Study On The Mechanism Of Qili Qiangxin Capsules Regulating Nrf2 And Inhibiting Ferroptosis To Improve Doxorubicin-induced Cardiomyocyte Injury

Background:Doxorubicin(DOX)is a common anthracycline antitumor drug,which can induce tumor cell apoptosis,inhibit tumor cell proliferation and angiogenesis through chemotherapy,and often shows dose-dependent cardiotoxicity during treatment.Since the heart is a highly energy-dependent organ,cardiomyocytes are terminally differentiated cells and cannot continue to divide and proliferate after birth.When the heart is affected by anthracyclines for a long time,cardiomyocyte damage will occur,and finally develop into accumulated myocardial damage and internal Imbalance in the ability of the heart to repair the original heart,which eventually leads to heart failure.Cardiomyocyte death occurs when doxorubicin induces cardiomyocyte injury,which directly leads to a decrease in the number of cardiomyocytes,resulting in the destruction of cardiac structure and function.As a new form of cell death,ferroptosis is an important manifestation of myocardial cell damage in heart failure,which can further aggravate the degree of myocardial cell damage.Among them,mitochondrial oxidative damage is an important mechanism for ferroptosis-induced myocardial cell damage.Doxorubicin-induced ferroptosis in cardiomyocytes is mainly due to mitochondrial oxidative stress injury leading to ROS production and lipid peroxidation formed by iron load.Therefore,mitigating doxorubicin-induced ferroptosis in cardiomyocytes is of great clinical significance.Nuclear factor erythroid 2-related factor 2(Nrf2)plays an important role in ferroptosis.Activation of Nrf2 signaling can reverse the process of ferroptosis to a certain extent,otherwise it will aggravate ferroptosis,so activation of Nrf2 signaling pathway inhibition of ferroptosis has attracted widespread attention as an emerging method to alleviate myocardial cell injury,and has become an important entry point for the treatment of cardiomyocyte injury.Qili Qiangxin Capsules has the therapeutic characteristics of "multi-component,multi-target,multi-channel".It is often used in the treatment of chronic heart failure and can significantly improve cardiomyocyte injury.This study further explored the intervention effect of Qili Qiangxin capsules on Nrf2-ferroptosis pathway in doxorubicin-induced cardiomyocyte injury model.Therefore,this study took H9c2 cardiomyocytes as the research object,intervened H9c2 cardiomyocytes with doxorubicin,and explored the biological characteristics of the H9c2 cardiomyocyte injury model,so as to provide a model for the in vitro study of cardiomyocyte injury.At the same time,the mechanism of Qili Qiangxin Capsules to reduce myocardial cell injury was further discussed,which provided a theoretical basis for in vitro research for clinical application.Objective:1.To analyze the advantages and limitations of doxorubicin at different action concentrations and different action times,and to observe the changes of cell proliferation ability,cell morphology and cell ROS level in H9c2 cardiomyocytes after doxorubicin intervention,so as to provide reference data and theoretical basis for the selection of H9c2 cardiomyocyte injury model in vitro in chronic heart failure.2.To observe the alteration of ferroptosis in a model of cardiomyocyte injury,and to explore the intervention effect of Qili Qiangxin capsules on ferroptosis.3.To observe the effects of agonists and inhibitors of Nrf2 pathway on cardiomyocyte injury model in heart failure and to explore whether Qili Qiangxin capsules inhibit ferroptosis in cardiomyocytes by activating Nrf2 pathway.Methods:1.The H9c2 cardiomyocytes were cultured in vitro for 2-5 times,and the proliferation of H9c2 cardiomyocytes was examined by the CCK-8 cell proliferation assay using 5 concentrations of doxorubicin at 6h,12h and 24h.The morphological changes of H9c2 cardiomyocytes induced by doxorubicin were also observed by light inverted microscopy,and the effect of doxorubicin on the ROS level of H9c2 cardiomyocytes was observed by the cell ROS assay kit,and the optimal concentration of doxorubicin was selected for subsequent experiments.2.Qili Qiangxin capsules were weighed and dissolved in complete medium and filtered,to prepare solutions of Qili Qiangxin capsules at concentrations of 25 mg/L,50 mg/L,100 mg/L,250 mg/L,500 mg/L,750 mg/L,1000 mg/L,1250 mg/L,1500 mg/L;carvedilol powder was weighed and dissolved in complete medium and filtered,to prepare solutions of carvedilol at concentrations of 1 μmol/L,2μmol/L,5μmol/L,10μmol/L,25μmol/L and 50μmol/L.The concentrations of Qili Qiangxin capsules and carvedilol that were not significantly toxic to cells were screened by CCK-8 assay kit.The effective concentrations of Qili Qiangxin capsules and carvedilol on the post-model cells were determined and the low,medium and high dose groups of Qili Qiangxin capsules were identified,and the proliferation of cells in each group was measured.The mitochondrial ultrastructure of the control group,model group,Qili’Qiangxin capsules dose group and carvedilol group was observed by electron microscopy.The differences in the GSH content of the cells in each group as determined by the GSH assay kit.Differences in the MDA content of the cells in each group as determined by the MDA assay kit.Differences in the SOD activity of each group of cells as determined by the SOD assay kit.Differences in ROS levels in each group of cells as observed by the ROS content assay kit.The differences in the area of iron deposition in the cells of each group as observed by Prussian blue staining.The expression of ferroptosis regulatory proteins(system xc-、GPX4)and Nrf2 in the doxorubicin-induced H9c2 cardiomyocyte injury model of heart failure was studied by Western-blot assay with different concentrations of Qili Qiangxin capsules.3.The Nrf2 agonist tBHQ powder was weighed and dissolved to prepare tBHQ solutions at concentrations of 1μmol/L,2μmol/L,5μmol/L,10μmol/L,25μmol/L,and 50μmol/L.The Nrf2 inhibitor ML385 powder was weigher and dissolved to prepare ML385 solutions at concentrations of 1μmol/L,2μmol/L,5μmol/L,10μmol/L,25μmol/L,and 50μmol/L.The concentrations of tBHQ and ML385 that were not significantly toxic to cells were screened by the CCK-8 assay kit.To determine the effective concentrations of tBHQ and ML385 on the cells after modeling.The experiments were divided into blank group(C),blank+agonist group(C+J),blank+inhibitor group(C+Y),model group(M),model+Qili Qiangxin capsules group(M+QLQX),model+agonist group(M+J),model+agonist+Qili Qiangxin capsules group(M+J+QLQX),model+inhibitor group(M+Y),model+inhibitor+Qili Qiangxin capsules group(M+Y+ QLQX),to determine the cell proliferation of each group after treatment.The alteration of the mitochondrial ultrastructure of the cells in each group after treatment as observed by electron microscopy.Differences in the GSH content of the cells in each group as determined by the GSH kit.Differences in the MDA content of the cells in each group as determined by the MDA assay kit.Differences in the SOD levels of the cells in each group as determined by the SOD assay kit.Differences in the ROS levels of the cells in each group as observed by the ROS assay kit.Differences in the area of iron deposition in the cells of each group as observed by Prussian blue staining.The expression of ferroptosis-related proteins system xc-,GPX4 and Nrf2 protein in a model of H9c2 heart failure cardiomyocyte injury induced by doxorubicin was investigated by Western-blot assays with Qili Qiangxin capsules through activation of the Nrf2 pathway.Results:1.The best modeling conditions for the H9c2 cardiomyocyte injury model were determined by CCK-8 cell proliferation assay,observation of cell growth status by optical inverted microscopy,and determination of cell ROS level by 0.5μmol/L doxorubicin intervention for 12h for subsequent experiments.2.Doxorubicin induced ferroptosis in cardiomyocytes.Compared with the blank group,doxorubicin significantly inhibited the proliferation of H9c2 cardiomyocytes,damaged the mitochondrial structure and function,increased the area of Prussian blue iron staining,increased the ROS and MDA levels in cardiomyocytes,decreased the SOD activity and GSH content in cardiomyocytes,and decreased the protein expression of system xc-,GPX4 and Nrf2,leading to oxidative damage and ferroptosis occurred.Compared with the model group,Qili Qiangxin capsules could promote the proliferation of H9c2 cardiomyocytes,improve the morphology of cardiomyocytes,protect the morphology and function of mitochondria,reduce the proportion of Prussian blue iron staining,reduce the ROS and MDA content of cardiomyocytes,and enhance the SOD activity and GSH content of cardiomyocytes;compared with the carvedilol group,Qili Qiangxin capsules could significantly enhance the SOD activity and GSH content of cardiomyocytes.This proved that Qili Qiangxin capsules could effectively reduce the oxidative damage of doxorubicin on H9c2 cardiomyocytes and protect the physiological function of cardiomyocytes.Qili Qiangxin capsules could reduce ferroptosis in cardiomyocytes.Compared with the model group,Qili Qiangxin capsules increased the protein expression of system xc-,GPX4 and Nrf2;compared with the carvedilol group,the high dose group of Qili Qiangxin capsules significantly up-regulated the protein expression of Nrf2.It was demonstrated that the reduction of oxidative damage and ferroptosis in cardiomyocytes by Qili Qiangxin capsules may be related to the upregulation of system xc-,GPX4 and Nrf2 expression,among which,the mechanism of the action of ferroptosis signaling pathway Nrf2 needs to be further investigated.3.Compared with the model group(M),the model+Qili Qiangxin capsules group(M+QLQX),the model+agonist group(M+J),and the model+agonist+Qili Qiangxin capsules group(M+J+QLQX)all increased H9c2 cardiomyocyte proliferation,protected mitochondrial function,reduced cellular iron deposition area,decreased H9c2 cardiomyocyte ROS and MDA content,and enhanced cardiomyocyte SOD activity,and the model+inhibitor group(M+Y)inhibited H9c2 cardiomyocyte proliferation,impaired mitochondrial function,increased Prussian blue iron deposition area,increased H9c2 cardiomyocyte ROS and MDA content,and decreased cardiomyocyte SOD activity and GSH content;compared with the model+inhibitor group,the model+inhibitor+Qili Qiangxin capsules group increased H9c2 cardiomyocyte proliferation and the structure and function of mitochondrial were significantly improved.The above results proved that Qili Qiangxin capsules and Nrf2 agonist tBHQ,could activate the Nrf2 signaling pathway,effectively reduce oxidative damage and ferroptosis in cardiomyocytes,and promote the recovery of cardiomyocyte function,while Nrf2 inhibitor ML385,could inhibit Nrf2 signaling pathway,increasing the level of oxidative stress in cardiomyocytes and further enhancing the degree of ferroptosis.Conclusion:1.The establishment of H9c2 cardiomyocyte injury model was accomplished by a concentration of 0.5μmol/L doxorubicin intervention for 12h,which can be used for the study of heart failure cell experiments.2.Doxorubicin can cause oxidative stress injury in H9c2 cardiomyocytes and induce the occurrence of ferroptosis;Qili Qiangxin capsules can reduce the oxidative damage induced by doxorubicin in H9c2 cardiomyocytes and inhibit the occurrence of ferroptosis.3.Qili Qiangxin capsules may upregulate the expression of system xc-and GPX4 through the Nrf2 signaling pathway,thereby reducing doxorubicin-induced ferroptosis in H9c2 cardiomyocytes.