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山东大学学报 (医学版) ›› 2022, Vol. 60 ›› Issue (8): 14-22.doi: 10.6040/j.issn.1671-7554.0.2022.0250

• 基础医学 • 上一篇    下一篇

葛根素对索拉非尼心肌毒性的保护及作用机制

姜卉1,魏甜2,李建平1,王聪3   

  1. 1.山东大学第二医院儿内科, 山东 济南 250033;2.山东大学第二医院胃肠外科 山东大学齐鲁医学院, 山东 济南 250033;3.山东第一医科大学第一附属医院心血管内科, 山东 济南 250015
  • 发布日期:2022-07-27
  • 通讯作者: 王聪. E-mail:congwang87@outlook.com
  • 基金资助:
    山东省自然科学基金青年基金(ZR2021QH026)

Protective effects and molecular mechanism of puerarin on sorafenib-induced cardiotoxicity

JIANG Hui1, WEI Tian2, LI Jianping1, WANG Cong3   

  1. 1. Department of Pediatrics, The Second Hospital, Cheeloo College of Medicine, Shandong University, Jinan 250033, Shandong, China;
    2. Department of Gastrointestinal Surgery, The Second Hospital, Cheeloo College of Medicine, Shandong University, Jinan 250033, Shandong, China;
    3. Department of Cardiology, The First Affiliated Hospital of Shandong First Medical University, Jinan 250014, Shandong, China
  • Published:2022-07-27

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摘要： 目的 探讨葛根素通过抑制心肌铁死亡对索拉非尼心肌毒性的保护及其分子作用机制。 方法 细胞实验:分离培养大鼠乳鼠原代心肌细胞,给予不同的药物处理,分为二甲基亚砜(DMSO)组、索拉非尼组、索拉非尼+Ferrostatin-1(Fer-1)组、索拉非尼+去铁胺组、索拉非尼+葛根素组,检测各组细胞活力和乳酸脱氢酶(LDH)释放,观察光镜下细胞形态,并检测脂质活性氧(ROS)水平以及脂质过氧化代谢产物丙二醛(MDA)水平;采用Western blotting法检测上述分组以及索拉非尼处理心肌细胞0、12、24、36、48、60、72 h后内质网应激标志物葡萄糖调节蛋白(GRP78)水平。给予不同的药物处理,将心肌细胞分为索拉非尼组、索拉非尼+葛根素组、索拉非尼+毒胡萝卜素组以及索拉非尼+葛根素+毒胡萝卜素,检测各组细胞活力、LDH释放、MDA水平,采用Western blotting法检测GRP78水平。小鼠实验:将40只C57BL/6J小鼠随机分为对照组、葛根素组、索拉非尼组、索拉非尼+葛根素组,采用ELISA法检测血浆肌酸激酶同工酶(CK-MB)水平,测算心脏质量/胫骨长度(HW/TL)、心肌组织切片Masson染色后测量心肌细胞横截面积(CSA),检测心肌组织ROS、MDA水平,采用Western blotting法检测心肌组织GRP78水平。 结果 细胞实验中索拉非尼组较DMSO组细胞活力降低、LDH释放增加,细胞空泡化,脂质ROS及MDA水平升高,GRP78蛋白上调,差异均有统计学意义(P均<0.01);与索拉非尼组相比,索拉非尼+ Fer-1组、索拉非尼+去铁胺组以及索拉非尼+葛根素组细胞活力升高、LDH释放减少、空泡化减轻,脂质ROS及MDA水平降低,且GRP78蛋白下调,差异均有统计学意义(P均<0.05);内质网应激诱导剂毒胡萝卜素和葛根素对GRP78水平、细胞活力、LDH释放、MDA水平的交互作用差异有统计学意义(P均<0.05)。小鼠实验中索拉非尼组较对照组CK-MB水平升高,HW/TL下降,CSA升高,脂质ROS和MDA水平升高,GRP78蛋白上调,差异均有统计学意义(P均<0.01);葛根素与索拉非尼间的交互作用差异有统计学意义(P均<0.05);与索拉非尼组相比,索拉非尼+葛根素组小鼠CK-MB水平下降、HW/TL升高,CSA下降,脂质ROS和MDA水平降低,GRP78蛋白下调,差异均有统计学意义(P均<0.05)。 结论 葛根素可以抑制索拉非尼引发的心肌铁死亡,对索拉非尼的心肌毒性具有良好的保护作用,毒胡萝卜素可拮抗葛根素的细胞保护和抑制脂质过氧化作用,葛根素通过清除脂质ROS抑制内质网应激可能是其背后主要的分子机制。

关键词: 索拉非尼, 心肌毒性, 葛根素, 铁死亡, 内质网应激

Abstract: Objective To elucidate the protective effects and molecular mechanism of puerarin on sorafenib-induced cardiotoxicity via inhibiting the ferroptosis of cardiomyocytes. Methods In the cell study, primary cardiomyocytes were isolated and treated with dimethyl sulfoxide(DMSO), sorafenib, sorafenib+deferoxamine, sorafenib+ferrostatin-1(Fer-1), and sorafenib+puerarin, respectively. The cell viability, lactate dehydrogenase(LDH)release, lipid ROS, and malondialdehyde(MDA)level were measured. The cell morphology was observed with microscopy. At 0, 12, 24, 36, 48, 60 and 72 h after treatment, cardiomyocytes were harvested and the classic marker of endoplasmic reticulum(ER)stress, glucose-regulated protein 78(GRP78)protein was detected with Western blotting. Cardiomyocytes were treated with sorafenib, sorafenib+puerarin, sorafenib+puerarin+thapsigargin, and sorafenib+puerarin+thapsigargin, respectively. The cell viability, LDH release, lipid ROS, MDA level and GRP78 level were measured. In the mice study, 40 C57BL/6J mice were divided into the control, puerarin, sorafenib, and sorafenib+puerarin groups. The level of serum creatine kinase MB isoenzyme(CK-MB)was determined with ELISA. The heart weight/tibia length(HW/TL)was evaluated. The cross-sectional area(CSA)was measured after Masson staining. The ROS, MDA and GRP78 levels of cardiac tissue were measured. Results In the cell study, compared to the DMSO group, the sorafenib group had decreased cell viability, increased LDH release, increased lipid ROS, MDA and GRP78 levels(all P<0.01). Compared with the sorafenib group, the sorafenib+Fer-1 group, sorafenib+deferroamine group and sorafenib+puerarin group had increased cell viability, decreased LDH release, decreased vacuolation, decreased lipid ROS, MDA and GRP78 levels(all P<0.05). The interactions between ER stress inducers thapsigargin and puerarin on GRP78 level, cell viability, LDH release and MDA level were statistically significant(all P<0.05). In the mice study, sorafenib administration caused elevated CK-MB level, decreased HW/TL, larger CSA of surviving cardiomyocytes, increased lipid ROS level and MDA content, and upregulated level of GRP78, which could all be inhibited by puerarin(all P<0.05). Compared with the sorafenib group, the sorafenib+puerarin group had decreased level of CK-MB, increased HW/TL, decreased CSA, decreased ROS, MDA and GRP78 levels(all P<0.05). Conclusion Puerarin inhibits sorafenib-induced ferroptosis of cardiomyocytes via inhibiting ER stress, which might be triggered by lipid ROS. Puerarin has a promising role in alleviating sorafenib-related cardiotoxicity.

Key words: Sorafenib, Cardiotoxicity, Puerarin, Ferroptosis, Endoplasmic reticulum stress

中图分类号: 

  • R541.9
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