Use of brain-type creatine kinase in preparation of drugs for preventing or treating Parkinson's disease

Abstract

The application discloses application of brain-type creatine kinase in preparation of a medicine for preventing or treating Parkinson's disease, belongs to the technical field of genetic engineering, and provides the application of brain-type creatine kinase in preparation of the medicine for preventing or treating Parkinson's disease, wherein the amino acid sequence of the brain-type creatine kinase is shown as SEQ ID NO. 6. The application of brain-type creatine kinase as a treatment target in screening of the medicine for preventing or treating Parkinson's disease. The application firstly finds that in a Parkinson's disease mouse model, reducing expression of brain-type creatine kinase (CKB) increases loss and death of dopaminergic neurons in the substantia nigra pars compacta of the Parkinson's disease mouse, and significantly aggravates motor dysfunction of the Parkinson's disease mouse; overexpression of CKB obviously reduces damage of Parkinson's disease neurons. The CKB protein is a new target for preventing and treating Parkinson's disease, can be used for screening and finding the medicine for preventing and treating Parkinson's disease, can inhibit death of dopaminergic neurons by improving CKB expression, and realizes the effect of preventing and treating Parkinson's disease.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and in particular relates to the application of brain-type creatine kinase in the preparation of drugs for the prevention or treatment of Parkinson's disease. Background Technology

[0002] Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta and the formation of Lewy bodies from the abnormal aggregation of α-synuclein. Clinical manifestations include motor symptoms such as resting tremor, bradykinesia, rigidity, and postural instability, as well as non-motor symptoms such as depression and cognitive impairment. With the increasing global aging population, the prevalence of Parkinson's disease is rising annually. Current treatment options for Parkinson's disease primarily include medication, surgery, and rehabilitation. While these methods can alleviate symptoms to some extent, they cannot stop or reverse the progression of the disease.

[0003] Levodopa (L-DOPA), a dopamine-like drug, is commonly used to improve motor function by supplementing dopamine in the brain. However, long-term use can lead to decreased efficacy and side effects such as dyskinesia. Anticholinergic drugs, such as trihexyphenidyl, treat motor dysfunction in PD patients symptomatically; there are currently no clinically available drugs that can fundamentally prevent or treat PD by inhibiting the death of dopaminergic neurons in the substantia nigra pars compacta. Surgical treatment, including deep brain stimulation (DBS), modulates abnormal neural activity by implanting electrodes in specific brain regions. It is suitable for some patients in the middle and late stages, but its widespread use is limited by surgical risks and high costs. Rehabilitation therapies, including physical therapy, occupational therapy, and speech therapy, can improve patients' quality of life, but they do not directly intervene in the disease itself.

[0004] In recent years, with the development of molecular biology and genomics technologies, scientists have made significant progress in the study of the pathogenesis of Parkinson's disease. Studies have shown that disorders of multiple signaling pathways and protein function are closely related to the occurrence and development of Parkinson's disease, providing a theoretical basis for finding new therapeutic targets. For example, mitochondrial dysfunction: mitochondrial DNA mutations and abnormal energy metabolism caused by oxidative stress are considered important pathogenic factors of Parkinson's disease; impaired autophagy-lysosomal pathway: abnormal aggregation of α-synuclein is closely related to impaired autophagy, and enhancing autophagy may help clear toxic protein aggregates; inflammatory response: chronic neuroinflammation triggered by microglial cell activation plays an important role in the pathological process of Parkinson's disease, and anti-inflammatory therapy may have potential value. Current research on therapeutic targets is insufficient; therefore, discovering new and more effective therapeutic targets is particularly important. Brain-type creatine kinase (CKB) is the most abundant CK subtype in brain tissue and plays an important role in regulating various physiological and pathological processes, but the role of CKB in Parkinson's disease has not yet been reported. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes the application of brain-type creatine kinase in the preparation of drugs for the prevention or treatment of Parkinson's disease. Brain-type creatine kinase is a novel target for the prevention and treatment of Parkinson's disease and can be used to screen and discover drugs for the prevention and treatment of Parkinson's disease. These drugs can play a role in preventing and treating Parkinson's disease by increasing the expression of brain-type creatine kinase and thereby inhibiting the loss and death of dopaminergic neurons.

[0006] To achieve the above objectives, the present invention provides the use of brain-type creatine kinase in the preparation of drugs for the prevention or treatment of Parkinson's disease, wherein the amino acid sequence of the brain-type creatine kinase is shown in SEQ ID NO.6.

[0007] Preferably, the drug reduces neuronal damage in Parkinson's disease by increasing the expression or activity of cerebral creatine kinase, thereby preventing or treating Parkinson's disease.

[0008] The present invention also provides a drug for the prevention or treatment of Parkinson's disease, the drug comprising one of brain-type creatine kinase, brain-type creatine kinase expression promoter, or brain-type creatine kinase activity promoter; the amino acid sequence of the brain-type creatine kinase is shown in SEQ ID NO.6.

[0009] Preferably, the drug reduces neuronal damage in Parkinson's disease by increasing the expression or activity of cerebral creatine kinase, thereby preventing or treating Parkinson's disease.

[0010] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0011] The present invention also provides the application of brain-type creatine kinase as a therapeutic target in screening drugs for the prevention or treatment of Parkinson's disease, wherein the amino acid sequence of the brain-type creatine kinase is shown in SEQ ID NO.6.

[0012] Preferably, the drug reduces neuronal damage in Parkinson's disease by increasing the expression or activity of cerebral creatine kinase, thereby preventing or treating Parkinson's disease.

[0013] The present invention also provides a drug for the prevention or treatment of Parkinson's disease, wherein the drug targets brain-type creatine kinase, increases the expression or activity of brain-type creatine kinase, reduces neuronal damage in Parkinson's disease, and prevents or treats Parkinson's disease; the amino acid sequence of the brain-type creatine kinase is shown in SEQ ID NO.6.

[0014] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0015] Compared with the prior art, the present invention has the following advantages and technical effects:

[0016] This invention is the first to discover that in a Parkinson's disease cell model, reducing brain-type creatine kinase (CKB) expression significantly aggravates neuronal damage in Parkinson's disease. Simultaneously, it is the first to discover that in a Parkinson's disease mouse model, reducing CKB expression increases the loss and death of dopaminergic neurons in the substantia nigra pars compacta, significantly aggravating motor dysfunction in Parkinson's disease mice; overexpression of CKB significantly alleviates neuronal damage in Parkinson's disease. CKB protein is a novel target for the prevention and treatment of Parkinson's disease and can be used to screen for and discover drugs for the prevention and treatment of Parkinson's disease. These drugs can inhibit dopaminergic neuronal death by increasing CKB expression, thereby achieving a preventive and therapeutic effect on Parkinson's disease and can be used to prepare drugs for the prevention and treatment of Parkinson's disease. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The graphs show the validation effects of knockdown and overexpression of CKB in neurons in vitro. In this graph, A is the validation effect of CKB, and B is the statistical graph of the relative expression level of CKB protein.

[0019] Figure 2 The images show the cell survival rate and lactate dehydrogenase release rate of in vitro knockdown of neurons to detect the effect of CKB on neuronal damage in Parkinson's disease. In this image, A is the cell survival rate and B is the lactate dehydrogenase release rate.

[0020] Figure 3 The images show the validation effects of Cre virus and LoxP virus-specific infection on mouse dopaminergic neurons in vivo. In this image, A is the validation effect of knocking out mouse dopaminergic neurons CKB in vivo, with a scale bar of 100 μm; B is the expression map of knocked-out mouse dopaminergic neurons CKB in vivo; and C is the statistical map of knocked-out mouse dopaminergic neurons CKB in vivo.

[0021] Figure 4 The images show the results of knocking down CKB expression in dopaminergic neurons in vivo, which exacerbates motor dysfunction in mice with Parkinson's disease. In the images, A is the result of the pole climbing experiment, B is the result of the suspension experiment, and C is the result of the rotarod experiment.

[0022] Figure 5 The graph shows the results of the increased dopaminergic neuron mortality in Parkinson's disease mice by knocking down CKB expression in dopaminergic neurons. In the graph, A is a representative image of Nissl staining in the substantia nigra pars compacta, with a scale bar of 100 μm; B is a representative image of immunofluorescence of tyrosine hydroxylase in the substantia nigra pars compacta, with a scale bar of 100 μm; C is a statistical graph of Nissl-stained positive cells in the substantia nigra pars compacta; and D is a statistical graph of tyrosine hydroxylase-positive cells in the substantia nigra pars compacta.

[0023] Figure 6 The images show the cell survival rate and lactate dehydrogenase release rate of neurons overexpressing CKB in vitro, which affect neuronal damage in Parkinson's disease. In the images, A represents the cell survival rate and B represents the lactate dehydrogenase release rate. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] Example 1

[0030] I. Establishment of in vitro models for knockdown and overexpression of neuronal brain-type creatine kinase (CKB) expression.

[0031] After seeding SH-SY5Y neurons (purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee), normal groups, knockdown control groups, CKB knockdown groups, overexpression control groups, and CKB overexpression groups were set up. CKB-siRNA was added to the CKB knockdown groups, and blank control CKB-siRNA was added to the knockdown control groups. The specific forward and reverse sequences of the siRNA are shown in Table 1 below.

[0032] Table 1 siRNA sequences

[0033]

[0034] The CKB overexpression group was given the CKB overexpression plasmid, while the overexpression control group was given the blank overexpression plasmid.

[0035]

[0036] The amino acid sequence of CKB (from the protein database uniprot, P12277) is shown in SEQ ID NO. 6, SEQ ID NO. 6: MPFSNSHNALKLRFPAEDEFPDLSAHNNHMAKVLTPELYAEL RAKSTPSGFTLDDVIQTGVDNPGHPYIMTVGCVAGDEESYEVFKDLFDPIIEDRHGGYKPSDEHKTDLNPDNLQGGDDLDPNYVLSSRVRTGRSIRGFCLPPHCSRGERRAIEKLAVEALSSLDGDLAGRYYALKSMTEAEQQQLIDDHFLFDKPVSPLLLASGMARDWP DARGIWHNDNKTFLVWVNEEDHLRVISMQKGGNMKEVFTRFCTGLTQIETLFKSKDYEFMWNPHLGYILTCPSNLGTGLRAGVHIKLPNLGKHEKFSEVLKRLRLQKRGTGGVDTAAVGGVFDVSNADRLGFSEVELVQMVVDGVKLLIEMEQRLEQGQAIDDLMPAQK.

[0037] The normal group was given an equal amount of transfection reagent. After 6 hours of transfection, the medium was replaced with fresh medium. After culturing for another 24 hours, the supernatant was discarded, and the cells were washed with PBS and fully lysed to extract cell proteins. Western blotting was used to detect the protein expression of CKB in the normal group, knockdown control group, CKB knockdown group, overexpression control group, and CKB overexpression group.

[0038] The results are as follows Figure 1 China A and Figure 1 As shown in Figure B, compared with the normal group, there was no significant change in CKB expression in the knockdown control group and the overexpression control group (p > 0.05); compared with the knockdown control group, CKB expression in the CKB knockdown group was significantly reduced (p < 0.01); compared with the overexpression control group, CKB expression in the CKB overexpression group was significantly increased. The results indicate that the CKB-siRNA used in this invention can significantly knock down neuronal CKB expression, and the CKB overexpression plasmid (CKB-oeRNA) can significantly increase neuronal CKB expression, which can be used for subsequent experiments.

[0039] Example 2

[0040] I. Effect of in vitro knockdown of neuronal CKB expression on increasing neuronal damage in Parkinson's disease (PD)

[0041] 1. Analyzing the effect of increased CKB expression in knockdown neurons on PD neuronal damage by detecting cell viability.

[0042] After seeding SH-SY5Y neurons, knockdown control normal group, knockdown control model group, and CKB knockdown model group were set up. CKB knockdown group was given CKB-siRNA (SEQ ID NO.1 and SEQ ID NO.2), and blank control CKB-siRNA (SEQ ID NO.3 and SEQ ID NO.4) was given to both knockdown control normal group and knockdown control model group.

[0043] Six hours after transfection, the medium was replaced with fresh medium, and the culture was continued for another 42 hours. MPP was then added to the knockdown control group and the CKB knockdown group. + A PD cell model was established by inducing cell culture at a final concentration of 1.0 mM. The control group was knocked down, and an equal volume of DMSO was added. After 24 h of culture, CCK-8 solution was added, and the cells were cultured for another 2 h. The absorbance at 450 nm was measured using a microplate reader, and cell viability was counted. Cell viability = (Experimental group absorbance - Blank solvent absorbance) / (Blank control group absorbance - Blank solvent absorbance) × 100%.

[0044] The results are as follows Figure 2 As shown in Figure A, compared with the normal knockdown control group, the cell survival rate of the knockdown control model group was significantly decreased (p < 0.01); while compared with the knockdown control model group, the cell survival rate of the CKB knockdown model group was significantly reduced (p < 0.01). These results indicate that knockdown of neuronal CKB expression in vitro can significantly increase PD neuronal damage.

[0045] 2. Analysis of the effect of increased CKB expression in knocked-down neurons on PD neuronal damage by detecting lactate dehydrogenase release rate.

[0046] After seeding SH-SY5Y neurons, knockdown control normal group, knockdown control model group, and CKB knockdown model group were set up. CKB knockdown model group was given CKB-siRNA (SEQ ID NO.1 and SEQ ID NO.2), while blank control CKB-siRNA (SEQ ID NO.3 and SEQ ID NO.4) was given to the knockdown control normal group and the knockdown control model group.

[0047] Six hours after transfection, the medium was replaced with fresh medium, and the culture was continued for another 42 hours. MPP was then added to the knockdown control group and the CKB knockdown group. +(Final concentration 1.0 mM) A PD cell model was established by inducing lactate dehydrogenase (PD) cell release. The control group was knocked down, and an equal volume of DMSO was added. After culturing for 24 h, lactate dehydrogenase release agent was added to the control group with maximum enzyme activity, and the cells were repeatedly pipetted. The cells were cultured for another 1 h. The wells were centrifuged at 1000 rpm for 5 min, and 60 μL of the supernatant was collected. The lactate dehydrogenase release rate was measured according to the kit instructions (purchased from Shanghai Beyotime Biotechnology Co., Ltd.). The absorbance at 490 nm was measured using a microplate reader, and the lactate dehydrogenase release rate was calculated. Lactate dehydrogenase release rate = (sample absorbance - blank solvent absorbance) / (maximum enzyme activity absorbance - background blank control absorbance) × 100%.

[0048] The results are as follows Figure 2 As shown in Figure B, a higher lactate dehydrogenase release rate indicates more severe cell damage. Compared with the knockdown control normal group, the lactate dehydrogenase release rate in the knockdown control model group was significantly increased (p < 0.01); while compared with the knockdown control model group, the lactate dehydrogenase release rate in the CKB knockdown model group was significantly increased (p < 0.01). These results further demonstrate that knocking down neuronal CKB expression in vitro can significantly increase PD neuronal damage.

[0049] III. Effects of in vivo knockdown of CKB expression in mouse dopaminergic neurons on motor dysfunction and dopaminergic neuronal death in PD mice

[0050] 1. Validation of the effects of Cre virus and LoxP virus-specific infection on mouse dopaminergic neurons in vivo.

[0051] Eight-week-old C57BL / 6 mice were acclimatized in an SPF-grade animal facility for 7 days, then anesthetized and fixed in a stereotaxic apparatus. Hair was removed from the head, and after disinfection with iodine, the scalp was surgically cut to expose the skull. The anterior and posterior fontanelles were located and aligned horizontally. Markings were made 3.16 mm posteriorly and 1.37 mm laterally from the anterior fontanelle as the origin. A small hole was gently drilled, and a microsyringe was inserted vertically downwards for 4.60 mm from the skull surface. A mixture of Cre virus and LoxP virus was injected bilaterally into the substantia nigra pars compacta. The Cre virus was rAAV-TH-Cre, and the LoxP virus was rAAV-DIO-EGFP-shRNA(scramble)-polyA. The functional sequence of the shRNA(scramble) was CCTAAGGTTAAGTCGCCCTCG (SEQ ID NO. 7). The needle was left in place for 3 minutes and then slowly withdrawn for 2 minutes to ensure sufficient diffusion of the solution. The scalp was then sutured, and erythromycin ointment was applied to prevent wound infection. After 21 days of continued feeding, the mice were anesthetized, followed by cardiac perfusion and brain harvesting. Brain tissue was fixed, dehydrated, embedded in paraffin, and sectioned. Brain slices (6 μm) from the substantia nigra pars compacta were collected, dewaxed, washed with PBS, blocked, incubated with tyrosine hydroxylase (TH) primary antibody, washed with PBS, incubated overnight with fluorescent secondary antibody (Cy3), washed with PBS, stained with DAPI, washed with PBS, and mounted. The co-labeling of tyrosine hydroxylase (TH) fluorescence and viral EGFP fluorescence in the substantia nigra pars compacta was then observed and analyzed under a microscope.

[0052] The results are as follows Figure 3 As shown in Figure A, tyrosine hydroxylase (TH)-positive cells are dopaminergic neurons, and EGFP-positive cells are rAAV-infected cells. A high degree of co-labeling between TH and EGFP fluorescence is observed. These results indicate that, based on the Cre-LoxP system, injection of Cre virus and LoxP virus into the substantia nigra pars compacta can specifically infect mouse dopaminergic neurons, and can be used for subsequent experiments.

[0053] 2. Validation of the effect of knocking down CKB expression in mouse dopaminergic neurons in vivo

[0054] Eight-week-old C57BL / 6 mice were acclimatized for 7 days in an SPF-grade animal facility, then anesthetized and fixed in a stereotaxic apparatus. Hair was removed from the head, and after disinfection with iodine, the scalp was surgically cut to expose the skull. The anterior and posterior fontanelles were located and adjusted to be completely horizontal. Markings were made 3.16 mm posteriorly and 1.37 mm laterally from the anterior fontanelle as the origin. A small hole was gently drilled with a cranial drill, and a microsyringe was inserted vertically downwards for 4.60 mm from the skull surface. Cre virus and LoxP virus were mixed and injected into the bilateral substantia nigra pars compacta. In the CKB knockdown model group, the Cre virus was rAAV-TH-Cre, and the LoxP virus was rAAV-DIO-EGFP-shRNA(CKB)-polyA, with the functional sequence of shRNA(CKB) being GCGAGGAGAGTTACGACGTA (SEQ ID NO.8). In the knockdown control normal group and the knockdown control model group, the Cre virus was rAAV-TH-Cre, and the LoxP virus was rAAV-DIO-EGFP-shRNA(scramble)-6polyA, with the functional sequence of shRNA(scramble) being CCTAAGGTTAAGTCGCCCTCG (SEQ ID NO.7). After injection, the needle was left in place for 3 minutes and then slowly withdrawn for 2 minutes to ensure that the solution spread fully. The scalp was then sutured and erythromycin ointment was applied to prevent wound infection. After 21 days of continued feeding, the mice were anesthetized, followed by cardiac perfusion, brain harvesting, and separation of the substantia nigra pars compacta brain tissue. Proteins were extracted from the substantia nigra pars compacta brain tissue of Parkinson's disease mice by pipetting with RIPA lysis buffer. The protein concentration of the samples was detected by BCA method, and the expression of CKB protein in the substantia nigra pars compacta brain tissue of the knockdown control group and the CKB knockdown group mice was detected by Western blotting.

[0055] The results are as follows Figure 3 China B and Figure 3 As shown in Figure C, the substantia nigra pars compacta is the main distribution area of ​​dopaminergic neurons. Compared with the knockdown control group, the CKB expression in the substantia nigra pars compacta of the CKB knockdown group was significantly decreased (p < 0.01). The results indicate that, based on the Cre-LoxP system, injecting Cre virus and LoxP virus into the substantia nigra pars compacta can knock down the CKB expression of mouse dopaminergic neurons, which can be used for subsequent experiments.

[0056] 3. Effects of knockdown of CKB expression in dopaminergic neurons in vivo on motor dysfunction and dopaminergic neuron death in Parkinson's disease mice

[0057] Eight-week-old C57BL / 6 mice were acclimatized for 7 days in an SPF-grade animal facility and then divided into three groups: a normal control group, a knockdown control model group, and a CKB knockdown model group, with 10 mice in each group. On day 1 of the experiment, the mice were anesthetized and fixed in a stereotaxic apparatus. Hair was removed from the head, and the skull was disinfected with iodine. The scalp was cut to expose the skull, and the anterior and posterior fontanelles were located and compared to each other until they were completely horizontal. The anterior fontanelle was used as the origin, and the positions were marked 3.16 cm posteriorly and 1.37 cm to the left and right. A skull drill was gently used to make a hole, and a microsyringe was inserted vertically downwards 4.60 cm from the surface of the skull. Cre virus and LoxP virus were mixed and injected bilaterally into the substantia nigra pars compacta. In the CKB knockdown model group, the Cre virus was rAAV-TH-Cre, and the LoxP virus was rAAV-DIO-EGFP-shRNA(CKB)-polyA, with the functional sequence of shRNA(CKB) being SEQ ID NO.8. In the knockdown control normal group and the knockdown control model group, the Cre virus was rAAV-TH-Cre, and the LoxP virus was rAAV-DIO-EGFP-shRNA(scramble)-6polyA, with the functional sequence of shRNA(scramble) being SEQ ID NO.7. The needles were left in place for 3 minutes after injection and then slowly withdrawn for 2 minutes to ensure sufficient diffusion of the solution. The scalp was then sutured, and erythromycin ointment was applied to prevent wound infection. Starting from day 24 of the experiment, mice in the knockdown control model group and the CKB knockdown model group were intraperitoneally injected with 30 mg / kg MPTP daily for 7 days to establish a Parkinson's disease mouse model. Mice in the knockdown control normal group were intraperitoneally injected with the same volume of physiological saline. On day 34 of the experiment, motor function behavioral tests were performed, including the pole climbing test, the suspension test, and the rotarod test. Subsequently, the mice were anesthetized, their hearts were perfused, and their brains were harvested. The brain tissue was fixed, dehydrated, embedded in paraffin, and sectioned. Brain slices (6 μm) of the substantia nigra pars compacta were collected, dewaxed, washed with PBS, blocked, incubated with tyrosine hydroxylase (TH) primary antibody, washed with PBS, incubated with fluorescent secondary antibody (Cy3) overnight, washed with PBS, stained with DAPI, washed with PBS, and mounted. Nissl staining and tyrosine hydroxylase (TH) immunofluorescence staining of the substantia nigra pars compacta were then observed and analyzed under a microscope.

[0058] Pole climbing experiment: A rough-surfaced wooden pole with a diameter of 1 cm and a height of 45 cm is vertically fixed inside the mouse cage. A cork ball with a diameter of 2.5 cm is glued to the top of the pole. The mouse is placed on the cork ball, and the time required for the mouse to climb from the cork ball to the mouse cage is recorded. The average value is calculated for three consecutive measurements.

[0059] Suspension test: The forepaws of mice are hung on a horizontal wire, and the time it takes for the mice to fall off the wire is recorded. The average value is calculated after three consecutive measurements.

[0060] Rotary bar experiment: Place the mouse on a rotating bar with a diameter of 4 cm, and then gradually increase the rotation speed from 0 r / min to 40 r / min, and then maintain the rotation speed at 40 r / min. Record the time required for the mouse to fall from the rotating bar after the rotating bar starts to rotate. Measure the average value of 3 consecutive measurements.

[0061] Nissl staining experiment: Brain slices (6μm) from the substantia nigra compacta were dewaxed, stained with Nissl, dehydrated, cleared, and mounted. The number of Nissl-positive cells in the substantia nigra compacta was then observed and counted under a microscope.

[0062] Tyrosine hydroxylase (TH) immunofluorescence assay: Brain tissue was fixed, dehydrated, embedded in paraffin, and sectioned. Brain slices (20 μm) from the substantia nigra compacta were collected, dewaxed, washed with PBS, blocked, incubated with tyrosine hydroxylase (TH) primary antibody, washed with PBS, incubated with red fluorescent secondary antibody, washed with PBS, and mounted. The number of tyrosine hydroxylase-positive cells in the substantia nigra compacta was then observed and counted under a microscope.

[0063] The results are as follows Figure 4 As shown in Figure A, a longer pole-climbing time indicates poorer motor function in mice. Compared with the knockdown control group, the pole-climbing time in the knockdown control model group was significantly increased (p < 0.01), while the pole-climbing time in the CKB knockdown model group was significantly decreased (p < 0.01). These results indicate that knockdown of CKB expression in dopaminergic neurons in mice can significantly aggravate motor dysfunction in Parkinson's disease mice. Figure 4 As shown in Figure B, shorter suspension time indicates poorer motor function in mice. Compared with the normal control group, the suspension time of the knockdown control model group was significantly reduced (p < 0.01), while the suspension time of the CKB knockdown model group was significantly increased (p < 0.01). These results further demonstrate that knocking down CKB expression in dopaminergic neurons in mice can significantly aggravate motor dysfunction in Parkinson's disease mice. Figure 4 As shown in Figure C, a shorter rotarod time indicates poorer motor function in mice. Compared with the knockdown control group, the rotarod time in the knockdown control model group was significantly reduced (p < 0.01), while the rotarod time in the CKB knockdown model group was significantly increased (p < 0.01). These results further demonstrate that knocking down CKB expression in dopaminergic neurons in mice can significantly aggravate motor dysfunction in Parkinson's disease mice.

[0064] like Figure 5 China A and Figure 5As shown in Figure C, a lower number of Nissl-positive cells in the substantia nigra pars compacta indicates more severe dopaminergic neuron death. Compared with the normal knockdown control group, the knockdown control model group showed a significant reduction in the number of Nissl-positive cells in the substantia nigra pars compacta (p < 0.01), while compared with the knockdown control model group, the CKB knockdown model group showed a significant reduction in the number of Nissl-positive cells in the substantia nigra pars compacta (p < 0.01). These results indicate that knockdown of CKB expression in mouse dopaminergic neurons significantly aggravates dopaminergic neuron death in Parkinson's disease mice. Figure 5 China B and Figure 5 As shown in Figure D, a lower number of tyrosine hydroxylase (TH) immunofluorescence-positive cells in the substantia nigra pars compacta indicates more severe dopaminergic neuron death. Compared with the normal knockdown control group, the number of tyrosine hydroxylase immunofluorescence-positive cells in the substantia nigra pars compacta of mice in the knockdown control group was significantly reduced (p < 0.01), while compared with the knockdown control group, the number of tyrosine hydroxylase immunofluorescence-positive cells in the substantia nigra pars compacta of mice in the CKB knockdown model group was significantly reduced (p < 0.01). These results further demonstrate that knocking down CKB expression in mouse dopaminergic neurons can significantly aggravate dopaminergic neuron death in Parkinson's disease mice.

[0065] IV. The effect of in vitro overexpression of neuronal CKB expression significantly reduces neuronal damage in Parkinson's disease.

[0066] 1. Analyzing the inhibitory effect of CKB overexpression on neuronal damage in Parkinson's disease by detecting cell viability in vitro.

[0067] After seeding SH-SY5Y neurons, three groups were established: a normal overexpression control group, an overexpression control model group, and a CKB overexpression model group. The overexpression control group received a blank control plasmid, and the CKB overexpression group received the CKB overexpression plasmid (SEQ ID NO. 5).

[0068] Six hours after transfection, the medium was replaced with fresh medium, and the culture was continued for another 42 hours. MPP was then added to each model group. + A Parkinson's disease cell model was established by inducing cell culture at a final concentration of 1.0 mM. An equal volume of DMSO was added to the normal group, and after 24 hours of culture, CCK-8 solution was added. Cells were cultured for another 2 hours, and the absorbance at 450 nm was measured using a microplate reader to count cell viability. Cell viability = (Experimental group absorbance - Blank solvent absorbance) / (Blank control group absorbance - Blank solvent absorbance) × 100%.

[0069] like Figure 6As shown in Figure A, compared with the overexpression control group, the cell survival rate of the overexpression control model group was significantly decreased (p < 0.01); while compared with the overexpression control model group, the cell survival rate of the overexpression CKB model group was significantly reduced (p < 0.01). These results indicate that in vitro overexpression of neuronal CKB can significantly reduce neuronal damage in Parkinson's disease.

[0070] 2. Analysis of the effect of increased CKB expression in overexpressed neurons on neuronal damage in Parkinson's disease by detecting lactate dehydrogenase release rate.

[0071] After seeding SH-SY5Y neurons, three groups were established: a normal overexpression control group, an overexpression control model group, and a CKB overexpression model group. The overexpression control group received a blank control plasmid, and the CKB overexpression group received the CKB overexpression plasmid (SEQ ID NO. 5).

[0072] Six hours after transfection, the medium was replaced with fresh medium, and the culture was continued for another 42 hours. MPP was then added to each model group. + A Parkinson's disease cell model was established by inducing lactate dehydrogenase release agent (final concentration 1.0 mM). An equal volume of DMSO was added to the normal group, and after 24 h of culture, the lactate dehydrogenase release agent was added to the control group with maximum enzyme activity, and the cells were repeatedly pipetted. The cells were cultured for another 1 h, and the wells were centrifuged at 1000 rpm for 5 min. 60 μL of the supernatant was collected, and the lactate dehydrogenase release rate was measured according to the manufacturer's instructions. The absorbance at 490 nm was measured using a microplate reader, and the lactate dehydrogenase release rate was calculated. Lactate dehydrogenase release rate = (sample absorbance - blank solvent absorbance) / (maximum enzyme activity absorbance - background blank control absorbance) × 100%.

[0073] like Figure 6 As shown in Figure B, a higher lactate dehydrogenase release rate indicates more severe cell damage. Compared with the overexpression control group, the lactate dehydrogenase release rate in the overexpression control model group was significantly increased (p < 0.01); while compared with the overexpression control model group, the lactate dehydrogenase release rate in the overexpression CKB model group was significantly decreased (p < 0.01). These results further demonstrate that in vitro overexpression of neuronal CKB can significantly reduce neuronal damage in Parkinson's disease.

[0074] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of brain-type creatine kinase in the preparation of drugs for the prevention or treatment of Parkinson's disease, characterized in that, The amino acid sequence of the brain-type creatine kinase is shown in SEQ ID NO.

6.

2. The application of brain-type creatine kinase as a therapeutic target in screening drugs for the prevention or treatment of Parkinson's disease, characterized in that, The amino acid sequence of the brain-type creatine kinase is shown in SEQ ID NO.

6.

3. The application according to claim 2, characterized in that, The drug reduces neuronal damage in Parkinson's disease by increasing the expression or activity of cerebral creatine kinase, thereby preventing or treating Parkinson's disease.

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