| 何飘;朴美虹;谢丽华;曾阳;王瑾茜;汪辛强;张程程;胡国恒;陈亚.从肝治心组方调控铁自噬保护缺血/再灌注损伤心肌细胞机制研究[J].,2024,44(2):183-192 |
| 从肝治心组方调控铁自噬保护缺血/再灌注损伤心肌细胞机制研究 |
| Mechanism Study of Conggan Zhixin Decoction for Ischemia/Reperfusion Injury of Myocardial Cells Based on Ferritinophagy |
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| DOI：10.7661/j.cjim.20240110.098 |
| 中文关键词: 从肝治心组方 缺血再灌注损伤 核受体共激活因子4 铁蛋白重链1 铁自噬 铁死亡 中药复方 |
| 英文关键词:Conggan Zhixin Decoction ischemia-reperfusion injury nuclear receptor coactivator 4 ferritin heavy chain 1 ferritinophagy ferroptosis Chinese herbal compound |
| 基金项目:全国名老中医药专家传承工作室建设项目（No.国中医药函人教函[2022]75号）;湖南省自然科学基金资助项目（No.2021JJ40418）;湖南中医药大学研究生创新课题立项项目（No.2022CX32） |
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| 中文摘要: |
| 目的 探讨从肝治心组方改善心肌缺血/再灌注损伤的机制。方法 将50只SD大鼠随机分为假手术组、模型组、从肝治心组方组、麝香保心丸组、地尔硫?组，每组10只，制备心肌缺血/再灌注损伤大鼠模型，干预组分别给予从肝治心组方[5.32 g/（kg·d）]、麝香保心丸[10.27 mg/（kg·d）]、地尔硫?[6.86 mg/（kg·d）]灌胃给药，假手术组和模型组给予等量生理盐水灌胃，干预14天后取材，HE染色观察心肌组织形态，透射电子显微镜观察心肌细胞线粒体形态，RT-PCR及Western Blot检测核受体共激活因子4（NCOA4）、铁蛋白重链1（FTH1）、前列腺素G/H合酶2（PTGS2）mRNA和蛋白表达情况。H9c2大鼠心肌细胞分为正常组、模型组、空白血清组、中药组、铁死亡抑制剂组（Ferrostatin-1）、铁死亡诱导剂组（Erastin）、中药诱导剂组，缺氧12 h复氧2 h造模，检测各组细胞存活率、活性氧（ROS）含量、线粒体膜电位水平、Fe2+水平，RT-PCR及Western Blot检测NCOA4、FTH1、PTGS2 mRNA和蛋白表达情况。结果 动物实验显示，模型组大鼠心肌纤维断裂或稀疏，可见心肌局灶性坏死，细胞间质浸润炎性细胞，心肌细胞内线粒体肿胀变大，纤维瘢痕组织增生；干预组病理改变减轻。与假手术组比较，模型组NCOA4、PTGS2 mRNA及蛋白表达升高，FTH1 mRNA及蛋白表达降低（P＜0.05，P＜0.01）。与模型组比较，各干预组NCOA4、PTGS2 mRNA及蛋白表达降低，FTH1 mRNA及蛋白表达升高（P＜0.05，P＜0.01）。细胞实验显示，与正常组比较，模型组细胞存活率、膜电位、FTH1 mRNA和蛋白表达降低，细胞内ROS、Fe2+水平、NCOA4、PTGS2 mRNA和蛋白表达升高（P＜0.05，P＜0.01）。与模型组比较，中药组和铁死亡抑制剂组细胞存活率、膜电位、FTH1 mRNA和蛋白表达升高，细胞内ROS、Fe2+水平、NCOA4、PTGS2 mRNA和蛋白表达降低（P＜0.01）；铁死亡诱导剂组细胞存活率、膜电位、FTH1 mRNA和蛋白表达降低，细胞内ROS、Fe2+水平、NCOA4、PTGS2 mRNA和蛋白表达升高（P＜0.05，P＜0.01）。与铁死亡诱导剂组比较，中药诱导剂组细胞存活率、膜电位、FTH1 mRNA和蛋白表达升高，细胞内ROS、Fe2+水平、NCOA4、PTGS2 mRNA和蛋白表达降低（P＜0.05，P＜0.01）。结论 从肝治心组方可能通过调控NCOA4/FTH1信号通路抑制铁自噬，从而减轻心肌细胞铁死亡，起到保护缺血/再灌注损伤心肌细胞的作用。 |
| 英文摘要: |
| Objective To investigate the mechanism of improving myocardial ischemia/reperfusion injury using Conggan Zhixin Decoction（CGZXD）. Methods Fifty SD rats were randomly divided into 5 groups：sham-operated group，model group，CGZXD group，Shexiang Baoxin Pill（SBP） group，Diltiazem group，with 10 rats in each group. A rat model of myocardial ischemia/reperfusion injury was prepared. The intervention group was given CGZXD [5.32 g/（kg·d）]，SBP [10.27 mg/（kg·d）]，and diltiazem [6.86 mg/（kg·d）] by gavage respectively. The sham-operated group and model group were given equal amounts of physiological saline by gavage. After 14 days of intervention，samples were taken，and the morphology of myocardial tissue was observed using HE staining. The morphology of myocardial cell mitochondria was observed using transmission electron microscopy. RT-PCR and Western Blot were used to detect the mRNA and protein expression of nuclear receptor co activator 4 （NCOA4），ferritin heavy chain 1 （FTH1），and prostaglandin G/H synthase 2 （PTGS2），respectively. H9c2 rat cardiomyocytes were divided into 7 groups：normal group，model group，blank serum group，Chinese medicine group， ferroptosis inhibitor group （Ferrostatin-1）， ferroptosis inducer group （Erastin），and Chinese medicine inducer group. After 12 hours of hypoxia and 2 hours of reoxygenation，the cell survival rate，reactive oxygen species（ROS） content，mitochondrial membrane potential level，and Fe2+ level were measured in each group. The mRNA and protein expression of NCOA4，FTH1，and PTGS2 were detected by RT-PCR and Western Blot，respectively. Results Animal experiments showed that in the model group of rats，myocardial fibers were broken or sparse，with focal myocardial necrosis，interstitial infiltration of inflammatory cells，swelling and enlargement of mitochondria in myocardial cells，and proliferation of fibrous scar tissue. The pathological changes in the intervention group were alleviated. Compared with the sham-operated group，the mRNA and protein expression of NCOA4 and PTGS2 increased in the model group，while the mRNA and protein expression of FTH1 decreased（P＜0.05，P＜0.01）. Compared with the model group，the mRNA and protein expression of NCOA4 and PTGS2 decreased in each intervention group，while the mRNA and protein expression of FTH1 increased （P＜0.05，P＜0.01）. Cell experiments showed that compared with the normal group，the model group exhibited a decreasing in cell survival rate，membrane potential，FTH1 mRNA and protein expression，and an increasing in intracellular ROS and Fe2+ levels，as well as NCOA4 and PTGS2 mRNA and protein expression（P＜0.05，P＜0.01）. Compared with the model group，both the Chinese medicine group and the ferroptosis inhibitor group demonstrated an increasing in cell survival rate，membrane potential，FTH1 mRNA and protein expression，while intracellular ROS，Fe2+ levels，NCOA4，and PTGS2 mRNA and protein expression decreased （P＜0.01）.The cell survival rate，membrane potential，FTH1 mRNA and protein expression decreased in the ferroptosis inducer group，while intracellular ROS，Fe2+ levels，NCOA4，PTGS2 mRNA，and protein expression increased （P＜0.05，P＜0.01）. Compared with the ferroptosis inducer group，the Chinese medicine inducer group showed an increase in cell survival rate，membrane potential，FTH1 mRNA and protein expression，while the intracellular ROS，Fe2+ levels，NCOA4，PTGS2 mRNA and protein expression decreased（P＜0.05，P＜0.01）. Conclusion CGZXD may inhibit ferritinophagy by regulating the NCOA4/FTH1 signaling pathway，thereby reducing ferroptosis in cardiomyocytes and protecting ischemia/reperfusion-injured cardiomyocytes. |
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