Neuroprotective medicine for Parkinson's disease and application of neuroprotective medicine

Abstract

The invention relates to a neuroprotective drug for Parkinson's disease and application thereof, and relates to the technical field of biological medicines. According to the invention, an SH-SY5Y nerve cell line is treated by MPP +, an MPP + induced PD cell model is established, N-[5-acetyl-1-[2-(5-hydroxy-1H-indole-3-yl) ethyl]-1H-pyrrole-3-yl] acetamide is used for intervention, cell viability, cytotoxicity level and mitochondrial membrane potential after intervention are detected, and the expression of the MPP + induced PD cell model is established. It is found that N-[5-acetyl-1-[2-(5-hydroxy-1H-indole-3-yl) ethyl]-1H-pyrrole-3-yl] acetamide can reduce SH-SY5Y cell damage and cytotoxicity induced by MPP < + >, and improve mitochondrial dysfunction caused by MPP < + > in SH-SY5Y cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a drug for protecting Parkinson's disease neurons and an application thereof. Background Art

[0002] Parkinson's disease (PD) is the second largest neurodegenerative disease in the world. Its main pathological features are degeneration, loss and Lewy bodies (LB) formation of dopamine (DA) neurons in the substantia nigra and striatum. Existing studies believe that the onset of PD is mainly related to oxidative stress, gene mutation, immune inflammatory response, microbial-gut-brain axis regulatory mechanism, defects and mitochondrial dysfunction. Among them, oxidative stress and mitochondrial damage play a core role in dopaminergic neurodegeneration. The activity of mitochondrial complex I in the brain of PD patients is reduced, and mitochondrial toxins such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and rotenone can induce DA neuron degeneration in the substantia nigra. At the same time, signs of oxidative damage, including DNA damage, lipid peroxidation and oxidized protein aggregates, have been found in postmortem brain tissue of PD patients. Moreover, multiple genes associated with familial PD, including PINK1, Parkin, LRRK2, SNCA, and DJ-1, are involved in the regulation of mitochondrial function, mitosis, and ROS production. In addition, a large amount of evidence shows that misfolded proteins accumulate in cells, especially α-synuclein (α-synuclein, α-syn) misfolding and abnormal aggregation further form insoluble amyloid proteins, namely Lewy bodies. This abnormal α-syn aggregate is difficult to be degraded after deposition in neurons, further mediating cytotoxic effects such as dysfunction of the protein degradation system, material transport disorders, and mitochondrial dysfunction, leading to neuronal death.

[0003] There are more than 10 million PD patients worldwide. Although PD has been discovered for 200 years and is one of the most common neurodegenerative diseases, the current clinical treatment for PD is mainly to relieve symptoms by taking levodopa, and the efficacy varies from person to person. Therefore, it is crucial to explore the mechanism of neuroprotection, find drugs that reverse or hinder PD neurodegeneration, and reduce dopaminergic neuron damage for the prevention and treatment of PD.

[0004] Toad venom is a traditional Chinese medicine. It is made from the dried white venom secreted from the skin glands and parotid glands of the Chinese giant toad or the black-framed toad of the Toad family. The Chinese Pharmacopoeia records that toad venom is pungent and warm in nature, has the functions of detoxification, analgesia, and awakening the mind. It is widely used as an anti-inflammatory drug, analgesic, cardiotonic, antibacterial drug and local anesthetic. The chemical composition of toad venom is complex and diverse. According to different separation and identification methods, its main chemical components have been determined to be bufadienolides, indole alkaloids, sterols and other compounds. Among them, indole derivatives are important alkaloid compounds with a broad spectrum of biological activities. They are widely used in the fields of anti-oxidation, anti-infection, anti-epileptic, anti-fungal and anti-tumor. Indole alkaloids have received widespread attention in drug research and development due to their special structure, strong drug-like properties, high selectivity and low toxicity and side effects.

[0005] At present, there is no report on the protective effect of N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide (abbreviated as Bufoserotonin C) on PD neuronal damage. Summary of the invention

[0006] In order to solve the above technical problems, the purpose of the present invention is to provide a drug for Parkinson's disease neuroprotection and its application. The main active ingredient of the drug of the present invention is N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide; MPP can be reduced + (MPTP metabolites)-induced SH-SY5Y cell damage and cytotoxicity, improving MPP in SH-SY5Y cells + The mitochondrial dysfunction induced by N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide plays a protective role in neuronal damage. + The induced neurotoxicity plays a protective role, providing new drugs and methods for the prevention and treatment of Parkinson's disease. It is expected to be used in the preparation of various neuroprotective drugs and has good application prospects.

[0007] The technical solution of the present invention to solve the above technical problems is as follows:

[0008] The purpose of the present invention is to provide the application of a drug for protecting the nerves of Parkinson's disease.

[0009] The beneficial effects of the present invention are as follows: the present invention establishes an in vivo cell model of PD, detects cell viability, cytotoxicity level, and mitochondrial membrane potential, and finds that N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide can reduce MPP +Induced SH-SY5Y cell damage and cytotoxicity, improved MPP in SH-SY5Y cells + The mitochondrial dysfunction induced by N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide plays a protective role in neuronal damage. + The induced neurotoxicity plays a protective role, providing new drugs and methods for the prevention and treatment of Parkinson's disease. It is expected to be used in the preparation of various neuroprotective drugs and has good application prospects.

[0010] Based on the above technical solution, the present invention can also be improved as follows.

[0011] Furthermore, the main active ingredient of the drug is indole alkaloid, and the indole alkaloid is N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide.

[0012] The beneficial effect of adopting the above further scheme is: N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide is referred to as Bufoserotonin C, and its molecular formula is C 18 H 19 N3O3, molecular weight is 325.36, the structural formula is as follows:

[0013]

[0014] Furthermore, the N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide plays a protective role in neuronal damage.

[0015] Furthermore, the N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide can reduce the MPP + Induced SH-SY5Y cell damage and cytotoxicity, improved MPP in SH-SY5Y cells + The mitochondrial dysfunction induced by mitochondria plays a protective role in neuronal damage.

[0016] Furthermore, the CAS number of the N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide is 1002722-87-3.

[0017] The second object of the present invention is to provide a drug with a neuroprotective effect on Parkinson's disease, wherein the main active ingredient of the drug is N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide.

[0018] Furthermore, the N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide is an indole alkaloid in toad venom.

[0019] Furthermore, the N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide reduces MPP in vitro + The effective concentration for induced SH-SY5Y cell damage and cytotoxicity was ≥2.5 μM.

[0020] Furthermore, the drug also includes pharmaceutically acceptable carriers and auxiliary components.

[0021] Furthermore, the dosage form of the drug is a clinically acceptable pharmaceutical preparation.

[0022] Furthermore, the dosage form of the drug includes any one of capsules, granules, tablets, pills, syrups, powders, granules, suppositories, drops, emulsions, solutions, and suspensions.

[0023] The beneficial effect of adopting the above further scheme is that the drug of the present invention can be prepared as a pharmaceutical composition according to methods known in the art, and can be prepared into any dosage form suitable for human or animal use by combining the drug of the present invention with one or more pharmaceutically acceptable solid or liquid excipients and / or adjuvants. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Bufoserotonin C improves MPP in the embodiment of the present invention + Figure 1 shows the viability of induced SH-SY5Y cells;

[0025] Figure 2 In the present invention, Bufoserotonin C reduces the level of MPP in SH-SY5Y cells. + induced neurotoxicity profile;

[0026] Figure 3 This is a fluorescence image of Mito-Tracker staining in an embodiment of the present invention;

[0027] Figure 4 This is a diagram showing that Bufoserotonin C can improve MPP+-induced mitochondrial damage in SH-SY5Y cells in an example of the present invention. DETAILED DESCRIPTION

[0028] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0029] Example 1: Application of Bufoserotonin C in the preparation of a neuroprotective drug for Parkinson's disease

[0030] 1. Preparation before the test

[0031] 1.1 Test materials

[0032] Human neuroblastoma cells (SH-SY5Y cells) were purchased from the Chinese Academy of Sciences; DMEM / F12 medium (Gibco, New York, USA); Bufoserotonin C (Chengdu Desit Biotechnology Co., Ltd.); DMSO (Shanghai Sulai Biotechnology Co., Ltd., China); CCK-8 detection kit (White Shark Biotechnology Co., Ltd., China); non-radioactive cytotoxicity detection kit (Promega, USA); mitochondrial membrane potential detection kit (Mito-Tracker red CMXR os; Bio-Tech, China)

[0033] 1.2 Test equipment

[0034] 5% CO2 cell culture incubator (Thermo3111; Thermo Fisher Scientific); continuous wavelength multifunctional microplate detector (Spark; TECAN); fluorescence microscope (Nikon Corporation, Japan)

[0035] 2 Test methods

[0036] 2.1 Cell culture and treatment

[0037] MPP + Dissolve it in ultrapure water to a concentration of 100 mM, and then dilute it to 2 mM with culture medium to obtain MPP +Solution; Bufoserotonin C was dissolved in DMSO to obtain Bufoserotonin C solution, with final concentrations of 2.5μM, 5μM, 10μM, 20μM and 40μM, respectively. SH-SY5Y cells were cultured in a 37°C, 5% CO2 cell culture incubator using DMEM / F12 medium supplemented with 10% fetal bovine serum. When the cell density reached 80-90%, 1mL of trypsin was used to digest the cells. After the cells were observed to be rounded under a microscope, 4mL of DMEM / F12 medium containing 10% FBS was added to terminate the digestion and the cells were counted. Then, 3×10 4 SH-SY5Y cells were seeded into 96-well plates at a rate of 2×10 5 SH-SY5Y cells were seeded into 24-well plates.

[0038] SH-SY5Y cells were inoculated in 96-well plates and cultured for 24 hours, then randomly divided into 6 groups. Each group was pretreated with 0 μM, 2.5 μM, 5 μM, 10 μM, 20 μM and 40 μM Bufosero tonin C solution for 24 hours. Subsequently, each group was treated with 2 mM MPP + The solution was continued to treat SH-SY5Y cells for 48 h, and then cell viability and lactate dehydrogenase (LDH) were detected. Another group without any treatment was set up as a blank control.

[0039] SH-SY5Y cells were inoculated in 24-well plates and cultured for 24 hours, then randomly divided into 4 groups. Each group was pretreated with 0 μM, 10 μM, 20 μM and 40 μM Bufoserotonin C solution for 24 hours. Subsequently, each group was treated with 2 mM MPP + The solution was continued to treat SH-SY5Y cells for 48 hours, and then Mito-Tracker staining was performed. Another group without any treatment was set up as a blank control.

[0040] The specific experimental steps for cell viability, lactate dehydrogenase (LDH) detection and Mito-Tracker staining detection are as follows:

[0041] 2.2 Cell viability assay

[0042] In this example, the CCK-8 experiment was used to detect the effect of Bufoserotonin C on MPP + Effects of induced damage on SH-SY5Y cells.

[0043] Use CCK-8 detection kit to evaluate cell viability: add 10 μL CCK-8 reagent to 100 μL culture medium and incubate for 1 hour at 37°C and 5% CO2. Read the absorbance value at 450nm using continuous wavelength multifunctional microplate reader. The results are as follows Figure 1 shown.

[0044] 2.3 Cytotoxicity assay

[0045] In this example, the effect of Bufoserotonin C on MPP was detected by LDH experiment. + Induced cytotoxicity in SH-SY 5Y cells.

[0046] Cytotoxicity detection uses a non-radioactive cytotoxicity detection kit. The operation method is carried out according to the instructions of the kit. The specific steps are to transfer 50μL of the culture medium of all samples to the detection plate. Add 50μL of LDH detection working solution to each well, mix well, incubate at room temperature in the dark for 30 minutes, and add 50μL of stop buffer. Measure the absorbance at 490nm. The results are as follows Figure 2 shown.

[0047] 2.4 Mito-Tracker staining

[0048] Mitochondrial membrane potential (MMP) is an important indicator for evaluating mitochondrial function and structural integrity. Mito-Tracker Red CMXRos (mitochondrial red fluorescent probe) can be used to specifically stain active mitochondria in cells to emit bright red fluorescence. When cells are damaged, the mitochondrial membrane potential decreases, and the mitochondrial red fluorescence gradually weakens or even disappears. This is used to evaluate the effect of Bufoserotonin C on MPP. + The effects of induced mitochondrial structure and function in SH-SY5Y cells.

[0049] Mito-Tracker experiment was used to detect the effect of Bufoserotonin C on mitochondrial membrane potential. Mitochondrial membrane potential was detected using a mitochondrial membrane potential detection kit: the cell culture medium was removed and washed once with PBS, 94 μL of reaction binding solution, 1 μL of Mito-Tracker Red CMXRos staining solution and 2.5 μL of Hoechst 33342 staining solution were added, and the mixture was gently mixed; incubated at room temperature in the dark for 30 min, and the cells were quickly imaged under a fluorescence microscope. Mito-Tracker Red CMXRos was red fluorescence and Hoechst 33342 was blue fluorescence. Scale bar = 100 μm (B) MitoTracker Red CMXRos fluorescence detection results.

[0050] The results are as follows Figure 3 , Figure 4 As shown, in the figure, the "+" sign indicates that a substance has been added, and the "-" sign indicates that no substance has been added.

[0051] 2.5 Statistical analysis

[0052] Data are presented as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism software 8.0 (GraphPad, California, USA). One-way ANOVA was used to evaluate differences between groups, followed by Tukey's multiple comparison test. P < 0.05 was considered statistically significant.

[0053] The results are as follows Figure 1-4 As shown:

[0054] (1) By Figure 1 It is found that Bufoserotonin C can effectively improve MPP + The effects of 2.5μM, 5μM, 10μM, 20μM and 40μM Bufoserotonin C intervention on the MPP of 2mM were examined. + The effect of treatment on the viability of SH-SY5Y cells. CCK-8 results showed that MPP + After treatment, cell viability decreased to 49.27%. As the concentration of Bufoserotonin C treatment increased, cell viability increased, and when the treatment concentration was 20 μM, cell viability increased from 49.27% ​​to 67.23%.

[0055] (2) From Figure 2 It can be seen that Bufoserotonin C can effectively rescue MPP + The cytotoxicity caused by 2.5, 5, 10, 20 and 40 μM Bufoserotonin C was tested for the effects of 2 mM MPP + The results of LDH detection showed that MPP + After treatment, LDH release increased to 273.38%. With the increase of Bufoserotonin C treatment concentration, LDH release decreased, and when the treatment concentration was 40 μM, LDH release decreased from 273.38% to 208.26%.

[0056] (3) By Figure 3 It can be seen that Bufoserotonin C can effectively improve MPP in SH-SY5Y cells +Compared with Bufoserotonin C treatment, MPP alone + The fluorescence intensity of the group was significantly reduced, indicating that MMP was depolarized; Bufoserotonin C can improve MPP in SH-SY5Y cells + Caused mitochondrial damage.

[0057] In summary, Bufoserotonin C has an in vitro + It has an effective protective effect on cell damage caused by toxicity.

[0058] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. Application of neuroprotective drugs in Parkinson's disease.

2. The use of the drug for protecting Parkinson's disease neurons according to claim 1, characterized in that: The main active ingredient of the drug is indole alkaloid, and the indole alkaloid is N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide.

3. The use of the drug for protecting Parkinson's disease neurons according to claim 2, characterized in that: The N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide plays a protective role in neuronal damage.

4. The use of the drug for protecting Parkinson's disease neurons according to claim 3, characterized in that: The N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide reduces the MPP + Induced SH-SY5Y cell damage and cytotoxicity, improved MPP in SH-SY5Y cells + The mitochondrial dysfunction induced by mitochondria plays a protective role in neuronal damage.

5. A drug for protecting Parkinson's disease neurons, characterized in that: The main active ingredient of the drug is N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide.

6. The drug for protecting Parkinson's disease neurons according to claim 5, characterized in that: The N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide is an indole alkaloid in toad venom.

7. The drug for protecting Parkinson's disease neurons according to claim 6, characterized in that: The N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide reduces MPP in vitro + The effective concentration for induced SH-SY5Y cell damage and cytotoxicity was ≥2.5 μM.

8. The drug for protecting Parkinson's disease neurons according to claim 6, characterized in that: The medicine also includes pharmaceutically acceptable carriers and auxiliary components.

9. The drug for protecting Parkinson's disease neurons according to claim 8, characterized in that: The dosage form of the drug is a clinically acceptable pharmaceutical preparation.

10. The drug for protecting Parkinson's disease neurons according to claim 9, characterized in that: The dosage form of the drug includes any one of capsules, granules, tablets, pills, syrups, powders, granules, suppositories, drops, emulsions, solutions and suspensions.

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