Application of traditional Chinese medicine monomer hypericin in preparation of related medicines for improving mitochondrial energy metabolism disorder and controlling oxidative stress to prevent and treat ischemic heart disease

Abstract

The invention discloses an application of a traditional Chinese medicine monomer hypericin in preparation of related medicines for improving mitochondrial energy metabolism disorder and controlling oxidative stress to prevent and treat ischemic heart disease. The invention provides an application of hypericin in preparation of a product for improving mitochondrial energy metabolism disorder. The mitochondrial energy metabolism disorder is caused by myocardial ischemia reperfusion injury. Researches show that the hypericin regulates energy metabolism and oxidative stress of mitochondria by up-regulating / activating an AMPK / PGC-1alpha signal channel, so that myocardial cell apoptosis is relieved, and finally the protection effect on myocardial ischemia-reperfusion injury is achieved. The invention provides a new thought and a new method for treating MIRI by traditional Chinese medicines.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to the use of a traditional Chinese medicine monomer, hypericin, in the preparation of drugs for improving mitochondrial energy metabolism disorders and controlling oxidative stress to prevent and treat ischemic heart disease. Background Art

[0002] Among cardiovascular diseases, ischemic heart disease is one of the most common types. Among them, acute myocardial infarction (AMI) is one of the main causes of death in patients with ischemic heart disease, with rapid disease progression, high mortality rate, serious complications, and poor short-term prognosis. At present, myocardial reperfusion therapy through percutaneous coronary intervention (PCI), thrombolytic drugs, and coronary artery bypass grafting has become a consensus to save AMI patients by restoring myocardial blood supply (reperfusion therapy) as soon as possible. Studies have shown that early treatment of acute myocardial infarction can restore blood supply to ischemic myocardium, thereby reducing the risk of death. However, relevant data show that after reperfusion therapy, patients still face a mortality rate of up to 40%. Other studies have shown that at the tissue level, myocardial ischemia reperfusion not only reduces the therapeutic effect of reperfusion and aggravates the degree of vascular lesions, but can also lead to sudden cardiac death or malignant arrhythmias. At the cellular level, reperfusion can lead to disordered energy metabolism of myocardial cells, myocardial cell necrosis, apoptosis and structural damage, which in turn aggravates myocardial damage, leading to the expansion of myocardial necrosis and even death. This shows that this method is a double-edged sword. During myocardial ischemia-reperfusion, both ischemia and reperfusion will cause damage to the myocardium, and the latter will even further aggravate myocardial damage. This phenomenon is called myocardial ischemia-reperfusion injury (MIRI). This phenomenon is an important clinical problem that needs to be solved urgently after the widespread application of cardiac interventional technology in recent years. Therefore, opening blood vessels to restore blood supply to ischemic myocardium while avoiding damage caused by reperfusion is of great significance in the prevention and treatment of ischemic heart disease.

[0003] At present, the treatment of reperfusion injury is divided into two categories, non-drug and drug intervention, among which non-drug intervention includes strategies such as pre-ischemic conditioning, post-ischemic conditioning and remote ischemic regulation. Studies have shown that drug therapy is still an important means of treating reperfusion injury. Currently, Western medicine preparations used to treat myocardial ischemia-reperfusion injury include atorvastatin, cyclosporine A, verapamil, pioglitazone, sultanide, allopurinol, trimetazidine, etc. However, it is not a specific drug for the treatment of MIRI. Clinical studies have shown that some cardioprotective drugs have shortcomings such as cardiotoxicity, insufficient intravenous administration or immature technology.

[0004] As a valuable legacy of traditional Chinese medicine, Chinese medicine has unique advantages in the prevention and treatment of MIRI due to its unique holistic concept and dialectical treatment. Studies have shown that traditional Chinese medicine and natural active ingredients are important research contents for the treatment of MIRI. Studies have shown that Chinese medicine ingredients such as Shenfu injection, Panax notoginseng stem and leaf saponins, Xuebijing injection, Rhodiola rosea, Ginkgo biloba extract Ginkgolide B, the active ingredient of Salvia miltiorrhiza, and ginsenoside Rb1 of ginseng play a protective role in MIRI. The above studies suggest that some research progress has been made in the treatment of MIRI with Chinese patent medicine and natural active ingredients. Natural medicine resources are rich and the development prospects are broad. Drug monomers can play multiple functions and have few toxic and side effects, which provides great possibilities for finding drugs with better therapeutic effects on myocardial ischemia-reperfusion injury.

[0005] Hypericin (Hyp) is a natural compound isolated from plants of the genus Hypericum. It was originally isolated from Hypericum perforatum, also known as St. John's wort. It has been found that Hyp has anti-inflammatory, anti-oxidative stress, anti-tumor, and immunomodulatory effects. In folk medicine, it is mainly used to relieve inflammation and promote wound healing. However, there are no reports on the possible protective effects of Hyp on myocardial ischemia-reperfusion injury and its related mechanisms.

[0006] In current research, the pathogenesis of MIRI involves mitochondrial energy metabolism disorders, oxidative stress, inflammation, calcium overload, ferroptosis and other aspects. Among them, mitochondrial energy metabolism disorders play a core role in the occurrence and development of MIRI. Under normal circumstances, the myocardium uses fatty acids, glucose, lactate, pyruvate and other substances to synthesize ATP to provide energy for the relaxation and contraction of the myocardium. However, under pathological conditions, many studies have shown that myocardial cell energy metabolism disorders run through the entire process from compensatory myocardial hypertrophy to heart failure, and the initiation of myocardial ischemia-reperfusion injury is also closely related to energy metabolism disorders. Other studies have shown that in the process of coronary heart disease, heart failure and other diseases, there are abnormalities in various links of energy metabolism, such as changes in substrate utilization and impaired oxidative phosphorylation. Liu et al. verified that the Wenyang Yixin method promotes mitochondrial transcription and biogenesis, increases oxidative phosphorylation levels, and improves myocardial ischemia-reperfusion injury. The effect of mitochondrial energy metabolism may be achieved through the adenosine5-monophosphate (AMP)-activated protein kinase (AMPK) / silent information regulator 1 (SIRT1) / peroxisome proliferator activated receptor-γcoactivator-1α (PGC-1α) energy signaling axis.

[0007] Oxidative stress is a key driver of myocardial ischemia-reperfusion injury. Studies have shown that the imbalance between the kinetic energy of normal scavenging enzyme systems such as superoxide dismutase, catalase and glutathione and the production of intracellular reactive oxygen species will lead to the toxic accumulation of reactive oxygen species (ROS) intermediates such as hydrogen peroxide. Studies have shown that excessive ROS can easily damage mitochondrial membranes, proteins and mitochondrial DNA, causing mitochondrial dysfunction, resulting in a reduction in mitochondrial DNA and other contents, causing damage to energy metabolism processes such as oxidative phosphorylation and a reduction in ATP synthesis. It can be seen that oxidative stress damage and mitochondrial energy metabolism disorders are mutually causal and aggravate myocardial damage. Other studies have shown that ROS induces various types of cell death (such as apoptosis and necrosis) and plays a key role in myocardial ischemia-reperfusion injury. Experimental studies have shown that activated AMPK can monitor mitochondrial function and cellular energy status. SIRT1 is a NAD-dependent +Deacetylase is involved in the regulation of energy homeostasis, cell aging, oxidative stress, etc. Studies have shown that overexpression of SIRT1 can prevent cardiomyocytes from MIRI-induced cell death and oxidative stress damage. Activated AMPK and SIRT1 regulate the activity of PGC-1α, further upregulating its expression. Li et al. found that cynaroside can attenuate MIRI by improving mitochondrial energy metabolism and reducing oxidative stress damage through the AMPK / SIRT1 / PGC-1α signaling pathway. The above studies show that reducing oxidative stress damage and alleviating mitochondrial energy metabolism disorders are worth exploring in myocardial ischemia-reperfusion injury. Summary of the invention

[0008] The present invention provides the use of a traditional Chinese medicine monomer, hypericin, in the preparation of drugs for improving mitochondrial energy metabolism disorders and controlling oxidative stress to prevent and treat ischemic heart disease.

[0009] In a first aspect, the present invention claims the use of hypericin in preparing a product for improving mitochondrial energy metabolism disorders. Furthermore, the mitochondrial energy metabolism disorder is caused by myocardial ischemia-reperfusion injury.

[0010] Furthermore, the main active ingredient or the only active ingredient of the product is hypericin.

[0011] Wherein, the product may be a medicine.

[0012] In a specific implementation case of the present invention, the improvement is a preventive improvement, that is, the medication is taken before the disease occurs.

[0013] In a second aspect, the present invention claims the use of hypericin in the preparation of a regulator for regulating (upregulating, activating) the AMPK / PGC-1α signaling pathway.

[0014] Furthermore, the AMPK / PGC-1α signaling pathway is the AMPK / PGC-1α signaling pathway in the body or cells of myocardial ischemia-reperfusion injury.

[0015] In the present invention, the up-regulation (activation) of the AMPK / PGC-1α signaling pathway can be specifically embodied as: an increase in the expression of p-AMPK protein and / or PGC-1α protein. The same applies below.

[0016] Furthermore, the main active ingredient or the only active ingredient of the regulator is hypericin.

[0017] In a specific implementation case of the present invention, the regulation is preventive regulation, that is, the medication is taken before the disease occurs.

[0018] In a third aspect, the present invention claims the use of hypericin in the preparation of a regulator for regulating (downregulating, inhibiting) the Bcl-2 / Bax / Caspase-3 signaling pathway.

[0019] Furthermore, the Bcl-2 / Bax / Caspase-3 signaling pathway is the Bcl-2 / Bax / Caspase-3 signaling pathway in the body or cells of myocardial ischemia-reperfusion injury.

[0020] In the present invention, the down-regulation (inhibition) of the Bcl-2 / Bax / Caspase-3 signaling pathway can be specifically embodied as: an increase in the expression of BCL-2 protein, and / or a decrease in the expression of Bax protein and / or cleaved-casepase3 protein. The same applies below.

[0021] Furthermore, the main active ingredient or the only active ingredient of the regulator is hypericin.

[0022] In a specific implementation case of the present invention, the regulation is preventive regulation, that is, the medication is taken before the disease occurs.

[0023] In a fourth aspect, the present invention claims protection for the use of hypericin as an activator of the AMPK / PGC-1α signaling pathway in the preparation of a product for improving myocardial ischemia-reperfusion injury.

[0024] Wherein, the product may be a medicine.

[0025] Furthermore, the main active ingredient or the only active ingredient of the product is hypericin.

[0026] In a fifth aspect, the present invention claims protection for the use of hypericin as a Bcl-2 / Bax / Caspase-3 signaling pathway inhibitor in the preparation of a product for improving myocardial ischemia-reperfusion injury.

[0027] Wherein, the product may be a medicine.

[0028] Furthermore, the main active ingredient or the only active ingredient of the product is hypericin.

[0029] In a specific implementation case of the present invention, the improvement is a preventive improvement, that is, the medication is taken before the disease occurs.

[0030] In a sixth aspect, the present invention claims the use of hypericin and AMPK inhibitor as or in the preparation of a positive control drug; the positive control drug is any of the following: (a1) As a positive control when screening drugs that prevent and / or treat myocardial ischemia-reperfusion injury by regulating (upregulating, activating) the AMPK / PGC-1α signaling pathway; (a2) As a positive control when screening drugs that alleviate cardiomyocyte injury by regulating (upregulating, activating) the AMPK / PGC-1α signaling pathway.

[0031] Further, in the application, the “screening of drugs for preventing and / or treating myocardial ischemia-reperfusion injury by regulating (upregulating, activating) the AMPK / PGC-1α signaling pathway” and the “screening of drugs for alleviating myocardial cell injury by regulating (upregulating, activating) the AMPK / PGC-1α signaling pathway” can be carried out according to a method comprising the following steps: (1) Setting up experimental groups: on the one hand, administering the drug to be tested alone to the animal model of myocardial ischemia-reperfusion injury (or the cell model of myocardial cell injury); on the other hand, administering the drug to be tested and the AMPK inhibitor in combination to the animal model (or the cell model); (2) Setting up a positive control group: on the one hand, administering hypericin alone to the animal model of myocardial ischemia-reperfusion injury (or the cell model of myocardial cell injury); on the other hand, administering hypericin and the AMPK inhibitor in combination to the animal model (or the cell model); (3) Testing and result determination: In the case where the positive control group shows that "the animal model (or the cell model) administered with hypericin alone shows upregulation (activation) of the AMPK / PGC-1α signaling pathway, but the upregulation (activation) of the AMPK / PGC-1α signaling pathway is weakened / reversed after the AMPK inhibitor is administered in combination with hypericin", if the animal model (or the cell model) administered with the test drug alone shows upregulation (activation) of the AMPK / PGC-1α signaling pathway, and the upregulation (activation) of the AMPK / PGC-1α signaling pathway is weakened / reversed after the AMPK inhibitor is administered in combination with the test drug, then the test drug is or is a candidate for the target drug to be screened, that is, a drug that can prevent and / or treat myocardial ischemia-reperfusion injury by regulating (upregulating, activating) the AMPK / PGC-1α signaling pathway (or reduce myocardial cell damage by regulating (upregulating, activating) the AMPK / PGC-1α signaling pathway).

[0032] In one embodiment of the present invention, the cardiomyocyte injury in (a2) is cardiomyocyte injury induced by hypoxia-reoxygenation (H / R).

[0033] In a seventh aspect, the present invention claims the use of hypericin and an AMPK inhibitor as or in the preparation of a positive control drug; the positive control drug is any of the following: (b1) As a positive control when screening drugs that prevent and / or treat myocardial ischemia-reperfusion injury by regulating (downregulating, inhibiting) the Bcl-2 / Bax / Caspase-3 signaling pathway; (b2) Used as a positive control when screening drugs that reduce cardiomyocyte injury by regulating (downregulating, inhibiting) the Bcl-2 / Bax / Caspase-3 signaling pathway.

[0034] Further, in the application, the “screening of drugs for preventing and / or treating myocardial ischemia-reperfusion injury by regulating (downregulating, inhibiting) the Bcl-2 / Bax / Caspase-3 signaling pathway” and the “screening of drugs for alleviating myocardial cell injury by regulating (downregulating, inhibiting) the Bcl-2 / Bax / Caspase-3 signaling pathway” can be carried out according to a method comprising the following steps: (1) Setting up experimental groups: on the one hand, administering the drug to be tested alone to the animal model of myocardial ischemia-reperfusion injury (or the cell model of myocardial cell injury); on the other hand, administering the drug to be tested and the AMPK inhibitor in combination to the animal model (or the cell model); (2) Setting up a positive control group: on the one hand, administering hypericin alone to the animal model of myocardial ischemia-reperfusion injury (or the cell model of myocardial cell injury); on the other hand, administering hypericin and the AMPK inhibitor in combination to the animal model (or the cell model); (3) Testing and result determination: In the case where the positive control group shows that "the animal model (or the cell model) administered with hypericin alone shows down-regulation (inhibition) of the Bcl-2 / Bax / Caspase-3 signaling pathway, but the down-regulation (inhibition) of the AMPK / PGC-1α signaling pathway is weakened / reversed after the AMPK inhibitor is administered in combination with hypericin", if the animal model (or the cell model) administered with the test drug alone shows down-regulation (inhibition) of the Bcl-2 / Bax / Caspase-3 signaling pathway, And after the AMPK inhibitor is administered in combination with the test drug, the downregulation (inhibition) of the Bcl-2 / Bax / Caspase-3 signaling pathway is weakened / reversed, then the test drug is or is a candidate for the target drug to be screened, that is, a drug that can prevent and / or treat myocardial ischemia-reperfusion injury by regulating (downregulating, inhibiting) the Bcl-2 / Bax / Caspase-3 signaling pathway (or reduce myocardial cell damage by regulating (downregulating, inhibiting) the Bcl-2 / Bax / Caspase-3 signaling pathway).

[0035] Regarding the sixth and seventh aspects mentioned above, it should be noted that screening out drugs that exert therapeutic effects through specific signaling pathways (understanding the drug's mechanism of action) is of great significance for guiding medication, especially combination medication.

[0036] In one embodiment of the present invention, the AMPK inhibitor is Compound C (also known as Dorsomorphin).

[0037] Experiments have shown that the Chinese medicine monomer hypericin can prevent and treat myocardial ischemia-reperfusion injury by upregulating / activating the AMPK / PGC-1α signaling pathway, regulating mitochondrial energy metabolism and oxidative stress, thereby alleviating myocardial cell apoptosis and ultimately playing a protective role. The present invention provides new ideas and methods for the treatment of MIRI with traditional Chinese medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Hyp can reduce the transverse death area and improve cardiac function in rats after MIRI. A. Chemical structure of hypericin; B is the electrocardiogram of rats in each group after 2 hours of reperfusion Figure II Schematic diagram of lead changes; C is the left ventricular echocardiogram of rats in each group; D is the electrocardiogram of rats in each group after 2 hours of reperfusion Figure II Quantification of the amplitude of ST segment elevation in lead changes; E is left ventricular ejection fraction (LVEF%); F is fractional shortening (LVFS%); G is left ventricular cardiac output (CO); H is heart rate (HR); I is left ventricular end-diastolic diameter (LVIDd); J is left ventricular end-systolic diameter (LVIDs); K is a quantitative diagram of TTC-stained infarct area; L is a schematic diagram of TTC-stained heart cross-sections of rats in each group; MN is the levels of serum myocardial injury markers LDH (M) and cTnI (N) in rats in each group detected by biochemical kits. ***P<0.001, **P<0.01, *P<0.05 vs sham group; ##P<0.01 #P<0.05, &&P<0.01, &P<0.05, ns no significant difference vs MIRI group.

[0039] Figure 2 Hyp can reduce myocardial pathological damage. A is HE staining of myocardial tissues of rats in each group; red arrows point to inflammatory cell infiltration; green arrows point to myocardial cell edema; blue arrows point to myocardial rupture and disorder; B is Sirius red staining and Masson staining of myocardial tissues of rats in each group; C is quantitative analysis of myocardial collagen fibers by Sirius red staining.

[0040] Figure 3To reveal that Hyp is closely related to mitochondrial energy metabolism regulated by the AMPK signaling pathway, rat myocardial tissues (n=3) from the Sham group, MIRI group, and MIRI+Hyp group were selected for proteomic analysis. A is the principal component analysis (PCA) of quantifiable proteins in the Sham, MIRI, and MIRI+Hyp groups. B is the comparative identification of significantly changed proteins in the Sham, MIRI, and MIRI+Hyp groups. C is the subcellular localization prediction of the identified proteins using WoLFPSORT. D is the clustering analysis of significantly changed proteins in the Sham, MIRI, and MIRI+Hyp groups according to the change trend. Each fold line of cluster 1 (n=67), cluster 2 (n=51), cluster 3 (n=182), cluster 4 (n=69), cluster 5 (n=26), and cluster 6 (n=73) represents the change trend of a protein. Proteins in clusters 1 and 3 were identified as being reversed or regulated by Hyp. E is the functional classification of proteins reversed or regulated by Hyp using the Gene Ontology (GO) and KEGG databases.

[0041] Figure 4 Figure 2 Effects of Hyp on mitochondrial energy metabolism disorders and protein expression levels in apoptosis-related signaling pathways in myocardial tissue of rats in each group. A is the relative content of ATP in rat myocardial tissue. B is the relative content of AMP in rat myocardial tissue. C, D, and G are the relative contents of SOD (C), MDA (D), and ROS (G) in rat serum, respectively. E is a schematic diagram of molecular docking of Hyp and AMPK. F and HL are Western blot analysis (F) to determine the protein expression levels of p-AMPK (H), PGC-1α (I), Bcl-2 (J), Bax (K), and Cleaved-Caspase-3 (L) in myocardial tissue of rats in Sham, MIRI, MIRI+L-Hyp, MIRI+H-Hyp, and MIRI+TMZ groups, respectively. n=6, ***P<0.001, **P<0.01, *P<0.05 vs Sham group; ##P<0.01, #P<0.05, &&P<0.01, &P<0.05, ns no significant difference vs MIRI group.

[0042] Figure 5Hyp can reduce myocardial cell damage. Among them, A is the effect of different concentrations of Hyp (2nM-2μM) on the viability of H9c2 cells. B is the effect of different concentrations of AMPK inhibitor Compound C (0.2μM-5μM) on the viability of H9c2 cells. C is the effect of different concentrations of Hyp (2nM-2μM) on the viability of H9c2 cells treated with H / R. D and F are the relative concentrations of LDH released by each group of cells. E is the effect of different hypoxia-reoxygenation time patterns on the viability of H9c2 cells. G is the morphology and cell number of each group of cells under an optical microscope. n=6, ** P <0.01, * P <0.05 vs CON group; ## P <0.01, # P <0.05 ns, no significant difference vs H / R group; & P <0.05 vs H / R+Hyp group.

[0043] Figure 6 Hyp can alleviate the mitochondrial energy metabolism disorder of cardiomyocytes in H / R. A is the relative content of ATP in H9c2 cells. B is the relative content of AMP in H9c2 cells. CD are the relative contents of SOD (C) and MDA (D) in H9c2 cells, respectively. E is the fluorescence image of ROS staining of H9c2 cells in each group. F is the fluorescence image of TUNEL staining of H9c2 cells in each group. G is the quantitative image of ROS staining fluorescence image of H9c2 cells in each group. H is the positive rate of TUNEL staining cells. n=6, ***P<0.001, **P<0.01, *P<0.05 vs CON group; #P<0.05 vs H / R group; &P<0.05 vs H / R+Hyp group.

[0044] Figure 7 The effects of Hyp on the protein expression levels of mitochondrial energy metabolism disorders and apoptosis-related signaling pathways in H9C2 cardiomyocytes in each group. Among them, AG are Western blot (A and B) analysis to determine the protein expression levels of p-AMPK (C), PGC-1α (D), Bcl-2 (E), Bax (F), and CleavedCaspase-3 (G) in H9c2 myocardial tissues in CON, H / R, H / R+Hyp, and H / R+Hyp+CC groups. n=6, **P<0.01, *P<0.05 vs CON group; ##P<0.01; #P<0.05 vs H / R group; &P<0.05 vs H / R+Hyp group. DETAILED DESCRIPTION

[0045] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0046] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0047] Example 1: Hypericin improves myocardial ischemia-reperfusion injury by regulating mitochondrial energy metabolism and oxidative stress 1. Animals Thirty clean-grade male SD rats aged 6-8 weeks, weighing approximately 180-220 g, were purchased from the Animal Experiment Center of Xinjiang Medical University, with the animal use license number: SYXK (Xin) 2023-0004. They were fed adaptively for 1 week, during which they were free to drink water and feed, with a 12-h day and night time, an ambient temperature of 18-25 °C, and a relative humidity of 50-60%.

[0048] 2. Drugs and Reagents Trimetazidine hydrochloride tablets, the specifications and approval numbers are: 20 mg, national medicine standard H20055465, products of Servier (Tianjin) Pharmaceutical Co., Ltd.; Hypericin (chemical structure as Figure 1The purity is ≥ 98%, batch number: M4375, which is a product of Abmole, USA; the rat ATP and AMP content detection kits are products of Beijing Andy Huatai Technology Co., Ltd.; the rat cardiac troponin I (cTnI) content and superoxide dismutase (SOD) activity detection kits are products of Wuhan FineTest Co., Ltd. and Nanjing Jiancheng Bioengineering Institute, respectively; the rat reactive oxygen species (ROS) The detection kit for ROS (recombinant proteins, ROS) was produced by Jiangsu Jingmei Biotechnology Co., Ltd.; the detection kit for malondialdehyde (MDA) content and lactate dehydrogenase (LDH) activity in rats, 2,3,5-triphenyltetrazolium chloride (TTC) staining solution, high-efficiency RIPA tissue / cell rapid lysis solution, dimethyl sulfoxide (DMSO), protease inhibitor (PMSF) and phosphatase inhibitor were all products of Beijing Solaibao Technology Co., Ltd.; AMPK and p-AMPK antibodies were products of CST; PGC-1α antibody was produced by Proteintech; BCL-2, Bax, and Caspase3 antibodies were produced by Abmart. AMPK inhibitor Compound C was produced by Abmole, USA.

[0049] 3. Experimental Methods 1. Animal grouping and drug administration Thirty rats were randomly divided into sham operation group (Sham group), myocardial ischemia-reperfusion injury group (MIRI group), low-dose hypericin group (MIRI+L-Hyp group), high-dose hypericin group (MIRI+H-Hyp group) and positive control drug trimetazidine group (MIRI+TMZ group), with 6 rats in each group.

[0050] Hypericin (Hyp) was dissolved in DMSO and diluted with saline to a final concentration of 0.15% (i.e., 0.15 g / 100 ml). Hyp low-dose (0.5 mg / kg) and high-dose (1 mg / kg) groups were intraperitoneally injected daily, the positive drug treatment group was given trimetazidine 10 mg / kg by gavage daily, and the Sham group and MIRI group were given an equal amount of saline by intraperitoneal injection. The treatment lasted for 14 days.

[0051] After the administration, each group of mice was modeled according to the following step 2.

[0052] 2. Construction of rat myocardial ischemia-reperfusion injury model (MIRI model) According to the preliminary experiment and the literature of Huang Weihua et al. [Huang Weihua. Effect of trimetazidine pretreatment on myocardial cell apoptosis and superoxide dismutase expression after myocardial ischemia-reperfusion in rats [D]. Zhengzhou: Zhengzhou University, 2021: 29-36.], the MIRI model was established, the left anterior descending coronary artery (LAD) was ligated, and the timing was started for 45 minutes when the ST segment of the electrocardiogram was observed to be continuously elevated. After 45 minutes of ischemia, the ligature was loosened and reperfused for 2 hours to end the experiment. The heart and serum were taken for subsequent experiments. The sham group (Sham group) rats had the same surgical steps as the MIRI group, but no ligation was performed.

[0053] 3. H / R model and grouping H9c2(2-1) rat cardiomyocytes (Procell CL-0089) were provided by Wuhan Procell Life Science Co., Ltd. (hereinafter referred to as H9c2). H9c2 cells were divided into 4 different experimental groups: control group (CON), H / R group, H / R+Hyp group, and H / R+Hyp+CC group.

[0054] In order to simulate the H / R model in vitro, H9c2 cells were first cultured in serum-free DMEM medium and cultured in a three-gas incubator at 37 °C, 1% O2, 5% CO2, and 94% N2 for 8 h. After hypoxia treatment, the medium was replaced with DMEM supplemented with 10% fetal bovine serum, and then the cells were cultured under normoxic conditions for 2 h. The control group (CON) was cultured continuously under normoxic conditions, and the other three groups were treated with H / R.

[0055] Drug treatment was as follows: Hyp was added to the culture medium 24 h before hypoxia, a concentration gradient was set (2 μM, 1000 nM, 800 nM, 400 nM, 200 nM, 20 nM, 2 nM), and the optimal Hyp concentration was selected for subsequent experiments; Compound C (1 μM) was added 1 h before hypoxia.

[0056] 4. Electrocardiogram After anesthesia, the rats in each group were immediately depilated and fixed, and connected to a ventilator after endotracheal intubation. Electrodes were placed at the ends of the rats' limbs to open the chest for modeling. The standard II lead electrocardiogram of the rats was observed and recorded in real time.

[0057] 5. Echocardiography After modeling, the left ventricular short axis shortening percentage (LVFS%), ejection fraction (LVEF%), heart rate (HR) and cardiac output (CO) of rats in each group were measured using a small animal ultrasound instrument.

[0058] 6. TTC staining The heart tissue was quickly removed, and 6 slices of about 2 mm were cut from the apex to the base of the heart. They were placed in 2% TTC solution and stained at 37 °C in the dark for 20 min. After staining, they were fixed with 4% paraformaldehyde for 24 h and photographed. The red area is normal myocardial tissue, and the white area is ischemic myocardium. Image J software was used to analyze and calculate the area of ​​ischemic infarction area and non-infarction area.

[0059] 7. HE staining Five heart tissues fixed in 4% paraformaldehyde were randomly selected from each group. Excess water was removed with filter paper, and the tissues were embedded in paraffin and made into 4 μm thick sections. The sections were stained and sealed according to the instructions of hematoxylin-eosin (HE) staining. Changes in myocardial tissue were observed under a microscope (400 times) and images were collected.

[0060] 8. Masson staining To observe the degree of myocardial fibrosis, 5 heart tissues fixed in 4% paraformaldehyde were randomly selected from each group, and the excess water was removed by filter paper, then embedded in paraffin and made into 4 μm thick slices. Masson staining and sealing of rat myocardial tissue were performed according to the instructions of Masson kit, and the changes of myocardial tissue were observed under a microscope (400 times) and images were collected. Blue represents collagen fibers and red represents muscle fibers.

[0061] 9. Sirius red staining To observe the degree of myocardial fibrosis, 5 heart tissues fixed in 4% paraformaldehyde were randomly selected from each group, and the excess water was removed by filter paper, then embedded in paraffin and made into 4 μm thick slices. The rat myocardial tissues were stained with Sirius red and sealed according to the instructions of the Sirius red staining kit. The changes of myocardial tissue were observed under a microscope (400 times) and images were collected. Red represents collagen fibers, and yellow represents muscle fibers.

[0062] 10. Detection of myocardial injury markers The procedure was performed according to the kit instructions (Beijing Solarbio, Beijing, China), and the prepared serum samples were added to measure the levels of LDH and cTnI. For H9c2 cardiomyocytes, the procedure was as follows: the cell suspension (10 5 / well), the cells on the plate were divided into 5 groups, namely Con group, H / R group, H / R+2 nM Hyp group, H / R+20 nM Hyp group, H / R+200 nM Hyp group, H / R+20 nM Hyp+CC group. After 24h and 1h of Hyp and CC drug treatment, the old culture medium was aspirated, washed with PBS and discarded. The H9c2 cells in the H / R group and the drug group were replaced with serum-free DMEM-L culture medium and subjected to hypoxia for 8 h in a hypoxic incubator (5% CO2, 94% N2, 1% O2, 37°C). The H9c2 cells in the Con group continued to be cultured in a conventional incubator for the same time. After the treatment, the culture supernatant was collected with a sterile tube, and the supernatant was collected after centrifugation at 3000 rpm for 20min. The LDH activity was determined according to the instructions of the kit.

[0063] 11. Oxidative stress detection The prepared serum samples were added according to the instructions of the kit to determine the levels of SOD, MDA, and ROS. H9c2 cardiomyocytes were processed and collected according to the instructions of the SOD kit and MDA kit. The absorbance was measured using an automatic microplate reader (Thermo Fisher Scientific, Waltham, MA, USA), and the contents of SOD and MDA in H9c2 cardiomyocytes were calculated.

[0064] 12. Energy metabolism index detection According to the instructions of the kit, the prepared tissue homogenate samples were added to determine the ATP and AMP levels; the cell processing operation was as follows: H9c2 cells were planted in a 6-well plate. After the modeling was completed, the ATP / AMP detection kit instructions were followed. The general steps are as follows: add lysis buffer to the cells for lysis, centrifuge, obtain the cell supernatant, take 1 / 4 for protein concentration determination, and the rest for ATP / AMP concentration detection. Then use a fluorescence microplate reader to detect the fluorescence value, and calculate the ATP / AMP concentration in the sample according to the standard curve. After that, the protein concentration of the sample was determined using the BCA kit. The corrected ATP / AMP value is the ATP / AMP concentration divided by the corresponding protein concentration, in nmol / L.

[0065] 13. Morphology and vitality detection Cell morphology was observed under a transmission microscope. 5000 cells were inoculated into a 96-well plate with the suspension (100 μL / well). The cells on the plate were divided into 8 groups, namely: control group (Con group), 2 μM Hyp group, 1000 nM Hyp group, 800 nM Hyp group, 400 nM Hyp group, 200 nM Hyp group, 20 nM Hyp group, and 2 nM Hyp group. After 24 h of drug treatment, the old culture medium was aspirated, washed with PBS, and discarded. 10 μL CCK-8 solution was added to each well, and no cells were added to the blank control well. After incubation for 2 h, the OD value was measured at a wavelength of 450 nm to compare the viability of rat H9c2 cardiomyocytes at hypericin concentrations of 2 μM, 1000 nM, 800 nM, 400 nM, 200 nM, 20 nM, and 2 nM, and to observe the effects of different concentrations of hypericin on the viability of rat H9c2 cardiomyocytes under conventional culture. In addition, the effects of different concentrations of Compound C (CC) on the viability of rat H9c2 cardiomyocytes were also detected. A concentration gradient of 5μM, 4μM, 3μM, 2μM, 1μM, 0.8μM, 0.6μM, 0.4μM, and 0.2μM CC groups was set for detection. After 1 hour of drug treatment, the old culture medium was aspirated, washed with PBS, and discarded. 10μL CCK-8 solution was added to each well, and no cells were added to the blank control well. After incubation for 2 hours, the OD value was measured at a wavelength of 450 nm to compare the viability of rat H9c2 cardiomyocytes when the concentration of Compound C was 5μM, 4μM, 3μM, 2μM, 1μM, 0.8μM, 0.6μM, 0.4μM, and 0.2μM CC groups, and the effects of different concentrations of Compound C on the viability of rat H9c2 cardiomyocytes under conventional culture were observed.

[0066] 14. ROS level detection experiment H9c2 cells in each group were expressed at 5×10 4 Cells were planted in six-well plates. After the corresponding treatment, the culture medium was discarded, serum-free culture medium containing DCFH-DA probe (DCFH-DA was 5 mmol / L, diluted in serum-free culture medium) was added, and the cells were incubated at 37°C for 15-30 min. The culture medium was removed, and PBS was washed three times. After that, 1 mL of PBS was added to observe the fluorescence intensity under an inverted fluorescence microscope. 15. TUNEL assay to detect cell apoptosis To detect the apoptosis of H9c2 cardiomyocytes, the cells were cultured at 7×10 3 The cells were inoculated at a density of 1.54 μg / mL in a 24-well plate, with 3 replicate wells in each group. After the cells were treated in groups, the culture medium was discarded and the cells were gently rinsed 3 times with PBS. The cells were treated according to the steps of the Tunel kit (Biyuntian, China), observed under a fluorescence microscope, and the relative fluorescence intensity was analyzed using Image J software.

[0067] 16. Molecular docking The use of molecular docking to explore the interaction patterns between receptors and drugs has become a hot area in computer-aided drug design. Molecular docking methods can not only be used to design drugs, but also to explore drug targets to further study the mechanism of action of related drugs. Therefore, the practical application value of molecular docking technology is relatively wide. According to the prediction of high-throughput proteomics, we assume that the drug target of Hyp exists in the AMPK signaling pathway. To verify this hypothesis, this experiment used molecular docking technology to preliminarily predict the possibility of interaction between Hyp and an important target protein (AMPK) in the AMPK signaling pathway. In order to evaluate the binding energy and interaction pattern between candidate drugs / small molecules and their targets, we used AutodockVina 1.2.2, a computer protein-ligand docking software. We obtained the molecular structure of Hypericin from the PubChem compound database (https: / / pubchem.ncbi.nlm.nih.gov / ). The 3D coordinates of the protein AMPK (PDB number, 7M74; resolution, 3.93Å) were downloaded from the PDB (http: / / www.rcsb.org / ). We first prepared the protein and ligand files. All protein and molecule files were converted to PDBQT format, all water molecules were removed, and polar hydrogen atoms were added. The grid box was centered to cover the domain of each protein and accommodate free molecular motion. The docking pocket was set to a 30 Å × 30 Å × 30 Å square pocket with a grid distance of 0.05 nm. Finally, our molecular docking study was performed by AutodockVina 1.2.2 (http: / / autodock.scripps.edu / ) for model visualization.

[0068] 17. Quantitative proteomics analysis Myocardial tissues of rats in each group were provided to Jingjie Bio (Hangzhou, China) for 4D label-free quantitative proteomics analysis. Briefly, samples were ground, lysed and centrifuged to obtain supernatants and their protein concentrations were measured. Protein samples were first precipitated with trichloroacetic acid and washed with precooled acetone, then re-dissolved with triethylamine borane and digested with trypsin to further obtain peptides. Peptides were desalted using Strata X SPE columns and separated using NanoElute ultra-high performance liquid system, then infused into a capillary ion source for ionization and mass spectrometry analysis using tims TOF Pro (tims: trapped ion mobility spectrometry; TOF: time of flight, Bruker, USA). Maxquant search engine (v1.6.15.0) was used to process mass spectrometry raw data, and the Mus_musculus_10090_SP_20201214 (fasta) database was used as a reverse bait database to search for matching proteins of tandem mass spectra. In addition, the reverse database was added to calculate the false discovery rate (FDR) caused by random matching, and contaminating proteins in the identified proteins were excluded to eliminate their influence. Trypsin / P was designated as the cleavage enzyme, and up to 2 missed cleavages were allowed. The minimum length of the peptide was set to 7 amino acid residues, while its maximum number of modifications was 5. The mass tolerance of the precursor ion was set to 20 ppm in the "first search" and 5 ppm in the "main search", while the mass tolerance of the fragment ion was 0.02 Da. The carbamide methyl group on cysteine ​​was designated as a fixed modification, and the oxidation on methionine and the acetylation of the N-terminus of the protein were designated as variable modifications. The FDR for protein and peptide identification was set to 1%. Then, the reproducibility of each group of samples was evaluated using the Pearson correlation coefficient, principal component analysis (PCA), and relative standard deviation. After sample qualitative analysis, the differentially expressed proteins were obtained, and the relative quantitative differences between the two groups were compared by T-test, and the corresponding p In addition, pProteins with a ratio of >1.5 were considered up-regulated, while proteins with a ratio of <1 / 1.5 were considered down-regulated, with a p value of ≤ 0.05. Based on the identified proteins, subcellular localization analysis was performed using the WoLF-PSORT database (https: / / www.genscript.com / wolf-psort.html); functional analysis was performed using Gene Ontology (GO) terms and the UniProt-GOA database (http: / / www.ebi.ac.uk / GOA / ); and pathway analysis was performed using the KEGG database (http: / / www.genome.jp / kegg / ). Meanwhile, enriched pathways were analyzed by two-tailed Fisher's exact test to determine the enrichment of differentially expressed proteins, and p values ​​≤ 0.05 were considered significant. In addition, the cluster membership of the identified proteins was visualized using the “heatmap.2” function in the “gplots” R-package, protein-protein interactions (PPIs) were analyzed using the STRING (V.11.0) database, and the PPI network was visualized using the R Package “networkD3” tool.

[0069] 18. Western blot was used to detect the expression levels of AMPK, p-AMPK, PGC-1α, Bcl-2, Bax, Caspase3, and Cleaved-Caspase3 proteins in myocardial tissue and H9C2 cardiomyocytes Proteins in myocardial tissues and cells were extracted by conventional methods, and the protein samples were denatured at high temperature after BCA protein quantification. SDS-PAGE gel electrophoresis was used and transferred to PVDF membranes, which were blocked with 5% skim milk at room temperature for 2 h and incubated with primary antibodies at 4 °C overnight. The next day, the secondary antibodies were incubated at room temperature for 1 h, and then the PVDF membrane was placed in an exposure instrument and developed with ECL developer. Finally, the grayscale value of each group of bands was analyzed by Image J software, and the protein expression was recorded and counted.

[0070] 19. Statistical processing GraphPad Prism 8.0 software was used to perform statistical analysis of the data. ) indicated that one-way ANOVA was used for comparison among groups, and LSD-t test was used for pairwise comparison.

[0071] 4. Results and Analysis 1. Hyp can reduce infarct size and improve cardiac function after MIRI in rats Myocardial ischemia reperfusion injury (MIRI) is a disease caused by reperfusion therapy of acute myocardial infarction. It has become an important factor affecting the prognosis of reperfusion therapy. Therefore, the classic left anterior descending coronary artery ligation and recanalization method was used to establish the MIRI model. Figure II The changes in the leads are used to evaluate whether the model is established successfully and the degree of ST segment elevation. Figure 1 As shown in B, the rats in each group were first connected to the electrocardiogram. The "ST" segment of the electrocardiogram of the rats in the MIRI group, the low-dose and high-dose hyperforin groups, and the positive control drug trimetazidine group was elevated, indicating that the rat myocardial ischemia model was successfully established. Compared with the Sham group, the "ST" segment elevation in the MIRI group increased; compared with the MIRI group, the "ST" segment elevation in the low-dose and high-dose hyperforin groups and the positive control drug trimetazidine group was reduced ( Figure 1 Middle D). Echocardiography was used to investigate the protective effect of Hyp on cardiac function after MIRI in rats ( Figure 1 C). Figure 1 As shown in EJ, the cardiac systolic function decreased after MIRI, LVEF, LVFS, CO, and HR all decreased significantly, LVIDs increased significantly, and the rats in the low-dose hyperforin group and the positive control drug trimetazidine group returned to normal. Among them, LVIDd increased in the MIRI group, while the rats in the low-dose hyperforin group and the positive control drug trimetazidine group showed a relatively decreasing trend, and the difference was not statistically significant. The above research results show that Hyp can improve the cardiac systolic function of MIRI rats. Its effect on the range of myocardial infarction area was evaluated by TTC staining (normal myocardial tissue is red area, myocardial infarction tissue is gray-white area). Compared with the Sham group, the myocardial infarction area of ​​MIRI rats increased significantly; compared with the MIRI group, the myocardial infarction area of ​​​​the low-dose hyperforin group and the positive control drug trimetazidine group was significantly decreased ( Figure 1 KL in the middle). LDH and cTnI are myocardial injury markers in serum. Compared with the Sham group, their concentrations in the MIRI group were observed to be increased; compared with the MIRI group, the concentrations of myocardial injury markers LDH and cTnI in the low-dose and high-dose hypericin groups and the positive control drug trimetazidine group were significantly decreased, and the differences were statistically significant ( Figure 1 Therefore, Hyp can reduce myocardial infarction area and myocardial damage, improve cardiac contractile function, and thus play a myocardial protective role.

[0072] 2. Hyp can reduce myocardial pathological damage HE staining, Sirius red staining and Masson staining were performed on the cross-sectional sections of the heart tissues of the rats in each group. The main purpose was to examine the histological changes in myocardial cells and tissue structure and myocardial fibrosis. The results of HE staining showed that ( Figure 2 Middle A), compared with the Sham group, the myocardial cells of the rats in the MIRI group showed nuclear fragmentation, nuclear dissolution, inflammatory cell infiltration, interstitial edema, and unclear muscle fiber boundaries; compared with the MIRI group, the pathological results of the rats in the low-dose and high-dose hypericin groups and the positive control drug trimetazidine group were greatly improved: the disorder of myocardial cell arrangement and myocardial damage were alleviated, the myocardial cells were arranged more neatly, myocardial apoptosis was reduced, and the inflammatory cell infiltration was significantly reduced. Figure 2 As shown in BC, Sirius red staining and Masson staining were further used to detect myocardial collagen deposition in the Sham group, MIRI group, and MIRI+H-Hyp group. Only a small amount of collagen fibers were found in the myocardial interstitium of the three groups. Compared with the Sham group, the accumulation of collagen fibers between myocardial cells in the MIRI group and the high-dose hypericin group increased slightly, and the difference was not statistically significant. In summary, these results indicate that Hyp can significantly reduce the pathological necrosis area of ​​myocardial tissue and improve myocardial disorder and inflammatory cell infiltration. At the same time, the results also show that the acute myocardial ischemia-reperfusion injury model failed to cause myocardial fibrosis and the effect of Hyp on improving cardiac fibrosis could not be known, which will be further explored in the long-term model in the future.

[0073] 3. Hyp can alleviate mitochondrial energy metabolism disorders The above only discusses the pharmacological effects of Hyp, but the mechanism of its protective effect is unknown. Therefore, in order to explore the protective mechanism of Hyp against MIRI, 4D label-free quantitative proteomics analysis was performed on myocardial tissues of different groups. A total of 25,393 peptides, 5,254 proteins, and 5,164 quantifiable proteins were identified in myocardial tissues. PCA analysis showed that the Sham group, MIRI group, and MIRI+H-Hyp group were relatively separated from each other, and the proteins in the MIRI group were partially reversed by Hyp ( Figure 3 In addition, quantitative analysis was performed on the significantly changed proteins, and the MIRI / Sham group included 48 up-regulated proteins and 70 down-regulated proteins, while the MIRI+H-Hyp / MIRI group included 229 up-regulated proteins and 54 down-regulated proteins ( Figure 3 Further analysis of the location of this significantly changed protein showed that the MIRI / Sham group was mainly distributed in mitochondria (7.63%), cytoplasm (27.97%), and nucleus (23.73%) ( Figure 3C). After Hyp pre-administration, they were mainly located in the cytoplasm (31.8%), nucleus (22.26%) and mitochondria (15.55%). This shows that the number of proteins localized in mitochondria increased, and further considered that mitochondria are a very important part of the mechanism. According to the trend of these differential protein changes, they were divided into 6 clusters ( Figure 3 (D). Specifically, the differentially expressed proteins in Cluster 3 were significantly downregulated in MIRI, but significantly upregulated in the MIRI+H-Hyp group. Cluster 1 showed the opposite trend. Compared with the MIRI group, the proteins in Clusters 2, 4, 5, and 6 did not show significant changes after Hyp treatment. Therefore, the changed proteins in Clusters 1 and 3 were regarded as important proteins rescued or regulated by Hyp. Crucially, GO and KEGG analysis of such proteins showed that their molecular functions were closely related to mitochondrial energy metabolism. Figure 3 As shown in E, KEGG pathway analysis of significantly changed proteins in the MIRI / sham group and the MIRI+H-Hyp / MIRI group showed that differentially expressed genes were significantly enriched in the AMPK pathway. Therefore, regulating the AMPK signaling pathway is the key to the therapeutic effect of Hyp on MIRI. Adenylate-activated protein kinase (AMPK) is a conserved serine / threonine protein kinase in eukaryotic organisms, which can regulate mitochondrial energy metabolism by sensing the intracellular ATP / AMP ratio. Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) can regulate mitochondrial biogenesis and mitochondrial energy metabolism. It can be regulated by the energy sensor AMPK, and phosphorylated AMPK can activate PGC-1α. Therefore, AMPK / PGC-1α plays an important role in mitochondrial energy metabolism. Under pathological conditions, many studies have shown that myocardial energy metabolism disorders run through the entire process from myocardial compensatory hypertrophy to heart failure, and the initiation of myocardial ischemia-reperfusion injury is also closely related to energy metabolism disorders. Therefore, improving mitochondrial energy metabolism disorders may be a key protective measure. In view of the above proteomics and previous research reports, it is proposed that improving mitochondrial energy metabolism disorders may be an important mechanism for the protective effect of Hyp on MIRI. In order to further evaluate the effect of Hyp on mitochondrial energy metabolism, ELISA kits were used to detect the content of ATP and AMP in rat myocardial tissue. Figure 4As shown in A and B, compared with Sham, the ATP content in myocardial tissue of rats in the MIRI group was significantly reduced, and compared with the MIRI group, the ATP content in the low and high hypericin groups and the trimetazidine group was significantly increased; while AMP showed the opposite trend; Studies have shown that ROS are produced during both ischemia and reperfusion, and excessive ROS can easily cause mitochondrial dysfunction, resulting in a reduction in mitochondrial DNA and other contents, resulting in damage to energy metabolism processes such as oxidative phosphorylation and a reduction in ATP synthesis. This shows that oxidative stress damage and mitochondrial energy metabolism disorders are mutually causal, thereby aggravating myocardial damage. Therefore, the oxidative stress-related indicators were detected, and the results showed that compared with the Sham group, the serum SOD activity of the rats in the MIRI group decreased; compared with the MIRI group, the serum SOD activity of the rats in the low-dose and high-dose hyperforin groups and the positive control drug trimetazidine group increased; compared with the Sham group, the serum MDA and ROS levels of the rats in the MIRI group increased; compared with the MIRI group, the serum MDA and ROS levels of the rats in the low-dose and high-dose hyperforin groups and the positive control drug trimetazidine group decreased significantly ( Figure 4 C, D, and G). All of the above indicate that the possible mechanism of Hyp in alleviating MIRI in rats is to improve mitochondrial energy metabolism disorders and alleviate oxidative stress damage.

[0074] 4. Effects of Hyp on mitochondrial energy metabolism disorders and protein expression levels in apoptosis-related signaling pathways in myocardial tissue of rats in each group Previous studies have shown that the AMPK / PGC-1α signaling pathway, as an energy sensing network, plays a vital regulatory role in mitochondrial energy metabolism and oxidative stress. It is further suggested that alleviating energy metabolism disorders and reducing oxidative stress damage by regulating the AMPK / PGC-1α signaling pathway may be an important mechanism for Hyp to play a protective role in rat MIRI. In order to reveal the potential mechanism, molecular docking was performed to test the interaction between Hyp and AMPK. First, the binding posture and interaction of Hypericin and AMPK were obtained by Autodock Vinav.1.2.2, and the binding energy between the two was calculated. The smaller the binding energy value, the easier it is for the active ingredient to bind to the protein. The binding energy ≤ -5.0 kcal / mol (1cal=4.4J) indicates that the binding activity between the two is good; ≤ -7.0 kcal / mol indicates that the binding activity between the two is very strong. The molecular docking results show that Hypericin and AMPK have strong binding activity, and the lowest binding energy is -11.362 kcal / mol. Finally, the results with the lowest binding energy were visualized using PyMol software ( Figure 4E). Then, the AMPK / PGC-1α signaling pathway was verified by Western blot. The results showed that compared with the Sham group, the expression levels of oxidative stress-related pathway proteins p-AMPK and PGC-1α in the MIRI group were significantly reduced. Compared with the MIRI group, the low-dose and high-dose hypericin groups and the positive control drug trimetazidine group had an up-regulating effect on the expression of oxidative stress-related pathway proteins p-AMPK and PGC-1α ( Figure 4 F, H and I in the middle). Many studies have confirmed that during myocardial ischemia-reperfusion, factors that directly or indirectly affect oxidative phosphorylation, such as decreased ATP content, destruction of the mitochondrial electron transport chain, decreased membrane potential, and opening of the membrane permeability transition pore, can all lead to decreased ATP production, insufficient energy, decreased myocardial contractile function, and can induce myocardial cell apoptosis. Therefore, apoptosis-related proteins were detected. The results showed that compared with the Sham group, the expression of anti-apoptotic protein BCL-2 in the MIRI group was significantly decreased, and the pro-apoptotic proteins Bax and cleaved-casepase3 were significantly increased; compared with the MIRI group, the low- and high-dose hypericin groups and the positive control drug trimetazidine group significantly upregulated the expression of BCL-2; and had a down-regulating effect on the expression of apoptosis-related proteins Bax and cleaved-casepase3 ( Figure 4 (F and JL).

[0075] 5. Hyp can reduce myocardial cell damage, and its mechanism is related to regulating AMPK signaling In order to observe the effect of Hyp on cardiomyocytes, the present invention uses a stable rat H9c2 cardiomyocyte cell line to conduct an in vitro classic hypoxia-reoxygenation (H / R) model to simulate myocardial ischemia-reperfusion injury in vivo. CCK-8 detection was used to conduct cytotoxicity tests and verify the hypoxia-reoxygenation (H / R) model. First, CCK-8 detection showed that after 24 hours of pretreatment with Hyp at different concentrations (0-2μM), cell viability decreased when the Hyp concentration was 1000nM. Therefore, the Hyp treatment concentration in the following experiments should be lower than 1000nM. At the same time, CCK-8 was used to detect the treatment concentration (0-5μM) of the AMPK inhibitor Compound C (CC). The test results showed that cell viability was optimal when the CC concentration was 1μM ( Figure 5 AB). Figure 5As shown in E, the time of the hypoxia-reoxygenation model was further determined. The two models were hypoxia for 6 hours and reoxygenation for 2 hours and hypoxia for 8 hours and reoxygenation for 2 hours. CCK-8 detection showed that the viability of rat cardiomyocyte H9c2 cells decreased in the first mode, but it was not statistically significant compared with the CON group. However, in the hypoxia for 8 hours and reoxygenation for 2 hours mode, its cell viability decreased significantly and the difference was statistically significant. Therefore, the latter was determined as the time mode for model establishment. Finally, CCK-8 detection showed that cell activity decreased after H / R, and pre-administration of Hyp could partially restore cell viability. Cell viability increased at concentrations of 200 nM, 20 nM, and 2nM, and there were significant differences, so as to select the optimal concentration in subsequent experiments ( Figure 5 C). The concentration of LDH secreted in the culture medium after H / R reflects the severity of myocardial cell damage. LDH secretion increased significantly after H / R. 20 nM and 200 nM Hyp pretreatment reduced the level of LDH in the culture medium. Therefore, 20 nM Hyp was selected as the optimal concentration for subsequent experiments ( Figure 5 However, after treatment with AMPK inhibitor CC, Figure 5 As shown in F, LDH secretion increased significantly, indicating that it reversed the protective effect of Hyp on H9c2 cardiomyocyte injury caused by H / R. Pretreatment with 20 nM Hyp under H / R conditions can restore the number and morphology of cardiomyocytes to a certain extent ( Figure 5 Middle G). These results suggest that Hyp can alleviate myocardial cell injury caused by hypoxia and reoxygenation, and its mechanism is related to the regulation of AMPK signaling.

[0076] 6. Hyp can alleviate mitochondrial energy metabolism disorders in the H / R model The heart contains a large number of mitochondria, which are involved in high-energy metabolism. Maintaining the normal structure and function of mitochondria is necessary for the normal physiological activities of the heart. Therefore, the proteomic results and the protective mechanism of Hyp against H9c2 cell damage caused by H / R were verified in an in vitro model, and the AMPK inhibitor CC was used to observe the changes in mitochondrial energy metabolism. Figure 6 In groups AD, E, and G), ATP content and SOD activity were lower than those in the CON group, while AMP, MDA, and ROS content increased and were higher than those in the CON group. Hyp pretreatment increased ATP content and SOD activity, and reduced AMP, MDA, and ROS content, but after adding CC, the effects of Hyp on improving mitochondrial energy metabolism disorders and alleviating oxidative stress were reversed or eliminated. Further in vitro verification of whether Hyp would affect the AMPK / PGC-1α signaling pathway revealed that the results were consistent with the proteomics and animal level results: Figure 7As shown in A, C, and D, the protein expression levels of p-AMPK and PGC-1α in the H / R group were significantly lower than those in the CON group; Hyp pretreatment increased the protein expression levels of p-AMPK and PGC-1α. However, after co-culture with CC, the regulatory effect of Hyp on AMPK / PGC-1α was also reversed. In H9c2 cardiomyocytes, compared with the CON group, the number of positive apoptotic cells in the H / R group increased, the expression of BCL-2 anti-apoptotic protein decreased, and Hyp pretreatment alleviated cell apoptosis and decreased the expression of pro-apoptotic proteins Bax and Cleaved-Caspase3. After inhibiting AMPK with CC, the anti-apoptotic protective effect of Hyp in H9C2 cells was weakened ( Figure 6 F and H, Figure 7 B, E, F, and G in the figure). It can be seen that Hyp improves mitochondrial energy metabolism and alleviates oxidative stress damage by upregulating the AMPK / PGC-1α signaling pathway, ultimately playing a protective role against myocardial cell damage caused by H / R.

[0077] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Application of hypericin in the preparation of products for improving mitochondrial energy metabolism disorders.

2. The use according to claim 1, characterized in that: The mitochondrial energy metabolism disorder is caused by myocardial ischemia-reperfusion injury.

3. Application of hypericin in the preparation of regulators for regulating AMPK / PGC-1α signaling pathway.

4. The use according to claim 3, characterized in that: The AMPK / PGC-1α signaling pathway is the AMPK / PGC-1α signaling pathway in the body or cells of myocardial ischemia-reperfusion injury.

5. Application of hypericin in the preparation of regulators for regulating the Bcl-2 / Bax / Caspase-3 signaling pathway.

6. The use according to claim 5, characterized in that: The Bcl-2 / Bax / Caspase-3 signaling pathway is the Bcl-2 / Bax / Caspase-3 signaling pathway in the body or cells of myocardial ischemia-reperfusion injury.

7. Application of hypericin as an activator of AMPK / PGC-1α signaling pathway in the preparation of products for improving myocardial ischemia-reperfusion injury.

8. Application of hypericin as a Bcl-2 / Bax / Caspase-3 signaling pathway inhibitor in the preparation of products for improving myocardial ischemia-reperfusion injury.

9. Use of hypericin and AMPK inhibitor as or in the preparation of a positive control drug; the positive control drug is any of the following: (a1) As a positive control when screening drugs that prevent and / or treat myocardial ischemia-reperfusion injury by regulating the AMPK / PGC-1α signaling pathway; (a2) As a positive control when screening drugs that alleviate cardiomyocyte injury by regulating the AMPK / PGC-1α signaling pathway.

10. Use of hypericin and AMPK inhibitor as or in the preparation of a positive control drug; the positive control drug is any of the following: (b1) As a positive control when screening drugs that prevent and / or treat myocardial ischemia-reperfusion injury by regulating the Bcl-2 / Bax / Caspase-3 signaling pathway; (b2) As a positive control when screening drugs that alleviate cardiomyocyte injury by regulating the Bcl-2 / Bax / Caspase-3 signaling pathway.

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