Mitochondrial transfer facilitator and its use in preparing drugs for preventing and treating mitochondrial dysfunction after CIRI

Abstract

The application relates to the technical field of medicines, and particularly discloses a mitochondrion transfer promoter and application of the mitochondrion transfer promoter in preparation of medicines for preventing and treating post-CIRI mitochondrion dysfunction, wherein the active component of the promoter is decocted liquid of blue cloth and pyrola calliantha, and the mass ratio of the blue cloth to the pyrola calliantha is 2-4:1-2. The mitochondrion transfer promoter is prepared by decocting and extracting the blue cloth and the pyrola calliantha, animal experiments are conducted, it is found that the mitochondrion transfer promoter can significantly improve CIRI induced by cerebral middle artery ischemia-reperfusion, reduce a neurological function defect score, reduce a cerebral infarction volume, and it is proved by HE and Nissl staining that the mitochondrion transfer promoter can effectively improve neuron morphology and reduce neuron death. The mitochondrion transfer promoter can improve mitochondrion function, reduce oxidative stress, and enhance blood-brain barrier endothelial function, and has important clinical significance and practical value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a mitochondrial transfer promoter and its application in the preparation of drugs for preventing and treating mitochondrial dysfunction after CIRI. Background Technology

[0002] Currently, the main clinical treatments for ischemic stroke (IS) include intravenous thrombolysis, mechanical thrombectomy, and improvement of collateral circulation to achieve vascular recanalization. However, after thrombolysis or interventional therapy, the blood reperfusion process can cause secondary pathological damage, namely cerebral ischemia-reperfusion injury (CIRI), which further aggravates nerve cell damage and hinders rehabilitation from ischemic stroke. This remains a pressing and unresolved clinical challenge.

[0003] Mitochondria, as the core organelles of eukaryotic cells, play vital physiological roles in supplying energy, metabolizing reactive oxygen species, storing calcium, and initiating programmed cell death. During intracellular intracellular resorption (CIRI), multiple factors, including oxidative stress, calcium overload, inflammatory responses, and excitatory amino acid toxicity, contribute to mitochondrial homeostasis imbalance. This imbalance is a key pathological link leading to subsequent neuronal pan-apoptosis (i.e., multiple forms of programmed cell death). Therefore, drug intervention and mechanistic studies targeting mitochondrial homeostasis, especially its quality control system (including kinetics and autophagy), are of significant theoretical and practical importance for mitigating secondary damage from CIRI and promoting neuronal function recovery. Notably, the transfer of healthy mitochondria between cells, as an emerging biological phenomenon, reveals a higher-level mitochondrial quality control mechanism in multicellular organisms that transcends the cellular level. This mechanism can directly replenish functional mitochondria and rapidly improve energy metabolism and redox balance in damaged neurons. Therefore, promoting mitochondrial transfer between nerve cells holds promise as a promising new therapeutic strategy to bypass complex intracellular damage pathways and directly salvage neuronal mitochondrial function. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a mitochondrial transfer promoter and its application in the preparation of drugs for preventing and treating mitochondrial dysfunction after CIRI. The promoter provided is precisely formulated to regulate mitochondrial quality control after reperfusion injury and is used to prevent and treat mitochondrial dysfunction after CIRI, which has significant clinical significance and practical value.

[0005] The technical solution of the present invention is a mitochondrial transfer promoter, wherein the active ingredients of the promoter are water extracts of *Lysimachia christinae* and *Pyrola rotundifolia*, wherein the mass ratio of *Lysimachia christinae* to *Pyrola rotundifolia* is 2~4:1~2.

[0006] Furthermore, the mass ratio of blue cloth to deer antler grass in the raw materials is 2:1.

[0007] Furthermore, in the preparation of the promoter, Blue Cloth and Deer Antler Grass are mixed in a certain ratio, and 8 to 10 times the total mass of the raw materials are added with water. The mixture is heated to boiling and then simmered over low heat until 4 to 5 times the volume of water is reduced. The mixture is then filtered, and the filtrate is the mitochondrial transfer promoter.

[0008] Furthermore, the blue cloth should be soaked for 20-60 minutes before decocting with the deer antler grass.

[0009] Furthermore, the promoter is made from the water extracts of *Ipomoea aquatica* and *Pyrola rotundifolia*, and its dosage form is either solid or liquid.

[0010] Furthermore, the solid dosage form is a tablet, capsule, granule, powder, or pill.

[0011] Furthermore, the liquid dosage form is a solution, suspension, emulsion, injection, or drop.

[0012] This invention also relates to the application of the aforementioned mitochondrial transfer promoter in the preparation of drugs for the prevention and treatment of cerebral ischemia-reperfusion injury.

[0013] Furthermore, this cerebral ischemia-reperfusion injury involves mitochondrial dysfunction.

[0014] This invention also relates to the application of the aforementioned mitochondrial transfer promoter in the preparation of drugs that activate the CD38 / Miro1-mediated astrocyte-neuronal mitochondrial transfer pathway and inhibit neuronal pan-apoptosis at multiple targets.

[0015] The present invention has the following beneficial effects: CIRI, as a major secondary pathological process following revascularization, falls under the category of "stroke" in Traditional Chinese Medicine (TCM) theory. It shares similarities with symptoms described in classical medical texts such as "hemiplegia" and "collapse." Its pathogenesis exhibits characteristics of deficiency in the root and excess in the branch, a mixture of deficiency and excess, often stemming from congenital insufficiency, aging, liver and kidney deficiency, yang hyperactivity transforming into wind, or internal injury due to overwork leading to qi and blood deficiency, poor blood circulation, resulting in organ dysfunction, qi and blood reversal, wind combined with phlegm and blood stasis, disturbing the brain orifices and invading the meridians. Modern TCM clinical practice shows that methods of invigorating qi and blood circulation, dispelling wind and unblocking the meridians, and tonifying the liver and kidneys have unique advantages in improving neurological deficits in ischemic stroke. From the perspective of their properties, flavors, meridian tropism, and synergistic effects, the combination of *Lysimachia christinae* and *Pyrola rotundifolia* is beneficial. *Lysimachia christinae* is cool in nature and pungent in taste, entering the liver, spleen, and lung meridians. It has the effects of invigorating qi and blood, nourishing blood and yin, and promoting qi and blood circulation. *Pyrola rotundifolia* is warm in nature and sweet and bitter in taste, entering the liver and kidney meridians. It can nourish the liver and kidneys, strengthen tendons and bones, and dispel wind and dampness. When used together, the combination of cold and warm properties harmonizes the liver, spleen, and kidney meridians, forming a combination characterized by "invigorating qi without promoting fire and invigorating blood without harming the body's vital energy." This combination can invigorate qi and blood, calm the liver and extinguish wind, and protect cerebrovascular health, thus preventing and treating CIRI (Cerebral Inflammation of Blood Regrowth).

[0016] This invention utilizes *Pyrrosia lingua* and *Pyrrosia lingua* to extract a mitochondrial transfer promoter. Animal experiments revealed that it significantly improves ischemia-reperfusion injury (CIRI) induced by middle cerebral artery ischemia-reperfusion, reduces neurological deficit scores, and decreases infarct volume. HE and Nissl staining further confirmed its effectiveness in improving neuronal morphology and reducing neuronal death. This mitochondrial transfer promoter improves mitochondrial function, reduces oxidative stress, and enhances blood-brain barrier endothelial function. Attached Figure Description

[0017] Figure 1 The effects of different doses of GJ-PD (GJ-PD) on neurological deficit scores in CIRI model C57 / BL6 mice were investigated: Sham: normal group, Model: model group, GJ-PD-1: GJ-PD (3.4 g / kg) group, GJ-PD-2: GJ-PD (6.8 g / kg) group, and GJ-PD-3: GJ-PD (13.6 g / kg) group.

[0018] Figure 2-3 The effect of different doses of GJ-PD on the infarct volume of CIRI model C57 / BL6 mice: Sham: normal group, Model: model group, GJ-PD-1: GJ-PD (3.4 g / kg) group, GJ-PD-2: GJ-PD (6.8 g / kg) group, GJ-PD-3: GJ-PD (13.6 g / kg) group.

[0019] Figure 4-7 The effects of different doses of GJ-PD on the motor function of CIRI model C57 / BL6 mice were investigated: Sham: normal group, Model: model group, GJ-PD-1: GJ-PD (3.4 g / kg) group, GJ-PD-2: GJ-PD (6.8 g / kg) group, and GJ-PD-3: GJ-PD (13.6 g / kg) group.

[0020] Figure 8 The effects of different doses of GJ-PD on the morphology of neurons in the ischemic cortex of CIRI model C57 / BL6 mice were investigated: Sham: normal group, Model: model group, GJ-PD-1: GJ-PD (3.4 g / kg) group, GJ-PD-2: GJ-PD (6.8 g / kg) group, and GJ-PD-3: GJ-PD (13.6 g / kg) group.

[0021] Figure 9-10The effect of different doses of GJ-PD on the number of neurons in the ischemic cortex of CIRI model C57 / BL6 mice was investigated: Sham: normal group, Model: model group, GJ-PD-1: GJ-PD (3.4 g / kg) group, GJ-PD-2: GJ-PD (6.8 g / kg) group, GJ-PD-3: GJ-PD (13.6 g / kg) group.

[0022] Figure 11 The effects of different doses of GJ-PD on β-catenin in the ischemic cortical region of CIRI model C57 / BL6 mice were investigated: Sham: normal group, Model: model group, GJ-PD-1: GJ-PD (3.4 g / kg) group, GJ-PD-2: GJ-PD (6.8 g / kg) group, and GJ-PD-3: GJ-PD (13.6 g / kg) group.

[0023] Figure 12 The effects of different doses of GJ-PD on the Claudin-5 cortex of the ischemic side in CIRI model C57 / BL6 mice were investigated: Sham: normal group, Model: model group, GJ-PD-1: GJ-PD (3.4 g / kg) group, GJ-PD-2: GJ-PD (6.8 g / kg) group, GJ-PD-3: GJ-PD (13.6 g / kg) group.

[0024] Figure 13 The effect of different doses of GJ-PD on mitochondrial ATP content in the ischemic cortical region of neurons in the CIRI model C57 / BL6 mice was investigated: Sham: normal A group, Model: model group, GJ-PD-1: GJ-PD (3.4 g / kg) group, GJ-PD-2: GJ-PD (6.8 g / kg) group, GJ-PD-3: GJ-PD (13.6 g / kg) group.

[0025] Figure 14 The effect of different doses of GJ-PD on the content of mitochondrial Complex I in the ischemic cortical region of neurons in the CIRI model C57 / BL6 mice was investigated: Sham: normal group, Model: model group, GJ-PD-1: GJ-PD (3.4 g / kg) group, GJ-PD-2: GJ-PD (6.8 g / kg) group, GJ-PD-3: GJ-PD (13.6 g / kg) group.

[0026] Figure 15The effect of different doses of GJ-PD on the mitochondrial ROS content of neurons in the ischemic cortex of CIRI model C57 / BL6 mice was investigated: Sham: normal group, Model: model group, GJ-PD-1: GJ-PD (3.4 g / kg) group, GJ-PD-2: GJ-PD (6.8 g / kg) group, GJ-PD-3: GJ-PD (13.6 g / kg) group.

[0027] Figure 16 The effect of different doses of GJ-PD on the mitochondrial LDH content of neurons in the ischemic cortex of CIRI model C57 / BL6 mice was investigated: Sham: normal group, Model: model group, GJ-PD-1: GJ-PD (3.4 g / kg) group, GJ-PD-2: GJ-PD (6.8 g / kg) group, GJ-PD-3: GJ-PD (13.6 g / kg) group.

[0028] Figure 17 The effects of different doses of GJ-PD on the ultrastructure and function of mitochondria in the ischemic cortex of C57 / BL6 mice with CIRI were investigated: Sham: normal group, Model: model group, GJ-PD-1: GJ-PD (3.4 g / kg) group, GJ-PD-2: GJ-PD (6.8 g / kg) group, and GJ-PD-3: GJ-PD (13.6 g / kg) group.

[0029] Figure 18-20 The effect of different doses of GJ-PD on the expression of mitochondrial transfer-related proteins (CD38, Miro1) in the ischemic cortex of CIRI model C57 / BL6 mice was investigated: Sham: normal group, Model: model group, GJ-PD-1: GJ-PD (3.4 g / kg) group, GJ-PD-2: GJ-PD (6.8 g / kg) group, GJ-PD-3: GJ-PD (13.6 g / kg) group.

[0030] Figure 21-29 The effects of different doses of GJ-PD on the expression of panapoptosis-related proteins (Bax, Bcl-2, Caspase 3, Caspase 1, GSDMD-N, MLKL, RIP3, ZBP1) in the ischemic cortex of CIRI model C57 / BL6 mice were investigated. The groups were: Sham (normal group), Model (model group), GJ-PD-1 (3.4 g / kg), GJ-PD-2 (6.8 g / kg), and GJ-PD-3 (13.6 g / kg).

[0031] Figure 30The effect of different concentrations of GJ-PD-containing plasma on the proliferation rate of co-cultured cells is shown on the vertical axis, which represents cell proliferation capacity, expressed as a percentage.

[0032] Figure 31 The effect of GJ-PD-containing plasma concentration on the proliferation rate of co-cultured cells is shown on the vertical axis, which represents cell proliferation capacity, expressed as a percentage.

[0033] Figure 32 The effect of GJ-PD-containing plasma on the ATP content of co-cultured cells is shown on the vertical axis, with the ATP content of cells expressed in μmol / g prot.

[0034] Figures 33-34 The effect of GJ-PD-containing plasma on the mitochondrial membrane potential of co-cultured cells.

[0035] Figure 35 To observe the effect of GJ-PD-containing plasma on the formation of intercellular tunnel nanotubes in co-cultured cells using scanning electron microscopy.

[0036] Figures 36-37 To observe the effect of GJ-PD-containing plasma on mitochondrial transfer between co-cultured cells using laser confocal microscopy.

[0037] Figures 38-40 The effect of GJ-PD-containing plasma on mitochondrial transfer-related proteins (CD38, Miro1) in co-cultured cells.

[0038] Figures 41-49 The effects of GJ-PD-containing plasma on panapoptosis-related proteins (Bcl-2, Caspase 3, Caspase 1, GSDMD, MLKL, RIP3, ZBP1, Bax) in co-cultured neurons. Detailed Implementation

[0039] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all raw materials and reagents used are commercially available.

[0040] Geum aleppicum: The dried whole herb of Geum aleppicum Jacq. or Geum japonicum Thunb. var. chinense Bolle of the Rosaceae family. It is cool in nature, sweet and bitter in taste, mainly enters the liver, spleen, lung and other meridians, and has various effects such as replenishing qi and strengthening the spleen, nourishing blood and yin, moistening the lung and resolving phlegm, promoting blood circulation and detumescence, regulating qi, and harmonizing blood. It has a good effect in treating diseases such as headache, dizziness, infantile convulsion, hypertension, lumbocrural pain and irregular menstruation. Modern pharmacological research has confirmed that the active ingredients contained in Geum aleppicum, such as carotene, tannin, aromatic bitter substances and volatile oil, have comprehensive pharmacological effects of antihypertensive, anticoagulant, anti-inflammatory, anti-tumor and anti-atherosclerosis, can effectively relieve hypertension symptoms, and show application potential in the treatment of cardiovascular and cerebrovascular diseases such as cerebral ischemia and myocardial ischemia.

[0041] Pyrola calliantha: The dried whole herb of Pyrola calliantha of the Pyrolaceae family, mostly growing beside valley streams or in shady and humid places under forests. This medicine is warm in nature, sweet and bitter in taste, and belongs to the liver and kidney meridians. It has the effects of dispelling wind-dampness, strengthening tendons and bones, promoting blood circulation and regulating menstruation, stopping bleeding, and relieving cough, and is commonly used to treat rheumatic arthralgia, weakness of the waist and knees, menorrhagia, hematemesis and epistaxis, chronic cough and fatigue, metrorrhagia and leukorrhagia, and traumatic bleeding. Modern pharmacological research has further confirmed that Pyrola calliantha has multiple biological activities such as anti-inflammatory, antibacterial, dilating cardiovascular and cerebrovascular, enhancing myocardial contractility and lowering blood pressure. It is mostly used in the treatment of diseases such as hypertension, coronary heart disease, chronic dysentery and cervical spondylosis in clinical practice, and also shows advantages in anti-tumor and anti-aging.

[0042] I. Preparation of mitochondrial transfer promoter A mitochondrial transfer promoter, the active ingredient of this promoter is the decoction of Geum aleppicum and Pyrola calliantha, and the mass ratio of Geum aleppicum to Pyrola calliantha is 2 - 4:1 - 2; in this embodiment, the mass ratio of Geum aleppicum to Pyrola calliantha is preferably 2:1 for preparing the mitochondrial transfer promoter.

[0043] Specifically in the preparation, mix Geum aleppicum and Pyrola calliantha according to the ratio, add 10 times the total mass of the raw materials of water, heat to boiling and then decoct with slow fire, decoct until the water volume is reduced to 5 times, and then filter. The filtrate is the mitochondrial transfer promoter.

[0044] In a more preferred case, it can be soaked for 20 - 60 minutes before decocting Geum aleppicum and Pyrola calliantha to ensure the extraction effect.

[0045] It should be noted that the mitochondrial transfer promoters used in the following animal experiments were the decoctions of *Lysimachia christinae* and *Pyrola rotundifolia*. However, in specific applications, the invention is not limited to the decoctions of *Lysimachia christinae* and *Pyrola rotundifolia*. Solid or liquid dosage forms of the aqueous extracts of *Lysimachia christinae* and *Pyrola rotundifolia* are all within the scope of protection of this invention. The solid dosage forms include, but are not limited to, tablets, capsules, granules, powders, or pills. Liquid dosage forms include, but are not limited to, solutions, suspensions, emulsions, injections, or drops.

[0046] II. Animal Experiments 1. Animal husbandry This study used 100 male C57BL / 6 mice as a model. All experimental animals met the international SPF grade standard (weight range 15-25 g) and were provided by Hunan Silek Jingda Experimental Animal Co., Ltd. (Experimental Animal Production License: SCXK (Xiang) 2019-0004; Quality Certificate No.: 430727241100651115, 430727241100746363, 430727241100983286). The management of experimental animals strictly followed national standards and was carried out in a standardized manner within the SPF-grade barrier system of Hunan University of Traditional Chinese Medicine: environmental parameters were kept constant at (22 ± 3)℃ temperature, (60 ± 5)% humidity, and 12 h diurnal rhythm. All individuals were allowed free access to food and water. This research protocol was reviewed by the Animal Experiment Ethics Committee of Hunan University of Traditional Chinese Medicine (ethics number: HNUCM21-2310-10), and the entire experiment followed the requirements of the "Regulations on the Management of Laboratory Animals" (the 3rd revised edition of the State Council Decree) and the "Guidelines for the Ethical Review of Laboratory Animal Welfare".

[0047] 2. Cell Culture Mouse hippocampal neurons (HT22) and mouse astrocytes (C8-D1A) purchased from Pronosei Biotechnology Co., Ltd. were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin and streptomycin at 37 ℃ in an incubator containing 5% CO2. The medium was changed every 2 days, and cells with stable growth and in the logarithmic growth phase were used for experiments.

[0048] 3. Experimental Grouping Animal groups: (1) Sham: normal group; (2) Model: MACO / R model group; (3) GJ-PD-1: GJ-PD (3.4 g / kg) group; (4) GJ-PD-2: GJ-PD (6.8 g / kg) group; (5) GJ-PD-3: GJ-PD (13.6 g / kg) group. (The mitochondrial transfer promoters used in GJ-PD-1, GJ-PD-2, and GJ-PD-3 were prepared using the method described in Example 1.) Cell group 1: (1) Control: co-culture group of normal HT22 and C8-D1A cells; (2) HT22: HT22 culture alone group; (3) OGD / R: OGD / R group after co-culture of HT22 and C8-D1A cells; (4) GJ-PD: OGD / R modeling after co-culture of HT22 and C8-D1A cells + GJ-PD drug-containing plasma administration group; (5) Cytochalasin D: OGD / R modeling after co-culture of HT22 and C8-D1A cells + Cytochalasin D (tunnel nanotube actin synthesis inhibitor) group.

[0049] Cell group 2: (1) Control: normal HT22 and C8-D1A cell co-culture group; (2) OGD / R: HT22 and C8-D1A cell co-culture OGD / R group; (3) GJ-PD: HT22 and C8-D1A cell co-culture OGD / R model + GJ-PD drug-containing plasma administration group; (4) Cytochalasin D: HT22 and C8-D1A cell co-culture OGD / R model + Cytochalasin D group.

[0050] 4. Establishment of MCAO / R model Sixty minutes after the last administration, mice were weighed, anesthetized with an intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg), and fixed in a supine position. The cervical surgical area was prepared and disinfected, and a 0.1 cm incision was made along the right side of the midline of the neck. The myofascia was microscopically dissected to expose the right common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA), avoiding vagus nerve injury. The proximal end of the CCA and the ECA were ligated, and the distal end of the ICA was clamped. A V-shaped stoma was made 3 mm proximal to the bifurcation, and a 0.18 mm diameter nylon suture was inserted retrogradely into the ICA, stopping upon encountering significant resistance (approximately 8 mm deep). The suture was fixed, the incision was sutured, and disinfected. Postoperatively, the mice were kept warm, and after 60 minutes of ischemia, the suture was withdrawn 5-8 mm before reperfusion. The mNSS score and tissue samples were taken 24 hours later. In the sham surgery group, only the blood vessels were exposed, without inserting a suture.

[0051] 5. Establishment of cell model The control group cells continued to be cultured in complete medium and placed in a 37 ℃, 5% CO2 incubator. For the other groups, OGD / R modeling was performed: the complete medium in the cell culture flasks was discarded, the cells were washed twice with 2 mL of 1% PBS, and then glucose-free DMEM basal medium (free of plasma and antibiotics) was added. The cells were then subjected to oxygen and glucose deprivation for 2 h in a triple-gas cell culture incubator at 37 ℃, 94% N2, 1% O2, and 5% CO2. After deprivation, the glucose-free DMEM medium was discarded, and complete medium containing drug-treated plasma was added. The cells were then returned to a conventional 37 ℃, 5% CO2 incubator for 24 h of further growth. The control group received 4 mL of complete medium containing blank plasma and was returned to a 37 ℃, 5% CO2 incubator for further culture.

[0052] 6. Experimental Procedure: (1) TTC staining: 24 h after reperfusion, 3 experimental mice were randomly selected from each group. They were deeply anesthetized by intraperitoneal injection of 1% sodium pentobarbital, followed by decapitation and brain removal. After brain removal, five brain slices of uniform thickness were prepared along the coronal plane. The brain tissue slices were immersed in 2% TTC staining solution prepared with PBS buffer and incubated at 37 ℃ in the dark for 30 min. After staining, the TTC staining solution was carefully aspirated, and an appropriate amount of 4% paraformaldehyde solution was added to completely immerse the brain slices. After fixation for 24 h, the brain slices were removed, arranged neatly in front-to-back order, and photographed to record the experimental results. White represents the infarct area, and red represents viable tissue. Finally, ImageJ image analysis software was used for infarct volume analysis.

[0053] (2) Open Field Experiment: An open field experiment was conducted after the animal intervention. The open field experiment required a quiet and dark environment. The ground in the system was evenly divided into 16 squares in a 4×4 pattern. The four middle squares were named the central area, and the rest were named the peripheral area. The animal's movement and exploration abilities were assessed by measuring the total movement distance, the movement distance in the central area, and the time spent in the central area. Before the experiment, the enclosure was disinfected and deodorized with alcohol. During the experiment, the animal's movement trajectory in the open field was recorded within 5 minutes. Each animal was tested only once, for 5 minutes. After each round of testing, the remaining excrement in the enclosure was cleaned, and the inner walls and bottom were wiped with 75% alcohol to ensure a clean experimental environment before testing the next animal.

[0054] (3) Transmission electron microscopy: Take 1 mm 3Immediately after immersion, cortical tissue samples were fixed in electron microscopy fixative at room temperature for 30 min, then transferred to 4 °C for 3-5 days. The samples were rinsed three times with 0.1 M PBS solution for 15 min each time. Next, they were fixed with 2% osmium tetroxide at room temperature for 1 h, and the EP (emulator) was wrapped in aluminum foil to protect from light. After osmium tetroxide fixation, the samples were rinsed three more times for 15 min each time. The tissues were then dehydrated sequentially in 50%, 70%, 90%, and 100% acetone for 20 min each time, followed by two applications of 100% acetone for 30 min each time. The samples were then placed in a pre-prepared soaking solution at room temperature overnight, followed by resin embedding and polymerization the next day. The embedded blocks were first placed in a 37 °C oven for 48 h, then transferred to a 60 °C oven for 24 h. After removing the embedded blocks, they were trimmed, sectioned into 50-70 nm ultrathin sections, and then mounted on a copper mesh and dried at 37 °C. After centrifugation in the dark, the uranium acetate was stained and washed three times with double-distilled water for 15 minutes each time. Then, it was placed in a sieve and dried overnight at 37°C. The ultrastructural changes of nerve cells in the cortical tissue of the ischemic-reperfused side of the brain were observed under a transmission electron microscope, and images were acquired.

[0055] (4) HE staining: Tissue fixation and preservation: The tissue was fixed in 4% paraformaldehyde at 4℃ for 72h; Dehydration treatment: Routine graded ethanol (70%, 80%, 90%, 95%, 100%) dehydration treatment; Embedding and sectioning: The dehydrated tissue was embedded in paraffin and cut into sections with a thickness of 5μm; Dewaxing and rehydration: The sections were treated with xylene I and II for 20 min each, and then immersed in anhydrous ethanol I and II for 5 min each. Then treated with 75% alcohol for 5 min, and finally rinsed with distilled water; Hematoxylin staining: After drying the sections, hematoxylin was stained for 3 min, washed with water, differentiated with hydrochloric acid ethanol differentiation solution, washed with water again and dried; Eosin staining: The sections were quickly stained with eosin staining solution for 1 s, washed with water and immediately dehydrated with anhydrous ethanol for 2 s; Mounting and observation: After drying the sections, they were mounted with neutral resin, observed under an optical microscope and images were acquired.

[0056] (5) Nissl staining: Section pretreatment: Immerse the sections in tar purple staining solution and stain in a constant temperature oven at 56 ℃ for 1 h; Differentiation and dehydration: After rinsing with pure water, differentiate in Nissl differentiation solution for 10 min. After observing under a microscope until the background is clear, immediately dehydrate with anhydrous ethanol; Mounting observation: After clearing with xylene, mount with neutral resin, dry, and count the number of surviving neurons in the ischemic cortex under an optical microscope.

[0057] (6) Detection of β-catenin, Claudin-5, and ROS content: Follow the instructions of the corresponding ELISA kits for β-catenin, Claudin-5, and ROS. Take the strips from the mouse β-catenin ELISA kit, add 50 μL of standard to the standard wells, and add 10 μL of sample and 40 μL of diluent to the sample wells. Except for the blank wells, add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each well and incubate at 37 ℃ for 60 min. After washing the plate 5 times, add 50 μL each of substrate A and B to each well and incubate at 37 ℃ in the dark for 15 min. Add 50 μL of stop solution and measure the OD value at 450 nm within 15 minutes.

[0058] (7) ATP content detection: Follow the instructions of the ATP detection kit (A095-1-1). Take blank tubes, standard tubes, test tubes, and control tubes, and add the corresponding amounts of 1 mmol / L standard solution, sample, reagent 1 (100 μL), reagent 2 (200 μL), reagent 3, and double-distilled water respectively. After mixing, incubate in a water bath at 37 ℃ for 30 min. Add 50 μL of reagent 4 and mix well. Centrifuge at 4000 rpm for 5 min and take 300 μL of supernatant. Add 500 μL of reagent 5, mix well, and let stand at room temperature for 2 min. Then add 500 μL of reagent 6, mix well, and let stand at 37 ℃ for 5-10 min. Zero the instrument with double-distilled water and measure the absorbance of each tube with a spectrophotometer at 636 nm and a light path of 0.5 cm. The cuvettes need to be cleaned multiple times before colorimetry to prevent phosphorus contamination.

[0059] (8) Complex I activity assay: Follow the instructions of the Mitochondrial Respiratory Chain Complex I / NADH-CoQ Reductase Activity Assay Kit (BC0510). Centrifuge the tissue supernatant at 11000g for 15 min at 4 ℃. Add 200 μL of Extraction Buffer I and 200 μL of Extraction Buffer II to the precipitate and sonicate (200W, 5 seconds / time, 10-second interval, repeated 15 times) for subsequent assays. Then perform enzyme activity assay: Preheat the UV spectrophotometer for more than 30 min, adjust to 340 nm, and zero with distilled water; preheat Reagent I at 37 ℃ for 15 min; add 50 μL of sample, 770 μL of Reagent I, 100 μL of working solution, and 80 μL of Reagent IV to a 1 mL quartz cuvette, mix well, and measure the absorbance value A1 at 340 nm for 10 s. After reacting at 37 ℃ for 1 min, measure the absorbance value A2 at 1 min 10 s. Calculate ΔA = A1 - A2.

[0060] (9) LDH content detection: According to the instructions of the lactate dehydrogenase (LDH) assay kit, accurately weigh the tissue, add 9 times the volume of physiological saline, mechanically homogenize under ice-water bath conditions, centrifuge at 4000 rpm for 10 min, and measure the protein concentration of the supernatant. Then take the supernatant and take the blank, standard, assay, and control wells, add the corresponding amount of double-distilled water, 0.2 μmol / mL sodium pyruvate standard solution, the sample to be tested, and reagent one, respectively. Add reagent two to the assay wells. After shaking and mixing, incubate at 37 ℃ for 15 min, add reagent three and incubate for another 15 min, add reagent four and mix well, and incubate at room temperature for 5 min. Measure the absorbance at 440 nm using an ELISA reader.

[0061] (10) Western blot detection of CD38, Miro1, Bax, Bcl-2, Caspase 3, Caspase 1, GSDMD-N, MLKL, RIP3, ZBP1 protein expression: After the mice in each experimental group were modeled and the samples were collected, the quantitative protein samples were denatured at 95 °C for 10 min and then electrophoresed and transferred to a membrane. Then, the samples were incubated with primary antibody at 4 °C overnight and secondary antibody at room temperature for 1 h. Finally, the samples were chemiluminescently developed using ECL substrate.

[0062] (11) CCK-8 assay for cell proliferation: Cells were seeded at a density of 5 × 10³ cells per well in 96-well plates and cultured routinely to the logarithmic growth phase. Each group was cultured in normal high-glucose medium followed by OGD / R treatment: the medium was replaced with sugar-free medium, and the cells were cultured for 2 hours in a tri-gas incubator containing 94% N2, 1% O2, and 5% CO2. The culture medium was then replaced with the corresponding treatment medium for 24 hours of normal culture. CCK-8 reagent was added to each well, and the cells were incubated in the dark for 4 hours. The OD value was measured at 450 nm using a microplate reader, and cell viability was calculated.

[0063] (12) Aspirate the culture medium from the culture plate and wash the cells twice with pre-cooled PBS buffer to remove residual serum and metabolites. Add 100 μL of lysis buffer to each well and incubate at room temperature in the dark for 10-15 min to allow the cells to lyse fully. Gently pipette the mixture to mix, and transfer the lysis buffer to a 1.5 mL centrifuge tube. Centrifuge at 12000 rpm / min for 5 min at 4 °C. Prepare the detection working solution according to the instructions, mixing the ATP detection buffer and luciferase in the correct ratio and storing in the dark. Add 90 μL of the detection working solution to each well of a 96-well plate, followed by 10 μL of cell lysis supernatant, and gently mix. Incubate at room temperature in the dark for 10 min to ensure the reaction proceeds fully. Use a fluorescence microplate reader to detect the fluorescence intensity of each well and record the data.

[0064] (13) JC-1 staining to detect mitochondrial membrane potential: Adjust the staining system according to cell density, using 0.5 mL of JC-1 staining solution per 500,000-1,000,000 cells. Quantitatively transfer 200× JC-1 stock solution, dilute at a volume ratio of 1:160, and vortex. Add 2 mL of 5× staining buffer to the above dilution, mix to obtain a final concentration of 1× working solution, and prepare fresh before use. Resuspend cells in 0.5 mL of complete culture medium; add 0.5 mL of JC-1 staining working solution, and invert several times to mix. Incubate at 37 ℃ for 20 min; during incubation, prepare an appropriate amount of JC-1 staining buffer (1×) by adding 4 mL of distilled water to 1 mL of JC-1 staining buffer (5×) and place it in an ice bath; after incubation at 37 ℃, centrifuge at 600g at 4 ℃ for 5 min to precipitate the cells; wash the cells twice with JC-1 staining buffer (1×); resuspend the cells in JC-1 staining buffer (1×) and then analyze them.

[0065] (14) Scanning electron microscopy observation of tunnel nanotube formation: After the cells adhering to the six-well plate were modeled and treated with drugs according to the experimental procedure, the complete culture medium was discarded, and the cells were rinsed twice with 2 mL of 0.1M PBS buffer. After discarding the PBS, 2 mL of electron microscopy fixative was added to each well and the cells were fixed at room temperature for 30 min. The pre-fixed samples were washed three times with a gradient of 0.1M PBS (pH 7.4) for 15 min each time. The samples were fixed with 1% osmium tetroxide and 0.1M phosphate buffer PB composite fixative at room temperature for 1.5 h. The samples were washed three times with PBS buffer for 15 min each time. The samples were dehydrated with a gradient of ethanol for 15 min each time. The samples were then permeated with isoamyl acetate for 15 min. The samples were loaded into a critical point desiccator and dried with supercritical liquid CO2. The samples were adhered to a conductive carbon substrate and Au-Pd alloy coating was performed by ion sputtering (30 s). The samples were then observed by scanning electron microscopy.

[0066] (15) C8-D1A cells and HT22 cells were stained with 200 nM MitoTracker Red CMXRos and 20 μM Celltracker Blue for 30 min, respectively. HT22 cells were washed twice after staining. The two cell types were co-cultured in a confocal dish at a ratio of 3:1 (total cell count approximately 10). 5 Once the cell adhesion density reached 80%, OGD / R modeling was performed. After 24 hours of reglycogenolysis and reoxygenation, the culture medium was discarded, the cells were washed with PBS, fixed with 4% paraformaldehyde for 10 minutes, and washed again. The cells were permeabilized with 0.5% Triton-X-100 for 5 minutes, washed, and then incubated in 50 μM FITC-phalloidin working solution containing 1% BSA for 30 minutes in the dark. Finally, the cell nuclei were counterstained with 100 nM DAPI for 30 seconds, washed, and mounted with an anti-fluorescence quencher.

[0067] (16) The WB bands were analyzed using ImageJ software for grayscale value analysis. The data results were compared with the corresponding internal parameters, and then normalized (i.e., the ratio of the target band in each group was compared with the mean ratio of the control group). All data were expressed as mean ± standard deviation. x (±s), the statistical method was one-way ANOVA with multiple groups. P <0.05 indicates that the difference is statistically significant.

[0068] Experimental results: in, Figure 1 To illustrate the effect of this invention on the neurological function scores of CIRI model C57 / BL6 mice: After pretreatment of CIRI mice with different doses of GJ-PD, the neurological function scores of the treated mice decreased with increasing dosage (**). P <0.01, * P <0.05). The results indicate that GJ-PD can effectively alleviate neurological function impairment caused by cerebral ischemia-reperfusion injury.

[0069] Figure 2-3 To investigate the effect of this invention on cerebral infarction volume in CIRI model C57 / BL6 mice: In the Sham group, no ischemic lesions were observed in brain slices, and the infarction volume percentage remained at zero; the Model group showed a significantly increased infarction volume compared to the Sham group (**). P <0.01). CIRI mice pretreated with GJ-PD showed a dose-dependent protective effect, with the GJ-PD-3 group showing the most significant effect (**). P <0.01). The results showed that GJ-PD could reduce the expansion of infarcts after cerebral ischemia-reperfusion, and its effect was positively correlated with the drug dosage.

[0070] Figure 4-7 The effect of this invention on the locomotor ability of CIRI model C57 / BL6 mice: Compared with the Sham group, the total locomotor distance of the Model group mice was significantly reduced (** P <0.01, the distance traveled in the central region is significantly reduced (** P <0.01), and the time spent exploring the central area was also significantly shortened (** P <0.01); Compared with the Model group, the total distance traveled by CIRI mice in the GJ-PD pre-drug group was significantly shorter throughout the observation area (*). P <0.05), the time and distance spent exploring the central region were significantly increased in CIRI mice in both the GJ-PD-2 and GJ-PD-3 groups (*). P<0.05). The results indicate that GJ-PD can improve the movement and autonomous spatial exploration ability of CIRI mice.

[0071] Figure 8 This invention investigated the effects of the present invention on the morphology of neurons in the ischemic cortex of CIRI model C57 / BL6 mice: In the Sham group, cortical neurons maintained normal morphological characteristics, with intact morphology, centrally located nuclei, and uniform cytoplasmic staining, without any pathological structural changes. In the Model group, the ischemic cortex showed extensive and severe neuronal damage, with disordered neuronal arrangement, increased intercellular spaces, intracellular cavitation, swelling and rupture, and pyknosis and deep staining of cell nuclei. The number and morphology of neurons in the GJ-PD pretreatment group were more normal than in the Model group. Cell vacuolation and nuclear pyknosis gradually decreased with increasing drug dosage, and the improvement effect was more significant in the GJ-PD-3 group.

[0072] Figure 9-10 To investigate the effect of this invention on the number of neurons in the ischemic cortex of CIRI model C57 / BL6 mice: In the Sham group of CIRI mice, Nissl bodies were evenly distributed in the neurons of the cortex, and the neurons were arranged in an orderly manner with large and regular nuclei. The number of neurons in the ischemic cortex of the Model group was significantly reduced compared to the Sham group (** P <0.01), widened intercellular spaces, and nuclear degeneration and condensation with vacuolar changes; compared with the Model group, GJ-PD pretreatment significantly improved the neuronal reduction induced by CIRI, and the number of cortical neurons on the affected side of mice in the GJ-PD pretreatment group was significantly increased (* P <0.05). The results showed that GJ-PD effectively reduced neuronal death in the cortical region of CIRI mice.

[0073] Figure 11 and Figure 12 The effects of this invention on the levels of β-catenin and Claudin-5 in the ischemic cortex of CIRI model C57 / BL6 mice: Compared with the Sham group, the levels of β-catenin and Claudin-5 in the Model group were significantly lower than those in the Sham group (** P <0.01), GJ-PD pretreatment significantly increased the levels of β-catenin and Claudin-5 in the ischemic brain tissue of CIRI mice (both * P <0.05). The results showed that the GJ-PD pre-treatment group could significantly reduce BBB damage, and the GJ-PD-3 group had a better effect.

[0074] Figure 13 and Figure 14The effects of this invention on ATP and Complex I content in the ischemic cortical region of CIRI model C57 / BL6 mice: The ATP content and Complex I activity in the Model group were significantly lower than those in the Sham group (** P <0.01,** P <0.01); Compared with the Model group, the GJ-PD pretreatment groups all showed a significant increase in ATP content in the ischemic tissue (*). P <0.05) and enhance Complex I activity (* P The difference was <0.05, and there were statistically significant differences among the three groups. The results indicate that the GJ-PD pretreatment group can significantly improve mitochondrial functional impairment, and the GJ-PD-3 group has a better effect.

[0075] Figure 15 and Figure 16 The effects of this invention on ROS and LDH content in the ischemic cortical region of CIRI model C57 / BL6 mice: Compared with the Sham group, the ROS and LDH content in the Model group tissues were significantly increased (** P <0.01,** P <0.01); Compared with the Model group, GJ-PD pretreatment significantly reduced ROS and LDH content (** P <0.01;** P The values ​​were <0.05), and there were statistically significant differences among the GJ-PD-1, GJ-PD-2, and GJ-PD-3 groups. The results indicate that the GJ-PD pretreatment group significantly reduced mitochondrial oxidative stress damage and neuronal injury.

[0076] Figure 17 This invention investigated the effects of the present invention on the ultrastructure and function of mitochondria in the ischemic cortical region of neurons in the CIRI model C57 / BL6 mice: In the Sham group, the number of organelles was moderate, and the mitochondrial cristae were generally normal; in the Model group, the number of mitochondria was reduced, mitochondria were swollen, and cristae were reduced or even disappeared, or arranged disorderedly. In the GJ-PD pretreatment group, the morphology of mitochondria in nerve cells gradually returned to normal, the number of mitochondria gradually increased, and the degree of mitochondrial cristae breakage gradually improved, with the GJ-PD-3 group showing more significant improvement.

[0077] Figure 18-20 The effects of this invention on the expression of CD38 and Miro1 proteins in the ischemic cortex of CIRI model C57 / BL6 mice: Compared with the Sham group, the expression of CD38 and Miro1 was increased in the Model group (*). P <0.05), the GJ-PD-2 and GJ-PD-3 pretreatment groups significantly increased CD38 (* P <0.05), GJ-PD pretreatment groups significantly increased Miro1 expression (*). P<0.05). The results showed that pre-drug administration of GJ-PD increased the expression of mitochondrial transfer-related proteins and promoted mitochondrial transfer to rescue CIRI.

[0078] Figure 21-29 The effect of this invention on the expression of panapoptotic-related proteins in the ischemic cortex of CIRI model C57 / BL6 mice: Compared with the Sham group, the expression of Bcl-2 in the Model group was significantly reduced ( P <0.01), the protein expression of Bax, Cleaved casepase-3, Casepase-1, GSDMD-N, RIP3, MLKL, and ZBP1 was significantly increased. P <0.05); among them, the expression of Bcl-2 was significantly increased in the GJ-PD-2 and GJ-PD-3 pretreatment groups (* P <0.05), the GJ-PD pretreatment group significantly reduced the expression of Casepase-1, GSDMD-N, RIP3, and MLKL (*). P <0.05), GJ-PD-2 and GJ-PD-3 pretreatment significantly reduced the protein expression of Bax, ZBP1, and Cleaved casepase-3 (*). P <0.05), and the GJ-PD-3 group showed the most significant effect. The results showed that GJ-PD pretreatment could significantly reduce neuronal pan-apoptosis in CIRI mice.

[0079] Figure 30 Effects of different concentrations of GJ-PD-containing plasma on the proliferation rate of co-cultured cells: Compared with the Control group, the cell proliferation capacity of the OGD / R group was significantly reduced (** P <0.01); Among the nine different concentrations of drug-containing plasma treatment groups, the group treated with 9 g / kg-10% drug-containing plasma showed the most significant increase in cell proliferation (**). P <0.01). Therefore, subsequent experiments used drug-containing plasma with a concentration of 9 g / kg-10%.

[0080] Figure 31 Effect of GJ-PD-containing plasma concentration on the proliferation rate of co-cultured cells: Compared with the co-cultured OGD / R group, the proliferation of HT22 cells cultured alone was significantly reduced after OGD / R modeling (**). P <0.01), the cell viability of the OGD / R co-cultured GJ-PD group was significantly increased ( P** <0.01).

[0081] Figure 32 The effect of this invention on the ATP content of co-cultured cells: Compared with the Control group, the ATP content of cells in the OGD / R model group was significantly decreased (**P <0.01); Compared with the co-cultured OGD / R group, the ATP content of HT22 cells cultured alone decreased significantly after OGD / R modeling (**). P <0.01), ATP content in the OGD / R co-culture GJ-PD treatment group was significantly restored (** P <0.01).

[0082] Figures 33-34 The effect of this invention on the mitochondrial membrane potential of co-cultured cells: Compared with the Control group, the mitochondrial membrane permeability of cells in the OGD / R group was significantly increased (** P <0.01 indicates a significant decrease in mitochondrial membrane potential, and compared with the OGD / R group, the proportion of JC-1 monomers in the GJ-PD group cells was significantly reduced (**). P <0.01 indicates a significant increase in mitochondrial membrane potential.

[0083] Figure 35 To observe the effect of this invention on the formation of intercellular tunnel nanotubes in co-cultured cells using scanning electron microscopy: Compared with the Control group, the overall structure of neurons in the OGD / R group was slightly worse, with irregularly elliptical cell bodies and a significant increase in microvilli on the cell surface. Astrocytes showed cell membrane damage, and TNTs with a diameter of approximately 80 nm were visible between cells, with many broken. Compared with the OGD / R group, the overall structure of neurons in the GJ-PD group was still acceptable, with abundant microvilli on the cell surface and intact cell membranes. Astrocytes showed slight cell membrane damage, forming pores of varying sizes, and TNT connections with a diameter of approximately 90 nm were visible between cells. These channels were long, numerous, and straight. Neuronal damage in the Cytochalasin D group was relatively obvious, with localized membrane rupture, forming pores of varying sizes, large-area expansion, and small cell protrusions. Astrocytes were flattened and elongated spindle-shaped, with TNT connections between cells and fewer channels.

[0084] Figures 36-37 To observe the effect of this invention on mitochondrial transfer between co-cultured cells using laser confocal microscopy: compared with the Control group, mitochondrial transfer was increased in the OGD / R group; compared with the OGD / R group, mitochondrial transfer from C8-D1A cells to HT22 neurons was significantly increased in the GJ-PD drug-treated group; mitochondrial transfer was decreased in the Cytochalasin D group; Figure 37In both the Control and OGD / R groups, a small number of red mitochondria were co-localized with F-actin. In the OGD / R group, F-actin-labeled intercellular junction TNTs were thickened, and a small number of labeled mitochondria were present within the channels. After administration of GJ-PD, an increase in the number of mitochondria transported from C8-D1A cells to HT22 neurons was observed, and the TNT channels were larger and more prominent. Co-localized mitochondria were significantly reduced in the Cytochalasin D group.

[0085] Figures 38-40 The effect of this invention on the expression of mitochondrial transfer-related proteins CD38 and Miro1 in co-cultured cells: Compared with the Control group, the expression of both CD38 and Miro1 proteins was increased in the OGD / R group (** P <0.01); compared with the OGD / R group, the GJ-PD group significantly increased the expression of CD38 and Miro1 (*). P <0.05).

[0086] Figures 41-49 The effect of this invention on the expression of pan-apoptotic proteins in co-cultured neurons: Compared with the OGD / R group, the GJ-PD group significantly increased the expression of Bcl-2 (*). P <0.05), GJ-PD significantly downregulated the expression of key proteins in different cell death pathways, including apoptosis, pyroptosis, and necrosis—Cleaved casepase-3, Casepase-1, GSDMD, RIP3, MLKL, ZBP1, and Bax (*). P <0.05), with the downregulation of key proteins regulating pyroptosis, Caspase-1 and GSDMD, being more significant (**). P <0.01).

[0087] Experimental conclusion: This invention reveals that the herbal pair *Gynostemma pentaphyllum* and *Hylocereus undatus* (GJ-PD) can significantly improve cerebral ischemia-reperfusion injury (CIRI) induced by middle cerebral artery ischemia-reperfusion, reduce neurological deficit scores, and decrease cerebral infarction volume. HE and Nissl staining confirmed its effective improvement of neuronal morphology and reduction of neuronal death. GJ-PD can improve mitochondrial function, alleviate oxidative stress, and enhance blood-brain barrier endothelial function. Further investigation revealed that its protective effect is closely related to the regulation of mitochondrial translocation. GJ-PD can upregulate the expression of CD38 and the mitochondrial adaptor protein Miro1 in astrocytes, mediating the translocation of mitochondria to damaged neurons via tunnel nanotubes (TNTs), thereby improving neuronal energy metabolism and inhibiting various cell death programs. Mitochondrial dysfunction and oxidative stress are key factors in triggering panapoptotic processes such as apoptosis, pyroptosis, and necroptosis. This invention demonstrates that GJ-PD, as a mitochondrial transfer promoter, enhances ATP synthesis and mitochondrial membrane potential in neurons on the one hand, and significantly upregulates the anti-apoptotic protein Bcl-2 on the other hand, while simultaneously downregulating the expression of pro-apoptotic proteins Bax and Caspase-3, the key pyroptosis executive protein GSDMD-N and its activating protein Caspase-1, as well as necroptosis-related proteins RIP3, MLKL, and ZBP1.

[0088] In summary, this invention demonstrates that the *Lysimachia christinae*-*Herba Epimedii* herbal pair, as a mitochondrial transfer promoter, exerts a neuroprotective effect by activating the CD38 / Miro1-mediated astrocyte-neuronal mitochondrial transfer pathway and inhibiting neuronal pan-apoptosis at multiple targets. This provides a novel mechanism of action and pharmacological strategy for the prevention and treatment of ischemic cerebrovascular diseases.

[0089] The above embodiments describe preferred embodiments of the present invention, but the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other way. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention. Therefore, the protection scope of this patent should be determined by the appended claims.

Claims

1. A mitochondrial transfer promoter, characterized in that, The active ingredient of this promoter is an aqueous extract of *Lysimachia christinae* and *Pyrola rotundifolia*, with a mass ratio of *Lysimachia christinae* to *Pyrola rotundifolia* of 2-4:1-2.

2. The mitochondrial transfer promoter according to claim 1, characterized in that: The mass ratio of blue cloth to deer antler grass in the raw materials is 2:

1.

3. The mitochondrial transfer promoter according to claim 1, characterized in that: When preparing the promoter, mix Blue Cloth and Deer Antler Grass in the specified ratio, add 8 to 10 times the total mass of the raw materials with water, heat to boiling, then simmer until 4 to 5 times the volume of water is reduced, then filter. The filtrate is the mitochondrial transfer promoter.

4. The mitochondrial transfer promoter according to claim 3, characterized in that: Soak the blue cloth and deer antler grass for 20-60 minutes before decocting.

5. The mitochondrial transfer promoter according to claim 3, characterized in that: The promoter is made from the water extracts of *Lysimachia christinae* and *Pyrola rotundifolia*, and its dosage form is either solid or liquid.

6. The mitochondrial transfer promoter according to claim 3, characterized in that: The solid dosage form is a tablet, capsule, granule, powder, or pill.

7. The mitochondrial transfer promoter according to claim 3, characterized in that: Liquid dosage forms include solutions, suspensions, emulsions, injections, or drops.

8. The use of the mitochondrial transfer promoter according to any one of claims 1 to 7 in the preparation of drugs for the prevention and treatment of cerebral ischemia-reperfusion injury.

9. The application according to claim 8, characterized in that: This brain ischemia-reperfusion injury involves mitochondrial dysfunction.

10. The use of the mitochondrial transfer promoter according to any one of claims 1 to 7 in the preparation of drugs that activate the CD38 / Miro1-mediated astrocyte-neuronal mitochondrial transfer pathway and inhibit neuronal pan-apoptosis at multiple targets.

Images