Application of DYRK1B as therapeutic target in preparation of medicine for treating sarcopenia

Abstract

The invention provides application of DYRK1B as a therapeutic target in preparation of a medicine for treating sarcopenia, and relates to the technical field of biomedicine, bispecific tyrosine phosphorylation regulatory kinase 1B (DYRK1B) is also called cerebellar related kinase (MIRK) and is one of DYRK kinase family members. It is generally expressed at a high level in skeletal muscle and testis. DYRK1B is a key regulatory factor of a signaling pathway for controlling proliferation and differentiation, thereby playing a key role in skeletal muscle differentiation, spermatogenesis and cancer. According to the application disclosed by the invention, the expression level of atrophic factors Atrogin-1 and Murf-1 in DYRK1B and C2C12 myoblasts after serum deprivation is knocked down through the expression change of the DYRK1B in gastrocnemius muscle tissues of a fasting induced mouse amyotrophy model and the expression change of the DYRK1B in the C2C12 myoblasts after serum deprivation by virtue of a specific verification method. The mouse skeletal muscle DYRK1B is overexpressed, and the exercise tolerance of the mouse is measured after fasting induction of skeletal muscle atrophy. The operation is simple and rapid, and the repeatability is good.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to application of DYRK1B as a therapeutic target in the preparation of a drug for treating sarcopenia. Background Art

[0002] Skeletal muscle is one of the most dynamic and plastic tissues in the human body, accounting for approximately 40% of total body weight and containing 50-75% of all body proteins. Skeletal muscle atrophy occurs in a variety of situations, such as disease states (heart failure, cancer, diabetes, sepsis, etc.), denervation damage, fasting, aging, and drug treatment. Skeletal muscle atrophy is mainly caused by excessive breakdown of muscle protein, which is usually accompanied by reduced protein synthesis, manifested as decreased skeletal muscle mass and dysfunction, leading to a decrease in the patient's quality of life and increased morbidity and mortality. Therefore, it is of great significance to find new and effective muscle factors as intervention targets for the treatment of muscle atrophy. Summary of the invention

[0003] The purpose of the present invention is to solve the technical problem of the lack of new and effective muscle factors as intervention targets for the treatment of muscular atrophy in the prior art.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

[0005] Application of dual specificity tyrosine phosphorylation-regulated kinase 1B (DYRK1B) as a therapeutic target in the preparation of drugs for the treatment of sarcopenia.

[0006] Preferably, the purpose of treating sarcopenia is achieved by increasing the expression of DYRK1B and reducing the expression of atrophy factors Atrogin-1 and Murf-1.

[0007] The present application also provides a drug for treating sarcopenia, wherein the drug uses DYRK1B as a target, and the drug is used to increase the expression of DYRK1B.

[0008] The present application also provides a method for detecting DYRK1B as a therapeutic target for sarcopenia, the detection method comprising detecting the expression of DYRK1B in the gastrocnemius muscle tissue of a fasting-induced mouse muscular atrophy model, detecting the expression of DYRK1B in C2C12 myoblasts after serum deprivation, knocking down DYRK1B, detecting the expression changes of Atrogin-1 and Murf-1 in serum-deprived C2C12 myoblasts, and overexpressing DYRK1B in mouse skeletal muscle by adenovirus, and detecting changes in exercise tolerance of mice after fasting-induced skeletal muscle atrophy.

[0009] Preferably, Western Blot detection is used to detect the expression of DYRK1B in the gastrocnemius muscle tissue of the fasting-induced muscular atrophy model in mice.

[0010] Preferably, the real-time fluorescence quantitative PCR detection method is used to detect the expression of DYRK1B in serum-deprived C2C12 myoblasts and the expression changes of Atrogin-1 and Murf-1 in serum-deprived C2C12 myoblasts after knocking down DYRK1B.

[0011] Compared with the prior art, this application has the following beneficial effects:

[0012] 1. The present invention verifies the expression changes of DYRK1B in the gastrocnemius muscle tissue of the fasting-induced mouse muscular atrophy model, the expression changes of DYRK1B in C2C12 myoblasts after serum deprivation, the knockdown of DYRK1B, and the expression levels of atrophy factors Atrogin-1 and Murf-1 in C2C12 myoblasts after serum deprivation through specific verification methods. Overexpression of mouse skeletal muscle DYRK1B, and measurement of mouse exercise tolerance after fasting-induced skeletal muscle atrophy. The operation is simple, fast, and reproducible;

[0013] 2. The present invention proves the application of DYRK1B in sarcopenia through specific verification experiments, which provides strong theoretical support for the clinical diagnosis and treatment of sarcopenia patients and has clinical significance and promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a schematic diagram of the protein expression of DYRK1B in the gastrocnemius muscle tissue of the fasting-induced muscular atrophy mouse model of the present invention;

[0015] Figure 2 This is a schematic diagram of protein quantification of DYRK1B in the gastrocnemius muscle tissue of the fasting-induced muscular atrophy model of mice;

[0016] Figure 3 Schematic diagram of the expression changes of DYRK1B at the mRNA level in serum-deprived C2C12 myoblasts of the present invention;

[0017] Figure 4 The present invention knocks down DYRK1B and changes the expression of Atrogin-1 and Murf-1 mRNA in serum-deprived C2C12 myoblasts;

[0018] Figure 5 This is a schematic diagram of detecting the movement distance of mice after the DYRK1B adenovirus is injected into the gastrocnemius muscle of mice of the present invention;

[0019] Figure 6 It is a schematic diagram of detecting the movement time of mice after the DYRK1B adenovirus is injected into the gastrocnemius muscle of the mice of the present invention. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below in conjunction with specific embodiments.

[0021] Application of dual specificity tyrosine phosphorylation-regulated kinase 1B (DYRK1B) as a therapeutic target in the preparation of drugs for the treatment of sarcopenia.

[0022] The dual specificity tyrosine phosphorylation regulated kinase 1B (DYRK1B), also known as microintestinal related kinase (MIRK), is a member of the DYRK kinase family. It is usually expressed at high levels in skeletal muscle and testis. DYRK1B is a key regulator of signaling pathways that control proliferation and differentiation, thereby playing a key role in skeletal muscle differentiation, spermatogenesis and cancer.

[0023] The present application also provides a drug for treating sarcopenia, which uses DYRK1B as a target and reduces the expression of atrophy factors Atrogin-1 and Murf-1 by increasing the expression of DYRK1B to achieve the purpose of treating sarcopenia.

[0024] Based on the above content, the present application also provides a detection method for DYRK1B as a therapeutic target for sarcopenia, the detection method comprising detecting the expression of DYRK1B in the gastrocnemius muscle tissue of a fasting-induced mouse muscular atrophy model, detecting the expression of DYRK1B in C2C12 myoblasts after serum deprivation, knocking down DYRK1B, detecting the expression changes of Atrogin-1 and Murf-1 in C2C12 myoblasts after serum deprivation, and overexpressing mouse skeletal muscle DYRK1B by adenovirus, and detecting changes in exercise tolerance of mice after fasting-induced skeletal muscle atrophy.

[0025] The following is an explanation of the above content in combination with specific verification experiments:

[0026] Example 1: Western Blot detection of DYRK1B expression in gastrocnemius muscle of mouse muscular atrophy model:

[0027] Materials and methods: Total protein was extracted from gastrocnemius muscle tissue of mice fed a normal diet or starved for 48 h (n=6), and 50 μg of total protein was used for Western Blot detection. Western Blot detection method includes total protein extraction, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), membrane transfer, blocking, incubation with primary antibody, incubation with secondary antibody, and development.

[0028] The specific steps are as follows:

[0029] S1: Total protein extraction

[0030] (1) Take 30 mg of gastrocnemius muscle tissue from two groups of mice, put it into a 2 mL EP tube, and store it at low temperature.

[0031] (2) Prepare protein extraction and lysis buffer (RIPA strong, phosphatase inhibitor, protease inhibitor) on ice.

[0032] (3) Add 200 μL of protein lysis buffer to each sample to fully lyse the muscle tissue and place on ice for 30 min.

[0033] (4) Centrifuge the sample for 15 min (12,000 rpm, 4°C), and transfer the supernatant to a new EP tube.

[0034] (5) Protein concentration was measured by BCA method.

[0035] (6) Adjust the protein concentration according to the measured protein concentration, and add water if it is insufficient.

[0036] (7) Add 5X Loading Buffer and place in a metal bath at 95°C for 5 min.

[0037] S2: SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

[0038] (1) Select a suitable electrophoresis tank and precast gel, and prepare the electrophoresis fluid and transfer fluid.

[0039] (2) Take an appropriate volume of sample for loading and take 4 μL of Maker as a control.

[0040] (3) Turn on the power and perform constant voltage electrophoresis at 110 V for 70 min.

[0041] S3: Transfer

[0042] (1) Prepare a PVDF membrane of corresponding size and activate it with formaldehyde for a few seconds.

[0043] (2) Cut the edge of the electrophoresis gel and transfer it to the transfer clip, paying attention to the positive and negative poles.

[0044] (3) Place the transfer tank in an ice bath and apply power at 300 mA for 90 min.

[0045] S4: Closed

[0046] (1) Prepare a blocking solution containing 0.5% skim milk in advance.

[0047] (2) Transfer the transferred PVDF membrane to the blocking solution and block it at room temperature for 120 min on a shaker at 20 rpm.

[0048] S5: Incubation with primary antibody

[0049] (1) The DYRK1B-specific antibody is shown as Antibody NO.1.

[0050] (2) The internal reference GAPDH-specific antibody is shown as Antibody NO.2.

[0051] (3) Dilute according to the antibody instructions and place on a shaker at 20 r / min at 4°C overnight.

[0052] S6: Incubation with secondary antibody

[0053] (1) Wash the membrane on a shaker at 70 r / min, 3 times, 10 min each time.

[0054] (2) Dilute the secondary antibody (Antibody NO.3 and Antibody NO.4) according to the instructions, place on a shaker at 20 r / min, and incubate at room temperature for 90 min.

[0055] S7: Development

[0056] (1) Wash the membrane on a shaker at 70 r / min, 3 times, 10 min each time.

[0057] (2) ECL developer is prepared in a 1:1 ratio and developed.

[0058] (3) Collect images.

[0059] S8: Results Analysis

[0060] According to the internal reference GAPDH expression, the gray value of DYRK1B was calculated, and the results are as follows Figure 1 As shown in 2, the protein content of DYRK1B was significantly reduced in the gastrocnemius muscle of muscular atrophy mice.

[0061] The following table shows the antibodies used in the above examples:

[0062] Antibody NO.1 Anti-DYRK1B Antibody CellSignalingTechnology,#567 Antibody NO.2 Anti-GAPDH Antibody Proteintech,60004-1-Ig Antibody NO.3 Anti-rabbitIgG,HRP-linkedAntibody CellSignalingTechnology,#7074 Antibody NO.4 Anti-mouseIgG,HRP-linkedAntibody CellSignalingTechnology,#7076

[0063] Example 2: PCR detection method to detect the expression of DYRK1B mRNA in C2C12 myoblasts after serum deprivation:

[0064] Materials and Methods

[0065] Total RNA was extracted from normal and serum-deprived C2C12 myoblasts (n=6), and 1 μg of RNA was reverse transcribed into cDNA and then subjected to real-time fluorescence quantitative PCR detection. The DYRK1B forward primer sequence is shown in SEQ ID NO.1, and the reverse primer sequence is shown in SEQ ID NO.2. The internal reference 18S forward primer sequence is shown in SEQ ID NO.3, and the reverse primer sequence is shown in SEQ ID NO.4.

[0066] The specific steps are as follows:

[0067] S1: The real-time fluorescence quantitative PCR reaction system is (the PCR detection method is two independent reactions to detect the expression of DYRK1B and internal reference 18S mRNA respectively, so the following forward primer and reverse primer are the specific primers used in each reaction respectively):

[0068]

[0069]

[0070] S2: Real-time fluorescence quantitative PCR reaction procedure:

[0071] 50℃ 2min 95℃ 10min 95℃ 15s 60℃ 30s 72℃ 30s 95℃ 15s 60℃ 1min 95℃ 15s

[0072] S3: After the reaction is completed, the amplification curve and melting curve of real-time fluorescence quantitative PCR are confirmed, and the relative quantitative value of DYRK1B is calculated according to the CT value using the internal reference gene 18S for standardization, and the difference in DYRK1B expression in C2C12 myoblasts after serum deprivation is calculated.

[0073] The results are as follows Figure 3 As shown, DYRK1B mRNA expression was decreased in C2C12 myoblasts after serum deprivation.

[0074] Example 3: Knockdown of DYRK1B, and detection of the expression levels of Atrogin-1 and Murf-1 in serum-deprived C2C12 myoblasts using PCR detection method:

[0075] Materials and methods: After si-DYRK1B or si-NC was administered for 24 hours, HBSS was used for 6 hours. Total RNA was then extracted from C2C12 cells, and 1 μg of RNA was reverse transcribed into cDNA and then subjected to real-time fluorescence quantitative PCR detection. The DYRK1B small interfering sequence is shown in SEQ ID NO.5. The Atrogin-1 forward primer sequence is shown in SEQ ID NO.6, and the reverse primer sequence is shown in SEQ ID NO.7. The Murf-1 forward primer sequence is shown in SEQ ID NO.8, and the reverse primer sequence is shown in SEQ ID NO.9. The internal reference 18s primer sequence is as shown above. The specific steps of the PCR detection method are shown in Example 2.

[0076] Please refer to the results Figure 4 , knocking down DYRK1B, the mRNA expression of atrophy factors Atrogin-1 and Murf-1 in C2C12 myoblasts was further reduced after serum deprivation.

[0077] Example 4: Overexpression of DYRK1B in skeletal muscle to detect exercise tolerance in a fasting-induced muscular atrophy mouse model:

[0078] Materials and methods: The DYRK1B adenovirus was injected orthotopically into the gastrocnemius muscle of mice. The mice were all C57B / 6J, with 6 mice in each group.

[0079] S1: According to the instructions for use of adenovirus, DYRK1B adenovirus was injected orally into the gastrocnemius muscle of mice, 10 μL / point, and 3-5 points were injected into the gastrocnemius muscle on each side. The adenovirus effect reached its peak 3-5 days after injection, and the mice were fasted for 48 hours before exercise tolerance test. The sequence of DYRK1B adenovirus is shown in SEQ ID NO.10.

[0080] S2: The mouse exercise tolerance was measured using a mouse treadmill, including exercise distance and exercise time. The mouse treadmill running speed started at 8m / min and increased by 2m / min every 4min. The inclination angle of the track started from 0° and increased by 1° every 4min, and was maintained at 14° due to the limitation of the treadmill. Before the exercise tolerance assessment, the mice were subjected to adaptive training, with the treadmill running speed of 8m / min, the inclination angle of the track of 0°, and the exercise time of 2min. Using a unified standard, the running distance and time of the mice were recorded when the mice were exhausted.

[0081] S3: Statistical analysis of the data obtained, the results are as follows Figure 5 , 6 As shown in Figure 2, the running distance and running time of mice in the DYRK1B adenovirus overexpression group were significantly greater than those of mice in the control group.

[0082] In combination with the above embodiments, the present application detected that the expression of DYRK1B was reduced in the mouse muscle atrophy model by Western Blot detection, suggesting that DYRK1B is meaningful in the occurrence and development of sarcopenia. Serum deprivation of C2C12 myoblasts found that the expression of DYRK1B mRNA was reduced; further studies found that knocking down DYRK1B further increased the expression of Atrogin-1 and Murf-1 atrophy factors in C2C12 myoblasts after serum deprivation. Further in-depth studies found that adenovirus overexpressed DYRK1B in mouse skeletal muscle, and the exercise tolerance of mice increased after fasting-induced skeletal muscle atrophy. It shows that DYRK1B plays a positive role in regulating skeletal muscle atrophy. This application proposes DYRK1B as a new therapeutic target for sarcopenia, providing a strong basis for the prediction and diagnosis of clinical sarcopenia patients, as well as providing a reference value for treatment.

Claims

1. Application of dual-specificity tyrosine phosphorylation-regulated kinase 1B (DYRK1B) as a therapeutic target in the preparation of drugs for the treatment of sarcopenia.

2. The use of DYRK1B as a therapeutic target in the preparation of a drug for treating sarcopenia according to claim 1, characterized in that: The purpose of treating sarcopenia is achieved by increasing the expression of DYRK1B and reducing the expression of atrophy factors Atrogin-1 and Murf-1.

3. A drug for treating sarcopenia, characterized in that: The drug takes DYRK1B as a target, and is used for increasing the expression of DYRK1B.

4. A method for detecting DYRK1B as a therapeutic target for sarcopenia, characterized in that: The detection method includes detecting the expression of DYRK1B in the gastrocnemius muscle tissue of a fasting-induced mouse muscular atrophy model, detecting the expression of DYRK1B in C2C12 myoblasts after serum deprivation, knocking down DYRK1B, detecting the expression changes of Atrogin-1 and Murf-1 in serum-deprived C2C12 myoblasts, and overexpressing mouse skeletal muscle DYRK1B through adenovirus, and detecting the changes in the exercise tolerance of mice after fasting-induced skeletal muscle atrophy.

5. A method for detecting DYRK1B as a therapeutic target for sarcopenia according to claim 4, characterized in that: Western Blot was used to detect the expression of DYRK1B in the gastrocnemius muscle tissue of the fasting-induced muscular atrophy mouse model.

6. A method for detecting DYRK1B as a therapeutic target for sarcopenia according to claim 4, characterized in that: Real-time fluorescence quantitative PCR was used to detect the expression of DYRK1B in serum-deprived C2C12 myoblasts and the expression changes of Atrogin-1 and Murf-1 in serum-deprived C2C12 myoblasts after knockdown of DYRK1B.

Images