Use of astragalol in the preparation of a product for treating or alleviating parkinson's disease

Abstract

The application discloses application of astragalol in preparation of products for treating or relieving Parkinson's disease. The application explores behavioral and pathological pharmacological effects of astragalol on PD through in-vitro and in-vivo PD models, evaluates intervention effects of the astragalol on inflammation levels, and analyzes differential metabolites and genes through metabolomics and transcriptomics. Enrichment pathway analysis is used to identify intervention signal channels and key genes, and results of the metabolomics analysis are verified. Finally, the molecular mechanism of the astragalol in playing a neuroprotective role on neural inflammation and PD is clarified, and a theoretical basis is provided for development of the astragalol as a new dietary supplement.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and in particular to the application of Astragaloside in the preparation of products for treating or relieving Parkinson's disease. BACKGROUND

[0002] Parkinson's disease (PD) is the second largest neurodegenerative disease in the world, second only to Alzheimer's disease. In recent years, the incidence of PD has been increasing year by year, and the age of onset is also becoming younger and younger. Therefore, exploring the treatment strategy of PD is a thorny problem all over the world. At present, the clinical treatment of PD mainly involves chemical drugs such as levodopa, which is used to compensate for the decrease or inhibit the degradation of dopamine concentration in the brain caused by Parkinson's disease. However, long-term use of drugs can easily lead to adverse reactions such as drug resistance, hindering the improvement of PD patients' symptoms. Due to the bottleneck of PD chemical drug exploration, the research of natural products has become an important source of new drug development. Therefore, exploring new natural products and finding potential drugs that are effective and stable for treating PD are challenging and practically significant for the research of PD.

[0003] The pathogenesis of PD involves multiple pathways, including abnormal aggregation of alpha-synuclein, oxidative stress, mitochondrial dysfunction, neuroinflammation, etc. Neuroinflammation is a common feature of various causes. In recent years, many effective substances have been found in natural products and their derivatives, such as ginsenosides, berberine, piperine, etc., which have significant improvement effects on neuroinflammation and neurodegenerative diseases. Therefore, potential drugs for treating neurodegenerative diseases based on neuroinflammation research have become a popular direction.

[0004] As a "food and medicine homologous substance", Astragalus can enhance human immunity and is widely used in food health and food hygiene fields. The main active ingredient of Astragalus, Astragaloside IV, has various pharmacological activities. Its aglycone structure ring, Astragaloside, is the only telomerase activator from natural sources, which has been successfully marketed as an anti-aging product. Astragaloside, as a conformational open ring compound of ring Astragaloside, is the product of acid hydrolysis of ring Astragaloside. However, due to the nature of the cyclic structure and the open ring structure, there are often great differences, and it cannot be determined whether Astragaloside can also play a role in relieving or treating PD symptoms. For example, strychnine is a natural product with strong neurotoxicity, but its metabolite has great differences in toxicity and other activities after ring opening, adding uncertainty to the ring opening of the bridge ring, and also challenging the research of structural diversity. SUMMARY

[0005] The purpose of the present application is to provide a product for treating or relieving Parkinson's disease and the application of Astragaloside in the preparation of the product for treating or relieving Parkinson's disease.

[0006] The present application claims the application of Astragaloside in the preparation of products for treating or relieving Parkinson's disease.

[0007] Further, the product is a pharmaceutical product.

[0008] Further, the treatment or alleviation of Parkinson's disease is manifested by:

[0009] 1) alleviating motor impairment caused by MPTP;

[0010] 2) reducing inflammatory factors in serum and brain tissue;

[0011] 3) improving abnormal aggregation of alpha-synuclein.

[0012] The present application provides a product for treating or alleviating Parkinson's disease, wherein the active ingredient of the product comprises astragalol.

[0013] Further, the product is a pharmaceutical product.

[0014] Further, the treatment or alleviation of Parkinson's disease is manifested by:

[0015] 1) alleviating motor impairment caused by MPTP;

[0016] 2) reducing inflammatory factors in serum and brain tissue;

[0017] 3) improving abnormal aggregation of alpha-synuclein.

[0018] In the present application, the behavioral and pathophysiological effects of AST on PD were explored through in vitro and in vivo PD models, the intervention effect of AST on the level of inflammation was evaluated, and the differential metabolites and genes were analyzed by metabolomics and transcriptomics. Enrichment pathway analysis was used to identify the intervention signal pathways and key genes, and the results of the omics analysis were verified. Finally, the molecular mechanisms of AST in playing a neuroprotective role in neuroinflammation and PD were elucidated, which provided a theoretical basis for the development of AST as a new drug. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Pharmacodynamic study of AST on in vitro and in vivo models of PD. Among them, (A) the chemical structure of AST; (B-C) cell survival rate and LDH expression of different concentrations of AST in PD in vitro model (MPP + induced SH-SY5Y cells); (D) experimental method of MPTP-induced PD model; (E-F) gait measurement; (G) pole climbing experiment; (H) immunofluorescence staining analysis of alpha-synuclein. Data are expressed as mean ± standard deviation of three independent repeated experiments * vs Control; # vs Model; * / # p<0.05,** / ## p<0.01, *** / ### p<0.001).

[0020] Figure 2 AST inhibited MPTP-induced neuroinflammation. (A-B) Inflammatory cytokines in brain tissue and serum were detected; (C-E) Immunofluorescence staining analysis of TH and Iba-1 expression; (F-I) Western Blot analysis of TH and Iba-1 expression. Data are expressed as mean ± standard deviation of three independent repeated experiments. (*p<0.05, **p<0.01, ***p<0.001).

[0021] Figure 3 AST regulated amino acid metabolic pathways. Among them, (A) PLS-DA analysis of Con and Mod with AST-H, Con and Mod, Mod and AST-G. (B) KEGG analysis related to differential metabolites. C. Venn diagram of metabolic differences between groups.

[0022] Figure 4 AST can up-regulate the expression of VDR gene. Among them, (A-C) Comparison of model with AST-H, KEGG, Go and differential gene expression; (D-F) qRT-PCR verification of Cnn1, VDR and Retnlg genes; (G, H) Western Blot detection of the expression level of VDR protein in brain tissue. Data are expressed as mean ± standard deviation of three independent repeated experiments (*p<0.05, **p<0.01, ***p<0.001).

[0023] Figure 5 AST can reduce the level of oxidative stress and inhibit ferroptosis. Among them, (A) Iron aggregation in brain tissue (Prussian blue staining); (B-F) Expression level of ferroptosis-related proteins in brain tissue; (G-I) Expression level of Nrf2 / HO-1 in brain tissue. (*p<0.05, **p<0.01, ***p<0.001).

[0024] Figure 6 Activity comparison chart between cycloastragenol and astragalol. DETAILED DESCRIPTION

[0025] The application will be further described in conjunction with the specific embodiments. The examples given are only to illustrate the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not in any way constitute a limitation on the application.

[0026] The experimental methods in the following examples are all conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0027] The quantitative experiments in the following examples are all set up in triplicate, and the results are averaged, unless otherwise specified.

[0028] Materials and methods

[0029] Chemical reagents and consumables. Astragalol (AST, CAS: 86541-79-9) and cycloastragenol (CAS: 78574-94-4) were purchased from Chengdu Epha Biotechnology Co., Ltd. MPTP and MPP + were purchased from Sigma-Aldrich. CCK-8 and LDH kits were purchased from Beijing Solabio Technology Co., Ltd. PCR experimental consumables were purchased from Shanghai Sangon Biotech Co., Ltd. IL-1β, IL-6, TNF-α kits were purchased from Wuhan Biaolinai Biotechnology Co., Ltd. Anti-TH, Anti-Iba-1, Anti-TfRC, Anti-FTH, Anti-Fpn, Anti-GPX4, Anti-p-Nrf2, Anti-HO-1, Anti-VDR and Anti-β-actin were purchased from Wuhan Sivier Biotechnology Co., Ltd.

[0030] Cell culture. SH-SY5Y was purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). Cells were cultured in DMEM containing 10% FBS at 37°C in a 5% CO2 incubator.

[0031] MPP + Induced cell activity in vitro. Cell survival rate was measured using CCK-8 assay. SH-SY5Y cells were treated with MPP + (3 mM) and different concentrations of AST (5, 10, 20, 40, 80, 100 μM) for 24 hours. SH-SY5Y cells were treated with MPP + (3 mM) and different concentrations of cycloastragenol (5, 10, 20, 40, 80, 100 μM) for 24 hours. Then, CCK-8 solution was added to each well, incubated at 37°C for 1 hour, and the absorbance was measured at 450 nm using a microplate reader. According to the detection requirements of the LDH detection kit, the supernatant of each group of cells was collected and added to the detection reagent. The absorbance value was measured at 450 nm, and the LDH content in each group was calculated.

[0032] Animals. All C57BL / 6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animal procedures were approved by the Animal Ethics Committee of Shanxi University of Chinese Medicine (AWE202307367). A total of 60 C57BL / 6 mice (male, 6-8 weeks, body weight 20±2 g) were used in this study and divided into 4 groups (n=15): control group, MPTP group, AST-L group (10 mg / kg), and AST-H group (30 mg / kg). The mouse PD model was established by intraperitoneal injection of MPTP, once a day for one week, with a concentration of 15 mg / kg on the first day; 20 mg / kg on the second day; and 30 mg / kg on the 3rd to 7th day. From the first day of preparation of the MPTP group, AST treatment was performed once a day for 14 days.

[0033] Behavioral experiments. Gait measurement. The DigiGait animal gait detection system was used to evaluate the gait behavior indicators of mice. The speed was set to 15 cm / s, and the stride and frequency of the mouse paw were recorded for 10 seconds continuously.

[0034] Rope climbing test. The mouse was placed on the experimental rod (1 cm in diameter and 60 cm in length), and the time the mouse stayed on the rod and the time it took to fall to the bottom were recorded. The test was repeated three times with an interval of 5 minutes.

[0035] Transcriptomic analysis of brain tissue. Brain tissues of the control group, model group, and AST-H group (n=6) were collected, and total RNA was extracted from each sample. Sequencing was commissioned by the company. The main steps included RNA library construction, sequencing, differential expression genes, and functional analysis (gene ontology analysis and pathway enrichment analysis), which were divided into control group vs. model group, model group vs. AST-H group. Then, the commonly differentially expressed genes between the model group and the AST-H group were screened for analysis.

[0036] Metabolomic analysis of brain tissue. Brain tissues of the control group, model group, and AST-H group (n=6) were collected, and sample processing and LC-MS analysis were commissioned by the company, including differential metabolite analysis identification, differential substance analysis (differential substance clustering analysis and KEGG analysis, etc.), univariate statistical analysis, and multivariate statistical analysis. Finally, the differential substances between the control group and the model group, and the model group and the AST-H group were obtained, and the metabolic pathways were analyzed.

[0037] Quantitative real-time PCR. Brain tissues of the control group, model group, and AST-H group were collected, total RNA was extracted using a spin column animal total RNA purification kit and reverse transcribed into cDNA. SGExcel fast SYBR mixture was mainly used for reactions on a fluorescence quantitative PCR instrument. GAPDH was used as an internal reference gene, and 2 -ΔΔCTMethod Analysis of relative gene expression levels. The above experiment was repeated three times for calculation and analysis. Based on the analysis of RNA-seq data, PD-related differentially expressed genes were identified, and the company was commissioned to design and synthesize primers. The primer sequences are shown in Table 1 below.

[0038] Table 1. Primer sequences of differentially expressed genes

[0039]

[0040] Inflammatory factor analysis. Brain tissue and serum of control group, model group and AST group were collected, and supernatant was extracted. ELISA analysis of inflammatory factors (IL-1β, IL-6 and TNF-α) was performed respectively, and absorbance value was measured at 450 nm to analyze the expression level of inflammatory factors.

[0041] Immunofluorescence analysis. 3 mice from each group were taken for immunofluorescence staining. The mice were anesthetized with isoflurane, and 10 mL of PBS solution and 30 mL of 4% paraformaldehyde were perfused through the abdominal aorta. The tissue was washed with PBS three times. After dehydration with different concentrations of sucrose gradient, OCT reagent was used for embedding. The brain tissue was frozen in liquid nitrogen, and coronal sections (10 μm) were cut using a low-temperature constant temperature microtome. The sections were dried for 24 hours and stored at -80°C. The sections were washed with PBS and the OCT embedding agent was removed. The sections were immersed in PBS containing 0.3% Triton X-100 and incubated at room temperature for 30 minutes. The first antibody (TH and Iba-1) was evenly added to the sections and incubated overnight. Then, secondary fluorescent antibody was incubated. Finally, DAPI was added and incubated for 5-10 minutes. Laser confocal microscope was used to observe and capture images, and Image-J software was used to analyze fluorescence intensity or cell count.

[0042] Prussian blue staining. After brain tissue sectioning, the paraffin was removed and immersed in Prussian blue staining solution for 20-30 minutes. Washed with distilled water, stained in nuclear fixation red staining solution for 5-10 minutes. After sealing the sections with glue, observe under an inverted fluorescence microscope, and take pictures at 200 μm and 100 μm magnification.

[0043] Protein expression analysis. Different groups of brain tissue and cell samples were collected and added to RIPA lysis buffer to extract total protein. The proteins were separated by SDS-PAGE (8%, 12% gel) and then transferred to a membrane. 5% skim milk was used to block the polyvinylidene fluoride membrane. Subsequently, the membrane was incubated with Anti-TH, Anti-Iba-1, Anti-TfRC, Anti-FTH, Anti-Fpn, Anti-p-Nrf2, Anti-HO-1, Anti-GPX4, Anti-VDR and Anti-β-actin (1:1000) at 4°C overnight. TBST was used to wash the membrane. The membrane was incubated with anti-rabbit / anti-mouse secondary antibody (1:5000). Finally, the gel imaging system captured the developed signal.

[0044] Statistical analysis. Statistical comparisons between groups were performed using one-way ANOVA followed by Tukey’s post hoc. Data are presented as the mean ± standard deviation of three independent experiments performed in triplicate. P values < 0.05 were considered statistically significant.

[0045] Results 1

[0047] AST can improve the in vitro and in vivo pharmacological indicators of the PD model. First, we detected the cell viability and LDH expression levels of AST in the in vitro PD model. The results showed that with the increase of AST concentration, the cell viability reached a peak at 40-80 μΜ, and the expression level of LDH decreased with the increase of cell viability, indicating that AST can reverse the MPP + induced neuronal damage and has a neuroprotective effect. Figure 1 B-C)

[0048] We constructed the MPTP-induced in vivo PD model and tested the pharmacological and pathological indicators of AST to improve PD. The results of behavioral experiments showed that MPTP induction can increase the step frequency and shorten the step length of mice in the gait test, which is consistent with the behavior of the PD model. After AST treatment, both step length and step frequency were significantly improved and tended to the control group. Figure 1 E-F) In the pole test, the time spent by model mice at the top significantly increased, indicating that the mice had cognitive and behavioral disorders. After AST treatment, the climbing speed of mice increased significantly. Figure 1 G) Abnormal aggregation of alpha-synuclein is a typical pathological symptom of PD. Further pathological examination of PD showed that after administration, AST promoted the elimination of alpha-synuclein. Figure 1 H) 2

[0050] AST can alleviate MPTP-induced neuroinflammation. Since IV is the precursor compound of AST, it has a significant inhibitory effect on neuroinflammation. Therefore, we investigated the therapeutic effect of AST on MPTP-induced neuroinflammation. First, ELISA analysis was used to detect the expression of inflammatory factors in serum and brain tissue. The results showed that AST reversed the increase in the expression of inflammatory factors in serum and brain tissue induced by MPTP, and was positively correlated with the dose of administration. Figure 2 A-B) In addition, the activation level of microglia in the substantia nigra pars compacta (SNpc) was analyzed by immunofluorescence staining and Western blotting. The results showed that the number of TH-positive cells in the SNpc was significantly reduced in the model group, and AST could restore the reduction of TH expression; Figure 2 C-E) At the same time, the model group caused an increase in the expression level of Iba-1, indicating that MPTP can stimulate the activation of microglia. After AST treatment, the expression of Iba-1 was down-regulated, indicating that AST can inhibit the inflammatory response induced by MPTP. Figure 2 F-I) 3

[0052] AST can affect neuroinflammation by regulating amino acid metabolic pathways. To further explore the pharmacological mechanism of AST in neuroinflammation, we used metabolomics to analyze the differential metabolites in brain tissue of different groups, and based on the differential metabolites, we analyzed the metabolic pathways. The results showed that in the multivariate statistical analysis and pairwise group analysis under PLS-DA analysis, there were obvious differences in the regions of different groups, indicating good differences between groups. Figure 3 A) In the differential metabolite analysis, compared with the control group, the model group caused 4 metabolites to be up-regulated and 4 metabolites to be down-regulated. After AST treatment, compared with the model group, CAG caused 7 metabolites to be up-regulated and 17 metabolites to be down-regulated. (Table 2 and Figure 3 C) In the KEGG analysis based on differential metabolites, arginine and proline metabolism, and amino acid and co-factor biosynthesis became the main metabolic pathways regulated by AST after administration. Figure 3 B) Studies have shown that amino acid metabolic pathways dominated by arginine and proline metabolism are particularly important in neurological diseases, especially in regulating the level of neuroinflammation. The biosynthesis of co-factors is related to the nutrition of neurons, and MPTP as a neurotoxic substance can significantly reduce the expression of neurotrophic factors such as BDNF. After AST treatment, it can nourish neurons and improve the function of nerve cells by restoring co-factors or neurotrophic substances. Therefore, the role of AST in improving neuroinflammation may be achieved through pathways such as amino acid metabolism and co-factor biosynthesis.

[0053] Table 2. Differential metabolites of Model and AST-H groups

[0054] 4

[0056] AST increased the expression of VDR gene, indicating its regulation of ferritin deposition signaling pathway. Transcriptomics was used to explain the neuroinflammatory pharmacological effects of AST at the mRNA level. According to the screening criteria (fold change > 2 or < 0.5, p < 0.05), the results of differentially expressed genes of Control + Model and Model + AST-H were obtained. In the control and model groups, 48 and 32 genes were up- and down-regulated, respectively. In Model + CAG-H, 39 and 8 genes were up- and down-regulated, respectively. Figure 4 C) By summarizing the differentially expressed genes, genes related to PD or neuroinflammation were screened for GO and KEGG analysis with a screening criterion of p < 0.05. Finally, we identified 7 differentially expressed genes related to the model group (Cd74, Mst1, Ccr2, Cnn1, Retnlg, VDR and Cd200r1) and verified them using fluorescent quantitative PCR technology. The results showed that the mRNA expression of VDR, Cnn1 and Retnlg was consistent with the transcriptome analysis. Figure 4 D-F) In gene function analysis, VDR is a vitamin D receptor that plays an important role in the nervous system by binding to various ligands, especially in the study of neurodegenerative diseases, it can mediate various physiological functions such as oxidative stress and ferroptosis. Cnn1 is a calmodulin protein, recent studies have found that its binding with Kdm6a enzyme can affect neuroinflammation and nerve function repair. The Retnlg gene is related to various physiological processes such as metabolic disorders, immune function and inflammation. In GO analysis, the results of Model vs AST-H include "response to external stimulus", "cellular response to chemical stimulus" and "signal receptor binding". Figure 4 B) For KEGG analysis, the model group and AST-H group were rich in "pathways of neurodevelopmental diseases" and other related diseases, as well as common inflammatory pathways such as IL-17, MAPK and HIF-1 signaling pathways. Figure 4 A) In addition, we noticed that there may be ferroptosis in the pathological process of the pharmacological effects of AST. Therefore, we conducted experimental verification around the VDR gene and the ferroptosis signaling pathway. In protein expression detection, the Western Blot results were consistent with the PCR results. The expression of VDR receptor decreased in the model group, while the expression level of VDR significantly increased after AST treatment. Figure 4 G-H) 5

[0058] AST exerts anti-neuroinflammatory effects by reducing oxidative stress levels and inhibiting ferroptosis pathways. We first analyzed the aggregation of iron ions in the brains of mice in different groups using Prussian blue staining. The results showed that after MPTP induction, the model group had significantly increased iron aggregation, with increased blue patches, and AST effectively reduced brain iron levels. Figure 5 A) In addition, the expression levels of ferritin-related proteins were detected by Western blotting. The results showed that compared with the model group, the expression of TfRC (iron transport protein) and FTH (iron storage protein) was significantly reduced after AST treatment, while the expression of Fpn (iron efflux protein) was up-regulated, indicating that AST can reverse MPTP-induced cellular iron storage, inhibit intracellular iron transport, and accelerate iron ion efflux. Glutathione peroxidase 4 (GPX4) is a key factor in the process of ferroptosis and can be used as one of the indicators to determine cellular ferroptosis. Therefore, in the protein expression results, it was found that the expression level of GPX4 was significantly reduced in the MPTP-induced model, leading to the accumulation of intracellular peroxide, while AST treatment can significantly up-regulate the expression level of GPX4. Figure 5 B-F)

[0059] To explore the effects of MPTP induction on oxidative stress, the Nrf2 / HO-1 signaling pathway is a key pathway for antioxidant systems in vivo. Nrf2 can repair the function of the glutathione system, and Nrf2 can regulate the expression of iron metabolism-related proteins in ferroptosis and improve ferroptosis. Therefore, we detected the phosphorylation level of Nrf2 and the expression of HO-1. The results showed that the phosphorylation level of Nrf2 and the expression of HO-1 protein were significantly reduced in the model group. After AST treatment, the expression of p-Nrf2 and HO-1 was significantly increased, indicating that AST can activate the Nrf2 / HO-1 signaling pathway in vivo and exert antioxidant effects. Figure 5 G-I) 6

[0061] It has been reported in the literature that cycloastragenol has cytotoxicity in vitro at high doses, which is a potential drawback of cycloastragenol. Through an in vitro cell model of Parkinson's disease (MPP+ induced SH-SY5Y cells), the activity comparison between cycloastragenol and astragalol (AST) was evaluated, and it was found that at a concentration of 20-40 μM, the activity of astragalol (AST) was slightly worse than that of cycloastragenol, but it is worth noting that in the LDH (lactate dehydrogenase detection, aimed to evaluate the potential toxicity of cells) test, at a concentration of 100 μM, the in vitro cytotoxicity of astragalol (AST) was significantly lower than that of cycloastragenol. This exploration further proves that the breakage of the bridge ring in the structure is beneficial to the change of the activity of the compound.

[0062] Conclusion

[0063] In summary, this study explored the potential application of AST in neurodegenerative diseases and found that AST can inhibit the progression of neuroinflammation and alleviate the symptoms of PD. In addition, through multi-omics analysis and verification experiments, ferroptosis is the key pathway of AST inhibiting neuroinflammation. AST can reduce the level of oxidative stress in cells, thereby regulating neuronal homeostasis and protecting neurons. This finding indicates the potential application of AST in neuroprotection, providing a scientific basis for the development of drugs or dietary supplements, and laying a data support for the comprehensive elucidation of the development and application of Astragalus membranaceus in "medicinal and edible homology".

[0064] The above has been described in detail. For those skilled in the art, the present application can be implemented within a wider range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present application and without unnecessary experiments. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In summary, according to the principle of the present application, this application intends to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which deviates from the range disclosed in this application. Some basic features can be applied within the scope of the following attached claims.

Claims

1. Use of astragalool in the preparation of medicines for treating or alleviating Parkinson's disease.

2. The use according to claim 1, characterized in that The treatment or alleviation of Parkinson's disease is embodied in: 1) Alleviate movement disorders caused by MPTP; 2) Reduce inflammatory factors in serum and brain tissue; 3) Improve the abnormal aggregation of α-synuclein.

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