Application of MAVS as a marker of myocardial ischemia-reperfusion injury

Abstract

The present invention proposes the application of MAVS as a marker of myocardial ischemia-reperfusion injury; discloses that MAVS, as a risk factor for promoting myocardial ischemia-reperfusion, can activate the TRAF6 / TAK1 / JNK non-classical pathway and induce apoptosis and inflammation of downstream cardiomyocytes, causing late adverse ventricular remodeling including decreased cardiac function, increased myocardial fibrosis, and decreased angiogenesis; after knocking down the expression of MAVS in the cardiomyocyte cell line HL-1, the apoptosis induced by hypoxia-reoxygenation can be significantly reduced, the stability of mitochondrial membrane potential can be increased, and the degree of decrease of protective reduced glutathione induced by hypoxia-reoxygenation can be reduced, thereby maintaining the stability of cardiomyocytes in a hypoxic environment; in a hydrogen peroxide-induced apoptosis model, MAVS knockdown can significantly inhibit the NF-κB inflammatory signaling pathway, showing a significant anti-inflammatory effect; the results from in vivo and in vitro show that inhibiting the expression of MAVS can inhibit myocardial ischemia-reperfusion injury, and has good application prospects for treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of MAVS as a marker of myocardial ischemia-reperfusion injury. Background Art

[0002] Coronary artery occlusion in coronary heart disease can cause myocardial infarction (MI). Coronary artery thrombolysis and interventional therapy are currently effective treatments for vascular recanalization. However, reperfusion after coronary artery recanalization in patients with myocardial infarction often causes myocardial damage, a second blow to the myocardium, also known as myocardial ischemia-reperfusion injury (MIRI). This injury often leads to complications and may be life-threatening, seriously restricting the postoperative recovery of patients with myocardial infarction. Mitochondrial damage to myocardial cells in the infarct area and peri-infarct cardiac tissue is one of the main mechanisms of MIRI. Mitochondria are the main source of ATP in myocardial cells and regulate a variety of cell functions. Mitochondrial proteins include outer membrane proteins and inner membrane proteins. Studies have shown that inner membrane proteins are mainly involved in the oxidative respiratory chain function and play an important role in MIRI, but mitochondrial outer membrane proteins have received less attention. Further exploration of risk factors in the occurrence and development of the disease has important clinical research value.

[0003] Mitochondrial antiviral signaling proteins (MAVS) are components of the mitochondrial outer membrane. MAVS consists of an N-terminal caspase recruitment domain, a proline-rich middle region, and a single transmembrane domain at the C-terminus. MAVS can be activated by viral RNA sensors such as melanoma differentiation-associated protein 5 (MDA5) and retinoic acid-inducible gene I (RIG-I), and participates in antiviral immune responses by promoting the expression of interferon-stimulated genes and inflammatory cytokines through the activation of nuclear factor κb (NF-κB) and interferon regulatory factor 3 (IRF3). In this process, MAVS binds to conformationally altered RIG-I through its CARD domain and forms prion-like aggregates on the mitochondrial outer membrane, triggering its activation. The proline-rich region in the middle of MAVS can bind to multiple members of the tumor necrosis factor (TNF)-related factor family, including TRAF2, TRAF3, TRAF5, and TRAF6, to promote downstream signal transduction. Among them, TRAF6 is associated with target cell apoptosis after viral infection. Although studies have shown that MAVS may cause myocardial hypertrophy and non-hypertrophic cardiac dysfunction by participating in lipid metabolism, the role of MAVS in MIRI is still unclear and deserves further study.

[0004] The mitogen activated protein kinase (MAPK) family plays important and diverse roles in signal transduction during cardiac development, physiological adaptation, and pathological conditions. MAPKs include four subfamilies, including extracellular signal-regulated kinases ERK (ERK1, ERK2), p38 kinase, large MAPK (BMK), and c-jun-NH2-terminal kinases (JNK1, JNK2, and JNK3). JNK, as a stress-activated protein kinase, can be activated by MAP3Ks during MIRI-induced cellular stress, DNA damage, inflammatory factors, and endoplasmic reticulum disorder. In addition, TGF-β-activated kinase 1 (TAK1), encoded by the Map3k7 gene, can regulate cardiomyocyte necrosis and affect ventricular remodeling during myocardial overload and significant stages of atherosclerosis in mice, but the role of TAK1 in MIRI remains unclear. Therefore, it is of great significance to study the factors involved in injury-related diseases in MIRI. Summary of the invention

[0005] The present invention proposes the application of MAVS as a marker of myocardial ischemia-reperfusion injury. This marker can provide guidance for related applications such as detection and diagnosis of myocardial ischemia-reperfusion injury, as well as prevention and / or treatment, and has good application prospects.

[0006] On one hand, the present invention proposes the use of MAVS as a marker in the preparation of a diagnostic kit for myocardial ischemia-reperfusion injury, wherein the kit predicts whether myocardial ischemia-reperfusion injury occurs by detecting the overexpression level of MAVS.

[0007] Another aspect of the present invention provides the use of MAVS as a marker in the preparation of drugs for the prevention and / or treatment of myocardial ischemia-reperfusion injury, wherein the drugs include MAVS inhibitors.

[0008] Furthermore, the MAVS inhibitor includes an adeno-associated virus that knocks down MAVS using a myocardial-specific promoter.

[0009] Preferably, the myocardium-specific promoter is cTNT.

[0010] Preferably, the adeno-associated virus is AAV9, which is used to improve the efficiency of targeting the heart.

[0011] Preferably, the vector after the myocardial-specific promoter knocks down MAVS is pAV-cTnT-shRNA-MAVS.

[0012] Furthermore, the preventive and / or therapeutic drug is used for systemic intravenous injection or intrapericardial injection.

[0013] Furthermore, the preventive and / or therapeutic drug also includes a pharmaceutically acceptable carrier.

[0014] Furthermore, the preventive and / or therapeutic drug is used to block the MAVS / TRAF6 / TAK1 / JNK signaling pathway.

[0015] Furthermore, the above-mentioned preventive and / or therapeutic drugs can reduce hypoxia-reoxygenation-induced cell apoptosis, increase mitochondrial membrane potential stability, and reduce the decrease of protective reduced glutathione induced by hypoxia-reoxygenation, thereby maintaining the stability of cardiomyocytes under stressful environments.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention proposes the application of MAVS as a marker of myocardial ischemia-reperfusion injury. This marker can provide guidance for the detection and diagnosis of myocardial ischemia-reperfusion injury, promote the development of drugs targeting MAVS and its application in the prevention or treatment of myocardial ischemia-reperfusion injury, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 The results in Example 1 show that hypoxia-reoxygenation can induce increased MAVS expression in cardiomyocyte mitochondria, and MAVS knockdown can reduce the susceptibility of cardiomyocytes to hypoxic injury.

[0020] Figure 2 This is the result of Example 1 that MAVS knockdown can reduce the susceptibility of cardiomyocytes to hypoxic injury.

[0021] Figure 3 These are the experimental results of Example 2 that MAVS overexpression can activate the NF-κB pathway and MAPK / JNK pathway in vitro.

[0022] Figure 4 This is the protective effect of MAVS knockdown on cardiomyocyte apoptosis induced by hydrogen peroxide in vitro in Example 3.

[0023] Figure 5 These are the experimental results of the effect of MAVS knockout on cardiac ischemia-reperfusion injury in vivo in Example 4.

[0024] Figure 6 This is the result of MAVS knockout alleviating the adverse ventricular remodeling caused by ischemia-reperfusion in Example 5.

[0025] Figure 7 These are the experimental results of Example 6 in which myocardial-specific knockout of MAVS alleviated the degree of MIRI injury.

[0026] Figure 8 These are the experimental results of Example 7 in which specific knockdown of myocardial MAVS alleviated adverse ventricular remodeling in the late stage of MIRI.

[0027] Fig. 9 This is the result of MAVS inducing cardiomyocyte apoptosis by activating the MAPK / JNK pathway through the TAK1 / TRAF6 axis in Example 8.

[0028] Fig.10 This is the result of myocardial-specific knockout of MAVS in Example 9 inhibiting MIRI injury-related signaling pathways in vivo.

[0029] Fig.11 This is the result of the effect of the JNK pathway inhibitor in Example 10 on the early stage of cardiac ischemia-reperfusion injury.

[0030] Fig.12 These are the experimental results of the effect of JNK pathway inhibitors on MIRI late-stage damage in Example 11. DETAILED DESCRIPTION

[0031] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] In one embodiment, it was determined that the occurrence of MIRI promoted the high expression of MAVS, which in turn aggravated the degree of injury of MIRI, and the feasibility of inhibiting MAVS expression as an intervention strategy for treating injury caused by cardiac ischemia-reperfusion was evaluated.

[0033] In another embodiment, an in vitro experiment was used to construct a cardiomyocyte cell line HL-1 that overexpressed and knocked down MAVS. In a hydrogen peroxide-stimulated cell apoptosis model, Western-blot was used to detect the expression of RIG-I, MAVS, TAK1, p-TAK1, TRAF6, IRF3, p-IRF3, TBK1, p-TBK1, P65, p-P65, JNK, p-JNK, PARP, Caspase3 shearing, BAX, and BCL2 apoptosis-related proteins. It was found that knocking down MAVS in cardiomyocytes HL-1 can significantly reduce the expression of apoptosis-related proteins and inhibit the activation of the inflammatory signaling pathway NF-κB pathway to a certain extent. By flow cytometry detection of apoptosis of HL-1 cell lines under hypoxia-reoxygenation conditions, as well as the expression of mitochondrial membrane potential TMRE and protective reduced glutathione GSH, it was found that knocking down MAVS can enhance the ability of cardiomyocytes to resist hypoxia-reoxygenation stress environments, which is reflected in reducing the proportion of cardiomyocyte apoptosis, inhibiting the decrease in mitochondrial membrane potential TMRE, and reducing the degree of decrease in the expression of GSH, thereby playing an effective protective role.

[0034] In vivo mouse experiments used systemic MAVS knockout and cardiomyocyte MAVS-specific knockdown mouse models to evaluate the effect of inhibiting MAVS expression on the disease in a myocardial ischemia-reperfusion injury model. Cardiac ultrasound, serum myocardial enzyme detection, transmission electron microscopy of heart tissue, EB+TTC double staining, TUNEL staining of myocardial tissue sections, H&E, Masson staining, and Western-blot experiments confirmed that systemic knockout or cardiomyocyte-specific knockdown of MAVS can effectively protect against myocardial ischemia-reperfusion injury, specifically in the inhibition of the TBK1 / IRF3 / NF-κB pathway and the RIG-I / MAVS-MAPK / JNK signaling pathway, reduced mitochondrial damage in myocardial tissue, reduced cell apoptosis in myocardial tissue, reduced tissue inflammation, reduced myocardial tissue fibrosis, and increased angiogenesis. At the same time, the small molecule inhibitor AS601245 was used to act on the MAPK-JNK signaling pathway to evaluate the effect of AS601245 on MIRI, and the results confirmed that AS601245 can effectively alleviate the degree of injury in MIRI.

[0035] In the above examples, it is revealed that during cardiac ischemia-reperfusion, MAVS is highly expressed and aggravates the degree of MIRI damage, while inhibiting MAVS expression can reduce myocardial tissue apoptosis and improve the damage caused by cardiac ischemia-reperfusion.

[0036] Example 1 MAVS knockdown reduces the susceptibility of cardiomyocytes to hypoxic injury

[0037] The experimental method is as follows: First, the HL-1 cardiomyocyte cell line was treated with 8 hours of hypoxia and 6 hours of reoxygenation, and the expression of MAVS was detected in the extracted mitochondria and whole cell lysates. Then, HL-1 cell lines with overexpression of MAVS and knockdown of MAVS were constructed and treated with hypoxia and reoxygenation, and cell apoptosis, mitochondrial membrane stability and intracellular reduced glutathione content were detected. The results of the susceptibility experiment are shown in Figure 2. Figure 1-2 shown.

[0038] Figure 1 Figure A shows that the HL-1 cell line induces increased expression of MAVS in cardiomyocytes under hypoxia and reoxygenation conditions, and the increased expression of MAVS is mainly concentrated in mitochondria. Figure B shows the successful construction of WB verification of HL-1 cell lines with overexpression of MAVS (MAVS-oe) and knockdown of MAVS (MAVS-sh), NC-oe and NC-sh are overexpression and knockdown controls, respectively. Figure C shows that the HL-1 cell line with MAVS knockdown significantly reduces cell apoptosis after 16 hours of hypoxia and 8 hours of reoxygenation. Figure 2 Figure A is the above Figure 1 Statistical analysis of apoptosis in HL-1 cell lines in B. Figure B shows that MAVS knockdown in HL-1 cell lines can reduce the degree of hypoxia-reoxygenation-induced decrease in mitochondrial membrane potential (TMRE). Figure 2 Statistical analysis of mitochondrial membrane potential (TMRE) of HL-1 cells in A. Figure D shows that the HL-1 cell line with MAVS knockdown can maintain the intracellular GSH level after hypoxia and reoxygenation, playing a protective role.

[0039] Example 2 MAVS can activate NF-κB pathway and MAPK / JNK pathway in vitro

[0040] The experimental method is as follows: HL-1 cell lines with overexpression and knockdown of MAVS were plated and protein samples were collected 72 hours later, and the NF-κB pathway and MAPK / JNK pathway were detected by western-blot. Figure 3 shown.

[0041] Figure 3 In the figure, A shows that MAVS overexpression can lead to increased expression of RIG-I, TAK1 and TRAF6 and enhanced TAK1 phosphorylation in HL-1 cell lines. B shows that MAVS overexpression can enhance the phosphorylation of TBK1, IRF3, and P65 in HL-1 cell lines. C shows that MAVS overexpression can enhance the phosphorylation of JNK and P38 in HL-1 cell lines. D shows that MAVS overexpression can lead to increased pro-apoptotic proteins PARP, BAX, cleaved caspase3 and decreased expression of anti-apoptotic BCL2 in HL-1 cells.

[0042] Example 3 MAVS knockdown can reduce cardiomyocyte apoptosis induced by hydrogen peroxide stimulation in vitro

[0043] The experimental method is as follows: HL-1 cell line was stimulated with 600 μM hydrogen peroxide for 8 hours and then cell proteins were extracted for Western-blot detection. The effect of MAVS knockdown on hydrogen peroxide-induced cardiomyocyte apoptosis in vitro was analyzed. The results are as follows Figure 4 shown.

[0044] Figure 4 Figure A shows that MAVS knockdown can inhibit the increased expression of RIG-I, TAK1 and TRAF6 in HL-1 cells after hydrogen peroxide stimulation. Figure B shows that MAVS knockdown can inhibit the enhanced phosphorylation of TBK1, IRF3, and P65 in HL-1 cells after hydrogen peroxide stimulation. Figure C shows that MAVS knockdown can inhibit the enhanced phosphorylation of JNK and P38 after hydrogen peroxide stimulation. Figure D shows that MAVS knockdown can reduce the expression of pro-apoptotic molecules PARP, BAX and cleaved caspase3 after hydrogen peroxide stimulation and increase the expression of anti-apoptotic protein BCL2, thereby protecting cardiomyocytes.

[0045] Example 4 MAVS knockout can reduce cardiac ischemia-reperfusion injury in vivo

[0046] The experimental method is as follows: First, WT mice were subjected to a 30-minute left anterior descending ischemia followed by a 6-hour reperfusion model, and MAVS expression was detected in the cardiac tissue of the infarcted area (IA), the area at risk (AAR), and the distal area (RA). Then, mice with systemic MAVS knockout (MAVS-KO) were used for the same modeling process, and cardiac ultrasound and tissue transmission electron microscopy were performed 6 hours after reperfusion, as well as serum myocardial enzyme spectrum detection, EB+TTC double staining was performed at 24 hours, and histopathological staining and TUNEL staining of myocardial tissue sections were performed at 3 days. The results are shown in Figure 2. Figure 5 shown.

[0047] Figure 5 In the figure, A shows that the expression of MAVS in the cardiac area at risk (AAR) and infarct area (IA) of WT mice increased compared with the expression of MAVS in the distal part of the heart and sham operation group 6 hours after ischemia-reperfusion surgery. B shows the echocardiogram of MAVS-KO mice and WT mice 6 hours after reperfusion. C shows that the cardiac contractile function of MAVS-KO mice 6 hours after reperfusion, including left ventricular ejection fraction (LVEF), left ventricular shortening fraction (LVFS), left ventricular end-systolic volume (LVESV), and left ventricular end-diastolic volume (LVEDV), was better than that of WT control mice. D shows that the left ventricular infarct volume (IA) of MAVS-KO mice was significantly reduced compared with WT after 2,3,5-triphenyltetrazolium chloride (TTC) + EB staining. E shows that the degree of inflammation in the myocardial tissue of MAVS-KO mice was milder than that of WT mice by H&E staining. F shows that the degree of mitochondrial damage in the infarct area of ​​the heart tissue of MAVS-KO mice was relatively milder 6 hours after reperfusion. Figure G shows that the percentage of damaged mitochondria in myocardial tissue sections of MAVS-KO mice was significantly reduced compared with WT. Figure H shows that TUNEL staining showed that the proportion of apoptotic cells in the heart tissue of MAVS-KO mice decreased compared with WT mice 6 hours after reperfusion. Figure I shows that 6 hours after reperfusion or sham surgery, the plasma myocardial enzyme levels of MAVS-KO mice decreased significantly compared with WT mice, showing a significant protective effect.

[0048] Example 5 MAVS knockout can alleviate adverse ventricular remodeling caused by ischemia-reperfusion

[0049] The experimental method is as follows: 14 days after the myocardial ischemia-reperfusion treatment of MAVS-KO mice, the mouse heart ultrasound test was performed, and the mouse body weight and heart weight were measured. The heart tissue sections were stained with WGA, Masson and CD31. The experimental results are as follows Figure 6 shown.

[0050] Figure 6Figure A shows that on the 14th day after reperfusion, the ratio of heart weight to body weight in MAVS-KO mice was reduced compared with WT mice, effectively inhibiting cardiac hypertrophy. Figure B WGA staining results showed that MAVS-KO mice could reduce cardiomyocyte enlargement on the 14th day after reperfusion. Figure C shows the cardiac ultrasound results of MAVS-KO mice and WT mice on the 14th day after reperfusion. Figure D cardiac ultrasound parameters showed that MAVS-KO mice had stronger cardiac contractile function on the 14th day after reperfusion compared with WT control mice. Figure E Masson staining showed that MAVS-KO mice could reduce the degree of fibrosis in the IA area of ​​myocardial tissue sections on the 14th day after reperfusion compared with WT mice. Figure F Immunofluorescence staining of the vascular endothelial cell marker CD31 in heart sections showed that the neovascularization of MAVS-KO mice on the 14th day was increased compared with WT control mice, alleviating adverse ventricular remodeling.

[0051] Example 6 Myocardial-specific knockout of MAVS can alleviate the degree of MIRI injury

[0052] The experimental method is as follows: WT mice were subjected to 30 min of ischemia followed by 6 h of reperfusion, primary cardiomyocytes and non-cardiomyocytes were isolated, and western-blot detection was performed. AAV9 (vector name pAV-cTnT-shRNA-MAVS) with a cardiomyocyte-specific promoter knocking down MAVS was used to perform 2×10 11 / 100μL dose was injected into the tail vein, and heart tissue was obtained three weeks later. Immunofluorescence of tissue sections was used to detect the efficiency of MAVS knockdown. The experimental mice were subjected to cardiac ischemia-reperfusion model, and cardiac ultrasound and tissue transmission electron microscopy were performed at 6 hours to observe the myocardial ultrastructure, and serum myocardial enzyme spectrum was detected. The expression of reactive oxygen species ROS in cardiac tissue was detected by flow cytometry. EB+TTC double staining was performed 24 hours after reperfusion, and tissue pathology and cardiomyocyte apoptosis TUNEL staining were performed 3 days later. The experimental results are as follows Figure 7 shown.

[0053] Figure 7In the figure, A shows that in primary cardiomyocytes and non-cardiomyocytes isolated from WT mice 6 hours after reperfusion, WB detected that the increased expression of MAVS was mainly in cardiomyocytes. Figure B shows immunofluorescence staining of myocardial tissue sections of mice with myocardial MAVS-specific knockdown (MAVS-sh) and control mice (PLKONC), indicating that MAVS was successfully knocked down in myocardial cells in vivo. Figure C shows that mouse echocardiography shows that MAVS-sh mice have stronger cardiac function than control (PLKONC) mice 6 hours after reperfusion. Figure D shows that the cardiac function parameters LVEF, LVFS, LVESV and LVEDV of MAVS-sh mice are stronger than those of PLKONC mice 6 hours after reperfusion. Figure E shows that TTC staining shows that MAVS-sh mice can effectively reduce the infarct area of ​​myocardial tissue. Figure F shows that the results of H&E staining show that MAVS-sh mice can significantly reduce the degree of necrosis and inflammation of myocardial tissue. Figure G shows that TUNEL staining results show that MAVS-sh can significantly reduce myocardial cell apoptosis on the third day after reperfusion. H is the statistical result of the percentage of apoptotic cells based on the staining results of Figure G. Figure I shows the results of transmission electron microscopy observation. The electron microscopy image shows that the proportion of damaged mitochondria in MAVS-sh mice decreased significantly compared with control mice 6 hours after reperfusion. Figure J shows the results of serum myocardial enzyme detection in mice 6 hours after reperfusion. The myocardial enzyme spectrum indicators of MAVS-sh mice were alleviated compared with those of control mice. Figure K shows that the reactive oxygen species (ROS) in the heart tissue of the MAVS-sh mouse group decreased significantly compared with the control mice 6 hours after reperfusion.

[0054] The above results indicate that the myocardial-specific knockdown MAVS mouse (MAVS-sh) model constructed using AAV9 successfully knocked down the expression of MAVS in cardiac tissue. Ultrasound results showed that mice with myocardial-specific knockout of MAVS had stronger cardiac function, reduced myocardial infarction area, reduced myocardial tissue necrosis and inflammation, reduced mitochondrial damage in myocardial cells, reduced myocardial cell apoptosis, decreased serum myocardial enzyme spectrum indicators, and effectively reduced the release of ROS in myocardial tissue after reperfusion injury.

[0055] Example 7 Myocardial-specific knockdown of MAVS can alleviate MIRI in the late stage

[0056] The experimental method is as follows: MAVS-sh mice were subjected to cardiac ischemia-reperfusion modeling. After 14 days of reperfusion, the mouse heart was tested by ultrasound, and the mouse body weight and heart weight were measured. Heart tissue sections were stained with WGA, Masson and CD31. The experimental results are shown in Figure 2. Figure 8 shown.

[0057] Figure 8Figure A shows that on the 14th day after reperfusion, the ratio of heart weight to body weight in MAVS-sh mice decreased compared with control mice, and cardiac hypertrophy was alleviated. Figure B WGA staining results showed that MAVS-sh mice could reduce the enlargement of myocardial cells on the 14th day after reperfusion. Figure C shows the cardiac ultrasound results of MAVS-sh mice and control mice on the 14th day after reperfusion. Figure D cardiac ultrasound showed that MAVS-sh mice had stronger cardiac contractile function than control mice on the 14th day after reperfusion. Figure E Masson staining showed that on the 14th day after reperfusion, the percentage of fibrosis in the IA area of ​​the myocardial tissue of MAVS-sh mice was significantly reduced compared with that of control mice. Figure F Immunofluorescence staining of CD31 in heart sections showed that on the 14th day, MAVS-sh mice had increased neovascularization compared with control mice, alleviating adverse ventricular remodeling.

[0058] Example 8 MAVS promotes cardiomyocyte apoptosis by activating the MAPK / JNK pathway through the TAK1 / TRAF6 axis

[0059] The experimental method is as follows: MAVS-KO mice and WT mice were subjected to cardiac ischemia-reperfusion model, and tissues were taken for protein extraction at 6 hours, 24 hours, and 3 days after reperfusion, and western-blot detection was performed. Fig. 9 shown.

[0060] Fig. 9 Figure A shows that the expression of RIG-I, TAK1 and TRAF6 in the heart tissue RA, AAR, IA and sham groups of MAVS-KO mice decreased 6 hours after reperfusion, and the phosphorylation of TAK1 was inhibited. Figure B shows that the phosphorylation of JNK in MAVS-KO mice was inhibited relative to WT mice 24 hours after reperfusion. Figure C shows that the phosphorylation of P38 and ERK in MAVS-KO mice decreased relative to WT mice 24 hours after reperfusion, but was not completely inhibited. Figure D shows that the phosphorylation of IRF3, TBK1 and P65 in MAVS-KO mice decreased relative to WT mice 3 days after reperfusion. Figure E shows that MAVS-KO can effectively reduce the expression of apoptosis-related proteins relative to WT controls after 3 days of perfusion, including the decreased expression of PARP, BAX and caspase3 and the enhancement of BCL2. Figure F represents a schematic diagram of the signaling pathway detected in AE.

[0061] The above results show that reperfusion modeling in WT mice can cause increased expression of RIG-I, TAK1 and TRAF6, enhanced phosphorylation of TBK1, IRF3, and P65, and enhanced phosphorylation of JNK, P38 and ERK in the MAPK pathway, while MAVS-KO mice showed a significant decrease, especially JNK phosphorylation was inhibited in MAVS-KO mice. At the same time, MAVS knockout significantly reduced the expression of pro-apoptotic proteins PARP, BAX and the cleavage of cleaved caspase3, and enhanced the expression of anti-apoptotic protein BCL2, protecting myocardial tissue.

[0062] Example 9 Myocardial-specific knockout of MAVS can inhibit MIRI injury-related signaling pathways

[0063] The experimental method is as follows: MAVS-sh mice and control PLKONC mice were subjected to cardiac ischemia-reperfusion model, and tissues were taken for protein extraction at 6 hours, 24 hours, and 3 days after reperfusion, and western-blot detection was performed. Fig.10 shown.

[0064] Fig.10 In the figure, the WB test results in Figure A showed that after 6 hours of reperfusion, the expression of RIG-I, TAK1 and TRAF6 in the myocardial tissue IA and AAR of MAVS-sh mice was significantly downregulated, and the phosphorylation of TAK1 was weakened. Figure B showed that in MAVS-sh mice, the phosphorylation of TBK1 in myocardial tissue was significantly decreased, but the phosphorylation of IRF3 was enhanced. Figure C showed that after 24 hours of reperfusion or sham surgery, the phosphorylation of JNK and P38 in the myocardial tissue of MAVS-sh mice was weakened to a certain extent. The results showed that MAVS-sh could significantly reduce the expression of apoptosis-related proteins PARP, BAX and cleaved caspase3 3 days after reperfusion, and increase the expression of anti-apoptotic protein BCL2 to a certain extent, playing a cardioprotective role.

[0065] The above experimental results show that after reperfusion modeling of myocardial-specific knockdown of MAVS mice, the expression of RIG-I, TAK1 and TRAF6 and the phosphorylation of TBK1, IRF3 and P65 are inhibited, and the phosphorylation of JNK and P38 in the MAPK pathway is reduced to a certain extent. At the same time, MAVS knockdown significantly reduces the expression of pro-apoptotic molecules PARP, BAX and the cleavage of caspase3, and enhances the expression of anti-apoptotic protein BCL2, showing the same protective effect as MAVS-KO mice.

[0066] Example 10 Protective effect of JNK pathway inhibition on the early stage of cardiac ischemia-reperfusion injury

[0067] The experimental method is as follows: AS601245 inhibitor was injected into the tail vein of WT mice at a dose of 15 mg / kg 30 minutes before and 3 hours after modeling. Heart tissue was taken for IP experiment 24 hours after reperfusion, and the phosphorylation degree of JNK was detected by western-blot. The group without drug injection was used as a control. Cardiac ultrasound and serum myocardial enzyme detection were performed 6 hours after reperfusion, EB+TTC double staining was performed 24 hours after reperfusion, and tissue pathological changes were detected 3 days after reperfusion, and heart sections were TUNEL stained and western-blot detected. The experimental results are shown in the figure. Fig.11 shown.

[0068] Fig.11 In the figure, A shows that WT mice were injected with the JNK inhibitor AS601245 before and after cardiac ischemia-reperfusion surgery. After 24 hours, the myocardial infarction area tissue was detected, and it was found that JNK phosphorylation was significantly inhibited. The results of EB+TTC staining in Figure B show that the myocardial infarction area of ​​the inhibitor-treated group was significantly reduced after 24 hours of treatment with or without AS601245 during reperfusion surgery in WT mice. Figure C shows that after AS601245 treatment, the cardiac function LVEF, LVFS, LVESV and LVEDV of WT mice 6 hours after reperfusion were enhanced to a certain extent compared with mice not treated with drugs. Figure D shows that 6 hours after reperfusion, the serum myocardial injury markers CK, CKMB and α-HB levels of WT mice treated with AS601245 were significantly decreased compared with the untreated group. The results of H&E staining in Figure E show that the area of ​​inflammatory cell infiltration and myocardial cell necrosis in myocardial tissue of WT mice treated with AS601245 was significantly reduced compared with that of untreated mice. Figure F shows that the TUNEL staining results of cardiac sections significantly reduced myocardial cell apoptosis in WT mice treated with AS601245. Figure G shows that on the third day after reperfusion, AS601245 treatment effectively reduced the expression of apoptosis-related proteins in the heart tissue of WT mice, including PARP, BAX and caspase3 cleavage and the enhancement of anti-apoptotic protein BCL2. Figure H shows the statistical analysis of protein expression in Figure G.

[0069] The above experimental results show that after the drug treatment of cardiac ischemia-reperfusion surgery in WT mice, JNK phosphorylation in the myocardial infarction area was significantly inhibited 24 hours later. In addition, the myocardial infarction area was reduced, tissue inflammation was alleviated, and it was significantly enhanced compared with the group without drug treatment. WB detection of apoptosis-related indicators found that the JNK inhibitor AS601245 can significantly reduce the expression of pro-apoptosis-related proteins PARP and BAX and reduce the shearing of cleaved caspase3, and enhance the expression of anti-apoptotic protein BCL2, protecting myocardial tissue.

[0070] Example 11 Protective effect of JNK pathway inhibitors on late MIRI damage

[0071] The experimental method is as follows: WT mice were subjected to cardiac ischemia-reperfusion modeling, and AS601245 was injected into the tail vein at 15 mg / kg 30 minutes before and 3 hours after modeling, and then injected daily at a dose of 15 mg / kg. Histopathological examination was performed on the 3rd day of reperfusion, and mouse heart ultrasound examination was performed on the 14th day of reperfusion, and Masson and CD31 staining was performed on the 14th day heart tissue sections. The experimental results are shown in Fig.12 shown.

[0072] Fig.12 In the figure, A shows that H&E staining shows that on the 3rd day after reperfusion, the area of ​​inflammatory cell infiltration and myocardial cell necrosis in the heart tissue of WT mice treated with AS601245 was reduced. B shows that on the 14th day after reperfusion, angiogenesis in the heart tissue of WT mice treated with AS601245 increased significantly. C shows that 14 days after reperfusion, the cardiac function of WT mice treated with AS601245 was improved to a certain extent. D shows that Masson staining shows that on the 14th day after reperfusion, the degree of fibrosis in the heart tissue of WT mice treated with AS601245 was significantly reduced.

[0073] The above experimental results show that the mice treated with inhibitors showed milder tissue inflammation and damage and stronger cardiac function than the control mice not treated with inhibitors. In addition, the inhibitor treatment can significantly reduce the degree of myocardial fibrosis, increase angiogenesis, and alleviate adverse ventricular remodeling.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of MAVS as a marker in the preparation of a drug for the prevention and / or treatment of myocardial ischemia-reperfusion injury, characterized in that: The drug includes a MAVS inhibitor for blocking the TRAF6 / TAK1 signaling pathway; the MAVS inhibitor includes an adeno-associated virus or liposome-encapsulated siRNA that knocks down MAVS with a myocardial-specific promoter.

2. The use according to claim 1, wherein the myocardium-specific promoter is cTNT.

3. The use according to claim 1, wherein the adeno-associated virus is AAV9.

4. The use according to claim 1, characterized in that: The drug is used for systemic intravenous injection or intracardiac injection.

5. The use according to claim 1, characterized in that: The medicament further includes a pharmaceutically acceptable carrier.

6. The use according to any one of claims 1 to 5, characterized in that: After the drug acts, it can reduce cell apoptosis induced by hypoxia and reoxygenation, increase the stability of mitochondrial membrane potential, and reduce the decline of protective reduced glutathione induced by hypoxia and reoxygenation, thereby maintaining the stability of myocardial cells under stressful environments.

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