WO2024055457A1 - Jwa多肽在制备抗帕金森病药物方面的应用 - Google Patents

Jwa多肽在制备抗帕金森病药物方面的应用 Download PDF

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WO2024055457A1
WO2024055457A1 PCT/CN2022/138737 CN2022138737W WO2024055457A1 WO 2024055457 A1 WO2024055457 A1 WO 2024055457A1 CN 2022138737 W CN2022138737 W CN 2022138737W WO 2024055457 A1 WO2024055457 A1 WO 2024055457A1
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disease
parkinson
mptp
jwa
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周建伟
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苏州明人医药生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • the present invention relates to the application of JWA polypeptide in the preparation of anti-Parkinson's disease drugs, and belongs to the technical field of central nervous system drugs.
  • Parkinson's disease is a common neurodegenerative disease. Its main pathological characteristics are: degeneration and loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc), and reduction of DA transmitters in the striatum. , glial cell proliferation, accompanied by the formation of intracellular eosinophilic Lewy bodies.
  • DA dopamine
  • SNc substantia nigra pars compacta
  • Clinical epidemiological survey results show that the prevalence of PD in people over 65 years old reaches more than 1%, and the prevalence of PD also shows an increasing trend with age. The risk of PD in people over 85 years old further increases to 4 %[1].
  • Parkinson's disease the second most common neurodegenerative disease in the world, affects the physical and mental health of countless patients.
  • PD patients mainly present with the following clinical symptoms: motor symptoms such as dyskinesia, muscle rigidity, and resting tremor, accompanied by non-motor symptoms such as hyposmia, sleep disorders, psychiatric symptoms, and cognitive impairment.
  • motor symptoms such as dyskinesia, muscle rigidity, and resting tremor
  • non-motor symptoms such as hyposmia, sleep disorders, psychiatric symptoms, and cognitive impairment.
  • the exact pathogenesis of PD remains unclear, so PD remains an incurable disease.
  • Levodopa L-Dopa
  • Dopamine dopamine
  • this therapy treats the symptoms but not the root cause and cannot cure PD or delay the further development of the disease.
  • MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
  • astrocytes are the most abundant glial cells and are an important part of the nervous system. In the human brain, the number of astrocytes is several times that of neurons [11]. Astrocytes are distributed in all brain regions and play an important "strategic position" in tightly integrating with neurons. Astrocytes have long been viewed as glial cells that merely provide structural support to neurons.
  • astrocytes can maintain the extracellular environment, stabilize communication between cells, and mediate the physiological and pathological states of neurons [12].
  • the function of astrocytes mainly relies on their ability to release and absorb molecules in the extracellular microenvironment, thereby protecting or damaging neurons. Therefore, elucidating the role of astrocytes in DA neuron damage caused by environmental toxicants is of great significance for scientifically formulating prevention and treatment strategies for neurotoxicity caused by environmental toxicants.
  • Astrocytes can influence neuronal survival through different molecular mechanisms. In a co-culture model of neurons and astrocytes, neurons were more resistant to oxidative damage when astrocytes were present. Glutamic acid is the most abundant excitatory amino acid (EAA) in the central nervous system. Glutamic acid-induced excitotoxicity is an important mechanism of neuronal death in central nervous system hemorrhage, trauma, and neurodegenerative diseases [13]. Part of the EAA in the synaptic cleft is degraded by enzymes, and the other part is taken back by EAA transporters (EAAT) located on the membranes of neurons and glial cells and is quickly inactivated.
  • EAA transporters EAA transporters
  • GLT-1 Various toxic molecules and growth factors can enhance the expression of GLT-1 and weaken the excitability by activating the NF- ⁇ B signaling pathway in astrocytes. Neurotoxicity. However, under the stimulation of the pro-inflammatory factor TNF- ⁇ , the activation of the astrocyte NF- ⁇ B signaling pathway did not increase the expression of GLT-1. Instead, NF- ⁇ B and N-myc increased the expression of GLT-1 at the GLT-1 promoter. Bind to each other and inhibit the expression of GLT-1. It can be seen that the expression of GLT-1 in astrocytes is crucial for the effects of neurotoxicity, and NF- ⁇ B activation is necessary to activate or inhibit GLT-1 [15].
  • Nrf2 nuclear factor E2-related factor
  • ARE antioxidant stress response elements
  • IFs intermediate filaments
  • IFs glial fibrillary acidic protein
  • astrocytes In various CNS diseases, the size of astrocytes increases excessively, changing from normal astrocytes to activated astrocytes. During this process, the expression of IFs, especially GFAP, increases. The expression of GFAP in the hypothalamus of PD patients is also significantly increased compared with normal people [22], suggesting that GFAP, a component of the astrocyte cytoskeleton, plays an important role in CNS diseases [23].
  • the above analysis shows that the selective toxic effects of environmental toxicants on nigrostriatal dopamine neurons involve complex mechanisms such as activation of the NF- ⁇ B signaling pathway, protein phosphorylation, and cytoskeletal changes.
  • the JWA gene (also known as ARL6IP5) is an environmental response gene that Zhou Jianwei and others first discovered and cloned from the retinoic acid-induced human bronchial epithelial (HBE) cell differentiation model and have been studied for a long time.
  • the protein encoded by it is a cytoskeletal binding protein. In normal cells, it can participate in processes such as regulating cell differentiation, responding to oxidative stress, and DNA repair.
  • Professor Zhou Jianwei leads the research team to conduct long-term research on the function and role of JWA gene in tumors and the nervous system. We used a Drosophila model in which the JWA gene was deleted and found that Drosophila with defects in JWA expression were less likely to develop tolerance to repeated exposure to ethanol [24].
  • Rat and cell models using antisense nucleic acids to inhibit JWA expression found that JWA maintains the stability of the opiate receptor DOR through the ubiquitin-proteasome pathway, thus having a direct regulatory effect on morphine dependence in rats [25].
  • JWA is involved in regulating signaling pathways such as NF- ⁇ B and MAPK.
  • NF- ⁇ B and MAPK signaling pathways
  • JWA is involved in regulating cellular aging by regulating the activity of NF- ⁇ B transcription factors.
  • the mechanism is that JWA regulates the degradation of IKK ⁇ through the ubiquitin proteasome pathway and inhibits Nuclear entry of p65.
  • H 2 O 2 induces the cellular nuclear factor NFI to bind to the CCAAT element in the proximal promoter region of JWA, thereby activating the expression of JWA in response to oxidative stress [26]; JWA regulates nuclear factors E2F1 and XRCC1 expression enhances the ability to repair DNA damage.
  • JWA inhibits the oxidative stress produced by paraquat and activates GSH and Nrf2 through MAPK and PI3K signaling pathways, effectively antagonizing paraquat-induced dopamine neuron damage in mice (see Figure 1, Panel A) [28]; for further research, Regarding the role of astrocyte JWA in the process of PD, we used astrocyte JWA knockout mice on a C57 background to construct a chronic model of MPTP, and further explored the molecules that astrocyte JWA affects the survival of DA neurons. Basically, it was found that deletion of the JWA gene in astrocytes can increase the sensitivity of mice to MPTP and paraquat.
  • the polypeptide JP1 screened by the previous research group based on JWA functional fragments targets the highly expressed integrin ⁇ V ⁇ 3 through its connected RGD sequence and then enters the cell. It negatively regulates the nuclear transcription factor SP1, downregulates the expression of ⁇ V ⁇ 3, and can effectively inhibit the growth of mouse melanoma. Transfer; further research found that this polypeptide can cross the blood-brain barrier [31].
  • JP1 can target integrin ⁇ V ⁇ 3 that is highly expressed on the surface of melanoma after being connected to the RGD sequence, it is still unclear whether the JP1 targeting peptide can be used to treat central nervous system diseases such as Parkinson's disease, and further exploration and research are urgently needed.
  • the inventor's research team has the latest research results and uses them to apply for a patent for this invention.
  • Pezzoli G, Cereda E Exposure to pesticides or solvents and risk of Parkinson disease. Neurology 2013,80(22):2035-2041.
  • Nrf2 target genes can be controlled by neuronal activity in the absence of Nrf2 and astrocytes.Proceedings of the National Academy of Sciences of the United States of America 2014,111(18):1818-1820.
  • the main purpose of the present invention is to propose the application of a JWA polypeptide in the preparation of anti-Parkinson's disease drugs in view of the problems existing in the existing technology, which can regulate the proliferation and activation of astrocytes/microglia in brain tissue. It can effectively inhibit the excessive activation of astrocytes/microglia, and can significantly increase the level of dopamine neuron antagonism and excitotoxicity, reduce neuron death, and provide new clinical drug possibilities for the treatment of Parkinson's disease. .
  • polypeptide characterized in that the use is used to prepare drugs for preventing or treating Parkinson's disease
  • amino acid sequence of the polypeptide is shown in I or II:
  • amino acid S is phosphorylated
  • X and Z are amino acids or amino acid sequences respectively;
  • Z is selected from one of (G) n -RGD and A-(G) n -RGD, n is an integer greater than or equal to 0, and the value range of n is 0-10.
  • the use is for the preparation of a medicament for preventing or treating specific conditions or symptoms of Parkinson's disease.
  • the specific disease or symptom of Parkinson's disease is: excessive activation or proliferation of astrocytes or microglia in brain tissue.
  • the specific disease or symptom of Parkinson's disease is: degeneration or loss of dopamine neurons in brain tissue.
  • the specific disease or symptom of Parkinson's disease is: hippocampal neuronal cells or glial cells are subjected to cytotoxic effects, resulting in weakened vitality.
  • the specific symptoms or symptoms of Parkinson's disease are: increased expression of inflammasomes and increased apoptosis in hippocampal neuronal cells or glial cells.
  • the specific disease or symptom of Parkinson's disease is: damage to mitochondria of hippocampal neuron cells or glial cells, resulting in an increase in membrane potential and an increase in intracellular reactive oxygen species levels.
  • the N-terminus of the polypeptide is modified by acetylation and the C-terminus is modified by amidation.
  • amino acid sequence of the polypeptide is FPGSDRF-RGD, wherein amino acid S is phosphorylated.
  • the drug includes a carrier, and the carrier is a pharmaceutically acceptable carrier.
  • the polypeptides involved in the present invention are part of the series of polypeptides recorded in the Chinese invention patent number CN201310178099X and authorization announcement number CN103239710B.
  • the inventor has confirmed through practical research that these polypeptides have therapeutic effects on Parkinson's disease. They can directly reach brain tissue astrocytes/microglia through targeted integrin molecules through blood-brain barriers and other barriers, and enter cells to regulate proliferation and Activation and other effects can effectively inhibit the excessive activation or proliferation of astrocytes/microglia; it can significantly increase the level of antagonistic excitatory neurotoxicity of dopamine neurons and reduce neuron death. Therefore, these polypeptides can be used as candidate molecules for the treatment or prevention of Parkinson's disease, or specific diseases or symptoms of Parkinson's disease, and can be used to prepare corresponding drugs, and have good application prospects.
  • Figure 1 is a corresponding drawing illustrating the content mentioned in the background of the present invention.
  • Figure 2 is a diagram showing the analysis results of JWA expression levels in whole blood samples of patients with early Parkinson's disease and healthy controls in Example 1 of the present invention.
  • the Ctrl group is a healthy control group
  • the PD group is patients with early Parkinson's disease.
  • Figure 3 is a diagram showing the analysis results of the relationship between JWA and NF- ⁇ B expression levels in whole blood samples of early-stage Parkinson's disease patients in Example 2 of the present invention.
  • Figure 4 is a diagram showing the analysis results of JWA and TH expression levels in brain tissue of Parkinson's disease patients in Example 3 of the present invention.
  • Figure 5 is a graph showing the results of TH and JWA protein expression levels in the brain tissue of wild-type C57BL/6 mice of different ages in Example 4 of the present invention.
  • Figure 6 is a graph showing the results of TH and JWA protein levels in the striatum tissue of wild-type C57BL/6 mice of different ages in Example 5 of the present invention.
  • FIG. 7 is a diagram showing the design scheme of JP1 treatment of MPTP-induced mouse Parkinson's disease model in Example 6 of the present invention.
  • the figure shows the schedule of MPTP modeling time and JP1 intervention time, as well as the administration method.
  • MPTP is administered by subcutaneous injection, while JP1 is administered by tail vein injection, both once a day; the administration time of JP1 is longer than that of MPTP 1 day in advance, and MPTP administration time is 2 hours after JP1.
  • Figure 8 is a graph showing the results of JP1 reversing MPTP-induced abnormal exploratory behavior of mice in PD in Example 7 of the present invention (open field experiment).
  • the solvent control group solvent + JP1 group
  • MPTP group MPTP + JP1 group.
  • Figure 9 is a graph showing the results of JP1 reversing the abnormal behavior of mice caused by MPTP (pole climbing and rotarod experiments) in Example 8 of the present invention.
  • the solvent control group solvent + JP1 group
  • MPTP group MPTP + JP1 group.
  • Figure 10 is a diagram showing the results of dopamine neuron loss (anti-TH IHC staining) in PD mice caused by JP1 antagonizing MPTP in Example 9 of the present invention.
  • the solvent control group solvent + JP1 group
  • MPTP group MPTP + JP1 group.
  • Figure 11 is a diagram showing the results of dopamine neuron loss (Nissl staining) in PD mice caused by JP1 antagonizing MPTP in Example 10 of the present invention.
  • the solvent control group solvent + JP1 group
  • MPTP group MPTP + JP1 group.
  • Figure 12 is a graph showing the results of astrogliosis (anti-GFAP IHC staining) in PD mice caused by JP1 antagonizing MPTP in Example 11 of the present invention.
  • the solvent control group solvent + JP1 group
  • MPTP group MPTP + JP1 group.
  • Figure 13 is a diagram showing the results of astrogliosis (anti-Iba1IHC staining) in PD mice caused by JP1 antagonizing MPTP in Example 12 of the present invention.
  • the solvent control group solvent + JP1 group
  • MPTP group MPTP + JP1 group.
  • Figure 14 is a graph showing the results of JP1 antagonizing the toxicity of rotenone (Rot) to the mouse hippocampal neuron cell line HT-22 and the human neuroblastoma cell line SH-SY5Y in Example 13 of the present invention.
  • Pictures A and B show the cell survival rate of HT-22 cells treated with Rot alone or Rot and JP1 together for 24 and 48 hours respectively;
  • Pictures C and D show SH-SYSY cells treated with Rot alone or Rot and JP1 together 24 respectively. and cell viability after 48 hours.
  • Figure 15 shows the abnormal expression of inflammasome (NLRP3) and apoptosis-related molecules (PARP1, Cleaved caspase1, 3, 9) in HT-22 and SH-SYSY cells caused by JP1 antagonizing rotenone (Rot) in Example 14 of the patent of the present invention.
  • NLRP3 inflammasome
  • PARP1 apoptosis-related molecules
  • Rot JP1 antagonizing rotenone
  • Figures 16 and 17 are respectively the results of mitochondrial damage (mitochondrial membrane potential JC1) and oxidative stress (reactive oxygen species ROS) in HT-22 and SH-SYSY cells caused by JP1 antagonizing rotenone (Rot) in Example 15 of the present invention.
  • JWA expression levels in whole blood samples from early-stage Parkinson's disease patients and healthy controls were analyzed based on data from international public databases.
  • This example is to detect the protein expression levels of TH and JWA in the brain tissue of wild-type C57BL/6 mice of different ages.
  • This example is to detect TH and JWA protein levels in the striatum tissue of wild-type C57BL/6 mice of different ages.
  • JP1 is a protocol designed to verify the effect of JP1 in treating MPTP-induced Parkinson's disease in mice.
  • the sequence of JP1 is FPGSDRF-RGD, in which the amino acid S is phosphorylated.
  • the design plan to verify the anti-PD effect of JP1 includes: dividing C57BL/6 mice into 4 groups, namely the solvent control group, the solvent + JP1 group, the MPTP group and the MPTP + JP1 group.
  • JP1 was administered via tail vein injection of mice at a dose of 150 mg/kg, once a day for 7 consecutive days; starting from the day after the administration of JP1, MPTP, a PD model poison, was injected subcutaneously into the back of the mice at a dose of It is 30 mg/kg, once a day and given 2 hours after JP1 administration.
  • Probenecid 250 mg/kg is administered intraperitoneally with a microsyringe 1 hour after MPTP injection (currently prepared), MPTP and probenecid Shujun was administered continuously for 5 days.
  • MPTP (30 mg/kg) was administered by subcutaneous injection on the back, and probenecid (250 mg/kg) was administered by intraperitoneal injection with a microsyringe 1 hour after MPTP injection (ready to use). Administer for 5 consecutive days.
  • JP1 was administered via tail vein injection of mice at a dose of 150 mg/kg, once a day for 7 consecutive days.
  • the starting point of timing is unified for each group, with the first dose of JP1 as day 1, and the first dose of MPTP as day 2.
  • the behavioral changes of the model mice were evaluated.
  • the model was terminated, brain tissue was taken, and brain slices were prepared for detection and analysis of PD-related molecular markers and evaluation of treatment effects.
  • This example is to evaluate the therapeutic effect of JP1 on reversing MPTP-induced abnormal exploratory behavior in mice with PD (open field experiment).
  • the open field test is a detection index used to verify the behavioral changes in the open field test of MPTP-treated PD mice.
  • the detection and evaluation time point is the 13th day after the start of administration in the design scheme of Example 6. Used to detect spontaneous activity and exploratory behavior in mice. PD model mice often show reduced spontaneous transport and inactive exploratory behavior, and are insensitive to new external environments.
  • This example is to evaluate the therapeutic effect of JP1 on reversing behavioral abnormalities in mice caused by MPTP (pole climbing and rotarod experiments).
  • Rod climbing and rotarod experiments are behavioral indicators used to verify the neural coordination ability of MPTP-treated PD mice.
  • the detection and evaluation time point is the 13th day after the start of administration in the design scheme of Example 6.
  • the rod-climbing test is used to evaluate basal ganglia-related movement disorders in mice, and the rotarod test mainly detects the coordination function of the central nervous system of mice.
  • PD can be manifested as prolonged pole climbing time, reduced coordination ability of pole rotation, and shortened pole climbing time.
  • This example is to evaluate the therapeutic effect of JP1 antagonizing MPTP-induced dopamine neuron loss in PD mice (anti-TH IHC staining).
  • TH is a monooxygenase, which is the rate-limiting enzyme that catalyzes the first step in a series of reactions for the body's own synthesis of L-Dopamine (DA).
  • DA L-Dopamine
  • This example is to evaluate the therapeutic effect of JP1 antagonizing MPTP-induced dopamine neuron loss in PD mice (Nissl staining).
  • Nissl granules in neuronal cytoplasm were stained to evaluate the number of dopamine neurons in the midbrain of MPTP-induced PD model mice.
  • the detection and evaluation time point is the 14th day after the start of administration in the design scheme of Example 6.
  • Nissl bodies are the site of neuronal protein synthesis. When neurons are stimulated and damaged, the number of Nissl bodies decreases. Therefore, high staining results are often used to observe neuronal damage.
  • This example is to evaluate the therapeutic effect of JP1 on antagonizing astrogliosis in PD mice caused by MPTP (anti-GFAP IHC staining).
  • Glial-derived fibrillary acidic protein GFAP is a specific molecular marker of astrocytes, and its expression level reflects the number and activation degree of brain astrocytes.
  • This example is to evaluate the therapeutic effect of JP1 on antagonizing MPTP-induced microglial proliferation in PD mice (anti-Iba1IHC staining).
  • Microglia are the main immune inflammatory cells in brain tissue.
  • Calcium ions are one of the most important signaling molecules known to regulate signals in all cells, including central nervous system (CNS) cells. Calcium ions exert signaling activities by binding to various calcium-binding proteins, many of which are divided into a large protein family, the EF chiral protein family.
  • Calcium adapter protein-1 (Iba1) is a 17 kDa EF chiral protein expressed in microglia, and its expression increases during the activation of these cells.
  • This example is to evaluate the protective effect of JP1 on the viability of mouse hippocampal neuronal cells HT-22 and human glioblastoma cells (SH-SYSY) induced by rotenone (Rot) respectively (CCK8 test).
  • This example is to evaluate the antagonistic effect of JP1 on the inflammatory apoptosis mechanism of HT-22 neuronal cells and SH-SYSY glial cells induced by rotenone (Rot) respectively.
  • This example is to evaluate the mitochondrial damage (mitochondrial membrane potential JC1) and its oxidative stress mechanism (reactive oxygen species ROS) in HT-22 and SH-SYSY cells caused by JP1 antagonizing rotenone (Rot).
  • mitochondrial damage mitochondrial membrane potential JC1
  • ROS reactive oxygen species
  • This example is to verify the anti-Parkinson's disease effect of JWA polypeptides other than JP1.
  • each JWA polypeptide shown in the table below is used for detection according to Examples 6 to 12.
  • the amino acid S of each JWA polypeptide is phosphorylated.
  • JWA polypeptides represented by JP1 on MPTP-induced mouse model Parkinson's disease.
  • JP1 JWA polypeptides represented by JP1
  • JWA polypeptides can pass through blood-brain and other barriers and directly reach brain tissue astrocytes/microglia through targeted integrin molecules, and enter the cells to regulate proliferation and activation, effectively inhibiting astrocytes/microglia.
  • Excessive activation or proliferation of glial cells and the resulting death of dopamine neurons; these JWA polypeptides can significantly increase the level of antagonistic excitotoxicity of dopamine neurons and reduce neuronal death; among them, they are maintained by reducing the loss of dopamine neurons It mediates neurobehavioral homeostasis.
  • these JWA peptides can be used as candidate molecules for the treatment or prevention of Parkinson's disease, and can be used to prepare drugs for the treatment or prevention of Parkinson's disease, and have good application prospects.
  • Main chemical reagents include: sodium chloride, acrylamide, ammonium persulfate, acrylamide, N',N',N',N'-tetramethylethylenediamine (TEMED), sodium bicarbonate, Sodium hydrogen phosphate, disodium hydrogen phosphate and sodium dodecyl sulfate (SDS).
  • Immunohistochemistry DAB chromogenic reagent (AR1000) was purchased from Wuhan Doctor's Bioengineering Co., Ltd. MPTP, probenecid and polylysine (Sigma-Aldrich, USA).
  • Peptides including JP1, etc.
  • Hybio Pharmaceutical Co., Ltd (Shenzhen, China) under standard GMP conditions. The purity is >98%, water-soluble, and the freeze-dried powder is stored at -20°C for a long time.
  • the primary anti-JWA monoclonal antibody was prepared by Beijing Jingtiancheng Biotechnology Co., Ltd. commissioned by our laboratory. Primary antibodies: GLT-1, GLAST, p-P65 antibody (Abcam, Cambridge, UK); Iba-1 antibody (Proteintech, Chicago, IL, USA); TH antibody (Sigma-Aldrich St. Louis, MO, USA); GFAP (SAB Signalway Antibody, Maryland, USA).
  • mice 2.5% chloral hydrate + 5% urethane, starting dose 0.1mL/20g, intraperitoneal administration of 0.2mL.
  • Formula 10mL NS+0.25g chloral hydrate+0.5g urethane.
  • Human neuroblastoma cell line SH-SY5Y and mouse hippocampal neuron cell line HT-22 were cultured in a medium containing 100 ⁇ g/ml streptomycin, 100 U/ml penicillin and 10% fetal bovine serum. Culture in DMEM culture medium at 37°C in an incubator with a 5% CO2 environment. Cells were treated with rotenone (Rot, 2.5 ⁇ M) and JWA targeting peptide JP1 (0, 25, 50, 100 ⁇ M) for 24 and 48 hours respectively.
  • Model cells were tested using CCK8 assay to detect cell viability, Western Blot to detect the expression levels of cellular inflammasome (NLRP3) and apoptosis-related proteins (PARP1, Cleaved caspase1, 3, 9), and immunofluorescence staining to detect cell mitochondrial membrane potential (JC1) and intracellular reactive oxygen species (ROS) levels.
  • CCK8 assay Western Blot to detect the expression levels of cellular inflammasome (NLRP3) and apoptosis-related proteins (PARP1, Cleaved caspase1, 3, 9)
  • PARP1 apoptosis-related proteins
  • JC1 cell mitochondrial membrane potential
  • ROS reactive oxygen species
  • mice 3-4 month old C57BL/6 mice weighing 25-30g were used in the experiment. All mice were maintained in the Experimental Animal Center of Nanjing Medical University (SPF environment). The mice were allowed to eat and drink freely. They were fed standard feed. The indoor temperature was maintained at (24 ⁇ 2)°C, the indoor humidity was 50-60%, and the ventilation was good. The light and dark times were 12 hours each day. Experiments were conducted at Nanjing Medical University All studies were conducted in accordance with the Nanjing Medical University Laboratory Animal Research Guidelines and approved by the Institutional Animal Care and Use Committee of Nanjing Medical University.
  • MPTP mouse PD model Preparation of MPTP mouse PD model: Prepare MPTP storage solution (200mg/kg) with sterile physiological saline and store it in a -80°C ultra-low temperature refrigerator. When used, thaw it on ice and dilute it 10 times with sterile physiological saline. MPTP (30 mg/kg) was administered by subcutaneous injection on the back, and probenecid (250 mg/kg) was administered by intraperitoneal injection with a microsyringe 1 hour after MPTP injection (ready to use). Administration was continued for 5 days, and mice were sacrificed uniformly on the 7th day after the last administration. The tissues required for relevant experiments were taken on ice for experiments.
  • mice in the control group were treated with sterile physiological saline and the same dose of probenecid.
  • the JP1 treatment group alone or the JP1 intervention group was diluted with sterile saline and injected intraperitoneally at a dose of 150 mg/kg, once a day for 7 consecutive days.
  • Model mice were treated with MPTP starting on the second day after JP1 injection.
  • MPTP was administered 2 hours after JP1 administration.
  • Probenecid 250 mg/kg was administered intraperitoneally with a microsyringe 1 hour after MPTP injection (currently prepared). , both MPTP and probenecid were administered continuously for 5 days.
  • JP1 was administered for the last time on the 7th day.
  • the model mice were subjected to behavioral testing and evaluation.
  • the mice were sacrificed the next day, i.e., the 14th day of the model, and brain tissue was collected. Brain slices were prepared for detection and evaluation of relevant molecular markers.
  • Cells were seeded in a 96-well plate at a density of 8,000 cells/well and allowed to adhere for 24 hours. Discard the culture medium, add prepared drugs of different concentrations, and culture the cells for 24 and 48 hours respectively. Discard the original culture medium of the 96-well plate, add a mixture of 10 ⁇ l CCK-8 reagent and 90 ⁇ l fresh culture medium to each well, and incubate in a 37°C, 5% CO2 incubator for about 2 hours.
  • the enzyme-linked immunoassay instrument uses a wavelength of 450 nm to read the absorbance value. Calculate cell survival rate (set 3 parallel samples in each group, repeated 3 times).
  • the cells are treated according to the test requirements, according to the instructions of the cell mitochondrial membrane potential detection and intracellular reactive oxygen species detection reagents (Nanjing Biyuntian), avoid light, and load the cells with the DCFH-DA probe (the ratio of probe to culture medium volume is 1: 1000), incubate in a cell culture incubator at 37°C for 20 minutes. Wash the cells three times with serum-free DMEM cell culture medium. Observe under a fluorescence inverted microscope. Observe 5 fields of view in each hole and take the average after recording.
  • the cell mitochondrial membrane potential detection and intracellular reactive oxygen species detection reagents Najing Biyuntian
  • the rotation speed of the rotary rod should be uniform (12 rpm) and the training time should not be too long (no more than 300 seconds).
  • the rotarod should be rotated at a constant speed (12 rpm). The rotation speed should be accelerated (5-20rpm), and the time the mouse stays on the rod is recorded.
  • Open-Field Test Put the mice in an open field to adapt for 15 minutes (the size of the open field is 20cm ⁇ 20cm ⁇ 15cm), and use Open field software to record the total distance crawled by the mice within 10 minutes.
  • mice were anesthetized by intraperitoneal injection of pentobarbital. After anesthesia, 100 ml of physiological saline and 100 ml of 4% paraformaldehyde were perfused through the left ventricle. Carefully peel off the skull and remove the brain tissue. The brain tissue is fixed with 4% paraformaldehyde and placed in a 4°C refrigerator overnight.
  • the thickness of the slices is 25 ⁇ m.
  • accurately find the area to be sliced. Take 1 for striatum septum 6 and 3 for midbrain septum. Take 1, wash the brain slices with PBS 3 times and finally collect it into a 1.5ml EP tube. Add PBS and glycerol (1:1) and store it in a -20°C refrigerator for long-term storage.
  • the primary antibodies mainly used in this experiment are JWA, tyrosine hydroxylase, anti-glial-derived acidic protein and glutamate transporter.
  • mice brain tissue The day after the behavioral test, collect the mouse brain tissue. Specific steps: After taking blood from the eyeballs, kill the mouse by cervical dissection. Carefully remove the mouse braincase and remove the brain tissue. Try to keep the brain tissue intact and make small symmetrical incisions. Mouse brain tissue was separated from the midbrain, striatum and hippocampus, weighed and added with protein lysis solution RIPA at a ratio of 1:8 and lysed in a refrigerator at 4°C for 30 minutes. Centrifuge at 12000 ⁇ g for 15 minutes at 4°C and collect the supernatant.
  • BCA bovine serum albumin
  • Cell protein preparation Aspirate the culture medium in the cell culture plate, rinse twice with pre-cooled PBS buffer or NaCl, add protein lysis solution, and place in a 4°C refrigerator for lysis for 30 minutes. Centrifuge at 12000g for 15 minutes at 4°C. Take 1 ⁇ l protein sample and measure the protein concentration according to the method for determining tissue protein concentration. The remaining protein supernatant is added to 6 ⁇ loading buffer by volume and denatured in a 100°C metal bath for 5 minutes. After aliquoting -20 Store at °C.
  • PVDF nitrocellulose membrane

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Abstract

提供了JWA多肽在制备抗帕金森病药物方面的应用，所述多肽的氨基酸序列如I或II所示：I：FPGSDRF-Z；II：X-FPGSDRF-Z；其中，氨基酸S经磷酸化修饰，X、Z分别为氨基酸或氨基酸序列；X选自F、(R) 9、(R) 9-F、6-氨基己酸、6-氨基己酸-F、6-氨基己酸-(R) 9、6-氨基己酸-(R) 9-F之一；Z选自(G) n-RGD、A-(G) n-RGD之一，n为大于或等于0的整数，且n的取值范围为0-10。上述多肽可作为针对帕金森病、或帕金森病的具体病症或病征进行治疗或预防的候选分子。

Description

JWA多肽在制备抗帕金森病药物方面的应用 技术领域
本发明涉及JWA多肽在制备抗帕金森病药物方面的应用，属于中枢神经系统药物技术领域。
背景技术
帕金森氏病(Parkinson’s disease，PD)为常见的神经退行性疾病，其主要病理特征为：黑质致密部(SNc)多巴胺(dopamine，DA)神经元变性、缺失，纹状体DA递质减少，胶质细胞增生，并且伴随着胞内嗜酸性路易小体形成。临床流行病学调查结果显示，65岁以上人群PD患病率达超过1％，并且随着年龄的增长PD的患病率也呈增加的趋势，85岁以上人群PD患病风险进一步增加到4％[1]。2010年，美国大约有630,000人被诊断患有PD，到2040年，确诊率可能翻倍；在美国，PD的经济负担在2010年超过144亿美元(每名患者约22,800美元)，预计在未来几十年内将大幅增长[2]。中国目前帕金森病患者人数已达到约200多万人，约占全球50％。同时，目前帕金森发病呈现年轻化趋势。虽然对PD的认识由来已久，但PD的发病机制尚不清楚，也缺乏行之有效的治疗办法，深入认识PD的病理机制并建立有效的治疗策略显得尤为重要。
帕金森病作为全球第二大神经退行性疾病，影响着无数患者的身心健康。PD患者主要出现以下临床症状：运动障碍、肌肉强直、静止性震颤等运动症状，并伴有嗅觉减退、睡眠障碍、精神症状、认知障碍等非运动症状。PD确切的发病机制尚不清楚，因此PD仍然是一种不能治愈的疾病。临床治疗上主要用左旋多巴(Levodopa，L-Dopa)弥补缺失的多巴胺(Dopamine，DA)，从而缓解PD的运动症状，但这种疗法治标不治本，无法治愈PD或延缓疾病的进一步发展，且长期服用还会引发一系列的并发症，如运动障碍(L-Dopa-induced dyskinesias，LIDs)、症状波动等。因此，亟需深入探究PD的致病机制，为开发治疗PD的新型疗法提供方向。
环境及遗传因素均是PD最为重要的致病因素。研究表明，常染色体显性遗传基因(SNCA、LRRK2)及常染色体隐性基因(PARK2、DJ1、PINK1、ATP13A2)可以通过线粒体功能紊乱、氧化应激、神经炎症、自噬不足及溶酶体功能障碍等多种机制引起DA神经元的死亡[3,4]。环境毒物的急性或慢性暴露可导致包括神经系统在内的人体多器官系统的损伤。在农业方面主要是农药的大量使用，如杀虫剂鱼藤酮和除草剂百草枯等；在工业方面主要是重金属元素的污染。1-甲基-4-苯基-1,2,3,6-四氢砒啶(MPTP)的暴露引起PD的发现，引起了对农药暴露与PD关系的研究，因为百草枯和鱼藤酮等农药均为MPTP的结构和功能类似物。流行病学研究表明农药暴露、务农和农村生活与患神经退行性疾病的风险相关，其高剂量暴露可增加多巴胺神经元的死亡，从而增加帕金森疾病的发生率[5]。鱼藤酮(Rotenone，RT)和百草枯(Paraquat，PQ)一直被视为有效的杀虫剂和除草剂而广泛应用于农业。但近年来，其对中枢神经的毒性作用以及可能与PD的潜在联系成为人们关注的焦点[6,7]。流行病学研究报道了长期使用RT和PQ与PD高发病率存在正相关[8]。动物和细胞水平的机制研究表明，二者与MPTP的代谢产物1-甲基-4-苯基吡啶离子(MPP +)的作用靶点相同，它们均可抑制线粒体电子转移链复合酶Ⅰ的活性，损害线粒体的功能，并引起氧化应激和炎症反应从而诱导PD的发生[9,10]。要有效降低环境毒物对多巴胺神经元的损伤，减少PD的发生，就必须对环境毒物致神经系统损伤的分子机制有深入了解，才能提出创新性的防治策略。
为了研究DA神经元损伤致神经退行性病变的机制，研究者把注意力主要集中在受损的神经元。然而，大量的研究表明邻近的神经胶质细胞在这些神经元死亡进程中也起着重要的作用。哺乳动物脑中，星形胶质细胞是含量最多的胶质细胞，是神经系统的重要组成部分，在人脑中星形胶质细胞的数量更是神经元的数倍[11]。星形胶质细胞在所有的脑区都有分布，并且与神经元紧密结合具有重要的“战略地位”。长期以来，星形胶质细胞被视为仅为神经元提供结构支持的胶质细胞。越来越多的研究表明，星形胶质细胞结构和功能的异常在多种神经性退行性疾病、脑损伤及中枢神经系统炎症方面都具有重要的作用。星形胶质细胞可以维持细胞外环境，稳定细胞之间的通信并介导神经元的生理及病理状态[12]。星形胶质细胞发挥其功能主要是依赖于它们可以释放和吸收细胞外微环境中的分子，从而起到对神经元的保护或损伤作用。因此，阐明环境毒物致DA神经元损伤中星形胶质细胞所扮演的角色，对于科学制定环境毒物引起神经毒性的防治策略具有非常重要的意义。
星形胶质细胞可以通过不同的分子机制影响神经元的存活。在神经元和星形胶质细胞联合培养模型中，当星形胶质细胞存在时，神经元对氧化损伤的抵抗能力增强。谷氨酸是中枢神经系统含量最多的兴奋性氨基酸(excitatory amino acid，EAA)。谷氨酸引起的兴奋毒性是中枢神经系统出血、创伤和神经退行性疾病等神经元死亡的重要机制[13]。突触间隙中的一部分EAA在酶的作用下被降解，另一部分被位于神经元和胶质细胞膜上的EAA转运体(EAA transporters，EAAT)摄回而被迅速灭活。当细胞间隙的谷氨酸含量过高时，可以与神经元表面的谷氨酸受体结合，引起神经元的兴奋性毒性，导致神经元的死亡。星形胶质细胞具有摄取谷氨酸的能力，其特异性的表达GLAST及GLT-1是谷氨酸摄取的关键分子。中枢神经系统中超过90％的谷氨酸都是通过GLT-1进行再摄取的[14]。一旦谷氨酸的摄取发生障碍，导致细胞间隙兴奋性氨基酸含量过高，即可引起神经兴奋性毒性从而导致PD在内的多种神经退行性疾病的发生。GLT-1的启动子上有多个NF-κB的结合位点，多种毒性分子及生长因子可以通过激活星形胶质细胞中的NF-κB信号通路增强GLT-1的表达，减弱兴奋性神经毒性。然而，在促炎因子TNF-α的刺激下，星形胶质细胞NF-κB信号通路被激活后并没有提高GLT-1的表达，反而，NF-κB与N-myc在GLT-1启动子上相互结合从而抑制了GLT-1的表达。由此可见，星形胶质细胞GLT-1的表达对于神经兴奋毒性的影响至关重要，而NF-κB活化是激活或抑制GLT-1所必需的[15]。
与神经元相比，胶质细胞拥有更强大的抗氧化酶合成系统，胞内抗氧化酶和Ⅱ型解毒酶的含量也远高于神经元。存在这种差别的原因之一是，与神经元相比，星形胶质细胞中的核因子E2相关因子(Nrf2)被优先激活[16,17]。Nrf2激活后可以与启动子上具有抗氧化应激反应元件(ARE)的基因结合，从而激活多种细胞保护性基因的表达。星形胶质细胞Nrf2的表达高低对整个神经系统抵御氧化损伤的能力起决定性作用，在小鼠MPTP模型中伴随着Nrf2的功能缺失。多种激酶能够引起Nrf2的激活，包括PKC、蛋白激酶CK2、PI3K、JNK、ERK等，因而磷酸化是调控Nrf2依赖性基因表达的关键[18-21]。在真核细胞中，微管、微丝和中间丝(IFs)共同组成细胞的骨架结构。在神经系统中，IFs主要存在于神经元和星形胶质细胞中。星形胶质细胞中的IFs包括巢蛋白、波形蛋白、联丝蛋白等，胶质纤维酸性蛋白(Glial fibrillary acid protein，GFAP)是最主要的IF。在多种CNS疾病中，星形胶质细胞体积过度增大，由常态的星形胶质细胞变为活化的星形胶质细胞。在此过程中，IFs特别是GFAP的表达量增加。PD病人与正常人相比其下丘脑GFAP的表达量也显著增加[22]，提示星形胶质细胞细胞骨架成分GFAP 在CNS疾病中发挥重要作用[23]。综上分析，环境毒物致黑质纹状体多巴胺神经元的选择性毒作用，涉及NF-κB信号通路激活、蛋白的磷酸化以及细胞骨架改变等复杂机制。
JWA基因(又名ARL6IP5)，是周建伟等率先从维甲酸诱导的人支气管上皮(HBE)细胞分化模型中发现和克隆并长期聚焦研究的环境应答基因，其编码蛋白是一种细胞骨架结合蛋白，在正常细胞可参与调节细胞分化、应答氧化应激、DNA修复等过程。周建伟教授带领课题组长期围绕着JWA基因在肿瘤及神经系统中的功能及作用展开研究。我们用剔除JWA基因的果蝇模型发现，JWA表达缺陷的果蝇不易对反复暴露乙醇产生耐受[24]。用反义核酸抑制JWA表达的大鼠和细胞模型发现，JWA通过泛素蛋白酶体途径维护鸦片受体DOR的稳定性，从而对大鼠吗啡依赖有直接的调节作用[25]。JWA参与调节NF-κB和MAPK等信号通路，在细胞水平上已证实JWA通过调控NF-κB转录因子活性参与调控细胞衰老等，其机制是JWA通过泛素蛋白酶体途径调节IKKβ的降解，且抑制p65的入核。在氧化应激条件下，H 2O 2诱导细胞内核因子NFI与JWA近端启动子区CCAAT元件结合，从而激活JWA的表达应答氧化应激[26]；JWA通过MAPK信号通路调节核因子E2F1和XRCC1表达而增强对DNA损伤的修复能力。
前期在以A129小鼠为背景的星形胶质细胞JWA基因剔除小鼠研究中发现，星形胶质JWA基因敲除之后对MPTP极其敏感，提示星形胶质细胞中的JWA是中枢神经系统抵御外界毒物刺激的关键分子[27]。JWA通过MAPK和PI3K信号通路抑制百草枯产生的氧化应激并激活GSH和Nrf2，有效拮抗百草枯所致的小鼠多巴胺神经元损伤(见图1的A图)[28]；为进一步研究在PD进程中星形胶质细胞JWA所扮演的角色，采用C57背景的星形胶质细胞JWA基因敲除小鼠构建MPTP慢性模型，并深入探索星形胶质细胞JWA影响DA神经元存活的分子基础，结果发现，星形胶质细胞JWA基因的缺失能够增加小鼠对MPTP及百草枯的敏感性。在机制研究中，我们首次发现JWA基因能通过MAPK/ERK及PI3K/Akt两条信号通路激活下游的CREB转录因子对下游的GLT-1产生调控，并最终引起细胞外间隙谷氨酸含量的改变(见图1的B图)[29]。此外，JWA还下调IKKβ从而抑制NFκB信号通路介导的神经炎症对PD的影响(见图1的C图)[30]。这些发现作为早期研究成果，为实现针对星形胶质细胞JWA为靶点的PD治疗奠定了基础。
前期课题组基于JWA功能片段筛选的多肽JP1，通过其连接的RGD序列靶向高表达整合素αVβ3后进入胞内，负调控核转录因子SP1，下调αVβ3表达，可有效抑制小鼠黑色素瘤生长、转移；进一步研究发现，该多肽可以穿过血脑屏障[31]。
虽然JP1连接RGD序列后能够靶向黑色素瘤表面高表达的整合素αVβ3，但是JP1靶向肽能否用于治疗中枢神经系统疾病如帕金森病等尚不清楚，亟待进一步探索和研究。对此，发明人课题组已有最新的研究成果，并以此来申请本发明专利。
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发明内容
本发明的主要目的是：针对现有技术存在的问题，提出一种JWA多肽在制备抗帕金森病药物方面的应用，能对脑组织星形胶质细胞/小胶质细胞发挥调控增殖和活化等作用，以有效抑制星形胶质细胞/小胶质细胞的过度活化，并且能显著提高多巴胺神经元拮抗兴奋性神经毒性水平，减少神经元死亡，为治疗帕金森病提供新的临床用药可能。
本发明解决其技术问题的技术方案如下：
一种多肽的用途，其特征是，所述用途为用于制备预防或治疗帕金森病的药物；
所述多肽的氨基酸序列如I或II所示：
I：FPGSDRF-Z；
II：X-FPGSDRF-Z；
其中，氨基酸S经磷酸化修饰，X、Z分别为氨基酸或氨基酸序列；
X选自F、(R) 9、(R) 9-F、6-氨基己酸、6-氨基己酸-F、6-氨基己酸-(R) 9、6-氨基己酸-(R) 9-F之一；
Z选自(G) n-RGD、A-(G) n-RGD之一，n为大于或等于0的整数，且n的取值范围为0-10。
优选地，所述用途为用于制备预防或治疗帕金森病的具体病症或病征的药物。
优选地，所述帕金森病的具体病症或病征为：脑组织星形胶质细胞或小胶质细胞的过度活化或增生。
优选地，所述帕金森病的具体病症或病征为：脑组织多巴胺神经元变性或缺失。
优选地，所述帕金森病的具体病症或病征为：海马神经元细胞或神经胶质细胞受到细胞毒性作用导致活力减弱。
优选地，所述帕金森病的具体病症或病征为：海马神经元细胞或神经胶质细胞的炎性小体表达增加以及细胞凋亡增加。
优选地，所述帕金森病的具体病症或病征为：海马神经元细胞或神经胶质细胞的细胞线粒体损伤而致膜电位增加以及胞内活性氧水平升高。
优选地，所述多肽的N端经乙酰化修饰、C端经酰胺化修饰。
优选地，所述多肽的氨基酸序列为FPGSDRF-RGD，其中，氨基酸S经磷酸化修饰。
优选地，所述药物包括载体，所述载体为药学上可接受的载体。
本发明涉及的多肽是专利号CN201310178099X、授权公告号CN103239710B的中国发明专利中记载的系列多肽中的一部分。发明人经实践研究证实这些多肽对帕金森病具有治疗效果，可通过血脑等屏障经靶向整合素分子直接到达脑组织星形胶质细胞/小胶质细胞，并进入细胞发挥调控增殖和活化等作用，有效抑制星形胶质细胞/小胶质细胞的过度活化或增生；能显著提高多巴胺神经元拮抗兴奋性神经毒性水平，减少神经元死亡。因此，这些多肽可作为针对帕金森病、或针对帕金森病的具体病症或病征进行治疗或预防的候选分子，用于制备相应药物，具有良好的应用前景。
附图说明
图1为本发明背景技术提及内容的相应附图。
图2为本发明实施例1中帕金森病早期患者与健康对照人群全血样本JWA表达水平的分析结果图。其中，Ctrl组为健康对照人群，PD组为早期帕金森病患者。
图3为本发明实施例2中帕金森病早期患者全血样本JWA与NF-κB表达水平关系的分析结果图。
图4为本发明实施例3中帕金森病患者脑组织JWA与TH表达水平的分析结果图。
图5为本发明实施例4中不同年龄野生型C57BL/6小鼠中脑组织TH和JWA蛋白质表达水平的结果图。
图6为本发明实施例5中不同年龄野生型C57BL/6小鼠纹状体组织TH和JWA蛋白质水平的结果图。
图7为本发明实施例6中JP1治疗MPTP诱导的小鼠帕金森病模型设计方案图。图中显示了MPTP造模时间和JP1干预时间的方案，以及给药处理方式，MPTP以皮下注射方式给药，而JP1以尾静脉注射方式给药，均每天1次；JP1比MPTP给药时间提前1天，MPTP给药时间在JP1之后2小时。
图8为本发明实施例7中JP1逆转MPTP所致小鼠PD探索性行为异常(旷场实验)的结果图。右图中，从左到右依次为溶剂对照组，溶剂+JP1组，MPTP组，MPTP+JP1组。
图9为本发明实施例8中JP1逆转MPTP所致小鼠行为异常(爬杆和转棒实验)的结果图。各图中，从左到右依次为溶剂对照组，溶剂+JP1组，MPTP组，MPTP+JP1组。
图10为本发明实施例9中JP1拮抗MPTP所致PD小鼠多巴胺神经元缺失(抗-TH IHC染色)的结果图。右图中，从左到右依次为溶剂对照组，溶剂+JP1组，MPTP组，MPTP+JP1组。
图11为本发明实施例10中JP1拮抗MPTP所致PD小鼠多巴胺神经元缺失(尼氏染色)的结果图。右图中，从左到右依次为溶剂对照组，溶剂+JP1组，MPTP组，MPTP+JP1组。
图12为本发明实施例11中JP1拮抗MPTP所致PD小鼠星形胶质细胞增生(抗-GFAP IHC染色)的结果图。右图中，从左到右依次为溶剂对照组，溶剂+JP1组，MPTP组，MPTP+JP1组。
图13为本发明实施例12中JP1拮抗MPTP致PD小鼠星形胶质细胞增生(抗-Iba1IHC染色)的结果图。右图中，从左到右依次为溶剂对照组，溶剂+JP1组，MPTP组，MPTP+JP1组。
图14为本发明实施例13中JP1拮抗鱼藤酮(Rot)致小鼠海马神经元细胞系HT-22和人神经母细胞瘤细胞系SH-SY5Y毒性所致细胞存活率的结果图。A图和B图分别是Rot单独处理或Rot与JP1共同处理HT-22细胞24和48小时后细胞存活率；C图和D图分别是Rot单独处理或Rot与JP1共同处理SH-SYSY细胞24和48小时后细胞存活率。
图15为本发明专利实施例14中JP1拮抗鱼藤酮(Rot)所致HT-22和SH-SYSY细胞炎症小体(NLRP3)、凋亡相关分子(PARP1，Cleaved caspase1、3、9)表达异常的结果图。
图16、17分别为本发明实施例15中JP1拮抗鱼藤酮(Rot)所致HT-22和SH-SYSY细胞线粒体损伤(线粒体膜电位JC1)和氧化应激(活性氧ROS)的结果图。
具体实施方式
下面参照附图并结合实施例对本发明作进一步详细描述。但是本发明不限于所给出的例子。各实施例所用的材料、方法、实验模型条件等内容附在各实施例之后，除此之外，如无特殊说明，所用材料、实验方法均为常规材料、常规实验方法。
实施例1
本实施例中，根据国际公开数据库的数据，分析帕金森病早期患者与健康对照人群全血样本JWA表达水平。
分析结果如图2所示：通过分析50例早期帕金森病患者和23例健康对照人群的血液样本JWA表达水平，结果表明早期PD患者血液中JWA表达水平与健康对照者相比明显降低，这提供了JWA是早期PD患者细胞血液中受干扰的分子的证据。
实施例2
本实施例中，根据国际公开数据库的数据，分析帕金森病早期患者全血样本JWA与NF-κB表达水平关系。
分析结果如图3所示：通过分析50例PD患者全血样本中JWA表达水平与炎症因子NF-κB表达水平的关系，结果表明PD患者JWA表达水平与NF-κB呈负相关关系。(Scherzer CR,Eklund AC,Morse LJ,Liao Z,Locascio JJ,Fefer D,Schwarzschild MA,Schlossmacher MG,Hauser MA,Vance JM,Sudarsky LR,Standaert DG,Growdon JH,Jensen RV,Gullans SR.Proc Natl Acad Sci U S A.2007 Jan 16；104(3):955-60.)
实施例3
本实施例中，根据国际公开数据库的数据，分析帕金森病干细胞JWA与TH表达水平。
分析结果如图4所示：分析12例病人来源的PD干细胞模型获得的JWA与TH表达水平数据，结果表明PD干细胞中JWA表达水平与TH表达水平呈显著正相关。(Ryan SD,Dolatabadi N,Chan SF,Zhang X et al.Isogenic human iPSC Parkinson's model shows nitrosative stress-induced dysfunction in MEF2- PGC1α transcription.Cell 2013 Dec 5；155(6):1351-64.)
实施例4
本实施例为检测不同年龄野生型C57BL/6小鼠中脑组织TH和JWA蛋白质表达水平。
检测结果如图5所示：对不同年龄正常野生型C57BL/6小鼠中脑组织进行蛋白质印迹分析检测，结果显示，随着小鼠年龄增长，小鼠中脑组织酪氨酸羟化酶(Tyrosine hydroxylase,TH)和JWA蛋白质表达水平均呈现逐渐下降的趋势，两个分子变大变化规律一致；TH表达水平降低表明多巴胺神经元逐渐减少，而JWA表达降低提示其抗氧化应激、抗炎和拮抗PD发生的功能逐渐减弱。
实施例5
本实施例为检测不同年龄野生型C57BL/6小鼠纹状体组织TH和JWA蛋白质水平。
检测结果如图6所示：对不同年龄正常野生型C57BL/6小鼠纹状体组织进行蛋白质印迹分析检测，结果显示，随着小鼠年龄增长，小鼠纹状体组织TH和JWA蛋白质表达均呈现逐渐降低趋势，两个分子表达变化规律一致。
实施例6
本实施例为设计验证JP1治疗MPTP诱导的小鼠帕金森病效果的方案。注：JP1的序列为FPGSDRF-RGD，其中，氨基酸S经磷酸化修饰。
如图7所示，验证JP1抗PD效果的设计方案包括：将C57BL/6小鼠分为4组，分别为溶剂对照组，溶剂+JP1组，MPTP组和MPTP+JP1组。
对于MPTP+JP1组：JP1经小鼠尾静脉注射给药，剂量为150mg/kg，每天1次，连续7天；在JP1给药次日起，给予小鼠PD模型毒物MPTP背部皮下注射，剂量为30mg/kg，每天1次且在JP1给药后2小时给予，丙磺舒(250mg/kg)在MPTP注射1h后用微量注射器进行腹腔注射给药(现用现配)，MPTP和丙磺舒均连续给药5天。
对于MPTP组：MPTP(30mg/kg)通过背部皮下注射的方式进行给药，丙磺舒(250mg/kg)在MPTP注射1h后用微量注射器进行腹腔注射给药(现用现配)。连续给药5天。
对于溶剂+JP1组：JP1经小鼠尾静脉注射给药，剂量为150mg/kg，每天1次，连续7天。
对于溶剂对照组：按MPTP组的给药方案，将MPTP等量替换为无菌生理盐水，其余均相同。
各组统一计时起点，以JP1第1次给药为第1天，并以MPTP第1次给药为第2天。在模型开始后第13天对模型小鼠行为学变化特征进行评估，并在第14天结束模型，取脑组织，制备脑片进行PD相关分子标志物检测分析和评估治疗效果。
实施例7
本实施例为评估JP1逆转MPTP所致小鼠PD探索性行为异常的治疗效果(旷场实验)。
旷场实验是用于验证MPTP处理的PD小鼠旷场实验行为学变化的检测指标。检测评估时间点是实施例6设计方案中开始给药的第13天。用于检测小鼠自发活动行为和探索性行为。PD模型小鼠常常表现为自发货都给减少和探索性行为不活跃，对外界新鲜环境不敏感。
结果如图8所示：与溶剂对照组比较，溶剂+JP1组表明，JP1处理对小鼠行自发活动和探索性行为有所增加；MPTP组表明，MPTP处理小鼠则自发活动和到旷场中心区域的探索性行为显著减少(P <0.05)；MPTP+JP1组表明，JP1干预能显著逆转MPTP处理导致的自发活动行为和探索性行为减少(P<0.01)。
实施例8
本实施例为评估JP1逆转MPTP所致小鼠行为异常的治疗效果(爬杆和转棒实验)。
爬杆和转棒实验是用于验证MPTP处理的PD小鼠神经协调能力的行为指标。检测评估时间点是实施例6设计方案中开始给药的第13天。爬杆测试用于评估小鼠基底神经节相关运动障碍，转棒实验主要检测小鼠中枢神经系统协调功能。PD可表现为爬杆时间延长和转棒协调能力降低，时间缩短。
结果如图9所示：与溶剂对照组比较，溶剂+JP1组表明，JP1单独处理对小鼠爬杆时间和从转棒掉落时间并无显著差异；MPTP组表明，MPTP处理后的PD模型小鼠则爬杆时间明显延长(P<0.01)，而从转棒掉落时间明显缩短；MPTP+JP1组表明，JP1干预能显著改善MPTP导致的小鼠爬杆和转棒行为变化(P<0.01)。
实施例9
本实施例为评估JP1拮抗MPTP所致PD小鼠多巴胺神经元缺失的治疗效果(抗-TH IHC染色)。
TH表达变化是用于验证MPTP处理的PD小鼠中脑黒质致密区多巴胺神经元的指标。检测评估时间点是实施例6设计方案中开始给药的第14天。TH是一种单加氧酶，它是催化生物体自身合成左旋多巴胺(L-Dopamine,DA)系列反应的第一步反应的限速酶。TH表达水平实际就反映脑内多巴胺神经元数量和递质水平，PD常导致TH显著减少。
结果如图10所示：与溶剂对照组比较，溶剂+JP1组表明，JP1单独处理对TH表达水平无明显影响；MPTP组表明，MPTP处理组TH表达水平显著下降(P<0.01)；MPTP+JP1组表明，JP1干预能使TH水平明显改善(P<0.01)。
实施例10
本实施例为评估JP1拮抗MPTP所致PD小鼠多巴胺神经元缺失的治疗效果(尼氏染色)。
针对神经元胞浆中尼氏颗粒进行染色以评估MPTP诱导的PD模型小鼠中脑多巴胺神经元数量。检测评估时间点是实施例6设计方案中开始给药的第14天。尼氏小体是神经元蛋白质合成场所，当神经元受到刺激和损伤后尼氏小体数量会减少。因此高染色结果常常用作观察神经元受损情况。
结果如图11所示：与溶剂对照组比较，溶剂+JP1组表明，JP1单独处理对中脑多按按神经元尼氏小体数量没有明显影响；MPTP组表明，MPTP处理后的PD小鼠则尼氏小体明显减少(P<0.01)；MPTP+JP1组表明，JP1干预后可显著逆转MPTP导致的多巴胺神经元尼氏小体数量减少(P<0.01)。
实施例11
本实施例为评估JP1拮抗MPTP所致PD小鼠星形胶质细胞增生的治疗效果(抗-GFAP IHC染色)。
针对PD模型特征以评估脑星形胶质细胞活化和增殖的胶质源性纤维酸性蛋白GFAP表达水平。检测评估时间点是实施例6设计方案中开始给药的第14天。胶质源性纤维酸性蛋白GFAP是星形胶质细胞的特异性分子标志物，其表达水平高低反映脑星形胶质细胞数量和活化程度。
结果如图12所示：与溶剂对照组比较，溶剂+JP1组表明，JP1单独处理对脑星形胶质细胞GFAP 表达水平无显著影响；MPTP组表明，MPTP处理的PD小鼠可见GFAP表达水平显著增加(P<0.01)；MPTP+JP1组表明，JP1干预可显著逆转MPTP导致的GFAP增加(P<0.01)。
实施例12
本实施例为评估JP1拮抗MPTP所致PD小鼠小胶质细胞增生的治疗效果(抗-Iba1IHC染色)。
用于验证MPTP所致PD模型中脑小胶质细胞激活的分子标志物表达水平。检测评估时间点是实施例设计方案中开始给药的第14天。小胶质细胞是脑组织中主要免疫炎症细胞。钙离子是已知的所有细胞(包括中枢神经系统(CNS)细胞)中最重要的信号调节信号分子之一。钙离子通过与各种钙结合蛋白结合发挥信号活动，其中许多钙结合蛋白被划分成一个大的蛋白家族，即EF手性蛋白家族。钙离子接头蛋白-1(Iba1)是一个17kDa的EF手性蛋白，在小胶质细胞中表达，并在这些细胞的活化过程中表达升高。
结果如图13所示：与溶剂对照组相比，溶剂+JP1组表明，JP1单独处理对中脑黒质致密区Iba-1表达水平无明显影响；MPTP组表明，MPTP处理的PD小鼠Iba-1表达显著增加(P<0.01)；MPTP+JP1组表明，JP1干预则可逆转MPTP导致的Iba-1增加(P<0.01)。
实施例13
本实施例为评估JP1分别对鱼藤酮(Rot)致小鼠海马神经元细胞HT-22和人神经胶质母细胞瘤细胞(SH-SYSY)活力(CCK8试验)的保护作用。
结果如图14所示，用Rot(2.5μM)单独处理或Rot(2.5μM)与JP1(25、50、100μM)共同处理HT-22细胞24和48小时后细胞存活率结果显示，与溶剂对照组比较，Rot处理后24小时两种细胞存活力均显著下降，Rot处理48小时后较24小时组两种细胞存活力活力下降更显著。JP1与Rot联合处理组结果显示JP1对Rot细胞毒性作用有明显保护作用，且呈现剂量依赖性。
实施例14
本实施例为评估JP1分别对鱼藤酮(Rot)致HT-22神经元细胞和SH-SYSY神经胶质细胞炎性凋亡机制的拮抗作用。
结果如图15所示，两种细胞分别用对照溶剂、Rot单独处理或ROT+JP1联合处理48小时，该结果表明，与溶剂对照组比较，Rot处理可显著激活和增加两种细胞的炎症小体NLRP3表达水平以及凋亡相关分子(PARP1，Cleaved caspase1、3、9)表达水平；而在Rot与JP1联合处理组，炎症小体和凋亡相关分子的表达水平均受到抑制，且呈现剂量依赖性。
实施例15
本实施例为评估JP1拮抗鱼藤酮(Rot)所致HT-22和SH-SYSY细胞线粒体损伤(线粒体膜电位JC1)及其氧化应激机制(活性氧ROS)。
结果如图16和图17所示，两种细胞分别用对照溶剂、Rot单独处理或ROT+JP1联合处理48小时，该结果表明，Rot处理(2.5μM，48小时)导致两种细胞严重线粒体膜损伤，胞内氧化应激(ROS)显著增加；而Rot与JP1(100μM，48小时)联合处理后，两种细胞的线粒体膜损伤和胞内氧化应激均显著减轻。
实施例16
本实施例为验证除JP1以外的JWA多肽的抗帕金森病效果。
本实施例采用下表所示的各JWA多肽按实施例6至12进行检测，各JWA多肽的氨基酸S经磷酸化修饰。
Figure PCTCN2022138737-appb-000001
受篇幅所限，本实施例未列出具体实验数据。所得实验数据表明，以上各JWA多肽按实施例6至12进行检测的结果均与JP1基本一致。
结论
由以上各实施例可知，本发明证实了以JP1为代表的系列JWA多肽对MPTP诱导的小鼠模型帕金森病的治疗效果。这些JWA多肽可通过血脑等屏障经靶向整合素分子直接到达脑组织星形胶质细胞/小胶质细胞，并进入细胞发挥调控增殖和活化等作用，有效抑制星形胶质细胞/小胶质细胞的过度活化或增生以及由此导致的多巴胺神经元死亡；这些JWA多肽能显著提高多巴胺神经元拮抗兴奋性神经毒性水平，减少神经元死亡；其中，通过减少多巴胺神经元的缺失而维持其介导的神经行为学稳态。此外，经这些JWA多肽干预可显著改善PD小鼠行为异常。因此，这些多肽可作为针对帕金森病进行治疗或预防的候选分子，用于制备治疗或预防帕金森病的药物，具有良好的应用前景。
以上各实施例所用的材料、方法、实验模型条件等如下文所示。
一、主要试剂及抗体及配置
主要化学试剂：国产AR级试剂包括：氯化钠、丙烯酰胺、过硫酸铵、丙烯酰胺、N’,N’,N’,N’－四甲基乙二胺(TEMED)、碳酸氢钠、磷酸二氢钠、磷酸氢二钠和十二烷基硫酸钠(SDS)。免疫组化DAB显色试剂(AR1000)购于武汉博士的生物工程有限公司。MPTP、丙磺舒(probenecid)及多聚赖 氨酸(美国Sigma-Aldrich公司)。多肽(包括JP1等)由GL Biochem(Shanghai)Ltd.和Hybio Pharmaceutical Co.,Ltd(中国深圳，在标准GMP条件下合成，纯度>98％，水溶性，冻干粉长期保存在-20℃。
一抗JWA单克隆抗体由本实验室委托北京京天成生物技术有限公司制备。一抗：GLT-1、GLAST、p-P65抗体(Abcam,Cambridge,UK)；Iba-1抗体(Proteintech,Chicago,IL,USA)；TH抗体(Sigma-Aldrich St.Louis,MO,USA)；GFAP(SAB Signalway Antibody,Maryland,USA)。
免疫组化试剂配制：4％多聚甲醛(PFA，paraformaldehyde)：称量PFA粉末4g，充分溶解于100mL PBS溶液中，过滤后使用。95％乙醇：用量筒，量取无水乙醇190mL，ddH 2O定容至200mL。80％乙醇：用量筒，量取无水乙醇160mL，ddH 2O定容至200mL。70％乙醇：用量筒，量取无水乙醇140mL，ddH 2O定容至200mL。PBST：1L ddH 2O+7g Na 2HPO 4·12H 2O+0.5g NaH 2PO 4·2H 2O+9g NaCl溶于1000mL水中。
小动物全身麻醉药配制：小鼠：2.5％水合氯醛+5％乌拉坦，起始剂量0.1mL/20g,腹腔给药0.2mL。配方：10mL NS+0.25g水合氯醛+0.5g乌拉坦。
二、细胞和动物来源和PD模型制备
1.细胞培养和处理：人神经母细胞瘤细胞系SH-SY5Y、小鼠海马神经元细胞系HT-22均培养于含100μg/ml链霉素、100U/ml青霉素和10％胎牛血清的DMEM培养液中，37℃，5％CO 2环境的培养箱中培养。鱼藤酮(Rot，2.5μM)和JWA靶向肽JP1(0、25、50、100μM)处理细胞时间分别为24和48小时。模型细胞分别用CCK8试验检测细胞活力、Western Blot检测细胞炎性小体(NLRP3)和凋亡相关蛋白(PARP1，Cleaved caspase1、3、9)表达水平、以及免疫荧光染色法检测细胞线粒体膜电位(JC1)和胞内活性氧(ROS)水平。
2.小鼠亚急性帕金森病模型：实验选用3-4月龄C57BL/6小鼠，体重25-30g。所有小鼠均饲养于南京医科大学实验动物中心(SPF环境)。小鼠自由饮食、饮水，饲喂的饲料为标准饲料，室内温度保持在(24±2)℃，室内湿度50-60％，通风良好，每日光照与黑暗时间各12h，于南京医科大学实验动物中心内饲养，所有研究均按照南京医科大学《实验动物研究指南》进行，并经南京医科大学机构动物护理和使用委员会批准。
MPTP小鼠PD模型的制备：用无菌生理盐水配置MPTP储存液(200mg/kg)并于-80℃超低温冰箱中储存，用时在冰上进行融化并用无菌生理盐水进行10倍稀释。MPTP(30mg/kg)通过背部皮下注射的方式进行给药，丙磺舒(250mg/kg)在MPTP注射1h后用微量注射器进行腹腔注射给药(现用现配)。连续给药5天，最后一次给药后的第7天统一对小鼠进行处死。于冰上取相关实验所需组织进行实验，对照组小鼠给予无菌生理盐水和同剂量丙磺舒处理。JP1单独处理组或JP1干预组，用无菌生理盐水稀释，按150mg/kg剂量腹腔注射，每天1次，连续7天。JP1注射后第2天开始MPTP处理模型小鼠，MPTP在JP1给药后2小时给予，丙磺舒(250mg/kg)在MPTP注射1h后用微量注射器进行腹腔注射给药(现用现配)，MPTP和丙磺舒均连续给药5天。JP1于第7天最后1次给药，停药后第6天(即模型第13天)，对模型小鼠进行行为学检测评估，次日即模型第14天处死小鼠，取脑组织，制备脑片进行相关分子标志物检测评估。
三、细胞毒性检测：
按照8000个/孔的密度将细胞接种于96孔板，贴壁24小时。弃培养液，加入配置好的不同浓度的药物，分别培养细胞24，48小时。弃去96孔板原有培养液，每孔加入10μl CCK-8试剂与90μl新鲜培养液的混合液，37℃，5％CO 2培养箱中培养2小时左右。酶联免疫检测仪取450nm波长读取吸光度值。计算细胞存活率(每组设3个平行样，重复3次)。
四、细胞线粒体膜电位和胞内活性氧检测
细胞根据试验要求处理后，根据细胞线粒体膜电位检测和胞内活性氧检测试剂说明书(南京碧云天)，避光，将细胞装载DCFH-DA探针(探针与培养液体积的比例为1：1000)，37℃细胞培养箱内孵育20min。用无血清DMEM细胞培养液洗涤细胞三次。荧光倒置显微镜下观察。每个孔观察5个视野，记录后取平均数。
五、小鼠行为学检测
在末次注射毒物的第2-3天对小鼠进行行为学的训练，为避免小鼠过度劳累训练项目需穿插进行，旷场实验不需要进行训练。爬杆实验(Pole Test)：在正式实验之前对小鼠进行爬杆训练，每天训练三次，中间需要让小鼠休息，不能过度劳累。正式开始实验时将小鼠的头向上放于杆的顶端(杆直径1cm、高50cm)记录小鼠从杆的顶端爬到杆底部的时间。转棒实验(Rotarod Test)：在正式测试之前需对小鼠进行训练，训练时转棒的转速要匀速转动(12rpm)且训练的时间不宜过长(不超过300sec)，正式测试实验时转棒转速要以加速的方式进行(5-20rpm)，记录小鼠在棒上停留的时间。开场实验(Open-Field Test)：把小鼠放于旷场内适应15min(旷场规格为20cm×20cm×15cm)，应用Open field软件记录小鼠在10min之内爬行的总距离。
六、脑组织冰冻切片制作及免疫组织化学染色
1.小鼠腹腔注射戊巴比妥进行麻醉，麻醉后经左心室灌注生理盐水100ml及4％多聚甲醛100ml。小心剥去脑壳取出脑组织，脑组织经4％多聚甲醛固定，放于4℃冰箱中过夜。
2.第2天把脑组织从4％多聚甲醛中取出放入20％蔗糖溶液中进行脱水，每天更换蔗糖溶液脱水3天后，把脑组织取出放入30％蔗糖溶液中进行梯度脱水，脱水3天并每天更换蔗糖溶液。
3.把脑组织进行包埋，并使用冰冻切片机对脑组织进行切片，切片厚度为25μm，按照小鼠脑图谱准确找出要切片的区域，纹状体隔6取1、中脑隔3取1，用PBS洗脑片3次并最终收集与1.5ml EP管中，加入PBS和甘油(1：1)可放于-20℃冰箱中长期保存。
4.脑组织免疫组织化学实验步骤
1)用PBS漂洗脑片，置于摇床上洗3次每次15min。用3％的H 2O 2处理脑片15-20min。
2)再次用PBS漂洗脑片置于摇床上洗3次每次15min。用含0.3％Triton的PBS配置BSA封闭液浓度为5％，并于室温封闭1h。
3)一抗室温孵育1h，4℃孵育过夜。本实验主要使用的一抗为JWA、酪氨酸羟化酶、抗胶质源性酸性蛋白及谷氨酸转运蛋白。
4)0.01M PBS漂洗3×10min后，加入辣根过氧化物酶标记的二抗(1:1000)室温孵育1h，再用PBS漂洗脑片，每次15min漂洗3次，DAB进行显示15min，梯度乙醇脱水中性树胶封片。脑片用体视学显微镜(Axiovert LSM510，Carl Zeiss Co.)拍照计数。
七、免疫印迹(Western blotting)
1.中脑及纹状体组织蛋白制备：
1)行为学检测之后第二天，采取小鼠脑组织，具体步骤：眼球取血之后对小鼠进行断颈处死，小心拔取小鼠脑壳取出脑组织尽量保持脑组织的完整，对称切开小鼠脑组织分离中脑、纹状体及海马组织，称重按照1:8的比例加入蛋白裂解液RIPA于4℃冰箱裂解30min。4℃ 12000×g离心15min，收取上清。
2)采用BCA蛋白定量法测定纹状体及中脑组织蛋白浓度，根据样本的数量配置BCA工作液(A：B为50：1)充分混匀，同时以5mg/ml的牛血清白蛋白(BSA)为标准蛋白作为对照，取10μl原液加入90μl NaCl。制备标注曲线：将标准品蛋白按梯度加入到96孔板中，加NaCl补充至20μl；取待测样品1μl加入到96孔板中，加NaCl至标20μl；在标注曲线各孔和待测样品各孔加入200μl BCA工作液并放入37℃培养箱中放置30min；于酶标仪中测定蛋白浓度。根据吸光度值和标准曲线计算出蛋白浓度。
3)蛋白上清按照体积比加入6×上样缓冲液(loading buffer)后于100℃金属浴中变性5min，分装后-20℃保存。
2.细胞蛋白制备：吸弃细胞培养板中的培养液，用预冷PBS缓冲液或NaCl漂洗两次后加入蛋白裂解液，放入4℃冰箱裂解30min。4℃ 12000g离心15min，取1μl蛋白样品按上述组织蛋白浓度测定的方法进行蛋白浓度测定，其余蛋白上清按体积加入6×上样缓冲液后100℃金属浴中变性5min，分装后-20℃保存。
3.SDS-PAGE制备：
1)根据蛋白质分子质量不同选择不同浓度的分离胶，分子量越大分离胶的浓度越低，在本实验中选择分离胶的为12.5％，其中30％丙烯酰胺的用量为2ml，1.5M Tris盐酸1.25ml，10％的SDS 50μl，10％的AP 28μl，TEMED 2.5μl，ddH 2O 1.675μl，总体5ml。用ddH 2O检测是否漏水后倒去ddH 2O并用滤纸吸干剩余水分。加入下层叫至适当高度并用100％酒精压平，置室温40min后倒多余掉酒精并用滤纸吸干。
2)配置4％浓缩胶，其中30％丙烯酰胺0.25g，0.5M Tris盐酸0.625ml，10％SDS 25μl,10％的AP 13μl,TEMED 1.3μl,ddH 2O 1.525μl，总体积为2.5ml。将浓缩胶倒入玻璃板中并根据需要插入10或15孔梳子，待干燥后进行下一步实验。
3)在电泳槽中入电泳液，拔掉梳子，在各孔中加入样品，每孔蛋白量约为40μg，电泳液加到电泳槽的最上方，接通电极，采取恒压60V电泳45min待样品压制分离胶后调整电压至90V继续电泳至溴酚蓝跑出下层胶后断开电源。小心取出分离胶放置于转移液中平衡15min。
4)根据蛋白大小裁剪适应大小的硝酸纤维素膜(PVDF)，采用湿转方法转印，依次放入海绵、滤纸、PVDF膜、分离胶、滤纸、海绵，确保胶与膜之间无气泡出现，固定夹板后放入到湿转槽中，倒入转移液并放入冰块，恒流200mA转印90min。
5)待转印结束后，将PVDF膜取出放入到5％的脱脂奶粉中或5％的BSA封闭液中，室温放 置于摇床上封闭1-2h。
6)按照抗体说明书要求比例稀释一抗，与PVDF膜共同孵育14h后回收一抗并用TBST洗涤5-6次，每次5min，洗涤结束后二抗孵育，二抗选用HRP标记的相应物种抗体，按比例稀释溶于5％脱脂奶粉中，室温孵育1-1.5h。结束后用TBST再次洗涤5-6次，每次5min。
7)按ECL发光液试剂盒说明书，将A液和B液按1:1体积混匀即可。通过成像系统进行发光检测，所得结果用Image J软件进行定量分析。
八、统计分析
统计数据采用SPSS 19.0软件，用mean±S.E.M表示，采用Two-way ANOVA或One-way ANOVA结合Turkey多重比较分析组间差异。P<0.05表示有统计学意义，P<0.01差异为极显著。
除上述实施例外，本发明还可以有其他实施方式。凡采用等同替换或等效变换形成的技术方案，均落在本发明要求的保护范围。

Claims (10)

  1. 一种多肽的用途，其特征是，所述用途为用于制备预防或治疗帕金森病的药物；
    所述多肽的氨基酸序列如I或II所示：
    I：FPGSDRF-Z；
    II：X-FPGSDRF-Z；
    其中，氨基酸S经磷酸化修饰，X、Z分别为氨基酸或氨基酸序列；
    X选自F、(R) 9、(R) 9-F、6-氨基己酸、6-氨基己酸-F、6-氨基己酸-(R) 9、6-氨基己酸-(R) 9-F之一；
    Z选自(G) n-RGD、A-(G) n-RGD之一，n为大于或等于0的整数，且n的取值范围为0-10。
  2. 根据权利要求1所述的用途，其特征是，所述用途为用于制备预防或治疗帕金森病的具体病症或病征的药物。
  3. 根据权利要求2所述的用途，其特征是，所述帕金森病的具体病症或病征为：脑组织星形胶质细胞或小胶质细胞的过度活化或增生。
  4. 根据权利要求2所述的用途，其特征是，所述帕金森病的具体病症或病征为：脑组织多巴胺神经元变性或缺失。
  5. 根据权利要求2所述的用途，其特征是，所述帕金森病的具体病症或病征为：海马神经元细胞或神经胶质细胞受到细胞毒性作用导致活力减弱。
  6. 根据权利要求2所述的用途，其特征是，所述帕金森病的具体病症或病征为：海马神经元细胞或神经胶质细胞的炎性小体表达增加以及细胞凋亡增加。
  7. 根据权利要求2所述的用途，其特征是，所述帕金森病的具体病症或病征为：海马神经元细胞或神经胶质细胞的细胞线粒体损伤而致膜电位增加以及胞内活性氧水平升高。
  8. 根据权利要求1至7任一项所述的用途，其特征是，所述多肽的N端经乙酰化修饰、C端经酰胺化修饰。
  9. 根据权利要求1至7任一项所述的用途，其特征是，所述多肽的氨基酸序列为FPGSDRF-RGD，其中，氨基酸S经磷酸化修饰。
  10. 根据权利要求1至7任一项所述的用途，其特征是，所述药物包括载体，所述载体为药学上可接受的载体。
PCT/CN2022/138737 2022-09-16 2022-12-13 Jwa多肽在制备抗帕金森病药物方面的应用 WO2024055457A1 (zh)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001030968A2 (en) * 1999-10-23 2001-05-03 The Johns Hopkins University School Of Medicine Glutamate transporter associated proteins and methods of use thereof
CN1951503A (zh) * 2005-10-17 2007-04-25 中国科学院上海生命科学研究院 细胞骨架样基因jwa在抑制帕金森兴奋性氨基酸毒性中的应用
CN103239710A (zh) * 2013-05-14 2013-08-14 南京医科大学 具有抗肿瘤活性的多肽及其用途
CN111714476A (zh) * 2019-03-21 2020-09-29 暨南大学 胡黄连素二聚体类似物衍生物在制备防治帕金森病的药物或保健品中的应用
CN114940702A (zh) * 2022-06-17 2022-08-26 周建伟 Jwa多肽在制备抗新生血管性眼病药物方面的应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001030968A2 (en) * 1999-10-23 2001-05-03 The Johns Hopkins University School Of Medicine Glutamate transporter associated proteins and methods of use thereof
CN1951503A (zh) * 2005-10-17 2007-04-25 中国科学院上海生命科学研究院 细胞骨架样基因jwa在抑制帕金森兴奋性氨基酸毒性中的应用
CN103239710A (zh) * 2013-05-14 2013-08-14 南京医科大学 具有抗肿瘤活性的多肽及其用途
CN111714476A (zh) * 2019-03-21 2020-09-29 暨南大学 胡黄连素二聚体类似物衍生物在制备防治帕金森病的药物或保健品中的应用
CN114940702A (zh) * 2022-06-17 2022-08-26 周建伟 Jwa多肽在制备抗新生血管性眼病药物方面的应用

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