Application of Shouhui Tongbian Capsules in the Preparation of Drugs for the Prevention and Treatment of Parkinson's Disease

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to the application of Shouhui Tongbian Capsules in the preparation of drugs for treating Parkinson's disease. Shouhui Tongbian Capsules can significantly improve the damage to dopamine neurons in the substantia nigra and striatum of Parkinson's disease mice with depression induced by MPTP and CUMS, and inhibit the activation of microglia and astrocytes; it not only alleviates the motor symptoms of Parkinson's disease mice, but also significantly improves the non-motor symptoms of Parkinson's disease mice, including mental disorders, and has a significant therapeutic effect on depression caused by Parkinson's disease.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to the application of Shouhui Tongbian Capsules in the preparation of drugs for the prevention and treatment of Parkinson's disease or non-motor symptoms of Parkinson's disease. Background Technology

[0002] The Shouhui Tongbian Capsule formula originates from clinical experience and consists of eight Chinese herbs: Polygonum multiflorum, Aloe vera, Cassia tora, Lycium barbarum, donkey-hide gelatin, ginseng, Atractylodes macrocephala, and Citrus aurantium. Guided by traditional Chinese medicine theory and incorporating clinical experience, this formula combines laxatives and detoxifying herbs with blood-nourishing and dryness-moistening herbs, along with qi-tonifying and spleen-strengthening herbs. The combination of these herbs is rational and precise, simultaneously nourishing qi and yin, and balancing tonification and purgation without causing stagnation or harming the body's vital energy. By tonifying qi and nourishing yin, it improves overall constitution and achieves the goal of treating constipation. This formula is particularly effective for constipation caused by internal accumulation of toxins and deficiency of yin fluids. It comprehensively improves gastrointestinal function, has a definite curative effect, works quickly, has no toxic side effects, and also has detoxifying, beautifying, weight-loss, and lipid-lowering effects. Clinical application has shown its definite efficacy, playing an important role in treating functional constipation. It is suitable for the elderly and patients with weak constitutions and has received high praise from both doctors and patients.

[0003] Parkinson's disease (PD), also known as Parkinson's syndrome, is a common neurodegenerative disease in middle-aged and elderly people. Its typical symptoms are divided into motor and non-motor symptoms. Non-motor symptoms include sensory disturbances, sleep disturbances, autonomic dysfunction, and mental disorders. Non-motor symptoms severely impact patients' quality of life and long-term prognosis. Currently, there are no effective treatments for the non-motor symptoms of Parkinson's disease, resulting in poor treatment outcomes.

[0004] The etiology and pathogenesis of depression in Parkinson's disease are not fully understood. Currently, it is clear that a single cause and mechanism cannot explain the condition, and it is more likely a result of multiple factors. On the one hand, similar to primary depression, neurotransmitter imbalances play a significant role; however, in Parkinson's disease depression, dopamine and norepinephrine play a more important role than serotonin in the pathogenesis. On the other hand, the psychogenic response of the disease itself also plays a role; patients are unable to accept the changes in their living abilities and lifestyles caused by Parkinson's disease, making them prone to emotional disturbances.

[0005] Treatment of depression in Parkinson's disease remains a significant challenge in clinical drug therapy. To date, there are no specific drugs for treating the disease. Among currently marketed anti-Parkinson's drugs, only the D3 receptor agonist Pramipexole has shown some therapeutic effect on depression associated with Parkinson's disease, but its side effects, such as kidney damage and sleep attacks, are considerable. SSRIs, used to treat primary depression, are less effective in treating depression in Parkinson's disease and may even worsen motor impairments and cause serotonin syndrome. Therefore, the development of new drugs is of great importance. Summary of the Invention

[0006] This invention provides the application of Shouhui Tongbian Capsules in the preparation of drugs for the prevention and treatment of Parkinson's disease.

[0007] Furthermore, the Parkinson's disease mentioned refers to Parkinson's motor symptoms.

[0008] Furthermore, the Parkinson's disease described herein is damage to dopamine neurons in the substantia nigra and / or striatum. Furthermore, the non-motor symptoms of Parkinson's disease described herein are mental disorders associated with Parkinson's disease; these mental disorders are mental states such as depression, anxiety, or sleep disturbances.

[0009] Furthermore, the non-motor symptoms of Parkinson's disease mentioned above are Parkinson's depression.

[0010] In some embodiments, the Parkinson's disease is an abnormal activation of microglia and / or astrocytes.

[0011] In some specific embodiments, the microglia are located in the substantia nigra and / or hippocampus.

[0012] In some specific embodiments, the astrocytes are located in the substantia nigra and / or hippocampus.

[0013] The drug described in this invention is administered to humans or non-human mammals, wherein the non-human mammals are mice, rats, monkeys, dogs, cattle, sheep, or chimpanzees.

[0014] The drug provided by the present invention is administered orally; wherein the oral administration method is to prepare a suspension by dissolving the powder of Shouhui Tongbian Capsules in a solution.

[0015] Specifically, the solution is a sodium carboxymethyl cellulose solution.

[0016] The dosage of the Shouhui Tongbian Capsules provided by this invention is 190-210 mg / kg / day.

[0017] Beneficial effects:

[0018] (1) Shouhui Tongbian Capsules can significantly improve the damage of dopamine neurons in the substantia nigra and striatum of Parkinson's mice caused by MPTP and inhibit the activation of microglia and astrocytes; it can not only alleviate the motor symptoms of Parkinson's disease mice, but also significantly improve the non-motor symptoms of Parkinson's disease mice, including mental disorders, and has a significant therapeutic effect on Parkinson's disease or non-motor symptoms of Parkinson's disease.

[0019] (2) Shouhui Tongbian Capsules can simultaneously improve dopamine neuron damage in animal models of Parkinson's disease and reduce depressive-like behavior in mice with comorbid depression, thus playing a dual therapeutic role in Parkinson's disease and / or Parkinson's disease depression.

[0020] (3) Shouhui Tongbian Capsules are a clinically marketed drug, which can greatly reduce the research costs in preclinical studies, save resources, and shorten the research and development cycle. All experimental data prove that Shouhui Tongbian Capsules have a good anti-Parkinson's disease effect and have potential clinical application prospects.

[0021] (4) No toxicity was observed in animal experiments with Shouhui Tongbian Capsules.

[0022] Detailed description

[0023] PD: Parkinson's disease

[0024] MPTP: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine

[0025] CUMS: Chronic, unpredictable, mild stimulation

[0026] CMC-Na: Sodium carboxymethyl cellulose

[0027] TH: Tyrosine hydroxylase

[0028] SPT: Sugar Water Preference Experiment

[0029] FST: Forced Swimming Experiment

[0030] TST: Tail Suspension Experiment

[0031] EPM: Elevated Cross Maze

[0032] OFT: Open Field Experiment

[0033] RIPA: Soluble proteins extracted from animal tissues and cells.

[0034] rpm: revolutions per minute, indicating the number of times the device rotates per minute.

[0035] min: minutes

[0036] WB: Western blot, protein immunoblotting

[0037] SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

[0038] um: micrometer Attached Figure Description

[0039] Figure 1 The present invention describes the preparation of an animal model of Parkinson's disease and the administration regimen.

[0040] Figure 2 In this embodiment of the invention, Shouhui Tongbian Capsules can alleviate the degeneration of dopaminergic neurons in the substantia nigra and striatum of Parkinson's disease mice; (A) and (B) TH immunohistochemistry detection of the effect of Shouhui Tongbian Capsules on the degeneration of dopaminergic neurons in the striatum and substantia nigra of Parkinson's disease mouse models; (C) and (D) Western Blot detection of the effect of Shouhui Tongbian Capsules on the expression of TH protein in the striatum and substantia nigra of Parkinson's disease mouse models with depression.

[0041] Figure 3 In this embodiment of the invention, Shouhui Tongbian Capsules can improve depressive-like behavior in mice with Parkinson's disease and depression; (A) and (B) results of forced swimming and tail suspension experiments show that Shouhui Tongbian Capsules can significantly improve the despair state of mice with Parkinson's disease and depression (one-way ANOVA. *P<0.05, **P<0.01, n=6); (C) results of sucrose preference experiment show that Shouhui Tongbian Capsules can significantly improve anhedonia in mice with Parkinson's disease and depression (one-way ANOVA. *P<0.05, **P<0.01, n=6); (D), (E), and (I) elevated cross maze experiment detects anxiety-like behavior in mice with Parkinson's disease and depression; (F), (G), and (H) open field experiment detects anxiety-like behavior in mice with Parkinson's disease and depression.

[0042] Figure 4 In this embodiment of the invention, Shouhui Tongbian Capsules can reduce the activation of astrocytes and microglia in the substantia nigra of mice with Parkinson's disease and depression; (A) GFAP immunofluorescence detection of astrocyte activation in the substantia nigra; (B) IBA1 immunofluorescence detection of microglia activation in the substantia nigra.

[0043] Figure 5 In this embodiment of the invention, Shouhui Tongbian Capsules can reduce the activation of astrocytes in the hippocampus of mice with Parkinson's disease and depression; (A) GFAP immunofluorescence detection of the activation of astrocytes in the hippocampus of the mouse model of Parkinson's disease and depression by Shouhui Tongbian Capsules; (B) Semi-quantitative analysis of GFAP immunofluorescence staining results was performed using ImageJ.

[0044] Figure 6In this embodiment of the invention, Shouhui Tongbian Capsules can reduce the activation of microglia in the hippocampus of mice with Parkinson's disease and depression; (A) IBA1 immunofluorescence detection of the effect of Shouhui Tongbian Capsules on the activation of astrocytes in the hippocampus of a mouse model with Parkinson's disease and depression; (B) Semi-quantitative analysis of IBA1 immunofluorescence staining results was performed using ImageJ (one-way ANOVA. *P<0.05, **P<0.01, n=3). Detailed Implementation

[0045] To enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments. The following descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0046] Example 1

[0047] 1. Preparation and administration regimen of a mouse model of Parkinson's disease

[0048] Thirty male C57BL / 6J mice aged 8-10 weeks were randomly divided into three groups after one week of adaptive feeding: control group, MPTP stimulation group (model group), and MPTP+CUMS+Shouhui Tongbian capsule treatment group, with 10 mice in each group.

[0049] (1) MPTP-induced subacute Parkinson's disease model

[0050] A subacute Parkinson's disease model was induced by intraperitoneal injection of MPTP (30 mg / kg). Both the model group and the treatment group received the medication once daily from day one to day five. Simultaneously, the control group received an equal dose of 0.9% sterile sodium chloride solution via intraperitoneal injection.

[0051] (2) Treatment with Shouhui Tongbian Capsules

[0052] Shouhui Tongbian capsule powder was prepared as a 10 mg / mL suspension using 0.5% CMC-Na (prepared with triple-distilled water) and administered by gavage at a dose of 200 mg / kg / day. The treatment group received the medication once daily from day 6 to day 19. Simultaneously, the control group and the model group received an equal volume of 0.5% CMC-Na by gavage.

[0053] (3) CUMS stimulation

[0054] From day 20 to day 40, both the model group and the treatment group were simultaneously given chronic, unpredictable, mild stimulation for three consecutive weeks. The specific methods were as follows: water deprivation (24h), food deprivation (24h), empty cage (24h), 4℃ cold swimming (5min), 45° cage tilting (12h), wet cage (24h), horizontal shaking (5min), tail clamping (1cm from the base of the tail, 1min), day-night reversal (24h), empty bottle stimulation (1h), and restraint (5h). Two different, unpredictable stimuli were randomly applied each day. Control group mice were normally fed and received no stimulation. The experimental design timeline for measurement and manipulation is as follows: Figure 1 As shown.

[0055] 2. Behavioral observation

[0056] After the CUMS stimulation of Parkinson's disease patients was completed and the treatment with Shouhui Tongbian capsules was finished, behavioral tests were conducted, including the Sugar Water Preference Test (SPT), Forced Swimming Test (FST), Suspended Tail Test (TST), Elevated Cross Maze (EPM), and Open Field Test (OFT).

[0057] 3. Western blot and immunohistochemical detection of tyrosine hydroxylase (TH) expression in the substantia nigra and striatum.

[0058] (1) Western blot detection of TH protein expression levels in substantia nigra and striatum

[0059] Mice were sacrificed after behavioral testing, and tissue samples were collected from the substantia nigra and striatum. RIPA lysis buffer, phosphatase inhibitors, and protease inhibitors were added, and the mixture was homogenized at low temperature. Then, the samples were centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was collected. After determining the protein concentration using the BCA method, the samples were mixed with the loading buffer at a ratio of 5:1 (protein sample: 5 × loading buffer). The centrifuge tubes were capped and incubated in a 100°C metal bath for 8 min to denature the proteins. The cooked samples were then added to the wells of a pre-prepared 10% SDS-PAGE gel for electrophoresis, and then transferred to a PVDF membrane. Changes in TH protein expression levels in different groups of substantia nigra and striatum were detected using Western blotting (WB) with primary and secondary antibodies.

[0060] (2) Immunohistochemical detection of TH expression levels in the substantia nigra and striatum

[0061] Mice were anesthetized after behavioral testing, fixed by perfusion with 4% paraformaldehyde, and then their whole brains were harvested. The brains were dehydrated using a sucrose gradient and embedded. Coronal slices of 30 μm thickness from the substantia nigra and striatum were obtained using a -20°C cryostat based on brain atlases. After washing the brain slices with PBS, endogenous peroxidase was blocked, serum was applied, and the slices were incubated with primary and secondary antibodies according to immunohistochemical principles. Color development was performed using a DAB kit. The slices were photographed under an optical microscope, and changes in TH-positive regions were statistically analyzed.

[0062] 4. Immunofluorescence detection of activation status of astrocytes and microglia in the substantia nigra.

[0063] (1) Activation of astrocytes and microglia in the substantia nigra region

[0064] Mice were anesthetized after behavioral testing, fixed by perfusion with 4% paraformaldehyde, and then their whole brains were harvested. The brains were dehydrated using a sucrose gradient and embedded. Coronal slices of 30 μm thickness from the substantia nigra region were cut using a -20°C cryostat based on brain spectroscopy. After washing with PBS, the brain slices were permeabilized with Triton X-100, blocked with BSA, immunostained (primary antibody binds to target epitopes; fluorescent secondary antibody recognizes primary antibody; DAPI stains nuclei), and mounted with a fluorescence quencher. The slices were then photographed under a fluorescence microscope to observe and analyze the activation status of astrocytes and microglia.

[0065] (2) Activation of astrocytes and microglia in the hippocampus

[0066] Take the embedded and fixed whole brain, and according to the brain spectrum, cut a 30 μm thick coronal slice from the hippocampus using a -20℃ cryostat. The remaining procedures are the same as (1).

[0067] 5. Experimental Results

[0068] (1) Shouhui Tongbian Capsules can alleviate the degeneration of dopamine neurons in the substantia nigra-striatal pathway in Parkinson's disease mice, such as Figure 2 As shown in the figures. (A) and (B) TH immunohistochemical detection of the effect of Shouhui Tongbian capsules on dopaminergic neuronal degeneration in the striatum and substantia nigra of a Parkinson's disease mouse model; the results showed that Shouhui Tongbian capsules could significantly reduce the loss of dopaminergic neurons and nerve fibers in the substantia nigra-striatal pathway induced by MPTP in Parkinson's disease mice (one-way ANOVA. *P<0.05, **P<0.01, n=3); (C) and (D) Western Blot detection of the effect of Shouhui Tongbian capsules on TH protein expression in the striatum and substantia nigra of a Parkinson's disease mouse model with depression. The results showed that Shouhui Tongbian capsules could significantly improve the loss of TH protein in the substantia nigra and striatum induced by MPTP (one-way ANOVA. *P<0.05, **P<0.01, n=3), and had a significant ameliorative effect on the motor symptoms of Parkinson's disease.

[0069] (2) Shouhui Tongbian Capsules can alleviate depressive-like behaviors in mice with Parkinson's disease and depression, such as... Figure 3 As shown in the figures: (A) and (B) The results of the forced swimming and tail suspension experiments showed that Shouhui Tongbian Capsules could significantly improve the behavioral despair state of Parkinson's disease and depression mice (one-way ANOVA. *P<0.05, **P<0.01, n=6); (C) The results of the sugar water preference experiment showed that Shouhui Tongbian Capsules could significantly improve the anhedonia phenomenon in Parkinson's disease and depression mice (one-way ANOVA. *P<0.05, **P<0.01, n=6); (D), (E), and (I) The elevated cross maze experiment detected anxiety-like behaviors in Parkinson's disease and depression mice, and the results showed that Shouhui Tongbian Capsules could improve anxiety-like behaviors in Parkinson's disease and depression mice. Compared with the model group, the Shouhui Tongbian Capsule treatment group showed a longer number of entries into the open arm and a longer time spent in the open arm (one-way ANOVA. *P<0.05, **P<0.01, n=6); Open field tests (F), (G), and (H) were used to detect anxiety-like behaviors in mice with Parkinson's disease and depression, and the results showed that Shouhui Tongbian Capsule could alleviate anxiety-like behaviors in mice with Parkinson's disease and depression; Compared with the model group, the Shouhui Tongbian Capsule treatment group showed a significant increase in the time spent in the central square area (one-way ANOVA. *P<0.05, **P<0.01, n=6).

[0070] (3) Shouhui Tongbian Capsules can reduce the activation of astrocytes and microglia in the substantia nigra of mice with Parkinson's disease and depression, such as... Figure 4 As shown in the figures: (A) GFAP immunofluorescence assay showed that Shouhui Tongbian capsules significantly reduced the activation of astrocytes in the substantia nigra of Parkinson's disease-complicated depression mice and reduced the inflammatory response (one-way ANOVA. *P<0.05, **P<0.01, n=3); (B) IBA1 immunofluorescence assay showed that Shouhui Tongbian capsules significantly reduced the activation of microglia in the substantia nigra of Parkinson's disease-complicated depression mouse models and reduced the inflammatory response in the brain (one-way ANOVA. *P<0.05, **P<0.01, n=3).

[0071] (4) Shouhui Tongbian Capsules can reduce the activation of astrocytes in the hippocampus of mice with Parkinson's disease and depression, such as... Figure 5As shown. (A) GFAP immunofluorescence assay to detect the activation of astrocytes in the hippocampus of a mouse model of Parkinson's disease with depression; (B) Semi-quantitative analysis of GFAP immunofluorescence staining results using ImageJ; The results showed that Shouhui Tongbian capsules could significantly reduce the activation of astrocytes in the hippocampus of a mouse model of Parkinson's disease with depression (one-way ANOVA. *P<0.05, **P<0.01, n=3).

[0072] (5) Shouhui Tongbian Capsules can reduce the activation of microglia in the hippocampus of mice with Parkinson's disease and depression, such as Figure 6 As shown in the figure. (A) Effect of IBA1 immunofluorescence assay on the activation of astrocytes in the hippocampus of a mouse model of Parkinson's disease with depression; (B) Semi-quantitative analysis of IBA1 immunofluorescence staining results using ImageJ (one-way ANOVA. *P<0.05, **P<0.01, n=3). The results indicate that Shouhui Tongbian capsules can significantly reduce the activation of microglia in the hippocampus of a mouse model of Parkinson's disease with depression.

[0073] It should be understood that the embodiments and implementations described herein are for illustrative purposes only and will inspire those skilled in the art to make various modifications or changes thereto, which will be included within the spirit and scope of this application. Furthermore, any element or limitation of any invention disclosed herein or any implementation thereof may be combined with any and / or all other elements or limitations disclosed herein (alone or in any combination) or any other invention or implementation thereof, and all such combinations are contemplated to be included within the scope of the invention, but are not limited thereto.

Claims

1. Use of Shouhao Tongbian Capsules in the preparation of a medicament for preventing or treating Parkinson's disease.

2. Use according to claim 1, wherein The Shouhao Tongbian Capsules improve the motor symptoms of Parkinson's disease.

3. Use according to claim 2, wherein the compound is ###0002### The motor symptoms of Parkinson's disease are damage to the dopaminergic neurons in the substantia nigra and / or the striatum.

4. The use according to claim 1, wherein The Shouhao Tongbian Capsules inhibit the activation of microglial cells and / or astrocytes.

5. The use according to claim 4, wherein the compound is ###0002### The microglial cells are located in the substantia nigra and / or the hippocampus, and the astrocytes are located in the substantia nigra and / or the hippocampus.

6. The use according to claim 1, wherein The medicament is administered to a human or non-human mammal, and the medicament is administered orally.

7. The use according to claim 1, wherein The Shouhao Tongbian Capsules are prepared into a suspension by mixing Shouhao Tongbian Capsule powder.

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