Use of achyranthes bidentata polypeptide combined with human umbilical cord mesenchymal stem cell-derived exosome in preparation of drug for preventing and / or treating parkinson's disease

Abstract

Provided in the present invention is the use of an Achyranthes bidentata polypeptide combined with a human umbilical cord mesenchymal stem cell-derived exosome in the preparation of a drug for preventing and / or treating Parkinson's disease. A Parkinson's disease (PD) model mouse is established using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and then an Achyranthes bidentata polypeptide is used in combination with a human umbilical cord mesenchymal stem cell-derived exosome to treat the mouse. It is found that the treatment can significantly relieve PD-related symptoms, for example, restoring the number of dopaminergic neurons in the substantia nigra, improving the expression of tyrosine hydroxylase in the midbrain, relieving motor and olfactory dysfunction of the PD model mouse, reducing the activation of microglia and astrocytes in the olfactory bulb and midbrain, and improving the activity of neurons in the olfactory bulb.

Description

Application of Achyranthes bidentata polypeptide combined with human umbilical cord mesenchymal stem cell exosomes in the preparation of drugs for the prevention and / or treatment of Parkinson's disease Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically the application of Achyranthes bidentata polypeptide combined with human umbilical cord mesenchymal stem cell exosomes in the preparation of drugs for the prevention and / or treatment of Parkinson's disease. Background Technology

[0002] Parkinson's disease (PD), also known as paralysis agitans, is the second leading cause of neurodegenerative diseases after Alzheimer's disease, severely impacting human quality of life. Statistics show that one in every 800 people worldwide suffers from Parkinson's disease, and by 2030, due to the accelerated aging process, the prevalence is projected to double, exceeding 9 million patients. Parkinson's disease is characterized by its progressive nature, multiple onset, and insidious nature, primarily manifesting as bradykinesia, rigidity, resting tremor, and postural instability. The main pathological feature of Parkinson's disease is the reduction of dopaminergic neurons in the substantia nigra. The cells in the brain that produce dopamine gradually lose their function in influencing the nervous system, limiting the patient's ability to control muscles. Clinical treatment for Parkinson's disease mainly involves dopamine replacement therapy. Long-term use of this therapy can cause various adverse reactions, such as anxiety, insomnia, and hallucinations. Therefore, the search for new drugs to treat PD has significant economic and social value.

[0003] Achyranthes bidentata root, the dried root of the plant Achyranthes bidentata BI. (family Amaranthaceae), is a commonly used traditional Chinese medicine. It possesses physiological functions such as promoting blood circulation and removing blood stasis, tonifying the liver and kidneys, strengthening tendons and bones, promoting diuresis, and guiding blood downwards. Achyranthes bidentata polypeptide is a bioactive substance isolated from Achyranthes bidentata. Further purification using high-performance liquid chromatography (HPLC) yielded a purified Achyranthes bidentata polypeptide fraction.

[0004] Human umbilical cord mesenchymal stem cells (hUCMCSs) are derived from human umbilical cord tissue and are characterized by easy extraction, strong self-renewal capacity, low immunogenicity, and the ability to secrete various functional factors. Therefore, hUCMCSs are considered the preferred mesenchymal stem cell source for clinical stem cell therapy and are widely used in various tissue repair applications. Exosomes are lipid bilayer vesicles with a diameter of 50-150 nm, produced and released by cells through active secretion. Exosomes contain various substances, including lipid molecules, proteins, and nucleic acids (including DNA, RNA, and various non-coding RNAs). Exosomes play a crucial role in intercellular communication and substance transport. Currently, increasing research indicates that stem cell exosomes possess physiological activities similar to stem cells.

[0005] The nasal cavity is richly supplied with capillary lymphatic vessels, and the surface of respiratory cells is covered with numerous microvilli, making it an ideal route for mucosal drug delivery. The nasal mucosa has high permeability and relatively few enzymes, resulting in lower degradation of protein-based drugs compared to the gastrointestinal mucosa. Furthermore, nasal administration allows drugs to reach the brain via the bipolar olfactory cells of the nasal epithelium along the olfactory nerve, bypassing the blood-brain barrier. Therefore, nasal administration is a highly effective method of drug delivery that acts on the central nervous system. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides the application of Achyranthes bidentata polypeptide combined with human umbilical cord mesenchymal stem cell exosomes in the preparation of drugs for the prevention and / or treatment of Parkinson's disease. It has been found that Achyranthes bidentata polypeptide combined with human umbilical cord mesenchymal stem cell exosomes can restore the number of dopaminergic neurons in the substantia nigra, increase the expression of tyrosine hydroxylase in the midbrain, alleviate motor and olfactory dysfunction in PD model mice, reduce the activation of microglia and astrocytes in the olfactory bulb and midbrain, and increase the activity of neurons in the olfactory bulb.

[0007] The technical solution provided by this invention is as follows:

[0008] This invention provides the application of Achyranthes bidentata polypeptide combined with human umbilical cord mesenchymal stem cell exosomes in the preparation of drugs for the prevention and / or treatment of Parkinson's disease.

[0009] Furthermore, the drug comprises 1 to 2 × 10 8 Human umbilical cord mesenchymal stem cell exosomes per kg and 50–100 μg / kg of Achyranthes bidentata polypeptide.

[0010] Furthermore, the Achyranthes bidentata polypeptide is prepared by the following method: The dried roots of Achyranthes bidentata are chopped, and the chopped dried roots are mixed with water and then boiled to obtain a mixed solution; Ammonium sulfate is added to the mixed solution for precipitation, centrifugation is performed, and the solution is freeze-dried to obtain a crude extract of Achyranthes bidentata polypeptide; The crude extract of Achyranthes bidentata polypeptide is dissolved in water to prepare a solution, and the solution is purified by high-performance liquid chromatography to obtain the Achyranthes bidentata polypeptide.

[0011] Furthermore, the cooking temperature is 80–85°C, and the cooking time is 40–60 minutes.

[0012] Furthermore, the high-performance liquid chromatography (HPLC) conditions are as follows: C18 reversed-phase column, mobile phase of water / acetonitrile containing 0.1% trifluoroacetic acid, water to acetonitrile volume ratio of 4:1, flow rate of 1.0 mL / min, and ultraviolet detector with wavelength of 220 nm.

[0013] Furthermore, the human umbilical cord mesenchymal stem cell exosomes are prepared by the following method: Primary human umbilical cord mesenchymal stem cells are passaged in a serum-containing culture medium. When the density of third-generation human umbilical cord mesenchymal stem cells reaches 80%, the culture medium is changed to serum-free culture medium and cultured for 48 hours. The supernatant is collected. The supernatant is then centrifuged at 400-500g to remove cells and cell debris, and then filtered through a 0.22μm filter. The filtered supernatant is treated with an exosome isolation kit, and the umbilical cord mesenchymal stem cell exosomes are obtained by separation and purification through specific buffer and centrifugation steps.

[0014] Furthermore, the applications include any of the following: improving motor dysfunction and olfactory perception; increasing the number of dopaminergic neurons in the brain; inhibiting abnormal activation of microglia and astrocytes in the brain; and enhancing the activity of olfactory bulb neurons.

[0015] Furthermore, the drug is administered via nasal administration.

[0016] Furthermore, the drug is a solution.

[0017] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0018] Beneficial effects

[0019] This invention treats mice with a combination of Achyranthes bidentata polypeptide and human umbilical cord mesenchymal stem cell exosomes, during which 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is used to induce a Parkinson's disease (PD) model in the mice. The results showed that this treatment significantly alleviated PD-related symptoms, such as restoring the number of dopaminergic neurons in the substantia nigra, increasing the expression of tyrosine hydroxylase in the midbrain, alleviating motor and olfactory dysfunction in PD model mice, reducing the activation of microglia and astrocytes in the olfactory bulb and midbrain, and increasing the activity of neurons in the olfactory bulb.

[0020] This invention allows drug delivery via the nasal cavity, which can reach the brain through the bipolar olfactory cells of the nasal olfactory epithelium along the olfactory nerve, thus avoiding the barrier effect of the blood-brain barrier on protein drugs. Attached Figure Description

[0021] Figure 1. Preparation of Achyranthes bidentata polypeptide. A: Preparation process; B: High performance liquid chromatography purification; C: Freeze-dried Achyranthes bidentata polypeptide.

[0022] Figure 2. Electron micrograph of exosomes from human umbilical cord mesenchymal stem cells. Arrows indicate exosomes. Scale bar: 200 nm.

[0023] Figure 3(A) PD model mouse model establishment and drug intervention process. ip: intraperitoneal injection; in: intranasal administration; (B) pole climbing test; (C) olfactory test. ***p<0.001, compared with the control group; #p<0.05,##p<0.01,###p<0.001, compared with the MPTP group; &p<0.05, compared with the MPTP + Achyranthes bidentata polypeptide group; $p<0.05, compared with the MPTP + exosome group.

[0024] Figure 4(A) Immunofluorescence assay showed that treatment with Achyranthes bidentata peptide combined with human umbilical cord stem cell mesenchymal exosomes increased the expression of tyrosine hydroxylase (TH) in the substantia nigra of the midbrain of PD model mice; scale bar: 50 mm. (B) Quantitative analysis of the number of TH-positive cells in the substantia nigra: ***p<0.001, compared with the control group; ##p<0.01, ###p<0.001, compared with the MPTP group; &&p<0.01, compared with the MPTP + Achyranthes bidentata peptide group; $p<0.05, compared with the MPTP + exosome group.

[0025] Figure 5(A) Immunofluorescence assay showed that treatment with Achyranthes bidentata peptide combined with human umbilical cord stem cell mesenchymal exosomes reduced the expression of Iba-1 in the substantia nigra of the brain in PD model mice; scale bar: 50 mm. (B) Quantitative analysis of Iba-1 fluorescence intensity in the substantia nigra: **p<0.01, compared with the control group; #p<0.05, compared with the MPTP group; &p<0.05, compared with the MPTP + Achyranthes bidentata peptide group; $p<0.05, compared with the MPTP + exosome group.

[0026] Figure 6(A) Immunofluorescence assay showed that treatment with Achyranthes bidentata peptide combined with human umbilical cord stem cell mesenchymal exosomes reduced the expression of GFAP in the substantia nigra of the brain in PD model mice; scale bar: 50 mm. (B) Quantitative analysis of GFAP fluorescence intensity in the substantia nigra: ***p<0.001, compared with the control group; #p<0.05, ##p<0.01, compared with the MPTP group; &p<0.05, compared with the MPTP+Achyranthes bidentata peptide group; $p<0.05, compared with the MPTP+exosome group.

[0027] Figure 7 shows that immunofluorescence assays revealed that treatment with Achyranthes bidentata peptide combined with human umbilical cord stem cell mesenchymal exosomes increased the expression of Phospho-S6 Ribosomal protein in the lateral and central olfactory bulb regions of PD model mice; red indicates Phospho-S6 Ribosomal protein; DAPI (blue) represents the cell nucleus. Scale bar: 25 mm. ***p<0.001, compared with the control group; #p<0.05, #p<0.01, ###p<0.001, compared with the MPTP group; &p<0.05, compared with the MPTP + Achyranthes bidentata peptide group; $p<0.05, compared with the MPTP + exosome group.

[0028] Figure 8 shows that immunofluorescence assays revealed that treatment with Achyranthes bidentata peptide combined with human umbilical cord stem cell mesenchymal exosomes reduced the expression of Iba-1 (red) in the lateral and central olfactory bulb regions of PD model mice; DAPI (blue) represents the cell nucleus. Scale bar: 25 mm. ***p<0.001, compared with the control group; #p<0.05, #p<0.01, ###p<0.001, compared with the MPTP group; &p<0.05, compared with the MPTP + Achyranthes bidentata peptide group; $p<0.05, compared with the MPTP + exosome group.

[0029] Figure 9 shows that immunofluorescence assays revealed that treatment with Achyranthes bidentata peptide combined with human umbilical cord stem cell mesenchymal exosomes reduced the expression of GFAP (red) in the lateral olfactory bulb region of PD model mice; DAPI (blue) represents the cell nucleus. Scale bar: 25 mm. **p<0.01, compared with the control group; #p<0.05, ##p<0.01, compared with the MPTP group; &p<0.05, compared with the MPTP + Achyranthes bidentata peptide group; $p<0.05, compared with the MPTP + exosome group. Detailed Implementation

[0030] The present invention will now be described in further detail with reference to the accompanying drawings:

[0031] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0032] All reagents and materials used in this example can be purchased routinely. The quantitative experiments involved in the examples were all repeated at least three times, and the results were averaged.

[0033] The dried roots of Achyranthes bidentata used were purchased from Yongjitang Pharmacy.

[0034] The primary human umbilical cord mesenchymal stem cells used were purchased from Nanjing Taisheng Biotechnology Co., Ltd.

[0035] The C57BL / 6J mice used were purchased from the Experimental Animal Center of Nantong University.

[0036] The 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) used was purchased from Sigma-Aldrich, catalog number M0896.

[0037] Example 1

[0038] 1. Preparation of Achyranthes bidentata polypeptide (Figure 1)

[0039] The dried roots of Achyranthes bidentata were cut into small pieces and decocted in a water bath (80-85℃, 40-60 min). The aqueous extract was subjected to two ammonium sulfate precipitations and high-speed centrifugation. The precipitate was freeze-dried to obtain the crude extract of Achyranthes bidentata polypeptides. The obtained crude extract of Achyranthes bidentata polypeptides was dissolved in water and further purified by high-performance liquid chromatography (HPLC). The chromatographic conditions were as follows: C18 reversed-phase column, mobile phase of water / acetonitrile containing 0.1% trifluoroacetic acid (4:1, v / v), flow rate of 1.0 mL / min, and UV detector (220 nm).

[0040] 2. Preparation of exosomes from human umbilical cord mesenchymal stem cells (Figure 2)

[0041] Cells were cultured in DMEM / F12 (Corning) containing 10% fetal bovine serum in a cell culture incubator at 37°C and 5% CO2 saturated humidity. When the cell density reached 80%, the cells were digested with 0.25% trypsin (Gibco) containing EDTA and passaged. When the density of human umbilical cord mesenchymal stem cells (HUC-C) reached 80% in the third generation, the culture medium was changed to serum-free medium and cultured for 48 hours. The supernatant was collected, and the supernatant was continuously ultracentrifuged at 400g for 10 min to remove dead cells and cell debris, then filtered through a 0.22 μm filter (Millipore). Exosomes were isolated from 15 ml of cell culture supernatant using the exoEasy Kit (QIAGEN). The filtered supernatant was transferred to a new tube, and an equal volume of buffer XBP was added. Then, a total of 30 ml of the mixture was added to an exoEasy rotating column and centrifuged at 500g for 1 min. After discarding the liquid, the rotating column was returned to the same collection tube. Repeat the above steps until no supernatant remains. Then, add 10 ml of buffer XWP to the spin column and centrifuge at 5000 g for 5 min to remove any residual buffer. Transfer the spin column to a new collection tube. Add 400 μl of buffer XE to the membrane and incubate for 1 min. Collect the eluent by centrifuging at 500 g for 5 min. Add the eluent to the spin column and incubate again for 1 min. Finally, collect the eluent after centrifuging at 5000 g for 5 min and store at -80°C until use.

[0042] Example 2

[0043] 1. Preparation and drug administration of Parkinson's disease animal model

[0044] Eight-week-old male C57BL / 6J mice were used to induce a PD model using intraperitoneal injection of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Prior to treatment, the mice underwent three days of behavioral training, including pole climbing. To avoid the influence of individual differences, mice with similar motor abilities were selected and divided into a control group and a model group. The model group mice were then injected intraperitoneally with MPTP (25 mg / kg / day) for one week, followed by behavioral evaluation (Figure 3A). Mice exhibiting a significant decrease in behavior compared to the control group (injected with the same volume of saline) were considered successfully induced PD models and used in subsequent experiments.

[0045] Successful PD model mice were divided into four groups: MPTP group, MPTP + Achyranthes bidentata peptide group, MPTP + exosome group, and MPTP + Achyranthes bidentata peptide / exosome group. Mice were treated via intranasal administration. Achyranthes bidentata peptide and exosomes were dissolved in PBS solution, with an exosome dose of 2 × 10⁻⁶. 8 Mice in the genus *Achyranthes bidentata* were administered at a dose of 100 μg / kg every 3 days for a total of 4 doses. Mice in the MPTP group were treated in the same manner and given the same volume of PBS solution.

[0046] 2. Pole Climbing Experiment

[0047] A pole with a diameter of 1 cm and a length of 50 cm was constructed, with a small cork ball of 1.5 cm in diameter attached to the top. Gauze was wrapped around the pole to increase friction. The pole was placed vertically, and mice were placed on the ball at the top of the pole. The time taken for the animal to start moving, turn completely head-down, and return to the bottom of the pole was recorded. Each test was conducted at 5-minute intervals, and the average value was taken for three tests. For three days prior to the tail vein injection, the mice were trained to climb the pole at the same time each day. Mice with significant differences in performance were removed to reduce experimental error.

[0048] 3. Olfactory Experiment

[0049] Mice were fasted for 24 hours beforehand. During this period, a small piece of cheese was placed in the cage to familiarize them with the smell and taste, enabling them to find the cheese during the experiment. A clean cage (42cm long, 24cm wide, 15cm high) was prepared, and cheese was buried sequentially in five locations on the clean bedding: the center, upper left, lower right, upper right, and lower left. The cheese should be buried 1cm below the bedding. The animal was gently placed in the center of the cage, and the time it took for the mouse to find the cheese was recorded. If the mouse did not find the cheese within 300 seconds, it was recorded as 300 seconds. The minimum and maximum values ​​were removed before statistical analysis.

[0050] 4. Immunofluorescence analysis of the substantia nigra and olfactory bulb

[0051] The experimental steps are as follows: (1) Mouse brains fixed overnight with 4% paraformaldehyde were dehydrated at 4°C with 20% and 30% sucrose, and removed after complete dehydration and settling; (2) After OCT embedding of brain blocks, continuous coronal sections of mouse substantia nigra were prepared using a cryostat, with a section thickness of 12 μm. The sections were dried at 37°C for later use; (3) Glass slides were washed three times with 0.01M PBS at room temperature for 10 min each time; (4) Blocking solution containing 5% BSA and 0.5% Triton X-100 was added and the slides were blocked at room temperature for 1 h; (5) The blocking solution was discarded, and the primary antibody diluted according to the ratio was added and incubated at 4°C overnight. Common primary antibody ratios: Chicken anti-TH (1:500, ab76442, Abcam), Rabbit anti-Iba-1 (1:500, ab7260, Abcam), Mouse anti-GFAP (1:500, MAB360, Millipore), Rabbit anti-pS6 Ribosomal Protein (1:50, 4858S, Cell Signaling Technology); (6) Wash 3 times with 0.01M PBS at room temperature for 10 min each time; (7) Add secondary antibody diluted with 0.01M PBS and incubate at room temperature in the dark for 90 min. Common secondary antibody ratios: Goat anti-rabbit IgG H&L (Alexa) 647)(1:500, A-31573, Invitrogen), Goat Anti-Chicken IgY H&L (Alexa) 555)(1:500, A-21437, Invitrogen), Goat anti-mouse IgG H&L (Alexa) 488)(1:500, A-11001, Invitrogen); (8) Wash 3 times with 0.01MPBS, at room temperature, protected from light, for 10 min each time; (9) Add anti-fluorescence quenching mounting solution containing DAPI to mount the slide; (10) Observe and photograph under a fluorescence microscope; (11) Use ImageJ software to measure the fluorescence intensity of the image.

[0052] 5. Experimental Results

[0053] The results showed that the combination of Achyranthes bidentata peptide and human umbilical cord mesenchymal stem cell exosomes significantly alleviated motor dysfunction in Parkinson's disease model mice. For example, in the pole climbing experiment, the time taken for the treated mice to complete the climb was significantly shorter than that of the MPTP-treated group (Figure 3B). Furthermore, this treatment significantly improved the olfactory function of Parkinson's disease model mice, as evidenced by a significantly shorter time to detect hidden food in the treated group (Figure 3C). These behavioral experimental results demonstrate that the combined treatment showed a more significant effect compared to treatment with Achyranthes bidentata peptide or exosomes alone. Histological analysis revealed a significant decrease in tyrosine hydroxylase (TH) expression in the substantia nigra of the brain in PD model mice, while treatment with Achyranthes bidentata peptide combined with human umbilical cord mesenchymal stem cell exosomes significantly increased TH expression, indicating an increase in the number of dopaminergic neurons (Figures 4A, B). Moreover, this treatment also inhibited the activation of microglia and astrocytes in the midbrain, as evidenced by a decrease in the number of Iba-1 and GFAP-positive cells (Figures 5A, B; 6A, B). Histological analysis of the olfactory bulb revealed that treatment with Achyranthes bidentata peptide combined with human umbilical cord mesenchymal stem cell exosomes significantly enhanced the expression of phosphorylated S6 ribosomal protein, indicating improved olfactory bulb neuronal activity (Figure 7). Simultaneously, microglial activation in the lateral and central regions of the olfactory bulb was inhibited, as evidenced by a decrease in Iba-1 positive cells after treatment (Figure 8). Furthermore, astrocyte activation in the lateral region of the olfactory bulb was also inhibited, as evidenced by a decrease in GFAP positive cells after treatment (Figure 9). These morphological results demonstrate that the combined treatment showed a more significant effect compared to treatment with Achyranthes bidentata peptide or exosomes alone.

[0054] Although the present invention has been described in detail above, it is not limited to the above embodiments. Those skilled in the art can make other modifications and improvements based on the inspiration of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. Application of Achyranthes bidentata polypeptide combined with human umbilical cord mesenchymal stem cell exosomes in the preparation of drugs for the prevention and / or treatment of Parkinson's disease.

2. The application according to claim 1, characterized in that, The drug comprises 1 to 2 × 10 8 Human umbilical cord mesenchymal stem cell exosomes per kg and 50–100 μg / kg of Achyranthes bidentata polypeptide.

3. The application according to claim 1 or 2, characterized in that, The Achyranthes bidentata polypeptide was prepared by the following method: Cut the dried root of Achyranthes bidentata into small pieces, then mix the cut dried root with water and steam it to obtain a mixture. Ammonium sulfate was added to the mixture to precipitate the precipitate, followed by centrifugation and freeze-drying to obtain crude extract of Achyranthes bidentata polypeptide. The crude extract of Achyranthes bidentata polypeptide was dissolved in water to prepare a solution, and then the solution was purified by high performance liquid chromatography to obtain the Achyranthes bidentata polypeptide.

4. The application according to claim 3, characterized in that, The cooking temperature is 80-85℃, and the time is 40-60 minutes.

5. The application according to claim 3, characterized in that, The high-performance liquid chromatography (HPLC) conditions are as follows: C18 reversed-phase column, mobile phase of water / acetonitrile containing 0.1% trifluoroacetic acid, water to acetonitrile volume ratio of 4:1, flow rate of 1.0 mL / min, and ultraviolet detector with wavelength of 220 nm.

6. The application according to claim 1 or 2, characterized in that, The human umbilical cord mesenchymal stem cell exosomes were prepared by the following method: Primary human umbilical cord mesenchymal stem cells were passaged in a serum-containing culture medium. When the density of third-generation human umbilical cord mesenchymal stem cells reached 80%, the culture medium was changed to serum-free culture medium and cultured for 48 hours. The supernatant was then collected. The supernatant was then centrifuged at 400–500 g to remove cells and cell debris, and then filtered through a 0.22 μm filter. The filtered supernatant was processed using an exosome isolation kit, and the umbilical cord mesenchymal stem cell exosomes were obtained by separation and purification through specific buffer and centrifugation steps.

7. The application according to claim 1, characterized in that, The application includes any of the following: Improve motor dysfunction and olfactory perception; Increase the number of dopaminergic neurons in the brain; Inhibits abnormal activation of microglia and astrocytes in the brain; Enhance the activity of olfactory bulb neurons.

8. The application according to claim 1, characterized in that, The drug is administered via nasal administration.

9. The application according to claim 1, characterized in that, The drug is a solution.

10. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

Images