Application of exercise-induced fatigue in regulation of exosome miRNA expression and reverse regulation of exosome miRNA expression by resveratrol

Abstract

The invention relates to the technical field of biology, in particular to application of exercise-induced fatigue in regulation and control of exosome miRNA expression and reverse regulation and control of exosome miRNA expression through resveratrol. According to the application of exercise-induced fatigue in regulation and control of exosome miRNA expression, the exercise-induced fatigue causes exosome miRNA differential expression of cells and tissues. The invention relates to application of resveratrol in reversing exosome miRNA differential expression of cells and tissues caused by exercise-induced fatigue. Exercise-induced fatigue can cause differential expression of exosome miRNA of primary myocardial cells, skeletal muscle cells and liver cells and differential expression of exosome miRNA of mouse myocardial tissues, skeletal muscle tissues and liver tissues, and resveratrol can reverse exosome miRNA differential expression caused by exercise-induced fatigue.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the application of sports fatigue in regulating the expression of exosome miRNA and reversal regulation of exosome miRNA expression by resveratrol. Background Art

[0002] After cells were treated with resveratrol, the miRNA expression profile in their exosomes changed significantly. For example, in SMB-S15 cells, resveratrol treatment was able to eliminate changes in miRNA expression in cell exosomes during PrP Sc, indicating that resveratrol can exert its biological effects by regulating exosomal miRNA expression. Although existing studies have revealed the association between exercise fatigue and exosomal miRNA expression, as well as the potential role of resveratrol in improving exercise fatigue and regulating miRNA expression, there is still a lack of systematic application solutions and technical means to effectively apply this mechanism to the prevention and treatment of exercise fatigue. Therefore, the present invention aims to provide a method for regulating exosomal miRNA expression based on resveratrol, in order to provide new technical means and theoretical basis for the reversal of exercise fatigue. Summary of the invention

[0003] In order to solve the above problems, the present invention provides the application of sports fatigue in regulating the expression of exosome miRNA and the reversal regulation of exosome miRNA expression by resveratrol.

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

[0005] The present invention provides the application of sports fatigue in regulating the expression of exosome miRNA;

[0006] The exercise fatigue causes differential expression of exosomal miRNA in cells and / or tissues.

[0007] Preferably, the cells include one or more of primary cardiomyocytes, skeletal muscle cells and liver cells;

[0008] The tissues include one or more of mouse myocardial tissue, skeletal muscle tissue and liver tissue.

[0009] Preferably, the exosomal miRNA includes exosomal mmu-miR-486b-3p_R+1.

[0010] Preferably, the cell model of sports-induced fatigue is induced by a hydrogen peroxide solution, the concentration of which is 300 μM; and the animal model of sports-induced fatigue is obtained by an exhaustive swimming experiment.

[0011] The present invention also provides the use of resveratrol in reversing the differential expression of exosome miRNA in cells and / or tissues caused by sports fatigue.

[0012] Preferably, in the cell experiment, the resveratrol is used at a concentration of 50 μM.

[0013] Preferably, in animal experiments, the resveratrol is used at a concentration of 10 mg / kg.

[0014] Preferably, the cells include one or more of primary cardiomyocytes, skeletal muscle cells and liver cells;

[0015] The tissues include one or more of mouse myocardial tissue, skeletal muscle tissue and liver tissue.

[0016] Preferably, the exosomal miRNA includes exosomal mmu-miR-486b-3p_R+1.

[0017] The present invention also provides the use of resveratrol in preparing an anti-sports fatigue product by reducing cell oxidative damage and / or improving cell antioxidant capacity.

[0018] Beneficial effects of the present invention:

[0019] Exercise fatigue can cause differential expression of exosomal miRNA in primary cardiomyocytes, skeletal muscle cells and liver cells, as well as differential expression of exosomal miRNA in mouse cardiomyocyte tissue, skeletal muscle tissue and liver tissue, while resveratrol can reverse the differential expression of exosomal miRNA caused by exercise fatigue.

[0020] Resveratrol has anti-fatigue effects, but its potential molecular mechanism remains to be studied. This experiment found that when sports fatigue occurs, it can cause differential expression of mouse exosome miRNA, and resveratrol can reverse the differential expression of exosome miRNA caused by sports fatigue. This will provide a theoretical basis for the development of more anti-fatigue health products, such as compounds with similar structures to resveratrol, or drugs that can also reverse the differential expression of these exosome miRNAs, which will be potential research objects for the development of new anti-fatigue products. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0022] Figure 1 The effect of Res on cellular ROS, MDA, and SOD; Note: A: H 2 O 2Effect on cell viability; B: Effect of Res on cell viability; C: Mcc ROS content; D: Msmc ROS content; E: Mlc ROS content; F: Mcc MDA content; G: MsmcMDA content; H: Mlc MDA content; I: Mcc SOD activity; J: Msmc SOD activity; K: Mlc SOD activity. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0023] Figure 2 For pharmacodynamic evaluation; Note: A: Screening of the best intervention concentration of Res; B: Changes in mouse body weight; C: Changes in mouse food intake; D: Exhaustive swimming time; E: MctT-AOC value; F: Msmt T-AOC value; G: MltT-AOC value; H: HE staining of Mct (scale bar is 1000μm-50μm); I: HE staining of Msmt (scale bar is 200μm-50μm); J: HE staining of Mlt (scale bar is 1000μm-50μm) *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0024] Figure 3 For identification of exosomes; Note: A: TEM results (scale is 200 nm); B: NTA results; C: Video was taken at a frame rate of 10 frames / s to observe particle movement; D: WB results; E: Mcc-Exo concentration; F: Msmc-Exo concentration; G: Mlc-Exo concentration; H: Mct-Exo concentration; I: Msmt-Exo concentration; J: Mlt-Exo concentration; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0025] Figure 4 To up-regulate exosomal miRNAs for exercise fatigue; Note: HC: Mcc-Exo; PHC: SF-Mcc-Exo; C_2_H: Mct-Exo; G_2_H: SF-Mct-Exo; BC: Msmc-Exo; PBC: SF-Msmc-Exo; C_2_B: Msmt-Exo; G_2_B: SF-Msmt-Exo; LC: Mlc-Exo; PLC: SF-Mlc-Exo; C_2_L: Mlt-Exo; G_2_L: SF-Mlt-Exo;

[0026] Figure 5Res reversed the changes in exosomal miRNAs induced by exercise fatigue; Note: PHE: Res-SF-Mcc-Exo; PHC: SF-Mcc-Exo; F_2_H: Res-SF-Mct-Exo; G_2_H: SF-Mct-Exo; PBE: Res-SF-Msmc-Exo; PBC: SF-Msmc-Exo; F_2_B: Res-SF-Msmt-Exo; G_2_B: SF-Msmt-Exo; PLE: Res-SF-Mlc-Exo; PLC: SF-Mlc-Exo; F_2_L: Res-SF-Mlt-Exo; G_2_L: SF-Mlt-Exo;

[0027] Figure 6 The sequencing results were verified by qRT-PCR; Note: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0028] Figure 7 is the expression of exosomal mmu-miR-486b-3p_R+1;

[0029] Figure 8 Evaluation of transfection efficiency and the effect of transfection on Res intervention in exercise-induced fatigue; Note: A: Transfection efficiency evaluation, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; BE: Effect of transfection on Res promoting cell proliferation and intervention in exercise-induced fatigue, compared with the Control group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; compared with the SF group, △ P<0.05, △△ P<0.01, △△△ P < 0.001, △△△△ P<0.0001; compared with SF+Res50μM group, # P<0.05, ## P<0.01, ### P < 0.001, #### P < 0.0001;

[0030] Fig. 9 It is a flow chart of the present invention. DETAILED DESCRIPTION

[0031] The present invention provides an application of sports fatigue in regulating the expression of exosomal miRNA; the sports fatigue causes differential expression of exosomal miRNA in cells and / or tissues. In the present invention, the cells preferably include one or more of primary cardiomyocytes, skeletal muscle cells and liver cells. In the present invention, the tissues preferably include one or more of myocardial tissue, skeletal muscle tissue and liver tissue. In the present invention, the exosomal miRNA preferably includes exosomal mmu-miR-486b-3p_R+1. In the present invention, the cell model of sports fatigue is preferably induced by a hydrogen peroxide solution, and the concentration of the hydrogen peroxide solution is preferably 300 μM. In the present invention, the animal model of sports fatigue is obtained by an exhaustive swimming experiment. In the present invention, the mouse model of sports fatigue is preferably obtained by an exhaustive swimming experiment.

[0032] The present invention also provides the use of resveratrol in reversing the differential expression of exosomal miRNA in cells and / or tissues caused by sports fatigue. In the present invention, in cell experiments, the use concentration of the resveratrol is preferably 50 μM. In the present invention, in animal experiments, the use concentration of the resveratrol is preferably 10 mg / kg. In the present invention, the cells preferably include one or more of primary cardiomyocytes, skeletal muscle cells and liver cells. The tissue preferably includes one or more of myocardial tissue, skeletal muscle tissue and liver tissue. In the present invention, the exosomal miRNA preferably includes exosomal mmu-miR-486b-3p_R+1.

[0033] The present invention also provides the use of resveratrol in the preparation of an anti-sports fatigue product by reducing cell oxidative damage and / or improving cell antioxidant capacity. In the present invention, the product preferably includes a medicine or a health product.

[0034] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1

[0036] (1) Experimental plan

[0037] ① In vitro experimental study on the intervention of resveratrol on sports-induced fatigue

[0038] Mouse primary cardiomyocytes (Mcc), mouse skeletal muscle cells (Msmc) and mouse liver cells (Mlc) were extracted and used as the control group. MTT method was used to detect H 2 O 2 The half inhibitory concentration of cells, H 2 O 2The sports fatigue (SF) cell model was constructed after 1 h of action, which was referred to as SF-Mcc, SF-Msmc and SF-Mlc, and was referred to as the SF group. The MTT method was used to screen the optimal Res intervention concentration, and resveratrol (referred to as Res) intervened in the SF group cells for 24 h, which was referred to as the SF+50μM group. The kit was used to detect the cell viability, ROS, MDA and SOD values ​​of each group. The cell culture supernatant of each group was collected, and the mouse cardiomyocyte exosomes (Mcc-Exo), mouse skeletal muscle cell exosomes (Msmc-Exo) and mouse liver cell exosomes (Mlc-Exo) were extracted by differential centrifugation.

[0039] ②In vivo experimental study on the intervention of resveratrol on exercise-induced fatigue

[0040] 5-6 week old C57BL / 6J male mice (license number SCXK (Ningxia Medical University) 2020-0001) were divided into Control and SF groups: gavage of equal volume of 0.5% sodium carboxymethyl cellulose (CMC-Na) every day, SF+Res10mg / kg: gavage of Res every day, Res was prepared with 0.5% CMC-Na, the dose was 10mg / kg, 0.1mL / 10g. There were 12 mice in each group, and the dosing cycle was 30 days. On the 31st day of the experiment, mice in the SF group and SF+Res10mg / kg group participated in the exhaustive swimming test, while the Control group did not participate, and the exhaustive swimming time of the mice was recorded. Sodium pentobarbital was injected intraperitoneally for anesthesia, and the mice were anesthetized by removing the eyeballs and taking blood for centrifugation to obtain serum. The mice were killed by cervical dislocation, and the mouse skin was disinfected with 75% alcohol. Mouse cardiac tissue (Mct), Mouse skeletal muscle tissue (Msmt) and Mouse liver tissue (Mlt) were quickly taken and divided into 3 parts: the first part was used for differential centrifugation to extract mouse myocardial tissue exosomes (Mct-Exo), mouse skeletal muscle tissue exosomes (Msmt-Exo) and mouse liver tissue exosomes (Mlt-Exo). The second part was drained with filter paper, frozen in liquid nitrogen, and then stored at -80℃ for the detection of T-AOC value by the kit. The third part was placed in a centrifuge tube containing tissue fixative and stained with hematoxylin-eosin (HE) by the kit.

[0041] ③Identification of exosomes

[0042] Transmission electron microscopy (TEM), nanoparticle analyzer (NTA) and WB experimental techniques were used to identify the extracted cell and tissue exosome samples.

[0043] ④ Sequencing of exosomal miRNAs and screening of differentially expressed exosomal miRNAs

[0044] The small RNA sequencing library was prepared using TruSeq Small RNA Sample Prep Kits. Single-end sequencing was performed using the high-throughput sequencing platform produced by Illumina. The sequencing data were quality controlled. ACGT101-miR (v4.2) was used to identify miRNAs, and then quantification and standardization of miRNA expression between groups were performed. The significantly differentially expressed exosomal miRNAs were screened from the two aspects of difference fold (fold_change>2 or fold_change<0.5, i.e. |log2FC|>1) and significance level (p_value<0.05).

[0045] ⑤Verify sequencing results

[0046] The screened differentially expressed exosomal miRNAs were reverse transcribed using the miRNA first-strand cDNA synthesis kit, and the sequencing results were verified by a fluorescent real-time quantitative experiment using the miRNA qRT-PCR kit.

[0047] ⑥Regulatory effect of Res on exosome mmu-miR-486b-3p_R+1

[0048] 5-6 week old C57BL / 6J male mice were divided into Control, SF and SF+Res10mg / kg groups. The treatment method was the same as "②". Mct, Msmt and Mlt were taken to extract Mct-Exo, Msmt-Exo and Mlt-Exo. The exosome mmu-miR-486b-3p_R+1 was reverse transcribed using the miRNA first-strand cDNA synthesis kit. The qRT-PCR experiment of exosome mmu-miR-486b-3p_R+1 was performed using the miRNA RT-PCR kit.

[0049] ⑦Effects of knockdown / overexpression of exosomal mmu-miR-486b-3p_R+1 on the effects of resveratrol on promoting cell proliferation and intervening in sports fatigue

[0050] Msmt cells were cultured, and Lipofectamine3000 was used to knock down and overexpress exosome mmu-miR-486b-3p_R+1 in cells, and the transfection effect was evaluated by qRT-PCR. Then Res was given synchronously, and the cell viability, ROS, MDA levels and SOD activity were detected by kits.

[0051] (2) Experimental results

[0052] ① Screening half inhibition concentration 300μM H 2 O 2As the optimal concentration for establishing the exercise-induced fatigue cell model ( Figure 1 Middle A). The Res concentration of 50 μM in the group with the highest cell viability was selected as the optimal intervention concentration ( Figure 1 After Res intervened in the sports fatigue cell model, the ROS and MDA contents of the three cells decreased (all p < 0.01), and the SOD activity increased (all p < 0.001), indicating that Res can reduce cell oxidative damage and play an anti-sports fatigue effect (see Figure 1 (in CJ).

[0053] ② The Res concentration of 10 mg / kg in the group with the longest exhaustive swimming time was selected as the optimal intervention concentration ( Figure 2 Middle A). After Res intervention in the exercise-induced fatigue animal model, there was no significant effect on the body weight and food intake of mice ( Figure 2 B, C), it prolonged the mice's exhaustive swimming time (p < 0.0001), extending it by 24.28% ( Figure 2 The T-AOC values ​​of the three tissues increased (all p<0.001, Figure 2 EG). The distance between the myocardial fibers and skeletal muscle fibers of mice becomes narrower, showing a certain striated structure and orderly arrangement ( Figure 3 A and B); the liver cells are relatively intact, the swelling is reduced, the structure is clear, the gaps between liver cells are reduced, and the arrangement is regular ( Figure 3 (C) This indicates that Res can improve the total antioxidant capacity of mice, improve tissue damage in mice, and exert an anti-exercise fatigue effect.

[0054] ③ Under the TEM field of view, the morphology of the extracted sample is a typical disc-shaped structure ( Figure 3 NTA showed that the particle size of the extracted samples was in the range of 30nm-200nm ( Figure 3 B, C). WB verified that the exosome positive proteins Alix, CD63, and CD9 were highly expressed in the extracted samples ( Figure 3 Middle D) indicates that the extracted sample is exosomes.

[0055] ④ Exercise fatigue caused 624 miRNAs to be differentially expressed in myocardial cells, 710 in myocardial tissue, 635 in skeletal muscle cells, 661 in skeletal muscle tissue, 658 in liver cells, and 840 in liver tissue (see Figure 4 ). This indicates that exercise fatigue can cause differential expression of Mcc-Exo, Msmc-Exo, Mlc-Exo, Mct-Exo, Msmt-Exo, and Mlt-Exo miRNAs.

[0056] (5) After Res intervention in the exercise-induced fatigue model, 25.96% of the changes in Mcc exosome miRNAs, 80.85% of the changes in Mct exosome miRNAs, 37.64% of the changes in Msmc exosome miRNAs, 64.90% of the changes in Msmt exosome miRNAs, 17.33% of the changes in Mlc exosome miRNAs, and 81.43% of the changes in Mlt exosome miRNAs induced by exercise fatigue were reversed. The most significantly differentially expressed were Msmc-Exo mmu-miR-486b-3p_R+1 and Msmt-Exo mmu-miR-486b-3p_R+1 ( Figure 5 ) 。

[0057] ⑥qRT-PCR verified the sequencing results, indicating that the sequencing results were credible ( Figure 6 ).

[0058] ⑦After Res intervened in the SF animal model, the expression of exosome mmu-miR-486b-3p_R+1 was upregulated (P<0.0001), indicating that Res can reverse the changes in exosome mmu-miR-486b-3p_R+1 caused by SF. Figure 7 .

[0059] ⑧The results showed that after transfection with mmu-miR-486b-3p_R+1mimics, the expression of mmu-miR-486b-3p_R+1 was upregulated (P<0.0001); after transfection with mmu-miR-486b-3p_R+1inhibitor, the expression of mmu-miR-486b-3p_R+1 was downregulated (P<0.0001), indicating that the transfection was successful ( Figure 8 Middle A). Compared with the SF+Res50μM group, the cell survival rate of the SF+mmu-miR-486b-3p_R+1mimics+Res50μM group was increased (P<0.0001), the ROS and MDA levels were decreased (P<0.05), and the SOD activity was increased (P<0.0001). The cell survival rate of the SF+mmu-miR-486b-3p_R+1inhibitor+Res50μM group was decreased (P<0.0001), the ROS and MDA levels were increased (P<0.0001), and the SOD activity was decreased (P<0.0001). This suggests that the effect of Res in promoting cell proliferation and intervening in sports fatigue decreased after knockdown of exosomal mmu-miR-486b-3p_R+1, but was enhanced after overexpression ( Figure 8 (in BE).

[0060] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of sports fatigue in regulating the expression of exosomal miRNA; The exercise fatigue causes differential expression of exosomal miRNA in cells and / or tissues.

2. The use according to claim 1, characterized in that: The cells include one or more of primary cardiomyocytes, skeletal muscle cells and liver cells; The tissue includes one or more of myocardial tissue, skeletal muscle tissue and liver tissue.

3. The use according to claim 1, characterized in that: The exosomal miRNA includes exosomal mmu-miR-486b-3p_R+1.

4. The use according to claim 1, characterized in that: The cell model of sports fatigue is induced by a hydrogen peroxide solution, the concentration of which is 300 μM; and the animal model of sports fatigue is obtained by an exhaustive swimming experiment.

5. Application of resveratrol in reversing the differential expression of exosomal miRNA in cells and / or tissues caused by exercise-induced fatigue.

6. The use according to claim 5, characterized in that: In the cell experiment, the resveratrol was used at a concentration of 50 μM.

7. The use according to claim 5, characterized in that: In animal experiments, the resveratrol was used at a concentration of 10 mg / kg.

8. The use according to claim 5, characterized in that: The cells include one or more of primary cardiomyocytes, skeletal muscle cells and liver cells; The tissue includes one or more of myocardial tissue, skeletal muscle tissue and liver tissue.

9. The use according to claim 5, characterized in that: The exosomal miRNA includes exosomal mmu-miR-486b-3p_R+1.

10. The use of resveratrol in the preparation of anti-sports fatigue products by reducing cell oxidative damage and / or improving cell antioxidant capacity.

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