CN113181171A - Application of silychristin in preparation of medicine for preventing and/or treating Parkinson's disease - Google Patents
Application of silychristin in preparation of medicine for preventing and/or treating Parkinson's disease Download PDFInfo
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- CN113181171A CN113181171A CN202110544867.3A CN202110544867A CN113181171A CN 113181171 A CN113181171 A CN 113181171A CN 202110544867 A CN202110544867 A CN 202110544867A CN 113181171 A CN113181171 A CN 113181171A
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- silychristin
- rotenone
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
- A61P25/16—Anti-Parkinson drugs
Abstract
An application of silychristin in preparing a medicament for preventing and/or treating Parkinson's disease relates to the field of Parkinson's disease medicaments. Experiments prove that silychristin can inhibit the killing effect of rotenone on human SH-SY5Y cells, silychristin can inhibit DNA double-chain damage caused by rotenone, silychristin can inhibit endoplasmic reticulum stress reaction caused by rotenone, and silychristin can inhibit protein aggregation caused by rotenone. Experiments prove that the silydianin can inhibit the damage of oxygen sugar deprivation (OGD) to neurons, and has the effects of preventing and/or treating the neuron damage and death after cerebral ischemia. The invention develops the new application of the silychristin and provides a new prevention and treatment way and a prevention and treatment method for preventing and treating the Parkinson's disease which is a common disease of the nervous system affecting the life quality of the old.
Description
Technical Field
The invention relates to the technical field of Parkinson disease medicines, and in particular relates to application of silychristin in preparation of a medicine for preventing and/or treating Parkinson disease.
Background
Silychristin (Silychritin) is a flavonoid monomer small molecule compound extracted from fruit of Silybum marianum (milk thistle) of Silybum of Compositae, and has a molecular formula: c25H22O10And molecular weight 482, white granular crystals. CAS number: 33889-69-9. Silychristin (Silychritin) has the advantages of easily-accessible raw materials and low cost.
Parkinson's Disease (PD) is a common nervous system degenerative disease, and is common in the elderly, with the average age of about 60 years, and the onset of juvenile Parkinson's disease below 40 years being rare. The prevalence rate of PD in people over 65 years old in China is about 1.7%. The clinical manifestations mainly include resting tremor, bradykinesia, muscular rigidity and postural gait disorder, and patients may be accompanied by non-motor symptoms such as depression, constipation and sleep disorder. The most prominent pathological change of parkinson's disease is the degenerative death of mesocerebral Dopaminergic (DA) neurons, which causes a marked reduction in striatal DA content and causes disease.
The diagnosis of Parkinson's disease depends primarily on medical history, clinical symptoms and signs. The general auxiliary inspection is mostly free from abnormal changes. Drug therapy is the most prominent treatment for parkinson's disease. Levodopa formulations remain the most effective drug. However, only symptoms can be improved, the progress of the disease cannot be stopped, and the disease cannot be cured, so that new preventive and therapeutic drugs are urgently needed for patients of the type. Research shows that degeneration and death of mesencephalic substantia nigra dopaminergic neuron are closely related to factors such as DNA damage, protein aggregation, endoplasmic reticulum stress response and the like, so that inhibition of neuronal death caused by DNA damage, protein aggregation and endoplasmic reticulum stress response is a main strategy for preventing and treating Parkinson's disease.
At present, the prevention and treatment effects of silychristin on nervous system diseases, especially on parkinson disease, are not reported.
Disclosure of Invention
The invention aims to provide a new application of silychristin, namely an application of silychristin in preparing medicines for preventing and/or treating Parkinson's disease. Specifically, silychristin can inhibit damage and killing effect of Rotenone (Rotenone) on neuron, and has effect of preventing and treating Parkinson disease.
The technical scheme adopted by the invention for solving the technical problem is as follows:
the invention provides application of silychristin in preparation of a medicament for preventing and/or treating Parkinson's disease.
In a preferred embodiment, the concentration of silychristin is 2.0 to 4.0 mmol/L.
In a more preferred embodiment, the concentration of silychristin is 2.0 mmol/L.
In a more preferred embodiment, the concentration of silychristin is 4.0 mmol/L.
The invention has the beneficial effects that: the invention is proved by example 1 that silychristin can inhibit the killing effect of rotenone on human SH-SY5Y cells. The invention is proved by example 2 that silychristin inhibits the up-regulation of rotenone-induced gamma-H2 AX expression in a concentration-dependent manner, silychristin significantly inhibits the increase of red spots in human SH-SY5Y cell nucleus, and silychristin significantly inhibits rotenone-induced cell nucleus tailing, thereby demonstrating that silychristin can inhibit DNA double-strand damage caused by rotenone. The invention is demonstrated in example 3 that silychristin can inhibit endoplasmic reticulum stress response caused by rotenone. The invention is demonstrated in example 4 that silychristin can inhibit protein aggregation caused by rotenone.
Experiments prove that the silydianin can inhibit the damage of oxygen sugar deprivation (OGD) to neurons, and has the effects of preventing and/or treating the neuron damage and death after cerebral ischemia.
The invention develops the new application of the silychristin and provides a new prevention and treatment way and a prevention and treatment method for preventing and treating the Parkinson's disease which is a common disease of the nervous system affecting the life quality of the old.
Drawings
FIG. 1 shows the results of silychristin inhibiting the killing effect of rotenone on human SH-SY5Y cells in example 1.
FIG. 2 shows the results of the inhibition of the DNA double strand break of human SH-SY5Y cells by rotenone by silychristin in example 2.
FIG. 3 shows the results of the inhibition of the DNA double strand break of human SH-SY5Y cells by rotenone by silychristin in example 2.
FIG. 4 shows the results of the inhibition of the DNA double strand break of human SH-SY5Y cells by rotenone by silychristin in example 2.
FIG. 5 shows the results of inhibition of stress response of endoplasmic reticulum caused by rotenone by silychristin in example 3.
FIG. 6 shows the results of the inhibition of protein aggregation by rotenone by silychristin in example 4.
Detailed Description
Experiments prove that the silychristin can inhibit the killing effect of the rotenone on neurons, and the silychristin and the rotenone have a dose-effect relationship. In vitro research experiments show that 2.0mmol/L and 4.0mmol/L silychristin is adopted to treat human SH-SY5Y cells and fetal rat cortical neurons for 1 hour, so that the neuron damage and death induced by rotenone can be obviously inhibited, and the mechanism of the inhibition is related to the inhibition of DNA breakage, protein aggregation and endoplasmic reticulum stress reaction.
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 inhibition of the killing action of Rotenone on human SH-SY5Y cells by Silibinin
Silychristin (purchased from Dorem) is prepared into mother liquor with DMSO when in use, and the administration is carried out after dilution.
Human SH-SY5Y cells (purchased from Gyka Biotechnology, China) were used to prepare a cell model of "Parkinson-induced neuronal damage" from rotenone (purchased from Sigma).
The human SH-SY5Y cells are inoculated in a DMEM medium in a 96-well plate, incubated overnight in a carbon dioxide incubator, respectively incubated with silychristin (2.0mmol/L and 4.0mmol/L) at different concentrations 1 hour earlier, rotenone (30 mu mol/L) is added, and the cell death rate is detected by an LDH (lactate dehydrogenase) release method after 8 hours.
The results of the experiment are shown in FIG. 1. The results show that: the use of silychristin alone has no killing effect on human SH-SY5Y cells, the use of rotenone alone has a killing rate of about 30.30% on human SH-SY5Y cells, and the inhibition effect of the silychristin on human SH-SY5Y cell death caused by the rotenone is concentration-dependent, namely, the killing effect of the rotenone on human SH-SY5Y cells is reduced to 27.05% and 24.00% along with the increase of the concentration of the silychristin. The results of the experimental data are shown in table 1.
TABLE 1
**: p <0.01, silychristin + rotenone compared to rotenone treated group alone.
The experiments prove that the silychristin can inhibit the killing effect of rotenone on human SH-SY5Y cells.
Example 2 inhibition of Rotenone by Silibinin against DNA double strand breaks in human SH-SY5Y cells
DNA is a carrier of genetic information, and double strand breaks in DNA are the basis for causing DNA lysis and cell death. To illustrate the protective effect of silychristin on cells, this experiment examined the effect of silychristin on rotenone-induced DNA double strand breaks.
(1) Human SH-SY5Y cells were seeded in DMEM medium in a 6cm diameter dish, incubated overnight with carbon dioxide, incubated with silychristin (2.0mmol/L and 4.0mmol/L) 1 hour earlier, followed by addition of rotenone (30. mu. mol/L) and collection of cells after 8 hours. The collected cells were homogenized, differentially centrifuged, and the cytoplasm fraction, the mitochondrial fraction, and the nuclear fraction were separated, and the protein concentration of each fraction was measured by BCA method, and subjected to Westernblotting analysis. The primary antibody adopted is a marker protein of DNA double-strand broken chain: gamma-H2 AX, and the internal reference is H2A.
The results of the experiment are shown in FIG. 2. The results show that: rotenone was able to significantly up-regulate the protein level of water γ -H2AX, but silychristin inhibited the rotenone-induced up-regulation of γ -H2AX expression in a concentration-dependent manner. This indicates that silychristin can inhibit DNA double strand damage caused by rotenone.
(2) To further illustrate that silychristin can inhibit DNA double-strand damage caused by rotenone, human SH-SY5Y cells were further inoculated in DMEM medium in a dish with a diameter of 3cm, incubated overnight with silychristin (4.0mmol/L) 1 hour earlier, then rotenone (30. mu. mol/L) was added, and the cells were fixed with ethanol 8 hours later. Performing immunocytochemistry staining, wherein the adopted primary antibody is a marker protein of DNA double-strand broken chain: γ -H2AX (red), nuclei were labeled with Hochest33258 (blue) and observed with a confocal laser microscope.
The results of the experiment are shown in FIG. 3. The results show that: under the action of rotenone, red spots in human SH-SY5Y cell nucleuses are obviously increased, which indicates that the expression of gamma-H2 AX is up-regulated; however, silychristin significantly inhibited the increase of red spots in the nucleus of human SH-SY5Y cells. This indicates that silychristin can inhibit DNA double strand damage caused by rotenone.
(3) In order to further illustrate that the silychristin can inhibit the DNA double-strand damage caused by rotenone, the inhibition effect of the silychristin on the DNA double-strand damage caused by the rotenone is also tested by a method of neutral single-cell gel electrophoresis (neutral comet assay). Firstly, inoculating human SH-SY5Y cells into a DMEM medium with a diameter of a 6cm culture dish, incubating the cells in a carbon dioxide incubator overnight, incubating the cells with silychristin (4.0mmol/L) 1 hour earlier, adding rotenone (30 mu mol/L), collecting the cells after 8 hours, mixing the cells with agarose, paving the mixed cells on a glass slide, splitting the mixed cells overnight, carrying out electrophoresis, staining the cells with ethidium bromide, and observing the cells by a fluorescence microscope.
The results of the experiment are shown in FIG. 4. The results show that: rotenone can induce cells to generate tailing phenomenon, and silychristin obviously inhibits the Rotenone from inducing nuclear tailing. This indicates that silychristin can inhibit DNA double strand damage caused by rotenone.
Example 3 inhibition of stress response of endoplasmic reticulum caused by rotenone by Silibinin
Endoplasmic reticulum stress response is one of the important pathological bases leading to parkinson's disease, and excessive endoplasmic reticulum stress can induce CHOP nuclear translocation, which in turn leads to cell death.
In order to illustrate the protective effect of silychristin on cells, the effect of silychristin on the stress response of endoplasmic reticulum induced by rotenone was examined by western blot analysis. Inoculating human SH-SY5Y cells into DMEM medium with a diameter of 6cm culture dish, incubating with carbon dioxide overnight, incubating with silychristin (2.0mmol/L and 4.0mmol/L) at different concentrations 1 hr earlier, adding rotenone (30. mu. mol/L), and collecting cells after 8 hr. The collected cells were homogenized, differentially centrifuged, the cell plasma fraction and the cell nucleus fraction were separated, the protein concentration of each fraction was measured by BCA method, and Westernblotting analysis was performed. The primary antibody adopted is marker proteins GRP78 and CHOP of endoplasmic reticulum stress response, the internal reference of cytoplasm is beta-Actin, and the internal reference of nucleus is H2A.
The results of the experiment are shown in FIG. 5. The results show that: the rotenone can not only remarkably up-regulate the expression levels of endoplasmic reticulum stress response marker proteins GRP78 and CHOP, but also promote the CHOP to enter the cell nucleus. However, silychristin inhibited rotenone-induced upregulation of GRP78 and CHIP expression, as well as nuclear translocation of CHOP, in a concentration-dependent manner. This indicates that silychristin can inhibit the endoplasmic reticulum stress response caused by rotenone.
Example 4 inhibition of protein aggregation by rotenone by Silibinin
Protein aggregation is one of the important pathological bases leading to parkinson's disease, and can lead to neuronal death.
In order to illustrate the protective effect of silychristin on cells, the effect of silychristin on rotenone-induced protein aggregation was examined by western blot analysis. Inoculating human SH-SY5Y cells into DMEM medium with a diameter of 6cm culture dish, incubating with carbon dioxide overnight, incubating with silychristin (2.0mmol/L and 4.0mmol/L) at different concentrations 1 hr earlier, adding rotenone (30. mu. mol/L), and collecting cells after 8 hr. The collected cells were homogenized, differentially centrifuged, the protein aggregate fraction was separated, the protein concentration was determined by BCA method, and western blotting was performed. The primary antibody is Ubiquitin, and the internal parameter is beta-Actin.
The results of the experiment are shown in FIG. 6. The results show that: under the action of rotenone, the ubiquitin-labeled protein was significantly increased, suggesting that rotenone induced protein aggregation. However, silychristin inhibited the increase in ubiquitin marker protein induced by rotenone in a concentration-dependent manner. This indicates that silychristin is able to inhibit protein aggregation caused by rotenone.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (4)
1. Application of silychristin in preparing medicine for preventing and/or treating Parkinson disease is provided.
2. The use according to claim 1, wherein the concentration of silychristin is 2.0 to 4.0 mmol/L.
3. The use according to claim 2, wherein the concentration of silychristin is 2.0 mmol/L.
4. The use according to claim 2, wherein the concentration of silychristin is 4.0 mmol/L.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202110544867.3A CN113181171A (en) | 2021-05-19 | 2021-05-19 | Application of silychristin in preparation of medicine for preventing and/or treating Parkinson's disease |
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| CN202110544867.3A CN113181171A (en) | 2021-05-19 | 2021-05-19 | Application of silychristin in preparation of medicine for preventing and/or treating Parkinson's disease |
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Application publication date: 20210730 |
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