Active peptide and application thereof

Abstract

The invention relates to an active peptide and application thereof. The amino acid sequence of the active peptide is as shown in SEQ ID No. 1 or SEQ ID No. 2.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of functional genes, in particular to active peptide drugs. BACKGROUND

[0002] Death and disability caused by neurodegenerative diseases are a serious challenge to global public health, among which Parkinson's disease is the fastest growing neurodegenerative disease. At present, the treatment of Parkinson's disease in the clinic adopts a multidisciplinary comprehensive treatment and whole-process management mode, but only can relieve symptoms and cannot achieve the purpose of cure. Therefore, the development of Parkinson's disease treatment drugs has important clinical significance.

[0003] The main pathological feature of Parkinson's disease is the loss of dopaminergic neurons in the substantia nigra compacta of the midbrain, which causes patients to have tremor and rigidity and other movement disorder symptoms. Studies have shown that the occurrence of Parkinson's disease is closely related to the decrease in the survival of dopaminergic neurons, the change in cell morphology and apoptosis caused by various factors. Compared with the current clinical dopamine replacement therapy, protecting dopaminergic neurons and reducing the apoptosis of dopaminergic neurons is a more fundamental treatment strategy. SUMMARY

[0004] One of the present application provides an active peptide, the amino acid sequence of which is shown in SEQ ID No. 1 or SEQ ID No. 2.

[0005] The second of the present application provides the use of the active peptide according to the first of the present application in the preparation of a drug for treating Parkinson's disease.

[0006] The third of the present application provides the use of the active peptide according to the first of the present application in the preparation of a drug for at least one of increasing the survival rate of dopaminergic neurons, protecting the morphology of dopaminergic neurons and protecting dopaminergic neurons from apoptosis.

[0007] The fourth of the present application provides the use of the active peptide according to the first of the present application in the preparation of a drug for at least one of increasing the cell survival rate of dopaminergic neurons, protecting the cell morphology of dopaminergic neurons and protecting dopaminergic neurons from apoptosis.

[0008] The present application has the beneficial effect that the active peptide-1 and the active peptide-2 of the present application can increase the cell survival rate of dopaminergic neurons, protect the cell morphology of dopaminergic neurons and protect dopaminergic neurons from apoptosis, and thus can be used for treating Parkinson's disease. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1 The cell survival rates of each group in Example 1 are shown.

[0010] Figure 2The neuronal morphology observed under the inverted fluorescence microscope in Example 2 is shown.

[0011] Figure 3 The staining of the neurons observed under the inverted fluorescence microscope in Example 3 is shown. DETAILED DESCRIPTION

[0012] The above content of the present application is further explained in detail in the form of preferred embodiments, but it does not constitute a limitation to the present application.

[0013] Unless otherwise specified, the reagents and instruments used in the examples of the present application can be purchased through commercial channels.

[0014] The amino acid sequence of the active peptide-1 is shown in SEQ ID No. 1, and the amino acid sequence of the active peptide-2 is shown in SEQ ID No. 2, which are synthesized by Beijing SAB Biotech Co., Ltd.

[0015] Active peptide-1 solution: The active peptide-1 is dissolved in pure water to prepare a solution of the active peptide-1 with a molar concentration of 0.5 millimole / liter.

[0016] Active peptide-2 solution: The active peptide-2 is dissolved in pure water to prepare a solution of the active peptide-2 with a molar concentration of 0.5 millimole / liter.

[0017] 6-Hydroxydopamine (6-OHDA) solution: 6-Hydroxydopamine is dissolved in physiological saline to prepare a solution of 6-OHDA with a molar concentration of 10 millimole / liter.

[0018] The human neuroblastoma cell line SH-SY5Y is purchased from the Cell Resource Center of the Chinese Academy of Sciences.

[0019] Parkinson's disease cell model: The neurotransmitter dopamine antagonist 6-OHDA can selectively destroy dopaminergic neurons. The 6-OHDA is used to act on SH-SY5Y cells to establish an in vitro Parkinson's disease cell model of dopaminergic neurons.

[0020] Culture medium: composed of MEM / F12 (1:1 volume ratio), 10% fetal bovine serum, 1% NEAA (a mixture of seven non-essential amino acids consisting of glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline and L-serine, GIBCO) and 1 millimole / liter of sodium pyruvate. Example 1: Effect of active peptide on the survival of SH-SY5Y cells treated with 6-OHDA

[0021] Active peptide-1 group: The SH-SY5Y cells in the logarithmic growth phase are resuspended with the culture medium, and 1×10 4SH-SY5Y cells were seeded in 96-well plates and cultured overnight. Then, active peptide-1 solution was added to a final concentration of 10 μmol / L for 24 h of pretreatment. Following this, 6-OHDA solution was added to a final concentration of 40 μmol / L, and incubation continued for another 24 h. Three replicates were set up. The absorbance (OD) at 570 nm was measured using the MTT assay.

[0022] Active peptide-2 group: Replace the active peptide-1 solution in the active peptide-1 group of this embodiment with the active peptide-2 solution, and perform the same operation as the active peptide-1 group. Set up three replicates. Measure the absorbance (OD) at 570 nm using the MTT assay.

[0023] Model group: SH-SY5Y cells in the logarithmic growth phase were resuspended in culture medium, and 1×10⁻⁶ cells were added to the model group. 4 SH-SY5Y cells were seeded in 96-well plates and cultured overnight. Then, the same volume of pure water as the active peptide-1 solution in the active peptide-1 group of this example was added for pretreatment for 24 h. Afterward, 6-OHDA solution was added to a final concentration of 40 μmol / L, and incubation continued for another 24 h. Three replicates were set up. The absorbance (OD) at 570 nm was measured using the MTT assay.

[0024] Control group: The active peptide-1 solution in the active peptide-1 group of this embodiment was replaced with the same volume of pure water for pretreatment for 24 h. The 6-OHDA solution in the active peptide-1 group of this embodiment was replaced with the same volume of physiological saline. All other operations were the same as those in the active peptide-1 group of this embodiment. Three replicates were set up. The absorbance (OD) at 570 nm was measured according to the MTT assay.

[0025] Calculate cell viability using the following formula.

[0026] Cell viability = OD value of each group / OD value of the control group × 100%.

[0027] Statistical analysis was performed on the cell viability of each group, and the results are as follows: Figure 1 . Figure 1 The results showed that both active peptide-1 and active peptide-2 could significantly inhibit 6-OHDA-induced SH-SY5Y cell damage and increase cell survival. Example 2: Protective effect of active peptides on the morphology of SH-SY5Y cells induced by 6-OHDA

[0028] Active peptide-1 group: SH-SY5Y cells in the logarithmic growth phase were resuspended in culture medium, and 1×10⁻⁶ peptides were added to each group. 5SH-SY5Y cells were plated in 24-well plates overnight, then active peptide-1 solution was added to make the final concentration of active peptide-1 10 micromole / liter, pre-treated for 24 h, then 6-OHDA solution was added to make the final concentration of 6-OHDA 40 micromole / liter, and incubated for another 24 h. Three replicates were set. The damage and morphology of neurons were observed under the bright field of inverted fluorescence microscope.

[0029] Active peptide-2 group: the active peptide-1 solution in the active peptide-1 group of this example was replaced by active peptide-2 solution, and the rest was the same as the operation of the active peptide-1 group of this example. Three replicates were set. The morphology of neurons was observed under the bright field of inverted fluorescence microscope.

[0030] Model group: SH-SY5Y cells in logarithmic growth phase were resuspended with culture medium, 1×10 5 SH-SY5Y cells were plated in 24-well plates overnight, then the same volume of pure water as the active peptide-1 solution in the active peptide-1 group was added for pre-treatment for 24 h, then 6-OHDA solution was added to make the final concentration of 6-OHDA 40 micromole / liter, and incubated for another 24 h. Three replicates were set. The damage and morphology of neurons were observed under the bright field of inverted fluorescence microscope.

[0031] Control group: the active peptide-1 solution in the active peptide-1 group of this example was replaced by the same volume of pure water as the active peptide-1 solution for pre-treatment for 24 h, and the 6-OHDA solution in the active peptide-1 group of this example was replaced by the same volume of normal saline as the 6-OHDA solution, and the rest was the same as the operation of the active peptide-1 group of this example. Three replicates were set. The damage and morphology of neurons were observed under the bright field of inverted fluorescence microscope.

[0032] The results of neurons observed under the bright field of inverted fluorescence microscope are shown in Figure 2 From the results, it can be seen that compared with the control group, the cells in the model group showed obvious morphological shrinkage and size reduction; while the active peptide-1 group and the active peptide-2 group reduced the 6-OHDA-induced cell morphological changes, and most of the cell morphology remained intact. Example 3: Protective effect of active peptide on 6-OHDA-induced apoptosis of SH-SY5Y cells

[0033] Annexin V-FITC and MitoTracker Red CMXRos were used to detect apoptosis and mitochondrial membrane potential. Green fluorescence labeled apoptotic cells, and red fluorescence labeled living cells that maintained mitochondrial membrane potential.

[0034] Active peptide-1 group: SH-SY5Y cells in the logarithmic growth phase were resuspended in culture medium, and 2×10⁻⁶ cells were added. 5 SH-SY5Y cells were seeded in 12-well plates and cultured overnight. Then, active peptide-1 solution was added to a final concentration of 10 μmol / L for 24 h of pretreatment. Next, 6-OHDA solution was added to a final concentration of 40 μmol / L, and incubation continued for another 24 h. 5 μL of Annexin V-FITC and 2 μL of Mito-Tracker Red CMXRos staining solution were added to each well and gently mixed. The cells were incubated at room temperature in the dark for 15 min. Three replicates were set up. Observation and photography were performed using an inverted fluorescence microscope.

[0035] Active peptide-2 group: In this embodiment, active peptide-1 is replaced with active peptide-2, and all other operations are the same as those for active peptide-1 group. Three replicates are set up. Observation and photography are performed using an inverted fluorescence microscope.

[0036] Model group: SH-SY5Y cells in the logarithmic growth phase were resuspended in culture medium, and 2×10⁻⁶ cells were added to the model group. 5 SH-SY5Y cells were seeded in 12-well plates and cultured overnight. Then, the same volume of pure water as the active peptide-1 solution in the active peptide-1 group of this example was added for pretreatment for 24 h. Next, 6-OHDA solution was added to bring the final concentration of 6-OHDA to 40 μmol / L, and incubation continued for another 24 h. 5 μL of Annexin V-FITC and 2 μL of Mito-Tracker Red CMXRos staining solution were added to each well and gently mixed. The cells were incubated at room temperature in the dark for 15 min. Three replicates were set up. Observation and photography were performed using an inverted fluorescence microscope.

[0037] Cell staining results observed under an inverted fluorescence microscope are shown in the figure. Figure 3 The results show that the number of apoptotic cells labeled with green fluorescence was significantly higher than that of live cells labeled with red fluorescence in the model group. In contrast, the active peptide-1 and active peptide-2 groups showed a significant decrease in apoptotic cells labeled with green fluorescence and a significant increase in live cells labeled with red fluorescence that maintained mitochondrial membrane potential.

Claims

1. An active peptide, the amino acid sequence of which is shown in SEQ ID No. 1 or SEQ ID No.

2.

2. Use of the active peptide according to claim 1 in the preparation of a medicament for treating Parkinson's disease.

3. Use of the active peptide according to claim 1 in the preparation of a medicament for at least one of increasing the survival rate of dopaminergic neurons, protecting the morphology of dopaminergic neurons, and protecting dopaminergic neurons from apoptosis.

4. Use of the active peptide according to claim 1 in the preparation of a medicament for at least one of increasing the cell survival rate of dopaminergic neurons, protecting the cell morphology of dopaminergic neurons, and protecting dopaminergic neuron cells from apoptosis.

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