药学学报, 2021, 56(4): 1070-1078
引用本文:
杨洁, 郑瑞芳, 都研文, 李海宁, 李少将, 刘砥威, 邢建国. 田蓟苷抗H9c2心肌细胞缺血再灌注损伤的作用机制研究[J]. 药学学报, 2021, 56(4): 1070-1078.
YANG Jie, ZHENG Rui-fang, DU Yan-wen, LI Hai-ning, LI Shao-jiang, LIU Di-wei, XING Jian-guo. The mechanism of tilianin against ischemia-reperfusion injury of H9c2 cells[J]. Acta Pharmaceutica Sinica, 2021, 56(4): 1070-1078.

田蓟苷抗H9c2心肌细胞缺血再灌注损伤的作用机制研究
杨洁1, 郑瑞芳2, 都研文2, 李海宁3, 李少将1, 刘砥威2*, 邢建国2*
1. 新疆医科大学, 新疆 乌鲁木齐 830011;
2. 新疆药物研究所, 新疆 乌鲁木齐 830002;
3. 石河子大学, 新疆 石河子 832000
摘要:
本研究利用泛caspase抑制剂Z-VAD-FMK联合缺氧/复氧(hypoxia-reoxygenation,H/R)损伤建立大鼠H9c2心肌细胞程序性坏死模型,并考察田蓟苷抗心肌缺血再灌注损伤的作用机制。采用CCK-8(cell counting kit-8)测定细胞活力;采用试剂盒法检测细胞培养上清中乳酸脱氢酶(lactate dehydrogenase,LDH)和细胞内超氧化物歧化酶(superoxide dismutase,SOD);Hoechst 33342/PI染色检测细胞死亡方式;采用DCFH-DA、BBcellProbeTMM61和JC-1探针依次检测细胞内活性氧(reactive oxygen species,ROS)、线粒体通透性转换孔(mitochondrial permeability transition pore,mPTP)开放和线粒体膜电位(mitochondrial membrane potential,MMP)改变;ELISA法检测细胞分泌肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)和白细胞介素-6(interleukin-6,IL-6)释放情况;分子对接技术锁定田蓟苷作用的分子靶标;Western blot检测相关蛋白表达。结果表明,与对照组比较,H/R损伤诱导的模型组细胞活力下降,程序性坏死率升高,LDH水平升高,SOD活性降低,ROS释放显著增多,mPTP开放,MMP降低,TNF-α、IL-1β以及IL-6水平升高,而田蓟苷则可剂量依赖性地改善这些因素变化。分子对接结果显示,田蓟苷可与钙/钙调蛋白依赖性蛋白激酶Ⅱ(calmodulin-dependent protein kinase Ⅱ,CaMKⅡ)结合。Western blot结果显示,与对照组比较,模型组细胞p-CaMKⅡ和磷酸化混合系结构域样蛋白(phospho-mixed lineage kinase domain-like protein,p-MLKL)表达水平显著升高;与模型组比较,田蓟苷则使这些蛋白表达水平降低。上述结果表明,田蓟苷能够保护H9c2心肌细胞,可能通过抑制CaMKⅡ苏氨酸Thr287位点磷酸化,抑制mPTP开放,保护线粒体功能以阻止程序性坏死进行,对H/R损伤治疗新方法的研究具有潜在价值。
关键词:    田蓟苷      心肌缺血再灌注损伤      程序性坏死      钙/钙调蛋白依赖性蛋白激酶II      线粒体膜通透性转换孔     
The mechanism of tilianin against ischemia-reperfusion injury of H9c2 cells
YANG Jie1, ZHENG Rui-fang2, DU Yan-wen2, LI Hai-ning3, LI Shao-jiang1, LIU Di-wei2*, XING Jian-guo2*
1. Xinjiang Medical University, Urumqi 830011, China;
2. Xinjiang Institute of Materia Medica, Urumqi 830002, China;
3. Shihezi University, Shihezi 832000, China
Abstract:
Z-VAD-FMK was combined with hypoxia-reoxygenation (H/R) injury to establish a necroptosis model of H9c2 cells to mimic the pathological changes of myocardial ischemia reperfusion injury (MIRI) in vitro and to study the effect and mechanism of tilianin against myocardial ischemia-reperfusion injury. A cell counting kit-8 (CCK-8) was used to detect cell viability, and commercial kits were used to detect lactate dehydrogenase (LDH) and superoxide dismutase (SOD) in the cell culture supernatant. Hoechst 33342/PI immunofluorescence staining was used to detect cell death. DCFH-DA, BBcellProbeTMM61, and JC-1 probes were used to detect reactive oxygen species (ROS), mitochondrial permeability transition pore (mPTP), and mitochondrial membrane potential (MMP), respectively. An enzyme-linked immunosorbent assay (ELISA) method was used to detect the release of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6). The results show that the cell viability, SOD activity, and MMP of the model group induced by H/R injury decreased, as compared with control group, but the necroptosis rate, LDH level, and ROS release increased significantly. Furthermore, mPTP of the model group cells opened, and TNF-α, IL-1β, and IL-6 levels were significantly higher. Molecular docking modeling showed that tilianin can bind to calmodulin-dependent protein kinase Ⅱ (CaMKⅡ), and Western blot results showed that compared with control group, the expression levels of p-CaMKⅡ and phospho-mixed lineage kinase domain-like protein increased in the model group, and tilianin could decrease the expression level of these proteins. The above results indicate that tilianin can protect H9c2 cells by inhibiting the phosphorylation of CaMKⅡ at threonine 287, protecting mitochondrial function, and inhibiting the opening of mPTP to prevent necroptosis. This study has value for research on new methods to treat H/R injury.
Key words:    tilianin    myocardial ischemia-reperfusion injury    necroptosis    calcium/calmodulin-dependent protein kinase II    mitochondrial permeability transition pore   
收稿日期: 2020-10-19
DOI: 10.16438/j.0513-4870.2020-1636
基金项目: 新疆维吾尔自治区自然科学基金(青年基金)资助项目(2020D01B50);新疆维吾尔自治区公益性科研院所基本科研业务经费资助项目(ky2019099).
通讯作者: 邢建国,Tel:13999178585,E-mail:xjguodd@163.com;刘砥威,E-mail:3498921775@qq.com
Email: xjguodd@163.com;3498921775@qq.com
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