摘要
目的基于氧化应激探索木犀草素对缺氧-复氧损害的H9c2心肌细胞的保护作用。方法在体外木犀草素培养后,再缺氧-复氧培养H9c2心肌细胞模拟缺血再灌注为实验组,以正常培养细胞为对照组,以仅缺氧-复氧培养细胞为缺氧-复氧组。观察细胞形态;流式检测细胞凋亡情况;生化检测细胞丙二醛(MDA)、8-羟基脱氧鸟苷(8-OHdG)、谷胱甘肽(GSH)含量;实时荧光定量PCR(qPCR)检测细胞γ-谷氨酰半胱氨酸合酶(γ-GCS)、血红素加氧酶(HO-1)、NAD(P)H醌氧化还原酶(NQO1)mRNA表达;蛋白质印迹法(Western Blot)检测细胞B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、Kelch样ECH关联蛋白1(Keap1)、核因子E2相关因子2(Nrf2)蛋白含量。结果缺氧-复氧诱导H9c2细胞凋亡[(8.14±1.13)比(72.37±6.98),t=15.733,P=0.000]及促进Bax蛋白表达[(0.38±0.04)比(1.01±0.09),t=11.079,P=0.000],抑制Bcl-2蛋白表达[(0.83±0.06)比0.43±0.04,t=9.608,P=0.001],木犀草素可浓度依赖的抑制细胞凋亡[(72.37±6.98)比(41.38±4.32)、(29.67±5.67),t1=6.539,P=0.003;t2=8.224,P=0.001]及Bax蛋白表达[(1.01±0.09)比(0.71±0.06)、(0.45±0.03),t1=4.979,P=0.008;t2=10.224,P=0.001],增加Bcl-2蛋白表达[(0.43±0.04)比(0.63±0.04)、(0.74±0.05),t1=6.124,P=0.004;t2=8.386,P=0.001];在氧化应激层面,木犀草素可降低由缺氧-复氧引起的MDA、8-OHdG升高[MDA:(1.73±0.12)nmol/mg比(1.12±0.11)nmol/mg、(0.82±0.06)nmol/mg、(0.63±0.04)nmol/mg,t1=5.879,P=0.004;t2=11.748,P=0.000;t3=15.062,P=0.000;8-OHdG:(316.37±21.53)ng/L比(253.26±19.33)ng/L、(211.45±20.98)ng/L、(189.15±18.01)ng/L,t1=3.778,P=0.019;t2=6.054,P=0.004;t3=7.850,P=0.001],升高由缺氧-复氧引起的GSH含量[(88.59±7.99)nmol/mg比(142.19±12.31)nmol/mg、(178.67±16.78)nmol/mg,t1=6.326,P=0.003;t2=8.395,P=0.001]以及γ-GCS、HO-1、NQO1 mRNA的表达下调[γ-GCS:(0.67±0.07)比(0.85±0.07)、(1.02±0.11),t1=3.149,P=0.035;t2=4.649,P=0.010;HO-1:(0.41±0.03)比(0.63±0.06)、(0.82±0.07)、(0.95±0.10),t1=5.
Objective To explore the protective effect of luteolin on H9c2 cardiomyocytes damaged by hypoxia-reoxygenation based on oxidative stress.Methods After in vitro treatment of luteolin,H9c2 cardiomyocytes treated with hypoxia-reoxygenation assay were used as the experimental group.Normal cultured cells were used as the control group,and only hypoxia-reoxygenated culture cells as the hypoxia-reoxygenated group.Cell morphology was observed using microscope;the cell apoptosis was detected by flow cytometry;Malondialdehyde(MDA),8-Hydroxy-2’-deoxyguanosine(8-OHdG)and glutathione(GSH)contents were detected by biochemical assay;mRNA expressions ofγ-glutamylcysteine synthetase(γ-GCS),heme oxygenase-1(HO-1)and NAD(P)H:quinone oxidoreductase 1(NQO1)were detected by quantitative real-time PCR(qPCR);B-cell lymphoma/leukemia gene-2(Bcl-2),Bcl-2 associated X protein(Bax),Kelch like ECH associated protein 1(Keap1)and nuclear factor erythroid 2-related factor 2(Nrf2)proteins were detected by Western Blot assay.Results Hypoxia-reoxygenation induced apoptosis of H9c2 cells[(8.14±1.13)vs.(72.37±6.98),t=15.733,P=0.000],promoted Bax protein expression[(0.38±0.04)vs.(1.01±0.09),t=11.079,P=0.000],inhibited Bcl-2 protein expression[(0.83±0.06)vs(.0.43±0.04),t=9.608,P=0.001].Luteolin inhibited apoptosis in a concentration-dependent manner[(72.37±6.98)vs(.41.38±4.32),(29.67±5.67),t1=6.539,P=0.003;t2=8.224,P=0.001]and Bax protein expression[(1.01±0.09)vs.(0.71±0.06),(0.45±0.03),t1=4.979,P=0.008;t2=10.224,P=0.001],increased Bcl-2 protein expression[(0.43±0.04)vs.(0.63±0.04),(0.74±0.05),t1=6.124,P=0.004;t2=8.386,P=0.001].At the level of oxidative stress,luteolin could reduce the increase of MDA and 8-OHdG caused by hypoxia-reoxygenation[MDA:(1.73±0.12)nmol/mg vs.(1.12±0.11)nmol/mg,(0.82±0.06)nmol/mg,(0.63±0.04)nmol/mg,t1=5.879,P=0.004;t2=11.748,P=0.000;t3=15.062,P=0.000;8-OHdG:(316.37±21.53)ng/L vs.(253.26±19.33)ng/L,(211.45±20.98)ng/L,(189.15±18.01)ng/L,t1=3.778,P=0.019;t2=6.054,P=0.004;t3=7.850,P=0.001],increase the
作者
刘琳
LIU Lin(Department of Coronary Heart Disease,Qinghai Cardiovascular and Cerebrovascular Disease Specialized Hospital,Xining,Qinghai 810000,China)
出处
《安徽医药》
CAS
2020年第6期1084-1089,I0002,共7页
Anhui Medical and Pharmaceutical Journal
