目的 探究黄芩素对缺氧/复氧诱导的心肌损伤保护作用以及活化受体相互作用蛋白激酶1(RIPK1)/RIPK3信号通路的影响。方法 体外培养H9c2细胞,分为空白组、模型组和实验组(黄芩素20 μmol·L-1)。实验组和模型组先用黄芩素或无血清培养基预处理12 h,然后100 μmol·L-1H2O2处理4 h造成氧化应激损伤。PI法和TUNEL法检测细胞坏死和凋亡情况。对于体内实验,将C57BL/6小鼠分为假手术组、模型组、阳性组(Nec-1 3 mg·kg-1)和3个实验组(黄芩素7.5、15和30 mg·kg-1)。除假手术组外,采用小鼠左冠状动脉侧支阻断法建立缺血/再灌注模型,术后次日给药,连续7 d。检测心肌梗死面积、乳酸脱氢酶(LDH)、肌酸激酶(CK)、肌酸激酶同工酶MB(CK-MB)、丙二醛(MDA)、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)水平。Western blot法检测RIPK1、RIPK3蛋白表达水平;免疫沉淀法检测H2O2作用于心肌细胞后RIPK1与RIPK3的相互作用。结果 MTT法确定黄芩素对H9c2细胞的无毒剂量为20 μmol·L-1后,进行后续实验。与模型组相比,实验组H9c2细胞PI阳性细胞指数[(19.26±3.35)% vs(12.82±4.30)%]和TUNEL阳性细胞指数[(25.75±5.70)% vs (13.80±5.47)%]则显著降低(P<0.05),同时细胞培养上清中LDH、MDA水平降低,SOD、GSH-Px水平则升高(P<0.05);RIPK1和RIPK3蛋白表达量也显著下调(P<0.05)。与模型组相比,黄芩素30 mg·kg-1组大鼠心肌组织梗死面积明显降低[(28.65±7.54)% vs (11.85±4.20)%](P<0.05);同时CK、CK-MB、LDH、SOD含量下降,SOD、GSH-Px水平则升高(P<0.05);RIPK1和RIPK3蛋白表达量下调(P<0.05),且RIPK1/RIPK3凋亡小体逐渐解聚。结论 黄芩素可保护缺氧/复氧诱导的心肌损伤,其机制可能与抑制RIPK1和RIPK3蛋白表达并干扰RIPK1/RIPK3坏死小体稳定性进而抑制心肌细胞程序性坏死有关。
Abstract
OBJECTIVE To study the effect of baicalein on protecting hypoxia/reoxygenation induced myocardial injury and receptor interacting protein kinase 1 (RIPK1)/RIPK3 pathway. METHODS H9c2 cells were cultured in vitro and divided into blank group, model group and baicalein group (baicalein 20 μmol·L-1). The model group and baicalein group were pretreated with baicalein or serum-free medium for 12 h, then treated with 100 μmol·L-1 H2O2 for 4 h, resulting in oxidative stress injury. PI method and TUNEL method were used to detect cell necrosis and apoptosis. In vivo, C57BL/6 mice were divided into sham group, model group, positive group (Nec-1 3 mg·kg-1) and three experimental groups (baicalein 7.5, 15 and 30 mg·kg-1). In addition to the sham operation group, I/R model was established by blocking the left coronary artery collateral in mice, and the drug was administered the next day after operation for 7 consecutive days. The myocardial infarction area, lactate dehydrogenase (LDH), creatine kinase (CK), creatine kinase isoenzyme MB (CK-MB), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) levels were measured. Western blot was used to detect the expression of RIPK1 and RIPK3, and immunoprecipitation was used to detect the interaction between RIPK1 and RIPK3. RESULTS After the nontoxic dose of baicalein on H9c2 cells was determined by MTT method as 20 μmol·L-1. Compared with the model group, PI positive cell index [(19.26±3.35)% vs (12.82±4.30)%] and TUNEL positive cell index [(25.75±5.70)% vs (13.80±5.47)%] were significantly higher in baicalein group than in the model group. Meanwhile, the levels of LDH and MDA in cell culture supernatant were decreased, SOD and GSH PX levels were increased (P<0.05); RIPK1 and RIPK3 protein expressions were also significantly down regulated (P<0.05). Compared with the model group, the infarct area of rats in baicalein 30 mg·kg-1 group was significantly decreased [(28.65±7.54)% vs (11.85±4.20)%] (P<0.05); the contents of CK, CK-MB, LDH and SOD decreased, while the levels of SOD and GSH PX increased (P<0.05); the protein expression of RIPK1 and RIPK3 was down-regulated (P<0.05), and RIPK1/RIPK3 necrosomes gradually disintegrated. CONCLUSION Baicalein can protect myocardial injury induced by hypoxia/reoxygenation. The mechanism may be related to the inhibition of RIPK1 and RIPK3 protein expression and interference with the stability of RIPK1/RIPK3 necrosomes, thus inhibiting the programmed necrosis of cardiomyocytes.
关键词
黄芩素 /
缺氧/复氧 /
RIPK1/RIPK3通路 /
程序性坏死 /
心肌损伤
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Key words
baicalein /
hypoxia/reoxygenation /
RIPK1/RIPK3 pathway /
programmed necrosis /
myocardial injury
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中图分类号：
R96
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参考文献
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脚注
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基金
山东省中医药科技发展计划项目资助(2017-229);山东省医药卫生科技发展计划项目资助(2017WS233)
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