Neuroprotective drugs for Parkinson's disease and their applications

Abstract

The present invention relates to a neuroprotective drug for Parkinson's disease and its application, and relates to the field of biomedicine technology. + Treatment of SH-SY5Y neural cell line to establish MPP + In a cell model of PD induced by PD, N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide was used for intervention. Cell viability, cytotoxicity and mitochondrial membrane potential were detected after intervention. It was found that N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide could reduce MPP + Induced SH‑SY5Y cell damage and cytotoxicity, improved MPP in SH‑SY5Y cells + Caused by mitochondrial dysfunction.

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 its application. Background Art

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disorder worldwide. Its primary pathological features are degeneration and loss of dopamine (DA) neurons in the substantia nigra and striatum, as well as the formation of Lewy bodies (LBs). Existing studies suggest that the pathogenesis of PD is primarily associated with oxidative stress, genetic mutations, immune inflammatory responses, regulatory mechanisms and defects in the microbiome-gut-brain axis, and mitochondrial dysfunction. Oxidative stress and mitochondrial damage play a central role in dopaminergic neurodegeneration. Mitochondrial complex I activity is reduced in the brains of PD patients, and mitochondrial toxins such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and rotenone can induce degeneration of DA neurons in the substantia nigra. Furthermore, markers of oxidative damage, including DNA damage, lipid peroxidation, and oxidized protein aggregates, have been found in postmortem brain tissue from PD patients. Furthermore, multiple genes associated with familial Parkinson's disease, including PINK1, Parkin, LRRK2, SNCA, and DJ-1, are involved in regulating mitochondrial function, mitosis, and ROS production. Furthermore, extensive evidence indicates that misfolded proteins accumulate intracellularly, particularly α-synuclein (α-syn), which misfolds and aggregates abnormally, further forming insoluble amyloid proteins known as Lewy bodies. Once deposited within neurons, these abnormal α-syn aggregates are resistant to degradation, further mediating cytotoxic effects such as dysfunction of the protein degradation system, impaired transport of substances, and mitochondrial dysfunction, leading to neuronal death.

[0003] Currently, there are over 10 million PD patients worldwide. Although PD has been recognized for 200 years and is one of the most common neurodegenerative diseases, current clinical treatment for PD mainly relies on levodopa to alleviate symptoms, and the efficacy varies from person to person. Therefore, exploring the relevant neuroprotective mechanisms, finding drugs that reverse or inhibit PD neurodegeneration, and reduce dopaminergic neuron damage are crucial for the prevention and treatment of PD.

[0004] Toad venom is a traditional Chinese medicine made from the dried white venom secreted by the skin and parotid glands of the Bufo bufo gargarizans (Bufo gargarizans), also known as the black-rimmed toad. The Chinese Pharmacopoeia states that toad venom is pungent and warm in nature, possessing detoxifying, analgesic, and invigorating properties. It is widely used as an anti-inflammatory, analgesic, cardiotonic, antibacterial, and local anesthetic. The chemical composition of toad venom is complex and diverse. Based on various isolation and identification methods, its main components have been identified as bufadienolides, indole alkaloids, sterols, and other compounds. Among them, indole derivatives are important alkaloid compounds with a broad spectrum of biological activities, finding widespread application in antioxidant, anti-infective, anti-epileptic, antifungal, and anti-tumor applications. Indole alkaloids, due to their unique structure, strong drug-like properties, high selectivity, and minimal toxicity and side effects, have garnered widespread attention in drug development.

[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 neuroprotective drug for Parkinson's disease 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; it can reduce MPP + (MPTP metabolites)-induced SH-SY5Y cell damage and cytotoxicity, improving MPP in SH-SY5Y cells + N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide can protect MPP from mitochondrial dysfunction. + It plays a protective role in the induced neurotoxicity, provides new drugs and methods for the prevention and treatment of Parkinson's disease, and is expected to be used in the preparation of various neuroprotective drugs, with 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 an application of a drug for protecting Parkinson's disease neurons.

[0009] The beneficial effects of the present invention are as follows: by establishing an in vivo cell model of PD, the present invention detected cell viability, cytotoxicity level, and mitochondrial membrane potential, and found 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 + N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide can protect MPP from mitochondrial dysfunction. + It plays a protective role in the induced neurotoxicity, provides new drugs and methods for the prevention and treatment of Parkinson's disease, and is expected to be used in the preparation of various neuroprotective drugs, with good application prospects.

[0010] On the basis of 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, and 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 β-actin 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 of induced SH-SY5Y cell damage and cytotoxicity was ≥2.5 μM.

[0020] Furthermore, the medicine 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 4 shows the viability of induced SH-SY5Y cells;

[0025] Figure 2 In the embodiment of the present invention, Bufoserotonin C reduced the MPP + 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 mitochondrial damage caused by MPP+ 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. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this field or the product instructions are used. Where the manufacturers of the reagents or instruments used are 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) was purchased from Chengdu Desite Biotechnology Co., Ltd. Bufoserotonin C was purchased from Chengdu Desite Biotechnology Co., Ltd. DMSO was purchased from Shanghai Sulai Biotechnology Co., Ltd. CCK-8 assay kit was purchased from White Shark Biotechnology Co., Ltd. Non-radioactive cytotoxicity assay kit was purchased from Promega Corporation. Mitochondrial membrane potential assay kit (Mito-Tracker red CMXROs, Beyotime Biotechnology, China) was purchased from

[0033] 1.2 Test instruments

[0034] 5% CO2 cell culture incubator (Thermo3111; Thermo Fisher Scientific); continuous wavelength multifunctional microplate reader (Spark; TECAN); fluorescence microscope (Nikon, 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 seeded 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 seeded 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 SH-SY5Y cells were treated with the solution for 48 h and then subjected to Mito-Tracker staining. 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 are as follows:

[0041] 2.2 Cell viability assay

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

[0043] Cell viability was assessed using the CCK-8 assay kit: 10 μL of CCK-8 reagent was added to 100 μL of culture medium and incubated at 37°C, 5% CO2 for 1 hour. The absorbance was read at 450 nm using a continuous wavelength multi-function microplate reader. The results were 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 assays were performed using a non-radioactive cytotoxicity assay kit. The procedure was performed according to the kit instructions. The specific steps were as follows: 50 μL of culture medium from all samples was transferred to the assay plate. 50 μL of LDH assay working solution was added to each well, mixed, and incubated in the dark at room temperature for 30 minutes. Then, 50 μL of stop buffer was added. The absorbance was measured at 490 nm. The results were 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. + Effects of induced mitochondrial structure and function in SH-SY5Y cells.

[0049] Mito-Tracker assays were used to examine the effect of Bufoserotonin C on mitochondrial membrane potential. Mitochondrial membrane potential was measured using a mitochondrial membrane potential assay kit: The cell culture medium was aspirated, and the cells were washed once with PBS. 94 μL of reaction binding buffer, 1 μL of Mito-Tracker Red CMXRos staining solution, and 2.5 μL of Hoechst 33342 staining solution were added and gently mixed. The cells were incubated at room temperature in the dark for 30 minutes, and the cells were immediately imaged under a fluorescence microscope. Mito-Tracker Red CMXRos fluorescence was detected as red fluorescence, and Hoechst 33342 fluorescence was detected as blue fluorescence. Scale bar = 100 μm. (B) MitoTracker Red CMXRos fluorescence assay 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 expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism software 8.0 (GraphPad, California, USA). Differences between groups were assessed using one-way analysis of variance (ANOVA) 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 Bufoserotonin C can effectively increase MPP + The effects of 2.5μM, 5μM, 10μM, 20μM and 40μM Bufoserotonin C intervention on the MPP of 2mM were examined. + Effect of treatment on SH-SY5Y cell viability. CCK-8 results showed that MPP + After treatment, cell viability decreased to 49.27%.With the increase of Bufoserotonin C treatment concentration, 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 detected by + 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, simple MPP + 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 conclusion, 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 will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. Use of Bufoserotonin C in the preparation of a drug for treating Parkinson's disease, characterized in that: Bufoserotonin C is N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide.

2. The use of Bufoserotonin C according to claim 1 in preparing a drug for treating Parkinson's disease, characterized in that Bufoserotonin C plays a protective role in neuronal damage.

3. The use of Bufoserotonin C according to claim 2 in the preparation of a medicament for treating Parkinson's disease, characterized in that: Bufoserotonin C reduces MPP + Induced SH-SY5Y cell damage and cytotoxicity, improved MPP in SH-SY5Y cells + The mitochondrial dysfunction induced by β-actin plays a protective role in neuronal damage.

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