CN119818509B - Application of astragalool in preparing products for treating or alleviating Parkinson's disease - Google Patents

Abstract

The present invention discloses the use of astragalool in the preparation of products for treating or alleviating Parkinson's disease. The present invention explores the behavioral and pathological pharmacological effects of astragalool on Parkinson's disease (PD) using in vitro and in vivo PD models, evaluates its intervention effects on inflammatory levels, and analyzes differential metabolites and genes through metabolomics and transcriptomics. Enrichment pathway analysis was used to identify intervention signaling pathways and key genes, and the results of omics analysis were validated. Ultimately, the molecular mechanism by which astragalool exerts its neuroprotective effects on neuroinflammation and PD is elucidated, providing a theoretical basis for the development of astragalool as a novel dietary supplement.

Description

Application of astragaloside in preparation of products for treating or relieving parkinsonism

Technical Field

The invention relates to the field of biological medicine, in particular to application of astragalus alcohol in preparing a product for treating or relieving parkinsonism.

Background

Parkinson's Disease (PD) is the second most neurodegenerative disease worldwide, next to alzheimer's disease. In recent years, the incidence of PD has increased year by year, and the age of onset has also been getting younger. Thus, exploring therapeutic strategies for PD is a troublesome problem worldwide. The current clinical treatment of PD mainly involves chemical drugs such as levodopa and the like, which are used for compensating the reduction of the concentration of dopamine in the brain or inhibiting the degradation of the dopamine in the brain caused by parkinsonism. However, long-term administration tends to cause adverse reactions such as drug resistance, and thus prevents improvement of symptoms in PD patients. Because of the bottleneck in PD chemical drug discovery, research into natural products has become an important source of new drug development. Thus, exploring new natural products and finding potential drugs that are effective and stable in treating PD has challenging and realistic implications for the study of PD.

The pathogenesis of PD involves a variety of pathways including abnormal aggregation of α -synuclein, oxidative stress, mitochondrial dysfunction, neuroinflammation, and the like. Neuroinflammation is a common feature of various etiologies. In recent years, many effective substances such as ginsenoside, berberine, piperine and the like are found in natural products and derivatives thereof, and have remarkable improving effect on neuroinflammation and neurodegenerative diseases. Thus, potential drugs for treating neurodegenerative disorders based on neuroinflammation studies have become a popular direction.

Astragalus as a kind of medicine and food homolog can strengthen human immunity and is widely used in the fields of food health and food sanitation. Astragalus saponin IV as main active component has various pharmacological activities. The aglycone structure of cycloastragaloside is the only telomerase activator from natural sources, and has been successfully marketed as an anti-aging product. The astragalus alcohol is used as an allosteric open ring compound of the cycloastragaloside and is a product of cycloastragaloside acid hydrolysis. However, due to the great difference between the nature of the cyclic structure and the nature of the ring-opened structure, it cannot be determined whether the astragalus alcohol can also exert an effect of alleviating or treating the PD symptoms. For example, strychnine is a natural product with strong neurotoxicity, but the activities such as toxicity and the like of the metabolic products after ring opening are greatly different, so that the activity uncertainty is added for ring opening of a bridged ring, and the structure diversity research is more challenging.

Disclosure of Invention

The invention aims to provide a product for treating or relieving parkinsonism and application of astragalus alcohol in preparing a product for treating or relieving parkinsonism.

The invention claims the application of astragalus alcohol in preparing a product for treating or relieving parkinsonism.

Further, the product is a pharmaceutical product.

Further, the treatment or alleviation of parkinson's disease is manifested in:

1) Alleviating MPTP-induced dyskinesias;

2) Reducing inflammatory factors in serum and brain tissue;

3) Improving abnormal aggregation of alpha-synuclein.

The invention provides a product for treating or relieving parkinsonism, and the active ingredients of the product comprise astragalus alcohol.

Further, the product is a pharmaceutical product.

Further, the treatment or alleviation of parkinson's disease is manifested in:

1) Alleviating MPTP-induced dyskinesias;

2) Reducing inflammatory factors in serum and brain tissue;

3) Improving abnormal aggregation of alpha-synuclein.

In the present invention, the behavioral and pathological pharmacological effects of AST on PD were studied by in vitro and in vivo PD models, their intervention on inflammatory levels was evaluated, and differential metabolites and genes were analyzed by metabolomics and transcriptomics. Enrichment pathway analysis was used to identify interfering signaling pathways and key genes, and the results of the genomic analysis were validated. Finally, the molecular mechanism of AST for protecting nerve inflammation and PD is clarified, and theoretical basis is provided for developing AST as new medicine.

Drawings

Fig. 1 is a pharmacodynamic study of AST on PD in vitro and in vivo models. Wherein, (A) chemical structure of AST, (B-C) cell viability and LDH expression of AST with different concentrations in an in vitro PD model (SH-SY 5Y cells induced by MPP ⁺), (D) experimental method of MPTP induced PD model, (E-F) gait measurement, (G) pole climbing experiment, and (H) immunofluorescence staining analysis of alpha-synuclein. Data are expressed as mean ± standard deviation of three replicates independently performed (^*vs Control;^#vs Model;^*/^#p<0.05,^**/^##p<0.01,^***/^###p<0.001).

Fig. 2 is an AST inhibiting MPTP-induced neuroinflammation. (A-B) detection of inflammatory factors in brain tissue and serum, (C-E) immunofluorescent staining analysis of TH and Iba-1 expression, and (F-I) Western Blot analysis of TH and Iba-1 expression. Data are expressed as mean ± standard deviation of three replicates performed independently. P <0.05, p <0.01, p < 0.001).

FIG. 3 is an AST-mediated amino acid metabolic pathway. Wherein, (A) PLS-DA analysis of Con and Mod and AST-H, con and Mod, mod and AST-G. (B) KEGG analysis associated with differential metabolites. Venn diagram of metabolite differences between group C.

FIG. 4 shows that AST can up-regulate VDR gene expression. Wherein, (A-C) comparison of the model with AST-H, KEGG, go and differential gene expression, (D-F) qRT-PCR verification of Cnn, VDR and Retnlg genes, and (G, H) Western Blot detection of brain tissue VDR protein expression level. Data are expressed as mean ± standard deviation of triplicate experiments performed independently (< p <0.05, < p <0.01, < p < 0.001).

Fig. 5 shows that AST can reduce the level of oxidative stress and inhibit iron death. Wherein, (A) iron aggregation of brain tissue (Prussian blue staining), (B-F) expression level of iron death-related protein in brain tissue, (G-I) expression level of Nrf2/HO-1 in brain tissue. P <0.05, p <0.01, p < 0.001).

FIG. 6 is a graph showing comparison of activities between cycloastragaloside and astragaloside.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Unless otherwise indicated, the quantitative tests in the examples below were all performed in triplicate, and the results averaged.

Materials and methods

Chemical reagents and consumables. Astragalol (AST, CAS: 86541-79-9), and cycloastragalosil (CAS: 78574-94-4) were purchased from Chengdu Biotechnology Co. MPTP ANDMPP ⁺ was purchased from Sigma-Aldrich. CCK-8 and LDH kits were purchased from Beijing Soy Bao technology Co. The PCR assay consumables were purchased from Shanghai Biotechnology Co. IL-1 beta, IL-6, TNF-alpha kit was purchased from Wohan Bei Yinlai 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 and from Wohan Sieve Biotechnology Co., ltd.

And (5) culturing the cells. SH-SY5Y was purchased from the cell bank of the national academy of sciences (Shanghai, china). Cells were cultured in DMEM containing 10% fbs in an incubator containing 5% co ₂ at 37 ℃.

MPP ⁺ induced in vitro cellular activity. Cell viability was measured using the CCK-8 assay. SH-SY5Y cells were treated with MPP ⁺ (3 mM) and AST (5, 10, 20, 40, 80, 100. Mu.M) at various concentrations for 24 hours. SH-SY5Y cells were treated with MPP ⁺ (3 mM) and with different concentrations of cycloastragenol (5, 10, 20, 40, 80, 100. Mu.M) for 24 hours. Then, CCK-8 solution was added to each well, incubated at 37℃for 1 hour, and absorbance was measured at 450nm using a microplate reader. According to the detection requirement of the LDH detection kit, collecting cell supernatants of each group, and adding the cell supernatants into a detection reagent. Absorbance values were measured at 450nm and LDH content in each group was calculated.

An animal. All C57BL/6 mice were purchased from beijing vernoniwa laboratory animal limited and all animal procedures were approved by the university of shanxi chinese medical laboratory ethical committee (AWE 202307367). The study used 60C 57BL/6 mice (males, 6-8 weeks, body weight 20.+ -.2 g) divided into 4 groups (n=15) of control, MPTP, AST-L (10 mg/kg) and AST-H (30 mg/kg). A PD model of the mice was established by intraperitoneal injection of MPTP once a day for a week, with a concentration of 15mg/kg on the first day, 20mg/kg on the second day, and 30mg/kg on days 3 to 7. AST treatment was once daily for 14 days starting from the first day of MPTP group preparation.

Behavioural experiments. Gait measurement. DigiGait animal gait detection system is used to evaluate the gait behavior index of the mice. The speed was set at 15cm/s and the steps and frequency of the paws of the different groups of mice were recorded for 10 seconds.

And (5) pole climbing test. The mouse was placed on an experimental bar (1 cm diameter, 60 cm length) and the time the mouse stopped on the bar and the time it was lowered to the bottom was recorded. The test article was repeated three times at 5 minute intervals.

Brain tissue transcriptomics analysis brain tissue of control, model and AST-H groups (n=6) was collected and total RNA was extracted from each sample. Sequencing is done by corporate commission. The method mainly comprises the steps of RNA library construction, sequencing, differential expression gene analysis and functional analysis (gene ontology analysis and pathway enrichment analysis), wherein the differential expression gene and the functional analysis are divided into a control group and a model group, and the model group and an AST-H group. The genes commonly differentially expressed between the model group and AST-H group were then screened for analysis.

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

Fluorescent quantitative PCR. Brain tissue of control, model and AST-H groups was collected, total RNA was extracted using spin column animal total RNA purification kit and reverse transcribed into cDNA. SGExcel Rapid SYBR mixtures are mainly used for reactions on fluorescent quantitative PCR instruments. GAPDH was used as an internal reference gene and relative gene expression levels were analyzed using the 2 ^-ΔΔCT method. The above experiment was repeated three times for calculation and analysis. Based on RNA-seq data analysis, PD-related differentially expressed genes were identified and primers were designed and synthesized by the company Limited. The primer sequences are shown in Table 1 below.

TABLE 1 primer sequences for differentially expressed genes

And (5) inflammatory factor analysis. Brain tissue and serum from control, model and AST groups were collected and supernatants were extracted. ELISA assays of inflammatory factors (IL-1. Beta., IL-6 and TNF-. Alpha.) were performed separately, and absorbance values were measured at 450nm to analyze the expression levels of inflammatory factors.

Immunofluorescence analysis. 3 mice were immunofluorescent stained for each group. Mice were anesthetized with isoflurane and perfused through the abdominal aorta with 10mL of PBS solution and 30mL of 4% paraformaldehyde. The tissue was washed three times with PBS. After gradient dehydration with sucrose at different concentrations, OCT reagents were used for embedding. Brain tissue was frozen in liquid nitrogen and coronal sections (10 μm) were cut using a cryostat microtome. The sections were dried for 24 hours and stored at-80 ℃. Sections were washed with PBS and OCT embedding medium was removed. The sections were immersed in PBS containing 0.3% Triton X-100 and incubated for 30 minutes at room temperature. The primary antibodies (TH and Iba-1) were added uniformly to the sections and incubated overnight. Then, incubation with secondary fluorescent antibody was performed. Finally, DAPI was added dropwise and incubated for 5-10 minutes. Images were observed and captured using a confocal laser microscope and fluorescence intensity or cell count was analyzed using Image-J software.

Prussian blue staining. After brain tissue sections, paraffin was removed and immersed in Prussian blue staining solution for 20-30 minutes. Washing with distilled water, and dyeing in nuclear fixation red staining solution for 5-10 min. After sealing the sections with glue, the sections were observed under an inverted fluorescence microscope and photographed at a magnification of 200 μm and 100 μm.

Protein expression analysis. Different groups of brain tissue and cell samples are collected and added into RIPA lysate to extract total proteins. Proteins were separated by SDS-PAGE (8%, 12% gel) and then subjected to transmembrane. 5% skim milk was used to block the polyvinylidene fluoride membrane. Subsequently, the membranes were incubated overnight at 4℃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, respectively. TBST is used to clean the membrane. Membranes were incubated with anti-rabbit/anti-mouse secondary antibody (1:5000). Finally, the gel imaging system captures the development signal.

And (5) carrying out statistical analysis. Group statistical comparisons were performed using one-way anova followed by Tukey posterior. Data are expressed as mean ± standard deviation of three replicates performed independently. P values <0.05 were considered statistically significant.

Results

1

AST may improve pharmacological index in vitro and in vivo for PD models. First, we examined AST cell viability and LDH expression levels in an in vitro PD model. The results show that with increasing concentration of AST, cell viability peaks at 40-80 μm, and the expression level of LDH decreases with increasing cell viability, indicating that AST can reverse MPP ⁺ -induced neuronal damage and has neuroprotective effect. (FIGS. 1B-C)

We constructed an MPTP-induced in vivo model of PD and tested AST for improving pharmacological and pathological indicators of PD. The behavior experiment result shows that MPTP induction can increase the step frequency of mice in gait experiments and shorten the step, which is consistent with the behavior state of a PD model. After AST treatment, both stride and stride frequency were significantly improved and trended towards the control group. (FIGS. 1E-F) in the pole-climbing test, the time that the mice of the model group remained at the top was significantly increased, indicating that the mice had cognitive and behavioral impairment. After AST treatment, the pole climbing speed of mice increased significantly. (FIG. 1G) abnormal aggregation of alpha-synuclein is a typical pathological symptom of PD. Further pathological examination of PD showed that AST promoted elimination of α -synuclein after administration. (FIG. 1H)

2

AST may alleviate MPTP-induced neuroinflammation. Since astragaloside IV is a precursor compound of AST, it has a remarkable inhibitory effect on neuroinflammation. Thus, we studied the therapeutic effect of AST on MPTP-induced neuroinflammation. First, ELISA assays were used to detect the expression of inflammatory factors in serum and brain tissue. The results indicate that AST reverses the increase in inflammatory factor expression caused by MPTP in serum and brain tissue and has a positive correlation with the dose administered. (FIGS. 2A-B) furthermore, the activation level of microglial cells in the substantia nigra compact part (SNpc) was analyzed by immunofluorescent staining and Western blotting. The results showed that in SNpc, the number of TH positive cells in the model group was significantly reduced, AST could restore the reduction in TH expression (FIG. 2C-E) while the model group caused an increase in Iba-1 expression level, indicating that MPTP could stimulate microglial activation. Expression of Iba-1 is down-regulated following AST treatment, indicating that AST can inhibit MPTP-induced inflammatory response. (FIGS. 2F-I)

3

AST may affect neuroinflammation by modulating amino acid metabolic pathways. To further explore the pharmacological mechanisms of AST in neuroinflammation, we analyzed differential metabolites in different groups of brain tissues using metabonomics and metabolic pathways based on the differential metabolites. The results showed that there was a clear difference between the regions of the different groups in the multivariate statistical analysis and the pairwise group analysis under PLS-DA analysis, indicating that the group-to-group differences were good. (FIG. 3A) in differential metabolite analysis, the model group resulted in up-regulation of 4 metabolites and down-regulation of 4 metabolites compared to the control group. After AST treatment, CAG resulted in up-regulation of 7 metabolites and down-regulation of 17 metabolites compared to the model group. (Table 2 and FIG. 3C) in the KEGG analysis based on differential metabolites, arginine and proline metabolism, amino acid and cofactor biosynthesis become the primary metabolic pathways regulated by AST after administration. (FIG. 3B) studies indicate that amino acid metabolic pathways, predominantly arginine and proline metabolism, are particularly important in neurological diseases, particularly in regulating neuroinflammatory levels. The biosynthesis of cofactors 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 neurons by recovering cofactors or neurotrophic substances. Thus, the role of AST in ameliorating neuroinflammation may be achieved through amino acid metabolism, cofactor biosynthesis, and the like.

TABLE 2 differential metabolites of model and AST-H groups

4

AST increased expression of the VDR gene, indicating that it regulates ferritin deposition signaling pathways. Transcriptomics is used to explain the neuroinflammatory pharmacologic effects of AST at the mRNA level. Based on the screening criteria (fold change >2 or <0.5, p < 0.05), the differentially expressed gene results of control + model and model + AST-H were obtained. In the control and model groups, there were 48 and 32 genes up-and down-regulated in total. In model+CAG-H, there were 39 and 8 genes up-and down-regulated in total. (FIG. 4C) by summarizing the differentially expressed genes, genes associated with PD or neuroinflammation were screened for GO and KEGG analysis, with p <0.05 as the screening criteria. Finally, we identified 7 differentially expressed genes (Cd 74, mst1, ccr2, cnn1, retnlg, VDR and Cd200r 1) associated with the model set and validated them using fluorescent quantitative PCR techniques. The results show that mRNA expression of VDR, cnn1 and Retnlg is consistent with transcriptome analysis. (FIGS. 4D-F) VDR is a vitamin D receptor which plays an important role in the nervous system by binding to various ligands in gene function analysis, and particularly in the study of neurodegenerative diseases, it can mediate various physiological functions such as oxidative stress and iron death. Cnn1 is a calmodulin protein, and recent studies have found that its binding to Kdm6a enzyme can affect neuroinflammation and nerve function repair. Retnlg genes are involved in various physiological processes such as metabolic disorders, immune functions and inflammation. In the GO assay, the results of ModelvsAST-H include "response to external stimuli", "cellular response to chemical stimuli" and "signal receptor binding". (FIG. 4B) for the KEGG analysis, the model and AST-H groups are rich in "neurogenic multiple disease pathways" and other related diseases, as well as common inflammatory pathways such as IL-17, MAPK, and HIF-1 signaling pathways. (fig. 4A) furthermore, we note that there may be iron death during the pathological course of AST exerting pharmacological effects. Thus, we have experimentally verified around the VDR gene and iron sagging signaling pathway. In the protein expression detection, the Western Blot results were consistent with the PCR results. Model group VDR receptor expression was reduced, while post AST treatment VDR expression levels were significantly up-regulated. (FIGS. 4G-H)

5

AST exerts an anti-neuroinflammatory effect by reducing the level of oxidative stress and inhibiting the iron death pathway. We first analyzed the aggregation of iron ions in the brains of mice of different groups using Prussian blue staining. The results show that after MPTP induction, the iron aggregation of the model group is obviously increased, blue spots are increased, and AST can effectively reduce the brain iron level. (FIG. 5A) furthermore, the expression level of ferritin-related protein was detected by Western blotting. The results show that TfRC (iron transporter) and FTH (iron storable protein) expression are significantly reduced after AST treatment compared to the model group, while Fpn (iron efflux protein) expression is up-regulated, indicating that AST can reverse MPTP-induced cellular iron storage, inhibit intracellular iron transport, and accelerate iron ion efflux. Glutathione peroxidase 4 (GPX 4) is a key factor in the iron death process and can be used as one of the indicators for determining cell iron death. Thus, in the protein expression results, it was found that in the MPTP-induced model, the expression level of GPX4 was significantly reduced, resulting in intracellular peroxide accumulation, whereas AST treatment could significantly up-regulate the expression level of GPX 4. (FIGS. 5B-F)

To explore the effects of MPTP induction on oxidative stress, the Nrf2/HO-1 signaling pathway is a critical pathway for the antioxidant system in vivo. Nrf2 can repair the function of a glutathione system, and simultaneously Nrf2 can regulate the expression of iron metabolism related proteins in iron death and improve iron death. Thus, we examined the level of phosphorylation of Nrf2 and the expression of HO-1. The results show that the model group has significantly reduced Nrf2 phosphorylation and reduced HO-1 protein expression. After AST treatment, the expression of p-Nrf2 and HO-1 is obviously increased, which shows that AST can activate Nrf2/HO-1 signal paths in vivo and play an antioxidant role. (FIG. 5G-I)

6

The in vitro cytotoxicity of the cycloastragal at high dosage is reported in the literature and is also a potential defect of the cycloastragal. The comparison of activities between cycloastragal and Astragal (AST) was evaluated by the parkinson in vitro cell model (mpp+ induced SH-SY5Y cells) and it was found that the activity of Astragal (AST) was slightly worse than that of cycloastragal at a concentration of 20-40 μm, but notably that in LDH (lactate dehydrogenase assay, objective evaluation of cytotoxicity) assay, the in vitro cytotoxicity of Astragal (AST) was significantly lower than that of cycloastragal at a concentration of 100 μm. This search may further prove that cleavage of the structurally bridged ring is beneficial for the altered activity of the compound.

Conclusion(s)

In summary, the present 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. Furthermore, iron death is a key pathway for AST to inhibit neuroinflammatory effects through multiple sets of chemical analysis and validation experiments. AST can reduce the level of oxidative stress in cells, thereby regulating neuronal homeostasis and protecting neurons. The discovery shows that the AST has potential application in neuroprotection, provides scientific basis for development of medicines or dietary supplements, and lays data support for comprehensively elucidating development and application of astragalus in medicine-food homology.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Claims (2)

1. Use of astragalool in the preparation of medicines for treating or alleviating Parkinson's disease. 2. The use according to claim 1, wherein 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.