Application of GHRH-A in the preparation of drugs for preventing and treating ischemic cerebral infarction

Abstract

The present invention belongs to the field of biomedicine, and mainly relates to the application of growth hormone releasing hormone receptor agonist MR-409 in the preparation of drugs for the prevention and treatment of ischemic cerebral infarction. MR-409 is a new type of GHRH receptor agonist. Previous studies have shown that it can alleviate the pathological damage of refractory diseases such as diabetes and myocardial ischemia by increasing the survival of a variety of bone marrow stem cells in ischemic lesions. In the present invention, the study found that MR-409 can significantly improve ischemic brain damage, survival rate and neurobehavioral performance in cerebral ischemia model mice, and its mechanism may be through increasing the proliferation, migration and survival of endogenous neural stem cells. This study provides a new strategy for the treatment of cerebral ischemia and has important clinical therapeutic significance. Provide a scientific theoretical basis for the future development of drugs that promote neuronal regeneration as a target for the treatment of cerebral ischemia.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the use of a growth hormone releasing hormone receptor agonist (GHRH-A) in the preparation of a drug for preventing and treating ischemic cerebral infarction, and mainly relates to the use of an artificially synthesized growth hormone receptor agonist MR-409 in the prevention and treatment of ischemic cerebral infarction (tMCAO) to protect nerve cells and reduce the sequelae of cerebral infarction. Background Art

[0002] Ischemic cerebral infarction (also known as stroke) is one of the main causes of death and permanent disability worldwide. At present, the treatment of ischemic cerebral infarction is mainly thrombolytic therapy and neuroprotective therapy. Although thrombolytic therapy can achieve vascular recanalization, the best time for thrombolytic therapy is within 6 hours of cerebral infarction. Clinically, most patients have missed the best time for thrombolytic therapy after arriving at the hospital. There is often irreversible neuronal loss during cerebral ischemia. Even after active rescue, patients still have sequelae of varying degrees of severity, such as sensory, motor and cognitive dysfunction. Neuroprotective agents provide some protective effects on nerve cells in the ischemic penumbra by improving brain metabolism, but their therapeutic effect is very limited; therefore, there is still no effective treatment for ischemic cerebral infarction in clinical practice. Repairing brain neuron loss caused by ischemia by effectively promoting neuronal regeneration is considered to be the most promising treatment for treating ischemic cerebral infarction and reducing the sequelae of cerebral infarction. It is well known that among all cell lines, nerve cells have the lowest regenerative capacity. In the past, it was believed that nerve cells lacked regenerative capacity. Since the existence of endogenous neural stem cells in brain regions such as SGZ and SVZ was found to be rich in endogenous neural stem cells, it is currently believed that endogenous neural stem cells in brain regions such as SGZ and SVZ may regenerate new neurons under appropriate conditions to play a repairing role. However, so far, a feasible method has not been found to effectively stimulate nerve stems to differentiate into new cell neurons and promote the regeneration of new neurons to treat ischemic cerebral infarction. Therefore, finding a new therapy to promote neuronal regeneration to treat ischemic cerebral infarction and reduce the sequelae of cerebral infarction has important clinical significance for the treatment and prognosis of cerebral infarction.

[0003] MR-409 is a new type of growth hormone releasing hormone analogue (GHRH-A) recently developed by Andrew V. Schally's team. It is not yet on the market. Andrew V. Schally won the 1977 Nobel Prize in Physiology or Medicine for discovering hypothalamic growth hormone releasing hormone and other peptides and clarifying their physiological functions. Growth hormone and growth hormone releasing hormone have important physiological functions, one of which is to promote the anabolism of tissues and cells, especially the anabolism of proteins, promote cell growth and development, and inhibit cell apoptosis. Based on these physiological effects, Professor Andrew V. Schally believes that some tumors may be able to block tumor growth by blocking the GHRH signaling pathway of some cells, while some senile diseases such as cardiovascular disease and neurodegenerative diseases may promote cell regeneration and boost the function of these cells by stimulating the GHRH signaling pathway to achieve the purpose of treating these diseases. Natural GHRH is easily degraded by enzymes in the body, has unstable effects, a short half-life, and stimulates the secretion of pituitary growth hormone, which may promote tumor growth and other side effects, so it is not suitable for clinical use. Andrew V. Schally's team replaced many of the coded amino acids in the GHRH peptide chain with artificially synthesized non-coded amino acids, making it more difficult to be degraded by proteases, thus having a longer half-life in the body and higher receptor affinity; in addition, GHRH-A can competitively inhibit the binding of endogenous GHRH to the receptor, thereby avoiding many side effects of endogenous GHRH, such as the occurrence of tumors. Andrew V. Schally synthesized a series of GHRH receptor agonists. After experimental studies, there are currently 7 GHRH-A products that have shown therapeutic effects in different animal experimental disease models, including type 1 diabetes pancreatic islet cell repair, myocardial infarction, lower limb ischemia, vascular calcification and diabetic retinopathy.

[0004] At present, the treatment of ischemic cerebral infarction is still mainly based on thrombolytic therapy and neuroprotective therapy, but both have their own limitations, and patients often have sequelae such as language or limb dysfunction after the acute phase. Some patients suffer from permanent disability. The occurrence of sequelae is mainly related to the irreversible loss of neurons during the ischemic period of cerebral infarction. Therefore, it is urgent to find a new and effective therapeutic drug, especially one that can promote neuronal regeneration to replace neurons lost by cerebral ischemia to reduce the sequelae of cerebral infarction. GHRH-A has the characteristics of promoting cell proliferation and inhibiting cell apoptosis. Preliminary experimental results have confirmed that MR-409 can improve a variety of refractory diseases including myocardial ischemia and pathological damage of pancreatic islet cells in type 1 diabetes. Some of its mechanisms may be related to its promotion of bone marrow-derived stem cells to enter the damaged lesions and increase survival. However, due to the particularity of nerve cell regeneration, the nerve cell regeneration rate is low, and nerve cell regeneration comes from endogenous neural stem cells in the brain, not bone marrow stem cells. Therefore, whether MR-409 can play the same therapeutic effect in ischemic cerebral infarction as in myocardial infarction is unpredictable and needs to be solved through new and different research programs. Summary of the invention

[0005] The purpose of the present invention is to provide a composition containing MR-409 for the treatment and prevention of ischemic stroke, which provides a new treatment strategy for the treatment of cerebral ischemia, especially for reducing the sequelae after ischemic cerebral infarction, and has important clinical therapeutic significance. This study also provides an important scientific theoretical basis for the future development of drugs that promote neuron regeneration as a target for regulating ischemic cerebral infarction.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] On the one hand, the present invention provides the use of GHRH-A in the preparation of a drug for preventing and treating ischemic cerebral infarction, and the technical key point is that the GHRH-A is MR409.

[0008] Furthermore, the drug is mainly administered in the form of injection.

[0009] On the other hand, the present invention provides a composition containing GHRH-A for preventing and treating ischemic cerebral infarction, the technical key point of which is that the GHRH-A is MR409.

[0010] The amino acid sequence of MR409 is: NMeTyr-D-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Orn-Leu-Leu-Gln-Asp-Ile-Nle-Asp-Arg-NHCH.

[0011] The dose of active ingredient MR409 is 5 μg or 10 μg.

[0012] Furthermore, the composition is mainly administered in the form of injection.

[0013] The concentration of the active ingredient MR409 was 0.5 μM or 1 μM.

[0014] Beneficial effects of the present invention: The present invention discloses the use of growth hormone releasing hormone receptor agonist MR-409 in the preparation of drugs for preventing and treating ischemic stroke. Experiments have confirmed that: 1) MR-409 can significantly alleviate brain atrophy in transient middle cerebral artery occlusion (tMCAO) model mice for 4 weeks; 2) MR-409 can significantly increase the brain weight of tMCAO model mice; 3) MR-409 can significantly reverse the neuronal loss in the hippocampus of tMCAO model mice for 4 weeks; 4) MR-409 can significantly improve the neurobehavioral performance of tMCAO model mice. 5) MR-409 can significantly increase the GFAP in the SGZ and SVZ brain regions of tMCAO model mice for 2 weeks. + / Nestin + / MCM2 + 6) MR-409 can significantly increase the expression of Tbr2 in the SGZ brain region of tMCAO model mice 2 weeks later. + / MCM2 + 7) MR-409 can significantly increase the BrdU expression in the hippocampal SGZ of tMCAO model mice 2 weeks later. + / DCX + and MCM2 in the SGZ and SVZ of the hippocampus + / DCX + Cell number and DCX + 8) MR-409 can significantly increase the BrdU expression in the SGZ and SVZ regions of the hippocampus of tMCAO model mice for 4 weeks. + / NeuN +Cell number; 9) MR-409 can significantly increase the proliferation and survival of NSCs after oxygen glucose deprivation / re-oxygenation (OGD / R) and inhibit their apoptosis. Its mechanism may be related to its increased expression of p-AKT and p-ERK proteins after cerebral ischemia. The preferred dose of MR-409 is 5 ug and 10 ug. The preferred administration method is subcutaneous injection every day after modeling. The above experimental results show that MR-409 can significantly improve brain damage in mice with ischemic cerebral infarction, increase the survival rate and improve the neurobehavioral performance of mice with ischemic cerebral infarction, and reduce the incidence of sequelae of cerebral infarction after ischemia. Its mechanism may be related to increasing endogenous neuron generation after cerebral ischemia. The above new uses of MR-409 provide new strategies for the treatment of cerebral ischemia and have important clinical therapeutic significance. It provides a scientific theoretical basis for the future development of drugs that promote neuronal regeneration as a target for the treatment of cerebral ischemia.

[0015] Compared with endogenous GHRH, synthetic GHRH-A has the advantages of low side effects, long half-life and stable action. Among these GHRH-A products, MR-409 has a better effect in the treatment of vascular calcification and myocardial infarction. Although Dr. Schally's team has reported that MR-409 has achieved certain effects in the treatment of experimental vascular calcification and myocardial infarction animal models. Compared with nerve cells, myocardial cells have better regeneration ability. After myocardial infarction, angiogenesis can be promoted by stimulating myocardial cell regeneration and bone marrow stem cells to differentiate into endothelial progenitor cells, thereby achieving partial self-repair. Compared with cerebral infarction, myocardial infarction has fewer sequelae and a low disability rate after the acute critical period. MR-409 promotes the healing of the myocardium after infarction by accelerating the repair process of myocardial cells. Compared with myocardial cells, nerve cells have low regeneration ability, and bone marrow stem cells cannot differentiate into neural stem cells and neurons. The regeneration of nerve cells mainly depends on the migration and repair of endogenous neural stem cells in the brain to the lesion area. Conventional treatment of ischemic cerebral infarction often leaves sequelae of neurological and limb dysfunction due to the difficulty in repairing damaged neurons. Therefore, compared with the treatment of myocardial infarction, there is more uncertainty as to whether MR-409 can treat cerebral infarction. In addition, MR-409 needs to pass through the blood-brain barrier to reach a low drug concentration in the brain, and the dosing regimen of MR-409 for the treatment of cerebral infarction needs to be further determined. Therefore, our results show that MR-409 can treat ischemic cerebral infarction by stimulating the proliferation of endogenous neural stem cells to promote neuronal regeneration and repair, and reduce the sequelae of cerebral infarction. These results will open up a new treatment approach for cerebral infarction.

[0016] Treatment with GHRH receptor agonist MR-409 can effectively reduce the mortality rate and increase the survival rate of mice in the ischemic cerebral infarction model caused by middle cerebral artery occlusion (see Figure 3 ). Therefore, this treatment regimen has the potential to reduce the mortality rate of patients with cerebral infarction.

[0017] MR-409 can increase the brain weight of ischemic cerebral infarction model mice ( Figure 1-2 ), improving hippocampal cavitation in model mice ( Figure 4 ), reducing brain atrophy in mice with ischemic cerebral infarction ( Figure 1-3 ). Therefore, the implementation of this new treatment regimen may effectively protect brain tissue damage in patients with ischemic cerebral infarction.

[0018] MR-409 can improve the neurobehavior of model mice, including improving learning and memory function after ischemic cerebral infarction ( Figure 6 ) and limb motor function ( Figure 5 ). Therefore, the implementation of this new treatment plan may effectively reduce the sequelae of patients with ischemic cerebral infarction.

[0019] MR-409 can increase the proliferation of neural stem cells in model mice ( Figure 7-8 ), neural precursor cells ( Fig. 9 ), proliferation and migration of neuroblasts and ( Figure 10-12 ) and the survival of newborn neurons ( Figure 13-14 ).

[0020] MR-409 can increase the survival and proliferation of ischemic neural stem cells cultured in vitro ( Figure 15-16 ), the mechanism of which may be related to its increased expression of p-AKT and p-ERK proteins after cerebral ischemia ( Fig.17 ).

[0021] Our results show that MR-409 can treat ischemic cerebral infarction by stimulating the proliferation of endogenous neural stem cells and promoting neuronal regeneration and repair, thereby reducing the sequelae of cerebral infarction. These results will open up a new treatment avenue for cerebral infarction. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 MR-409 treatment (28 days) reduces brain atrophy in tMCAO (middle cerebral artery ligation)-induced ischemic stroke mice.

[0023] Figure 2 MR-409 treatment (28 days) increased brain weight in tMCAO mice with ischemic cerebral infarction.

[0024] Figure 3 MR-409 treatment (28 days) increased the survival rate of mice with tMCAO-induced ischemic cerebral infarction.

[0025] Figure 4 MR-409 treatment (28 days) reduced the hippocampal cavitation induced by tMCAO ischemic cerebral infarction.

[0026] Figure 5 MR-409 treatment (28 days) improved the sensorimotor function of tMCAO model mice at different time points.

[0027] Figure 5 In the figure, Figure A is the measurement of the neurological function score of mice; Figure B is the measurement of the number of ipsilateral turns of mice; Figure C is the measurement of the rotarod residence time of mice; and Figure D is the measurement of the forelimb pulling force of mice.

[0028] Figure 6 MR-409 improves the learning and memory functions of tMCAO model mice at different time points of ischemia.

[0029] Figure 6 In the figure, Figure A shows the swimming distance of mice in the water maze; Figure B shows the escape latency of mice; Figure C shows the time mice stay in the target quadrant; and Figure D shows the number of times mice cross the platform.

[0030] Figure 7 MR-409 treatment promotes the proliferation of neural stem cells in the SGZ region of the hippocampus of tMCAO mice.

[0031] Figure 7 In the figure, green fluorescence represents the neural stem cell marker GFAP, red fluorescence represents the neural stem cell marker Nestin, and gray fluorescence represents the endogenous proliferative cell marker MCM2.

[0032] Figure 8 MR-409 treatment promotes the proliferation of neural stem cells in the SVZ brain region of tMCAO mice.

[0033] Figure 8 In the figure, green fluorescence represents the neural stem cell marker GFAP, red fluorescence represents the neural stem cell marker Nestin, and gray fluorescence represents the endogenous proliferative cell marker MCM2.

[0034] Fig. 9 MR-409 treatment promotes the proliferation of neural precursor cells in the SGZ region of the hippocampus of tMCAO mice.

[0035] Fig. 9 In the figure, red fluorescence represents the neural progenitor cell marker Tbr2, and green fluorescence represents the endogenous proliferative cell marker MCM2.

[0036] Fig.10 MR-409 treatment promotes the transient proliferation and migration of neuroblasts in the SGZ region of the hippocampus of tMCAO mice.

[0037] Fig.10 In the figure, red fluorescence represents the neuroblast marker DCX, and green fluorescence represents the exogenous proliferative cell marker BrdU.

[0038] Fig.11 MR-409 treatment promotes the proliferation and migration of neuroblasts in the SGZ region of the hippocampus of tMCAO mice.

[0039] Fig.11 In the figure, red fluorescence represents the neuroblast cell marker DCX, and green fluorescence represents the endogenous proliferative cell marker MCM2.

[0040] Fig.12 MR-409 treatment promotes the proliferation and migration of neuroblasts in the SVZ brain region of tMCAO mice.

[0041] Fig.12 In the figure, red fluorescence represents the neuroblast cell marker DCX, and green fluorescence represents the endogenous proliferative cell marker MCM2.

[0042] Fig.13 MR-409 treatment increases neuronal survival in the SGZ region of the hippocampus of tMCAO mice.

[0043] Fig.13 In the figure, green fluorescence represents the exogenous proliferative cell marker BrdU, and blue fluorescence represents the mature neuron marker NeuN.

[0044] Fig.14 MR-409 increases the survival of newborn neurons in the brain area of ​​tMCAO mice.

[0045] Fig.14 In the figure, green fluorescence represents the exogenous proliferative cell marker BrdU, and blue fluorescence represents the mature neuron marker NeuN.

[0046] Fig.15 Effects of MR-409 on synaptic plasticity in tMCAO mice

[0047] Fig.15 In the figure, Figures A and B are schematic diagrams of synaptic structures in the CA1 region of the mouse hippocampus detected by electron microscopy; Figure C is a schematic diagram of Golgi-stained neurons; Figures D and E are schematic diagrams of Golgi-stained dendritic spines; Figures F and G are schematic diagrams of semi-quantitative results of dendritic spines.

[0048] Fig.16 MR-409 inhibits apoptosis of neural stem cells cultured in vitro under oxygen-glucose deprivation. Green fluorescence indicates TUNEL-labeled positive cells.

[0049] Fig.17MR-409 promotes the proliferation of oxygen-glucose-deprived neural stem cells cultured in vitro, and red fluorescence indicates BrdU-labeled positive cells.

[0050] Fig.18 MR-409 increases the survival rate of neural stem cells cultured in vitro under oxygen-glucose deprivation.

[0051] Fig.19 MR-409 increases the expression of AKT and ERK proteins in oxygen-glucose-deprived neural stem cells cultured in vitro.

[0052] Fig. 20 The expression of GHRH receptor in the mouse neurogenesis specific brain regions SVZ and SGZ and in vitro cultured neural stem cells.

[0053] Fig. 20 In the figure, Figure A is a schematic diagram of the expression of GHRH receptors in specific brain areas of mouse neurogenesis; Figure B is a schematic diagram of the expression of GHRH receptors in neural stem cells cultured in vitro. DETAILED DESCRIPTION

[0054] The following combination Figures 1 to 20 , the contents of the present invention are described in detail through specific embodiments.

[0055] The present invention relates to the use of growth hormone releasing hormone receptor agonist MR-409 in the preparation of drugs for preventing and treating ischemic stroke:

[0056] 1. Experimental materials:

[0057] C57BL / 6 mice, male, weighing 22-25g, animal qualification certificate number: purchased from the Experimental Animal Center of Shenyang Medical College. Adult SD rats, neural stem cells from the hippocampus. MR-409 was provided by the laboratory of Professor Andrew Schally (1977 Nobel Prize winner in Medicine) at the University of Miami (Miami, FL, USA), purity, prepared with 10% 1,2-propylene glycol to the required concentration for use.

[0058] 、Experimental methods:

[0059] 2.1 Experimental groups and drug administration

[0060] 1) C57BL / 6 mice weighing 22-25 g were used as experimental animals and were divided into sham operation group, model group, MR-409 low-dose group (5 μg each time) and MR-409 high-dose group (10 μg each time).

[0061] 2) Dosage: In the MR-409 low-dose group, each mouse was given a single dose of 5 μg of the test drug [MR-409 was dissolved in DMSO solution to prepare a stock solution with a concentration of 100 mg / ml. Before use, it was diluted with 10% polyethylene glycol to a working concentration of 0.1 mg / ml (the final concentration of DMSO was <1%)]; in the MR-409 high-dose group, each mouse was given a single dose of 10 μg of the test drug; the sham operation group and the model group were given an equal volume of normal saline.

[0062] 3) Administration: Subcutaneous injection.

[0063] 4) Administration time: The MR-409 group was administered immediately after cerebral ischemia modeling and then once a day at the same time. The treatment time was 14-18 days.

[0064] Model preparation

[0065] 1) Animal experiment: cerebral ischemia-reperfusion model

[0066] Before MCAO surgery, mice were fasted but not watered for 12 h, anesthetized with isoflurane (4% induction, 2% maintenance), fixed in supine position, and a 1 cm midline incision was made in the neck to expose the right carotid CCA, external carotid artery ECA, and internal carotid artery ICA. The proximal ends of the ECA and CCA were ligated with sutures, and the ICA was temporarily clamped with a micro artery clamp. The ECA and its branches were ligated and separated, the CCA was clamped with an artery clamp, a small incision was made at the beginning of the ECA stump, and the prepared thread plug was gently inserted through this incision until a slight resistance was felt and the thread plug was fixed with a prepared thread. The depth of thread plug insertion was about 8 mm from the bifurcation of the ECA. After the operation, the CCA artery clamp was opened, the skin was sutured, and the thread plug was left outside. After 1 hour of ischemia and reperfusion, the mice were anesthetized with isoflurane, the neck suture incision was opened, the thread plug was gradually pulled out until it completely left the ECA, the ECA stump was ligated, the neck incision was sutured again, and iodine was applied for disinfection. After surgery, the animals were irradiated with an electric heating lamp for 4 h to maintain their body temperature. They were fasted but not watered for 24 h and kept in single cages. In the sham operation group, the head of the suture was only advanced to 3 mm above the bifurcation in the direction of the ICA, and the rest of the surgical procedure was the same as above.

[0067] 2) In vitro experiment: Cellular oxygen glucose deprivation / reperfusion model

[0068] Neural stem cell culture

[0069] (1) Hippocampal isolation and neurosphere culture

[0070] 1) Take a SD rat that is within 24 hours of birth, soak it in 75% ethanol for 5 minutes, remove the brain tissue completely under sterile conditions, and quickly place it in a pre-cooled plate containing Dissection solution. Separate the hippocampus and remove the meninges and blood vessels. Use microtweezers to cut it into pieces of about 1 mm3, merge the bilateral hippocampal tissues and transfer them to a centrifuge tube containing pre-cooled Dissection solution, and mark them.

[0071] 2) Centrifuge at 250 × g for 5 min.

[0072] 3) Discard the supernatant, add 500 μL / tube Accutase and incubate in a 37°C incubator for 3 min to digest the tissue.

[0073] 4) Remove the supernatant Accutase, add 200 μL SFM, and pipette up and down 30 to 40 times at full volume. When pipetting, place the tip of the pipette against the bottom of the tube to obtain maximum shear force. The action should be decisive, and each time the aspiration should be full, while trying to avoid bubbles.

[0074] 5) After moderate perfusion, add SFM complete medium and filter through a 70 μm cell strainer.

[0075] 6) Centrifuge at 250×g for 3-5 minutes.

[0076] 7) Discard the supernatant, add 200 μL SFM complete medium, and resuspend the tissue by pipetting to separate the cells into single cells.

[0077] 8) Add SFM complete medium, mix the cell suspension thoroughly, and inoculate the suspension into a 6-well plate at 3-4 mL / well. Place the cell suspension of both hippocampi in one well.

[0078] 9) Gently shake the culture plate in a cross shape to ensure that the cells are evenly distributed. Observe the cell morphology under a microscope.

[0079] 10) Culture in a 5% CO2, 37°C incubator for 7-10 days. During this period, differentiated cells will die, while NSCs and some progenitor cells will begin to proliferate and form neurospheres.

[0080] (2) Neurosphere subculture

[0081] 1) Transfer the culture medium containing neurospheres to a 15 mL centrifuge tube (you can rinse the bottom of the well plate with complete culture medium to obtain as many cells as possible).

[0082] 2) Depending on the size and number of the neurospheres, centrifuge at 250 × g for 5 min.

[0083] 3) Carefully remove the supernatant, add 200 μL Accutase, resuspend the cell pellet, and incubate at 37°C for 3 min.

[0084] 4) Add 800 μL of complete medium to dilute and centrifuge at 500 × g for 5 min.

[0085] 5) Carefully remove the supernatant, add 200 μL SFM complete medium, and thoroughly pipette to form a single-cell suspension.

[0086] 6) Subculture at a ratio of 1:2 to 1:5 according to the cell density and inoculate into 6-well plates.

[0087] 7) Culture in a 5% CO2, 37℃ incubator. After 5-7 days, the new neurospheres can be passaged again.

[0088] (3) Adherent culture and induced differentiation of neural stem cells

[0089] 1) Cells that have been passaged for more than 3 generations can be cultured adherently. Pre-coat a 24-well slide with a mixed solution containing 100 μg / mL PDL / PLL and 5 μg / mL Laminin.

[0090] 2) The remaining steps are the same as 2.2.2 (2) for cell culture.

[0091] 3) After culturing for 3 to 5 days, the cells can be used for immunofluorescence identification or drug experiments.

[0092] 4) The steps of inducing differentiation are as follows: the complete medium is still used for the first 1-2 days of adherent culture, and the culture time can be extended depending on the cell density; then the SFM complete medium is replaced with 1% FBS and half the FGF, without EGF. For the second medium change, the above medium with all FGF removed is used. The medium is changed by half every three days. After 14 days of differentiation culture, it can be used for immunocytochemistry experiments or drug experiments.

[0093] Preparation of oxygen glucose deprivation / reoxygenation model

[0094] NSCs that have been passaged for more than 3 generations are cultured adherently on culture plates / dishes. After 3-5 days of culture, the culture medium is replaced with sugar-free DMEM and placed in a 37°C hypoxic chamber. A mixed gas of 95% N2 and 5% CO2 is introduced to create the OGD model. The cells are then transferred to normal oxygen concentration and continue to be cultured in SFM complete culture medium to simulate the cell ischemia-reperfusion injury model.

[0095] 2.3. Neurological function testing after cerebral ischemia

[0096] 1) Neurological function score

[0097] Modified Neurological Severity Score (mNss) was used to test the neurological function of animals at different time points after surgery.

[0098] 2) Rotarod test

[0099] One week before the test, the mice were trained at a speed of 4-20 rpm, and the maximum speed lasted for 60 s. Each mouse was trained three times a day, and the basal value was recorded on the 7th day. During the test, each mouse was tested three times, and the longest retention time was taken; when the mouse's continuous retention time exceeded 60 s, the test was terminated, and 60 s was taken as the final score. The retention time of the rotarod and the statistical analysis were performed.

[0100] 3) Tensile test

[0101] Place the mouse's two forelimbs on the pull rod of the dynamometer, keep the mouse's body level with the table, and slowly and evenly pull the mouse backward until the two forelimbs leave the pull rod of the dynamometer. Repeat the recording three times and take the maximum value for statistical calculation.

[0102] 4) Corner test

[0103] The corner in the experiment is composed of a 30-degree angled flat plate with a small gap at the junction of the two plates to attract mice into the corner. During the experiment, the mouse was placed between the two plates, facing the corner. When the mouse entered the corner, its whiskers on both sides would sense the presence of the obstacle at the same time, and then it would raise its forelimbs and turn to face the entry end. Each mouse repeated the test 10 times, with one minute interval between each time. The number of times the mouse deviated to the left or right was recorded.

[0104] 5) Morris water maze test

[0105] The Morris water maze system includes a black stainless steel circular pool (1.5 m in diameter, 45 cm in height, and water temperature of 20°C), a cylindrical black platform with a diameter of 10 cm, a camera system, and an Etho Vision XT 8.0 software analysis system. Four marking points were set at equal distances on the upper edge of the pool, namely the east, west, south, and north. The pool was divided into four equal quadrants by these four marking points and the projection points on the bottom of the pool. The cylindrical black platform was located 1.5 to 2 cm below the horizontal plane of the target quadrant. Titanium dioxide was used to dye the water in the pool white during the experiment. The experiment lasted for 5 days, including positioning and navigation experiments and space exploration experiments. The first 3 days were the training phase, and each mouse was trained 3 times a day, with an interval of 120 s between each training. In the experiment, mice were gently placed in the water from different quadrants facing the pool wall, and the time from entering the water to finding the platform was recorded within 120 seconds, i.e., the escape latency. When the rat found the platform, it was allowed to stay on the platform for 10 seconds; if the rat did not find the platform within 90 seconds, it could be induced to stay on the platform for 10 seconds, and the escape latency was recorded as 90 seconds. The fourth day was the positioning navigation experiment detection stage, and the escape latency of the rat was detected within 90 seconds. The fifth day was the spatial exploration experiment, and the platform was removed, and the swimming time of the rat in the quadrant where the platform was located and the number of times the rat crossed the platform position were recorded within 90 seconds.

[0106] 2.4. Immunofluorescence detection:

[0107] The brains of mice were removed after PFA cardiac perfusion, and then fixed with PFA and gradient precipitation for OTC frozen sections. The brain slices were washed with PBS washing buffer 3 times for 5 min each time, and the water marks on the slides were carefully wiped off. Lines were drawn around the brain slices with a histochemical pen, and the serum working solution was incubated on the brain slices for 1 h at 37°C. After that, the serum working solution was discarded, the liquid at the edge of the brain slices was carefully wiped off, and the primary antibody was incubated at 4°C overnight in an appropriate proportion. The next day, the incubated brain slices were washed with PBS washing buffer 3 times, the water marks on the slides were carefully wiped off, and the lines were filled around the brain slices with a histochemical pen. After incubating the fluorescent secondary antibody working solution at room temperature for 1 h, the incubated brain slices were washed with PBS washing buffer 3 times, and the water marks on the slides were carefully wiped off. The slides were sealed with a quenching sealing agent, and the fluorescence was observed under a fluorescence microscope.

[0108] 2.5 Protein immunoblotting detection:

[0109] (1) Extraction of total cell protein

[0110] The adherent cells were added with RIPA lysis buffer, ultrasonically disrupted on ice for 1 min, centrifuged at 4°C, 12,000×g for 15 min, and the supernatant was taken. The protein concentration was measured by the BCA method. The extracted supernatant was diluted with 5X loading buffer and heat denatured in a 95°C metal bath for 10 min.

[0111] (2) SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and staining

[0112] Prepare 12% separation gel and 5% stacking gel, respectively, and cast SDS-PAGE (6×8×0.1 cm). After installing the electrophoresis tank, add electrophoresis buffer and load the sample, 30 μg / well of total tissue protein and 5 μg / well of marker. Perform constant voltage electrophoresis, and run the sample at 60 V in the stacking gel. After bromophenol blue migrates to the separation gel interface, run the sample at 120 V in the separation gel until bromophenol blue migrates to the bottom of the gel, and turn off the power. Remove the gel and perform electrotransfer.

[0113] (3) Immunoblotting

[0114] 1) Activate the PVDF membrane in methanol for 15 seconds, then soak the membrane, wet transfer filter paper and sponge in transfer buffer for 15 minutes;

[0115] 2) After taking out the SDS polyacrylamide gel, fold the gel into a "sandwich" shape and insert it into the transfer cell, with one side of the gel facing the cathode, and transfer at a constant current of 260 mA at 4°C for the corresponding time;

[0116] 3) The transferred PVDF membrane was rinsed in TBST and blocked with 5% skimmed milk powder at room temperature for 1 hour;

[0117] 4) Dilute the primary antibody to an appropriate concentration with 5% skim milk powder and incubate overnight at 4°C;

[0118] 5) Rinse the membrane 4 times with TBST, 5 minutes each time;

[0119] 6) Dilute the secondary antibody to an appropriate concentration with 5% skim milk powder and incubate at room temperature for 1 hour;

[0120] 7) Rinse the membrane three times with TBST, 10 minutes each time;

[0121] 8) Add ECL reagent to the membrane protein surface in a dark room, react for 1 minute, place in an exposure box, cover with X-ray film, and expose for a certain period of time;

[0122] 9) Take out the film and place it in the developer. When the stripes appear, rinse it with tap water, place it in the fixer, and finally rinse it with tap water, dry it, and scan the image for storage.

[0123] 2.6 Golgi staining

[0124] After the mice were killed by cervical dislocation, the whole brain tissue was directly taken out and washed with PBS buffer in a cell culture dish. The brain tissue was then immersed in Golgi staining solution (Fdneurotech, PK401, A:B=1:1) and kept in the dark for 2-3 weeks at room temperature. Then, 100 μm thick coronal continuous sections of the hippocampus were made in solution C using an oscillating slicer, and the brain slices were attached to gelatin-coated anti-desorption slides using a brush and air-dried in the dark for 48 h. The slices were rinsed with ultrapure water, immersed in Golgi staining solution (D:E: ultrapure water=1:1:2), rinsed again with ultrapure water, dehydrated in 50%, 75%, 95% and 100% ethanol solutions (3 times / 5 min each), permeabilized with xylene, sealed with neutral gum, and sliced ​​using an optical microscope. Then, ImageJ software was used for image processing and analysis, and the dendrites of various parts of the hippocampal CA1 region were selected to analyze the neuronal development and count the dendritic spine density.

[0125] 2.7 Projection electron microscopy

[0126] Mice were anesthetized with 4% chloral hydrate (0.1 ml / 10 g) and then the chest was opened. The brain was quickly removed after perfusion with precooled saline and 2% PFA-2.5% glutaraldehyde phosphate buffer. The hippocampus was separated and cut into 1 mm3 tissue blocks and fixed in precooled 2.5% glutaraldehyde solution. Then, the tissue blocks were rinsed with phosphate buffer and fixed with 1% osmium acid solution for 80 min. After rinsing with ultrapure water, dehydration with gradient acetone, and epoxy resin infiltration embedding, tissue blocks were prepared. Ultrathin sections of 70 nm thickness were prepared using an ultrathin slicer and then scooped onto a copper grid. The tissue was stained with 3% uranyl acetate and lead citrate. The sections were finally rinsed with ultrapure water and sampled using a transmission electron microscope.

[0127] 3. Experimental results:

[0128] 1) Effects of MR-409 on brain atrophy, brain weight, survival rate and hippocampal morphology in tMCAO mice 28 days after tMCAO

[0129] C57BL / 6 mice were induced by tMCAO (1 hour of ischemia followed by 28 days of reperfusion). At the same time, MR-409 (5, 10 μg / day / mouse, sc) was administered on the day of modeling for 4 consecutive weeks. The brains of the animals in each group were removed by decapitation on the 28th day after modeling to observe brain atrophy. Figure 1-4 As shown in the figure, after tMCAO surgery, the right hemisphere showed obvious atrophy, brain weight decreased significantly, and hippocampal cavitation appeared. After low-dose and high-dose MR-409 was given, the atrophy of the right hemisphere caused by the model was significantly alleviated, brain weight was increased, the survival rate of model mice was improved, and the hippocampal cavitation phenomenon was improved. This suggests that MR-409 can significantly improve cerebral ischemic damage in tMCAO model mice. ## p<0.01, ### p <0.001 vs. sham group; * p <0.5, ** p <0.01 vs. Vehicle group. Scale bar is 200 μm.

[0130] 2) Effects of MR-409 on neurological function and learning and memory function in tMCAO mice

[0131] C57BL / 6 mice were induced by tMCAO (1 hour of ischemia followed by 28 days of reperfusion). At the same time, MR-409 (5, 10 μg / day / mouse, sc) was administered on the day of modeling for 4 consecutive weeks. Neurological function-related behavioral tests were performed on the 3rd, 7th, 14th, 21st, and 28th days after modeling. Figure 5-6 As shown, in the neurological function score experiment, MR-409 can significantly reduce the neurological function scores at different time points after tMCAO; in the corner turning experiment, MR-409 can significantly reduce the number of ischemic mice deflecting to the ischemic ipsilateral side; in the rotarod test experiment, MR-409 significantly increased the rotarod residence time of the model mice, and in the tension test experiment, MR-409 significantly increased the forelimb tension of the model mice; in the Morris water maze experiment, MR-409 significantly reduced the swimming distance of the model mice, significantly reduced the escape latency of the model mice, significantly increased the residence time of the model mice in the target quadrant, and significantly increased the number of times the model mice crossed the platform. The above results indicate that MR-409 can significantly improve the neurological function and learning and memory ability of tMCAO model mice. # p <0.05, ## p <0.01, ### p <0.001 vs. sham group; * p <0.05, ** p <0.01, *** p <0.001 vs. Vehicle group.

[0132] 3) Effect of MR-409 on the proliferation of neural stem cells in tMCAO model mice

[0133] C57BL / 6 mice were induced by tMCAO (1 hour of ischemia followed by 14 days of reperfusion). At the same time, MR-409 (5, 10 μg / day / mouse, sc) was administered for 14 consecutive days on the day of modeling. The brains were removed by decapitation on the 14th day after modeling, and the co-staining of GFAP, Nestin and MCM2 in the SGZ and SVZ regions of the hippocampus was observed using a confocal fluorescence microscope. Figure 7-8 As shown, green fluorescence is GFAP+ Cells, red fluorescence is Nestin + Cells, gray fluorescence is MCM + The low-dose and high-dose MR-409 groups could significantly increase the GFAP expression in the SGZ and SVZ regions of the hippocampus of the model mice. + / Nestin + / MCM2 + Cell number. This indicates that MR-409 can increase the proliferation of neural stem cells in tMCAO model mice. The scale bar is 40 μm.

[0134] 4) Effect of MR-409 on the proliferation of neuroblasts in tMCAO model mice

[0135] C57BL / 6 mice were induced by tMCAO (1 hour of ischemia followed by 14 days of reperfusion). At the same time, MR-409 (5, 10 μg / day / mouse, sc) was administered on the day of modeling for 14 consecutive days. The brains were removed by decapitation on the 14th day after modeling, and then co-staining of Tbr2 and MCM2 in the hippocampal SGZ brain region was observed using a confocal fluorescence microscope. The experimental results are shown in Fig. 9 As shown, red fluorescence is Tbr2-labeled neuroblasts, green fluorescence is endogenous proliferative cell marker MCM2, and blue fluorescence is nuclear dye DAPI. The low-dose and high-dose MR-409 groups can significantly increase the expression of Tbr2 in the SGZ brain region of the hippocampus of model mice. + / MCM2 + This indicates that MR-409 can increase the proliferation of neuroblasts in tMCAO model mice. The scale bar is 40 μm.

[0136] 5) Effects of MR-409 on the proliferation and migration of neuroblasts in tMCAO model mice

[0137] C57BL / 6 mice were tMCAO-induced (1 h of ischemia followed by 14 days of reperfusion). At the same time, MR-409 (5, 10 μg / day / mouse, sc) was given for 2 consecutive weeks on the day of modeling. The brains were removed by decapitation on the 14th day after modeling. BrdU (300 mg / kg) was injected intraperitoneally into the mice 2 hours before decapitation. Confocal fluorescence microscopy was then used to examine the co-staining of BrdU and DCX in the SGZ of the hippocampus and the co-staining of MCM2 and DCX in the SGZ and SVZ of the hippocampus. The experimental results are shown in Figure 2. Figure 10-12 As shown, the red fluorescent labeled cells are DCX + The green fluorescence of cells is respectively the exogenous and endogenous marker BrdU for proliferating cells. + and MCM2 +The blue fluorescence is the nuclear dye DAPI. + / DCX + and MCM2 + / DCX + The number of cells increased significantly, and DCX + Cells migrate to the molecular layer of the hippocampus; MCM2 in the SVZ + / DCX + The number of cells increased significantly, and DCX + Compared with the tMCAO group, low-dose and high-dose MR-409 administration could further increase the BrdU + / DCX + and MCM2 + / DCX + Cell number and further increase DCX + The number of cells that migrated to the molecular layer of the hippocampus and the striatum. This suggests that MR-409 can promote the proliferation and migration of neural stem cells in the SGZ region of the hippocampus of mice with tMCAO model for 14 days. The scale bar is 40 μm.

[0138] 6) Effects of MR-409 on synaptic plasticity in tMCAO model mice

[0139] C57BL / 6 mice were induced by tMCAO (1 h of ischemia followed by 28 days of reperfusion). At the same time, MR-409 (5, 10 μg / day / mouse, sc) was given on the day of modeling for 4 consecutive weeks. The brains were removed by decapitation on the 28th day after modeling. Brain tissues from the CA1 region of the hippocampus were taken for electron microscopy and Golgi staining. The experimental results are shown in Figure 2. Fig.15 As shown in the figure, Figure A and Figure B are the synaptic structures in the CA1 region of the hippocampus. The results show that in the sham operation group, the synaptic structure is intact, the postsynaptic dense band is darkly stained, and the synaptic cleft is clear. In the tMCAO group, the postsynaptic dense band widens, the synaptic cleft narrows, the synaptic vesicles disappear, and the synaptic curvature flattens. After the administration of MR-409, the synaptic plasticity changes caused by tMCAO can be significantly improved. Figure C is the experimental result of Golgi staining. As shown in the figure, the hippocampal structure is intact in the sham operation group, the neuronal morphology is normal, and the axon and dendritic branches are clear. In the tMCAO group, neurons in the CA1 and CA3 regions of the hippocampus are missing, and the dendritic branches are reduced. Figures D and E are the results of dendrite magnification. The pictures show that in the tMCAO group, the density of dendritic spines is significantly reduced, and the administration of MR-409 can significantly reverse the loss of dendritic spines caused by the model. Figures F and G are the semi-quantitative results of dendritic spines. ***p<0.01 vs. sham group; ##p<0.01, ###p<0.001 vs. tMCAO group

[0140] 7) Effect of MR-409 on neuronal survival in tMCAO model mice

[0141] C57BL / 6 mice were tMCAO-induced (1 h of ischemia followed by 28 days of reperfusion). At the same time, MR-409 (5, 10 μg / day / mouse, sc) was given on the day of modeling for 4 consecutive weeks. BrdU (50 mg / kg, ip) was given on the first day after modeling, twice a day for 5 consecutive days. The brain was removed by decapitation on the 28th day after modeling, and then BrdU and NeuN co-staining was performed in the SGZ and SVZ brain regions of the hippocampus ipsilateral to the ischemic side using a confocal fluorescence microscope. The experimental results are shown in Figure 2. Fig.13 , 14 As shown, cells labeled with green fluorescence are BrdU + Cells labeled with blue fluorescence are NeuN + 28 days after tMCAO, BrdU + / NeuN + The number of cells increased significantly, and the total NeuN + Compared with the tMCAO group, low-dose and high-dose MR-409 administration could further increase the number of BrdU + / NeuN + The cell number and total NeuN + The number of cells was 28 days after tMCAO. This indicates that MR-409 can promote the survival of newborn neurons in the SGZ and SVZ regions of the hippocampus. The scale bar is 40 μm.

[0142] 8) Effects of MR-409 on proliferation, survival and apoptosis of neural stem cells induced by OGD / R

[0143] NSCs of passage 3 or above were cultured for 3 days, treated with OGD for 4 h, then reoxygenated and treated with MR-409 (0.5 μM, 1 μM) for 72 h. The cells were fixed with PFA, treated with proteinase K, and stained with BrdU. Figure 15-18 As shown, red fluorescence indicates BrdU-labeled neural stem cells, and green fluorescence indicates TUNEL-labeled neural stem cells. + The number of cells and total NSCs was significantly reduced. + The number of cells increased significantly, and the BrdU +The number of cells and the total number of NSCs. At the same time, the survival rate of NSCs was detected by MTT method. The results showed that after OGD / R, the survival rate of cells was significantly lower than that of the blank group, and the survival rate of NSCs was increased in a dose-dependent manner after administration of MR-409. The results of Western blot experiments showed that MR-409 could increase the expression of p-AKT / AKT and p-ERK / ERK proteins in a dose-dependent manner. The above results suggest that MR-409 can increase the proliferation and survival of OGD / R neural stem cells and inhibit their apoptosis, and its mechanism may be related to its increased expression of p-AKT and p-ERK proteins after cerebral ischemia. ** p <0.01 vs. con group; ## p <0.01, ### p <0.001 vs. mod group. Scale bar is 20 μm.

[0144] 9) Expression of GHRH receptors in specific brain regions of neurogenesis and in vitro cultured neural stem cells

[0145] The hippocampal SVZ and SGZ brain regions, positive control hypothalamic brain tissue, and negative control skeletal muscle were obtained from C57BL / 6 mice. Western blot was used for detection. The results are shown in Fig. 20 As shown in A, GHRH receptors are highly expressed in the hypothalamus, but almost not in the skeletal muscle tissue. However, GHRH receptors are highly expressed in the SVZ and SGZ, the specific brain regions for neurogenesis. At the same time, Western blot was used to examine the expression of GHRH receptors in neural stem cells cultured in vitro, with Hela cells as the positive control and MCF7 cells as the negative control. The experimental results are shown in Fig. 20 B shows that GHRH receptors are highly expressed in Hela cells, but almost not in MCF7 cells. However, GHRH receptors are also highly expressed in neural stem cells.

[0146] The above examples show that MR-409 can improve cerebral ischemic damage in tMCAO model mice, increase the survival rate of model mice and improve hippocampal cavitation; MR-409 can improve the neurobehavioral performance of tMCAO model mice; MR-409 can increase the proliferation of neural stem cells in tMCAO model mice; MR-409 can increase the proliferation of neuroblasts in tMCAO model mice; MR-409 can increase the proliferation and migration of neuroblasts in tMCAO model mice; MR-409 can increase the survival of newborn neurons in tMCAO model mice; MR-409 can improve the synaptic plasticity of the hippocampus of tMCAO model mice; MR-409 can increase the proliferation and survival of neural stem cells treated with OGD / R and inhibit their apoptosis; its mechanism may be related to its increased expression of p-AKT and p-ERK proteins in cerebral ischemia, and there are highly expressed GHRH receptors in specific brain areas of neurogenesis and neural stem cells cultured in vitro.

Claims

1. A use of GHRH-A in the preparation of a medicament for treating ischemic cerebral infarction caused by transient middle cerebral artery occlusion, characterized in that: The GHRH-A is MR409, and the amino acid sequence of MR409 is: NMeTyr-D-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Orn-Leu-Leu-Gln-Asp-Ile-Nle-Asp-Arg-NHCH3.

Images