Use of gastrodin in preparation of SCA3 disease treatment drugs

Abstract

The application discloses application of gastrodin in preparation of SCA3 disease treatment drugs, and the gastrodin is a compound extracted from Gastrodia elata Blume of the Orchidaceae family and has therapeutic effects on neurasthenia, headache and the like. Researches show that the gastrodin can significantly reduce inclusion bodies caused by mutant ataxin-3, improve soluble ataxin-3 protein levels, has no toxic side effects on HEK293T cells, and regulates the specific molecular mechanism of inclusion bodies formed by ataxin-3 through an AKT / ERK signal axis. The application aims to reveal the application of the gastrodin in playing a neuroprotective effect on SCA3 diseases, enrich new uses of old drugs, and provide a new idea for prevention and treatment of other neurodegenerative diseases.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical applications, specifically relating to the application of gastrodin in the preparation of drugs for treating SCA3 diseases. Background Technology

[0002] Spinocerebellar ataxia type 3 / Machado-Joseph Disease (SCA3 / MJD) is an autosomal dominant inherited neurodegenerative disease caused by abnormal amplification of the polyglutamine (polyQ) chain in the ataxin-3 protein encoded by the ATXN3 gene. Normally, the number of polyQ repeat sequences in healthy individuals ranges from 12 to 43, while in SCA3 patients, this number increases to 51 to 91. SCA3 is a rare disease with an incidence of approximately (3-5) per 100,000. However, as the most common subtype of spinocerebellar ataxia (SCAs), it accounts for approximately 63% of the SCAAs population in my country, indicating a significant number of patients. Although gene therapy targeting ATXN3 is one of the most promising methods to halt the progression of SCA3, many challenges remain regarding safety, specificity, and delivery. Because SCA3 has a late onset and slow progression, lifelong medication may be necessary. Treatment drugs need to have minimal side effects and be able to cross the blood-brain barrier. Currently, treatment is only symptomatic and lacks effective drugs, making it difficult to meet clinical needs.

[0003] Normal ataxin-3 protein is a cytoplasmic protein. Ataxin-3 protein containing aberrantly amplified polyQ molecules enters the nucleus under the stimulation of various pathogenic factors. Within the nucleus, it recruits various components, including transcription factors, ubiquitin proteases, chaperone proteins, or RNA, to form intranuclear inclusion bodies (a hallmark pathological feature of SCA3). This leads to abnormal nuclear transcription, protein homeostasis imbalance, energy metabolism disorders, and transport disorders, ultimately resulting in neuronal cell death. Therefore, like many other pathogenic proteins in neurodegenerative diseases, inclusion bodies formed by mutant ataxin-3 are an important early pathological feature and pathogenesis of SCA3. Elucidating its molecular regulatory mechanism will help reduce the number of intranuclear inclusion bodies, improve neuronal apoptosis, and alleviate SCA3 ataxia symptoms. Thus, inhibiting ataxin-3 inclusion bodies is a promising direction for targeted drug development. However, current research in this area is lacking. Summary of the Invention

[0004] The purpose of this invention is to provide the application of gastrodin in the preparation of drugs for the treatment of SCA3 disease, which has a significant protective effect on the prevention and treatment of SCA3 disease and provides a new approach for the treatment of neurodegenerative diseases.

[0005] The technical solution adopted in this invention is the application of gastrodin in the preparation of drugs for treating SCA3 diseases.

[0006] The invention is further characterized by:

[0007] The application of gastrodin in the preparation of drugs for the treatment of SCA3 disease, with the concentration of gastrodin used in the drugs ranging from 5μM to 100μM.

[0008] Gastrodin exerts a neuroprotective effect in SCA3 disease through the AKT / ERK signaling axis.

[0009] The beneficial effects of this invention are:

[0010] This invention provides gastrodin that significantly inhibits inclusion bodies induced by mutant ataxin-3, has no toxic side effects on HEK293T cells, and exerts a significant protective effect against SCA3 disease by influencing the AKT / ERK signaling axis to regulate inclusion body formation. This enriches the new uses of existing drugs and provides new ideas for the prevention and treatment of other neurodegenerative diseases. Attached Figure Description

[0011] Figure 1 This is a schematic diagram of the SCA3 pathogenesis process mentioned in this invention;

[0012] Figure 2 This is a schematic diagram illustrating the construction results of the SCA3 cell model in this invention;

[0013] Figure 3 This is a schematic diagram illustrating the effects of two methods in this invention on the activity of SCA3 cells;

[0014] Figure 4 This is a schematic diagram illustrating the effect of gastrodin on the inclusion bodies formed by the pathogenic protein ataxin-3 in this invention;

[0015] Figure 5 This is a schematic diagram illustrating the effect of gastrodin on the soluble protein level of the pathogenic protein ataxin-3 in this invention;

[0016] Figure 6 This is a schematic diagram illustrating the changes in the AKT signaling pathway in different cell models in this invention;

[0017] Figure 7 This is a schematic diagram illustrating the changes in the ERK signaling pathway in different cell models in this invention;

[0018] Figure 8This is a schematic diagram illustrating the changes in the P38 signaling pathway in different cell models in this invention;

[0019] Figure 9 This is a schematic diagram illustrating the changes in the p65 signaling pathway in different cell models in this invention;

[0020] Figure 10 This is a schematic diagram illustrating the effect of gastrodin on the AKT signaling pathway in different cell models in this invention;

[0021] Figure 11 This is a schematic diagram illustrating the effect of gastrodin on the ERK signaling pathway in different cell models in this invention;

[0022] Figure 12 This is a schematic diagram illustrating the effect of gastrodin on the P38 signaling pathway in different cell models in this invention;

[0023] Figure 13 This is a schematic diagram illustrating the effect of gastrodin on the p65 signaling pathway in different cell models in this invention;

[0024] Figure 14 This is a schematic diagram illustrating the changes in the antioxidant capacity of gastrodin in cells during the present invention. Detailed Implementation

[0025] The present invention will now be described in detail with reference to specific embodiments.

[0026] The present invention relates to the application of gastrodin in the preparation of drugs for treating SCA3 disease.

[0027] The application of gastrodin in the preparation of drugs for the treatment of SCA3 disease, with the concentration of gastrodin used in the drugs ranging from 5μM to 100μM.

[0028] Gastrodin exerts a neuroprotective effect in SCA3 disease through the AKT / ERK signaling axis.

[0029] The theoretical basis of this invention is: Figure 1 As shown, mutant ataxin-3 enters the cell nucleus and forms inclusion bodies under the stimulation of various pathogenic factors, thereby affecting intranuclear transcription and other normal physiological functions, leading to apoptosis, and consequently affecting the normal function of the nervous system. Therefore, inhibiting the formation of inclusion bodies from mutant ataxin-3 and apoptosis to protect nerve cells is a promising research direction for drug targets in the treatment of SCA3 disease.

[0030] The detection principle employed in this invention is as follows: the effects of gastrodin on cell viability or cytotoxicity in an SCA3 cell model are detected using both CCK-8 and MTT methods. By constructing an SCA3 cell model, the effect of gastrodin on inclusion bodies formed by the pathogenic protein ataxin-3 is analyzed using a combination of microfiltration and Western blot methods. The microfiltration method uses a cellulose acetate membrane; under pressure, the ataxin-3 monomer can pass through this membrane, while inclusion bodies formed by ataxin-3 remain on the membrane. The number of ataxin-3 inclusion bodies is detected using an ataxin-3 antibody via Western blot. Furthermore, the effects of gastrodin on SDS-soluble proteins (soluble ataxin-3 protein levels, generally referring to monomers) and SDS-insoluble proteins (protein aggregates) are verified by cleaving proteins of different sizes with SDS / formic acid. The target proteins of gastrodin are primarily studied using existing literature and Western blot methods.

[0031] This application mainly describes the neuroprotective effect of gastrodin in SCA3 disease from several aspects, and the specific implementation process is as follows:

[0032] The DMSO used in the following examples is dimethyl sulfoxide.

[0033] 1. Constructing an SCA3 cell model

[0034] HEK293T cells are commonly used and widely applied as human cells in SCA3 disease research, and they have high transfection efficiency. Therefore, this cell line was used for subsequent experiments. HEK293T cells were transiently transfected with wild-type and mutant ataxin-3 plasmids (ataxin-3-15Q wild-type, ataxin-3-77Q moderately mutant, and ataxin-3-148Q severely mutant) to construct SCA3 cell models with different mutation types (HEK293-ataxin-3 cell models). Human embryonic kidney cells containing endogenous ataxin-3 were designated as group A, wild-type ataxin-3-15Q transfected human embryonic kidney cells as group B, moderately mutant ataxin-3-77Q transfected human embryonic kidney cells as group C, and moderately mutant ataxin-3-148Q transfected human embryonic kidney cells as group D. The different plasmids were transfected into cells. After 48 hours, the cells expressed ataxin-3 protein containing different amounts of polyglutamine. Cells were collected and lysed, and the protein was extracted for Western blot analysis. The antibody used to detect ataxin-3 protein was 1H9. Figure 2As shown in the figure. The results of this embodiment indicate that overexpression of ataxin-3 protein containing different polyglutamines was detected in cell models with different mutation types, and the SCA3 cell model was successfully constructed.

[0035] The different SCA3 cell models constructed above are summarized in Table 1:

[0036] Table 1

[0037]

[0038] 2. Two methods were used to demonstrate the effect of gastrodin on the activity of SCA3 cells.

[0039] This embodiment uses both CCK-8 and MTT assays to detect the effects of gastrodin on the activity and cytotoxicity of SCA3 cells. Figure 3 Cell viability was assessed using the easy-to-use CCK-8 assay. Group C cells were seeded into 6-well plates (cell count up to 5 × 10⁶). 5 Cells were transfected (cells / well), and 24 hours later, the cells were transferred to 96-well plates (cell count increased to 1×10⁶ cells / well). 5 Cells / well were collected and treated with gastrodin at concentrations of 5 μM, 10 μM, 20 μM, 50 μM, and 100 μM after 2 hours. CCK-8 solution was added after 2 hours of treatment, and the effects of gastrodin on ataxin-3 overexpression were measured at 2, 4, 6, 24, and 48 hours after 10 minutes of treatment. 77CAG Effects of HEK293T cell viability Figure 3 (Middle AE). CCK-8 results showed that concentrations of gastrodin ranging from 5 μM to 100 μM had an effect on the expression of ataxin-3. 77CAG The activity of HEK293T cells was not affected, but the activity was enhanced at a concentration of 100 μM after 4 hours of treatment.

[0040] The MTT assay was used to determine cytotoxicity. Group C cells were seeded into 6-well plates (cell count up to 5 × 10⁶). 5 Cells were transfected (cells / well), and 24 hours later, the cells were transferred to 96-well plates (cell count increased to 1×10⁶ cells / well). 5 Cells / well were collected and treated with gastrodin at concentrations of 5 μM, 10 μM, 20 μM, 50 μM, and 100 μM after 24 hours. MTT solution was added after 4 hours of treatment, followed by centrifugation. The effects of different concentrations of gastrodin on ataxin-3 expression at 4 hours were then analyzed. 77CAG The study investigated the effects of 5-100 μM gastrodin on the viability of HEK293T cells during a 4-hour treatment period. 77CAGHEK293T cells did not show cytotoxicity. Furthermore, a concentration of 5 μM gastrodin showed a trend towards increasing cell viability after 4 hours of treatment. Figure 3 (F). All results were performed in triplicate, independent experiments. Statistical analysis was conducted using the standard error of the mean and the t-test. P < 0.05 was considered statistically significant (*), P < 0.01 was considered statistically significant (**), and P < 0.001 was considered extremely significant (***).

[0041] The above results indicate that gastrodin at concentrations of 5-100 μM is very safe for different SCA3 cell models, and at certain concentrations and durations of action, it can enhance cell activity.

[0042] 3. The effect of gastrodin on the inclusion bodies formed by the pathogenic protein ataxin-3.

[0043] Seed group D cells into six-well plates (cell count up to 5 × 10⁶). 5 After 72 hours, the cell model was divided into a drug treatment group and a negative control group. The drug treatment group was treated with a final concentration of 50 μM gastrodin, and the negative control group was treated with the same volume of DMSO. After 24 hours of drug treatment, the cells were collected, resuspended in PBS (containing protease inhibitors), and the cell samples were sonicated twice for 30 seconds each time. SDS with a final concentration of 2% was added, and the samples were heated at 95°C for 5 minutes to completely denature the proteins. Cellulose acetate membranes were pretreated by immersion in 1xTBST (containing 2% SDS to a final concentration). A microfiltration device was installed, and 100 μg of protein sample was added to the micropores. The filter was opened, and the membrane was washed three times with PBS until all sample was adsorbed onto the membrane. The microfiltration device was then closed, and the membrane was removed and incubated overnight in blocking buffer at 4°C. Incubation with primary antibody, secondary antibody, and ECL color development was then performed. The effect of gastrodin on inclusion bodies formed by the pathogenic protein ataxin-3 was detected using a filter trap assay and Western blot. The antibody used to detect ataxin-3 protein was 1H9. N=3 (three independent replicate experiments). Statistical analysis was performed using a t-test. P<0.05 was considered statistically significant (*), P<0.01 was considered statistically significant (**), and P<0.001 was considered extremely significant (***). Results are as follows: Figure 4 As shown in Figure A, gastrodin significantly inhibited the formation of "toxic" inclusion bodies at a concentration of 50 μM.

[0044] Furthermore, the SDS / formic acid cleavage method was used for validation. Treatment with Triton X-100, SDS, and formic acid cleaved proteins of different sizes, thus examining the effect of gastrodin on SDS-soluble proteins (soluble ataxin-3 protein levels, generally referring to monomers) and SDS-insoluble proteins (protein aggregates). The results showed that gastrodin increased the levels of ataxin-3 monomers and small protein aggregates, but reduced the number of large protein aggregates. Figure 4 (B) The results are consistent with those obtained by the previous method.

[0045] 4. Effect of gastrodin on the level of soluble ataxin-3, a pathogenic protein.

[0046] In this embodiment, two effective concentrations of gastrodin, 10 μM and 50 μM, were used for treatment for 2 hours to detect the effect of gastrodin on the level of soluble ataxin-3, a pathogenic protein. Cells from groups A, B, and C were seeded (cell count up to 5 × 10⁶). 5 Cells per well were transfected into six-well plates (Table 2). Forty-eight hours after transfection, the drug-treated groups were treated with 10 μM and 50 μM gastrodin, while the negative control group received the same volume of DMSO. The treatment time was 2 hours. Cell lysate proteins were collected, and the effect of gastrodin on the expression level of soluble ataxin-3 protein was detected using Western blot. Results are shown below. Figure 5 As shown.

[0047] Table 2

[0048]

[0049] like Figure 5 As shown, gastrodin treatment of a cell model (HEK293T) containing endogenous ataxin-3 for 2 hours had no significant effect on the soluble protein level of ataxin-3. However, at concentrations of 10 μM and 100 μM, it significantly affected the cell model (HEK293T-Ataxin-3) overexpressing mutant ataxin-3. 77CAG ), and the effect of a concentration of 100 μM on the overexpression of wild-type ataxin-3 (HEK293T-Ataxin-3). 15CAG After 2 hours of treatment in the cell model, gastrodin significantly increased the level of soluble ataxin-3 protein, indicating that gastrodin restored the level of soluble ataxin-3 protein by degrading ataxin-3 inclusion bodies (the monomeric toxicity of this protein is less than that of the inclusion bodies).

[0050] The antibody used in the Western blot experiment to detect ataxin-3 protein was 1H9. Actin was used as the internal control protein. Statistical analysis was performed using the standard error of the mean and t-tests. P < 0.05 was considered statistically significant (*), P < 0.01 was considered statistically significant (**), and P < 0.001 was considered extremely significant (***).

[0051] 7. Changes in the AKT signaling pathway in different cell models.

[0052] Next, this invention investigates the molecular mechanism by which gastrodin exerts its neuroprotective effect through the following signaling pathways. Protein kinase B (PKB), also known as Akt, plays an important role in cell apoptosis and survival. Studies have shown that gastrodin may regulate the AKT signaling pathway. Cells from groups A, B, and C were seeded (cell number up to 5 × 10⁶). 5 Cells (10 cells / well) were transferred to six-well plates. Forty-eight hours after transfection, without any drugs or DMSO treatment, cell lysates were collected. Western blot was used to detect the effect of different ataxin-3 protein expression levels on phosphorylated AKT and AKT proteins. Results are as follows: Figure 6 As shown in the figure. Western blot results indicated that, compared to endogenous ataxin-3, phosphorylated AKT (activated AKT at phosphorylation site S473) was downregulated in cells overexpressing wild-type ataxin-3, while total AKT protein was upregulated. Conversely, in cells overexpressing mutant ataxin-3, phosphorylated AKT (activated AKT at phosphorylation site S473) was also downregulated, but total AKT protein was downregulated relative to wild-type ataxin-3. This suggests that ataxin-3 can inhibit AKT pathway activation, and that mutant ataxin-3 can downregulate AKT expression.

[0053] 8. Changes in the ERK signaling pathway in different cell models.

[0054] Extracellular regulated protein kinases (including ERK1 and ERK2) are normally located in the cytoplasm, but upon activation, they enter the nucleus and play roles in various physiological functions such as cell growth and development. Studies have shown that gastrodin may regulate the ERK signaling pathway. Cells in groups A, B, and C (cell count up to 5 × 10⁶) were seeded. 5Cells (10 cells / well) were transferred to six-well plates. Forty-eight hours after transfection, without any drugs or DMSO treatment, cell lysates were collected. Western blot analysis was used to detect the effects of different ataxin-3 protein expression levels on phosphorylated ERK1 / 2 and ERK1 / 2 proteins. Results are as follows: Figure 7 As shown in the figure. Western blot results indicated that, relative to endogenous ataxin-3, the total ERK1 / 2 protein level remained unchanged in cells overexpressing wild-type or mutant ataxin-3. However, in cells overexpressing mutant ataxin-3, phosphorylated ERK1 / 2, i.e., activated ERK1 / 2 (phosphorylation sites Thr202 / Tyr204), was downregulated. This suggests that mutant ataxin-3 can inhibit ERK1 / 2 protein activation, or that cells protect themselves by regulating ERK1 / 2 protein activation.

[0055] 9. Changes in the P38 signaling pathway in different cell models.

[0056] Mitogen-activated protein kinase (p38MAPK, p38) plays a role in apoptosis, cellular stress, and other functions. Studies have shown that gastrodin may regulate the p38 signaling pathway. Cells in groups A, B, and C were seeded (cell count up to 5 × 10⁶). 5 Cells (10 cells / well) were transferred to six-well plates. Forty-eight hours after transfection, without any drugs or DMSO treatment, cell lysates were collected. Western blot was used to detect the effect of different ataxin-3 protein expression levels on phosphorylated P38 and P38 proteins. Results are as follows: Figure 8 As shown in the figure. Western blot results showed that, relative to endogenous ataxin-3, the total P38 protein did not change in cells overexpressing wild-type or mutant ataxin-3, but in cells overexpressing mutant ataxin-3, phosphorylated P38, i.e., activated P38 (phosphorylation sites Thr180 / Tyr182), showed a decreasing trend.

[0057] 10. Changes in the P65 signaling pathway in different cell models.

[0058] P65, also known as RelA, is a component of the nuclear factor kappa-B (NF-κB) transcription factor family. Cells from groups A, B, and C were seeded (cell count up to 5 × 10⁻⁶). 5 Cells (10 cells / well) were transferred to six-well plates. Forty-eight hours after transfection, without any drugs or DMSO treatment, cell lysates were collected. Western blot analysis was used to detect the effect of different ataxin-3 protein expression levels on P65 protein. Results are as follows: Figure 9As shown in the figure. Western blot results indicated that, relative to endogenous ataxin-3, P65 protein levels remained unchanged in cells overexpressing wild-type ataxin-3, but were downregulated in cells overexpressing mutant ataxin-3. The experiments suggest that P65 protein levels decrease under pathogenic conditions, and mutant ataxin-3 may have an inhibitory effect on it.

[0059] 11. Effects of gastrodin on the AKT signaling pathway in different cell models.

[0060] Cells from groups A, B, and C were seeded (cell count up to 5 × 10⁻⁶). 5 Cells (10 cells / well) were transferred to six-well plates. Forty-eight hours after transfection, the drug-treated group received 50 μM gastrodin, while the negative control group received the same volume of DMSO. The treatment time was 2 hours. Cell lysates were collected, and Western blot was used to detect the effect of gastrodin on phosphorylated AKT and AKT protein. Results are as follows: Figure 10 As shown in the figure. Western blot results showed that, in the three cell models, gastrodin had no effect on AKT phosphorylation levels relative to the DMSO treatment group, but it could increase AKT protein levels in cells overexpressing mutant ataxin-3, suggesting that gastrodin may play a beneficial role in the SCA3 cell model by restoring AKT protein levels.

[0061] 12. Effects of gastrodin on the ERK signaling pathway in different cell models.

[0062] Cells from groups A, B, and C were seeded (cell count up to 5 × 10⁻⁶). 5 Cells (10 cells / well) were transferred to six-well plates. Forty-eight hours after transfection, the drug-treated group received 50 μM gastrodin, while the negative control group received the same volume of DMSO. The treatment time was 2 hours. Cell lysates were collected, and Western blot was used to detect the effect of gastrodin on phosphorylated ERK1 / 2 and ERK1 / 2 proteins. Results are as follows: Figure 11 As shown in the figure. Western blot results showed that, in the three cell models, compared with the DMSO treatment group, gastrodin could downregulate the phosphorylation level of ERK1 / 2 in HEK293T cells, and could also downregulate the ERK1 / 2 protein level in the three cell models.

[0063] 13. Effects of gastrodin on the P38 signaling pathway in different cell models.

[0064] Cells from groups A, B, and C were seeded (cell count up to 5 × 10⁻⁶). 5Cells (1 cell / well) were transferred to six-well plates. Forty-eight hours after transfection, the drug-treated group received 50 μM gastrodin, while the negative control group received the same volume of DMSO. The treatment time was 2 hours. Cell lysates were collected, and Western blot was used to detect the effects of gastrodin on phosphorylated P38 and P38 proteins in the three cell models. Results are as follows: Figure 12 As shown in the figure. Western blot results indicated that, compared to the DMSO control group, the total P38 protein level remained unchanged in cells overexpressing wild-type or mutant ataxin-3. However, in HEK293T cells, gastrodin tended to downregulate phosphorylated P38, i.e., activated P38 (phosphorylation sites Thr180 / Tyr182). Moreover, gastrodin downregulated P38 protein levels in HEK293T cells and cells overexpressing wild-type ataxin-3, indicating that gastrodin plays a role in the P38 pathway, but its effect on the SCA3 cell model overexpressing mutant ataxin-3 was minimal.

[0065] 14. Effects of gastrodin on the P65 signaling pathway in different cell models.

[0066] Cells from groups A, B, and C were seeded (cell count up to 5 × 10⁻⁶). 5 Cells (1 cell / well) were transferred to six-well plates. Forty-eight hours after transfection, the drug-treated group received 50 μM gastrodin, while the negative control group received the same volume of DMSO. The treatment time was 2 hours. Cell lysate proteins were collected, and the effect of gastrodin on P65 protein in the three cell models was detected using Western blot. Results are shown below. Figure 13 As shown in the figure. Western blot results showed that, compared with the DMSO control group, gastrodin could increase the P65 protein level in cells overexpressing wild-type ataxin-3, but had no effect on other cell models, indicating that gastrodin plays a role in the P65 pathway, but has little effect on the SCA3 cell model overexpressing mutant ataxin-3.

[0067] 15. The antioxidant capacity of gastrodin on cells.

[0068] Spread untransfected HEK293T cells (cell count up to 1×10⁻⁶) 6Cells were added to each well of a 6-well plate and allowed to grow for 2-3 days until 80% cell coverage was achieved. The drug treatment groups received 10 μM, 50 μM, and 100 μM gastrodin, while the negative control group received the same volume of DMSO. The blank control group received no treatment. The incubation period was 24 hours, and cells were collected to determine antioxidant capacity. Before use, the ABTS stock solution was diluted 50-fold with PBS to obtain the ABTS working solution. The ABTS stock solution, after being kept at room temperature and protected from light for 14 hours, was diluted 50-fold with PBS (180 μl ABTS stock solution + 820 μl PBS) to obtain the ABTS working solution. 200 μl of ABTS working solution was added to each well of a 96-well plate; 10 μl of distilled water was added to the blank control well; 10 μl of DMSO was added to the negative control well; 10 μl of Trolox standard solutions of various concentrations were added to the standard curve wells; and 10 μl of each group of cell samples was added to the sample wells and mixed well. The plates were incubated at room temperature for 6 minutes, and the A405 value was measured using a microplate reader. The inhibition rate of each group of cell samples was substituted into the standard curve to calculate the corresponding Trolox concentration. Then the total antioxidant capacity of the sample (mmol / g) = the corresponding Trolox concentration (mM) / protein concentration (mg / ml).

[0069] like Figure 14 As shown, gastrodin at a concentration of 10 μM can enhance the antioxidant capacity of cells for 24 hours.

[0070] Through the above-described method, this invention demonstrates the application of gastrodin in the preparation of therapeutic drugs for SCA3 disease. Gastrodin is a compound extracted from the orchid *Gastrodia elata*, and it has therapeutic effects on neurasthenia and headaches. Studies have shown that gastrodin can significantly reduce inclusion bodies induced by mutant ataxin-3, increase soluble ataxin-3 protein levels, has no toxic side effects on HEK293T cells, and regulates the specific molecular mechanism of ataxin-3 inclusion body formation through the AKT / ERK signaling axis. This invention aims to reveal the neuroprotective application of gastrodin in SCA3 disease, enrich the new uses of existing drugs, and provide new ideas for the prevention and treatment of other neurodegenerative diseases.

Claims

1. The application of gastrodin in the preparation of drugs for treating SCA3 diseases, characterized in that, The concentration of gastrodin in the drug is between 5 μM and 100 μM; gastrodin exerts a neuroprotective effect in SCA3 disease through the AKT / ERK signal axis.

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