A drug for assisting the prevention and treatment of Alzheimer's disease

Abstract

The present invention provides a drug for assisting the prevention and treatment of Alzheimer's disease, belonging to the technical field of geriatric medicine. The core active ingredient of the drug for assisting the prevention and treatment of Alzheimer's disease provided by the present invention is siRNA of non-coding RNA LINC01735, and the use of the siRNA can effectively inhibit the decrease in nerve cell activity and cell apoptosis caused by β-amyloid protein. Therefore, the siRNA of LINC01735 can be used to prepare drugs for assisting the prevention and treatment of Alzheimer's disease to reduce the nerve damage caused by β-amyloid protein.

Description

Technical Field

[0001] The invention belongs to the technical field of geriatric medicine, and in particular relates to a medicine for assisting the prevention and treatment of Alzheimer's disease. Background Art

[0002] Alzheimer's disease, also known as Alzheimer's disease, is a chronic brain dysfunction disease. The early behavioral changes of Alzheimer's disease are mild memory disorders or personality changes. The patient's symptoms gradually worsen 5-10 days after the onset of the disease. The patient will eventually develop severe dementia symptoms and be unable to take care of himself. This not only affects the patient's quality of life, but also brings a heavy burden to the patient's family and society. With the approach of an aging society, the prevalence and incidence of Alzheimer's disease patients are constantly increasing. Therefore, it is urgent to study the mechanism and preventive and therapeutic drugs of Alzheimer's disease.

[0003] At present, the pathogenesis of Alzheimer's disease is not very clear, but the main recognized pathogenesis is that after the deposition of β-amyloid protein (Aβ) forms senile plaques, it induces neuronal oxidative stress, leading to neuronal cell damage or even death, and then leads to progressive degeneration of cognitive function. Therefore, effectively inhibiting the neuronal cell damage caused by β-amyloid protein will help the treatment of Alzheimer's disease. Summary of the invention

[0004] The purpose of the present invention is to provide a drug for assisting in the prevention and treatment of Alzheimer's disease, which can prevent and treat Alzheimer's disease by reducing nerve cell damage caused by β-amyloid protein.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides a drug for assisting in the prevention and treatment of Alzheimer's disease, wherein the core active ingredient of the drug is siRNA of LINC01735.

[0007] Preferably, the transcription sequence of LINC01735 is as shown in SEQ ID NO.1; the sense strand of the siRNA is as shown in SEQ ID NO.4, and the antisense strand of the siRNA is as shown in SEQ ID NO.5.

[0008] Preferably, the medicament further comprises a pharmaceutically acceptable additive.

[0009] Preferably, the pharmaceutically acceptable additives include carriers, binders, and lubricants.

[0010] In a second aspect, the present invention provides an application of a small interfering RNA in the preparation of a drug for preventing and treating Alzheimer's disease caused by β-amyloid protein, wherein the small interfering RNA is siRNA of LINC01735.

[0011] Preferably, the transcription sequence of LINC01735 is as described in SEQ ID NO.1.

[0012] Preferably, the sense strand of the siRNA is shown as SEQ ID NO.4, and the antisense strand of the siRNA is shown as SEQ ID NO.5.

[0013] In a third aspect, the present invention provides a use of a small interfering RNA in the preparation of a drug for inhibiting neuronal apoptosis caused by β-amyloid protein, wherein the small interfering RNA is siRNA of LINC01735.

[0014] Preferably, the transcription sequence of LINC01735 is as described in SEQ ID NO.1.

[0015] Preferably, the sense strand of the siRNA is shown as SEQ ID NO.4, and the antisense strand of the siRNA is shown as SEQ ID NO.5.

[0016] The beneficial effects of the present invention are:

[0017] The present invention uses β-amyloid protein to treat nerve cells and discovers a non-coding RNA LINC01735 with a significantly increased expression level. After inhibiting LINC01735 in cells by siRNA, the present invention finds that it can significantly inhibit the decrease in cell activity and cell apoptosis caused by β-amyloid protein. Therefore, the siRNA of LINC01735 can be used to prepare drugs for auxiliary prevention and treatment of Alzheimer's disease to reduce nerve damage caused by β-amyloid protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a result diagram showing the changes in LINC01735 expression levels in neural cells caused by β-amyloid protein treatment;

[0019] Figure 2 A result diagram showing the knockdown effect of si-LINC01735 designed for the present invention on LINC01735;

[0020] Figure 3 This is a result diagram showing the role of LINC01735 in the decrease of neuronal cell activity caused by β-amyloid protein;

[0021] Figure 4This is a result diagram showing the role of LINC01735 in neuronal apoptosis caused by β-amyloid protein;

[0022] Figure 5 This figure shows the effect of LINC01735 on the upregulation of pro-apoptotic proteins in neurons caused by β-amyloid protein. DETAILED DESCRIPTION

[0023] The present invention is further described in detail below in conjunction with specific embodiments and drawings. Unless otherwise specified, the drugs, reagents, instruments, equipment, etc. used in the present invention can be purchased through commercial channels, and the molecular biology experimental operations such as PCR amplification involved are conventional experimental operations in the field or are performed in accordance with the product instructions of the corresponding reagents unless otherwise specified.

[0024] Long non-coding RNA is a type of RNA without coding function, which is longer than 200bp. Existing studies have shown that it plays an important role in organisms and regulates gene expression at multiple levels. The present invention studies the function of the non-coding gene LINC01735 in nerve cells and explores its role in nerve cell damage caused by β-amyloid protein.

[0025] Example 1: Detection of the effect of β-amyloid protein on LINC01735 expression in neural cells

[0026] 1. Collect and count the SH-SY5Y neurons in the logarithmic growth phase, and dilute them to 1×10 using DMEM / F-12 complete medium. 5 celIs / mL, the cells were seeded in a 6-well plate and cultured in a cell culture incubator overnight;

[0027] 2. After the cells were completely attached, the culture medium was removed, and complete culture medium was added to the control group, and Aβ25-35 and complete culture medium were added to the Aβ25-35 group. The final concentration of Aβ25-35 was 20 μM, and 3 replicates were set for each treatment group;

[0028] 3. After culturing in a cell culture incubator for 24 hours, remove the culture medium and add TRIZOL reagent to extract RNA;

[0029] 4. After reverse transcription of RNA into cDNA using a reverse transcription kit, PCR and gel electrophoresis were performed to detect LINC01735 expression using primers designed according to the transcription sequence of LINC01735 (as shown in SEQ ID NO.1) (upstream primer as shown in SEQ ID NO.2, downstream primer as shown in SEQ ID NO.3). GAPDH (3-phosphoglyceraldehyde dehydrogenase) was used as the internal reference gene. The results are shown in Figure 1shown.

[0030] from Figure 1 We can see that after treating nerve cells with Aβ25-35, the expression of LINC01735 in the cells was significantly increased compared with the Control group. This result shows that when β-amyloid protein causes neural damage to nerve cells, it will lead to an increase in the expression level of LINC01735 in nerve cells. Therefore, we speculate that inhibiting the expression of LINC01735 can inhibit the neural damage caused by β-amyloid protein.

[0031] To this end, we designed and synthesized siRNA of LINC01735, named si-LINC01735, whose positive chain sequence is shown in SEQ ID NO.4, and its antisense chain sequence is shown in SEQ ID NO.5.

[0032] Example 2: Detection of the inhibitory effect of si-LINC01735

[0033] 1. Collect and count the SH-SY5Y neurons in the logarithmic growth phase, and dilute them to 1×10 using DMEM / F-12 complete medium. 5 celIs / mL, the cells were seeded in a 6-well plate and cultured in a cell culture incubator overnight;

[0034] 2. After the cells are completely attached, remove the culture medium, transfect the si-NC group with si-NC (negative control siRNA), and transfect the si-LINC01735 group with si-LINC01735. After transfection, place the cells in a cell culture incubator for continued culture;

[0035] 3. 48 hours after transfection, remove the cell culture plate, remove the culture medium, and add TRIZOL reagent to extract RNA;

[0036] 4. After reverse transcription of RNA into cDNA using a reverse transcription kit, PCR and gel electrophoresis were performed to detect LINC01735 expression. The results were as follows Figure 2 shown.

[0037] from Figure 2 It can be seen that after transfection of si-LINC01735, the expression of LINC01735 in nerve cells was significantly inhibited, indicating that the si-LINC01735 designed and synthesized in the present invention can significantly inhibit the expression of LINC01735 and can be used in subsequent experiments.

[0038] Example 3: Detecting the role of LINC01735 in the decrease of neuronal cell activity caused by β-amyloid protein

[0039] 1. Collect and count the SH-SY5Y neurons in the logarithmic growth phase, and dilute them to 1×10 using DMEM / F-12 complete medium. 5 celIs / mL, cells were seeded in 96-well cell culture plates;

[0040] 2. After the cells are completely attached, remove the culture medium and treat the cells according to the following groups:

[0041] Control group: transfected with si-NC, and the complete medium was replaced after 24 h;

[0042] si-NC group: after transfection with si-NC, the medium was replaced with complete medium containing Aβ25-35 24 h later, and the final concentration of Aβ25-35 was 20 μM;

[0043] si-LINC01735 group: transfected with si-LINC01735, and 24 hours later, the medium was replaced with complete medium containing Aβ25-35, and the final concentration of Aβ25-35 was 20 μM;

[0044] 3. After treatment, discard the culture medium, add 100 μl of complete culture medium containing 10% CCK-8, place in the cell culture incubator and continue incubation for 2-3 hours, place on the microplate reader to measure OD450 and calculate cell activity based on the control.

[0045] The experimental results are as follows Figure 3 As shown in the figure, it can be seen that culturing neurons in a culture medium containing Aβ25-35 will lead to a decrease in cell activity; when si-LINC01735 is transfected into the cells, the decrease in activity caused by Aβ25-35 is inhibited to a certain extent, which means that the use of si-LINC01735 can effectively inhibit the decrease in neuronal activity caused by β-amyloid protein.

[0046] Example 4: Detection of the role of LINC01735 in neuronal apoptosis caused by β-amyloid protein

[0047] 1. Count the SH-SY5Y cells transfected with si-NC and si-LINC01735 and dilute them to 1×10 using DMEM / F-12 complete medium. 5 celIs / mL, sterilized small round glass slides were placed in 24-well plates, and the cells were inoculated in 24-well cell culture plates and cultured overnight;

[0048] 2. The culture medium was removed, and the complete culture medium of SH-SY5Y cells transfected with si-NC in 3 wells was replaced with new complete culture medium (Control group), and the complete culture medium containing Aβ25-35 was replaced in SH-SY5Y cells transfected with si-NC (si-NC group) and 3 wells transfected with si-LINC01735 (si-LINC01735 group). The final concentration of Aβ25-35 was 20 μM.

[0049] 3. After 24 hours of treatment, wash with PBS three times, 5 minutes each time;

[0050] 4. Add 4% paraformaldehyde to fix at room temperature for 15 minutes, discard the fixative, and wash with PBS three times, 5 minutes each time;

[0051] 5. Add Hoechst 33342 staining solution to the cell slide, incubate at room temperature in the dark for 5 minutes, then aspirate the staining solution, let it rest in PBS for 5 minutes, and seal the slide with an anti-fluorescence quenching sealing agent;

[0052] 6. Observe and take photos under a fluorescence microscope.

[0053] The experimental results are as follows Figure 4 As shown in the figure, it can be seen that after treatment with Aβ25-35, the number of apoptotic cells in the si-NC group increased significantly. When LINC01735 was knocked down in the cells, the number of apoptotic cells in the si-LINC01735 group was significantly less than that in the si-NC group, indicating that knocking down LINC01735 can inhibit the apoptosis of neurons caused by β-amyloid protein.

[0054] Example 5: Detection of the application of LINC01735 in the upregulation of pro-apoptotic proteins in neurons caused by β-amyloid protein

[0055] 1. Collect and count the SH-SY5Y neurons in the logarithmic growth phase, and dilute them to 1×10 using DMEM / F-12 complete medium. 5 celIs / mL, the cells were seeded in a 6-well plate and cultured in a cell culture incubator overnight;

[0056] 2. After the cells are completely attached, remove the culture medium and treat the cells according to the following groups:

[0057] Control group: transfected with si-NC, and the complete medium was replaced after 24 h;

[0058] si-NC group: after transfection with si-NC, the medium was replaced with complete medium containing Aβ25-35 24 h later, and the final concentration of Aβ25-35 was 20 μM;

[0059] si-LINC01735 group: transfected with si-LINC01735, and 24 hours later, the medium was replaced with complete medium containing Aβ25-35, and the final concentration of Aβ25-35 was 20 μM;

[0060] 3. After 24 hours of treatment, the culture medium was removed and protein lysis buffer was added to extract protein samples of each group;

[0061] 4. Prepare 12% protein electrophoresis gel, assemble the electrophoresis rack, add protein samples and protein marker indicators from each group for electrophoresis;

[0062] 5. After the electrophoresis is completed, assemble the electrotransfer clip, add electrotransfer solution and place in an ice box for electrotransfer;

[0063] 6. After electroporation, transfer the PVDF membrane to the prepared blocking solution and block for 1 hour;

[0064] 7. After washing the membrane with TBST, incubate with diluted primary antibody at 4°C overnight;

[0065] 8. After washing the membrane with TBST, incubate with secondary antibody for 1 hour, wash the membrane again, and then develop.

[0066] The experimental results are as follows Figure 5 As shown in the figure, it can be seen that knocking down LINC01735 significantly reduced the increased expression of cleaved-Caspase3 caused by Aβ25-35, indicating that knocking down LINC01735 can effectively regulate the apoptosis caused by Aβ25-35 through the pro-apoptotic protein cleaved-Caspase3.

Claims

1. A drug for assisting the prevention and treatment of Alzheimer's disease, characterized in that: The core active ingredient of the drug is siRNA of LINC01735; The transcription sequence of LINC01735 is as shown in SEQ ID NO.1; the positive strand of the siRNA is as shown in SEQ ID NO.4, and the antisense strand of the siRNA is as shown in SEQ ID NO.

5.

2. The drug according to claim 1, characterized in that The medicament further includes pharmaceutically acceptable additives.

3. Use of a small interfering RNA in the preparation of a drug for preventing and treating Alzheimer's disease caused by β-amyloid protein, characterized in that: The small interfering RNA is siRNA of LINC01735; The transcription sequence of LINC01735 is as described in SEQ ID NO.1; The sense strand of the siRNA is shown in SEQ ID NO.4, and the antisense strand of the siRNA is shown in SEQ ID NO.

5.

4. Use of small interfering RNA in the preparation of a drug for inhibiting neuronal apoptosis caused by β-amyloid protein, characterized in that: The small interfering RNA is siRNA of LINC01735; The transcription sequence of LINC01735 is as described in SEQ ID NO.1; The sense strand of the siRNA is shown in SEQ ID NO.4, and the antisense strand of the siRNA is shown in SEQ ID NO.5.

Images