Use of aucubin in the preparation of a drug for treating ischemic encephalopathy

Abstract

The present application provides the use of aucubin or the combination of aucubin and ACA in the preparation of a medicament for treating ischemic brain injury, in particular acute cerebral ischemia-reperfusion injury. The present application first discovers that aucubin or the combination of aucubin and ACA can increase the blood flow of the ischemic side after ischemic brain injury in mice; significantly reduce the cerebral infarction volume; improve the cognitive and motor function of mice after ischemic brain injury; reduce the degree of injury caused by cerebral ischemia, significantly improve the degree of neuronal necrosis of brain tissue after ischemia; significantly increase the survival rate of HT22 cells after oxygen-glucose deprivation and reperfusion injury, reduce intracellular Ca 2+ fluorescence intensity, and inhibit cell apoptosis.

Description

Field of Invention:

[0001] This application pertains to the fields of stroke treatment and natural medicines. Specifically, this application provides the use of aucubin in the preparation of a medicament for treating ischemic encephalopathy. Background technology:

[0002] Stroke is characterized by high morbidity, high disability rate, high mortality rate, and high recurrence rate. Approximately 2 million new stroke patients are diagnosed annually in China, with 75%-85% of these being ischemic stroke (IS). Ischemic stroke, also known as cerebral infarction, refers to localized softening or necrosis of brain tissue due to impaired blood supply to the brain, resulting in ischemia and hypoxia. Numerous studies have shown that China has one of the highest incidence rates of ischemic stroke in the world, and its stroke recurrence rate is higher than the global average. With such a large patient population, ischemic stroke has become a significant medical and economic burden in China. The pathogenesis of ischemic stroke is complex. Currently, intravenous thrombolysis is one of the most effective interventions and is used in many countries and regions worldwide. However, due to its narrow therapeutic window, the risk of hemorrhagic transformation, and the possibility of permanent neurological damage, only a small number of patients currently benefit from it. Therefore, restoring blood supply to the brain as early as possible, promoting post-injury repair, reducing post-injury sequelae, and finding safe and effective neuroprotective agents have always been hot topics in the fields of brain science and neuromedicine, and also have important social value and research significance.

[0003] Aucubin is an iridoid compound. It is mainly extracted from plants such as Eucommia ulmoides, Plantago asiatica, and Rehmannia glutinosa. Numerous studies have confirmed its antioxidant, anti-photoaging, and collagen-promoting effects, making it a potential natural anti-aging protectant. Previous literature has reported that aucubin has a certain protective effect against traumatic brain injury (TBI). Researchers, through constructing in vitro neuronal oxidative stress models and in vivo TBI models, found that in the in vitro neuronal oxidative stress model, aucubin can exert antioxidant and neuronal apoptosis-inhibiting effects through the Nrf2-ARE pathway. Simultaneously, in the TBI model, aucubin can also promote Nrf2 nuclear translocation within neurons, activate the Nrf2-ARE antioxidant pathway, inhibit oxidative stress responses, reduce inflammation, decrease neuronal loss, alleviate cerebral edema, and improve motor and cognitive functions in mice, thus exerting a neuroprotective effect. Furthermore, studies have indicated that aucubin has a certain protective effect against hemorrhagic brain injury, subarachnoid hemorrhage, and diabetic encephalopathy. However, there are currently no reports on whether aucubin has a protective effect against ischemic brain injury. Therefore, developing it into a drug for the treatment of ischemic brain injury has extremely high value and social significance. Summary of the Invention

[0004] The purpose of this invention is to provide the use of aucubin in the preparation of drugs for treating ischemic brain injury through pharmacological studies of aucubin.

[0005] On the one hand, this application provides the use of aucubin in the preparation of drugs for treating ischemic brain injury.

[0006] On the other hand, this application provides the use of aucubin and TRPM2 inhibitors in the preparation of drugs for treating ischemic brain injury.

[0007] Furthermore, the TRPM2 inhibitor is ACA.

[0008] Furthermore, the ischemic brain injury is acute cerebral ischemia-reperfusion injury.

[0009] Furthermore, the single dose of aucubin is limited to a dose that does not cause central nervous system depression.

[0010] Furthermore, the single application dose of aucubin is 20-80 mg / kg; preferably 40-80 mg / kg; more preferably 80 mg / kg.

[0011] Furthermore, the single-dose administration of aucubin and ACA is 5-50 mg / kg and 1-40 mg / kg, respectively; preferably 1-20 mg / kg and 2-10 mg / kg; more preferably 15 mg / kg and 5 mg / kg.

[0012] Furthermore, the drug is in oral or injectable form.

[0013] Furthermore, the drug is an injectable dosage form.

[0014] Furthermore, the treatment of ischemic brain injury includes increasing blood flow to the ischemic side of the mouse after ischemic brain injury, reducing the volume of cerebral infarction, and inhibiting the degree of neuronal necrosis in the brain tissue after ischemia.

[0015] Furthermore, the treatment of ischemic brain injury includes reducing the neurological deficit score after ischemic brain injury in mice and improving cognitive and motor function after ischemic brain injury in mice.

[0016] Furthermore, the treatment for ischemic brain injury includes improving mitochondrial dysfunction.

[0017] Furthermore, aucubin increases cell survival after oxygen-glucose deprivation-reperfusion injury.

[0018] Furthermore, aucubin reduces intracellular calcium levels after oxygen-glucose deprivation-reperfusion injury. 2+ concentration.

[0019] Furthermore, aucubin reduces the degree of apoptosis in cells following oxygen-glucose deprivation-reperfusion injury.

[0020] The structural formula of aucubin described in this application is shown in (1). It is also known as β-D-glucopyranoside, English name Aucubin, abbreviated as AU, and CAS number 479-98-1. These names and numbers can be used interchangeably.

[0021]

[0022] The ACA structure described in this application is shown in formula (2), also known as N-(p-amylcinnamoyl)anthranilic acid, with the English name N-(p-Amylcinnamoyl)anthranilic Acid and CAS number 110683-10-8. These names and numbers can be used interchangeably.

[0023]

[0024] The drug described in this application can be used to treat various mammals, including humans, and other mammals besides humans include, but are not limited to, rats, rabbits, and dogs.

[0025] The drug dosage forms available in this application include injectable and oral dosage forms, and specific dosage forms include, but are not limited to, tablets, capsules, oral liquids, injections, powder injections, and transdermal drug delivery preparations, with injections being particularly preferred.

[0026] In addition to aucubin, the pharmaceutical products described in this application may also include various pharmaceutically acceptable excipients and formulations, including but not limited to coating materials, solvents, solubilizers, stabilizers, binders, antioxidants, pH adjusters, flavoring agents, etc., especially various excipients for injectable formulations.

[0027] The medicament of the present invention may contain traditional Chinese medicine or health products for treating ischemic brain injury, or the medicament of the present invention may be used in combination with such traditional Chinese medicine or health products or treatment methods. The traditional Chinese medicine or health products include, but are not limited to, anti-infective drugs, gonadotropin drugs, antioxidant drugs, drugs that improve energy metabolism and blood circulation, and natural extracts.

[0028] Beneficial effects:

[0029] This invention is the first to demonstrate that aucubin has a therapeutic effect on neuronal damage caused by ischemic brain injury and can be used to prepare therapeutic drugs for ischemic brain injury. Aucubin, and combinations of aucubin and ACA, significantly reduced neurological function scores in mice after ischemic brain injury; significantly increased blood flow to the ischemic side in mice after ischemic brain injury; significantly reduced infarct volume; significantly improved cognitive and motor function in mice after ischemic brain injury; aucubin can reduce the degree of damage caused by cerebral ischemia, significantly improve the degree of neuronal necrosis in brain tissue after ischemia; and significantly increase the survival rate of HT22 cells after oxygen-glucose deprivation-reperfusion injury and reduce intracellular calcium. 2+ Fluorescence intensity, inhibition of cell apoptosis. Attached Figure Description

[0030] Figure 1A , 1B Effects of aucubin on cerebral infarction volume in mice with cerebral ischemia-reperfusion injury Note: Compared with the Sham+NS group. ### P<0.001; compared with the MCAO / R group, *** P<0.001.

[0031] Figure 2A , Figure 2B , Figure 2C Effects of aucubin on blood flow perfusion in the ischemic cerebral cortex of mice with cerebral ischemia-reperfusion injury Compared with the Sham+NS group, ### P<0.001; compared with the MCAO / R+NS group, *** P<0.01.

[0032] Figure 3 Effects of aucubin on neurological function scores in mice with cerebral ischemia-reperfusion injury Compared with the Sham+NS group, ### P<0.001; compared with the MCAO / R+NS group, * P<0.05.

[0033] Figure 4 To investigate the effect of aucubin on escape latency in mice with cerebral ischemia-reperfusion injury in the Morris water maze-orientation navigation experiment. Compared with the Sham+NS group, ### P<0.001; compared with the MCAO / R+NS group, *** P<0.001.

[0034] Figure 5 To investigate the effect of aucubin on the number of times mice with cerebral ischemia-reperfusion injury can traverse a platform in a space exploration experiment. Compared with the Sham+NS group, # P<0.05; compared with the MCAO / R+NS group, ** P<0.01.

[0035] Figure 6 The target quadrant ratio of mice in each group during the Morris water maze-space exploration experiment. Compared with the Sham+NS group, ### P<0.001; compared with the MCAO / R+NS group, ** P<0.01, *** P<0.001.

[0036] Figure 7 The movement trajectory of mice in the Morris water maze-space exploration experiment

[0037] Figure 8 Effects of aucubin on pathological changes in the CA3 region of the ischemic hippocampus in mice with cerebral ischemia-reperfusion injury (200×)-HE staining.

[0038] Figure 9 Effects of aucubin on pathological changes in the CA1 region of the ischemic hippocampus in mice with cerebral ischemia-reperfusion injury (100×)-HE staining.

[0039] Figure 10 Effects of aucubin on pathological changes in the CA3 region of the ischemic hippocampus in mice with cerebral ischemia-reperfusion injury (200×)-Nissl staining.

[0040] Figure 11 Effects of aucubin on pathological changes in the CA1 region of the ischemic hippocampus in mice with cerebral ischemia-reperfusion injury (100×)-Nissl staining.

[0041] Figure 12 Effects of aucubin on the ultrastructure of mitochondria in hippocampal neurons of the ischemic side of the brain in mice with cerebral ischemia-reperfusion injury. Transmission electron microscopy.

[0042] Figure 13 Effects of aucubin and the TRPM2 inhibitor ACA on neurological deficit scores in mice with ischemic brain injury. Compared with the Sham+NS group, ### P<0.001; compared with the MCAO / R+NS group, ** P<0.01.

[0043] Figure 14 Effects of aucubin and the TRPM2 inhibitor ACA on cerebral infarction volume in mice with ischemic brain injury. Compared with the Sham+NS group, ### P<0.001; compared with the MCAO / R+NS group, * P<0.05.

[0044] Figure 15 Effects of aucubin and the TRPM2 agonist H2O2 on neurological deficit scores in mice with ischemic brain injury. Compared with the Sham+NS group, ### P<0.001; compared with the MCAO / R+NS group, * P<0.05, compared with the MCAO / R+AU (80mg / kg) group, + P<0.05.

[0045] Figure 16 Effects of aucubin and TRPM2 agonist H2O2 on cerebral infarction volume in mice with ischemic brain injury Compared with the Sham+NS group, ### P<0.001; compared with the MCAO / R+NS group, *** P<0.001, compared with the MCAO / R+AU group, + P<0.05.

[0046] Figure 17 Effects of aucubin on the survival rate of HT22 cells after oxygen-glucose deprivation-reperfusion injury Compared to the Control group, ### P<0.001; compared with the OGD / R group, *** P<0.001.

[0047] Figure 18A , 18B Intracellular Ca2+ in HT22 cells following aucubin-induced oxygen deprivation-reperfusion injury 2+ Impact Compared to the Control group, ### P<0.001; compared with the OGD / R group *** P<0.001.

[0048] Figure 19A , 19B Effects of aucubin on apoptosis in HT22 cells after oxygen-glucose deprivation-reperfusion injury Compared to the Control group, ### P<0.001; compared with the OGD / R group *** P<0.001. Detailed Implementation

[0049] The present invention will be described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0050] Example 1 Animal Experiment Protocol

[0051] Laboratory animals:

[0052] SPF-grade male adult mice weighing 26-32g were bred at the Experimental Animal Center of Ningxia Medical University (Animal incubation qualification batch number: SCXK(Ning)2020-0001). Animal husbandry followed laboratory animal husbandry standards, with a 12-hour controlled day-night cycle in the rearing room and free access to water.

[0053] Experimental reagents and instruments:

[0054] The main drugs and reagents used in this experiment included: aucubin (purchased from Shanghai Yuanye Biotechnology Co., Ltd., purity ≥98%); nimodipine (purchased from Bayer AG, Germany); physiological saline (purchased from Tianjin Damao Chemical Reagent Factory); model suture plug (purchased from Rewards Biotech); TTC (purchased from Sigma-Aldrich); HE kit (purchased from Wuhan Sewell Biotechnology Co., Ltd.); and Nissl staining kit (purchased from Wuhan Sewell Biotechnology Co., Ltd.).

[0055] The main instruments involved in this experiment include: a laser speckle blood flow imaging system (Parry GmbH, Sweden); a full-wavelength microplate reader (purchased from Hunan Thermo Fisher Scientific Technology Co., Ltd.); an electric thermostatic water bath (purchased from Shanghai Precision Experimental Equipment Co., Ltd.); and a high-speed low-temperature centrifuge (Endorphin, Germany).

[0056] Grouping and administration of experimental animals:

[0057] Male ICR mice weighing 26-32g were raised in a clean-range environment for one week. They were then randomly assigned to four groups (n=6 per group) using a weight-gradient sampling method: sham-operated group, model group, model group + aucubin (20mg / kg, 40mg / kg, 80mg / kg) group, and model group + nimodipine 1.4mg / kg group. The aucubin groups and nimodipine groups received the medication at a fixed time each morning for one week. The sham-operated group and model group received the same volume of saline at the same time each day, with all other parameters remaining the same.

[0058] Example 2: Animal Experiment Procedures and Results

[0059] Observe the neuroprotective effect of aucubin on mice with ischemic brain injury.

[0060] Experimental grouping and drug administration:

[0061] Male ICR mice were randomly divided into the following 7 groups: Sham+NS group, Sham+AU (80 mg / kg) group, MCAO / R+NS group, MCAO / R+AU (20, 40, 80 mg / kg) group, and MCAO / R+Nim (1.4 mg / kg) group. The MCAO / R+AU (20, 40, 80 mg / kg) group and the MCAO / R+Nim group were administered the drug via intraperitoneal injection at the same time every day for 7 consecutive days; the other groups were given the same volume of physiological saline at the same time point, with an administration interval of 24 hours.

[0062] (I) Effect of aucubin on cerebral infarction volume in mice with ischemic brain injury

[0063] Experimental methods:

[0064] Twenty-four hours after modeling in each group of mice, tissue samples were collected. Mice were anesthetized with chloral hydrate, and brain tissue was quickly removed by decapitation. The brain tissue was rinsed with physiological saline to remove surface impurities, transferred to a pre-cooled brain trough, and flash-frozen at -80°C for 3-5 minutes. Five 2mm coronal sections of the mouse brain were prepared and stained with 2% TTC staining solution. The sections were incubated at 37°C for approximately 10 minutes. Normal brain tissue appeared red, while the infarcted tissue appeared white. The TTC staining solution was recovered. A suitable amount of 4% formaldehyde solution was added and the sections were incubated overnight at 4°C. The brain slices were then arranged in order on a graduated horizontal plate and photographed. The infarct volume on the ischemic side was detected using Image-Proplus software.

[0065] Experimental results:

[0066] The results are shown in Table 1 and Figure 1A , Figure 1B As shown in the figure, compared with the Sham+NS group, the cerebral infarction volume of mice in the MCAO / R+NS group was significantly increased (P<0.001); compared with the MCAO / R+NS group, the cerebral infarction volume of mice in the MCAO / R+Nim group was significantly decreased (P<0.001); and the cerebral infarction volume of mice in the MCAO / R+AU intervention group was significantly decreased (P<0.001).

[0067] Table 1. Effects of aucubin on infarct volume in ischemic brain injury in mice.

[0068]

[0069] Note: Compared with the Sham+NS group. ### P<0.001; compared with the MCAO / R+NS group, *** P<0.001

[0070] (II) Effects of aucubin on cerebral cortical blood perfusion in mice with ischemic brain injury

[0071] Experimental methods:

[0072] After 24 hours of ischemia, mice were anesthetized, the hair on the skin surface of the parietal bone of the mouse skull was removed, the skin was longitudinally cut after disinfection to fully expose the periosteum, and a small amount of physiological saline was dripped onto the periosteum. The laser speckle blood flow imaging scanner was set to the top of the mouse brain to mark the changes in cerebral blood flow in the cerebral cortex, and the data were recorded and exported for analysis.

[0073] Experimental results

[0074] As shown in Table 2 and Figure 2A , Figure 2B , Figure 2C As shown, 24 hours after cerebral ischemia-reperfusion, laser speckle blood flow imaging results showed that, compared with the sham-operated group, the cerebral blood flow in the ischemic side of the model group mice was significantly reduced (P<0.001); compared with the model group, after treatment with aucubin, the cerebral blood flow in the ischemic side of the model group mice was significantly increased (P<0.05, P<0.01, P<0.001).

[0075] Table 2. Effects of aucubin on blood perfusion in the ischemic cerebral cortex of mice.

[0076]

[0077] Note: Compared with the Sham+NS group. ### P<0.001; compared with the MCAO / R+NS group, *** P<0.01

[0078] (III) Effects of aucubin on neurological function in mice with ischemic brain injury

[0079] Experimental methods:

[0080] After the last administration, the body weight of mice in each group was recorded, and the experiment was conducted using a double-blind method. Mice in each group were induced to develop neurological function 1 hour after administration, and the Longa score was used to assess neurological function.

[0081] A mouse MCAO / R model was established using the Zea-Longa suture tether method. One hour after drug administration on day 7, mice were anesthetized with an intraperitoneal injection of 4% chloral hydrate. The mice were then fixed in a supine position on the operating table. Neck fur was clipped, and the area was disinfected with iodine. The skin was incised along the midline of the neck, and the muscles and the left common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were bluntly dissected. The CCA was temporarily clamped, the distal end of the ECA was ligated, and a slipknot was tied at the CCA. The suture tether was inserted approximately 8-10 mm into the ICA, stopping when resistance was encountered. The suture tether was then fixed, and the mouse wound was sutured. The mice were placed on a 37°C warming blanket to maintain their body temperature. The suture tether was removed 1.5 hours later, and the mice were fully awake and then housed in their normal environment. In the sham-operated group and the sham-operated + aucubin (80 mg / kg) group, only the common carotid artery was exposed, without ligation; all other procedures were the same as MCAO / R. Experimental results:

[0082] The results are shown in Table 3 and Figure 3 As shown, compared with the sham-operated group, the neurological function scores of the model group were significantly increased (P<0.001); the neurological function scores of mice treated with aucubin (20 mg / kg, 40 mg / kg, 80 mg / kg) were significantly decreased (P<0.05, P<0.01, P<0.001). The nimodipine treatment group also significantly reduced the neurological function scores of mice (P<0.001). These findings suggest that aucubin has a certain protective effect against ischemic brain injury in mice.

[0083] Table 3. Effects of aucubin on neurological function scores in mice with ischemic brain injury.

[0084]

[0085] Note: Compared with the Sham+NS group. ### P<0.001; compared with the MCAO / R+NS group, * P<0.05 (IV)

[0086] Effects of aucubin on cognitive and motor function in mice after ischemic brain injury

[0087] Experimental methods:

[0088] Learning and Memory Ability - Morris Water Maze Orientation and Navigation Experiment: Starting on the second day after model creation, mice in the sham-operated group, model group, aucubin group, and nimodipine group underwent the water maze experiment, a total of 5 times, completed within 5 days. Before the formal experiment, the experimental animals were placed in the pool for 60 seconds to acclimatize. During the training process, the platform positions remained unchanged, and the animals were placed into the water to swim sequentially from four entry points. In the formal experiment, an entry point was randomly selected, and the animal was placed in the pool facing the pool wall. The time it took for the mouse to climb onto the platform (i.e., escape lateney) was recorded. In the first few training sessions, if the mouse could not find the platform within 60 seconds, it was guided to climb onto the platform with a thin stick and stay there for 10 seconds; this escape lateney was recorded as 60 seconds. Afterward, the mouse was removed and its fur was dried with a hair dryer. All experimental mice were trained continuously for 5 days, once at each entry point, 4 times a day, with a 15-minute rest between training sessions. The arithmetic mean of the 4 escape lateney times for each day was used for analysis. (2) Spatial probe test: Two hours after the final positioning and navigation experiment (day 5), the platform was removed from the pool, and the mice underwent a 60-second exploration experiment. The mice were placed into the pool from the entry point on the opposite side of the quadrant where the platform was located, and the number of times they crossed the platform and the ratio of movement time in the target quadrant were recorded to evaluate the mice's spatial memory ability.

[0089] Experimental results:

[0090] As shown in Table 4-1, aucubin improved learning and memory deficits in mice with ischemic-reperfusion brain injury. Compared with the Sham group, the Vehicle group showed an increased escape latency on days 2-5 of the experiment (P<0.01, P<0.001). Figure 4 The number of platform crossings decreased (P<0.01, P<0.001). Figure 5 The time spent crossing the quadrant where the platform is located decreased in space exploration experiments (P<0.05, P<0.01). Figure 6 The camera-tracked mouse movements showed that the Sham mice swam close to the platform, while the Vehicle group mice moved erratically, either along the pool wall or away from the platform. Figure 6 In comparison, the LBP group mice showed a reduced escape latency on days 3-5 of the experiment (P<0.01), an increased number of platform crossings in the spatial exploration experiment (P<0.001), a prolonged time spent crossing the quadrant containing the platform (P<0.05, P<0.01), and more regularized movement trajectories. Figure 7 ).

[0091] Table 4-1 Effects of aucubin on escape latency, time spent in the target quadrant, and number of platform crossings in mice with ischemic brain injury.

[0092]

[0093] Note: Compared with the Sham+NS group. ### P<0.001; compared with the MCAO / R+NS group, * P<0.05, ** P<0.01, *** P<0.001

[0094] (V) Effects of aucubin on pathological changes in the hippocampus of mice with ischemic brain injury (HE staining to observe pathological changes in the ischemic side of the brain tissue in mice)

[0095] Experimental methods:

[0096] Hematoxylin-eosin staining (HE staining) was performed on paraffin sections of tissue. The sections were baked in a 70°C oven for 2 hours, then sequentially immersed in xylene I (10 min), xylene II (5 min), anhydrous ethanol I (1 min), anhydrous ethanol II (1 min), 95% ethanol (30 s), 80% ethanol (30 s), and 70% ethanol (30 s), washed three times with water, then immersed in hematoxylin for 5 min, washed three times with water, and finally immersed in 1% hydrochloric acid-alcohol solution for 20 s, washed with water, and then in eosin solution. After soaking in ethanol for 2 minutes and rinsing quickly with water, the paraffin sections were then soaked in 80% ethanol (30 seconds), 95% ethanol (30 seconds), anhydrous ethanol I (1 minute), anhydrous ethanol II (3 minutes), xylene I (2 minutes), and xylene II (2 minutes) in sequence. Finally, the sections were mounted with neutral resin. After HE staining of paraffin sections of brain tissue from newborn rats in each group, they were observed under a microscope. Each section was taken at a ×200 field of view, and 3-6 regions in each field of view were photographed and recorded.

[0097] Experimental results:

[0098] like Figure 8-9 As shown, a single intraperitoneal injection of aucubin exhibited a bidirectional effect on the structural and pathological changes in brain tissue induced by ischemia-reperfusion injury in mice. Compared with the hypoxic-ischemic model group, with increasing aucubin dosage, the pathological changes in the hippocampal CA3 and CA1 regions of the ischemic brain were weakened, the number of neurons increased, edema and vacuolar changes were reduced, and nuclear condensation and margination were weakened. This suggests that aucubin (80 mg / kg) has the effect of alleviating the pathological damage to brain tissue cellular structure after ischemia-reperfusion injury in mice.

[0099] (VI) Effects of aucubin on pathological changes in the hippocampus of mice with ischemic brain injury (Nissl staining to observe necrosis of the ischemic side of the brain tissue in mice)

[0100] Experimental methods:

[0101] Nissl staining was performed by baking paraffin sections in a 70°C oven for 2 hours. The slides were then sequentially immersed in xylene I (10 min), xylene II (5 min), anhydrous ethanol I (1 min), anhydrous ethanol II (1 min), 95% ethanol (30 s), 80% ethanol (30 s), and 70% ethanol (30 s), followed by 3 washes with water. The slides were then incubated in Nissl staining solution at 50°C for 1 hour, washed 3 times, and immersed in 1% hydrochloric acid-alcohol solution for 20 seconds, washed again, and finally mounted with neutral resin. After Nissl staining, the paraffin sections of mouse brain tissue from each group were observed under a microscope. Each section was examined at ×200, and 3-6 regions from each field of view were photographed and recorded.

[0102] Experimental results:

[0103] like Figure 10-11 As shown, a single intraperitoneal injection of aucubin exhibited a bidirectional effect on the structural necrosis of brain tissue induced by ischemia-reperfusion injury in mice. In the model group, with increasing aucubin dosage, the pathological changes in the CA3 and CA1 regions of the ischemic hippocampus gradually weakened, neurons became more regular, and the number of Nissl bodies gradually increased. The least pathological damage to neuronal tissue was observed when aucubin was administered (80 mg / kg), suggesting that aucubin has a protective effect against neuronal necrosis in the brain tissue following ischemia-reperfusion injury in mice.

[0104] (VII) Effects of aucubin on the ultrastructure of mitochondria in hippocampal neurons of mice with ischemic brain injury: Experimental method:

[0105] Twenty-four hours after model establishment, mice were injected intraperitoneally with 4% chloral hydrate. After anesthesia, the mice were quickly decapitated and the brains were removed. The ischemic brain tissue was cut into 1mm sections. 3 Small fragments were soaked in 2.5% glutaraldehyde solution at 4°C for 12 hours, then transferred to 0.1M phosphate buffer for 12 hours, followed by soaking in 2% osmium tetroxide for 2 hours. After dehydration in a gradient of alcohols, they were embedded in epoxy resin. Ultrathin sections (70 nm) were prepared after embedding, stained with lead citrate and uranium acetate, and finally the ultrastructure of mitochondria in hippocampal neurons was observed under a transmission electron microscope, and photographs were taken and recorded.

[0106] Experimental results:

[0107] like Figure 12As shown, transmission electron microscopy was used to observe the effects of aucubin on the ultrastructure of neurons in mice with ischemic brain injury. The results showed that the ultrastructure of mitochondria in the Sham+NS group remained unchanged, exhibiting rod-shaped or oval morphology, with clear mitochondrial membranes and cristae. In the MCAO / R+NS group, the number of mitochondria decreased, edema was observed, and the morphology was predominantly round. Mitochondrial cristae were broken, dissolved, or disappeared, exhibiting a cloud-like appearance, and the matrix became transparent, showing vacuolar changes. After treatment with AU and nimodipine, the number of mitochondria increased, the membrane structure was clearer, predominantly oval and short rod-shaped, and a small number of mitochondrial cristae decreased or broke.

[0108] Example 3: Animal-level investigation of the neuroprotective mechanism of aucubin on ischemic brain injury in mice. Experimental grouping and administration:

[0109] Male ICR mice were randomly divided into the following 8 groups: Sham+NS group, MCAO / R+NS group, MCAO / R+AU (80 mg / kg) group, MCAO / R+AU (80 mg / kg) group + ACA (5 mg / kg) group, MCAO / R+ACA (5 mg / kg) group, MCAO / R+H2O2 (80 mg / kg) group, MCAO / R+AU (80 mg / kg) + H2O2 (80 mg / kg) group, and MCAO / R+Nim group. They were administered the drug via intraperitoneal injection at the same time every day for 7 consecutive days. The remaining groups were given the same volume of physiological saline at the same time point, with an administration interval of 24 hours.

[0110] Preparation of a mouse model of ischemic brain injury:

[0111] A mouse MCAO / R model was established using the suture tether method. One hour after drug administration on day 7, mice were anesthetized and fixed in a supine position on the operating table. Neck fur was clipped, and the area was disinfected with iodine. The skin was incised along the midline of the neck, and the muscles and the left common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were bluntly dissected. The CCA was temporarily clamped with an arterial clamp, and the distal end of the ECA was ligated with a slipknot. The suture tether was inserted approximately 8-10 mm into the ICA, stopping when resistance was encountered. After securing the suture tether, the mouse wound was sutured. The mouse was placed on a 37°C warming blanket to maintain its body temperature. The suture tether was removed 1.5 hours later, and the mice were fully awake and then housed in their normal environment.

[0112] Neurological function score:

[0113] Twenty-four hours after model establishment, the neurological function of mice in each group was evaluated using the Zea-Longa score. The scores were as follows: 0 points: no neurological dysfunction, free movement; 1 point: the forelimb on the contralateral side of ischemia could not be fully extended; 2 points: circling towards the contralateral side of ischemia; 3 points: falling towards the contralateral side of ischemia; 4 points: loss of consciousness, unable to walk.

[0114] Measurement of cerebral infarction volume:

[0115] Twenty-four hours after model construction, the brain was decapitated under anesthesia. The brain tissue was placed in a pre-cooled brain trough and then placed in a -80°C freezer for 3-5 minutes. Five slices were cut along the coronal plane and placed in 2% TTC staining solution. The slices were incubated at 37°C for 10-15 minutes, gently turned with forceps, and then incubated overnight with 4% paraformaldehyde. The images were photographed with a digital camera, and the data were analyzed and processed using ImageJ software.

[0116] (I) Effects of aucubin and TRPM2 inhibitor ACA on neurological deficit scores in mice with ischemic brain injury

[0117] Experimental results:

[0118] As shown in Table 4-2 and Figure 13 As shown, compared with the Sham+NS group, the neurological deficit scores of mice in the MCAO / R+NS group were significantly increased (P<0.001); compared with the MCAO / R+NS group, the neurological function scores of the MCAO / R+AU (15mg / kg) group and the MCAO / R+ACA (5mg / kg) group showed no significant changes; compared with the MCAO / R group, the neurological function scores of the MCAO / R+AU (15mg / kg)+ACA (5mg / kg) group were significantly decreased (P<0.01).

[0119] Table 4-2 Effects of aucubin and TRPM2 inhibitor ACA on neurological function scores in mice with ischemic brain injury

[0120]

[0121] Note: Compared with the Sham+NS group. ### P<0.001; compared with the MCAO / R+NS group, ** P<0.01

[0122] (II) Effects of aucubin and TRPM2 inhibitor ACA on cerebral infarction volume in mice with ischemic brain injury

[0123] Experimental results:

[0124] As shown in Table 5 and Figure 14As shown, compared with the Sham+NS group, the cerebral infarction volume of mice in the MCAO / R+NS group was significantly increased (P<0.001); compared with the MCAO / R group, the cerebral infarction volume of the MCAO / R+AU (15mg / kg) group and the cerebral infarction volume of the MCAO / R+ACA (5mg / kg) group were not significantly different; compared with the MCAO / R group, the cerebral infarction volume of the MCAO / R+AU (15mg / kg)+ACA (5mg / kg) group was significantly reduced (P<0.05).

[0125] Table 5. Effects of aucubin and TRPM2 inhibitor ACA on cerebral infarction volume in mice with ischemic brain injury.

[0126]

[0127] Note: Compared with the Sham+NS group. ### P<0.001; compared with the MCAO / R+NS group, *** P<0.001

[0128] (III) Effects of aucubin and TRPM2 agonist H2O2 on neurological deficit scores in mice with ischemic brain injury

[0129] Experimental results:

[0130] As shown in Table 6 and Figure 15 As shown, compared with the Sham+NS group, the neurological function score of mice in the MCAO / R group was significantly increased (P<0.001); compared with the MCAO / R+NS group, the neurological function score of mice in the MCAO / R+AU (80mg / kg) group was decreased (P<0.05); compared with the MCAO / R+AU (80mg / kg) group, the neurological function score of mice in the MCAO / R+AU (80mg / kg)+H2O2 (80mg / kg) group was increased (P<0.05).

[0131] Table 6. Effects of aucubin and TRPM2 agonist H2O2 on neurological function scores in mice with ischemic brain injury.

[0132]

[0133] Note: Compared with the Sham group ### P<0.001; compared with the MCAO / R group * P<0.05, compared with the MCAO / R+AU group, + P<0.05

[0134] (iv) Effects of aucubin and TRPM2 agonist H2O2 on cerebral infarction volume in mice with ischemic brain injury

[0135] Experimental results:

[0136] As shown in Table 7 and Figure 16 As shown, compared with the Sham+NS group, the cerebral infarction volume in the MCAO / R+NS group was significantly increased (P<0.001); compared with the MCAO / R+NS group, the cerebral infarction volume in the MCAO / R+AU (80 mg / kg) group was significantly decreased (P<0.001); compared with the MCAO / R+AU (80 mg / kg) group, the cerebral infarction volume in the MCAO / R+AU (80 mg / kg)+H2O2 (80 mg / kg) group was significantly increased (P<0.05).

[0137] Table 7. Effects of aucubin and TRPM2 agonist H2O2 on cerebral infarction volume in mice with ischemic brain injury.

[0138]

[0139] Note: Compared with the Sham+NS group. ### P<0.001; compared with the MCAO / R+NS group, *** P<0.01; compared with MCAO / R+AU, + P<0.01

[0140] Example 4: Exploring the neuroprotective mechanism of aucubin on ischemic brain injury in mice at the cellular level. This included grouping of cells for the cell experiment and establishing an oxygen-glucose deprivation-reperfusion (OGD / R) injury model.

[0141] Cells were divided into blank group, control group, OGD / R group, and OGD / R+AU (0.01, 0.1, 1, 10 μM) group, and the operation was as follows:

[0142] (1) Digest the cells and adjust the cell density to 10. 5 Inoculate at 1 / mL according to the grouping requirements and transfer to a 37℃ CO2 incubator for further culture;

[0143] (2) After 24 hours, the culture medium for each group of cells that required oxygen and sugar deprivation treatment was replaced with sugar-free culture medium;

[0144] (3) Place the cells in a preheated 37°C incubator and introduce 95% N2 + 5% CO2 gas for about 10 minutes to check for leaks. Then start timing to allow the cells to be hypoxic for 4.5 hours. After the cells are hypoxic for the timed period, replace the OGD / R group, blank group and control group with complete culture medium at the same time. The OGD / R + AU (0.01, 0.1, 1, 10 μM) groups are given the corresponding drug-containing culture medium. Then place them in the incubator and continue to culture for 24 hours before harvesting.

[0145] Cell passage:

[0146] (1) Place the purchased cells in a CO2 incubator (37℃, 5% CO2) and incubate for 3 hours. When the cell density is about 85%, digest and passage them.

[0147] (2) Prepare complete culture medium, PBS solution, double antibiotics and trypsin in advance and preheat them in a 37°C water bath.

[0148] (3) Remove the culture flask, aspirate the old culture medium, add 3ml PBS to wash the cells, discard the PBS solution, and repeat the operation twice;

[0149] (4) Add 1.5 ml of trypsin to digest until all cells are detached, and immediately add 3 ml of complete culture medium to stop the digestion;

[0150] (5) Transfer the digested culture medium to a 5ml centrifuge tube and centrifuge at 1000g for 5min. Discard the supernatant and add 5ml of complete culture medium. Mix well.

[0151] (6) Take 6 60mm culture dishes, add the cell suspension evenly to each dish, add 3ml of complete culture medium to each dish, and gently pipette to mix.

[0152] (7) Place in an incubator with 5% CO2 and 37°C for incubation.

[0153] (I) Effect of aucubin on the survival rate of HT22 cells after oxygen-glucose deprivation-reperfusion injury

[0154] Experimental methods:

[0155] (1) After cell passage, 100 μL of PBS was added around the perimeter of a 96-well plate, and the cell suspension concentration was adjusted to 1 × 10⁵ with complete culture medium for plating. Except for the blank group and the control group, all groups were subjected to oxygen-glucose deprivation treatment, followed by reoxygenation and reglucose treatment. At the same time, AU was added to the drug treatment group, while the model group, blank group and control group were added with the same volume of complete culture medium and cultured for 24 hours.

[0156] (2) Discard the old culture medium, add freshly prepared CCK-8 solution (CCK-8: culture medium = 1:9) to each well, incubate in a 5% CO2 incubator at 37°C for 1.5 hours, and measure the absorbance (OD value) at 450 nm.

[0157] (3) Calculation formula: Cell viability % = (OD value of experimental wells - OD value of blank wells) / (OD value of control wells - OD value of blank wells);

[0158] Experimental results:

[0159] As shown in Table 8 and Figure 17The experimental results showed that the survival rate of the OGD / R group was significantly lower than that of the Control group (P<0.001); compared with the OGD / R group, the cell survival rate of the AU (0.01μM, 0.1μM, 1μM and 10μM) groups was significantly higher (P<0.001).

[0160] Table 8. Effects of aucubin on HT22 cell survival after oxygen-glucose deprivation-reperfusion injury.

[0161]

[0162]

[0163] Note: Compared to the Control group, ### P<0.001; compared with the OGD / R group, *** P<0.01

[0164] (II) Intracellular Ca2+ in HT22 cells after aucubin-induced glucose deprivation-reperfusion injury 2+ Impact

[0165] Experimental methods:

[0166] (1) After cell passage, take a 12-well plate and adjust the cell suspension concentration to 1×10⁻⁶ with complete culture medium. 2 Add 1 ml of cell suspension to each well and incubate in a 5% CO2 incubator at 37°C for 24 hours.

[0167] (2) Except for the blank group and the control group, all groups were subjected to oxygen and sugar deprivation treatment, followed by reoxygenation and resupply treatment. At the same time, AU was added to the drug treatment group, while the model group and the normal group were added to the same volume of complete culture medium and cultured for 24 hours.

[0168] (3) Discard the old culture medium and wash three times with PBS;

[0169] (4) Add Fluo-4 staining solution (to completely cover the sample), and incubate in a 5% CO2 incubator at 37°C for 40 min;

[0170] (5) Discard the Fluo-4 staining solution, wash three times with PBS, and observe under a microscope.

[0171] Experimental results:

[0172] As shown in Table 9 and Figure 18A , 18B The experimental results show that, compared with the Control group, the Ca2+ level in the OGD / R group was lower. 2+ Fluorescence intensity was significantly upregulated (P<0.001); compared with the OGD / R group, the Ca2+ level in cells of the AU (1 μM) group was significantly increased. 2+Fluorescence intensity was significantly downregulated (P<0.001).

[0173] Table 9. Effects of aucubin on intracellular Ca2+ in HT22 cells after paraglycemic deprivation-reperfusion injury. 2+ Impact

[0174]

[0175]

[0176] Note: Compared to the Control group, ### P<0.001; compared with the OGD / R group, *** P<0.01

[0177] (III) Effects of aucubin on apoptosis in HT22 cells after oxygen-glucose deprivation-reperfusion injury

[0178] Experimental methods:

[0179] (1) After cell passage, take a 12-well plate and adjust the cell suspension concentration to 1×10⁻⁶ with complete culture medium. 2 Add 1 ml of cell suspension to each well and incubate in a 5% CO2 incubator at 37°C for 24 hours.

[0180] (2) Except for the blank group and the control group, all groups were subjected to oxygen and sugar deprivation treatment, followed by reoxygenation and resupply treatment. At the same time, AU was added to the drug treatment group, while the model group and the normal group were added to the same volume of complete culture medium and cultured for 24 hours.

[0181] (3) Discard the old culture medium and wash three times with PBS;

[0182] (4) Fix the cells with 4% paraformaldehyde for 30 minutes and wash them three times with PBS;

[0183] (5) Permeabilize the cells with 0.3% Triton-X-100 PBS and incubate at room temperature for 5 min, then wash twice with PBS.

[0184] (6) Add 50 μL of TUNEL staining solution evenly to each sample and incubate at 37°C in the dark for 60 minutes (cover the test sample with an anti-evaporation membrane to prevent the TUNEL staining solution from evaporating). Then wash 3 times with PBS and resuspend in 500 μL of PBS.

[0185] (7) Observe and collect the slides under a fluorescence microscope, and then calculate the number of TUNEL positive cells in each group using ImageJ software.

[0186] Experimental results:

[0187] As shown in Table 10 and Figure 19A , 19BThe experimental results showed that, compared with the Control group, the fluorescence intensity of the OGD / R group was significantly enhanced and the number of positive cells was significantly increased (P<0.001); compared with the OGD / R group, the fluorescence intensity of the cells in the AU (1μM) group was significantly downregulated and the number of positive cells was significantly reduced (P<0.001).

[0188] Table 10 Effects of aucubin on apoptosis in HT22 cells after oxygen-glucose deprivation-reperfusion injury.

[0189]

[0190] Note: Compared to the Control group, ### P<0.001; compared with the OGD / R group, *** P<0.001.

Claims

1. Use of aucubin and TRPM2 inhibitor in the preparation of a drug for treating ischemic brain injury; wherein the TRPM2 inhibitor is N-(p-pentylcinnamyl)-o-aminobenzoic acid; wherein the ischemic brain injury is acute cerebral ischemia-reperfusion injury; and wherein the weight ratio of aucubin to TRPM2 inhibitor in the drug is 3:

1.

2. The application according to claim 1, wherein the drug is an oral or injectable dosage form.

3. The application according to claim 2, wherein the drug is an injectable dosage form.

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