Application of mitochondrial uncoupling agent in injury caused by ischemia reperfusion

Abstract

The invention relates to application of a mitochondrial uncoupling agent to preparation of a medicine for treating injury caused by ischemia reperfusion. The injury caused by ischemia reperfusion is cerebral apoplexy or ischemic myocardial injury. The medicine prepared from the mitochondrial uncoupling agent can radically inhibit ROS outbreak by reducing mitochondrial membrane potential and reduce neuron cell area loss and apoptosis, thereby promoting protection of neurons in an ischemic penumbra region; in addition, apoptosis in ischemic myocardial injury can be reduced, and a new strategy is provided for treatment of diseases such as cerebral arterial thrombosis or ischemic myocardial injury.

Description

Technical Field

[0001] This application belongs to the pharmaceutical field, specifically relating to the use of mitochondrial uncoupling agents in ischemia-reperfusion injury. Background Technology

[0002] Ischemic stroke is the second leading cause of death worldwide. It results from reduced cerebral blood flow, leading to brain tissue damage. Blockage of blood vessels prevents blood flow to the brain, causing ischemia (cerebral infarction) and brain tissue damage. It is characterized by high incidence, high recurrence rate, high disability rate, and high mortality rate. Ischemic stroke has a particularly severe impact on the brain because it is one of the most oxygen-sensitive organs. This may be due to the brain's high energy requirements, limited glycolytic capacity, and reliance on oxidative phosphorylation. The stroke area is divided into the infarct core and the penumbra. Blood flow in the infarct core is less than 20% of normal, while blood flow in the ischemic penumbra is 30%–40% of normal. After an ischemic stroke, energy and redox metabolic disorders leading to mitochondrial damage are key to irreversible penumbra damage. In recent years, endovascular treatment has made significant progress, significantly improving recanalization rates. However, although reperfusion has a certain therapeutic effect, and suitable patients can now undergo drug and surgical bridging to open occluded blood vessels, only about 46% of patients can complete the treatment, which depends on thrombolysis and thrombectomy within the time window (4.5 hours), but the mortality rate is as high as 15.3%. Therefore, it is crucial to protect neurons in the damaged area from ischemia-reperfusion injury.

[0003] Hypoxia induced by ischemia can also increase proton levels in the mitochondrial intermembrane space, further triggering a burst of reactive oxygen species (ROS) that damages neurons. Protons in mitochondria can be transferred to the intermembrane space via specific respiratory chain complexes, returning to the cytoplasm without producing ATP; this process is called proton leakage, which generates heat without generating ATP. Proton leakage is closely related to ROS generated by the mitochondrial respiratory chain. The production of mitochondrial superoxide in mitochondria is highly dependent on the proton gradient Δp. Proton leakage has a role in reducing ROS, thereby protecting mitochondrial integrity. Although there is limited direct research on accumulated protons, studies on uncoupling proteins have been conducted. Uncoupling proteins (UCPs) are members of a transport protein family present in the inner mitochondrial membrane and can form proton leakage on the inner mitochondrial membrane. UCP protein expression is tissue-specific, with UCP4 and UCP5 primarily expressed in the central nervous system. UCP1 is expressed in brown adipose tissue, uncoupling substrate oxidation and electron transport from ATP production and generating heat. UCP2 is widely expressed in mammalian tissues, including brain tissue. UCP2 and UCP3 only exert their uncoupling effects when specifically activated. UCP2 also plays a role in releasing reactive oxygen species from the mitochondrial matrix into the mitochondrial extracellular space; therefore, the expression level of UCP2 protein is positively correlated with neuronal survival in stroke and traumatic brain injury. Studies by Emanuela Viggiano et al. have shown that UCP5 can protect against stroke-related cortical spreading depression (CSD).

[0004] Current understanding suggests that oxidative stress occurs during ischemic stroke, with a rapid increase in ROS production at the onset of acute ischemia, and reperfusion can trigger a surge in ROS. Besides the brain, other studies have shown that oxidative stress also occurs in other tissues or organs such as the kidneys, retina, and heart during ischemia. NADPH oxidation plays a crucial role in neurological diseases; ROS generated by inhibition of glial cell NADPH oxidase can also damage neurons. NADPH oxidation is rapid, with superoxide production reaching its peak within 30 minutes. Hypoxia can activate NADPH oxidation, and NADPH participates in oxygen sensing through NADPH oxidase. Existing research indicates that GSH generated from NADPH can improve myocardial ischemia-reperfusion injury. Therefore, developing drugs to combat oxidative stress and intervene in stroke treatment is essential.

[0005] Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) is a novel free radical scavenger that inhibits both ·OH-dependent and ·OH-independent lipid peroxidation by quenching hydroxyl radicals (·OH). In Japan, edaravone has been successfully used clinically to treat acute stroke and has subsequently been used to treat neonatal hypoxic-ischemic encephalopathy. Edaravone's free radical scavenging properties reduce lipid peroxidation and oxidative DNA damage; it also exerts an anti-inflammatory effect by reducing microglial cell aggregation in the penumbra, and retains good neuroprotective effects even 6 hours after ischemia-reperfusion injury. Later, edaravone was also used clinically to treat amyotrophic lateral sclerosis (ALS), a neurodegenerative disease. However, subsequent studies found that edaravone's effect was not significant. The reasons include: First, edaravone scavenge hydroxyl radicals, which are groups or atoms with unpaired electrons formed when the covalent bonds within a compound molecule break under specific conditions. These include various types such as hydrogen radicals (H·), chlorine radicals (Cl·), hydroxyl radicals (OH·), methyl radicals (CH3·), and tetramethylpiperidine oxygen radicals. However, determining which type of free radical causes brain tissue damage during a stroke still requires further investigation. First, it is determined that the types of free radicals generated during stroke need to be eliminated to effectively exert a neuroprotective effect. Second, due to the burst of ROS during reperfusion, antioxidant drugs can directly exert their effects by resisting oxidative stress caused by reperfusion, but this requires strict control of the administration time; if the optimal time is missed, the protective effect of antioxidants may be weakened. Third, due to the existence of the blood-brain barrier, many drugs cannot enter the brain, so the reason for the weaker protective effect of edaravone may be related to the efficiency of drug entry into the brain. Finally, the inflammatory response after stroke is also an important factor in brain injury, and antioxidant drugs can exert a neuroprotective effect by reducing the level of ROS and weakening the inflammatory response.

[0006] In summary, there is an urgent need in this field for a drug that can effectively alleviate ischemia-reperfusion injury, especially nerve damage in the ischemic penumbra of the brain. Summary of the Invention

[0007] In order to address the problems existing in the prior art, the present invention provides the use of mitochondrial uncoupling agents in the preparation of medicaments for treating ischemia-reperfusion injury, wherein the ischemia-reperfusion injury is stroke or ischemic myocardial injury.

[0008] Compared to existing technologies, the beneficial effects of this application are at least as follows: This application innovatively discovers that mitochondrial uncoupling agents can alleviate ischemia-reperfusion injury, particularly neuronal damage in the ischemic penumbra. Therefore, drugs prepared using mitochondrial uncoupling agents can reduce mitochondrial membrane potential, thereby inhibiting ROS bursts at the source, reducing neuronal cell area loss and apoptosis, thus protecting neurons in the ischemic penumbra without adverse effects on blood vessels, providing a new strategy for the treatment of ischemic stroke and other diseases. Attached Figure Description

[0009] To gain a more detailed understanding of the foregoing features of this disclosure, reference can be made to the aspects illustrated in the accompanying drawings for a more specific description of the disclosure, which has been briefly outlined above. However, it is worth noting that the drawings illustrate only typical aspects of this disclosure and should not be construed as limiting its scope, as other equally valid aspects may be acknowledged.

[0010] Figure 1 The study demonstrates that BAM15 can alleviate the increase in mitochondrial membrane potential at various stages of reperfusion. Specifically, it shows the mitochondrial membrane potential and corresponding statistical graphs of the negative control group (control) without OGD treatment and the OGD-treated group (OGD) and OGD-BAM15-treated group after different reperfusion times (15 min, 1 h, 2 h, and 4 h after oxygen-glucose deprivation). The fluorescence intensity of CMTMRos dye indicates mitochondrial membrane potential, mito-dendra indicates mitochondrial dynamic changes, MAP2 is a neuron-specific marker staining, and %MMP represents the normalized mitochondrial membrane potential relative to the negative control group.

[0011] Figure 2 The effects of BAM15 on neuronal survival and proton levels in the mitochondrial matrix after reperfusion are shown. Specifically, the area and statistical plots of neurons in the negative control group (without OGD treatment), the OGD treatment group (OGD), the OGD-BAM15 treatment group, and the OGD-Genipin treatment group after oxygen-glucose deprivation reperfusion treatment are displayed, along with apoptosis detection results and mitochondrial matrix pH statistics. DIV8 represents neurons cultured for 8 days, DIV28 represents neurons cultured for 28 days, MAP2 is a neuron-specific marker staining, and %mitoPH represents the mitochondrial pH normalized relative to the negative control group.

[0012] Figure 3 The effects of BAM15 on total ATP, mitochondrial ATP, and total GSH in neurons after reperfusion are shown. Specifically, Figure 3 A, Figure 3 B shows the results of total ATP levels in neurons for each group, indicating that BAM15 does not reduce total ATP levels in neurons; Figure 3 C Figure 3D shows the neuronal mitochondrial ATP levels in each group, indicating that BAM15 has little effect on mitochondrial ATP. Figure 3 E showed neuronal area loss, indicating that simply increasing ATP levels is not sufficient to exert a neuroprotective effect, and BAM15 does not rely on increasing ATP to exert its neuroprotective effect. Figure 3 F, Figure 3 G shows a statistical graph of the total GSH level of neurons in each group.

[0013] Figure 4 This demonstrates that BAM15 can effectively reduce cerebral ischemia-reperfusion injury in animal models, specifically, Figure 4 A shows a schematic diagram of the tMCAO modeling method, in which brain tissue samples were collected 24 h after reperfusion; Figure 4 B- Figure 4 C shows the results of cerebral infarction volume measured by TTC staining 24 h after reperfusion; Figure 4 D- Figure 4 E shows the effect of BAM15 on neuronal survival in brain tissue after reperfusion; Figure 4 F, Figure 4 G shows the effect of BAM15 on the expression of UCP2 in neurons of brain tissue after reperfusion; Figure 4 H shows a schematic diagram of the tMCAO modeling method, in which brain tissue samples were collected 72 h after reperfusion; Figure 4 I- Figure 4 J shows the results of the infarct volume 72 h after reperfusion; Figure 4 K- Figure 4 L shows the effect of BAM15 on intracerebral vessels after reperfusion.

[0014] Figure 5 The results showed that F16 can alleviate the increase in mitochondrial membrane potential and reduce neuronal mortality during ischemia-reperfusion, but does not affect total ATP production. Specifically, Figure 5 A- Figure 5 D shows that F16 has a certain effect on reducing mitochondrial ATP, but does not affect the production of total ATP. The red and green fluorescence represent the mito-GoAtem probe, and the bright field shows the neurons. Figure 5 E, Figure 5 F shows that F16 reduces the pH in the mitochondrial matrix of neurons after reperfusion, where red fluorescence represents the mito pH probe, bright field shows neurons, and %mitoPH represents the normalized mitochondrial pH relative to the negative control group. Figure 5 G, Figure 5 H shows that F16 can effectively alleviate the increase in mitochondrial membrane potential induced by reperfusion, where %MMP represents the normalized mitochondrial membrane potential relative to the negative control group. Figure 5 K, Figure 5 L showed that F16 can increase neuronal survival.

[0015] Figure 6 The results showed that F16 can reduce the infarct volume and protect neurological function in a mouse model of ischemic stroke. Specifically, Figure 6 A, Figure 6 B shows the results of TTC staining to determine the infarct volume 24 h after reperfusion; Figure 6 C shows the neurological function scores of mice in an acute ischemic stroke model.

[0016] Figure 7 The results showed that BAM15 can reduce cardiomyocyte apoptosis during ischemia-reperfusion.

[0017] Figure 8 This demonstrates that BAM15 does not affect the integrity of cerebral hemisphere vessels.

[0018] For ease of understanding, the same reference numbers are used where possible to denote common elements in the diagrams. These diagrams are not drawn to scale and may have been simplified for clarity. It is conceivable that elements and features of one aspect can be beneficially incorporated into other aspects without further elaboration. Detailed Implementation

[0019] Specific embodiments of the present application will now be described in more detail with reference to the accompanying drawings. While specific embodiments of the present application are shown in the drawings, it should be understood that the present application can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0020] It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. This specification and claims do not distinguish components based on differences in terminology, but rather on differences in function. The terms "comprising" or "including" used throughout the specification and claims are open-ended and should be interpreted as "comprising but not limited to." The following descriptions in the specification are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims.

[0021] The term “or” is used in the claims to mean “and / or” unless it is explicitly stated that it refers only to an alternative or that the alternatives are mutually exclusive, although the content of this application supports the definition of referring only to an alternative and “and / or”. As used herein, “another” can mean at least a second or more.

[0022] Throughout this application, the term “about” is used to indicate an attribute, condition, or value within ±10% of the referenced attribute, condition, or value.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter pertains. Before a detailed description of the invention, the following definitions are provided to better understand it.

[0024] The terms "ischemia-reperfusion injury" and "ischemia-reperfusion injury" are used interchangeably. It refers to the phenomenon where, after a period of ischemia (interruption of blood supply), the restoration of blood flow (reperfusion) to a tissue or organ leads to more severe tissue damage and functional impairment. This phenomenon is commonly seen in myocardial infarction, stroke, organ transplantation, and shock resuscitation. The occurrence of ischemia-reperfusion injury involves multiple complex pathophysiological processes, primarily including: energy metabolism disorders and ATP depletion, a burst of oxygen free radicals during reperfusion (oxidative stress), calcium overload, exacerbated inflammatory response, microvascular damage, and no-reflow phenomenon.

[0025] The term "mitochondrial uncoupling agent" refers to a compound that disrupts the coupling between the electron transport chain and ATP synthesis in mitochondria. This disruption occurs by creating proton leakage in the matrix and intermembrane space, leading to the disappearance of the proton gradient across the inner mitochondrial membrane. This uncoupling effect can stimulate the flexibility of mitochondrial respiration and metabolism, thereby regulating cellular energy metabolism and potentially offering therapeutic benefits for various diseases. The inventors discovered that some mitochondrial uncoupling agents can help alleviate ischemia-reperfusion injury, particularly neurological damage in the ischemic penumbra of the brain, by reducing mitochondrial membrane potential and thus inhibiting the burst of oxygen free radicals. This led to the completion of this invention.

[0026] Therefore, in one aspect, the present invention provides the use of mitochondrial uncoupling agents in the preparation of medicaments for treating ischemia-reperfusion injury.

[0027] In some implementations, the ischemia-reperfusion injury is a stroke, such as an acute stroke.

[0028] In some preferred embodiments, the ischemia-reperfusion injury is neurological damage in the ischemic penumbra.

[0029] Common mitochondrial uncoupling agents include 2,4-dinitrophenol (DNP), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), niclosamide, and DNP derivatives. However, due to reasons such as high toxicity and poor selectivity, not all mitochondrial uncoupling agents can be used for disease treatment.

[0030] Among them, BAM15 (N 5 N 6 -bis(2-fluorophenyl)-[1,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine (CAS No. 210302-17-3) is a novel mitochondrial uncoupling agent with the structure shown in formula (I): .

[0031] The inventors were surprised to discover that BAM15 can effectively alleviate mitochondrial stress in stroke patients and reduce neuronal apoptosis. Specifically, BAM15 can lower mitochondrial membrane potential, inhibiting ROS bursts at the source and reducing neuronal cell area loss and apoptosis.

[0032] Furthermore, compared to other uncoupling agents, BAM15 exhibits lower cytotoxicity and fewer off-target effects on plasma membrane depolarization. The therapeutic potential of BAM15 has been investigated for the treatment of obesity, type 2 diabetes, and renal ischemia-reperfusion injury. However, those skilled in the art will recognize that drugs effective for renal ischemia-reperfusion injury do not necessarily apply to cerebral ischemia-reperfusion injury (e.g., stroke). Although renal and cerebral ischemia-reperfusion injury share significant overlap in their pathological mechanisms (e.g., oxidative stress, inflammatory response, apoptosis), and therefore some drugs may be effective for both, many drugs suitable for renal ischemia-reperfusion injury are not suitable for cerebral ischemia-reperfusion injury, or have extremely limited efficacy in cerebral ischemia-reperfusion injury, considering the differences in their applicable scenarios, significant organ-specific differences, and the presence of the blood-brain barrier (possibly due to poor blood-brain barrier penetration). For example, some non-selective calcium channel blockers (such as verapamil and diltiazem) can be used for renal ischemia-reperfusion injury, but they may inhibit intracellular calcium homeostasis, affecting neurotransmitter release and neuronal function. Furthermore, some drugs (such as verapamil) have vasodilatory effects on cerebral blood vessels, potentially exacerbating cerebral edema or increasing intracranial pressure; therefore, they cannot be used for cerebral ischemia-reperfusion injury. Similarly, some nitric oxide (NO) synthase inhibitors (such as L-NAME and aminoguanidine) can inhibit NO production in renal ischemia-reperfusion injury, potentially aggravating damage by increasing oxidative stress (such as the formation of peroxynitrite) or promoting inflammatory responses, thus exerting a renal protective effect. However, in cerebral ischemia-reperfusion injury, NO typically has vasodilatory, microcirculatory-improving, and anti-inflammatory effects; inhibiting its production may lead to cerebral vasoconstriction, worsening cerebral ischemia-reperfusion injury. In addition, some NO synthase inhibitors may not effectively penetrate the blood-brain barrier or inhibit the neuroprotective function of endogenous NO in the brain. Therefore, although there have been reports that BAM15 can be used to alleviate renal ischemia-reperfusion injury, it may not necessarily be effective for cerebral ischemia-reperfusion injury, considering many factors (such as blood-brain barrier penetration and its impact on the brain environment).

[0033] In some implementations, the ischemia-reperfusion injury does not include ischemia-reperfusion kidney injury.

[0034] In addition, F16 (C 16 H 15 IN2 (CAS No. 36098-33-6) is also a novel mitochondrial uncoupling agent, with the structure shown in formula (II): .

[0035] F16 was previously mainly used in anti-cancer research, where it was found to have high tumor selectivity. It can selectively uncouple cancer cell mitochondria, disrupt energy metabolism, and induce apoptosis, making it a potential candidate molecule for anti-cancer treatment. However, the inventors were surprised to discover that F16 can also effectively alleviate mitochondrial stress in stroke patients and reduce neuronal apoptosis through a mechanism similar to that of BAM15.

[0036] Furthermore, considering the numerous obstacles to their application in cerebral ischemia-reperfusion injury, the inventors also evaluated the safety of BAM15 and F16 in this area, finding that: 1) BAM15 and F16 can maintain the neuronal area in cerebral ischemia-reperfusion injury, exhibiting neuroprotective effects; 2) the application of BAM15 and F16 does not affect vascular integrity and does not cause severe vasodilation or vasoconstriction. Therefore, BAM15 and F16 have been verified to have sufficient safety for use in cerebral ischemia-reperfusion injury.

[0037] Furthermore, the inventors applied BAM15, as shown in Formula (I), to the study of ischemic myocardial injury and found that BAM15 can also reduce myocardial cell apoptosis during ischemia-reperfusion.

[0038] Therefore, in some implementations, the ischemia-reperfusion injury is ischemic myocardial injury.

[0039] In some embodiments, the mitochondrial uncoupling agent includes BAM15 as shown in formula (I), F16 as shown in formula (II), C12TPP, FR85P1, or a combination thereof.

[0040] In some preferred embodiments, the mitochondrial uncoupling agent comprises BAM15 as shown in formula (I), F16 as shown in formula (II), or a combination thereof.

[0041] In some embodiments, the mitochondrial uncoupling agent is administered in the drug at a dose of 1 mg / kg to 5 mg / kg.

[0042] In some embodiments, the administration of the drug includes intravenous administration, intramuscular injection, local administration, intraperitoneal injection, intracranial injection, intrapleural administration, intracranial administration, pulmonary administration, subcutaneous administration, sublingual administration, oral administration, nasal administration, interventional administration, implantation administration, patch administration, transdermal administration, film administration, or rectal administration.

[0043] In some embodiments, the dosage form of the drug is selected from injections, powder for injection, drops, patches, tablets, granules, sublingual tablets, microneedles, effervescent tablets, solutions, emulsions, liposomes, suspensions, ointments, creams, transdermal absorbers, transmucosal absorbers, lozenges, drops, pills, capsules, powders, liniments, granules, and syrups.

[0044] In some implementations, the drug is administered during the acute blood loss period or the recovery period.

[0045] The term "acute blood loss phase" refers to the stage in ischemic stroke or ischemic myocardial injury where the thrombus has not been cleared and the blood vessels are not yet open.

[0046] The term "recovery period" refers to the recovery phase of brain or myocardial tissue after thrombus removal and restoration of blood supply.

[0047] In some embodiments, the drug further includes pharmaceutically acceptable excipients selected from excipients, binders, disintegrants, lubricants, diluents, solubilizers, suspending agents, isotonic agents, pH adjusters, buffers, stabilizers, colorants, flavoring agents, and taste agents.

[0048] In some implementations, the drug is administered in combination with other therapeutic agents that can be used for ischemia-reperfusion injury.

[0049] The methods for obtaining various biological materials described in the embodiments are merely to provide experimental methods for specific disclosure purposes and should not be construed as limiting the sources of biological materials in this application. In fact, the sources of biological materials used are wide-ranging, and any biological material that can be obtained without violating laws and ethical standards can be substituted and used according to the suggestions in the embodiments.

[0050] Example Example 1 1. In vitro culture methods for cortical neurons (1) Before cell seeding, sterile 25 mm diameter round glass slides were placed in a 35 mm culture dish in a clean bench, and a mixture of 2 mL poly-D-lysine hydrobromide (100 μg / mL, Sigma-Aldrich) and laminin (Roche) was added and coated in a 37°C, 5% CO2 cell culture incubator for 2 h.

[0051] (2) Dissection: The brains of SD rat pups less than 12 h after birth were separated. The meninges were peeled off under a microscope with curved forceps, and the cortical nerve tissue was removed from the brain with curved forceps. The tissue was then cut into pieces and placed in a 15 mL centrifuge tube.

[0052] (3) Digestion: Add 5 mL of papain (Worthington) dissolved in HBSS (Gibco) to the 15 mL centrifuge tube, gently pipette and mix the precipitated cells in the centrifuge tube; then, place the centrifuge tube horizontally in a 37℃, 5% CO2 incubator for 40 min, then pipette the cell slurry 10-15 times with a 5 mL pipette, and finally centrifuge the centrifuge tube at 300 rpm for 5 min.

[0053] (4) Preparation of neuronal cell suspension: Take out the digestion solution and resuspend the cells in 5 mL of prepared papain inhibitor (600 µl inhibitor (Worthington) + 4.4 mL HBSS) solution. Then, centrifuge the suspension at 300 rpm for 10 min. After centrifugation, discard the supernatant and add neuronal basal culture medium (containing Neurobasal-A Medium, B27 Supplement, GlutaMAX Supplement, fetal bovine serum and penicillin-streptomycin) to resuspend the cells to obtain primary neuronal cell suspension.

[0054] (5) Inoculation of cortical neuronal cells: Take 10 μL of well-mixed cortical neuronal cell suspension for cell counting, and inoculate at a rate of 4 × 10⁻⁶ cells / mL. 5 One cell per 3.5 cm culture dish was seeded, neuronal basal culture medium was added, and the dish was placed in a cell culture incubator at 37°C, 5% CO2, and 75% humidity.

[0055] 2. Lentiviral Packaging and Infection Following the manufacturer's instructions, multiple probe genes (including the ATP fluorescent probe E8 GoAtem (addgene, 51958), the mitochondrial ATP fluorescent probe mito GoAtem, the total GSH fluorescent probe, the mitochondrial dynamic change fluorescent probe mito-dendra2, the mitochondrial pH probe mitoPH, and the SoNar fluorescent probe) were cloned into the pFUGW plasmid vector. Then, the lentiviral vector (pFUGW) carrying the probe genes, the lentiviral packaging vectors psPAX2, and pMD2.G cDNA were co-transfected into HEK293T cells at a ratio of 5:3:2 to produce lentivirus. The supernatants containing the viruses with different probe genes were collected separately, filtered through a 0.45 μm filter, and stored at -80℃ for later use.

[0056] In the cortical neuron cells prepared in 1.(5), according to 6×10 5 One cell per 1 mL lentivirus solution was added to the prepared lentivirus to obtain transfected cells with different fluorescent probes, which were then cultured for subsequent experiments.

[0057] 3. Establish a cellular oxygen-glucose deprivation (OGD) injury model. Replace the culture medium in the culture dish of the cells cultured in 1.(5) or the transfected cells in 2.(2) with OGD buffer (each liter of OGD buffer contains 8.01g NaCl, 0.37g KCl, 0.1g MgCl2·6H2O, 0.05g KH2PO4, 0.04g Na2HPO4, 0.34g NaHCO3, 25ml 1M HEPES, 0.2ml MgSO4·7H2O, and 0.5ml CaCl2·2H2O).

[0058] The petri dish was placed in a hypoxic chamber, which was brought into a hypoxic state by replacing the gas in the chamber with a mixture of 94% nitrogen, 1% oxygen and 5% carbon dioxide (for 5 minutes, at 25 L / min).

[0059] The hypoxia chamber was placed in a 37°C incubator and kept there for 4 hours to deprive oxygen and glucose.

[0060] 4. Membrane potential detection after reperfusion BAM15 treatment: The culture medium in the culture dish containing oxygen-glucose-deprived cortical neurons transfected with the mito-dendra2 fluorescent probe was replaced with neuronal basal medium, and 1 µL of DMSO was added (as the OGD reperfusion control group), or 0.1 µM, 0.2 µM, and 2 µM of BAM15 (as the OGD-BAM15 treatment groups). At 15 min, 1 h, 2 h, and 4 h of reperfusion, 30 nM of CMTMRos dye (Invitrogen, M7510) was added and incubated for 15 min, followed by elution of the remaining dye with OGD buffer and fixation with paraformaldehyde for 15 min. After fixation, the slide was rinsed three times with PBS, then MAP2 (BD, 556320) primary antibody working solution (diluted 1000 times with 1% BSA) was added, and after overnight incubation, it was washed with PBS, then the secondary antibody (Invitrogen, A32723) working solution (diluted 1000 times with 1% BSA) was added, and after PBS washing, the slide was mounted with mounting medium and placed on a confocal microscope to detect fluorescence.

[0061] F16 treatment: Replace the culture medium in the culture dish containing oxygen-glucose-deprived cortical neurons transfected with the mito-dendra2 fluorescent probe with neuronal basal medium, and add 1 µL of DMSO (as the OGD reperfusion control group), or 0.2 µM F16 or 2 µM F16 (as the OGD-F16 treatment group), and reperfuse for 25 minutes. Then add 200 nM TMRE dye (Invitrogen, T669) to each dish, incubate at 37°C for 15 minutes, then wash off the remaining dye with OGD buffer and detect fluorescence under a confocal microscope.

[0062] The results of BAM15 processing are shown below. Figure 1 .from Figure 1 It can be seen that BAM15 can effectively alleviate the effects of reperfusion at different times, such as 15 minutes of reperfusion (e.g.) Figure 1 A, Figure 1 (as shown in B), then perfusion for 1 hour (as shown in B) Figure 1 C Figure 1 As shown in D), reperfusion for 2 hours (as shown in D) Figure 1 E, Figure 1 (as shown in F) and reperfusion for 4 hours (as shown in F) Figure 1 G, Figure 1 The increase in mitochondrial membrane potential induced by (as shown in H). The results of F16 treatment are shown in... Figure 5 G, Figure 5 H. As can be seen from this figure, F16 can effectively alleviate the increase in mitochondrial membrane potential caused by reperfusion.

[0063] 5. Effects of BAM15 and F16 on neuronal survival and proton levels in the mitochondrial matrix after reperfusion Neuronal cells were incubated in a cell culture incubator at 37°C and 5% CO2 for a period of time, and then subjected to oxygen-glucose deprivation treatment as described above in vitro, followed by reperfusion treatment: For BAM15 treatment, physiological saline (as OGD control group) or 0.1 µM, 0.2 µM, and 2 µM BAM15 (as OGD-BAM15 treatment group) or 100 µM genipin (a UCP2 inhibitor) (as OGD-Genipin treatment group) were added to the basal culture medium containing neuronal cells, and the cells were cultured for 24 hours for reperfusion treatment; For F16 treatment, 1 µL of DMSO (as OGD control group) or 0.2 µM F16 or 2 µM F16 (as OGD-F16 treatment group) were added to the basal culture medium containing neuronal cells, and the cells were cultured for 24 hours for reperfusion treatment.

[0064] After reperfusion, neurons were fixed with paraformaldehyde, washed three times with PBS, and then incubated overnight with MAP2 (BD, 556320) primary antibody working solution (diluted 1000-fold with 1% BSA). After washing with PBS, secondary antibody (Invitrogen, A32723) working solution (diluted 1000-fold with 1% BSA) was added, followed by PBS washing. The cells were then mounted with mounting media and examined under a confocal microscope or subjected to subsequent TUNEL staining. For MAP2-stained neurons, the area of ​​the neurons was statistically analyzed using ImageJ to represent neuronal viability.

[0065] TUNEL staining working solution (Beyotime, C1090) was added to the neuronal cells and incubated for 1 hour. After washing with PBS, the cells were mounted with DAPI-containing mounting medium and examined under a confocal microscope.

[0066] Figure 2 AD shows the effect of BAM15 treatment on neurons. As can be seen from the figure, whether for young neurons (cultured for 8 days (DIV8), such as... Figure 2 A, Figure 2 (As shown in B) or older neurons (cultured for 28 days (DIV28), such as Figure 2 C Figure 2 As shown in D), BAM15 significantly alleviated the reduction in neuronal area caused by ischemia-reperfusion injury, while genipin treatment further aggravated the reduction in neuronal area, indicating that BAM15 can increase neuronal survival after ischemia-reperfusion injury.

[0067] Figure 2 E and Figure 2 Figure F shows the effect of BAM15 treatment on neuronal apoptosis. These figures demonstrate that, compared to the OGD control group, BAM15 treatment (0.1 µM and 0.2 µM treatments) significantly reduced apoptotic signals, while genipin treatment significantly increased apoptotic signals, further proving that BAM15 can reduce apoptosis and increase neuronal survival. Figure 5 K and Figure 5 Figures L show the effect of F16 treatment on neuronal apoptosis. As can be seen from these figures, similar to BAM15, F16 can increase neuronal survival.

[0068] 6. Effects of BAM15 and F16 on proton levels in the mitochondrial matrix after reperfusion Cells transfected with the mitoPH fluorescent probe were cultured in a 37°C, 5% CO2 cell culture incubator. Once the cells reached a suitable state, they underwent oxygen-glucose deprivation in vitro, followed by reperfusion treatment. The procedures for both oxygen-glucose deprivation and reperfusion were the same as above. Immediately after reperfusion (24 hours later), the cells were examined for fluorescence using a confocal microscope.

[0069] The results of BAM15 processing are shown below. Figure 2 G. As can be seen from these figures, ischemia-reperfusion increases the pH in the mitochondrial matrix (decreases the proton level), while BAM15 can significantly increase the proton level in the mitochondrial matrix. This means that BAM15 promotes the return of protons from the intermembrane space to the mitochondrial matrix through uncoupling, thus alleviating the increase in mitochondrial membrane potential.

[0070] The results of F16 processing are shown below Figure 5 E, Figure 5 As can be seen from these figures, ischemia-reperfusion increases the pH of the mitochondrial matrix (decreases the proton level), while F16, as a mitochondrial uncoupling agent, can promote the return of protons from the intermembrane space to the mitochondrial matrix through uncoupling, thereby decreasing the pH of the mitochondrial matrix (increasing the proton level), and this effect increases with increasing F16 concentration.

[0071] 7. Detection of total intracellular ATP and mitochondrial ATP in neurons under different conditions Cells transfected with either the E8 GoAtem fluorescent probe (for detecting neuronal ATP) or the mito GoAtem fluorescent probe (for detecting mitochondrial ATP) were cultured in a 37°C, 5% CO2 cell culture incubator. Once the cells reached a suitable state, they underwent in vitro oxygen-glucose deprivation injury, followed by reperfusion treatment. The procedures for both oxygen-glucose deprivation and reperfusion were the same as described above. Immediately after reperfusion (24 hours), the cells were examined under a confocal microscope for fluorescence detection.

[0072] To further illustrate whether changes in ATP can exert a neuroprotective effect, the inventors also used creatine and phosphocreatine, which provide energy, for further verification. Specifically, neurons were cultured for 7 days in a cell culture incubator at 37°C and 5% CO2, and then subjected to the oxygen-glucose deprivation treatment described above in vitro. Simultaneously, creatine or phosphocreatine at a final concentration of 200 µM was added to the neurons. After the oxygen-glucose deprivation was completed, the neurons were washed and then subjected to a 24-hour reperfusion treatment (i.e., the neurons were cultured in normal culture medium for 24 hours). After reperfusion, neurons were fixed with paraformaldehyde, washed three times with PBS, and then incubated overnight with MAP2 (BD, 556320) primary antibody working solution (diluted 1000 times with 1% BSA). After washing with PBS, secondary antibody (Invitrogen, A32723) working solution (diluted 1000 times with 1% BSA) was added, followed by PBS washing, mounting with mounting medium, and examination under a confocal microscope. The area of ​​neurons was statistically analyzed using ImageJ to represent neuronal viability.

[0073] The results of the effect of BAM treatment on total ATP in neuronal cells are shown in the figure. Figure 3 A, Figure 3 B. As can be seen from these figures, although BAM15 is an uncoupling agent, it does not decrease the total ATP level in neurons; in fact, it increases it. This means that BAM15 does not inhibit ATP production. The results of the effect of BAM treatment on mitochondrial ATP are shown in... Figure 3 C Figure 3 D. As can be seen from these two figures, although BAM15 is a mitochondrial uncoupling agent, it has little effect on mitochondrial ATP.

[0074] In addition, from Figure 3 E shows that increasing ATP by adding creatine and phosphocreatine does not alleviate the loss of neuronal area caused by oxygen-glucose deprivation injury. This indicates that simply increasing ATP levels cannot effectively exert a neuroprotective effect, and BAM15 does not rely on increasing ATP to exert its neuroprotective effect.

[0075] The results of the effect of F16 treatment on total intracellular ATP in neurons are shown in Figure 5 A- Figure 5D. As can be seen from these figures, F16, as a mitochondrial uncoupling agent, has a certain effect on reducing mitochondrial ATP, and the effect of reducing mitochondrial ATP becomes stronger with the increase of F16 concentration. However, F16 has no effect on the total ATP level in neurons. In fact, the total ATP level in neurons treated with F16 is higher than that in neurons treated with OGD only but without F16. This may be because F16 protects neurons undergoing ischemia-reperfusion, reducing neuronal cell damage, and therefore has a stronger effect of cytoplasmic ATP compensating for mitochondrial ATP, thereby increasing the total ATP level in neurons.

[0076] 8. Detection of glutathione (GSH) under various conditions GSH / GSSG is a key redox small molecule pair, and its changes can mediate downstream ferroptosis or alter intracellular redox homeostasis, potentially participating in the regulation of cell fate. To further verify the specific mechanism of action of BAM15, GSH levels were measured under various conditions.

[0077] Cells transfected with the total GSH fluorescent probe (for detecting intracellular total GSH) were cultured in a 37°C, 5% CO2 cell culture incubator. Once the cells reached a suitable state, they underwent oxygen-glucose deprivation injury in vitro, followed by reperfusion treatment. The procedures for glucose-oxygen deprivation and reperfusion were the same as above. Immediately after reperfusion (24 hours later), the cells were examined under a confocal microscope for fluorescence detection.

[0078] The results of the effect of BAM treatment on GSH are shown below. Figure 3 F, Figure 3 As can be seen from these figures, BAM15 has little effect on intraneuronal GSH levels. This indicates that BAM15 does not affect cell fate by regulating GSH / GSSG.

[0079] Example 2 1. tMCAO Surgical Method (1) Weigh 12-week-old male C57 mice in each intervention group, and try to control the weight between 26.5 g and 27.5 g, and select sutures corresponding to the weight.

[0080] (2) Inducing anesthesia in mice to undergo tMCAO (Transient Middle Cerebral Artery Occlusion) model, placing them in an anesthesia induction box, adjusting the anesthesia system parameters to 2% isoflurane concentration and 1.5 L / min oxygen flow rate, and observing the mice at the same time. If the mice are found to have significantly reduced activity and no response to painful stimuli, the maintenance anesthesia stage can be entered.

[0081] (3) Subsequently, in order to keep the mouse's body temperature relatively constant, the mouse's back was placed on an electric heating pad to maintain the mouse's body temperature at 36.5℃.

[0082] (4) Place the mouse's head inside the mask, with the skull level with the ground, to ensure unobstructed breathing. Adjust the ventilator parameters to maintain the isoflurane concentration between 1.4% and 1.5% and the oxygen flow rate at 0.4 L / min during the operation to keep the mouse under anesthesia.

[0083] (5) Expose the neck, administer continuous inhalation anesthesia, shave the neck with a rat razor, then disinfect with iodine-soaked cotton balls three times, and remove the iodine with 75% ethanol.

[0084] (6) Make a vertical incision along the midline of the neck with small scissors, about 1.2 cm long. Separate the subcutaneous tissue and neck muscles along the intermuscular spaces until the left common carotid artery with obvious pulsation can be seen below the muscles. Further separate along the course of the blood vessels to expose the common carotid artery, internal carotid artery and external carotid artery. Then carefully separate the thin membrane tissue around the artery and the vagus nerve accompanying the common carotid artery with ophthalmic temporal technique. Carefully separate the common carotid artery, which is about 0.3 cm long. The external carotid artery, internal carotid artery and common carotid artery are distributed in a "Y" shape.

[0085] (7) After the blood vessels are separated, ligate the common carotid artery (the closer to the tail, the better) and the external carotid artery (near the bifurcation). Leave a spare suture before the bifurcation of the common carotid artery and loosen the knot. Temporarily clamp the internal carotid artery with an arterial clamp. Make a "V" shaped incision with ophthalmic scissors about 0.15 mm from the bifurcation of the common carotid artery, slightly lift the tail end of the common carotid artery, grasp the suture plug with ophthalmic forceps, and gradually insert it into the internal carotid artery. To prevent blood backflow, tighten the reserved suture when it is about to reach the position of the arterial clamp. Release the arterial clamp, slightly lift the external carotid artery, so that the extracranial segment and intracranial segment of the internal carotid artery are as straight as possible, and continue to insert the suture plug. Stop inserting when you feel resistance. This means that the suture plug has reached the origin of the middle cerebral artery.

[0086] (8) After the suture plug is inserted, tighten and fix the ligature reserved in the previous step, and cut off the exposed reserved suture. Clean the neck with sterile saline, then align the subcutaneous tissue and skin, suture layer by layer, and finally disinfect with iodine cotton balls 3 times.

[0087] (9) Apply cocaine gel to relieve pain in mice, apply erythromycin eye ointment to prevent infection, stop anesthesia, and the operation is over.

[0088] (10) After the surgery, put the mice into a sterile cage to rest. They will wake up in about 5 minutes. After that, you can lift the tail to observe the movement of the rat's limbs to determine whether the modeling was successful.

[0089] (11) After inserting the suture 54 min, anesthetize the mouse again. At this time, inject the mouse intraperitoneally with 1 mg / kg of BAM15 or physiological saline (solvent), or 1 mg / kg of F16 or DMSO (solvent). Disinfect the neck skin, open a small incision, leave a suture in the common carotid artery, tie a slipknot, carefully untie the knot that fixes the suture, and after inserting the suture 60 min, use ophthalmic forceps to hold the suture and slowly pull it out.

[0090] (12) Tighten the reserved knots, sew them together layer by layer, and disinfect.

[0091] (13) For mice in the sham surgery group, only the artery was dissected without inserting a suture plug, and then the wound was sutured, and the mice were put back into the cage and given sufficient food and water.

[0092] (14) After the mouse wakes up, lift the mouse by its tail and stand it upside down. If the mouse's right forelimb is found to be clenched into a fist, or the mouse leans to the right when walking, and the symptoms need to last for 24 hours, then the tMCAO model is considered to have been successfully established.

[0093] 2. Determination of cerebral infarction volume using TTC staining method Mice were euthanized 24 hours after the embolism was removed. The whole brain was dissected and placed in a mold. The cerebellum and olfactory bulb were discarded, and a coronal slice of about 2 mm thickness was cut. The brain slice was quickly placed in 2% triphenyltetrazolium chloride (TTC) phosphate buffer solution and incubated at 37°C in the dark for 15-20 min. During incubation, the slice was turned over every 3-5 min. The brain slice was then removed, washed with PBS, and soaked in 4% PFA (paraformaldehyde) overnight. The brain slice was then removed, washed with PBS, and photographed.

[0094] In addition, the inventors also performed neurological functional assessments on mice with acute ischemic stroke according to the Clark scoring criteria, as shown in Table 1.

[0095]

[0096] Table 1. Clark Scoring Criteria The results of the effect of BAM15 on cerebral infarction are shown in Figure 4 B- Figure 4 C and Figure 4 I- Figure 4 The results of the effect of J,F16 on cerebral infarction are shown in Figure 6 A and Figure 6 B. As can be seen from these figures, BAM15 can effectively reduce reperfusion time to short (24 hours) (e.g.) Figure 4 B. Figure 4 (as shown in C) and mice with long reperfusion times (72 hours) (e.g. Figure 4 I, Figure 4F16 can also significantly reduce the cerebral infarction volume in mice (as shown in J).

[0097] Furthermore, using the Clark scoring system, the inventors discovered that F16 significantly reduced the neurological function scores of mice with acute ischemic stroke, demonstrating a neuroprotective function. Figure 6 C).

[0098] 3. Magnetic resonance imaging (MRI) measurement of cerebral infarction volume Mice that were successfully modeled were anesthetized with isoflurane, and their body temperature was maintained at 37 degrees Celsius and their respiratory rate was 100-120 breaths / min. A uMR 9.4T instrument was used, with voxel size of 0.10*0.10*0.50 mm, 30 slices in transverse, 30 slices in sagittal, and 30 slices in coronal planes, and slice thickness of 0.5 mm.

[0099] The results of MRI analysis of the effect of BAM15 on cerebral infarction volume are shown below. Figure 4 I- Figure 4 As can be seen from these two figures, BAM15 can effectively reduce the infarct volume in mice. The results obtained by MRI and TTC staining are consistent, indicating that BAM can protect neurons from ischemia-reperfusion injury.

[0100] 4. Effects of BAM15 on neuronal survival in brain tissue after reperfusion Mouse brain tissue collected 24 hours after tMCAO surgery was fixed with paraformaldehyde, washed three times with PBS, and sectioned coronally at a thickness of 30 μm using a cryostat (Leica CM1950, catalog number: 1405201950). The sections were then incubated overnight with MAP2 (BD, 556320) primary antibody working solution (diluted 1000-fold with 1% BSA), washed with PBS, and then mounted with secondary antibody (Invitrogen, A32723) working solution (diluted 1000-fold with 1% BSA). The sections were then examined under a confocal microscope. For MAP2-stained neurons, the area of ​​the neurons was statistically analyzed using ImageJ to represent neuronal viability.

[0101] The results of the effect of BAM15 treatment on neuronal survival in brain tissue after reperfusion were shown. Figure 4 D- Figure 4 E. As can be seen from these figures, BAM15 can increase neuronal survival in brain tissue after ischemia-reperfusion injury.

[0102] 5. Effect of BAM15 on UCP2 expression in neurons of brain tissue after reperfusion The mouse brain tissues collected 24 hours after tMCAO were fixed with paraformaldehyde and coronally sectioned using a cryostat (Leica CM1950, catalog number: 1405201950) at a thickness of 30 μm. After rinsing 3 times with PBS, the working solutions of primary antibodies MAP2 (BD, 556320) and UCP2 (proteintech / Sanying Bio, 11081-1-AP) (diluted 1000-fold with 1% BSA) were added, incubated overnight, washed with PBS, and then the working solutions of secondary antibodies (invitrogen, A32723; Thermo Fisher, A32794) (diluted 1000-fold with 1% BSA) were added. After washing with PBS, the sections were mounted with a mounting medium containing DAPI and examined under a confocal microscope. The stained area of UCP2 was counted using imageJ.

[0103] The results of the effect of BAM15 on the expression level of UCP2 in neurons of the post-reperfusion brain tissue are shown in Figure 4 F, Figure 4 G. It can be seen from these figures that BAM15 has no significant effect on the expression level of UCP2, indicating that as an uncoupler, BAM15 plays a neuroprotective role through its own uncoupling effect rather than by promoting the expression of uncoupling proteins.

[0104] 6. Effect of BAM15 on blood vessels in the post-reperfusion brain 1) The mouse brain tissues collected 24 hours after tMCAO were fixed with paraformaldehyde and coronally sectioned using a cryostat (Leica CM1950, catalog number: 1405201950) at a thickness of 30 μm. After rinsing 3 times with PBS, the working solutions of primary antibodies MAP2 (BD, 556320) and CD31 (Abclonal, A0378) (diluted 1000-fold with 1% BSA) were added, incubated overnight, washed with PBS, and then the working solutions of secondary antibodies (invitrogen, A32723; Thermo Fisher, A32794) (diluted 1000-fold with 1% BSA) were added. After washing with PBS, the sections were mounted with a mounting medium containing DAPI and examined under a confocal microscope.

[0105] 2) Twenty micrograms of lectin (dissolved in 200 μl of physiological saline) was injected into the tail vein of mice 24 hours after tMCAO to label blood vessels. The mouse brain tissues were collected 20 minutes later and fixed with paraformaldehyde. Coronally sectioned using a cryostat (Leica CM1950, catalog number: 1405201950) at a thickness of 30 μm, after washing with PBS, the sections were mounted with a mounting medium and examined under a confocal microscope, and the blood vessel morphology was counted using imageJ.

[0106] The results of the effects of BAM15 on cerebral vessels after reperfusion were shown. Figure 4 K- Figure 4 L and Figure 8 A- Figure 8 B. As can be seen from these figures, BAM15 has no significant effect on the expression of the vascular endothelial cell marker CD31, nor does it affect vascular integrity.

[0107] The above results indicate that BAM15 does not affect cerebral blood vessels, further demonstrating that BAM15 exerts its neuroprotective effect as an uncoupling agent through its own uncoupling action rather than by altering cerebral blood vessels.

[0108] Example 3 1. In vitro culture method for cardiomyocytes (H9C2) H9C2 cardiomyocytes (purchased from Yurisheng) were cultured in DMEM (containing 1.5 g / L NaHCO3) (catalog number: iCell-128-0001) supplemented with 10% high-quality fetal bovine serum and 1% penicillin-streptomycin, and placed in a cell culture incubator at 37°C, 5% CO2, and 70%-80% humidity until the density reached 80%.

[0109] 2. Establish a cellular oxygen-glucose deprivation injury model. Replace the culture medium of the cells obtained in step 1 with OGD buffer (each liter of OGD buffer contains 8.01g NaCl, 0.37g KCl, 0.1g MgCl2·6H2O, 0.05g KH2PO4, 0.04g Na2HPO4, 0.34g NaHCO3, 25ml 1M HEPES, 0.2ml MgSO4·7H2O, and 0.5ml CaCl2·2H2O).

[0110] The petri dish was placed in a hypoxic chamber, which was brought into a hypoxic state by replacing the gas in the chamber with a mixture of 94% nitrogen, 1% oxygen and 5% carbon dioxide (for 5 minutes, at 25 L / min).

[0111] The hypoxia chamber was placed in a 37°C incubator and kept there for 4 hours to deprive oxygen and glucose.

[0112] 3. Detection of apoptosis after reperfusion BAM15 treatment: The culture medium for oxygen-glucose deprived cardiomyocytes was replaced with DMEM medium, and 1 µM MDMSO was added (as the OGD reperfusion control group), or 0.1 µM and 0.2 µM BAM15 (as the OGD-BAM15 treatment group). Twenty-four hours after reperfusion, cardiomyocytes were fixed with paraformaldehyde for 30 minutes, washed three times with PBS, incubated with TUNEL staining working solution (Beyotime, C1090) for 1 hour, washed with PBS, mounted with DAPI-containing mounting medium, and examined under a confocal microscope.

[0113] Figure 7 A- Figure 7 Figure B shows the effect of BAM15 treatment on cardiomyocyte apoptosis during ischemia-reperfusion. These figures demonstrate that, compared to the OGD control group, BAM15 treatment (0.1 µM and 0.2 µM treatments) significantly reduced apoptotic signaling through uncoupling, thereby increasing cardiomyocyte survival.

[0114] Although the embodiments of this application have been described above in conjunction with the accompanying drawings, this application is not limited to the specific embodiments and application fields described above. The specific embodiments described above are merely illustrative and instructive, not restrictive. Those skilled in the art can make many other forms based on the guidance of this specification and without departing from the scope of protection of the claims of this application, and these are all within the scope of protection of this application.

[0115] References Campbell BCV, Khatri P. Stroke. Lancet. 2020 Jul 11;396(10244):129-142. doi: 10.1016 / S0140-6736(20)31179-X. PMID: 32653056. Gold L, Lauritzen M. Neuronal deactivation explains decreasedcerebellar blood flow in response to focal cerebral ischemia or suppressed neocortical function. Proc Natl Acad Sci US A. 2002 May 28;99(11):7699-704.doi: 10.1073 / pnas.112012499. PMID: 12032346; PMCID: PMC124326. Kristián T. Metabolic stages, mitochondria and calcium in hypoxic / ischemic brain damage. Cell Calcium. 2004 Sep-Oct;36(3-4):221-33. doi:10.1016 / j.ceca.2004.02.016. PMID: 15261478. Erecińska M, Silver IA. ATP and brain function. J Cereb Blood FlowMetab. 1989 Feb;9(1):2-19. doi: 10.1038 / jcbfm.1989.2. PMID: 2642915. Erecińska M, Silver IA. Ions and energy in mammalian brain. ProgNeurobiol. 1994 May;43(1):37-71. doi: 10.1016 / 0301-0082(94)90015-9. PMID:7972852. Kalogeris T, Baines CP, Krenz M, Korthuis RJ. Cell biology ofischemia / reperfusion injury. Int Rev Cell Mol Biol. 2012;298:229-317. doi:10.1016 / B978-0-12-394309-5.00006-7. PMID: 22878108; PMCID: PMC3904795. Choi WJ, Li Y, Wang RK. Monitoring Acute Stroke Progression: Multi-Parametric OCT Imaging of Cortical Perfusion, Flow, and Tissue Scattering ina Mouse Model of Permanent Focal Ischemia. IEEE Trans Med Imaging. 2019 Jun;38(6):1427-1437. doi: 10.1109 / TMI.2019.2895779. Epub 2019 Jan 31. PMID:30714910; PMCID: PMC6660833. Brenna S, Altmeppen HC, Mohammadi B, Rissiek B, Schlink F, Ludewig P,Krisp C, Schlüter H, Failla AV, Schneider C, Glatzel M, Puig B, Magnus T.Characterization of brain-derived extracellular vesicles reveals changes incellular origin after stroke and enrichment of the prion protein with apotential role in cellular uptake. J Extracell Vesicles. 2020 Aug 27;9(1):1809065. doi: 10.1080 / 20013078.2020.1809065. PMID: 32944194; PMCID:PMC7480459. Goyal M, Menon BK, van Zwam WH, Dippel DW, Mitchell PJ, Demchuk AM, Dávalos A, Majoie CB, van der Lugt A, de Miquel MA, Donnan GA, Roos YB, BonafeA, Jahan R, Diener HC, van den Berg LA, Levy EI, Berkhemer OA, Pereira, MRempel, Millán, MRempel, Davis SM, Roy D, Thornton J, Román LS, Ribó M, Beumer D,Stouch B, Brown S, Campbell BC, van Oostenbrugge RJ, Saver JL, Hill MD, JovinTG; HERMES collaborators. Endovascular thrombectomy after large-vesselischaemic stroke: a meta-analysis of individual patient data from fiverandomised trials. Lancet. 2016 Apr 23;387(10029):1723-31. doi: 10.1016 / S0140-6736(16)00163-X. Epub 2016 Feb 18. PMID: 26898852. Boveris A, Valdez LB, Zaobornyj T, Bustamante J. Mitochondrialmetabolic states regulate nitric oxide and hydrogen peroxide diffusion to thecytosol. Biochim Biophys Acta. 2006 May-Jun;1757(5-6):535-42. doi: 10.1016 / j.bbabio.2006.02.010. Epub 2006 Mar 20. PMID: 16615992. Ramsden DB, Ho PWL, Ho JWM, Liu HF, So DHF, Tse HM, et al. Humanneuronal uncoupling proteins 4 and 5 (UCP4 and UCP5): structural properties,regulation, and physiological role in protection against oxidative stress andmitochondrial dysfunction. Brain Behav. 2012;2(4):468–78. Nicholls DG, Snelling R, Rial E. Proton and calcium circuits acrossthe mitochondrial inner membrane. Biochem Soc Trans. 1984 Jun;12(3):388-90.doi: 10.1042 / bst0120388. PMID: 6329839. Fleury C, Neverova M, Collins S, Raimbault S, Champigny O, Levi-Meyrueis C, Bouillaud F, Seldin MF, Surwit RS, Ricquier D, Warden CH.Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia.Nat Genet. 1997 Mar;15(3):269-72. doi: 10.1038 / ng0397-269. PMID: 9054939. Richard D, Clavel S, Huang Q, Sanchis D, Ricquier D. Uncouplingprotein 2 in the brain: distribution and function. Biochem Soc Trans. 2001Nov;29(Pt 6):812-7. doi: 10.1042 / 0300-5127:0290812. PMID: 11709080. Mattiasson G, Shamloo M, Gido G, Mathi K, Tomasevic G, Yi S, WardenCH, Castilho RF, Melcher T, Gonzalez-Zulueta M, Nikolich K, Wieloch T.Uncoupling protein-2 prevents neuronal death and diminishes brain dysfunctionafter stroke and brain trauma. Nat Med. 2003 Aug;9(8):1062-8. doi: 10.1038 / nm903. Epub 2003 Jul 13. PMID: 12858170. Viggiano E, Monda V, Messina A, Moscatelli F, Valenzano A, Tafuri D,Cibelli G, De Luca B, Messina G, Monda M. Cortical spreading depressionproduces a neuroprotective effect activating mitochondrial uncouplingprotein-5. Neuropsychiatr Dis Treat. 2016 Jul 11;12:1705-10. doi: 10.2147 / NDT.S107074. PMID: 27468234; PMCID: PMC4946829. Zhang K, Tu M, Gao W, Cai X, Song F, Chen Z, Zhang Q, Wang J, Jin C,Shi J, Yang X, Zhu Y, Gu W, Hu B, Zheng Y, Zhang H, Tian M. Hollow PrussianBlue Nanozymes Drive Neuroprotection against Ischemic Stroke via AttenuatingOxidative Stress, Counteracting Inflammation, and Suppressing Cell Apoptosis.Nano Lett. 2019 May 8;19(5):2812-2823. doi: 10.1021 / acs.nanolett.8b04729.Epub 2019 Apr 4. PMID: 30908916. Tian Y, Su Y, Ye Q, Chen L, Yuan F, Wang Z. Silencing of TXNIPAlleviated Oxidative Stress Injury by Regulating MAPK-Nrf2 Axis in IschemicStroke. Neurochem Res. 2020 Feb;45(2):428-436. doi: 10.1007 / s11064-019-02933-y. Epub 2019 Dec 19. PMID: 31858374. Lorenzano S, Rost NS, Khan M, Li H, Batista LM, Chutinet A, Green RE,Thankachan TK, Thornell B, Muzikansky A, Arai K, Som AT, Pham LD, Wu O,Harris GJ, Lo EH, Blumberg JB, Milbury PE, Feske SK, Furie KL. Earlymolecular oxidative stress biomarkers of ischemic penumbra in acute stroke.Neurology. 2019 Sep 24;93(13):e1288-e1298. doi: 10.1212 / WNL.0000000000008158.Epub 2019 Aug 27. PMID: 31455665; PMCID: PMC7011868. Zhang D, Feng Y, Pan H, Xuan Z, Yan S, Mao Y, Xiao X, Huang X, ZhangH, Zhou F, Chen B, Chen X, Liu H, Yan X, Liang H, Cui W. 9-Methylfascaplysinexerts anti-ischemic stroke neuroprotective effects via the inhibition ofneuroinflammation and oxidative stress in rats. Int Immunopharmacol. 2021Aug;97:107656. doi: 10.1016 / j.intimp.2021.107656. Epub 2021 Apr 23. PMID:33895476. Kim YN, Jung HY, Eum WS, Kim DW, Shin MJ, Ahn EH, Kim SJ, Lee CH,Yong JI, Ryu EJ, Park J, Choi JH, Hwang IK, Choi SY. Neuroprotective effectsof PEP-1-carbonyl reductase 1 against oxidative-stress-induced ischemicneuronal cell damage. Free Radic Biol Med. 2014 Apr;69:181-96. doi: 10.1016 / j.freeradbiomed.2014.01.006. Epub 2014 Jan 17. PMID: 24440593. Rodrigo R, Fernández-Gajardo R, Gutiérrez R, Matamala JM, Carrasco R,Miranda-Merchak A, Feuerhake W. Oxidative stress and pathophysiology ofischemic stroke: novel therapeutic opportunities. CNS Neurol Disord DrugTargets. 2013 Aug;12(5):698-714. doi: 10.2174 / 1871527311312050015. PMID:23469845. Liu SS. Cooperation of a "reactive oxygen cycle" with the Q cycle andthe proton cycle in the respiratory chain--superoxide generating and cyclingmechanisms in mitochondria. J Bioenerg Biomembr. 1999 Aug;31(4):367-76. doi:10.1023 / a:1018650103259. PMID: 10665526. Indo HP, Davidson M, Yen HC, Suenaga S, Tomita K, Nishii T, HiguchiM, Koga Y, Ozawa T, Majima HJ. Evidence of ROS generation by mitochondria incells with impaired electron transport chain and mitochondrial DNA damage.Mitochondrion. 2007 Feb-Apr;7(1-2):106-18. doi: 10.1016 / j.mito.2006.11.026.Epub 2006 Dec 13. PMID: 17307400. Andrianova NV, Zorov DB, Plotnikov EY. Targeting Inflammation andOxidative Stress as a Therapy for Ischemic Kidney Injury. Biochemistry(Mosc). 2020 Dec;85(12):1591-1602. doi: 10.1134 / S0006297920120111. PMID:33705297. Li SY, Fu ZJ, Lo AC. Hypoxia-induced oxidative stress in ischemicretinopathy. Oxid Med Cell Longev. 2012;2012:426769. doi: 10.1155 / 2012 / 426769. Epub 2012 Oct 17. PMID: 23125893; PMCID: PMC3483772. Lee D, Kim KY, Shim MS, Kim SY, Ellisman MH, Weinreb RN, Ju WK.Coenzyme Q10 ameliorates oxidative stress and prevents mitochondrialalteration in ischemic retinal injury. Apoptosis. 2014 Apr;19(4):603-14. doi:10.1007 / s10495-013-0956-x. PMID: 24337820; PMCID: PMC3938850. Rezvan A. Telomeres, oxidative stress, and myocardial infarction. EurHeart J. 2017 Nov 1;38(41):3105-3107. doi: 10.1093 / eurheartj / ehx305. PMID:28637256; PMCID: PMC5837205. Neri M, Fineschi V, Di Paolo M, Pomara C, Riezzo I, Turillazzi E,Cerretani D. Cardiac oxidative stress and inflammatory cytokines responseafter myocardial infarction. Curr Vasc Pharmacol. 2015;13(1):26-36. doi:10.2174 / 15701611113119990003. PMID: 23628007. Havryliuk SO, Chekman IS, Horchakova NO, Ivanov SV, Oliĭnyk SA,Baraboĭ VA. [Role of oxidative stress in pathogenesis of atherosclerosis andischemic heart disease]. Ukr Biokhim Zh (1999). 2005;77(6):16-23. Ukrainian.PMID: 19618738. Brand MD, Goncalves RL, Orr AL, Vargas L, Gerencser AA, Borch JensenM, Wang YT, Melov S, Turk CN, Matzen JT, Dardov VJ, Petrassi HM, Meeusen SL,Perevoshchikova IV, Jasper H, Brookes PS, Ainscow EK. Suppressors ofSuperoxide-H2O2 Production at Site IQ of Mitochondrial Complex I Protectagainst Stem Cell Hyperplasia and Ischemia-Reperfusion Injury. Cell Metab.2016 Oct 11;24(4):582-592. doi: 10.1016 / j.cmet.2016.08.012. Epub 2016 Sep 22.PMID: 27667666; PMCID: PMC5061631. Gao HM, Zhou H, Hong JS. NADPH oxidases: novel therapeutic targetsfor neurodegenerative diseases. Trends Pharmacol Sci. 2012 Jun;33(6):295-303.doi: 10.1016 / j.tips.2012.03.008. Epub 2012 Apr 11. PMID: 22503440; PMCID:PMC3477578. Zhang J, Malik A, Choi HB, Ko RW, Dissing-Olesen L, MacVicar BA.Microglial CR3 activation triggers long-term synaptic depression in thehippocampus via NADPH oxidase. Neuron. 2014 Apr 2;82(1):195-207. doi:10.1016 / j.neuron.2014.01.043. Epub 2014 Mar 13. PMID: 24631344. Pei Z, Pang H, Qian L, Yang S, Wang T, Zhang W, Wu X, Dallas S,Wilson B, Reece JM, Miller DS, Hong JS, Block ML. MAC1 mediates LPS-inducedproduction of superoxide by microglia: the role of pattern recognitionreceptors in dopaminergic neurotoxicity. Glia. 2007 Oct;55(13):1362-73. doi:10.1002 / glia.20545. PMID: 17654704. Qin L, Liu Y, Wang T, Wei SJ, Block ML, Wilson B, Liu B, Hong JS.NADPH oxidase mediates lipopolysaccharide-induced neurotoxicity andproinflammatory gene expression in activated microglia. J Biol Chem. 2004 Jan9;279(2):1415-21. doi: 10.1074 / jbc.M307657200. Epub 2003 Oct 24. PMID:14578353. Bedard K, Krause KH. The NOX family of ROS-generating NADPH oxidases:physiology and pathophysiology. Physiol Rev. 2007 Jan;87(1):245-313. doi:10.1152 / physrev.00044.2005. PMID: 17237347. Deep G, Kumar R, Jain AK, Dhar D, Panigrahi GK, Hussain A, Agarwal C,El-Elimat T, Sica VP, Oberlies NH, Agarwal R. Graviola inhibits hypoxia-induced NADPH oxidase activity in prostate cancer cells reducing theirproliferation and clonogenicity. Sci Rep. 2016 Mar 16;6:23135. doi: 10.1038 / srep23135. PMID: 26979487; PMCID: PMC4793251. Gao J, Ding XS, Zhang YM, Dai DZ, Liu M, Zhang C, Dai Y. Hypoxia / oxidative stress alters the pharmacokinetics of CPU86017-RS throughmitochondrial dysfunction and NADPH oxidase activation. Acta Pharmacol Sin.2013 Dec;34(12):1575-84. doi: 10.1038 / aps.2013.94. Epub 2013 Oct 14. PMID:24122013; PMCID: PMC4002573. Barioni NO, Derakhshan F, Tenorio Lopes L, Onimaru H, Roy A, McDonaldF, Scheibli E, Baghdadwala MI, Heidari N, Bharadia M, Ikeda K, Yazawa I,Okada Y, Harris MB, Dutschmann M, Wilson RJA. Novel oxygen sensing mechanisms of the spinal cord involved in cardiorespiratory responses to hypoxia. SciAdv. 2022 Mar 25;8(12):eabm1444. doi: 10.1126 / sciadv.abm1444. Epub 2022 Mar25. PMID: 35333571; PMCID: PMC8956269. [ PubMed ] Zhu HL, Stewart AS, Taylor MD, Vijayasarathy C, Gardner TJ, SweeneyHL. Blocking free radical production via adenoviral gene transfer decreasescardiac ischemia-reperfusion injury. Mol Ther. 2000 Nov;2(5):470-5. doi:10.1006 / mthe.2000.0193. PMID: 11082320. Houkin K, Nakayama N, Kamada K, Noujou T, Abe H, Kashiwaba T.Neuroprotective effect of the free radical scavenger MCI-186 in patients withcerebral infarction: clinical evaluation using magnetic resonance imaging andspectroscopy. J Stroke Cerebrovasc Dis. 1998 Sep-Oct;7(5):315-22. doi:10.1016 / s1052-3057(98)80049-9. PMID: 17895107. Ohta Y, Takamatsu K, Fukushima T, Ikegami S, Takeda I, Ota T, Goto K,Abe K. Efficacy of the free radical scavenger, edaravone, for motor palsy ofacute lacunar infarction. Intern Med. 2009;48(8):593-6. doi: 10.2169 / internalmedicine.48.1871. Epub 2009 Apr 15. PMID: 19367054. Buonocore G, Perrone S, Bracci R. Free radicals and brain damage inthe newborn. Biol Neonate. 2001;79(3-4):180-6. doi: 10.1159 / 000047088. PMID:11275648. Noor JI, Ikeda T, Mishima K, Aoo N, Ohta S, Egashira N, Iwasaki K,Fujiwara M, Ikenoue T. Short-term administration of a new free radicalscavenger, edaravone, is more effective than its long-term administration forthe treatment of neonatal hypoxic-ischemic encephalopathy. Stroke. 2005 Nov;36(11):2468-74. doi: 10.1161 / 01.STR.0000185653.49740.c6. Epub 2005 Oct 6.PMID: 16210562. Zhang N, Komine-Kobayashi M, Tanaka R, Liu M, Mizuno Y, Urabe T.Edaravone reduces early accumulation of oxidative products and sequentialinflammatory responses after transient focal ischemia in mice brain. Stroke.2005 Oct;36(10):2220-5. doi: 10.1161 / 01.STR.0000182241.07096.06. Epub 2005Sep 15. PMID: 16166574. Yamashita T, Abe K. Update on Antioxidant Therapy with Edaravone:Expanding Applications in Neurodegenerative Diseases. Int J Mol Sci. 2024 Mar3;25(5):2945. doi: 10.3390 / ijms25052945. PMID: 38474192; PMCID: PMC10932469. He Q, Ma Y, Fang C, Deng Z, Wang F, Qu Y, Yin M, Zhao R, Zhang D, GuoF, Yang Y, Chang J, Guo ZN. Remote ischemic conditioning attenuates blood-brain barrier disruption after recombinant tissue plasminogen activatortreatment via reducing PDGF-CC. Pharmacol Res. 2023 Jan;187:106641. doi:10.1016 / j.phrs.2022.106641. Epub 2022 Dec 29. PMID: 36587812. Xu J, Wang A, Meng X, Yalkun G, Xu A, Gao Z, Chen H, Ji Y, Xu J, GengD, Zhu R, Liu B, Dong A, Mu H, Lu Z, Li S, Zheng H, Chen X, Wang Y, Zhao X,Wang Y; TASTE Trial Investigators†. Edaravone Dexborneol Versus EdaravoneAlone for the Treatment of Acute Ischemic Stroke: A Phase III, Randomized,Double-Blind, Comparative Trial. Stroke. 2021 Mar;52(3):772-780. doi:10.1161 / STROKEAHA.120.031197. Epub 2021 Feb 16. PMID: 33588596。

Claims

1. Use of mitochondrial uncoupling agents in the preparation of drugs for the treatment of ischemia-reperfusion injury.

2. The use as described in claim 1, wherein, The ischemia-reperfusion injury is a stroke, such as an acute stroke; preferably, the ischemia-reperfusion injury is a neurological injury in the ischemic penumbra of the brain.

3. The use as described in claim 1 or 2, wherein, The injury caused by ischemia-reperfusion is ischemic myocardial injury.

4. The use as described in any one of claims 1 to 3, wherein, The mitochondrial uncoupling agents include BAM15 as shown in formula (I), F16 as shown in formula (II), C12TPP, FR85P1, or combinations thereof: 。 5. The use as described in any one of claims 1 to 4, wherein, The mitochondrial uncoupling agent is administered in the drug at a dose of 1 mg / kg to 5 mg / kg.

6. The use as described in any one of claims 1 to 5, wherein, The administration methods of the drug include intravenous administration, intramuscular injection, local administration, intraperitoneal injection, intracranial injection, intrapleural administration, intracranial administration, pulmonary administration, subcutaneous administration, sublingual administration, oral administration, nasal administration, interventional administration, implantation administration, patch administration, transdermal administration, film administration, or rectal administration.

7. The use as described in any one of claims 1 to 6, wherein, The dosage form of the drug is selected from injections, powder for injection, drops, patches, tablets, granules, sublingual tablets, microneedles, effervescent tablets, solutions, emulsions, liposomes, suspensions, ointments, creams, transdermal absorbers, transmucosal absorbers, lozenges, drops, pills, capsules, powders, liniments, granules, and syrups.

8. The use as described in any one of claims 1 to 7, wherein, The drug also includes pharmaceutically acceptable excipients selected from excipients, binders, disintegrants, lubricants, diluents, solubilizers, suspending agents, isotonic agents, pH adjusters, buffers, stabilizers, colorants, flavoring agents, and taste agents.

9. The use as described in any one of claims 1 to 8, wherein, The drug is administered during the acute blood loss period or the recovery period.

10. The use as described in any one of claims 1 to 9, wherein, The drug is administered in combination with other therapeutic agents that can be used for injuries caused by ischemia-reperfusion.

Images