain Fatty Acids in Neurodegenerative Diseases [J]. Chinese General Practice, 2023, 26(24): 3061-3066.
[7]	ZHAO Wei, TANG Rongjie, YANG Shanshan, YANG Fang, SUN Feng, LIAN Qiufang. The Role and Molecules Mechanism of Klotho in Renal Injury in Salt-sensitive Hypertension [J]. Chinese General Practice, 2023, 26(24): 3042-3049.
[8]	ZHAO Xiaolong, NIU Zhiping, TAN Jidong, HAN Donghui, YANG Fa, WEN Weihong, WANG Anhui, QIN Weijun. Association of Oxidative Stress and Energy Metabolism with Sperm Quality in Chinese Male Adults [J]. Chinese General Practice, 2023, 26(11): 1318-1324.
[9]	WANG Nan, Motuziuk OLEKSANDR, Mishchenko IRYNA, ZHOU Yingting. Levels of Relevant Inflammatory Factors and Oxidative Stress Markers in Alcoholic Myopathy: a Meta-analysis [J]. Chinese General Practice, 2022, 25(33): 4123-4129.
[10]	Shaokun WANG, Shiqiang WANG, Yijie WANG, Zhihan XU. Research Progress on the Effect of Exercise and Neurobiological Mechanism on Depression [J]. Chinese General Practice, 2022, 25(27): 3443-3451.
[11]	Zhimin DING, Kaiqi SU, Jing GAO, Tingyu CHEN, Yixuan FENG, Xiaodong FENG. Research Progress on the Relationship between Mitochondrial Dysfunction and Post-stroke Depression [J]. Chinese General Practice, 2022, 25(12): 1528-1532.
[12]	LYU Yanhang，WU Shanshan，WANG Zhenchang，YE Xuejin，FU Yanqing，DUAN Guijiao，SU Xiaowen，NONG Xiaoxin. Clinical，Serum Inflammatory Cytokine，Immune，and Oxidative Stress Responses to Rougan Huaxian Granules with Transplantation of Bone Mesenchymal Stem Cells in Decompensated Cirrhosis Patients　 [J]. Chinese General Practice, 2021, 24(3): 355-362.
[13]	XI Yue，WANG Yuanyuan，CUI Xiuling. Protective Effects and Mechanism of α-lipoic Acid in Graves' Disease Complicated with Liver Damage [J]. Chinese General Practice, 2019, 22(8): 931-935.
[14]	ZHANG Xuyang，WANG Xinjun，FU Xudong，ZHOU Shaolong，MA Jian，LIU Feifei，MENG Enping. Levels of HMGB1，TLR2 and TLR4 in Serum and Their Clinical Significance in Patients with Mild Traumatic Brain Injury Combined with Mild Cognitive Impairment [J]. Chinese General Practice, 2019, 22(6): 673-677.
[15]	HAN Guangtao，LI Haohuan，CAI Weisong，ZHANG Yubiao. Research Progress on High Cholesterol and Osteoarthritis　 [J]. Chinese General Practice, 2019, 22(32): 4015-4019.