Application of geitogenin to prevention or treatment of neurodegenerative diseases

Abstract

The invention discloses an application of geitogenin in prevention or treatment of neurodegenerative diseases, in-vitro cell experiments and animal model researches prove that the geitogenin can significantly down-regulate the expression level of HSD17B10 or inhibit the enzymatic activity of HSD17B10; therefore, the nerve cell survival is improved, the neuroinflammation is reduced, the epileptic seizure frequency is reduced, and the cognitive function is improved. Furthermore, it is proved that the Gemtogenin can significantly reduce the HSD17B10 protein level in vitro and in vivo, improve cognitive impairment and reduce the epileptic seizure frequency, and the Gemtogenin has good safety and application prospects. According to the invention, the new application of the geitogenin in preparing the medicine for preventing or treating the neurodegenerative diseases and the epilepsy is provided for the first time, and a new way and means are provided for treating the neurodegenerative diseases and the epilepsy.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to the application of gitogenin in preventing or treating neurodegenerative diseases. BACKGROUND

[0002] Neurodegenerative diseases are a class of central nervous system diseases caused by the gradual degeneration and loss of neurons, including Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington's disease (HD) and the like. Its pathological mechanism involves multiple factors such as Aβ deposition, abnormal phosphorylation of tau protein, impaired mitochondrial function and neuroinflammatory response. HSD17B10 (17β-hydroxysteroid dehydrogenase type 10) is a mitochondrial multifunctional enzyme that can bind to Aβ and participate in neurotoxic signaling, and is highly expressed in the brain tissue of AD patients, which is a key factor for early disturbance of the dynamic balance of neuroprotective steroids and leading to mitochondrial dysfunction in AD. Inhibition of HSD17B10 can improve mitochondrial function, restore neurosteroid homeostasis and alleviate Aβ-related toxic reactions, and therefore is considered as a potential therapeutic target. Epilepsy is a common chronic brain disease characterized by repeated seizures caused by abnormal discharge of neurons, and its mechanism involves neurotransmitter imbalance, ion channel disorder, mitochondrial dysfunction and neuroinflammation. Studies have shown that abnormal expression of HSD17B10 may promote the occurrence and development of seizures, and regulation of the activity of the enzyme is expected to improve the course of epilepsy.

[0003] Gitogenin is a steroidal saponin compound derived from plants containing steroidal saponins (such as Cucurbitaceae and Liliaceae), which has similar structure to steroid hormones and has anti-inflammatory, immunoregulatory, anti-tumor and other biological activities. There is no direct application of gitogenin in the regulation of HSD17B10, the treatment of neurodegenerative diseases or epilepsy reported so far. SUMMARY

[0004] The present application provides a new use of gitogenin in the preparation of a drug for preventing or treating neurodegenerative diseases and epilepsy, especially for Alzheimer's disease, Parkinson's disease, frontotemporal dementia and epilepsy and other diseases related to abnormal expression of HSD17B10. Through in vitro cell experiments and animal model studies, it is proved that gitogenin can significantly down-regulate the expression level of HSD17B10 or inhibit its enzyme activity, thereby improving the survival of neural cells, reducing neuroinflammation, reducing the frequency of seizures and improving cognitive function. Further, it is proved that gitogenin can significantly reduce the protein level of HSD17B10 in vitro and in vivo, improve cognitive impairment and reduce the frequency of seizures, and has good safety and application prospect.

[0005] The application first proposes a new use of gitosapogenin in the preparation of a medicine for preventing or treating neurodegenerative diseases and epilepsy, and provides a new way and means for treating neurodegenerative diseases and epilepsy.

[0006] The application also provides a medicine composition for the new use of gitosapogenin in the preparation of a medicine for preventing or treating neurodegenerative diseases and epilepsy.

[0007] Technical scheme: In order to achieve the above technical purpose, the application of gitosapogenin in the preparation of a medicine for preventing or treating neurodegenerative diseases.

[0008] The application of gitosapogenin in the preparation of a medicine for preventing or treating epilepsy.

[0009] Among them, the neurodegenerative disease is Alzheimer's disease, Huntington's disease (HD), frontotemporal dementia or Parkinson's disease.

[0010] Among them, the gitosapogenin can down-regulate the expression level of HSD17B10 or inhibit the enzyme activity thereof, so as to improve the survival of nerve cells, reduce nerve inflammation, and improve cognitive function in the preparation of a medicine for preventing or treating neurodegenerative diseases or epilepsy.

[0011] Among them, the gitosapogenin reduces the HSD17B10 protein level, improves cognitive impairment, and reduces the frequency of seizures and neuron damage, and is used in the preparation of a medicine for preventing or treating neurodegenerative diseases or epilepsy.

[0012] The application of gitosapogenin combined with a medicine for improving cognitive impairment in the preparation of a medicine for preventing or treating neurodegenerative diseases and epilepsy.

[0013] Among them, the medicine for improving cognitive impairment includes but is not limited to any one or more of donepezil, rivastigmine, galantamine, and memantine.

[0014] The medicine composition for preventing or treating neurodegenerative diseases and epilepsy in the application contains gitosapogenin as an active ingredient and a pharmaceutically acceptable carrier.

[0015] Among them, the dosage form of the medicine composition is preferably a capsule, a powder, a tablet, a granule, a pill, an injection, a syrup, an oral liquid, an inhalant, an ointment, a suppository or a patch.

[0016] The application of the medicine composition in the preparation of a medicine for preventing or treating neurodegenerative diseases and epilepsy.

[0017] The application discloses application of gitosapogenin in preparation of medicines for preventing or treating neurodegenerative diseases and epilepsy.

[0018] The application first discovers that gitosapogenin can specifically down-regulate the expression or activity of HSD17B10, and verifies the effect of improving cognitive function, reducing the frequency of epileptic seizures and neuron damage through animal experiments, thereby providing a brand-new drug development direction for treatment of the diseases.

[0019] Beneficial effects: compared with the prior art, the application has the following advantages:

[0020] The application first proposes the application of gitosapogenin in preparation of medicines for preventing or treating neurodegenerative diseases and epilepsy, and the application utilizes gitosapogenin to down-regulate the expression or activity of HSD17B10, and acts on early pathological links of diseases, thereby significantly improving cognitive function in animal models. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 In the in-vitro cell experiment, the influence of gitosapogenin with different concentrations on the expression level of HSD17B10 in a 10 muM a beta 1-42 induced PC12 cell neurotoxicity model is shown, and the gitosapogenin treatment group has an inhibitory effect compared with the model group.

[0022] Figure 2 In the in-vitro cell experiment, the influence of gitosapogenin with different concentrations on the expression level of HSD17B10 in 293T cells overexpressing HSD17B10 is shown, and the gitosapogenin treatment group has an inhibitory effect compared with the model group.

[0023] Figure 3 In the in-vitro cell experiment, the toxic effect of gitosapogenin with different concentrations on PC12 cells is shown, and no significant cytotoxic side effect of gitosapogenin in the effective dose range is observed.

[0024] Figure 4 The figure shows the body weight change of an Alzheimer's disease model mouse intragastrically administered with gitosapogenin, and there is no significant difference between the gitosapogenin treatment group and the model group.

[0025] Figure 5 The figure shows the comparison of escape latency of an Alzheimer's disease model mouse intragastrically administered with gitosapogenin in a water maze experiment, and there is a significant difference between the gitosapogenin treatment group and the model group.

[0026] Figure 6 Figure 6 is a comparison chart of the target quadrant residence time of Alzheimer's disease model mice administered gitorogenin by gavage in the water maze experiment; showing that the gitorogenin treatment group has a significant difference compared with the model group.

[0027] Figure 7 Figure 7 is a graph showing the expression of HSD17B10 protein levels in the brain tissue of Alzheimer's disease model mice administered gitorogenin by gavage. It shows that the gitorogenin treatment group has a significant difference compared with the model group. DETAILED DESCRIPTION

[0028] The content of the present application is specifically illustrated by the following examples. In the present application, the following examples are described in order to better illustrate the present application, and are not intended to limit the scope of the present application. Various changes and modifications can be made to the present application without departing from the spirit and scope of the present application.

[0029] Gitorogenin (MedChemExpress LLC, #HY-N2574).

[0030] aβ1-42 (Bi Yun Tian Biotechnology Co., Ltd., #P9001).

[0031] Rat adrenal pheochromocytoma differentiated cell line PC12 cells were purchased from Wuhan Punsai Life Science Co., Ltd., CL-0412.

[0032] 293T cells were purchased from Wuhan Punsai Life Science Co., Ltd., CL-0005.

[0033] 6-month-old APP / PS1 double transgenic mice were purchased from Changzhou Cavenx Laboratory Animal Warrant Co., Ltd., strain number: C000111.

[0034] Example 1

[0035] The rat adrenal pheochromocytoma differentiation cell line PC12 cell line was cultured in DMEM medium containing 10% FBS and 1% penicillin / streptomycin at 37°C in a 5% CO2 incubator. When the cell density reached 80%-90% of the area of the culture dish, the cells were subcultured at a ratio of 1:3. The cells were in good condition, and when the cell density reached 80%-90% of the area of the culture dish, the cells were trypsinized, resuspended after centrifugation, and inoculated in a 6-well plate at 37°C. After 24 h of cell adhesion, different concentrations (1, 5, 10 μM) of gitosaponarin were used to pretreat PC12 cells for 2 h, and then 10 μM αβ1-42 was used to induce a neurotoxicity model in PC12 cells for 12 h at 37°C in a cell incubator. The PC12 cells were washed with PBS three times, the cell pellets were collected, and the cells were lysed with RIPA buffer containing 1 mM PMSF and phosphatase inhibitors. The cells were resuspended, shaken on ice for 30 min, and then collected for protein. Western blot was used to detect the expression changes of HSD17B10 in PC12 cells. The results of Western blot showed that gitosaponarin at a concentration of 1, 5, 10 μM could inhibit the expression of HSD17B10 Figure 1 ).

[0036] Example 2

[0037] After overexpression of HSD17B10 in 293T cell lines, 5, 10 μM gitosaponarin was used to treat 293T cells for 12 h, and the expression changes of HSD17B10 were detected.

[0038] Firstly, the transfection reagent was configured: 2ul Lipofectamine 2000 was added to an EP tube with 50ul opti-MEM, and gently blown, and reacted at room temperature for 5min; a new EP tube was taken to add 0.8ul DNA and 50ul opti-MEM; the liquids in the two EP tubes were mixed, gently blown, and reacted at room temperature for 20min; 400ul opti-MEM was added after 20min, and mixed evenly by gently blowing. Secondly, when the 4-5 generation 293T cells after resuscitation had a fusion degree of 85-95%, they were digested with 0.25% trypsin and inoculated into a new twelve-hole plate, and cultured in a 37℃ incubator for 24h with high glucose DMEM culture solution containing 10% FBS; after 24h, the original culture solution in the culture plate was discarded, and washed once with 1xPBS, 500uL of the prepared transfection reagent was added, and placed in a 37℃ incubator for transfection for 4h; after 4h, the transfection reagent in the culture plate was discarded, and washed once with 1xPBS, and 1ml of DMEM culture solution containing 10% FBS was added to each hole to incubate the cells for 20h. Then 1ml of culture solution containing 5, 10μM concentration of gitosaponigenin was added to each hole, and incubation was continued for 12h. Finally, the cell protein was collected and Western blot detection was performed. The results showed that gitosaponigenin could inhibit the expression of HSD17B10 Figure 2 ).

[0039] Example 3

[0040] The rat adrenal medulla pheochromocytoma differentiation cell line PC12 cell line was cultured in DMEM culture medium containing 10% FBS and 1% penicillin / streptomycin in a 37℃, 5% CO2 incubator. The cells were in good condition, and when the cell density reached 80%-90% of the area of the culture dish, the cells were digested with 0.25% trypsin, centrifuged, resuspended, and inoculated in a 96-well plate at 37℃. After 24h of cell adhesion, different concentrations (0.01, 0.1, 1, 10, 100μM) of gitosaponigenin were added to treat PC12 cells. After 48h of treatment, 10μL of CCK-8 reagent was added to the 96-well plate, and incubated in a 37℃ cell incubator. The absorbance value at 450nm was read every 1h by an enzyme marker for a total of 4h. The average value of the control was set as 100% (reference). The relative fold change of the control was calculated. The results showed that the IC 50 value of gitosaponigenin was >100μM, with no obvious biological toxicity Figure 3 ).

[0041] Example 4

[0042] The 6-month-old C57BL / 6J wild-type male mice (WT) are used as a control group, the 6-month-old APP / PS1 double transgenic mice are used as an AD model group and a drug administration group (low dose and high dose), and gitosapogenin is continuously administered at 5 or 10 mg / kg by gavage for 2 weeks. The water maze experiment training is performed on day 9, and the gavage is continuously performed until the test on day 14. The water maze device mainly consists of a circular pool (diameter 150 cm x height 60 cm), an escape platform (diameter 10 cm), a data acquisition system and an analysis system. The circular pool can be divided into four quadrants, and the escape platform is always located at the center of the third quadrant. At the beginning of the experiment, water is added to the pool until the water level is 1 cm higher than the escape platform, and 0.25 g / L titanium dioxide powder is added to make the water opaque. In the training stage, the mouse is placed facing the inner wall, and is randomly introduced into the pool from each of the four quadrants. If the rat finds the escape platform within one minute, it is allowed to rest on the platform for 15 seconds; if it is unsuccessful, it is gently guided to the platform to rest for 15 seconds, and the continuous training is performed for 5 days. The exploration experiment starts from day 6. In the exploration experiment, the mouse is placed in the pool from the first quadrant, and the mouse is given 1 minute to find the platform, during which the movement trajectory and the time spent positioning the platform of the mouse are observed, recorded and analyzed. The body weight of the Alzheimer's disease model mouse administered with gitosapogenin does not change significantly compared with the control group, indicating that gitosapogenin has no obvious biological toxicity Figure 4 ). The Morris water maze experiment shows that the escape latency of the mice in the administration group is significantly shortened Figure 5 ), the platform quadrant residence time is increased Figure 6 ), and the learning and memory ability is improved Figure 5 ). The Western blot result shows that the HSD17B10 protein level in the brain tissue is significantly decreased compared with the model group Figure 7 ).

[0043] In summary, the present application finds that gitosapogenin can significantly down-regulate or inhibit the expression and activity of HSD17B10 and improve the learning and memory ability of AD mice in in vivo and in vitro experiments. Compared with the prior art, the present application uses gitosapogenin from a natural source, and no obvious toxic side effects are observed within the effective dose range, which has the potential for long-term safe application. At the same time, the present application not only proposes a new use of gitosapogenin in the prevention and treatment of neurodegenerative diseases and epilepsy, but also provides a new idea and direction for the drug research and development of HSD17B10 related diseases. Therefore, the present application provides an innovative and clinically transformative approach and means for the treatment of neurodegenerative diseases and epilepsy.

Claims

1. Use of gitosapogenin in the preparation of a medicament for preventing or treating neurodegenerative diseases.

2. Use of gitosapogenin in the preparation of a medicament for preventing or treating epilepsy.

3. Use according to claim 1, characterized in that, The neurodegenerative disease is Alzheimer's disease, Huntington's disease (HD), frontotemporal dementia or Parkinson's disease.

4. Use according to claim 1 or 2, characterized in that, The gitosapogenin can down-regulate the expression level of HSD17B10 or inhibit the enzyme activity thereof, thereby improving the survival of nerve cells, reducing neural inflammation, and improving cognitive function in the preparation of a medicament for preventing or treating neurodegenerative diseases or epilepsy.

5. The use according to claim 1 or 2, characterized in that, The gitosapogenin reduces the level of HSD17B10 protein, improves cognitive impairment, and reduces the frequency of seizures and neuronal damage, and is used in the preparation of a medicament for preventing or treating neurodegenerative diseases or epilepsy.

6. Use of gitosapogenin in combination with a drug for improving cognitive impairment in the preparation of a medicament for preventing or treating neurodegenerative diseases and epilepsy.

7. Use according to claim 6, characterized in that, The drug for improving cognitive impairment includes, but is not limited to, any one or more of donepezil, rivastigmine, galantamine, and memantine.

8. A pharmaceutical composition for preventing or treating a neurodegenerative disease and epilepsy, wherein the pharmaceutical composition is prepared by mixing the compound or a pharmaceutically acceptable salt thereof according to claim 1 and a pharmaceutically acceptable carrier. The pharmaceutical composition contains gitosapogenin as an active ingredient and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition of claim 8, wherein, The dosage form of the pharmaceutical composition is preferably a capsule, a powder, a tablet, a granule, a pill, an injection, a syrup, an oral liquid, an inhalant, an ointment, a suppository, or a patch.

10. Use of the pharmaceutical composition of claim 8 in the preparation of a medicament for preventing or treating neurodegenerative diseases and epilepsy.

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