Application of homoplantaginin in preparation of medicine for preventing and treating neurodegenerative diseases

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of homoplantaginin to preparation of a medicine for preventing and treating neurodegenerative diseases. The invention discloses multiple effects of homoplantaginin on the aspects of inhibiting NLRP3 inflammasome related inflammatory response, relieving neuron oxidative damage and maintaining neuron mitochondrial homeostasis. Pharmacological experiment results show that the homoplantaginin not only significantly improves learning and memory impairment of the Alzheimer's disease and reduces A beta plaque deposition, but also effectively improves the viability of dopaminergic neurons, and shows an obvious relieving effect on motor and non-motor symptoms of the Parkinson's disease. Furthermore, the homoplantaginin is low in dosage, high in blood-brain barrier permeability, high in safety and wide in application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to an application of homoplanin in the preparation of a drug for preventing and treating neurodegenerative diseases. Background Art

[0002] Neurodegenerative diseases (NDDs) are a large spectrum of diseases driven by the progressive decline of nerve cell structure and function. Millions of people worldwide are suffering from the disease. With the increase in the average human life expectancy and the intensification of the aging trend, the prevalence of NDDs is showing a worrying trend of continuous growth. The pathological characteristics of NDDs include: aggregation of misfolded proteins, dysfunction of synapses and neuronal networks, imbalance of protein homeostasis, abnormal cytoskeletal structure, altered energy metabolism patterns, DNA and RNA defects, neuroinflammation and neuronal death. This series of pathological changes ultimately leads to impairment of memory, cognition, movement, sensation and / or language, seriously affecting the quality of life of patients. Therefore, strengthening the study of the pathogenesis of NDDs and finding effective prevention and treatment methods have become the hot spots of current research.

[0003] NDDs include Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease, amyotrophic lateral sclerosis, frontotemporal dementia, Lewy body dementia, etc. A large number of studies have shown that factors such as mitochondrial dysfunction, chronic inflammation, redox homeostasis imbalance and aging seriously affect the function and survival of neurons, and play a key driving role in the pathogenesis of NDDs. Developing new disease-modifying therapies targeting these factors is crucial for the prevention and treatment of NDDs.

[0004] Litchi grass, also known as snow primrose, is the whole herb of Salvia plebeia R.Br., a plant of the genus Salvia in the Lamiaceae family. It is cool in nature and bitter in taste. In traditional Chinese medicine, it can treat hemoptysis, hematuria, metrorrhagia, sore throat, carbuncle, and hemorrhoids. Homoplantaginin (HOM) is the main active ingredient of Litchi grass. It belongs to the flavonoids. The chemical structure is shown below. It is a yellow crystal that is easily soluble in methanol and ethanol. Modern pharmacological studies have shown that HOM has anti-inflammatory, antioxidant, liver protection, hypoglycemic, antibacterial, antitussive and expectorant effects, improves endothelial insulin resistance and anti-tumor effects. However, there is no relevant literature report on the study of HOM in neurological diseases. In particular, its regulatory effects on neuronal function and survival and its application potential in the preparation of drugs for the prevention and treatment of neurodegenerative diseases are still unclear. Therefore, in-depth neurological research based on the multiple pharmacological properties of HOM is conducive to the development of new candidate drugs for neuroprotection and the treatment of neurodegenerative diseases.

[0005] Summary of the invention

[0006] The purpose of the present invention is to provide a new use of homophyllium glycosides, namely, the regulatory effect of homophyllium glycosides on neuronal function and survival, and its use in the preparation of drugs for preventing and treating neurodegenerative diseases, including but not limited to Alzheimer's disease and Parkinson's disease.

[0007] In order to achieve the above-mentioned purpose, the present invention provides a method for inhibiting the activation of NLRP3 inflammasome and the inflammatory response mediated by homoplanin.

[0008] Preferably, the homophytoside can inhibit apoptosis and oxidative stress damage of nerve cells.

[0009] Preferably, the homophytoside can protect the mitochondrial function of nerve cells damaged by L-glutamate.

[0010] Preferably, the homophytoside significantly improves the behavioral disorder of AD mice induced by AlCl3 and D-gal, and inhibits the deposition of Aβ plaques in the hippocampus and cortex of AD mice.

[0011] Preferably, the homoplanin can alleviate chronic inflammation and redox homeostasis imbalance in AD mice and inhibit apoptosis of nerve cells.

[0012] Preferably, the homoplanin can alleviate cell senescence in AD mice.

[0013] Preferably, the homophyllidine significantly improves the movement disorder of PD mice induced by the neurotoxin MPTP, and reduces the dopaminergic neuron damage in PD mice.

[0014] The beneficial effects of the present invention are:

[0015] Plantagoside forms hydrogen bonds with Ala228, Arg578, Glu629, and Tyr632 in the active pocket of NLRP3, indicating that it has a strong affinity for NLRP3.

[0016] Homophytoside may alleviate the inflammatory response by inhibiting the activation of NLRP3 inflammasome.

[0017] Homoplanin can alleviate L-Glu-induced apoptosis in PC12 cells.

[0018] Homophytoside can alleviate L-Glu-induced oxidative damage and mitochondrial dysfunction.

[0019] The improvement effect of homoplanin on AD-like behavior may be partly due to its inhibition of Aβ deposition.

[0020] Homophytoside has anti-inflammatory and antioxidant properties, which can inhibit the apoptosis of nerve cells, exerting neuroprotective and anti-AD-like symptom effects.

[0021] Homophytoside treatment can reduce the activity of β-Gal. Homophytoside has anti-aging properties, which may partly contribute to the improvement of AD symptoms.

[0022] Homophytoside has an effective effect on PD-like behavioral disorders and decreased motor coordination, similar to the positive drug levodopa. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 The diagram shows the binding pattern of homoplanin to NLRP3 receptor and its inhibitory effect on inflammatory response mediated by NLRP3 inflammasome; Figure 1 A is a three-dimensional simulation diagram of molecular docking of homoplanin and NLRP3 (PDB: 7PZC), the co-crystal ligand MCC950 in the NLRP3 protein structure is represented by a gray line, and neohesperidin is represented by a yellow line; Figure 1 B is a local enlarged image of the interaction between MCC950 and the NLRP3 active pocket; Figure 1 C is a local magnified image of the interaction between homoplanin and the active pocket of NLRP3; Figure 1 D is the effect of homoplanin on the survival of J774A.1 cells; Figure 1 E and F show the effects of homoplanin on the levels of IL-1β and IL-18 in the supernatant of J774A.1 cells under LPS / ATP stimulation. Figure 1 G and H show the protein expressions of IL-1β (p17), caspase-1 (p20) and NLRP3 in J774A.1 cells under homoplanin treatment.

[0025] Figure 2 Homophytoside alleviates L-Glu-induced apoptosis in PC12 cells; Figure 2 A shows the effect of different concentrations of high-plantagin treatment on the viability of PC12 cells; Figure 2 B shows the protective effect of homoplanin on PC12 cell injury induced by L-Glu; Figure 2 C and D show the effects of homoplanin on the expression of apoptosis-related proteins Bcl-2, Bax and caspase-3 in PC12 cells stimulated by L-Glu.

[0026] Figure 3 Plantagogue-containing glycosides reduce L-Glu-induced oxidative damage in PC12 cells; Figure 3 A and B are the MDA content and SOD activity of PC12 cells induced by L-Glu after 3 h of homoplanin pretreatment; Figure 3 C and D are the detection of mitochondrial membrane potential of PC12 cells induced by L-Glu after 3 h of homoplanin pretreatment.

[0027] Figure 4 Homophytoside significantly improved the behavioral disorders of AD mice induced by AlCl3 and D-gal; Figure 4 A is the representative movement trajectory of mice in each group in the open field test; Figure 4 B and C are the total distance moved by mice in the open field test and the time spent in the central area; Figure 4 D and E are the spontaneous alternation rate and the time the mice spend in the novel arm in the Y-maze experiment; Figure 4 F is the representative swimming trajectory of mice in the water maze experiment; Figure 4 G–I are the escape latency, number of platform crossings, and time spent in the target quadrant in the water maze experiment.

[0028] Figure 5 Plantagogue glycosides significantly reduced the deposition of Aβ plaques in the hippocampus and cortex of AD mice. Figure 5 A and B are thioflavin S staining to detect Aβ plaque deposition in the hippocampus and cortex; Figure 5 C is the ELISA to measure the content of Aβ1-42 in hippocampus and cortex homogenates.

[0029] Figure 6 Plantagogue glycosides improve the inflammatory response and oxidative stress of AD mice and inhibit the apoptosis of hippocampal and cortical neurons. Figure 6 A is the levels of IL-6, IL-1β, and IL-10 factors in mouse serum; Figure 6 B and C are the MDA concentration and SOD activity in mouse serum; Figure 6 D–F show the protein expressions of IL-1β (p17), caspase-1 (p20), Bcl-2, Bax, and caspase-3 in the hippocampus and cortex.

[0030] Figure 7 Treatment with psyllium root extract can alleviate cellular senescence in AD mice. Figure 7 A, B are representative images and quantification of SA-β-Gal-positive cells in the mouse hippocampus and cortex, scale bar, 2.5 μm; Figure 7 C is the ELISA method to detect β-Gal activity in the mouse hippocampus and cortex; Figure 7D and E are Western blot analyses of the protein expression of aging indicators p21 and SIRT1 in the mouse hippocampus and cortex.

[0031] Figure 8 Plantagogue glycoside can improve the movement disorder of MPTP-induced PD mice; Figure 8 A is the structural formula of homoplanin; Figure 8 B is the experimental design and behavioral paradigm; Figure 8 C is the total movement distance in the open field test, the falling latency in the rotating rod test, and the head turning time and the total time from the top of the pole to the bottom of the pole in the climbing pole test.

[0032] Fig. 9 Plantagogue glycosides can reduce the dopaminergic neuron damage in PD mice; Figure 7 A and B are representative fluorescence images and density analysis of TH-positive nerve fibers in the mouse striatum, scale bar is 0.5 mm; Figure 7 C and D are representative images and quantification of TH-positive dopaminergic neurons in the substantia nigra pars compacta of mice; Figure 7 E is Western blot analysis of the protein expression of TH, a dopaminergic neuron marker, in the striatum and substantia nigra pars compacta of mice. DETAILED DESCRIPTION

[0033] The technical solution of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] Example 1: Diagram of the binding pattern of homoplanin to NLRP3 receptor and its inhibitory effect on inflammatory response mediated by NLRP3 inflammasome.

[0035] 1. Molecular docking of psyllium glycosides with NLRP3 inflammasome.

[0036] Refer to the method reported in the literature (Luo, L. et al. molecular Brain, 2020, 13 (1): 15). The molecular docking study of homoplanin and NLRP3 was carried out using the protein crystal structure of the complex of NLRP3 inflammasome and MCC950 (selective NLRP3 inhibitor) (PDB: 7pzc). The docking software was the CDOCKER module of Discoverystudio 2021 (DS, Accelrys Inc., san Diego, CA, USA). The NLRP3 protein was optimized using the "Macromolecules" module. The A chain and the corresponding ligand were retained, and the remaining chains and ligands were deleted. Neohesperidin was optimized using the "Prepare Ligands" and "Minimize Ligands" modules. During the docking process, the active site was defined with MCC950 in the crystal structure of the complex protein as the center, and the radius of the active sphere was The CDOCKER calculation was performed without changing the default settings. The results showed that the RMSD between the dominant conformation and the original conformation in the complex protein crystal structure was The binding mode is consistent with the key amino acid Arg578. Using this condition to dock homoplanin, it was found that homoplanin formed hydrogen bonds with Ala228, Arg578, Glu629, and Tyr632 in the NLRP3 active pocket, indicating that it has a strong affinity for NLRP3.

[0037] 2. The inhibitory effect of psyllium on inflammatory response mediated by NLRP3 inflammasome.

[0038] J774A.1 cells were cultured in DMEM medium (AF29498406, HyClone) supplemented with 10% fetal bovine serum (2206993CP, Gibco) and 1% penicillin-streptomycin mixture (20191028, Solarbio) at 37°C with 5% CO 2incubator. Homophytoside was dissolved in DMSO, and the CCK-8 method was used to detect the effect of 0.4-200 μM homophytoside on the viability of J774A.1 cells. In order to evaluate the effect of homophytoside on NLRP3 inflammasome activation and its mediated inflammatory response, J774A.1 cells were pretreated with homophytoside for 12 hours, then stimulated with 500nM LPS for 4 hours, and then treated with 5mM ATP for 1 hour. Refer to the method reported in the literature (Luo, L. et al. molecular brain, 2020, 13 (1): 15). The results of the ELISA test showed that LPS / ATP stimulation increased the levels of IL-1β and IL-18 in the cell supernatant, while treatment with homophytoside (2, 10 and 50 μm) reduced the secretion of the two in a dose-dependent manner.

[0039] In addition, Western blot was used to analyze the expression of target proteins: cells from each group were collected and RIPA lysis buffer (containing protease and phosphatase inhibitors) was added, ultrasonically crushed on ice, centrifuged at 4°C, 12000rpm for 20min, and the supernatant was collected for quantification by BCA method. 5× loading buffer was added and the sample was boiled at 100°C for 10min to completely denature the protein. After cooling, 20μg of sample was loaded and separated by SDS-PAGE gel electrophoresis. After constant flow transfer, it was blocked in 5% skim milk powder for 1h. The corresponding primary antibodies: IL-1β (p17), caspase-1 (p20), NLRP3 and β-actin (ACTB) were used for incubation at 4°C overnight. The next day, after rinsing with TBST 3 times, the corresponding secondary antibody was added according to the resistance of the primary antibody, incubated at room temperature for 1h, rinsed with TBST 3 times, and chemiluminescence was performed. The results showed that psyllium treatment significantly reduced the protein expression of mature IL-1β (p17) and activated caspase-1 (p20), while there was no significant change in the protein level of NLRP3, indicating that psyllium treatment may alleviate the inflammatory response by inhibiting the activation of NLRP3 inflammasome.

[0040] Example 2: Homoplanin can alleviate L-Glu-induced apoptosis of PC12 cells.

[0041] The PC12 neuronal cell line was cultured under the conditions of Example 1, and the CCK-8 method was used to detect that 12.5-200 μM of homoplanin had no significant damage to the viability of PC12 cells. A neuronal cell injury model was established using 25mM L-Glu to evaluate the protective effect of homoplanin on neuronal cells. The results showed that pretreatment with homoplanin (12.5-200 μM) for 3h could increase cell viability in a dose-dependent manner. In addition, the results of Western blot experiments showed that homoplanin upregulated the expression of anti-apoptotic protein Bcl-2 in PC12 cells stimulated by L-Glu, and downregulated the expression of apoptosis-related proteins Bax and caspase-3. These results suggest that homoplanin can alleviate L-Glu-induced PC12 cell apoptosis.

[0042] Example 3: Homoplanin can alleviate L-Glu-induced oxidative damage in PC12 cells.

[0043] Compared with the control group, the MDA content in the L-Glu injury group increased and the SOD enzyme activity decreased. 50 μM homophyllidine treatment can reduce the MDA concentration in L-Glu-induced PC12 cells and enhance the SOD enzyme activity, indicating that homophyllidine has an antioxidant effect. In addition, mitochondrial membrane potential is a negative potential difference caused by the different ion concentrations on both sides of the mitochondrial inner membrane, which is a sensitive indicator for evaluating mitochondrial function. The JC-1 fluorescent probe was used to indicate the changes in mitochondrial membrane potential. The results showed that in the control cells, JC-1 formed a red fluorescent polymer, while L-Glu stimulation caused the mitochondrial membrane potential to collapse, and JC-1 existed in the form of a green fluorescent monomer. 50 μM homophyllidine treatment reversed this change, which was specifically manifested by an increase in the red / green fluorescence intensity ratio. These results indicate that homophyllidine can alleviate L-Glu-induced oxidative damage and mitochondrial dysfunction.

[0044] Example 4: Homophytoside significantly improves AD-like behavioral disorders in mice.

[0045] 1. Experimental animals and grouping.

[0046] The experimental animals were SPF-grade male C57BL / 6 mice, 6-8 weeks old, purchased from the Experimental Animal Center of the Air Force Medical University. They were acclimatized to the environment one week before the experiment and kept in an animal room with a constant temperature (24±2°C), constant humidity (50-60%), and a light-dark cycle of 12:12h. They were allowed to have free access to water and food. All experimental procedures were in accordance with the regulations of the Animal Use and Management Committee of the Air Force Medical University.

[0047] The mice were randomly divided into four groups: control group, homophyllose group, AlCl 3 There were 8 rats in each group.3 AlCl3 (ig) and D-gal (ig) were dissolved in 0.9% saline and given once a day for 70 days to establish a joint AD model. Donepezil hydrochloride (4 mg / kg, ig) or homoplanin (50 mg / kg, ig) was given from day 41, once a day for 30 days. The control group was given an equal volume of 0.9% saline. Behavioral tests were performed on day 71.

[0048] 2. Behavioral testing

[0049] (1) Open field test: The animals were placed in an open field reaction box (40×40×40 cm 3 ) and a digital camera was set up 2m above the center, with a field of view covering the entire open field. The animal's movement trajectory was recorded for 15 minutes. The inner wall and bottom of the box were cleaned in time during each experiment to prevent the remaining odor of the animal from affecting the next test result. Finally, the total distance the mouse moved and the time it stayed in the central area were counted using the software.

[0050] (2) Y-maze test: The Y-maze apparatus consists of three identical arms, each arm is 40×3×15 cm long and arranged at an angle of 120°. Mice were subjected to spontaneous alternation and forced alternation tests in the Y-maze to evaluate the differences in spatial working memory and spatial reference memory. First, the mouse was placed in one of the three arms and allowed to explore all arms for 8 min without interference. As the mouse entered the three arms in sequence, alternation occurred, and the spontaneous alternation rate (%) was calculated as (number of alternations) / (total arm entries-2)×100%. In the forced alternation test, the mouse was allowed to explore 2 of the 3 arms for 3 min, while the third arm was blocked. After 2 h, the blocked arm was opened and the mouse was allowed to explore all three arms for 3 min in order to compare the time spent in the new arm and the familiar arm.

[0051] (3) Morris water maze test: The Morris water maze test system for mice consists of two parts: an ABS spray-coated cylindrical pool and a VisuTrack animal behavior analysis system. The pool is 120 cm in diameter and 50 cm in height. It is evenly divided into four quadrants (I, II, III, and IV) in the four directions of east, south, west, and north. A circular platform with a diameter of 10 cm is hidden 2 cm below the water surface and can be placed in the center of any quadrant. The water depth in the pool is 45 cm, and the water temperature is maintained at 25±1°C. Place the mice in the water and record the time it takes them to find the underwater platform. During the training process, if the platform is not found within 60 seconds, the experimenter guides the mouse to find it and let it stay on the platform for 10 seconds. Each group of mice was trained 4 times a day for 4 consecutive days. After the platform was removed on the 5th day, the mice were placed in the water at any water entry point for a 60 s free swimming test. The swimming trajectory was recorded, and the escape latency, number of platform crossings, and time spent in the target quadrant were analyzed.

[0052] 4. Experimental results

[0053] First, the open field test was used to evaluate whether psyllium can improve the motor disorders and anxiety-like behaviors of AD mice. Compared with the normal group, the total movement distance and the time spent in the central area of ​​the mice in the AD model group were significantly reduced, indicating that their motor ability was impaired and anxiety-like behaviors appeared. Psyllium treatment significantly increased the total movement distance and the time spent in the central area of ​​AD mice, while there was no significant difference in donepezil hydrochloride treatment. In the Y-maze test, AD mice showed obvious spatial working memory and spatial reference memory dysfunction, manifested as a decrease in spontaneous alternation rate and exploration time in the new arm. Psyllium can improve these two memory abilities, but donepezil hydrochloride only has a significant effect on the latter. Similarly, similar results were observed in the Morris water maze test. Specifically, the memory ability of AD mice decreased and the escape latency was significantly increased, while treatment with psyllium or donepezil hydrochloride significantly reduced the escape latency of D mice. In addition, after the platform was removed, the mice in the homophyllidine-treated group showed more platform crossing times and time in the target quadrant compared with the model group, but donepezil hydrochloride treatment had no significant effect. These data indicate that homophyllidine shows a superior effect in improving AD-like behavioral disorders in mice compared with donepezil hydrochloride.

[0054] Example 5: Homophytoside significantly reduced the deposition of Aβ plaques in the hippocampus and cortex of AD mice.

[0055] Aβ plaque deposition is one of the core pathological features of AD. Thioflavin S staining was used to identify Aβ plaques in brain tissue. 25μm paraffin sections of brain tissue were mounted on slides and dried overnight. The sections were rehydrated in PBS and stained with 1% Thioflavin S for 8 minutes at room temperature. The samples were washed three times with 50% ethanol and then washed with PBS, and photographed and analyzed using an Olympus FluoViewFV1000 microscope. The results showed that 50mg / kg of high plantagogue glycoside treatment significantly reduced the number of Aβ plaques in the hippocampus and cortex of AD mice. The same results were obtained using the ELISA method, that is, high plantagogue glycoside significantly reduced the levels of Aβ1-42 in the hippocampus and cortical homogenates of AD mice. These findings suggest that the improvement of high plantagogue glycoside on AD-like behavior may be partly due to its inhibition of Aβ deposition.

[0056] Example 6: Homophytoside can improve the inflammatory response and oxidative stress of AD mice and inhibit the apoptosis of hippocampal and cortical neurons.

[0057] The anti-inflammatory, antioxidant and anti-apoptotic effects of psyllium were further confirmed in vivo. The experimental results showed that the proinflammatory factors IL-6 and IL-1β in the serum of AD mice were significantly increased, while the level of anti-inflammatory cytokine IL-10 was significantly decreased. Psyllium treatment can effectively reverse the chronic inflammatory response of AD mice. Importantly, psyllium treatment also reduced the protein expression of mature IL-1β (p17) and activated caspase-1 (p20) in the hippocampus and cortex of AD mice, indicating that the anti-inflammatory effect of psyllium is closely related to the inhibition of NLRP3 inflammasome activation. In addition, psyllium treatment significantly reduced the redox imbalance of AD mice, as manifested by a significant decrease in the MDA content in serum and an increase in SOD activity. Furthermore, AlCl 3 Combined treatment with D-gal induced significant apoptosis in the hippocampus and cortex, characterized by increased levels of caspase-3 and Bax proteins and decreased expression of Bcl-2 protein. Treatment with homophyllidine effectively reversed these changes. These results collectively indicate that homophyllidine has anti-inflammatory and antioxidant properties, which in turn inhibits neuronal apoptosis, exerting neuroprotective and anti-AD-like symptom effects.

[0058] Example 7: Homophytoside can alleviate cell senescence in AD mice.

[0059] Cellular senescence is characterized by irreversible cell cycle arrest, in which cells maintain metabolic activity and secrete proinflammatory factors. Cellular senescence is closely related to a variety of age-related diseases, including AD and PD. Based on the aforementioned ability of homophyllidine to inhibit the secretion of proinflammatory factors in J774A.1 cells and AD mice, as well as previous reports of its anti-photoaging effects (Guo, Y. et al. Photodermatol Photoimmunol Photomed, 2023, 39(5): 466), we further evaluated whether homophyllidine could inhibit cellular senescence in AD mice. Frozen sections of mouse brain tissue were stained with cell senescence β-galactosidase staining solution, and by counting SA-β-Gal-positive cells, it was found that senescent cells in the hippocampus and cortex of AD mice increased significantly. Treatment with homophyllidine can reduce the number of SA-β-Gal-positive senescent cells in the hippocampus and cortex of AD mice. Consistently, the ELISA test results also suggested that treatment with homophyllidine can reduce the activity of β-Gal compared with AD mice. In addition, Western blot results showed that the expression of aging-related proteins p21 and SIRT1 in the hippocampus and cortex tissues of AD mice increased and decreased, respectively. However, treatment with psyllium was able to effectively reverse these changes, indicating that it has anti-aging properties, which may partly contribute to the improvement of AD symptoms.

[0060] Example 8: Homophytoside can improve movement disorders in MPTP-induced PD mice.

[0061] 1. Experimental animals and grouping.

[0062] The experimental animals were SPF-grade male C57BL / 6 mice, 8-10 weeks old, purchased from the Experimental Animal Center of the Air Force Medical University. They were acclimatized to the environment one week before the experiment and kept in an animal room with a constant temperature (24±2°C), constant humidity (50-60%), and a light-dark cycle of 12:12h. They were allowed to have free access to water and food. All experimental procedures were in accordance with the regulations of the Animal Use and Management Committee of the Air Force Medical University.

[0063] Mice were randomly divided into seven groups (6 mice in each group): control group, homophytoside (50 mg / kg) group, MPTP group, MPTP+homophytoside (2 mg / kg) group, MPTP+homophytoside (10 mg / kg) group, MPTP+homophytoside (50 mg / kg) group and MPTP+levodopa (L-DOPA) group. Mice were intraperitoneally injected with MPTP (25 mg / kg, dissolved in saline) for 7 consecutive days. One hour after the injection, the mice in the treatment group were gavaged with different doses of homophytoside or positive control drug L-DOPA (75 mg / kg) once a day for 14 consecutive days. Homophytoside and L-DOPA were dissolved in saline containing 10% DMSO, 40% PEG 300 and 5% Tween 80. The mice in the control group were intraperitoneally injected with an equal amount of saline and gavaged with an equal volume of the above-mentioned carrier. Mice in the homophyllidine (50 mg / kg) group were intraperitoneally injected with an equal amount of saline and intragastrically administered with homophyllidine for 14 days. After the final treatment, all mice underwent behavioral tests.

[0064] 2. Behavioral testing.

[0065] (1) Open field test: refer to Example 4.

[0066] (2) Rotarod test: In the rotarod test, mice were placed on the SA102 rotarod apparatus, and the rotarod was accelerated from 4 rpm / min to 40 rpm / min within 300 s. The time the mice stayed on the rotarod was recorded. The experiment required 3 days of pre-training, and 3 trials were conducted on the test day, with 30 minutes interval between each trial to obtain the average time of stay.

[0067] (3) Pole test: The mouse is placed head-up on top of a 1-cm-diameter, 55-cm-high pole. The time it takes for the mouse's head to turn downward and the total time it takes to reach the bottom of the pole are measured. The test is performed every 2 minutes, and the average of 3 trials is calculated. The animals need to be pre-trained for 3 days before the test.

[0068] 4. Experimental results.

[0069] The results of the open field test showed that treatment with psyllium can significantly increase the total distance traveled by PD mice, indicating that it can improve the motor disorders of PD mice. The motor coordination ability was tested using the rotating rod and climbing pole tests. The results showed that PD mice had obvious motor coordination dysfunction, which was manifested by a shortened latency period of the rotating rod falling and a prolonged time and total time of turning the head on the climbing pole. Treatment with psyllium improved these changes in a concentration gradient-dependent manner, suggesting that psyllium has an effective effect on PD-like behavioral disorders and decreased motor coordination ability, similar to the positive drug levodopa.

[0070] Example 9: Homophytoside can reduce dopaminergic neuron damage in PD mice.

[0071] 1. Immunofluorescence staining was used to detect the number of dopaminergic neurons.

[0072] Refer to the method reported in the literature (Luo, L. et al. molecular brain, 2020, 13 (1): 15). The brain tissue of each group of mice was fixed in 4% paraformaldehyde overnight, and then dehydrated in 20%-30% sucrose in a gradient. After OCT embedding, 20 μm continuous frozen sections were performed. Coronal sections containing striatum and substantia nigra were collected and blocked with 10% goat serum containing 0.3% Triton X-100 at room temperature for 2 hours. The blocking solution was discarded, and the sections were incubated with the primary antibody of dopaminergic neuron marker TH at 4°C overnight, washed 3 times with PBS, and incubated with mouse anti-rabbit fluorescent secondary antibody at room temperature for 2 hours. The cell nuclei were stained with DAPI for 5 minutes. The sections were then sealed with an anti-fluorescence quencher, observed under a microscope and photographed.

[0073] 2. Experimental results.

[0074] Immunofluorescence staining showed that the number of TH-positive nerve fibers in the striatum of PD mice was significantly reduced, while high-planting glycoside treatment increased the density of TH-positive nerve fibers in the striatum of PD mice in a dose-dependent manner. In addition, compared with the PD model group, high-planting glycoside treatment also increased the number of TH-positive dopaminergic neurons in the substantia nigra compacta of mice, and its effect was similar to or slightly stronger than that of levodopa. Western blot analysis showed that high-planting glycoside treatment could upregulate the TH protein content in the homogenate of the striatum and substantia nigra compacta of mice. These results all indicate that high-planting glycoside improves the loss of dopaminergic neurons in PD mice, which provides strong scientific support for the movement disorder of high-planting glycoside in PD mice.

[0075] In summary, in some embodiments of the present invention, the administration method of homoplanin is intragastric injection, but it should not be limited to oral administration.

[0076] In some embodiments of the present invention, the homophytoside dosage is 2-50 mg / kg.

[0077] The invention provides a pharmaceutical composition comprising homoplanin and a pharmaceutical carrier, wherein the pharmaceutical composition is a preparation prepared by conventional or special preparation technology and is administered orally or non-orally.

[0078] The present invention provides a method for preventing and treating neurodegenerative diseases, comprising administering homophyllium or the aforementioned pharmaceutical composition to a patient with a neurodegenerative disease. The neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, frontotemporal dementia, Lewy body dementia, and other diseases with memory and cognitive dysfunction and motor impairment.

[0079] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. Application of psyllium glycosides in the preparation of drugs for preventing and treating neurodegenerative diseases.

2. The use according to claim 1, characterized in that: The neurodegenerative disease includes at least one of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, frontotemporal dementia, and Lewy body dementia.

3. The use according to claim 1, characterized in that: Homophytoside is used to improve NLRP3 inflammasome-related neuroinflammatory response, inhibit neuronal oxidative damage and neuronal cell aging, and maintain neuronal mitochondrial homeostasis and function.

4. The use according to claim 1, characterized in that: Homophytoside is used to inhibit excessive deposition of Aβ plaques and enhance learning and memory abilities.

5. The use according to claim 1, characterized in that: Homophytoside is used to regulate the growth and development of dopaminergic neurons, increase the secretion level of dopamine, and restore motor coordination ability.

6. The use according to claim 1, characterized in that: The medicine is a pharmaceutical composition, which is prepared from homoplanin and one or more pharmaceutically acceptable excipients or carriers.

7. The use according to claim 6, characterized in that: The pharmaceutical composition is in any clinically acceptable dosage form, including oil, granules, tablets, powders, capsules, pills, powders, oral liquids, sols, sprays, and atomizers.

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