络风宁2号方抑制心肌细胞铁死亡对心力衰竭的药效和机制研究

【作者】 李海燕；

【导师】 王显；

【作者基本信息】 北京中医药大学 ， 中西医结合临床（专业学位）， 2022， 博士

【摘要】 背景:心力衰竭目前仍然位于全球发病率和死亡率的前列,随着社会经济发展,我国心血管疾病患病人数逐年上升,心衰患病人数推算高达890万,为社会带来的经济负担日益加重,成为我国面临的巨大公共卫生问题之一。中医药在防治心血管疾病中具有显著的优势。王显教授基于络风内动学说及多年临床经验拟定的络风宁2号方对心衰患者症状及生活质量均有显著改善,但目前具体作用机制并不明确。随着现代医学研究的发展,对心衰病理生理机制深入研究,心肌细胞作为终末分化细胞,除了传统的细胞凋亡、坏死等心肌细胞数量减少外,近年来新的程序性死亡方式-铁死亡逐渐出现在人们的视野,其主要特征是细胞内铁超载诱导脂质过氧化物生成增多导致细胞死亡,有报道称这种死亡方式也可能存在于心衰的发生发展中,在AHA/ACC/HFSA最新发布的心力衰竭管理指南中,也将铁超载列入到心衰的发病原因之一。因此我们从铁死亡角度探索络风宁2号方对心衰疾病心肌细胞的影响,明确其可能的保护机制,为今后新药研发提供新的理论依据。目的:本研究通过观察临床心衰患者中铁死亡相关指标变化,确定铁死亡和心衰疾病之间相关性;在动物层面进一步评价络风宁2号方对心衰大鼠的药效;通过细胞实验探索络风宁2号方调节铁死亡保护心肌细胞的作用机制及靶点,为络风宁2号方的新药研发提供重要科学依据,为中医药防治心血管疾病提供新的思路。方法:第一部分 铁死亡相关因子与心力衰竭间的相关性研究通过连续纳入一段时间内在东直门医院心内科住院患者的病例资料,根据是否患有心衰将研究对象分为观察组和对照组,检测两组患者人群血液中丙二醛（MDA）、还原型谷胱甘肽（GSH）和亚铁离子(Fe²⁺)含量变化,探索铁死亡和心衰之间的关系,并确定其是否是独立预测因素。第二部分 络风宁2号方干预心梗后心衰大鼠的药效评价研究研究通过结扎大鼠冠脉前降支构建心梗后心衰动物模型,判断成模后给予络风宁2号方灌胃,观察其对大鼠心衰的治疗效果以及铁死亡指标变化情况。第三部分 络风宁2号方对Ang Ⅱ损伤的心肌细胞的保护作用及机制研究实验一选择H9C2细胞进行体外培养:探索不同密度（1×10⁴、2×10⁴、3×10⁴、4×10⁴、5×10⁴、8×10⁴个/ml）H9C2心肌细胞体外培养的最佳生长条件;实验二Ang Ⅱ诱导的H9C2心肌细胞肥大模型的建立:设置不同浓度（10-8M、10-7M、10-6、10-5M）血管紧张素Ⅱ（AngⅡ）刺激H9C2细胞,通过CCK-8检测观察AngⅡ对细胞活力的影响,ELISA法测上清BNP、罗丹明鬼笔环肽染色法测细胞面积观察Ang Ⅱ对细胞功能和形态影响,选择最佳造模浓度;实验三 探讨络风宁2号方在体外对Ang Ⅱ诱导的心肌细胞损伤的保护作用:通过CCK-8观察不同浓度的络风宁2号方对H9C2心肌细胞活力的影响,ELISA法测细胞上清BNP水平、流式细胞检测细胞内ROS水平,探索络风宁2号方对细胞的保护作用;实验四络风宁2号方对Ang Ⅱ诱导的肥大心肌细胞铁死亡相关途径影响及机制研究:应用TBA、ELISA、Western blot、免疫荧光、透射电镜等多种技术方法探索络风宁2号方对Ang Ⅱ诱导损伤过程中脂质过氧化物MDA生成情况及GPX4、ACSL4、FTH1、SLC39A14蛋白的影响,观察铁死亡相关途径关键分子表达情况,阐明络风宁2号方通过铁死亡途径改善心肌损伤的作用机制。结果:第一部分 铁死亡相关因子与心力衰竭间的相关性分析心衰患者较非心衰患者血液中MDA、Fe²⁺水平含量增高,GSH水平降低（p<0.05）,提示铁死亡相关指标和心衰疾病相关;多因素逻辑回归和逐步回归分析,将包含MDA和Fe²⁺在内的与心衰疾病有关的6个因素作为变量构建预测模型并进行验证,绘制受试者工作特征曲线（ROC）并计算曲线下面积（AUC）为0.972。第二部分 络风宁2号方干预心力衰竭大鼠的药效评价研究采用结扎冠脉前降支14天可以建立稳定的大鼠心梗后心衰模型,左室射血分数（EF）和左室短轴缩短率（FS）明显降低（p<0.05）,络风宁2号方干预14天后可以明显改善心脏结构,提高EF、FS值（p<0.05）。和假手术组相比,模型组LPO有升高趋势,MDA显著升高（p<0.05）,GSH有降低趋势,GPX4显著降低（p<0.05）,和模型组比,络风宁2号方组LPO和MDA有降低趋势,GPX4有升高趋势,GSH显著升高（p<0.05）。第三部分 络风宁2号方对Ang Ⅱ损伤的心肌细胞保护作用及机制研究实验一 H9C2细胞体外培养:根据不同密度及时间点的细胞活力值绘制生长曲线提示3 × 104/mL生长最佳,选择该种板密度用于后续实验研究。实验二 Ang Ⅱ诱导的H9C2大鼠心肌细胞肥大模型的建立:Ang Ⅱ在刺激24小时内使H9C2细胞表面积增大,释放BNP增加（p<0.05）,刺激48小时细胞面积减少,相对24小时细胞活力降低,因此确定10-6 M浓度是Ang Ⅱ刺激H9C2细胞造成心肌细胞肥大模型的最佳浓度。实验三 探讨络风宁2号方对肥大心肌的保护作用:络风宁2号方最低干预剂量为200 μg/ml,可以使Ang Ⅱ刺激的肥大心肌细胞释放的BNP和活性氧有降低趋势（p>0.05）,铁死亡抑制剂效果相同,最佳干预浓度为0.1 μM。实验四络风宁2号方对肥大心肌细胞铁死亡相关途径影响及机制研究:（1）脂质过氧化物生成（MDA）:TBA结果显示和对照组相比,模型组MDA水平升高,络风宁2号方和铁死亡抑制剂组可以使MDA产生有降低趋势,24和48小时一致（p>0.05）;（2）氨基酸代谢相关（GPX4）:ELISA法检测GPX4,和对照组相比,Ang Ⅱ刺激24小时模型组升高（p<0.05）,络风宁2号方和铁死亡抑制剂组比模型组趋势降低（P>0.05）;Ang Ⅱ刺激48小时模型组GPX4降低,络风宁2号方和铁死亡抑制剂比模型组水平升高,WB检测GPX4结果相同;（3）脂代谢相关（ACSL4）:WB和免疫荧光检测不同时间点细胞中ACSL4蛋白水平,和对照组相比,模型组均升高（p<0.05）,络风宁2号方和铁死亡抑制剂均可以使之降低（p<0.05）,24和48小时结果一致;（4）铁代谢相关:WB测细胞内SLC39A14蛋白水平,和对照组相比,模型组SLC39A14表达量增高（p<0.05）,络风宁2号方组和铁抑制组均可以使其表达下降（p<0.05）,24和48小时结果一致;WB和免疫荧光检测细胞内FHT1水平,在AngⅡ刺激24小时,和对照组相比,FTH1表达量增高,而络风宁2号方组和铁抑制剂组可以均可以使之降低,但仍高于正常对照组,在Ang Ⅱ干预48小时后,和同时间点对照组相比,模型组FTH1表达下降显著,差异具有统计学意义（p<0.05）,和同时间点模型组相比,络风宁2号方组和铁抑制剂组均可以使其表达显著增加,差异具有统计学意义（p<0.05）。结论:1铁死亡相关指标MDA、GSH、Fe²⁺和心衰疾病的发生密切相关,其中MDA和Fe²⁺可以作为预判心衰疾病发生的独立预测因子。2络风宁2号方可能通过增加GSH、GPX4和降低LPO、MDA抑制铁死亡,改善心衰大鼠心脏功能。3络风宁2号方可以改善Ang Ⅱ诱导的肥大心肌,其机制可能通过增加GPX4、FTH1、减少ACSL4、SLC39A14等多途径抑制铁死亡保护心肌。更多还原

【Abstract】 Background:Heart failure is still in the forefront of global morbidity and mortality.With the development of social economy,the number of patients with cardiovascular diseases in China is increasing year by year,and the number of patients with heart failure is estimated to be as high as 8.9 million,which brings increasing economic burden to the society and becomes one of the huge public health problems facing China.Traditional Chinese medicine（TCM）has significant advantages in the prevention and treatment of cardiovascular diseases.Professor Wang Xian formulated Luofengning No.2 formula based on the theory of internal action of"endogenous collateral wind" and years of clinical experience,which significantly improved the symptoms and quality of life of patients with heart failure,but the specific mechanism of action is not clear at present.With the development of modern medical research,in-depth study of pathophysiological mechanisms of heart failure,myocardial cells as terminal differentiation,in addition to the traditional cell apoptosis and necrosis of myocardial cells decreased,the new way of programmed death-ferroptosis in recent years gradually appear in people’s field of vision,the main feature is intracellular iron overload induced increased formation of lipid peroxide leads to cell death,It has been reported that this mode of death may also be involved in the development of heart failure,and the AHA/ACC/HFSA recently published guidelines for the management of heart failure also listed iron overload as a cause of heart failure.Therefore,from the perspective of ferroptosis,we explored the effect of Luofengning No.2 formula on cardiac myocytes of heart failure disease,clarified its possible protective mechanism,and provided new theoretical basis for future drug research and development.Objective:In this study,the correlation between ferroptosis and heart failure disease was determined by observing the changes of ferroptosis related indicators in patients with clinical heart failure.To further evaluate the effect of Luofengning No.2 formula on heart failure rats in animal level.Cell experiments were conducted to explore the mechanism and target of Luofengning No.2 formula in regulating ferroptosis and protecting cardiac cells,providing important scientific basis for the research and development of new drugs and new ideas for the prevention and treatment of cardiovascular diseases by TCM.Methods:Part Ⅰ Analysis of the correlation between ferroptosis related factors and heart failureIn this study,the data of inpatients in the Department of Cardiology of Dongzhimen Hospital were collected over a period of time,according to whether the patients with heart failure will be the research object is divided into observation group and control group,the detection of two groups of patients with the crowd in the blood malondialdehyde（MDA）and reduced glutathione（GSH）and ferrous ions(Fe²⁺)content changes,to explore the relationship between ferroptosis and heart failure,And determine whether it is an independent predictor.Part Ⅱ Efficacy evaluation of Luofengning No.2 formula for heart failure after Myocardial infarction in ratsIn this study,an animal model of heart failure after myocardial infarction was established by ligation of anterior descending coronary artery in rats.After the model was determined,Luofengning No.2 formula was given intragastric administration to observe the therapeutic effect of Luofengning No.2 formula on heart failure and the changes of ferroptosis index in rats.Part Ⅲ Protective effect and mechanism of Luofengning No.2 formula on myocardium damaged by Ang ⅡExperiment 1:Selection of H9C2 cells for in vitro culture:To explore the optimal growth conditions of H9C2 cardiomyocytes with different densities（1×10⁴,2×10⁴,3×10⁴,4×10⁴,5×10⁴,8×10⁴/mL）.Experiment 2:Establishment of H9C2 myocardial hypertrophy model induced by Ang Ⅱ:H9C2 cells were stimulated by angiotensin Ⅱ（Ang Ⅱ）at different concentrations（10-8M,107M,10-6,10-5M）,and the effect of Ang Ⅱ on cell viability was observed by CCK-8 detection.ELISA method was used to measure BNP and rhodamine goptidine ring peptide staining method was used to measure the cell area to observe the effects of Ang Ⅱ on cell function and morphology,and the optimal concentration was selected for modeling.Experiment 3:To investigate the protective effect of Luofengning No.2 formula on AngⅡ-induced myocardial cell injury in vitro:The effect of Luofengning No.2 formula on H9C2 myocardial cell viability was observed by CCK-8,BNP level in cell supernatant was measured by ELISA,ROS level in cells was measured by flow cytometry,and the protective effect of Luofengning No.2 formula on cells was explored.Experimental 4:Effect of Luofengning No.2 formula on ferroptosis pathway of Ang Ⅱinduced hypertrophic cardiomyocytes and its possible mechanism:TBA,ELISA,Western blot,immunofluorescence,transmission electron microscopy and other techniques were used to explore the effects of Luofengning No.2 formula on the generation of lipid peroxidase MDA and the protein expression levels of GPX4,ACSL4,FTH1 and SLC39A14 in Ang Ⅱ induced injury.The expression of key molecules related to ferroptosis pathway was observed to clarify the mechanism of Luofengning No.2 formula in improving myocardial injury through ferroptosis pathway.Results:Part Ⅰ Analysis of the correlation between ferroptosis related factors and heart failureThe levels of MDA and Fe²⁺ in blood of patients with heart failure were higher than those of patients without heart failure,and the levels of GSH were lower than those of patients without heart failure（P<0.05）,suggesting that ferroptosis related indexes were related to heart failure disease.Multivariate logistic regression and stepwise regression analysis were conducted.Six factors related to heart failure disease including MDA and Fe²⁺were used as variables to construct and verify the prediction model.Receiver operating characteristic curve（ROC）was drawn and the area under the curve（AUC）was calculated to be 0.972.Part Ⅱ Efficacy evaluation of Luofengning No.2 formula for heart failure after Myocardial infarction in ratsAfter 14 days of ligation of anterior descending coronary artery,a stable model of heart failure after myocardial infarction was established in rats,and left ventricular ejection fraction（EF）and left ventricular short axis shortening rate（FS）were significantly decreased（P<0.05）.After 14 days of intervention,Luofengning No.2 formula could significantly improve the cardiac structure and increase the value of EF and FS（P<0.05）.Compared with sham operation group,LPO and MDA in model group were significantly increased（P<0.05）,GSH and GPX4 were significantly decreased（P<0.05）.Compared with model group,LPO and MDA in Luofengning No.2 formula group were significantly decreased,GPX4 was significantly increased（P<0.05）.Part Ⅲ Protective effect and mechanism of Luofengning No.2 formula on myocardium damaged by Ang ⅡIn Experiment 1:In vitro culture of H9C2 cells:According to the cell viability values at different densities and time points,the growth curve was drawn and 3×10⁴/mL was the best growth.The density of this plate was selected for subsequent experimental studies.Experiment 2:Establishment of H9C2 rat myocardial hypertrophy model induced by AngⅡ:Ang Ⅱ increased H9C2 cell surface area and BNP release within 24 h of stimulation（P<0.05）,but decreased H9C2 cell area within 48 h of stimulation and decreased cell viability relative to 24 h of stimulation.Therefore,10-6 M concentration was determined to be the optimal concentration of H9C2 cell hypertrophy induced by Ang Ⅱ.Experiment 3:Objective to investigate the protective effect of Luofengning No.2 formula on hypertrophic myocardium:the lowest intervention dose of Luofengning No.2 formula was 200 μg/mL,which could reduce BNP and reactive oxygen species（ROS）released by hypertrophic myocardium stimulated by Ang Ⅱ（P>0.05）.The effect of ferroptosis inhibitor was the same,and the optimal intervention concentration was 0.1 μM.Experimental 4:Effect and mechanism of Luofengning No.2 formula on ferroptosis pathway of hypertrophic cardiomyocytes:（1）Lipid peroxide production（MDA）:TB A results showed that compared with the control group,MDA level in model group was increased,and Luofengning No.2 formula and ferroptosis inhibitor group could reduce MDA production,24 and 48 hours consistent（P>0.05）.（2）Amino acid metabolism correlation（GPX4）:COMPARED with the control group,GPX4 was detected by ELISA.Compared with the control group,GPX4 was increased in 24 h Ang Ⅱ stimulation model group（P<0.05）,and lower in Luofengning No.2 formula and ferroptosis inhibitor group（P>0.05）.GPX4 was decreased in the model group after 48 hours of Ang Ⅱ stimulation,and the levels of Luofengning No.2 formula and ferroptosis inhibitor were higher than those in the model group.（3）Lipid metabolism correlation（ACSL4）:WB and immunofluorescence detected ACSL4 protein level in cells at different time points,and compared with the control group,the ACSL4 protein level in model group was increased（P<0.05）.Luofengning No.2 formula and ferroptosis inhibitor could reduce the ACSL4 protein level（P<0.05）,and the results were consistent at 24 and 48 h.（4）Iron metabolism:The protein level of SLC39A14 in cells was measured by WB.Compared with the control group,the expression of SLC39A14 in model group was increased（P<0.05）,and the expression of SLC39A14 in Luofengning No.2 formula group and iron inhibition group was decreased（P<0.05）,and the results were consistent at 24 and 48 hours.WB and immunofluorescence detection of intracellular FHT1 level,24 h after Ang Ⅱstimulation,compared with the control group,FTH1 expression level increased,while Luofengning No.2 formula group and iron inhibitor group can both reduce,but still higher than the normal control group,48 h after Ang Ⅱ intervention,compared with the control group at the same time point,The expression of FTH1 in the model group decreased significantly,and the difference was statistically significant（P<0.05）.Compared with the model group at the same time point,the expression of FTH1 in the Luofengning No.2 formula group and the iron inhibitor group was significantly increased,and the difference was statistically significant（P<0.05）.Conclusion:1 The ferroptosis related indexes MDA,GSH and Fe²⁺ are closely related to the occurrence of heart failure disease,and MDA and Fe²⁺can be used as independent predictors of heart failure disease.2 Luofengning No.2 formula may inhibit ferroptosis by increasing GSH,GPX4 and decreasing LPO and MDA,and improve cardiac function of rats with heart failure after MYOCARDIAL infarction.3 Luofengning No.2 formula can improve myocardium hypertrophy induced by Ang II,and its mechanism may inhibit ferroptosis by increasing GPX4,FTH1,decreasing ACSL4,SLC39A14 and other ways to protect myocardium.更多还原