| 刘琳.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[J].安徽医药,2020,24(6):1084-1089. |
| 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 |
| The protective effect of luteolin on H9c2 cardiomyocytes damaged by hypoxia?reoxygenation based on oxidative stress |
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| DOI：10.3969/j.issn.1009?6469.2020.06.006 |
| 中文关键词: 心肌再灌注损伤/病因学 木犀草素 氧化性应激 肌细胞，心脏 H9c2细胞株 |
| 英文关键词: Myocardial reperfusion injury/etiology Luteolin Oxidative stress Myocytes,cardiac H9c2 cell line LuteolincanactivatetheNrf2?anti?oxidativeresponseelem, |
| 基金项目: |
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| 中文摘要: |
| 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( 41.3，8±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＝5.945，P＝0.004；t2＝9.624，P＝0.001；t3＝9.126，P＝0.001；NQO1:(0.51±0.04)比( 0.73±0.06)、(0.82±0.08)、(1.15±0.12)t1＝5.284，P＝0.006；t2＝ 6.003，P＝0.004；t3＝8.764，P＝0.001］；进一步发现木犀草素可导致 Nrf2蛋白的进一步升高［(0.62±0比( 0.85±0.07)、(0.97± .05)，0.08)t1＝4.631，P＝0.010；t2＝6.426，P＝0.003］以及 Keap1进一步下调［(0.72±0.04)比( 0.28±0.02)、(0.15±0.01)，t1＝17.041， P＝00；t2＝23.945，P＝0.000］。结论木犀草素可作用于 Nrf2?(抗氧化响应元件) ARE通路，激活该信号通路后，可增加 .00，H9c2细胞中抗氧自由基酶的表达量，并降低由缺氧 -复氧培养所引起的 H9c2细胞的氧化应激水平，抑制心肌细胞凋亡，保护心肌细胞免受缺氧-复氧损伤。 |
| 英文摘要: |
| 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 assaywere 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 cy? tometry；Malondialdehyde(MDA)，8?Hydroxy?2’?deoxyguanosine(8?OHdG)and glutathione(GSH)contents were detected by bio? chemical assay；mRNA expressions of γ?glutamylcysteine synthetase(γ?GCS)，heme oxygenase?1(HO?1)and NAD(P)H:quinoneoxidoreductase 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］，inhib? ited Bcl?2 protein expression［(0.83±006)vs.(0.43±0.04)，t＝9.608， tosis in a concentration?de? pendent 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 expres? sion［( 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.，P＝0.001］.Luteolininhibitedapop，)，［( 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 couldreducetheincreaseofMDA，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 down?regulation of GSH content［( 88.59±7.99)nmol/mg vs.(142.19±12.31)nmol/mg，(178.67±16.78)nmol/mg，t1＝6.326，P＝0.003；t2＝8.395，P＝0.001］and γ?GCS，HO?1，NQO1 mRNA expressions caused by hy? poxia?reoxygenation［γ?GCS:(0.67±0.07)vs.(0.85±0.07)(1.02±0.11)t1＝3.194，P＝0.035；t2＝4.649，P＝0.010；HO?1:(0.41±0.03)vs.(0.63±0.06)，(0.82±0.07)，(0.95±0.10)t1＝5.945＝0.004；t2＝9624，P＝0.001；t3＝9.126，P＝0.001；NQO1:(0.51±0.04) vs.(0.73±0.06)(0.82±0.08)(1.15±0.12)，t1＝54，P＝0.006；t2＝6.003，P＝0.004；t3＝8.764，P＝0.001］.Further findings indicat? increase in Nrf2 protein［(0.62±0.05)vs.(0.85±0.07)，(0.97±0.08)，t1＝4.631，P＝0.010；t2＝6.426， P＝0.003］and a further down?regulation in Keap1 protein［( 0.72±0.04)vs.(0.28±0.02)(0.15±0.01)，t1＝17.041，P＝0.000；t2＝P28，edthatluteolinca，usedafurther，23.945，P＝0.000］.Conclusion ent(ARE)signaling pathway，increase the expression of anti?oxidative free radical enzymes in cardiomyocytes，reduce the oxidative stress level of cardiomyocytes induced by hypoxia?reoxygenation，inhibit cardiomyocyte apoptosis，and protect cardiomyocytes from the injury of hypoxia?reoxygenation. |
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