Application of APEX1 gene in preparation of medicine for preventing or treating Parkinson's disease

Abstract

The invention provides application of an APEX1 gene in preparation of a medicine for preventing or treating Parkinson's disease, and belongs to the technical field of biomedicine.The APEX1 can be combined with Nrf2 to promote expression of a downstream antioxidant element ARE, so that active oxygen accumulation induced by mitochondrial membrane potential change is resisted, and then dopaminergic neuron death is inhibited. The APEX1 gene is applied to preparation of a medicine for preventing and treating the Parkinson's disease, and the Parkinson's disease is expected to be fundamentally improved or treated.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to the application of APEX1 gene in preparing drugs for preventing or treating Parkinson's disease. Background Art

[0002] Parkinson's disease (PD), also often referred to as "tremor paralysis", is currently the second largest neurodegenerative disease in the world and the fastest growing neurological disease, mostly seen in the middle-aged and elderly population after the age of 60. In 1990, the number of Parkinson's patients was about 2.5 million, which more than doubled to 6.2 million by 2015. It is expected that by 2040, due to the aging of the population, this number will double again to 12.9 million, which will bring heavy economic burdens and social development pressures to society. The accumulation of reactive oxygen species in the substantia nigra and lipid peroxide-induced apoptosis of dopaminergic neurons are important pathological mechanisms of PD. At present, the pathogenesis of PD is not fully understood, and there is a lack of effective treatments. Most of the main drugs and targets for the treatment of PD are aimed at improving Parkinson's disease by promoting the secretion of dopamine by dopaminergic neurons, but this strategy is "exhausting the pond to catch fish", which can only relieve the symptoms of Parkinson's disease and produce strong side effects, and cannot fundamentally improve or treat Parkinson's disease.

[0003] In the existing PD treatment program, dopamine replacement therapy (such as levodopa and dopamine receptor agonists) can also be used to alleviate movement symptoms such as bradykinesia, rigidity and resting tremor caused by PD. There are also research reports that promoting the high expression of some proteins or drug-induced protein expression can promote neuronal survival and improve Parkinson's disease. For example, adamantane can promote dopamine release from dopaminergic neurons to improve Parkinson's disease.

[0004] Although various existing drugs can help patients effectively relieve Parkinson's-like symptoms, these drugs usually have many side effects, such as gastrointestinal symptoms, orthostatic hypotension, insomnia and movement disorders, and these drugs can only alleviate Parkinson's-related symptoms but cannot fundamentally treat Parkinson's disease. Summary of the invention

[0005] In order to solve the problem that existing drugs are difficult to fundamentally treat Parkinson's disease, the present invention provides the use of the APEX1 gene in the preparation of drugs for preventing or treating Parkinson's disease. APEX1 can bind to Nrf2 and promote the expression of the downstream antioxidant element ARE, thereby resisting the accumulation of reactive oxygen species induced by changes in mitochondrial membrane potential. It is used to prepare drugs for preventing and treating Parkinson's disease, which is expected to fundamentally improve or treat Parkinson's disease.

[0006] The present invention is achieved through the following technical solutions:

[0007] The present invention provides application of APEX1 gene in preparing medicine for preventing or treating Parkinson's disease.

[0008] Based on the same inventive concept, the present invention provides the use of an APEX1 gene overexpression reagent or an overexpression kit in the preparation of a drug for preventing or treating Parkinson's disease.

[0009] Further, the APEX1 gene overexpression reagent comprises an APEX1 gene overexpression vector;

[0010] Alternatively, the APEX1 gene overexpression kit comprises at least one of an APEX1 gene, an overexpression vector and an APEX1 gene overexpression vector.

[0011] Preferably, the overexpression vector includes a pEnter vector (human gene C-terminus fused with F l ag & 6×His tag, containing a puromycin eukaryotic resistance gene, containing a kanamycin prokaryotic resistance gene);

[0012] The APEX1 gene overexpression vector is prepared by homologous recombination between the pEnter vector and the APEX1 gene.

[0013] Based on the same inventive concept, the present invention provides a drug for preventing or treating Parkinson's disease, wherein the active ingredient of the drug includes an APEX1 gene overexpression agent or an APEX1 gene expression promoter.

[0014] Based on the same inventive concept, the present invention provides the use of APEX1 gene as or in the preparation of Nrf2 activator.

[0015] Based on the same inventive concept, the present invention provides the use of an APEX1 gene overexpression reagent or an overexpression kit in the preparation of an Nrf2 activator.

[0016] Based on the same inventive concept, the present invention provides the use of the APEX1 gene as or in the preparation of an inhibitor of TH, the rate-limiting enzyme in dopamine synthesis.

[0017] Based on the same inventive concept, the present invention provides the use of APEX1 gene in inhibiting changes in mitochondrial membrane potential.

[0018] Based on the same inventive concept, the present invention provides the use of APEX1 gene in resisting mitochondrial dysfunction or inhibiting mitochondrial apoptosis.

[0019] Based on the same inventive concept, the present invention provides the use of APEX1 gene as or in the preparation of ARE gene expression promoter.

[0020] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0021] 1. Use of the APEX1 gene of the present invention in the preparation of drugs for preventing or treating Parkinson's disease. In Parkinson's disease, changes in mitochondrial membrane potential induce the accumulation of reactive oxygen species, thereby leading to apoptosis cascade signals, causing the death of dopaminergic neurons to induce Parkinson's disease. The inventors have found through testing that the APEX1 protein can bind to Nrf2 and promote the expression of the downstream antioxidant element ARE, thereby resisting the accumulation of reactive oxygen species induced by changes in mitochondrial membrane potential and inhibiting the death of dopaminergic neurons. From the perspective of antioxidant activity, the present invention applies APEX1 to the preparation of drugs for preventing and treating Parkinson's disease, which is expected to fundamentally improve or treat Parkinson's disease.

[0022] 2. Use of the APEX1 gene of the present invention in the preparation of drugs for preventing or treating Parkinson's disease. The inventors have found that APEX1 has a positive regulatory effect on Nrf2, and inhibits mitochondrial apoptosis by regulating Nrf2 to activate downstream antioxidant elements, thereby protecting dopaminergic neurons from Parkinson's disease. The present invention promotes the expression of APEX1 protein and then promotes the expression of Nrf2 downstream antioxidant gene ARE, thereby resisting mitochondrial apoptosis induced by intracellular oxidative stress accumulation, improving the survival of dopaminergic neurons, and then improving Parkinson's disease and the quality of life of patients. This plays a very important role in improving the symptoms of Parkinson's patients and alleviating socioeconomic pressures. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0024] Figure 1 Schematic diagram of APEX1 binding to Nrf2 to promote the expression of antioxidant element ARE.

[0025] Figure 2 This is a diagram for detecting the binding between APEX1 and Nrf2.

[0026] Figure 3 APEX1 overexpression inhibits MPP + Expression of TH, the rate-limiting enzyme for dopamine synthesis, in the induced cellular Parkinson's disease model: Figure A shows the successful extraction and verification of the human APEX1 high-expression plasmid; Figure B shows the successful expression of the exogenous APEX1 plasmid and the alleviation of the decreased APEX1 expression; Figure C shows that high expression of APEX1 improves the decreased expression of TH in the MPP+-induced cellular PD model.

[0027] Figure 4 APEX1 overexpression inhibits MPP+ Induction of apoptosis in a cellular Parkinson's disease model.

[0028] Figure 5 APEX1 overexpression inhibits MPP + Alterations in mitochondrial membrane potential in a cellular Parkinson's disease model induced by

[0029] Figure 6 This is the plasmid map of the pEnter vector.

[0030] Figure 7 This is the APEX1 high expression plasmid map. DETAILED DESCRIPTION

[0031] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0032] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0033] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0034] The overall idea of ​​the present invention is as follows:

[0035] At present, the main drugs and targets for the treatment of PD are to improve Parkinson's disease by promoting the secretion of dopamine by dopaminergic neurons, but this strategy is "draining the pond to catch fish", which can only relieve the symptoms of Parkinson's disease and produce strong side effects, and cannot fundamentally improve the treatment of Parkinson's disease. Therefore, the inventors believe that Parkinson's disease can only be treated by fundamentally resisting the oxidative stress damage of dopaminergic neurons. In order to improve the current Parkinson's disease induced by mitochondrial dysfunction caused by the accumulation of oxidative stress, the inventors have conducted a lot of exploratory studies and found that APEX1 can regulate the expression of Nrf2 downstream antioxidant genes to resist mitochondrial apoptosis-induced neuronal cell death. Nrf2 activators have neuroprotective effects in PD models, and promoting the transcription of Nrf2 can inhibit mitochondrial dysfunction, suggesting that Nrf2 may be a target for PD treatment to improve mitochondrial function. The inventors have found through research that APEX1 has a positive regulatory effect on Nrf2, that there is a physical interaction between APEX1 and Nrf2, and that APEX1 is crucial for the activation of Nrf2 downstream antioxidant elements. Therefore, the inventors believe that APEX1 inhibits mitochondrial apoptosis by regulating Nrf2 to activate downstream antioxidant elements, thereby protecting dopaminergic neurons from Parkinson's disease.

[0036] APEX1 is a multifunctional protein that plays a very important role in DNA repair. In addition, APEX1 can also regulate the binding affinity of transcription factors to DNA by controlling the redox state of its DNA binding domain, thereby regulating its transcriptional activity. At present, the molecular mechanism by which APEX1 regulates transcription factors to resist oxidative stress is not very clear.

[0037] In Parkinson's disease, changes in mitochondrial membrane potential (PTP) induce the accumulation of reactive oxygen species (ROS), which leads to the apoptotic cascade, causing neuronal death and Parkinson's disease. The interaction between APEX1 and Nrf2 can promote the expression of downstream antioxidant elements (ARE), thereby resisting the accumulation of reactive oxygen species induced by changes in mitochondrial membrane potential and improving Parkinson's disease (such as Figure 1 ). The present invention promotes the expression of APEX1 protein to resist mitochondrial apoptosis induced by oxidative stress accumulation in neuronal cells, thereby improving the survival of dopaminergic neurons.

[0038] From the perspective of anti-oxidation, the present invention promotes the expression of downstream antioxidant genes through Nrf2, thereby inhibiting neuronal cell death, improving Parkinson's disease, and improving the quality of life of patients, and is expected to fundamentally improve or treat Parkinson's disease.

[0039] The research on APEX1 by the present invention can enrich and supplement the neuromolecular biological mechanism of Parkinson's disease, provide a theoretical basis for future drug research on Parkinson's disease, and play a very important role in improving the symptoms of Parkinson's disease patients and alleviating social and economic pressure. With the aggravation of the aging population in my country, the incidence of Parkinson's disease has greatly increased. Therefore, the present invention has a good application prospect and promotes economic and social development.

[0040] The application of the APEX1 gene of the present application in the preparation of drugs for preventing or treating Parkinson's disease will be described in detail below in combination with examples and experimental data.

[0041] Example 1

[0042] In this example, the binding between APEX1 and Nrf2 was detected by immunoprecipitation. Figure 2 shown.

[0043] The specific detection steps are as follows:

[0044] SH-SY5Y cell proteins were extracted using cold RIPA lysis buffer and then centrifuged at 14,000×g for 15 minutes at 4°C. 3μg of APEX1 primary antibody and 20μl of protein A / G agarose were added to the supernatant and incubated at 4°C overnight. The next day, after centrifugation at 14,000×g for 5 minutes, the pellet was washed three times with pre-cooled PBS, and then the beads were boiled in 2× loading buffer (100-mM Tris-HCl [pH 6.8], 4% SDS, 1% bromophenol blue, 20% glycerol, and 2% β-mercaptoethanol). The supernatant was then collected and finally subjected to Western blot analysis.

[0045] like Figure 2 As shown: SH-SY5Y cell protein was extracted, and the Co-IP experiment results showed that APEX1 was successfully captured using the APEX1 primary antibody. At the same time, Nrf2 was also pulled down. The above results indicate that there is an interaction between APEX1 and Nrf2.

[0046] Example 2

[0047] This example tests the effect of high expression of APEX1 on the expression of TH, the rate-limiting enzyme in dopamine synthesis, as follows:

[0048] 1. Model construction

[0049] An in vitro cellular Parkinson's disease model was constructed by stimulating SH-SY5Y cells with the Parkinson's virus substance MPP+4.88mM for 24h.

[0050] 2. APEX1 high expression

[0051] Use APEX1 high expression plasmid (such as Figure 7 ) were transfected into SH-SY5Y cells to overexpress APEX1 in the Parkinson's disease model.

[0052] Specific steps:

[0053] ① Culture cells: SH-SY5Y cells in logarithmic growth phase were taken and cultured at 1×10 6 The cells were seeded at a density of 100 cells / well in a 6-well plate and cultured until the cells expanded to 40%-50% for transfection.

[0054] ②Cell transfection: Replace the culture medium in the six-well plate with serum-free DMEM / F12 culture medium. Prepare two EP tubes, marked as APEX1 and Lipo3000, and then add 250mL of Pt i-MEM culture medium to the two EP tubes respectively. Add APEX1 high expression plasmid and P3000 to one EP tube marked as APEX1, and add liposome Lipo3000 to another EP tube marked as Lipo3000, and mix well. Add the liquid in the EP tube marked as APEX1 to another EP tube marked as Li po3000, and mix gently. Add the transfection solution of APEX1 high expression plasmid encapsulated by Lipo3000 in the EP to the prepared 6-well plate, and culture at 37°C, 5% CO2 for 24h. After 6h, replace the culture medium in the six-well plate with serum culture medium (90% DMEM / F12 + 10% fetal bovine serum). The empty group also performs the above operation for transfection to form a control.

[0055] ③ Adding drugs: Add drugs 24 hours after transfection.

[0056] ④ Protein extraction: Protein can be extracted 24 hours after drug addition, and the expression of APEX1 protein can be detected by Western Blot.

[0057] 3. Western blot was used to detect the effect of high expression of APEX1 on the expression of TH, the rate-limiting enzyme for dopamine synthesis.

[0058] ① Protein extraction: Lyse cells using RIPA buffer, sonicate, and centrifuge at 15,000 rpm for 15 min at 4°C. Collect the supernatant, add 2× loading buffer, and boil in boiling water for 5 min to denature the protein.

[0059] ② BCA concentration determination: Dilute the 5 mg / mL standard to 2.5 mg / mL, 1.25 mg / mL, and 0.625 mg / mL in sequence. According to the number of standards and samples, prepare the BCA working solution by adding 50 volumes of BCA reagent and 1 volume of Cu reagent, and mix thoroughly. Then add 4 microliters of the standard and the sample to be tested to a 96-well plate, and then add 200 microliters of BCA working solution in sequence, and incubate at 37°C for 30 minutes. Use an enzyme reader to measure the OD value at 562nm, and calculate the protein concentration based on the standard curve.

[0060] ③Prepare gel: Install sandwich vertical glass plates and tighten them to prepare separation gel. Pour the separation solution into the gap between the two glass plates, leaving space for the injection of concentrated gel (the length of the teeth of the comb plus 0.5 cm), and carefully inject a layer of water or isopropanol (about 2-3 mm high) on the surface of the gel to prevent air from entering the gel solution and affecting polymerization. After the separation gel is completely polymerized (about 30 minutes), pour out the covering water layer, and then use filter paper to carefully absorb the remaining water. Be careful not to let the filter paper touch the gel surface. Prepare concentrated gel. And directly inject the concentrated gel solution on the polymerized separation gel, and immediately insert a clean comb into the solution. After polymerization, prepare to add samples. The specific formula is as follows:

[0061]

[0062] ④ Loading samples: After the concentrated gel is polymerized, pull out the comb and add samples in the predetermined order.

[0063] ⑤Electrophoresis: Connect the electrophoresis device to the power supply, adjust the voltage to 180V, and perform electrophoresis for 1h.

[0064] ⑥ Transfer: Immerse the glue in the transfer solution, cut the PVDF membrane according to the size of the glue, and activate it by soaking it in formaldehyde for 15 seconds. Assemble the transfer device (1 layer of sponge, 2 layers of filter paper film, 1 layer of sponge pad), place the transfer tank in an ice bath, add the transfer solution, turn on the power supply, adjust the voltage to 100V, and the time is 1h.

[0065] ⑦Immunohybridization color development: Wash the membrane with TPBS for 5 minutes at room temperature with shaking. Place the membrane in 10% skim milk blocking solution and shake at room temperature for 1 hour. Wash with TPBS 3 times (5 minutes each time). Prepare the primary antibody and incubate at 4℃ overnight. Add the secondary antibody of appropriate dilution, incubate at room temperature for 1 hour with slow shaking. Wash with TPBS 3 times (10 minutes each time). Then place the membrane in the chemiluminescence imaging system instrument to observe and save the photographic results.

[0066] 4. Discussion of results

[0067] The results are as follows Figure 3As shown: A human APEX1 high-expression plasmid with a tagged protein was used to transfer the APEX1 high-expression plasmid into SH-SY5Y cells for 24 hours, and then stimulated with MPP+ for 24 hours. Western Blot detection results showed that APEX1 high expression restored the MPP+-induced decrease in APEX1 and TH expression. These results indicate that high expression of APEX1 alleviates PD.

[0068] Example 3

[0069] This example tests the effect of high expression of APEX1 on MPP + The effects of induced apoptosis in the cellular Parkinson's disease model are as follows:

[0070] 1. The steps of constructing an in vitro cell Parkinson's disease model and overexpressing APEX1 are the same as those in Example 1.

[0071] 2. Flow cytometry was used to detect the effect of APEX1 high expression on MPP + Effects of induced apoptosis in a cellular Parkinson's disease model.

[0072] SH-SY5Y cells with good growth status were taken and operated according to the instructions of the reactive oxygen detection kit. The fluorescent probe DCFH-DA was used to measure the cell peroxide level, and the fluorescence signal value change of the strong green fluorescent substance DCF was detected by flow cytometry. After the logarithmic phase SH-SY5Y cells were centrifuged at 3500r / min for 5min in a 4℃ centrifuge, they were washed three times with pre-cooled phosphate buffer, and the cell apoptosis rate was detected using the Annexin V-FI TC / PI cell apoptosis detection kit.

[0073] 3. Results and Discussion

[0074] The results are as follows Figure 4 As shown: In the cell model of PD, MPP+ induced apoptosis of SH-SY5Y cells, and high expression of APEX1 alleviated cell apoptosis in the MPP+-induced cell PD model.

[0075] Example 4

[0076] This example tests the effect of high expression of APEX1 on MPP + Effects on mitochondrial membrane potential in the induced cellular Parkinson's disease model are as follows:

[0077] 1. The steps of constructing an in vitro cell Parkinson's disease model and overexpressing APEX1 are the same as those in Example 1.

[0078] 2. Use mitochondrial membrane potential kit to detect the effect of APEX1 high expression on MPP + Effects of induced mitochondrial membrane potential in a cellular Parkinson's disease model.

[0079] (1) For one well of a six-well plate, remove the culture medium, wash the cells once with PBS or other appropriate solution if necessary according to the specific experiment, and add 1 mL of cell culture medium. The cell culture medium may contain serum and phenol red.

[0080] (2) Add 1 mL of JC-1 staining working solution and mix thoroughly. Incubate in a cell culture incubator at 37°C for 20 minutes.

[0081] (3) During the incubation period, prepare an appropriate amount of JC-1 staining buffer (1×) by adding 4 mL of distilled water to every 1 mL of JC-1 staining buffer (5×) and place it in an ice bath.

[0082] (4) After incubation at 37°C, remove the supernatant and wash twice with JC-1 staining buffer (1×).

[0083] (5) Add 2 mL of cell culture medium, which may contain serum and phenol red.

[0084] (6) Observe under a fluorescence microscope or a laser confocal microscope.

[0085] 3. Results and Discussion

[0086] The results are as follows Figure 5 As shown: In the cell model of PD, MPP+ induced mitochondrial dysfunction in SH-SY5Y cells and caused changes in mitochondrial membrane permeability. High expression of APEX1 alleviated the changes in mitochondrial membrane permeability caused by mitochondrial dysfunction in the cell PD model induced by MPP+, thereby exerting a neuroprotective effect.

[0087] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0088] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0089] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Application of APEX1 gene in the preparation of drugs for preventing or treating Parkinson's disease.

2. Use of an APEX1 gene overexpression reagent or overexpression kit in the preparation of drugs for preventing or treating Parkinson's disease.

3. The use according to claim 2, characterized in that: The APEX1 gene overexpression reagent comprises an APEX1 gene overexpression vector; Alternatively, the APEX1 gene overexpression kit comprises at least one of an APEX1 gene, an overexpression vector and an APEX1 gene overexpression vector.

4. The use according to claim 3, characterized in that: The overexpression vector includes a pEnter vector; The APEX1 gene overexpression vector is prepared by homologous recombination between the pEnter vector and the APEX1 gene.

5. A drug for preventing or treating Parkinson's disease, characterized in that: The active ingredients of the medicine include an APEX1 gene overexpression reagent or an APEX1 gene expression promoter.

6. Use of APEX1 gene as or in the preparation of Nrf2 activator.

7. Application of APEX1 gene overexpression reagent or overexpression kit in the preparation of Nrf2 activator.

8. Use of the APEX1 gene as or in the preparation of an inhibitor of TH, the rate-limiting enzyme in dopamine synthesis.

9. Application of APEX1 gene in inhibiting changes in mitochondrial membrane potential.

10. Application of APEX1 gene in resisting mitochondrial dysfunction or inhibiting mitochondrial apoptosis.

11. Use of the APEX1 gene as or in the preparation of an ARE gene expression promoter.

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