Method for promoting skeletal muscle development and application

Abstract

The invention discloses a method for promoting skeletal muscle development and application, and belongs to the technical field of biology. The invention provides a method for promoting skeletal muscle development, an AMPK / p38MAPK pathway is regulated and controlled by activating PPARalpha, the way of activating the PPARalpha is to give a PPARalpha agonist GW7647, and the regulation and control of the AMPK / p38MAPK pathway is shown as reduction of AMPK and P38 phosphorylation levels. Experiments show that the expression of MyoD, MyoG and Pax7 in skeletal muscle can be remarkably improved by activating PPARalpha, the phosphorylation level of AMPK and P38 is inhibited, and therefore muscle fiber proliferation and myoblast proliferation are promoted. The technology can be applied to livestock breeding to improve meat yield or develop drugs for treating skeletal muscle atrophy, and has remarkable economic and clinical values.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for promoting skeletal muscle development and application thereof. Background Art

[0002] Skeletal muscle, the largest muscle tissue in the animal body, accounts for approximately 40% of a healthy animal's body weight. Besides supporting and enabling movement, it also serves as the body's primary energy metabolism and the largest source of protein for humans. Muscle tissue production depends on factors such as the increase in skeletal muscle fiber numbers during embryonic development, the expansion of muscle fiber size after birth, and the composition of skeletal muscle fiber types. Therefore, in-depth research on the mechanisms of skeletal muscle development can reveal key factors influencing muscle growth, such as gene regulation, hormonal effects, nutritional supply, and environmental conditions. These findings provide a scientific basis for optimizing livestock production. Furthermore, research on skeletal muscle development can help address common clinical problems, such as muscle atrophy and growth stagnation. Therefore, understanding the molecular mechanisms involved in skeletal muscle development is crucial for increasing the economic value of animals, promoting the development of the livestock industry, and treating skeletal muscle-related diseases.

[0003] PPARα is a subtype of peroxisome proliferator-activated receptors (PPARs). In addition to its regulatory role in glucose and lipid metabolism, PPARα has also been reported to play a role in tissue regeneration. Studies have shown that PPARα can regulate liver size and regeneration by activating the YAP-TEAD signaling pathway and promote liver regeneration by regulating lipid metabolism through mTORC2. Existing research has largely focused on the role of PPARα in glucose and lipid metabolism and liver regeneration, but its functional mechanism in skeletal muscle development remains unclear. Summary of the Invention

[0004] The present invention aims to provide a method and use for promoting skeletal muscle development to address the problems of the prior art. The present invention aims to provide a new mechanism of action for skeletal muscle development research, a theoretical basis for further research into the function and molecular mechanism of PPARα in skeletal muscle development and differentiation, new breeding strategies for improving meat yield and quality in animal husbandry and veterinary production practices, and a theoretical basis for new therapeutic targets for skeletal muscle-related diseases in clinical practice.

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

[0006] The present invention provides a method for promoting skeletal muscle development by regulating the AMPK / p38 MAPK pathway through activating PPARα.

[0007] Furthermore, the method of activating PPARα is to administer the PPARα agonist GW7647.

[0008] Furthermore, the regulation of the AMPK / p38 MAPK pathway is manifested as a decrease in the phosphorylation levels of AMPK and P38.

[0009] Furthermore, the promotion of skeletal muscle development includes increasing the cross-sectional area of ​​muscle fibers or enhancing the proliferation capacity of muscle satellite cells.

[0010] The present invention also provides a method for detecting skeletal muscle development, which detects the expression levels of MyoD, MyoG or Pax7 by Western blot or qRT-PCR.

[0011] The present invention also provides the use of a PPARα agonist in preparing a product for increasing the meat yield of livestock, wherein the PPARα agonist is GW7647.

[0012] The present invention also provides the use of a PPARα agonist in preparing a drug for treating skeletal muscle atrophy, muscle regeneration disorder or growth stagnation, wherein the agonist is GW7647.

[0013] The present invention discloses the following technical effects:

[0014] The present invention first analyzed the changes in the expression level of PPARα in the gastrocnemius muscles of young, adult and aged mice. The results showed that compared with young mice, the expression of PPARα in the gastrocnemius muscles of aged mice was significantly reduced, indicating that PPARα may be a key gene involved in skeletal muscle development. - / - ) phenotypic changes and expression levels of skeletal muscle development-related genes and AMPK, P38 in the two groups. The results showed that compared with WT mice, PPARα - / - The cross-sectional area of ​​the gastrocnemius muscles of mice was reduced, and the expression of skeletal muscle development-related genes MyoD, MyoG, and Pax7 was significantly decreased, indicating that PPARα knockout significantly inhibited skeletal muscle development. Furthermore, phosphorylation levels of AMPK and P38 were significantly increased after PPARα knockout. Furthermore, the PPARα agonist GW7647 was administered to proliferating C2C12 myoblasts to activate PPARα and assess cell proliferation and the expression levels of skeletal muscle development-related genes, AMPK, and P38. The results showed that PPARα activation significantly increased the expression of MyoD, MyoG, and Pax7, indicating that PPARα activation promotes C2C12 myoblast proliferation. Furthermore, phosphorylation levels of AMPK and P38 were significantly decreased after PPARα activation. These results suggest that PPARα regulates skeletal muscle development by affecting the AMPK / p38 MAPK pathway, providing a new theoretical basis for the development of new breeding strategies.

[0015] This study reveals for the first time how PPARα regulates skeletal muscle development by inhibiting the AMPK / p38 MAPK pathway. This mechanism provides a new strategy for increasing meat yield in livestock production through gene editing or drug intervention, and also offers a potential target for the clinical treatment of diseases such as muscle atrophy. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 The morphological changes of gastrocnemius muscles in mice at different age stages; A: HE staining of gastrocnemius muscles in young, adult, and aged mice (250 μM); BC: Immunofluorescence detection and quantitative analysis of P21 expression in gastrocnemius muscles in mice at different age stages (200 μM); DE: Immunofluorescence detection and quantitative analysis of P53 expression in gastrocnemius muscles in mice at different age stages (200 μM); Note: *P<0.05, **P<0.01, ***P<0.001;

[0018] Figure 2 Figure 3. Effects of aging on the expression of PPARα in mouse skeletal muscle. A: PCR detection of PPARα expression in skeletal muscle. Lane M: DL500 DNA marker. Lane 1: negative control (DEPC water). Lane 2: positive control (liver tissue cDNA). Lane 3: target band (gastrocnemius muscle cDNA). B: Real-time quantitative PCR detection of PPARα expression. CD: Western blot detection of PPARα protein expression and quantitative analysis. E: Immunofluorescence detection of PPARα expression in gastrocnemius muscle at different developmental stages (100 μM). Note: *P < 0.05, **P < 0.01, ***P < 0.001.

[0019] Figure 3 The effect of knocking out PPARα on skeletal muscle development; A: WT and PPARα - / - HE staining of mouse gastrocnemius muscle (100 μM); B: WT and PPARα - / - Statistical analysis of cross-sectional area of ​​mouse gastrocnemius muscle; C: WT and PPARα - / - Body composition analysis of mice; D: qRT-PCR detection of WT and PPARα - / - Mouse skeletal muscle development-related genes; E: Western blot detection of WT and PPARα- / - Expression of PPARα, MyoD, and MyoG in mouse gastrocnemius muscle; F: Relative quantitative statistical graph of PPARα, MyoD, and MyoG proteins; Note: *P<0.05, **P<0.01, ***P<0.001;

[0020] Figure 4 Knockout of PPARα significantly reduced the positive areas of MyoD and Pax7 in gastrocnemius muscle; AB: WT and PPARα - / - Immunofluorescence detection and quantitative analysis of MyoD in gastrocnemius muscle of mice (100 μM); CD: Immunofluorescence detection and quantitative analysis of Pax7 in gastrocnemius muscle of WT and PPARα- / - mice (100 μM); Note: *P<0.05, **P<0.01, ***P<0.001;

[0021] Figure 5 To knock out the effect of PPARα on the phosphorylation levels of P38 and AMPK; A: Western blot detection of WT and PPARα - / - Expression and quantitative analysis of P38 and AMPK in mouse gastrocnemius muscle; B: Quantitative analysis of protein bands in Figure A; Note: *P<0.05, **P<0.01, ***P<0.001;

[0022] Figure 6 qRT-PCR was used to detect the expression of PPARα and CPT-1α in cells treated with different concentrations of GW7647;

[0023] Figure 7 Western blot analysis of MyoD in C2C12 cells after treatment with GW7647;

[0024] Figure 8 Western blot was used to detect the expression of MyoD in C2C12 cells after treatment with GW7647;

[0025] Figure 9 Western blot analysis of the expression of P38 and AMPK in proliferating C2C12 cells treated with GW7647 (A) and quantitative analysis of their protein bands (B); Note: *P<0.05, **P<0.01, ***P<0.001;

[0026] Figure 10 Statistical analysis of EdU detection in C2C12 cells after treatment with different concentrations of GW7647 (A) and qRT-PCR detection of MyoD and MyoG expression in C2C12 cells after treatment with GW7647 (B and C);

[0027] Figure 11 Quantitative analysis of MyoD in C2C12 cells after GW7647 treatment (A) and quantitative analysis of Pax7 in C2C12 cells after GW7647 treatment (B);

[0028] Figure 12 The proliferation of C2C12 cells after treatment with different concentrations of GW7647 was detected by EdU. Scale bar = 100 μm.

[0029] Figure 13 Immunofluorescence detection of MyoD and Pax7 in C2C12 cells after GW7647 treatment. Scale bar = 500 μm. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] The present invention studies the effect and mechanism of action of PPARα on skeletal muscle development. First, 8 young mice (6-8 weeks old), 8 adult mice (3-4 months old) and 8 old mice (18-20 months old) were used to explore the expression changes of PPARα in skeletal muscle at different developmental stages. HE staining was used to observe the morphological differences of the gastrocnemius muscles of mice of different ages. Immunofluorescence staining was used to detect the expression changes of P21 and P53 in the gastrocnemius muscles of mice of different ages to determine the age differences of the mice. Conventional PCR reaction was used to detect the expression of PPARα in skeletal muscle. Western blot, real-time fluorescence quantitative PCR (qRT-PCR) and immunofluorescence staining (IF) were used to detect the expression changes of PPARα in the gastrocnemius muscles of mice of different ages. PPARα systemic gene knockout mice were further constructed to explore its effect on skeletal muscle development. qRT-PCR, Western blot and immunofluorescence staining were used to detect the expression of PPARα in WT and PPARα knockout mice (PPARα - / - ) in the gastrocnemius muscle. The effects of a PPARα agonist (GW7647) on C2C12 cell proliferation were then assessed using EdU, immunofluorescence, qRT-PCR, and Western blot. Furthermore, Western blot was used to examine protein expression of downstream signaling pathways, including p-P38 and p-AMPK, in both tissues and cells to determine the effects of PPARα on skeletal muscle development and its underlying mechanisms.

[0036] PPARα knockout mice (PPARα - / - ) was purchased from The Jackson Laboratory, USA.

[0037] Example 1

[0038] 1. HE staining analysis of gastrocnemius muscle morphology in young, adult, and aged mice

[0039] (1) Experimental materials

[0040] 4% paraformaldehyde fixative (Solarbol), hematoxylin staining solution (G1005), and eosin staining solution (G1001) were all from Wuhan Saiweier Biotechnology Co., Ltd.

[0041] (2) Experimental methods

[0042] ① The gastrocnemius muscle tissue was fixed in 10 volumes of 4% paraformaldehyde solution for 24 hours, dehydrated with graded alcohol, and then transparentized with xylene. The tissue was immersed in melted paraffin and embedded to obtain a tissue embedded block. The tissue was then sectioned (8 μm), unfolded, retrieved, and dried to obtain paraffin sections.

[0043] ②Three sections were selected from each group for HE staining. Follow the steps below:

[0044] a. Dewaxing: Immerse sections in xylene I and II for 20 minutes, then dewax in ethanol I and II for 10 minutes each, followed by 95%, 90%, 80%, and 70% alcohol for 5 minutes each. Finally, wash with distilled water twice, 5 minutes each.

[0045] b. Nuclear staining: Stain with hematoxylin for 2 minutes, observe the effect under a light microscope, and then rinse with tap water to reverse the blueing;

[0046] c. Cytoplasmic staining: Sections were soaked in 70% and 80% alcohol for 5 minutes, followed by staining with eosin for 5 seconds.

[0047] d. Mounting: Dehydrate and make the slides transparent by immersing in 95% alcohol I and II, anhydrous ethanol I and II, and xylene I and II, for 5 minutes each. Wipe dry and then mount the slides with a neutral resin.

[0048] ③ After drying naturally, observe and capture images under a microscope and analyze them.

[0049] 2. Immunofluorescence staining analysis of the expression of P21, P53, and PPARα in the gastrocnemius muscle of mice at different developmental stages, as well as the expression changes of MyoD and Pax7 after PPARα knockout

[0050] (1) Experimental materials

[0051] 4% paraformaldehyde solution, 0.4% Triton X-100, 50× sodium citrate antigen retrieval solution (C1032, Solebol), 3% blocking goat serum (SL038, Solebol), primary antibodies: P21 (28248-1-AP, 1:200, Mitaka), P53 (60283-2-lg, 1:200, Mitaka), PPARα (66826-1-Ig, 1:200, Mitaka), MyoD (NB100-56511, 1:50, Novus), Pax7 (AB_528428, 1:50, DSHB), fluorescent secondary antibody goat anti-rabbit (A32723, 1:1000, Thermo Fisher), fluorescent secondary antibody goat anti-mouse (1:1000, Thermo Fisher) Fisher), and anti-fluorescence fading mounting medium containing DAPI (S2110, Solebro).

[0052] (2) Experimental methods

[0053] ① Drying: Bake the cut paraffin sections on a 60°C drying machine for 1 hour to prevent affecting subsequent operations;

[0054] ② Dewaxing: Immerse the sections in xylene I and II for 20 min, then in anhydrous ethanol I and II, 95%, 85%, and 75% alcohol for 5 min each, and finally wash with distilled water for 2 × 5 min (2 times, 5 min each time);

[0055] ③ Antigen retrieval: Immerse the sections completely in 1× sodium citrate antigen retrieval solution. First, retrieval in a microwave oven at medium heat (P80) for 8 minutes, then stop heating and let it stand for 8 minutes. Finally, heat at medium-low heat (P50) for 7 minutes to complete retrieval, and let it cool to room temperature naturally.

[0056] ④ Permeabilization: Wash the cooled sections in PBS for 3 x 5 min (3 times, 5 min each time) on a shaker. Add 50 μL of 0.4% Triton-X 100 to each tissue and permeabilize at room temperature in the dark for 1 hour.

[0057] ⑤ Blocking: Wash the sections in PBS for 3 x 5 min on a shaker. Add 50 μL of 3% goat serum to each tissue and block for 30 min at room temperature.

[0058] ⑥ Primary antibody incubation: dilute the primary antibody with PBS, add 20 μL of primary antibody to each tissue, and incubate at 4°C for 16 h;

[0059] ⑦ Secondary antibody incubation: The sections were rewarmed at room temperature for 30 minutes, washed with PBS for 3×5 minutes, and 20 μL of fluorescent secondary antibody corresponding to the species of the primary antibody at a dilution of 1:1000 was added to each tissue, and incubated at room temperature in the dark for 1 hour;

[0060] ⑧Seal the slides: After washing with PBS for 3×5 min, add anti-fluorescence quenching sealing medium containing DAPI, cover with a coverslip, and obtain the results under a microscope.

[0061] 3. C2C12 cell culture and culture with the PPARα agonist GW7647

[0062] (1) Experimental materials

[0063] DMEM (SH30022.01, Cytiva), fetal bovine serum (Yobibio), penicillin-streptomycin mixture (P1400, Solebro), PBS buffer (Seville), 0.25% trypsin-EDTA digestion solution (T1300, Solebro), dimethyl sulfoxide (D2650, Sigma), PPARα agonist (GW7647, MCE).

[0064] (2) Experimental methods

[0065] 1) Cell recovery

[0066] a. Preparation: Open the water bath and set it to 37°C in advance; pre-cool the centrifuge; add complete culture medium to the culture dish to create the culture environment.

[0067] b. Thaw cells: Use tweezers to remove the cryovial from the liquid nitrogen tank and immediately place it in a 37°C water bath, shaking continuously. Once the cells are completely thawed, aspirate the cells from the cryovial into a centrifuge tube. Add 4 ml of stop buffer, mix thoroughly, and centrifuge at 1000 g for 10 minutes.

[0068] c. Culturing cells: After centrifugation, discard the liquid in the centrifuge tube and add 1.5 mL of culture medium. Pipet and evenly distribute the culture medium onto the plate. Gently shake the plate in a ⅓-shaped pattern. Observe under a microscope to ensure that the cells are evenly distributed in the dish. Place the plate in an incubator. The next day, change the medium and subculture the cells based on their condition and number.

[0069] 2) Cell passaging

[0070] a. Observe cell density: Take cells out of the incubator and observe their growth. When cells are in good condition and the density reaches 100%, they can be passaged.

[0071] b. Wash cells: Discard old culture medium, add 1 mL of PBS and shake to wash, repeat twice.

[0072] c. Cell Digestion: Discard PBS, add 1 mL of trypsin, gently shake and evenly distribute, and place in an incubator for 2 minutes. Remove the tube and pipette to remove the cells. Transfer the tube to a centrifuge tube and quickly add culture medium (trypsin: culture medium = 1:3) to terminate the digestion. Mix thoroughly by gently pipetting and centrifuge (1000g, 10 minutes).

[0073] d. Cultivate cells: Add complete culture medium to the culture dish during centrifugation to create a culture environment. After centrifugation, discard the supernatant and add new culture medium. Mix thoroughly by pipetting. Then distribute the cells onto the culture dish that has been added with culture medium in advance. Shake the plate evenly. Observe the cells under a microscope for even spreading. Then place the cells in the incubator for culture.

[0074] 3) Cultivate C2C12 cells with the PPARα agonist GW7647

[0075] The cultured cells were evenly plated in a six-well plate. After 12 h of culture, gradient concentrations of GW7647 (0.1, 1, 5, and 10 μM) were added to the culture medium. RNA was extracted after 24 h of culture. The expression changes of PPARα and its downstream CPT-1α were detected, and the optimal concentration was determined to be 1 μM before subsequent culture.

[0076] The cultured cells were evenly plated in a six-well plate. After culturing for 12 hours, 1 μM GW7647 was added to the culture medium. RNA was extracted after culturing for 24 hours, and protein was extracted after culturing for 48 hours for the detection of development-related genes.

[0077] 4. Immunofluorescence staining analysis of the expression changes of MyoD and Pax7 in C2C12 cells after activation of PPARα

[0078] Experimental methods

[0079] ① The cultured cells were evenly spread in a twelve-well plate. After culturing for 12 hours, GW7647 at a concentration of 1 μM was added to the culture medium and cultured for 48 hours before subsequent operations.

[0080] ②Discard the culture medium in the well plate and add PBS to wash three times, each time for 5 minutes.

[0081] ③Discard PBS, add 4% paraformaldehyde fixative at room temperature for 30 minutes, and wash three times with PBS, each time for 5 minutes.

[0082] ④ Discard PBS, add 0.4% TritonX-100 to permeabilize at room temperature in the dark for 30 minutes, and add PBS to wash three times, each time for 5 minutes.

[0083] ⑤ Discard PBS, add 3% goat serum to block at room temperature for 1 hour, and then add PBS to wash three times, each time for 5 minutes.

[0084] ⑥Discard PBS, add corresponding primary antibody, and incubate the plate at 4℃ for 12h.

[0085] ⑦ Rewarm for 30 minutes, wash with PBS three times, 5 minutes each time, and prepare the corresponding secondary antibody in the dark.

[0086] ⑧Discard PBS, add corresponding secondary antibody, and incubate at room temperature in the dark for 1 hour.

[0087] ⑨ Drop the DAPI-containing mounting medium into the well plate, observe and take pictures under a fluorescence microscope.

[0088] 5. EdU staining to detect cell proliferation

[0089] (1) Experimental reagents

[0090] Dual-luciferase gene reporter assay kit (11402ES60, Yisheng Biotechnology).

[0091] (2) Experimental methods

[0092] ① EdU labeling of cells: First, prepare 1× EdU working solution with a ratio of 10 mM EdU: serum-free medium = 1:1000. Discard the old medium in the well plate, wash twice with PBS, add 1× EdU working solution, and incubate in a cell culture incubator for 2 hours.

[0093] ② Fix the cells: After incubation, discard the old culture medium, add 4% paraformaldehyde fixative, and fix at room temperature for 15 minutes.

[0094] ③ Permeabilize cells: After fixation, discard the cell fixative and wash with PBS for 3×5 minutes. Then add 0.4% TritonX-100 and incubate at room temperature in the dark for 15 minutes. Then discard the permeabilization solution and wash with PBS three times, each time for 5 minutes.

[0095] ④ Prepare the Click reaction solution according to the sequence in Table 1.

[0096] Click reaction solution was added, incubated at room temperature in the dark for 30 min, the reaction solution was discarded, and the cells were washed with PBS for 3×5 min.

[0097] ⑤ Nuclear staining: discard PBS, add 1×DAPI working solution and incubate at room temperature in the dark for 30 minutes, then add PBS to wash 3×5 minutes.

[0098] ⑥ Observe the cells under a fluorescence microscope and take pictures, and perform quantitative analysis using Image J.

[0099] Table 1 Preparation of Click reaction solution

[0100]

[0101] 6. PCR verification of PPARα expression in mouse gastrocnemius muscle

[0102] The PCR reaction system is shown in Table 2. DEPC (diethyl pyrocarbonate) water was used as a negative control, liver tissue cDNA was used as a positive control, and gastrocnemius muscle tissue cDNA was used as the target. PCR reactions were performed according to the following protocol: pre-denaturation at 95°C for 30 s, denaturation at 95°C for 40 s, annealing at 60°C for 30 s (35 cycles), and extension at 72°C for 40 s. PCR products were electrophoresed on a 1% agarose gel at 220 V and visualized under UV light.

[0103] Table 2 PCR reaction system

[0104]

[0105] 7. qRT-PCR analysis of the expression changes of PPARα, PPARα, MyoD and MyoG after PPARα knockout, and CPT-1α, PPARα, MyoD and MyoG after PPARα activation in the gastrocnemius muscle of mice at different developmental stages

[0106] (1) Experimental materials

[0107] Trizol (9109, Takara), chloroform and isopropanol (Tianjin Beichen Fangzheng Reagent Factory), mRNA reverse transcription kit (#22107, Tolo Biotechnology Co., Ltd.), SYBR Green qPCR Mix (MQ1020, Mona Biotech).

[0108] (2) Experimental methods

[0109] ①Total RNA extraction

[0110] For tissue samples, add 1 mL of Trizol reagent and three grinding beads to the grinding tube containing the tissue and grind at 50 Hz for 5 minutes in a high-throughput tissue grinder (if the suspension is not completely ground, increase the grinding time appropriately) to obtain a tissue mixture. For cells, discard the culture medium, wash three times with PBS, add an appropriate amount of Trizol reagent, grind thoroughly, and collect into a centrifuge tube. Extract RNA from the collected tissue and cell mixture using the following steps:

[0111] Before extraction, pre-cool the chloroform, isopropanol, and 70% anhydrous ethanol in a 4°C refrigerator. Pre-cool the centrifuge. Sterilize all equipment in the centrifuge and place in a clean bench for UV irradiation for 30 minutes.

[0112] b. Add 200 μL of chloroform to the above mixture, mix well, let it stand on ice for 10 minutes, and centrifuge at 12,000 g in a 4°C centrifuge for 15 minutes;

[0113] c. Carefully aspirate the supernatant into a new centrifuge tube, add 500 μL of isopropanol, mix thoroughly, and let stand on ice for 10 minutes. Then, centrifuge at 12,000 g for 10 minutes.

[0114] d. After centrifugation, discard the supernatant and aspirate the excess liquid with a pipette. A white RNA precipitate will be present at the bottom of the tube.

[0115] e. Add 1 mL of 70% anhydrous ethanol, wash the tube walls, and centrifuge at 7500 g for 5 minutes at 4°C.

[0116] f. Use a pipette to suck out the supernatant and dry until the precipitate becomes transparent;

[0117] g. Add an appropriate amount of DEPC water according to the size of the RNA pellet to dissolve it and mix thoroughly by pipetting;

[0118] h. Aliquot 3 μL of RNA sample for RNA concentration and PCR testing of RNA quality;

[0119] Reverse transcription was performed to synthesize cDNA according to the RNA concentration and stored at -80°C for future use.

[0120] ②PCR reaction verification primers

[0121] Use Primer 5 to design the required primers (Table 3). PCR was used to verify the primers; the system is shown in Table 4. After adding the above system, the PCR reaction was performed using a pre-denaturation cycle of 94°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds (35 cycles), extension at 72°C for 30 seconds, and termination extension at 72°C for 5 minutes. The PCR products were spotted onto a 1% agarose gel and electrophoresed at 220V. The results were observed under UV light. A single positive band for the target gene indicated successful primer design and that subsequent experiments were suitable.

[0122] Table 3 Primer sequences used in the experiment

[0123]

[0124]

[0125] Table 4 PCR reaction

[0126]

[0127] ③RNA reverse transcription

[0128] Reverse transcription was performed using the ToloScript ALL-in-one RT EasyMix for qPCR Reverse Transcription Kit. The specific steps are as follows: Calculate the volume based on the RNA concentration and add the reaction mixture according to Table 5. Add all reagents to a centrifuge tube by pipetting and mix thoroughly. Briefly centrifuge to the bottom of the tube. Reverse transcription was performed in a PCR instrument using a cycler program of 50°C for 15 minutes and 85°C for 5 seconds.

[0129] Table 5 Reaction system

[0130]

[0131] ④qRT-PCR reaction

[0132] After the prepared cDNA was diluted, real-time fluorescence quantitative PCR was performed according to the following reaction system (Table 6). The reaction was carried out in the following procedure: pre-denaturation at 95℃ for 1min → denaturation at 95℃ for 10s, annealing at 60℃ for 30s (40 cycles) → extension at 95℃ for 15s. -ΔΔ Ct method analysis results.

[0133] Table 6 Real-time fluorescence quantitative PCR reaction

[0134]

[0135]

[0136] 8. Western blot analysis was performed to examine the expression changes of PPARα in the gastrocnemius muscle of mice at different developmental stages, PPARα, MyoD, MyoG, AMPK and P38 after PPARα knockout, and MyoD, AMPK and P38 after PPARα activation.

[0137] (1) Experimental materials

[0138] RIPA lysis buffer (P0013B, Beyotime), PMSF protease inhibitor (CR2311102, Seville), phosphatase inhibitor (MCE), BCA protein concentration assay kit (P0011, Beyotime), 5× loading buffer (CW0052, Kangwei Century), protein marker (26616, Thermo Fisher), Tris Buffered Saline (TBS, Power, G0001, Seville), 0.22 μM PVDF membrane (Merck Millipore). Primary antibodies: GAPDH (10494-1-AP, 1:25,000, Mitsubishi Corporation), PPARα (66826-1-Ig, 1:2,000, Mitsubishi Corporation), MyoD (ab203383, 1:2,000, abcam), MyoG (ab1835, 1:2,000, abcam), AMPK (2532S, 1:2,000, CST) and p-AMPK (2532S, 1:1,500, CST), P38 (8690S, 1:2,000, CST) and p-P38 (4511T, 1:2,000, CST); secondary antibodies: goat anti-rabbit (L3012, 1:25,000, SAB), goat anti-mouse (SA00001-1, 1:25,000, Mitsubishi Corporation).

[0139] (2) Experimental methods

[0140] ① Extraction of total protein

[0141] For tissue samples, 1 mL of tissue lysis buffer (RIPA) containing 10 μL of protease inhibitors and 10 μL of phosphatase inhibitors was added to the grinding tube containing the tissue. The sample was ground in a high-throughput tissue grinder at 50 Hz for 5 minutes to obtain a completely broken tissue sample and oscillated to mix. For cells, the cells were rinsed three times with PBS, and the corresponding RIPA lysis buffer (containing pre-added protease inhibitors and phosphatase inhibitors) was added according to the cell volume. The cells were ground and pipetted down and placed in a centrifuge tube. The cells were broken in an ultrasonic disruptor at 150W with a 5s on / 10s off program for 3 minutes to ensure complete cell lysis. The obtained tissue and cell mixture was allowed to stand on ice for 30 minutes and then centrifuged at 10,000g in a 4°C centrifuge for 10 minutes. The supernatant was aspirated as the total protein sample.

[0142] ② Protein concentration detection and denaturation

[0143] The protein concentration was determined according to the instructions of the BCA protein concentration assay kit. The protein samples for electrophoresis were then prepared according to the following system (Table 7), denatured by boiling at 99°C for 10 min, and stored in a -80°C refrigerator.

[0144] Table 7 Protein sample preparation system

[0145]

[0146] ③ Western blotting

[0147] Preparation of WB gel: Select appropriate concentration of separation gel according to the size of the target protein to be detected for the preparation of WB gel. The formula is shown in Table 8.

[0148] First, clean the glass plate and gel preparation rack with running water and distilled water. After clamping the glass plate, place it on a horizontal table and add distilled water to check for leaks. During this period, prepare the separation gel according to the recipe table. After confirming that the glass plate does not leak, pour out the distilled water and use filter paper to absorb the moisture between the glass plates. Add the separation gel and press it with distilled water. Let it stand for 30 minutes. After solidification, pour out the upper layer of distilled water and absorb the moisture with filter paper. Add the prepared concentrated gel and quickly insert the comb to avoid the generation of bubbles. Let it stand for 20 minutes to solidify before electrophoresis.

[0149] Electrophoresis: Add the calculated volume of denatured protein sample and 3 μL protein marker vertically and slowly into the spotting wells, and then perform electrophoresis at 80V.

[0150] Transfer: Activate the PVDF membrane by soaking it in methanol for 3 minutes and pre-cooling the transfer drawer to 4°C. After electrophoresis, transfer the protein bands from the gel to the PVDF membrane using a semi-dry transfer apparatus at 1.3A for 10 minutes.

[0151] Table 8 Separation gel preparation system

[0152]

[0153] Blocking: The PVDF membrane printed with protein bands was blocked with 5% skim milk powder in a shaker at 37°C for 1 h.

[0154] Primary antibody incubation: After blocking, wash the milk powder with TBST, then add the antibody of interest and incubate overnight on a shaker at 4°C.

[0155] Secondary antibody incubation: On the second day, after recovering the primary antibody, wash the membrane three times with TBST, each time for 10 minutes, then add the diluted secondary antibody and incubate at 37℃ for 1 hour.

[0156] Exposure: After the end, wash the membrane with TBST 6 times, 5 minutes each time, and then expose it with a 1:1 prepared luminescent solution.

[0157] Image J image processing software was used to measure the grayscale value and area of ​​protein bands and calculate protein expression. Significance analysis was performed by dividing the target protein expression level by the internal reference protein expression level.

[0158] 9. Data Analysis

[0159] All experimental data were analyzed and processed using GraphPad Prism 5.0 software. Data are presented as mean ± standard deviation (mean ± SD). Statistical significance was determined using a two-tailed t-test. P < 0.05 (*) indicates a significant difference; P < 0.01 (**) indicates an extremely significant difference; and P < 0.001 (***) indicates a very significant difference.

[0160] 10. Results and Analysis

[0161] (1) Morphological changes of gastrocnemius muscles in young, adult, and aged mice

[0162] HE staining and immunofluorescence staining for cell cycle and apoptosis markers P21 and P53 were used to observe their morphological differences to determine the mouse models at different developmental stages. The results showed that the cross-sectional area of ​​the gastrocnemius muscle decreased with age; and the degree of compactness of the arrangement of muscle fibers was also very different. The muscle fibers of young mice were tightly arranged, those of adult mice were relatively loose, and those of old mice were the loosest. Immunofluorescence results showed that P21 and P53 were expressed in the nuclei of skeletal muscle cells at all age stages, and their expression levels gradually increased with age. In general, the present invention determined the age differences among the three groups of mice by observing the morphological differences of the gastrocnemius muscles of young, adult, and aging mice through HE staining and immunofluorescence staining ( Figure 1 ).

[0163] (2) PPARα expression in mouse skeletal muscle decreases with age

[0164] The present invention first detected the expression of PPARα in skeletal muscle by conventional PCR using liver cDNA as a positive control. Electrophoresis results showed that the PPARα band corresponding to the lane containing gastrocnemius cDNA was clear and had no tailing, verifying the expression of PPARα in the gastrocnemius muscle of mice. Western blot and real-time quantitative PCR were used to detect the expression of PPARα in the gastrocnemius muscle of young, adult, and elderly mice. The results showed that the protein and mRNA expression levels of PPARα in the gastrocnemius muscle of young, adult, and elderly mice showed a gradual downward trend, and the levels of PPARα in the gastrocnemius muscle of young and elderly mice differed significantly, indicating that PPARα expression significantly decreases with age. To further determine the expression changes of PPARα in the gastrocnemius muscle of mice at different ages, the present invention performed immunofluorescence staining for PPARα in gastrocnemius muscle sections. The results showed that PPARα was expressed in both the cytoplasm and nucleus of the gastrocnemius muscle. In addition, the positive areas in the gastrocnemius muscle of young mice were more than those of adult mice, and the positive areas in the gastrocnemius muscle of old mice were the least. Immunofluorescence results further confirmed that the expression level of PPARα decreased with age ( Figure 2 ).

[0165] (3) Knockout of PPARα inhibits skeletal muscle development

[0166] The present invention prepared and bred PPARα knockout mice (PPARα - / - ) and wild-type mice (WT) to study the effect of PPARα on skeletal muscle development. First, HE staining was used to observe and analyze that knocking out PPARα resulted in a decrease in the cross-sectional area of ​​the gastrocnemius muscle in mice. - / - Body composition analysis of mice showed that PPARα - / - The body weight of mice increased significantly; WT and PPARα - / - The gastrocnemius muscle samples of mice were tested for the expression of skeletal muscle development-related indicators by qRT-PCR and Western blot. The results showed that compared with WT mice, PPARα - / - The mRNA and protein levels of MyoD and MyoG in the gastrocnemius muscle of mice were significantly reduced, indicating that PPARα knockout inhibited the development of skeletal muscle ( Figure 3 ).

[0167] (4) Knockout of PPARα significantly reduced the positive areas of MyoD and Pax7 in gastrocnemius muscle

[0168] In order to further determine the effect of PPARα knockout on skeletal muscle development, the present invention used immunofluorescence to detect WT and PPARα - / - The expression of MyoD and Pax7 in gastrocnemius muscle of mice showed that PPARα - / - The positive areas of MyoD and Pax7 in the gastrocnemius muscle of mice were significantly reduced. The above results indicate that the number of muscle satellite cells was significantly reduced and their proliferation ability and myogenic differentiation potential were significantly decreased, indicating that the growth and development of the gastrocnemius muscle of mice was inhibited after the knockout of PPARα. Figure 4 ).

[0169] (5) Knockout of PPARα activates P38 and AMPK phosphorylation levels

[0170] In order to determine the mechanism of PPARα regulating skeletal muscle development, the present invention detected WT and PPARα by Western blot. - / - The expression of AMPK and P38 MAPK signaling pathways in mouse gastrocnemius muscle showed that the phosphorylation levels of P38 and AMPK in gastrocnemius muscle increased significantly after PPARα knockout. The above results indicate that PPARα affects skeletal muscle development by affecting the phosphorylation levels of AMPK and P38, and the effect on the P38 signaling pathway is more significant. Figure 5 ).

[0171] (6) Activation of PPARα promotes C2C12 myoblast proliferation by inhibiting P38 and AMPK signaling pathways

[0172] To validate the effects of PPARα on skeletal muscle development in cells, the PPARα agonist GW7647 was administered to proliferating C2C12 myoblasts. qRT-PCR assays were used to assess the expression of PPARα and its downstream regulator, CPT-1α, following administration of GW7647 at varying concentrations. Results showed that expression of PPARα and its downstream regulator, CPT-1α, increased gradually over the 0.1 μM to 1 μM concentration range. Furthermore, at concentrations exceeding 1 μM, expression of PPARα and its downstream regulator, CPT-1α, decreased with increasing GW7647 concentrations. Furthermore, the effects of varying GW7647 concentrations on C2C12 cell proliferation were assessed using EdU assays. Results showed that a 1 μM concentration significantly increased the number of proliferating C2C12 cells. To further investigate the effect of PPARα activation on C2C12 cell proliferation, the present invention performed qRT-PCR, Western blot, and immunofluorescence assays. The results showed that activation of PPARα significantly increased the mRNA levels of MyoD and MyoG, as well as the protein level of MyoD. Indirect cell fluorescence revealed that the percentage of MyoD and Pax7-positive areas in C2C12 cells significantly increased after culturing with 1 μM GW7647. These results demonstrate that culturing C2C12 cells with an appropriate concentration (1 μM) of GW7647 during their proliferation phase activates PPARα and promotes the proliferation of C2C12 myoblasts.

[0173] Western Blot analysis was used to verify the expression of AMPK and P38 MAPK signaling pathways in C2C12 cells during the proliferation phase after PPARα activation. The results showed that the phosphorylation levels of P38 and AMPK in C2C12 cells were significantly reduced after PPARα activation, suggesting that PPARα activation promotes C2C12 cell proliferation by inhibiting the phosphorylation levels of AMPK and P38. Figure 6-Figure 13 ).

[0174] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for promoting skeletal muscle development, characterized in that: Regulates the AMPK / p38 MAPK pathway by activating PPARα.

2. The method according to claim 1, characterized in that The method of activating PPARα is to administer the PPARα agonist GW7647.

3. The method according to claim 1, characterized in that The regulation of the AMPK / p38 MAPK pathway is manifested as a decrease in the phosphorylation levels of AMPK and P38.

4. The method according to claim 1, wherein The promotion of skeletal muscle development includes increasing the cross-sectional area of ​​muscle fibers or enhancing the proliferation capacity of muscle satellite cells.

5. A skeletal muscle development detection method, characterized in that: The expression levels of MyoD, MyoG, or Pax7 were detected by Western blot or qRT-PCR.

6. Use of a PPARα agonist in the preparation of a product for increasing meat yield in livestock, characterized in that: The PPARα agonist is GW7647.

7. Use of a PPARα agonist in the preparation of a medicament for treating skeletal muscle atrophy, muscle regeneration disorder or growth stagnation, characterized in that: The agonist is GW7647.

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