Application of Nrf3 inhibitor in preparation of medicine for preventing and treating myocardial infarction

Abstract

The invention discloses application of an Nrf3 inhibitor in preparation of a medicine for preventing and treating myocardial infarction. The research finds that the heart specific knockout Nrf3 can improve the myocardial function, inhibit myocardial fibrosis and reduce myocardial cell apoptosis after ischemia reperfusion (I / R) injury; the recovery of mitochondrial reactive oxygen species (ROS) can weaken the beneficial effect of Nrf3 gene deletion on the cardiac function and reconstruction after myocardial infarction (MI); the heart-specific knock-down Pitx2 can weaken the beneficial effect of heart-specific Nrf3 gene deletion on cardiac function and pathological reconstruction after myocardial infarction; the Nrf3 inhibitor or Pitx2 overexpression can significantly improve the cardiac function after myocardial infarction, and reduce mitochondrial ROS generation and myocardial cell apoptosis; therefore, the targeted Nrf3-Pitx2-mitochondrial ROS signal axis may represent a novel myocardial infarction treatment method.

Description

Technical Field

[0001] The present invention relates to the technical field of application of Nrf3 inhibitors, and in particular to the application of Nrf3 inhibitors in the preparation of drugs for preventing and treating myocardial infarction. Background Art

[0002] Acute myocardial infarction refers to acute myocardial ischemic necrosis, which is mostly caused by a sharp reduction or interruption of coronary blood supply on the basis of coronary artery disease, resulting in severe and persistent acute ischemia of the corresponding myocardium. The cause of acute myocardial infarction is usually secondary to thrombosis on the basis of rupture and erosion of unstable coronary plaques, leading to persistent and complete occlusion of the coronary artery.

[0003] One of the most striking features of cardiac injury in patients with myocardial infarction (MI) is the death of a large number of cardiomyocytes (CMs) due to lack of oxygen and energy supply. The extent of cardiomyocyte death is a key determinant of short-term cardiac rupture and long-term pathological cardiac remodeling. Cardiac injury produces a large amount of reactive oxidative stress (ROS), and the vast majority of cellular ROS (90%) comes from the mitochondrial compartment. Mitochondrial ROS can directly or indirectly cause CM death, so inhibiting excessive mitochondrial ROS during myocardial injury is considered an effective method to inhibit CM death and improve myocardial function. However, drugs that effectively and specifically target mitochondrial ROS production in the damaged heart are still lacking and unmet medical needs.

[0004] Nrf3 (nuclear factor erythroid 2-related factor 3) is the most recently discovered member of the Cap'N'Collar (CNC)-basic leucine zipper (bZip) transcription factor family, which also includes Nrf1 and Nrf2. The CNC domain contributes to the unique DNA binding specificity of these transcription factors. Nrf3 has been reported to transcriptionally repress or activate its target genes by directly binding to the antioxidant response element (ARE) within the promoter region of the target genes.

[0005] Specifically, it has been documented that Nrf3 inhibits the expression of antioxidant genes such as NAD(P)H:quinone oxidoreductase e1 (Nqo1) and peroxiredoxin 6, but promotes the transcription of Nox4 (NADPH oxidase 4) and Pla2g7 (phospholipase A2, group 7), suggesting that it has a cell-dependent transcriptional regulatory role. Unlike its two homologs, the current understanding of the pathophysiological role of Nrf3 is limited. Nrf3 is considered to be an oncogene that confers a selective growth advantage to cancer cells and promotes cancer malignancy-related processes, including metastasis and angiogenesis.

[0006] However, the functional role of Nrf3 in lipid homeostasis has recently been confirmed, which supports its impact in lipid-related diseases. In addition, studies have confirmed the key role of Nrf3-mediated ROS in the differentiation of stem cells into smooth muscle cells, indicating that Nrf3, unlike Nrf2, participates in the regulation of redox signaling by promoting rather than inhibiting the generation of ROS. However, little is known about the functional involvement of Nrf3 and Nrf3-mediated ROS signaling in CM death and pathological cardiac remodeling after myocardial infarction. Summary of the invention

[0007] To solve the above problems, the present invention provides the use of Nrf3 inhibitors in the preparation of drugs for preventing and treating myocardial infarction, aiming to find a drug that can specifically target the production of mitochondrial ROS in damaged hearts.

[0008] To achieve the above objectives, the present invention provides the use of an Nrf3 inhibitor in the preparation of a drug for preventing and treating myocardial infarction and heart damage diseases.

[0009] Preferably, the prevention and treatment of myocardial infarction and heart damage diseases includes at least one of the following 1) to 4):

[0010] 1) Promote Pitx2 expression; 2) Inhibit mitochondrial reactive oxygen species production; 3) Inhibit CM cell apoptosis induced by ischemia-reperfusion injury; 4) Improve cardiac function.

[0011] The use of the Nrf3 inhibitor of the present invention in the preparation of a drug for preventing and treating myocardial infarction has the following beneficial effects: studies have found that cardiac-specific knockout of Nrf3 can improve myocardial function, inhibit myocardial fibrosis, and reduce myocardial cell apoptosis after ischemia-reperfusion (I / R) injury; and restoration of mitochondrial reactive oxygen species (ROS) can weaken the beneficial effects of Nrf3 gene deletion on cardiac function and remodeling after myocardial infarction (MI); cardiac-specific knockdown of Pitx2 can weaken the beneficial effects of cardiac-specific Nrf3 gene deletion on cardiac function and pathological remodeling after myocardial infarction (MI); Nrf3 inhibitors or Pitx2 overexpression can significantly improve cardiac function after myocardial infarction, reduce mitochondrial ROS generation and CM apoptosis; therefore, targeting the Nrf3-Pitx2-mitochondrial ROS signaling axis may represent a new treatment method for myocardial infarction.

[0012] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The results of the experiment to explore the expression level of Nrf3 in the hearts of humans and mice with myocardial infarction;

[0014] Figure 2To explore the experimental results of the effect of global knockout of Nrf3 on myocardial infarction;

[0015] Figure 3 To explore the experimental results of the effects of cardiac-specific knockout of Nrf3 on cardiac function and cardiomyocyte apoptosis after myocardial infarction;

[0016] Figure 4 To investigate the effects of cardiac-specific knockout of Nrf3 on myocardial fibrosis and mitochondrial superoxide production;

[0017] Figure 5 To investigate the potential role of CM-specific Nrf3 in myocardial I / R injury;

[0018] Figure 6 To restore the experimental results of the effects of mitochondrial reactive oxygen species (ROS) on cardiac function and remodeling after myocardial infarction caused by Nrf3 gene deficiency;

[0019] Figure 7 The experimental results show that Nrf3 inhibits the expression of Pitx2;

[0020] Figure 8 The strategies for constructing the wild-type (WT) Pitx2 promoter and the mutant Pitx2 promoter lacking the predicted binding motifs;

[0021] Fig. 9 To explore the experimental results of the mechanism of Nrf3 inhibiting the expression of Pitx2 and the regulatory effect of hnRNPK-DNMT1;

[0022] Fig.10 The experimental results show the effects of cardiac-specific knockdown of Pitx2 on cardiac function and pathological remodeling after myocardial infarction caused by cardiac-specific Nrf3 gene deletion.

[0023] Fig.11 These are the experimental results of the effects of Pitx2 overexpression on cardiac function, mitochondrial ROS generation and CM apoptosis after myocardial infarction. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages disclosed in the embodiments of the present invention more clearly understood, the embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the embodiments of the present invention and are not intended to limit the embodiments of the present invention. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0025] Embodiment 1

[0026] Single cell / nucleus RNA sequencing (Sc / SnRNA-seq) of normal human heart was downloaded from the Heart Cell Atlas (www.heartcellatlas.org) and analyzed to establish uniform manifold approximation and projection map, such as Figure 1 As shown in part A.

[0027] Figure 1 Part A in the middle shows that there are 11 major cell populations in the healthy human heart, including atrial myocytes, ventricular myocytes, fibroblasts, endothelial cells, smooth muscle cells, pericytes, myeloid cells, lymphoid cells, mesothelial cells, adipocytes and neurons.

[0028] Nrf3 expression in 11 major cell populations in the healthy human heart Figure 1 As shown in part B, Nrf3 is mainly expressed in CMs of healthy human hearts, including atrial and ventricular CMs, inferring the role of Nrf3 in CM homeostasis.

[0029] To investigate the potential role of Nrf3 in cardiac injury, cardiac tissues from necrotic areas (ischemic zone (IZ) and border zone (BZ)) of patients with myocardial infarction, unaffected left ventricular myocardium (remote zone, RZ) of patients with acute myocardial infarction, human heart specimens at the late stage of myocardial infarction (fibrotic zone, FZ), and cardiomyocytes from healthy human hearts (CTRL) were collected from online archives (https: / / cellxgene.czisci-ence.com / collections / 8191c283-0816-424b-9b61-c3e1d6258a77) for snRNA-seq analysis.

[0030] Since Nrf3 is mainly expressed in CMs, CMs were extracted from different cardiac regions and the expression level of Nrf3 was detected. Figure 1 As shown in part C. Figure 1 The violin plots in part C show that the expression level of Nrf3 in BZ CMs cells was significantly higher than that in other cells in other cardiac zones of healthy hearts or human myocardial infarction hearts.

[0031] To confirm the above observations, cardiac tissue specimens from the BZ and RZ regions of patients undergoing surgical ventricular repair after myocardial infarction or heart transplantation were collected and subjected to RT-qPCR and immunofluorescence (IF) staining to measure the Nrf3 fluorescence density. The Nrf3 fluorescence density in the BZ and RZ regions was compared. The results are shown in Figure 2. Figure 1 As shown in parts D to F.

[0032] Figure 1Part D is the test result of RT-qPCR, which shows that the expression levels of Nrf3, Caspase 7, γ-H2AX (γ-H2AX) and Pitx2 in BZ are significantly increased.

[0033] Figure 1 Part E in the middle shows the results of HE and immunofluorescence (IF) staining of cardiac tissue in the BZ and RZ regions. Figure 1 Part F in the middle shows the fluorescence density of Nrf3 in the BZ and RZ regions, and each data point in part F represents the average expression level of Nrf3 in each sample (BZ, n = 5; RZ, n = 3). The results showed that Nrf3 was mainly expressed and increased in CMs within the BZ of human infarcted myocardium.

[0034] Myocardial infarction was induced by sham surgery or permanent ligation of the left anterior descending coronary artery (LAD) in C57BL / 6 mice. Myocardial marginal zone cardiac tissues were collected on day 1 after ligation and analyzed by RT-qPCR, IF staining, and Western blot. Statistical significance was assessed using a two-tailed unpaired t-test with Welch correction.

[0035] The results of RT-qPCR analysis of sham group and simulated myocardial infarction group are shown in Figure 1 As shown in the middle G part, the IF staining results and Nrf3 fluorescence density analysis are as follows Figure 1 As shown in part H (white arrows indicate Nrf3-positive CMs), Western blot analysis was performed as shown in Figure 1 As shown in Part I.

[0036] RT-qPCR, IF staining and Western blot detection showed that the expression levels of Nrf3 mRNA and protein in the heart tissue of mice after injury were significantly increased. Similarly, IF staining also confirmed that Nrf3 was mainly expressed and increased in the BZ area of ​​the heart of mice with myocardial infarction.

[0037] Interestingly, IF staining showed that Nrf3 was mainly localized in CM nuclei within the BZ of infarcted myocardium. Collectively, these data suggest a pathological role for Nrf3 in myocardial infarction.

[0038] Embodiment 2

[0039] Experimental design Figure 2 As shown in Part A, wild-type (WT) and Nrf3 knockout (Nrf3-KO) mice underwent sham surgery or left anterior descending coronary artery (LAD) ligation to induce myocardial infarction and were analyzed 28 days later.

[0040] like Figure 2Part B shows the survival curves of the sham-operated (sham) and simulated myocardial infarction (MI) mice in the WT group and the Nrf3-KO group. The numbers in brackets in the figure indicate the number of mice used in each group.

[0041] like Figure 2 Part C shows representative echocardiograms of cardiac function analysis of sham and simulated myocardial infarction (MI) mice in the WT and Nrf3-KO groups.

[0042] like Figure 2 Part D in the middle shows left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular internal diameter (LVID; d), left ventricular posterior wall thickness at end-diastole (LVPW; d), and interventricular septum thickness at end-diastole (IVS; d), respectively. (d) is presented here.

[0043] like Figure 2 Part E shows representative images of HE, Masson, and PSR staining of sham and simulated myocardial infarction (MI) mice in the WT and Nrf3-KO groups at 28 days.

[0044] like Figure 2 Part F shows the quantitative analysis of cardiac fibrosis area in sham-operated (sham) and simulated myocardial infarction (MI) mice in the WT and Nrf3-ko groups.

[0045] Compared with WT mice, Nrf3-KO mice had a higher survival rate after myocardial infarction, and significantly reduced left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular posterior wall thickness at end diastole (LVPW; d), and interventricular septum thickness at end diastole (IVS; d). However, left ventricular inner diameter at end diastole increased (LVID; d). Histological analysis on day 28 after myocardial infarction showed that Nrf3-KO mice had smaller cardiac scars compared with WT mice. There were no significant differences in all indicators between WT and Nrf3-KO mice under sham surgery. It can be seen that global knockout of Nrf3 can improve cardiac function and inhibit cardiac fibrosis after myocardial infarction.

[0046] Embodiment 3

[0047] Experimental design Figure 3 As shown in Part A, the control group (Nrf3 flox ) and cardiomyocyte (CM)-specific knockout of Nrf3 (Nrf3 △CM ) mice underwent sham surgery or left anterior descending coronary artery (LAD) ligation to induce myocardial infarction (MI) and were analyzed 28 days later.

[0048] like Figure 3 Part B shows Nrf3flox Sham group, Nrf3 flox MI group, Nrf3 △CM Sham and Nrf3 △CM Survival curves of mice in the MI group. The numbers in brackets indicate the number of mice used in each group.

[0049] like Figure 3 Part C shows Nrf3 flox Sham group, Nrf3 flox MI group, Nrf3 △CM Sham and Nrf3 △CM Representative echocardiograms of cardiac function analysis in mice in the MI group.

[0050] like Figure 3 Part D in the middle shows the echocardiographic analysis of left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular internal diameter (LVID; d), left ventricular posterior wall thickness (LVPW; d), and interventricular septum thickness (IVS; d).

[0051] like Figure 3 Part E shows Nrf3 flox Sham group, Nrf3 flox MI group, Nrf3 △CM Sham and Nrf3 △CM Representative images of HE, Masson, and PSR staining in mice in the MI group 28 days after surgery.

[0052] like Figure 3 Part F shows Nrf3 flox Sham group, Nrf3 flox MI group, Nrf3 △CM Sham and Nrf3 △CM Quantification of cardiac fibrosis area in mice in the MI group 28 days after surgery.

[0053] The results showed that Nrf3 flox Compared with the MI group, Nrf3 △CM The MI group had a higher survival rate and significantly reduced left ventricular ejection fraction (LVEF), fractional shortening (LVFS), left ventricular posterior wall thickness at end diastole (LVPW; d), and interventricular septum thickness at end diastole (IVS; d). However, left ventricular internal diameter at end diastole (LVID; d) increased. Histological analysis at day 28 after myocardial infarction showed that Nrf3 flox Compared with mice, Nrf3 △CM The mice had smaller cardiac scars. flox Nrf3 △CMThere were no significant differences in all indicators of mice, indicating that inhibiting Nrf3 can protect mice from myocardial infarction, improve cardiac function, and improve adverse cardiac remodeling caused by myocardial infarction.

[0054] Embodiment 4

[0055] All BZ and CTRL CMs were extracted from the snRNA-seq dataset in Example 1, and gene set enrichment analysis (GSEA) was performed on the marker gene sets of BZ and CTRL subgroups. The results showed that the ROS pathway was upregulated in BZ and downregulated in CTRL CMs.

[0056] In addition, previous studies have shown that Nrf3 regulates the production of ROS during stem cell differentiation, and mitochondrial ROS is at the central stage of myocardial infarction damage. Increased mitochondrial ROS leads to excessive apoptosis and death of CMs after myocardial infarction, impairing cardiac remodeling and deteriorating cardiac function.

[0057] Experimental design Figure 3 As shown in Part A, the control group (Nrf3 flox ) and cardiomyocyte (CM)-specific knockout of Nrf3 (Nrf3 △CM ) mice underwent sham surgery or left anterior descending coronary artery (LAD) ligation to induce myocardial infarction (MI).

[0058] For in vitro and in vivo studies with DHE and MitoSOX, cells or fresh heart tissue were homogenized and stained with 5 μmol / L DHE and 5 μmol / L MitoSOX for 30 min and 10 min, respectively. Chemiluminescence was recorded using a Spark multimode microplate reader (Tecan Group Ltd, Switzerland), and the signal was normalized by Hoechest counts or tissue weight.

[0059] like Figure 4 Part A shows Nrf3 flox Mice and Nrf3 △CM Immunofluorescence (IF) staining images of mitochondrial superoxide (MitoSOX) in myocardial tissue of mice 24 hours after myocardial infarction and relative fluorescence analysis of MitoSOX. flox Compared with mice, Nrf3 △CM The level of mitochondrial ROS generation in mouse myocardial tissue was reduced.

[0060] like Figure 4 Part B shows Nrf3 flox Mice and Nrf3 △CMImmunofluorescence (IF) staining of mice with dihydroethidium (DHE) 24 hours after myocardial infarction and relative fluorescence intensity analysis after DHE staining. flox Compared with mice, Nrf3 △CM The relative fluorescence intensity of mice after DHE staining was significantly reduced, indicating that the level of mitochondrial ROS was low.

[0061] right Figure 4 The results of A and B are verified:

[0062] Nrf3 flox Mice and Nrf3 △CM Heart homogenates were collected from mice 24 hours after myocardial infarction and stained with DHE to detect the relative level of reactive oxygen species (ROS). Figure 4 As shown in part C.

[0063] Mitochondria were isolated from the heart 24 hours after myocardial infarction, and mitochondrial ROS were analyzed using MitoSOX Red. Figure 4 As shown in part D.

[0064] like Figure 4 Part E shows Nrf3 flox Mice and Nrf3 △CM IF staining and TUNEL staining of cardiac troponin I (cTnI) and TUNEL in mice 24 hours after myocardial infarction + Analysis of percentage of CMs.

[0065] like Figure 4 Part F shows Nrf3 flox Mice and Nrf3 △CM IF staining of cardiac troponin I (cTnI) and γ-H2AX in mice 24 hours after myocardial infarction + Analysis of percentage of CMs.

[0066] like Figure 4 Part G in the middle shows Nrf3 flox Mice and Nrf3 △CM IF staining of cardiac troponin I (cTnI) and cleaved caspase3 in mice 24 hours after myocardial infarction + Analysis of percentage of CMs.

[0067] like Figure 4 The H part in the middle shows Nrf3 flox Mice and Nrf3 △CM IF staining of cardiac troponin I (cTnI) and Ki67 in mice 24 hours after myocardial infarction+ Analysis of percentage of CMs.

[0068] The results showed that Nrf3 △CM Injury-induced CM apoptosis in mice was significantly reduced, and Nrf3 △CM TUNEL in mice + ,γ-H2AX + and cleaved caspase 3 + However, Nrf3 flox Mice and Nrf3 △CM Mouse Ki67 + There was no significant difference in the percentage of CMs, suggesting that Nrf3 had no significant effect on CM proliferation after myocardial infarction.

[0069] Embodiment 5

[0070] Most patients with myocardial infarction undergo coronary angioplasty and stent placement to restore blood supply to the infarcted heart, which causes cardiac I / R injury. flox and Nrf3 △CM Myocardial I / R surgery was performed in mice to investigate the potential role of CM-specific Nrf3 in myocardial I / R injury. Figure 5 As shown in part A, the control group (Nrf3 flox ) and cardiomyocyte (CM)-specific knockout of Nrf3 (Nrf3 △CM ) Mice were subjected to I / R injury induced by ligation of the left anterior descending coronary artery for 30 minutes followed by reperfusion, and the mice were analyzed at the indicated time points.

[0071] like Figure 5 Part B shows Nrf3 flox Mice and Nrf3 △CM Representative echocardiograms of mice 28 days after induced I / R injury.

[0072] like Figure 5 Part C shows Nrf3 flox Mice and Nrf3 △CM Left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular internal diameter (LVID; d), left ventricular posterior wall thickness (LVPW; d), and interventricular septum thickness (IVS; d) in mice 28 days after induced I / R injury.

[0073] Depend on Figure 5 As shown in B and C, Nrf3 flox Compared with the group, Nrf3 △CM The cardiac function and ventricular remodeling were significantly improved in the two groups.

[0074] like Figure 5 As shown in the D part, it is Nrf3 flox Mice and Nrf3 △CM Analysis of Nrf3 by triphenyltetrazolium chloride (TTC) staining of cardiac cross-sectional sections 24 hours after I / R injury in mice △CM Smaller infarct size was observed in mice.

[0075] like Figure 5 Part E shows Nrf3 flox Mice and Nrf3 △CM Representative images of HE, Masson and PSR staining and quantitative analysis of cardiac fibrosis area 28 days after I / R injury in mice. flox Compared with the group, Nrf3 △CM The area of ​​cardiac fibrosis in mice was significantly reduced.

[0076] like Figure 5 Part F shows Nrf3 flox Mice and Nrf3 △CM Heart tissues collected 24 hours after I / R injury were induced in mice and stained with cardiac troponin I (cTnI) and γ-H2AX or TUNEL for immunofluorescence (IF). Figure 5 Part G in the middle is the quantification of IF staining.

[0077] IF staining showed Nrf3 △CM The apoptosis of CMs in mice was significantly reduced, and Nrf3 △CM TUNEL in mice + and γ-H2AX + The percentage of CMs decreased.

[0078] The above experimental results show that CM-specific knockout of Nrf3 can improve cardiac function and reduce CM apoptosis in I / R-injured mice.

[0079] Embodiment 6

[0080] The control group (Nrf3 flox ) and cardiomyocyte (CM)-specific knockout of Nrf3 (Nrf3 △CM ) mice were subjected to left anterior descending coronary artery (LAD) ligation to induce myocardial infarction (MI). flox and Nrf3 △CM Mice were intraperitoneally injected with PBS or mitochondrial targeted oxidant (MitoPQ) (0.1 μg / g body weight) for 7 consecutive days. Figure 6 As shown in part A.

[0081] like Figure 6 Part B shows Nrf3 flox +PBS group, Nrf3 flox +MitoPQ group, Nrf3 △CM +PBS group, Nrf3 △CM Survival curve of mice in the +MitoPQ group after 28 days.

[0082] like Figure 6 Part C shows Nrf3 flox +PBS group, Nrf3 flox +MitoPQ group, Nrf3 △CM +PBS group, Nrf3 △CM Echocardiographic analysis of left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular internal diameter (LVID; d), left ventricular posterior wall thickness (LVPW; d), and interventricular septum thickness (IVS; d) in mice in the +MitoPQ group after 28 days. Each dot in the figure represents one mouse, n = 6 or 7 mice per group.

[0083] like Figure 6 As shown in the D part, it is Nrf3 flox +PBS group, Nrf3 flox +MitoPQ group, Nrf3 △CM +PBS group, Nrf3 △CM Cleaved caspase 3 staining images and quantitative analysis of cardiac fibrosis area in mice in the +MitoPQ group after 28 days. n = 5 mice per group, scale bar = 500 μm.

[0084] like Figure 6 Part E shows Nrf3 flox +PBS group, Nrf3 flox +MitoPQ group, Nrf3 △CM +PBS group, Nrf3 △CM Heart tissues collected from mice in the +MitoPQ group after 28 days were subjected to immunofluorescence (IF) staining images and quantitative analysis of IF staining using cardiac troponin I (cTnI) and γ-H2AX.

[0085] like Figure 6 Part F shows Nrf3 flox +PBS group, Nrf3 flox +MitoPQ group, Nrf3 △CM +PBS group, Nrf3 △CM Heart tissues collected from mice in the +MitoPQ group after 28 days were subjected to immunofluorescence (IF) staining images and quantitative analysis of IF staining using cardiac troponin I (cTnI) and TUNEL.

[0086] The results showed that after PBS injection, Nrf3 flox Compared with the group, Nrf3 △CM The survival rate of mice increased. flox The survival rate of mice in the +MitoPQ group was the lowest, and Nrf3 flox + PBS group mice and Nrf3 △CM The survival rate of mice in the +MitoPQ group was comparable. flox In contrast, Nrf3 △CM The cardiac function of mice was improved and the area of ​​fibrosis was reduced; however, Nrf3 flox and Nrf3 △CM There was no significant difference between the mice in the +MitoPQ group. IF staining showed that Nrf3 flox Compared with the mice in the +PBS group, Nrf3 △CM MitoPQ increased Nrf3 in mice with decreased CM apoptosis and mitochondrial ROS production flox Importantly, after administration of MitoPQ to mice, the observed flox Nrf3 △CM The differences between the mice disappeared.

[0087] The above experimental results indicate that Nrf3 promotes MI-induced CM apoptosis and cardiac injury by regulating the production of mitochondrial ROS.

[0088] Embodiment 7

[0089] H9C2 cells were infected with Nrf3 overexpression vectors (Ad-flag-Nrf3 or AdNrf3). Cell lysate was immunoprecipitated with IgG control. Immunoprecipitated DNA was subjected to DNA sequencing (ChIP-seq) analysis. Representative views of ChIP-seq data for the Pitx2 gene locus are shown in Figure 2. Figure 7 As shown in part A, Nrf3 is significantly enriched in the promoter region of the Pitx2 gene.

[0090] Neonatal rat ventricular myocytes (NRVMs) infected with IgG (control) or Ad-flag-Nrf3 were exposed to H2O2 (200 μM; 12 h) and then subjected to ChIP assay. Figure 7 As shown in part B, it was further confirmed that Nrf3 was enriched at the promoter of the Pitx2 gene.

[0091] Based on the Pitx2 DNA sequence motif detected by ChIP-seq and predicted by STREME of The MEME Suite34, a potential Nrf3 binding element close to -2100 was found near the transcription start site of the Pitx2 gene, and two luciferase reporter plasmids were constructed. The construction strategy is as follows Figure 8 Shown are the wild-type (WT) Pitx2 promoter and a mutant Pitx2 promoter lacking the predicted binding motif.

[0092] NRVMs cells infected with control (IgG, AdCtrl) or Ad-flag-Nrf3 (AdNrf3) vectors were transfected with wild-type (WT) or mutant (Mut) Nrf3 binding sites reported by the Pitx2 gene promoter as indicated, and then luciferase activity analysis was performed. The results are shown in Figure 7 As shown in part C. Luciferase activity assay data showed that overexpression of Nrf3 in CMs significantly reduced the activity of the WTPitx2 gene promoter, but failed to inhibit the activity of the mutant Pitx2 gene promoter. This indicates that the Nrf3 binding element is required for Nrf3-mediated Pitx2 gene transcription.

[0093] RT-qPCR analysis of Pitx2 mRNA in NRVMs cells infected with AdCtrl or AdNrf3 treated with H2O2 Figure 7 As shown in part D.

[0094] NRVMs transfected with control (siCtrl) or Nrf3-specific siRNA (siNrf3) were treated with H2O2. Western blot analysis and semiquantitative data of Pitx2 protein are shown in Figure 7 As shown in part E,

[0095] The RNA sequence of siNrf3 is:

[0096] ratNrf3-sense,5′-GUGGACCAUAGGACUAUAUTT-3′,

[0097] ratNrf3-anti-sense,5′-AUAUAGUCCUAUGGUCCACTT-3′.

[0098] The siCtrl in the control group was a nonsense RNA sequence.

[0099] It can be seen that in NRVMs cells treated with H2O2, Nrf3 overexpression significantly reduced the expression of Pitx2 ( Figure 7 D), while Nrf3 knockdown significantly upregulated the protein and mRNA levels of Pitx2 ( Figure 7E and F).

[0100] The control group (Nrf3 flox ) and cardiomyocyte (CM)-specific knockout of Nrf3 (Nrf3 △CM Myocardial infarction (MI) was induced by ligation of the left anterior descending coronary artery (LAD) in mice. Figure 7 Part G in the middle shows the expression of Nrf3 28 days after myocardial infarction. flox and Nrf3 △CM Western blot analysis and semi-quantitative data analysis of Pitx2 protein expression in mouse myocardium. Figure 7 Part H shows the expression of Nrf3 28 days after myocardial infarction. flox and Nrf3 △CM RT-qPCR analysis of mouse myocardial Pitx2 mRNA. The results showed that it was related to Nrf3 flox Compared with mice, Nrf3 △CM The expression of Pitx2 was increased in mouse heart tissue, confirming the role of Nrf3 in suppressing Pitx2 gene regulation in myocardial infarcted hearts.

[0101] To further confirm the direct inhibitory effect of Nrf3 on Pitx2 gene transcription in myocardial tissue after myocardial infarction, AAV9 vectors containing Nrf3 and Flag-tagged proteins were injected into mice to induce myocardial injury.

[0102] Mice were randomly injected with control (AAV9-ctrl) or Nrf3 overexpression (AAV9-flag-Nrf3) adeno-associated virus 9 (AAV9) vectors via the tail vein. Four weeks later, the mice underwent left anterior descending coronary artery ligation to induce myocardial infarction. One day later, heart tissues were collected for Western blot analysis, RT-qPCR analysis, and ChIP analysis.

[0103] Western blot analysis results Figure 7 As shown in Part I, the RT-qPCR analysis results are shown in Figure 7 The results of ChIP analysis are shown in Figure 5(J) and (K).

[0104] Western blot and RT-qPCR analysis data showed that Nrf3 overexpression in damaged heart tissue significantly reduced Pitx2 expression. ChIP experiments showed that Nrf3 directly bound to the Pitx2 gene promoter and Nrf3 overexpression was significantly enriched in damaged heart tissue.

[0105] The above experimental results indicate that Pitx2 is a functional target of Nrf3 in the context of cardiac injury.

[0106] Embodiment 8

[0107] First, the promoter region of Pitx2 gene was analyzed, and a large number of CpG islands were found in the promoter region of Pitx2 gene using MethPrimer online software, such as Fig. 9 As shown in part A.

[0108] NRVMs were transfected with control group (siCtrl or AdCtrl), Nrf3-specific siRNA (siNrf3), or Nrf3 overexpression vector (AdNrf3). Genomic DNA was extracted and bisulfite-converted, followed by PCR analysis.

[0109] like Fig. 9 Part B shows the PCR analysis results of NRVMs transfected with siCtrl and siNrf3; Fig. 9 Part C in the middle shows the PCR analysis results of NRVMs transfected with AdCtrl and AdNrf3. The data of bisulfite PCR analysis with specific primers showed that Nrf3 knockdown significantly reduced the methylation level of the promoter region of the Pitx2 gene, while Nrf3 overexpression had the opposite effect.

[0110] NRVMs were infected with control (AdCtrl) or Nrf3 overexpressing (AdNrf3) adenovirus and then incubated with H2O2 using Pierce TM The immunoprecipitation experiment was performed using the Classic Magnetic IP / Co-IP Kit (Thermo Fisher Scientific, 88804). The immunoprecipitated complexes were separated by SDS-PAGE and then stained with Coomassie Brilliant Blue R-250 to visualize the protein bands. The protein complexes were incubated and precipitated with anti-Flag antibody, and then Western blot analysis was performed with Nrf3, hnRNPK, and DNMT1 antibodies. The protein bands and analysis results are shown in Figure 2. Fig. 9 As shown in part D.

[0111] NRVMs were infected with control (AdCtrl) or Nrf3 overexpressing (AdNrf3) adenovirus and then incubated with H2O2. TM The immunoprecipitation experiment was performed using the Classic Magnetic IP / Co-IP Kit (Thermo Fisher Scientific, 88804). The immunoprecipitated complexes were separated by SDS-PAGE and subsequently stained with Coomassie Brilliant Blue R-250 to visualize the protein bands. The protein complexes were incubated and precipitated with anti-hnRNPK antibody, and then Western blot analysis was performed using Nrf3, hnRNPK, and DNMT1 antibodies. The protein bands and analysis results are shown in Figure 2. Fig. 9 As shown in part E.

[0112] Co-immunoprecipitation (co-IP) experiments verified the interaction between Nrf3, hnRNPK and DNMT1 in H2O2-treated NRVMs.

[0113] In NRVMs co-transfected with control (AdCtrl) or Nrf3 overexpression vector (AdNrf3) and / or DNMT1 knockdown siRNA and exposed to H2O2 stimulation, the CpG island in the promoter region of Pitx2 gene was identified by sulfite PCR. Fig. 9 As shown in Part F, Western blot analysis and semi-quantitative data of Pitx2 protein in NRVMs are shown in Fig. 9 As shown in part G.

[0114] The results showed that overexpression of Nrf3 alone significantly increased and decreased the methylation level of the Pitx2 gene promoter region and the expression of Pitx2 protein; while the simultaneous downregulation of DNMT1 in NRVMs offset the regulatory effects of Nrf3 overexpression on Pitx2 gene promoter methylation and protein expression.

[0115] Embodiment 9

[0116] like Fig.10 As shown in Part A, the control group (Nrf3 flox ) and Nrf3 cardiomyocyte (CM)-specific knockout mice (Nrf3 △CM ) were randomly injected with a control group (AAV-Ctrl) or adeno-associated virus 9 (AAV9)-cnt-vector specific for Pitx2 gene (AAV9-cnt-shPitx2, AAV-shPitx2). Four weeks later, myocardial infarction was induced by ligation of the left anterior descending coronary artery in the mice, and various analyses were performed.

[0117] The survival curves of mice in each group after myocardial infarction was induced in mice. Fig.10 As shown in Part B, the numbers in brackets indicate the number of mice used in each group.

[0118] Due to the role of Pitx2 in myocardial injury Nrf3 △CM The expression of Figure 7 G and H), and thus a CM-specific AAV9-cTNT-shPitx2 vector (AAV-shPitx2) was generated to selectively silence the expression of Pitx2 in CM by western blot, RT-qPCR, and IF staining analysis.

[0119] The RNA sequence of shPixt2 is:

[0120] Pitx2-sense,5′-GUACGUUUAUAGGGACACATT-3′;

[0121] Pitx2-anti-sense,5′-UGUGUCCCUAUAAACGUACTT-3′.

[0122] Nrf3 injected with control (AAV-Ctrl) vector flox Compared with mice injected with AAV-Ctrl vector, Nrf3 △CM MI-induced mortality in mice was reduced when Nrf3 was injected with AAV-shPitx2 vector flox There was a trend toward increased MI-induced mortality in mice. Importantly, Nrf3 △CM Pitx2 knockdown in mice increased mortality similar to that of Nrf3 injected with AAV-Ctrl flox Mouse

[0123] After inducing myocardial infarction in mice, the changes of left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular internal diameter (LVID; d), left ventricular posterior wall thickness (LVPW; d), and interventricular septum thickness (IVS; d) were observed in each group of mice. Fig.10 As shown in part C.

[0124] and Fig.10 Similar results were shown in Figure 2B. When AAV-Ctrl vector was administered, Nrf3 △CM Cardiac function in mice is better than that in Nrf3 flox The mice showed improvement, whereas this improvement was lost when the mice were given AAV-shPitx2.

[0125] like Fig.10 Part D shows representative images of Masson staining and quantification of cardiac fibrosis area in each group of mice 28 days after surgery. Scale bar = 500 μm. flox Compared with mice treated with AAV-Ctrl vector, Nrf3 △CM Cardiac fibrosis was reduced in mice treated with AAV-shPitx2, but Nrf3 flox Mice and Nrf3 △CM The mice had comparable levels of cardiac fibrosis.

[0126] IF staining of cardiac troponin I (cTnI) and γ-H2AX and γ-H2AX 24 hours after myocardial infarction in mice + CMs percentage analysis Fig.10 As shown in the middle E part; IF staining of cardiac troponin I (cTnI) and TUNEL +CMs percentage analysis Fig.10 As shown in the middle F part; IF staining and IF staining quantification of cardiac MitoSOX are shown in Fig.10 As shown in the middle G. The results show that reducing the level of Pitx2 in cardiomyocytes abolishes the reduction of cardiomyocyte apoptosis and ROS production after myocardial infarction caused by cardiac-specific Nrf3 gene deletion.

[0127] The above experimental results indicate that cardiac-specific knockdown of Pitx2 will weaken the beneficial effects of cardiac-specific Nrf3 gene deletion on cardiac function and pathological remodeling after myocardial infarction (MI).

[0128] Embodiment 10

[0129] To further validate the cardioprotective effect of Pitx2 activation after myocardial infarction, a CM-specific AAV9-cTNT-Pitx2 vector (AAV9-Pitx2) was generated and injected into mice for cardiac overexpression. Fig.11 As shown in part A.

[0130] Control group (Nrf3 flox ) and cardiomyocyte (CM)-specific knockout of Nrf3 (Nrf3 △CM ) mice were randomly given a control group (AAV9-Ctrl) or adeno-associated virus 9 (AAV9)-ctnt vector for cardiac-specific overexpression of Pitx2 (AAV9-ctnt-Pitx2, AAV9-Pitx2) by tail vein injection. Four weeks later, myocardial infarction was induced by left anterior descending coronary artery ligation in mice, and various analyses were performed.

[0131] Echocardiograms of mice in each group after myocardial infarction were induced. Fig.11 As shown in part B, the left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS) of mice in each group were Fig.11 As shown in part C.

[0132] like Fig.11 Part D in the middle shows representative images of PSR staining of mice in each group 28 days after surgery and quantification of the area of ​​cardiac fibrosis in mice. Scale bar = 500 μm.

[0133] IF staining of cardiac troponin I (cTnI) and γ-H2AX was performed in mice 24 hours after myocardial infarction. Fig.11 As shown in the middle E part; IF staining of cardiac troponin I (cTnI) and TUNEL is shown in Fig.11 As shown in the middle F part; IF staining of cardiac MitoSOX as shown Fig.11 As shown in part G.

[0134] and Fig.11 The corresponding γ-H2AX in E + CMs percentage analysis Fig.11 As shown in the H part; Fig.11 The corresponding TUNEL in F + CMs percentage analysis Fig.11 As shown in Part I; Fig.11 Quantitative analysis of MitoSOX IF staining corresponding to G in Figure 2 Fig.11 As shown in part J.

[0135] Compared with control mice, mice with cardiac-specific deletion of Nrf3 or overexpression of Pitx2 showed improved cardiac function, reduced cardiac fibrosis, decreased CM apoptosis, and reduced mitochondrial ROS production ( Fig.11 In addition, we observed non-significant synergistic effects of cardiac-specific Nrf3 inhibition and Pitx2 activation on all parameters examined ( Fig.11 Chinese BJ).

[0136] The above experimental results indicate that overexpression of Pitx2 after myocardial infarction can significantly improve cardiac function, reduce mitochondrial ROS generation and CM apoptosis.

[0137] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. Application of Nrf3 inhibitors in the preparation of drugs for preventing and treating myocardial infarction and heart damage diseases.

2. The use according to claim 1, characterized in that: The prevention and treatment of myocardial infarction and heart damage diseases includes at least one of the following 1) to 4): 1) Promote Pitx2 expression; 2) Inhibit mitochondrial reactive oxygen species production; 3) Inhibit cardiomyocyte apoptosis induced by ischemia-reperfusion injury; 4) Improve myocardial function.

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