Application of OH-CATH30 in the preparation of drugs for treating skeletal muscle atrophy

Abstract

The present invention relates to the application of OH‑CATH30 in the preparation of drugs for treating skeletal muscle atrophy, and belongs to the field of biomedicine technology. The OH‑CATH30 is a king cobra antimicrobial peptide with a molecular weight of 4693.71Da and an isoelectric point of 12.08. The skeletal muscle atrophy is skeletal muscle atrophy caused by cachexia. The present invention first discovered that OH‑CATH30, as a small molecule antimicrobial peptide prepared from king cobra, can improve skeletal muscle atrophy. Experiments have confirmed that OH‑CATH30 can effectively increase the expression of myosin MyHC in C2C12 myotubes induced by 4T1 cell culture supernatant, and increase the diameter of myotubes. In addition, in a cachexia mouse model, exogenous injection of OH‑CATH30 can increase the mouse's grip strength and significantly improve skeletal muscle atrophy caused by cachexia.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of OH-CATH30 in preparing a drug for treating skeletal muscle atrophy. Background Art

[0002] Skeletal muscle is an important tissue in the body, accounting for a large proportion. These muscles are mainly composed of myofiber cells, which are arranged together to form myofibrils and are wrapped by myofiber membranes. Myofiber is the basic unit of skeletal muscle and also the smallest functional unit.

[0003] Skeletal muscle atrophy is common in cachectic patients, elderly patients and patients with muscular dystrophy. Among them, cachexia in cachectic patients is induced by malignant tumors, chronic heart failure, chronic obstructive pulmonary disease, chronic kidney disease, rheumatoid arthritis and other diseases. Skeletal muscle atrophy is manifested as increased protein catabolism and insufficient anabolism, leading to severe weight loss and muscle loss. The occurrence of skeletal muscle atrophy not only reduces the quality of life of patients and shortens their survival, but also seriously affects the implementation of the treatment plan for the patient's underlying disease, reduces the sensitivity of drug treatment, and increases the occurrence of complications.

[0004] OH-CATH30 is a new antimicrobial peptide extracted from king cobra. It has strong, broad-spectrum, salt-independent antimicrobial activity and no obvious hemolytic activity, making it a new systemic antimicrobial agent. At present, literature has reported that OH-CATH30 has an antibacterial effect on human pathogens such as Escherichia coli and Staphylococcus aureus.

[0005] So far, no reports have been found on the application of OH-CATH30 in the treatment of skeletal muscle atrophy. Summary of the invention

[0006] The purpose of the present invention is to solve the deficiencies of the prior art and provide the use of OH-CATH30 in the preparation of a drug for treating skeletal muscle atrophy.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] Application of OH-CATH30 in the preparation of drugs for treating skeletal muscle atrophy.

[0009] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0010] Preferably, the medicine is in the form of tablets, capsules, granules, powders, pills or injections.

[0011] Preferably, the skeletal muscle atrophy is cachexia-induced skeletal muscle atrophy.

[0012] The invention also provides a skeletal muscle atrophy drug, wherein the active ingredient includes OH-CATH30.

[0013] The king cobra antimicrobial peptide OH-CATH30 used in the present invention is composed of 34 amino acids; its molecular weight is 4693.71Da and its isoelectric point is 12.08.

[0014] The present invention detects the expression of myosin MyHC in myotubes differentiated from mouse myoblasts C2C12 for 6 days by Western-Blot, and finds that the expression level of MyHC in myotubes induced by 4T1 cell supernatant after OH-CATH30 treatment is significantly higher than that in myotubes induced by 4T1 cell supernatant without OH-CATH30 treatment, and the difference is statistically significant; in addition, the diameter of myotubes is detected by hematoxylin-eosin staining, and it is found that the diameter of myotubes treated with OH-CATH30 is significantly higher than that of myotubes not treated with OH-CATH30.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention first discovered that OH-CATH30, a small molecule antimicrobial peptide prepared from king cobra, can improve skeletal muscle atrophy. Experiments have confirmed that OH-CATH30 can effectively increase the expression of myosin MyHC in C2C12 myotubes induced by 4T1 cell culture supernatant and increase the diameter of myotubes. In addition, in a cachexia mouse model, exogenous injection of OH-CATH30 can increase the mouse's grip and significantly improve skeletal muscle atrophy caused by cachexia. The present invention first reveals the potential application of OH-CATH30 in improving skeletal muscle atrophy caused by cachexia and other conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a flow chart of the grouping test processing for Western-Blot detection;

[0018] Figure 2 The protein expression of MyHC was determined by Western-Blot;

[0019] Figure 3 The figure shows the myotube diameter measured by hematoxylin-eosin staining, where the scale bar is 100 μm;

[0020] Figure 4 Flow chart of group experiment processing for the study of skeletal muscle atrophy in mice;

[0021] Figure 5 The figure is the result of monitoring the weight of mice; a is the percentage of the weight of mice in each group to the initial weight; b is the cumulative food intake of mice in each group;

[0022] Figure 6 This is the result of the mouse grasping function test;

[0023] Figure 7 The results of hematoxylin-eosin staining are shown in Figure 1. a is a representative picture of hematoxylin-eosin staining of the gastrocnemius muscle of each group of mice; b is a statistical diagram of the cross-sectional area of ​​the gastrocnemius muscle fibers; the scale bar is 20 μm, **P<0.01, ***P<0.001, ****P<0.0001. DETAILED DESCRIPTION

[0024] The present invention is further described in detail below in conjunction with embodiments.

[0025] Those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the art or the product specifications are used. If the manufacturer of the materials or equipment used is not specified, they are all conventional products that can be purchased.

[0026] In this specific embodiment, the manufacturer and model of FBS is Viva Cell 2910-0500; the manufacturer and model of DMEM high-glucose culture medium is Viva Cell C3113-0500; the manufacturer and model of horse serum is Biosimilars 6281S03.

[0027] The DMEM high-glucose medium containing 10% FBS is prepared by adding 5 mL of FBS to 45 mL of DMEM high-glucose medium; the differentiation medium containing 2% horse serum is prepared by adding 1 mL of horse serum to 49 mL of DMEM high-glucose medium.

[0028] Example 1 Study on the expression level of myosin heavy chain MYHC in myotubes differentiated from mouse myoblasts C2C12 for 6 days induced by the supernatant of mouse breast cancer cells 4T1 cells

[0029] Western-Blot was used to detect the expression of myosin MYHC in myotubes differentiated from mouse myoblasts C2C12 for 6 days induced by 4T1 cell supernatant.

[0030] 1. Experimental cells and culture methods:

[0031] Mouse myoblasts (C2C12) were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences. Mouse myoblasts were washed twice with 37°C 1× PBS buffer; 0.25% trypsin (Biological Industry) was added for digestion for 2 min, and DMEM high-glucose medium (Viva Cell C3113-0500) containing 10% FBS (Viva Cell 2910-0500) was added to terminate the digestion. The medium was aspirated with a pipette and the cells were repeatedly blown to obtain a cell suspension, and all the liquid was collected in a 15 mL centrifuge tube. The 15 mL centrifuge tube was centrifuged at 900 rpm for 5 min at room temperature, and the supernatant was discarded and placed in a new culture dish; then 10 mL of DMEM high-glucose medium containing 10% FBS was added to the new culture dish, and the cell density reached 70%-80% and the cells were passaged again.

[0032] 2. Experimental methods:

[0033] 2.1 Method for obtaining 4T1 cell supernatant

[0034] 4T1 cells were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences. 4T1 cells were cultured in a DMEM high-glucose medium containing 10% FBS by volume at 37°C in an incubator with 5% CO2 by volume. When the cell density reached 70%-80%, the medium was replaced with serum-free DMEM medium. After 24 hours of culture, the cell culture medium was carefully transferred from the culture dish to a centrifuge tube using a pipette. The centrifuge tube was centrifuged at 1000 rpm and room temperature for 10 minutes to remove the cell debris and obtain the 4T1 cell supernatant.

[0035] 2.2 Western-Blot detection

[0036] C2C12 cells were plated in 6-well plates at 1×10 5 The cells were plated evenly at a density of 100 cells. After the C2C12 cells adhered to the wall, a differentiation medium (gibco 2209550) containing 2% horse serum (Bio-Mingsai 6281S03) was added and placed in a 37°C, CO2 incubator for 6 days to differentiate the C2C12 cells into myotubes. When the myotube differentiation rate reached 70%-80% or more, the cells were grouped for experimental treatment.

[0037] Control group: discard differentiation medium, add 2 mL differentiation medium containing 2% horse serum by volume, and treat for 48 h;

[0038] 4T1 cell supernatant group: discard differentiation medium, add 2 mL 4T1 cell supernatant, and treat for 48 h;

[0039] 4T1 cell supernatant + OH-CATH30 2.5 μg / mL group: discard the differentiation medium, add 2 mL of the mixture of 4T1 cell supernatant and OH-CATH30 (2.5 μg / mL) and treat for 48 h.

[0040] 4T1 cell supernatant + OH-CATH30 5 μg / mL group: The differentiation medium was discarded, and 2 mL of a mixture of 4T1 cell supernatant and OH-CATH30 (5 μg / mL) was added and treated for 48 h.

[0041] Western-Blot was used to detect the expression of myosin MyHC in myotubes of mouse myoblasts C2C12 differentiated for 6 days. The experimental flow chart is as follows Figure 1 shown.

[0042] 2.3 Hematoxylin-eosin staining

[0043] C2C12 cells were plated in 12-well plates at 1×10 4 The cells were plated evenly at a uniform density, and differentiation medium (gibco 2209550) containing 2% horse serum (Bio-Mingsai 6281S03) was added, and the cells were cultured in an incubator at 37°C and 5% CO2. The differentiation medium was changed every day, and the cells were differentiated for 6 days, and the cell growth and differentiation status were observed. The area of ​​differentiated myotubes was observed under an optical microscope to account for more than 70%-80% of the area of ​​a single cell well plate, and grouping experiments were performed;

[0044] Control group: discard differentiation medium, add 2 mL differentiation medium containing 2% horse serum by volume, and treat for 48 h;

[0045] 4T1 cell supernatant group: discard differentiation medium, add 2 mL 4T1 cell supernatant, and treat for 48 h;

[0046] 4T1 cell supernatant + OH-CATH30 2.5 μg / mL group: discard the differentiation medium, add 2 mL of the mixture of 4T1 cell supernatant and OH-CATH30 (2.5 μg / mL) and treat for 48 h.

[0047] 4T1 cell supernatant + OH-CATH30 5 μg / mL group: The differentiation medium was discarded, and 2 mL of a mixture of 4T1 cell supernatant and OH-CATH30 (5 μg / mL) was added and treated for 48 h.

[0048] After 48 hours of group experiment treatment, discard the culture medium in the 12-well plate, wash twice with 1×PBS solution, and add 1mL of anhydrous methanol to each well to fix the cells for 10 minutes. Wash the fixed myotubes three times with 1×PBS solution, each time for 10 minutes, and preheat the hematoxylin in a 60℃ water bath in advance. Add the preheated hematoxylin to the surface of the myotubes and incubate for 10 seconds. Wash off the dye with double distilled water, and add 1% hydrochloric acid ethanol by volume for differentiation for 10 seconds. Use double distilled water to terminate differentiation. Add 200μL of eosin and incubate for 5 minutes. Wash off the eosin with double distilled water, add 1mL of 1×PBS solution, and then observe and take pictures under an upright optical microscope.

[0049] 3. Experimental results:

[0050] (1) Western-Blot test results are as follows Figure 2 As shown. Figure 2 It can be seen that the protein expression of glyceraldehyde-3-phosphate dehydrogenase GAPDH is constant in tissues and is highly expressed in a variety of tissues and cells. Therefore, it is used as an internal reference protein in Western-Blot experiments and as a control for determining the relative level of myosin MYHC. Compared with the control group, the expression level of myosin MYHC in C2C12 cells in the 4T1 cell supernatant group was significantly reduced, indicating that 4T1 cell supernatant can induce atrophy of C2C12 myotubes; moreover, after exogenous addition of OH-CATH30, that is, in the 4T1 cell supernatant + OH-CATH30 group, the expression of myosin MYHC in C2C12 myotubes induced by 4T1 cell supernatant can be increased, and the difference is statistically significant, indicating that OH-CATH30 can alleviate the atrophy of mouse myoblast C2C12 myotubes induced by 4T1 cell supernatant.

[0051] (2) Hematoxylin-eosin staining results are as follows Figure 3 As shown. Figure 3 It can be seen that compared with the control group, the diameter of C2C12 cell myotubes in the 4T1 cell supernatant group was significantly decreased, indicating that 4T1 cell supernatant induced atrophy of C2C12 myotubes; in the 4T1 cell supernatant + OH-CATH30 group, after exogenous addition of OH-CATH30, the diameter of C2C12 myotubes induced by 4T1 cell supernatant can be significantly increased, indicating that OH-CATH30 can alleviate the atrophy of mouse myoblast C2C12 myotubes induced by 4T1 cell supernatant.

[0052] Example 2: Study on skeletal muscle atrophy in mice after treatment with OH-CATH30 in 4T1 cell supernatant

[0053] 1. Selection of experimental animals and feeding conditions:

[0054] SPF grade 8-week-old BALB / c mice, weighing 16g-18g, were purchased from Jiangsu Jicui Yaokang Biological Co., Ltd. and were raised in an SPF environment under the conditions of 12 hours of light / 12 hours of darkness, temperature controlled at 18-23°C, and humidity maintained at 40%-60%, and were fed normal mouse chow.

[0055] 2. Experimental methods:

[0056] (1) After one week of adaptive feeding, the mice were randomly divided into a blank control group, a 4T1 group, and a 4T1+OH-CATH30 group, with 10 mice in each group.

[0057] The 4T1 tumor model was constructed as follows: 8-week-old BALB / c mice were intraperitoneally injected with 4T1 cell supernatant one week in advance, with an injection volume of 0.1 mL / mouse. The concentration of 4T1 cells in the injected 4T1 cell supernatant was 1×10 7 / mL. Then the group test treatment was carried out;

[0058] 4T1 group: One week after the injection of 4T1 cell supernatant, PBS was injected, 100 μL / mouse, once every four days; (PBS injection was used as a blank control for OH-CATH30)

[0059] 4T1+OH-CATH30 group: One week after the injection of 4T1 cell supernatant, OH-CATH30 was injected at a dose of 5 mg / kg / 100 μL, once every four days;

[0060] In addition, 8-week-old BALB / c mice that did not receive any experimental treatment were selected as the blank control group, which served as a benchmark for evaluating the effects of the experimental intervention;

[0061] The experimental process is as follows Figure 4 shown.

[0062] (2) Blood samples and skeletal muscle samples were collected from mice on day 28. A portion of the skeletal muscle tissue was embedded in an embedding agent, cut into 7 μM thick slices, and stained with hematoxylin-eosin to detect skeletal muscle atrophy in mice. The remaining tissue was quickly frozen in liquid nitrogen and stored at -80°C for subsequent experiments.

[0063] Among them, the hematoxylin-eosin staining method is as follows:

[0064] In the first step, take the slices out of the -80°C refrigerator, place them at room temperature for 30 minutes, and preheat the hematoxylin in a 60°C water bath.

[0065] In the second step, 4% paraformaldehyde was added dropwise onto the slice surface and fixed for 10 min.

[0066] In the third step, add the preheated hematoxylin in the first step to the surface of the slice and incubate for 10 minutes. Rinse with running water for 5 seconds, and add 1% hydrochloric acid ethanol to differentiate for 30 seconds. Rinse with running water for 5 seconds to terminate the differentiation, and place the slice in 50℃ warm water for 30 seconds to turn blue. Wipe the surface of the slice dry, add 200μL eosin, and incubate for 2 minutes. Rinse with running water for 5 seconds, and dehydrate in 80% ethanol for 1 minute. Use 95% ethanol and anhydrous ethanol for 2 minutes and 4 minutes respectively. Place the slice in xylene for 5 minutes to make it transparent.

[0067] The fourth step was to seal the slides with neutral gum and then observe and photograph them under an upright optical microscope. Image J software was used to statistically process the images and calculate the cross-sectional area of ​​the gastrocnemius muscle fibers.

[0068] (3) Grip strength test. The grip strength test of mice was conducted using a grip strength tester purchased from Nanjing Calvin Biological Company. Nine control mice, ten 4T1 mice, and nine 4T1+OH-CATH30 mice were subjected to grip strength adaptation for five days. After the 26th day of the experiment, the three groups of mice that had adapted to grip strength were subjected to grip strength tests. During the test, the mice were allowed to grasp the iron bar with their four limbs, and the tail of the mouse was grasped by hand and pulled back. The peak grip strength was recorded and repeated five times. The maximum and minimum values ​​were removed and the average was calculated.

[0069] 3. Experimental results:

[0070] (1) Mouse weight monitoring

[0071] The experimental results are as follows Figure 5 As shown. In terms of physiological indicators, compared with the blank control group mice, the weight of the 4T1 group mice was significantly reduced, and the food intake decreased, indicating that the 4T1 tumor affects the mice's feeding and thus their weight. After the simultaneous injection of OH-CATH30, the weight of the mice can be significantly increased. This shows that the injection of OH-CATH30 can improve the mice's food intake and increase their weight.

[0072] (2) Mouse grip strength test

[0073] The experimental results are as follows Figure 6 As shown. In terms of motor ability, the gripping force of mice in the 4T1 group was significantly reduced compared with the blank control group, indicating that 4T1 affects the gripping force of mice. In the 4T1 + OH-CATH30 group, it was found that the gripping force of mice was significantly improved after the simultaneous injection of OH-CATH30. This indicates that the injection of OH-CATH30 can improve the motor ability after the injection of 4T1 cell supernatant.

[0074] (3) Hematoxylin-eosin staining results

[0075] The experimental results are as follows Figure 7As shown. Hematoxylin-eosin staining results showed that the skeletal muscle morphology of mice in the blank control group was normal, with no obvious damage and central nuclei, while the skeletal muscle of mice in the 4T1 group was disordered, with obvious damage and central nuclei. In the 4T1+OH-CATH30 group, it was found that the simultaneous injection of OH-CATH30 could improve the skeletal muscle disorder caused by the injection of 4T1 cell supernatant.

[0076] The cross-sectional area of ​​muscle fibers was calculated and it was found that the average cross-sectional area of ​​muscle fibers in the 4T1 group of mice decreased significantly, and the cross-sectional area was between 500 and 2000 μm. 2 Increased small muscle fibers in the range of 3000-4500 μm 2 In the 4T1 + OH-CATH30 group, it was found that after the injection of OH-CATH30, the size of the larger muscle fibers increased by 3000-4500 μm. 2 Larger muscle fibers within the range, improving skeletal muscle atrophy.

[0077] The above experimental results show that OH-CATH30 can significantly improve skeletal muscle atrophy in mice induced by injection of 4T1 cell supernatant.

[0078] In summary, at the cellular level, exogenous addition of OH-CATH30 can inhibit the activity of 4T1 cell supernatant and improve C2C12 cell myotube atrophy. In vitro injection of OH-CATH30 can significantly improve skeletal muscle atrophy in mice induced by injection of 4T1 cell supernatant.

[0079] Based on this, the present invention provides the use of OH-CATH30 in the preparation of drugs for treating skeletal muscle atrophy.

[0080] The medicine also includes pharmaceutically acceptable excipients.

[0081] The medicine is in the form of tablets, capsules, granules, powders, pills or injections.

[0082] The skeletal muscle atrophy is skeletal muscle atrophy caused by cachexia.

[0083] Also provided is a skeletal muscle atrophy drug, wherein the active ingredient comprises OH-CATH30.

[0084] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. Application of OH-CATH30 in the preparation of drugs for the treatment of skeletal muscle atrophy caused by breast cancer.

2. The use of OH-CATH30 according to claim 1 in the preparation of a drug for treating skeletal muscle atrophy caused by breast cancer, characterized in that: The medicine also includes pharmaceutically acceptable excipients.

3. The use of OH-CATH30 according to claim 1 in the preparation of a drug for treating skeletal muscle atrophy caused by breast cancer, characterized in that: The medicine is in the form of tablets, capsules, granules, powders, pills or injections.

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