目的 建立大鼠H9c2心肌細(xì)胞缺氧/復(fù)氧（H/R）損傷模型，考察圣草次苷改善心肌缺血再灌注損傷的作用機(jī)制。方法 利用無糖無血清培養(yǎng)基結(jié)合厭氧（94%N₂、5%CO₂、1%O₂）處理H9c2心肌細(xì)胞4h后，更換新鮮完全培養(yǎng)基再放入正常孵箱復(fù)氧24h，制備H/R損傷模型。在造模前12h給予圣草次苷（5、10、20μg·mL^-1），細(xì)胞活力及乳酸脫氫酶（LDH）檢測(cè)實(shí)驗(yàn)中選擇燈盞乙素（20μg·mL^-1）作為陽性藥，對(duì)照組及模型組給予等體積DMSO。MTT法測(cè)定細(xì)胞存活率；試劑盒檢測(cè)細(xì)胞培養(yǎng)上清液中LDH水平；試劑盒檢測(cè)細(xì)胞內(nèi)丙二醛（MDA）水平和超氧化物歧化酶（SOD）、過氧化氫酶（CAT）、谷胱甘肽過氧化物酶（GSH-Px）活力；TUNEL染色檢測(cè)細(xì)胞凋亡；DCFH-DA和JC-1探針分別檢測(cè)細(xì)胞內(nèi)活性氧（ROS）和線粒體膜電位改變；Western blotting檢測(cè)核蛋白中轉(zhuǎn)錄因子（NF）-E2相關(guān)因子2（Nrf2）和總蛋白中血紅素氧合酶1（HO-1）、γ-谷氨酰半胱氨酸連接酶（GCL）水平。結(jié)果 與對(duì)照組比較，H/R損傷誘導(dǎo)的模型組細(xì)胞存活率明顯下降，凋亡明顯增加，LDH水平明顯升高，細(xì)胞內(nèi)MDA水平明顯升高，SOD、CAT、GSH-Px活力明顯降低，ROS釋放明顯增多，線粒體膜電位明顯降低，差異均有統(tǒng)計(jì)學(xué)意義（P＜0.01）；與模型組比較，圣草次苷可劑量相關(guān)性地改善上述變化，其中10、20μg·mL^-1組均差異顯著（P＜0.01）。Western blotting結(jié)果顯示，與對(duì)照組比較，模型組細(xì)胞Nrf2核轉(zhuǎn)位以及HO-1、GCL表達(dá)水平無顯著變化；與模型組比較，圣草次苷10、20μg·mL^-1顯著增加Nrf2核轉(zhuǎn)位及HO-1、GCL表達(dá)水平（P＜0.01）。結(jié)論 圣草次苷能夠保護(hù)H/R誘導(dǎo)的H9c2心肌細(xì)胞損傷，其可能通過激活Nrf2抗氧化信號(hào)通路，增加細(xì)胞內(nèi)源性抗氧化能力，抑制氧化應(yīng)激損傷，保護(hù)線粒體功能以阻止細(xì)胞凋亡的發(fā)生。

Objective The hypoxia/reoxygenation (H/R) injury model of rat H9c2 cardiomyocytes was established to investigate the mechanism of Eriocitrin (ERI) in improving myocardial ischemia-reperfusion injury.Methods H9c2 cardiomyocytes were treated with sugar-free serum-free medium combined with anaerobic (94% N₂, 5% CO₂, 1% O₂) for 4 h, and then replaced with fresh complete medium and reoxygenated in normal incubator for 24 h to prepare H/R injury model. ERI (5, 10, and 20 μg·mL^-1) treatment from 12 h before modeling. Breviscapine (20 μg·mL^-1) was selected as the positive drug for cell viability and lactate dehydrogenase (LDH) detection. The control group and model group were treated with equal volume DMSO. Cell viability was measured by MTT assay. The levels of LDH, malondialdehyde (MDA) and the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) were detected by the kit. Cell apoptosis were detected by TUNEL staining. DCFH-DA and JC-1 probes were used to detect the changes of intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP). The expression of nuclear protein NF-E2-related factor 2 (Nrf2) and expression of heme oxygenase-1 (HO-1) and glutamate cysteine ligase (GCL) expression in total protein was detected by western blotting.Results Compared with control group, H/R decreased cell viability and increased apoptosis rate and LDH level significantly (P <0.01). Besides, H/R decreased MMP and increased ROS production and the level of MDA (P <0.01). The activities of SOD, CAT and GSH-Px decreased significantly (P <0.01). ERI could improve these changes in a dose-dependent manner compared with model group, and there were significant differences in 10 and 20 μg·mL^-1 groups (P <0.01). Western blotting results showed that compared with control group, the nuclear translocation of Nrf2 and the expression levels of HO-1 and GCL in the model group had no significant changes. Compared with model group, ERI 10 and 20 μg·mL^-1 significantly increased Nrf2 nuclear translocation and the expression levels of HO-1 and GCL (P <0.01).Conclusions ERI protected against H/R induced H9c2 injury and apoptosis by increasing the endogenous antioxidant capacity of cells, inhibiting oxidative stress damage, and protecting mitochondrial function, which may be mediated by Nrf2 signaling pathway.

R285.5

中國醫(yī)學(xué)科學(xué)院醫(yī)學(xué)與健康科技創(chuàng)新工程(2021-I2M-1-031)