Study On The Efficacy And Mechanism Of Luofengning No. 2 Prescription In Inhibiting Myocardial Cell Ferroptosis In Heart Failur

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.