Application of liquiritigenin in preparation of medicine for relieving muscle injury caused by exercise fatigue
Glycyrrhizin addresses muscle damage caused by exercise fatigue by regulating PI3K/Akt protein expression and inhibiting inflammatory factors and Caspase 3, thus significantly improving the exercise capacity and endurance of mice.
Patent Information
- Application Number
- CN202511243858.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-02
- Publication Date
- 2025-11-04
AI Technical Summary
Current technologies have failed to effectively mitigate muscle damage caused by exercise fatigue, especially the expression of inflammatory factors and cell apoptosis in muscles during exercise.
Glycyrrhizin slows down muscle damage by affecting the expression of PI3K/Akt protein, inhibiting the expression of inflammatory factors such as IL-1β, IL-6, and TNF-α, and suppressing the expression of Caspase 3.
Glycyrrhizin significantly prolonged the time to exhaustion, enhanced endurance, reduced muscle damage, improved muscle tissue structure, reduced the concentration of inflammatory factors, and inhibited muscle cell apoptosis in mouse models.
Smart Images
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the field of Liquiritigenin application, and particularly relates to application of Liquiritigenin in preparation of a medicine for relieving muscle injury caused by exercise fatigue. BACKGROUND
[0002] Liquiritigenin is a representative component of dihydroflavones isolated from licorice plants, and is a flavone of licorice. At present, a plurality of compounds have been isolated from flavones of licorice, mainly including isoflavones, liquiritin, Liquiritigenin and the like. Among them, Liquiritigenin is a main active component of flavones of licorice. Liquiritigenin has pharmacological effects of eliminating free radicals, resisting oxidation, resisting tumors, participating in enzyme reactions, resisting bacteria, protecting liver, estrogen-like effects, resisting rheumatism and resisting mental depression. SUMMARY
[0003] The application aims to provide a new use of Liquiritigenin, i.e. application of Liquiritigenin in skeletal muscle injury.
[0004] The technical scheme adopted by the application to solve the technical problem is as follows:
[0005] Application of Liquiritigenin in preparation of a medicine for relieving muscle injury caused by exercise fatigue.
[0006] Preferably, Liquiritigenin relieves muscle injury caused by exercise fatigue by inhibiting expression of inflammatory factors.
[0007] Preferably, the inflammatory factors are at least one of IL-1β, IL-6 and TNF-α.
[0008] Preferably, Liquiritigenin inhibits expression of inflammatory factors by affecting expression of PI3K / Akt protein.
[0009] Preferably, Liquiritigenin up-regulates expression of PI3K / Akt protein.
[0010] Preferably, Liquiritigenin relieves muscle injury caused by exercise fatigue by inhibiting apoptosis of muscle cells.
[0011] Preferably, Liquiritigenin inhibits apoptosis of muscle cells by affecting expression of PI3K / Akt protein and inhibiting expression of Caspase 3.
[0012] Preferably, Liquiritigenin up-regulates expression of PI3K / Akt protein.
[0013] Glycyrrhizin can alleviate muscle damage caused by exercise-induced fatigue in two ways. On the one hand, glycyrrhizin can alleviate muscle damage caused by exercise-induced fatigue by up-regulating the PI3K / Akt protein ratio and reducing the expression of inflammatory factors. On the other hand, glycyrrhizin can inhibit muscle cell apoptosis by up-regulating the PI3K / Akt protein ratio and inhibiting the expression of Caspase 3. In mice, glycyrrhizin can further prolong the exhaustive exercise time of mice, enhance the endurance of mice, and improve the exercise capacity of mice on the basis of improving the endurance of mice through exercise. BRIEF DESCRIPTION OF DRAWINGS
[0014] Figure 1 is a schematic diagram of the forelimb and hindlimb grip strength results of the mice in groups A, B and C.
[0015] The comparison between groups A and B is p<0.001, and the comparison between groups C and A is p<0.001.
[0016] Figure 2 is the determination result of IL-1β content in the serum (a), ileum (b) and skeletal muscle (c) of mice.
[0017] In figure (a), the comparison between groups A and C is p<0.0001, and the comparison between groups D and C is p<0.001; in figure (b), the comparison between groups C and A is p<0.0001, and the comparison between groups D and C is p<0.001; in figure (c), the comparison between groups C and A is p<0.0001, and the comparison between groups D and C is p<0.001.
[0018] Figure 3 is the determination result of IL-6 content in the serum (a), ileum (b) and skeletal muscle (c) of mice.
[0019] In figure (a), the comparison between groups C and A is p<0.001, and the comparison between groups D and C is p<0.001; in figure (b), the comparison between groups C and A is p<0.001, and the comparison between groups D and C is p<0.001; in figure (c), the comparison between groups C and A is p<0.0001, and the comparison between groups D and C is p<0.001.
[0020] Figure 4 is the determination result of TNF-α content in the serum (a), ileum (b) and skeletal muscle (c) of mice.
[0021] In figure (a), the comparison between groups C and A is p<0.001, and the comparison between groups D and C is p<0.001; in figure (b), the comparison between groups C and A is p<0.001, and the comparison between groups D and C is p<0.05; in figure (c), the comparison between groups C and A is p<0.001, and the comparison between groups D and C is p<0.001.
[0022] Figure 5Effect of Liquiritigenin on the Histopathological Changes of Skeletal Muscle in Mice. (Mean ± SD, n = 3, Magnification, 20.0x)
[0023] Figure 6 Effect of Liquiritigenin on the Immunofluorescence Sections of Skeletal Muscle in Mice. (Mean ± SD, n = 3, Magnification, 63.0x) DETAILED DESCRIPTION
[0024] The technical solutions and technical effects of the present application are further described in detail below in combination with the drawings of the present application.
[0025] I. Experimental Animals: The experimental animals were purchased from the Experimental Animal Center of Ningxia Medical University, male C57 BL / 6J, four weeks old, body weight (20 g ± 2 g), SPF level, feeding conditions including standard feed, pure water, room temperature maintained at (24 ± 2) °C, humidity at 50%-60%.
[0026] II. Test Method
[0027] 1. Preparation of Test Drugs
[0028] The preparation of the liquiritigenin solution was performed every three days according to the actual weight of the test mice, and the prepared solution was stored in a 4 °C refrigerator for storage and standby. The specific preparation method of the liquiritigenin solution was as follows: 1 mg of liquiritigenin was added to 1 m of 0.5% carboxymethylcellulose sodium physiological saline.
[0029] 2. Test Grouping
[0030] Sixty male test mice, four weeks old, body weight (20 ± 2) g, SPF level, were normally raised. After three days of adaptive feeding and daily adaptive swimming for 20 min, the mice that could not swim were eliminated, and then they were randomly divided into four groups according to the body weight, i.e., A (quiet control group contol), B (quiet exhaustion control group), C (fatigue model group model), and D (liquiritigenin administration group liquiritigenin), with 15 mice in each group.
[0031] According to the daily weight change of the test mice, the test mice in each group were administered at 1:00 pm every day. The mice in group D were given the liquiritigenin solution by gavage, and the gavage amount was 10 mg per kilogram of body weight. The amount of liquiritigenin in the 10 mg / kg liquiritigenin solution was 10 mg. The mice in groups A, B, and C were respectively given the same volume of 0.5% carboxymethylcellulose sodium solution by gavage, and the gavage was continuously performed for six weeks. The clean bedding was changed every day, the clean water bottle was changed every day, and the body weight of the mice was measured and recorded every day.
[0032] 3. Mouse Grip Strength Test
[0033] Group A was only given intragastrically without grip training, and groups C and D were given grip training at the sixth week of drug administration. Grip training was performed at 2:00 pm every day. The mice were placed on the grid, and the tail of the experimental mouse was gently pulled to ensure that the mouse gripped the top of the grid and the torso remained horizontal. The mouse was pulled until it could not maintain the grip, and the metal bar fell off the grip. The maximum grip strength of the mouse on the screen was recorded. This process was repeated five times, with a 3-minute rest interval between each test. The grip test was performed on the seventh day of training, and the average performance of the mice was expressed as the average of three grip tests.
[0034] 4. Exhaustive swimming test in mice
[0035] After the grip test, the mice in groups B, C, and D were given exhaustive swimming tests three days later. The mice were placed in a swimming tank with a water depth of 30 cm and a water temperature of 25 ± 2°C. The exhaustive criteria were that the mouse's head was completely submerged in water for 10 seconds and could not float to the surface, and the mouse could not complete the righting reflex when placed on a flat surface. After exhaustion, the mouse was quickly pulled out of the water and dried with a towel.
[0036] 5. Mouse sampling
[0037] On the second day after exhaustion, groups C and D were given 100 minutes of swimming training. Blood was collected from the mice in groups A, B, C, and D by enucleation. The samples were incubated at room temperature for 2 hours, centrifuged at 3000 rpm and 4°C for 15 minutes, and the supernatant was stored at -80°C. The skeletal muscle and proximal ileum of the mouse were dissected and cleaned with physiological saline. The physiological saline was absorbed with filter paper, and the sample was then quickly frozen in liquid nitrogen and stored in a -80°C freezer for long-term preservation.
[0038] III. Main indicators
[0039] 1. Exhaustion time of mice (Table 1)
[0040]
[0041] Note: P < 0.001 for group C compared to group B, and P < 0.05 for group D compared to group C.
[0042] As shown in Table 1, the exhaustive swimming time of the mice in the glycyrrhizin administration group was longer than that of the exhaustive control group and the fatigue model group. The exhaustive swimming time of the fatigue model group was longer than that of the exhaustive control group because the grip test increased the endurance of the mice. The exhaustive swimming time of the mice in the glycyrrhizin administration group was longer than that of the fatigue model group. Therefore, glycyrrhizin solution can further prolong the exhaustive swimming time of mice based on the improvement of their own endurance, enhance their endurance, and thus improve their exercise capacity.
[0043] 2. Forelimb and hindlimb grip strength of mice
[0044] From Figure 1 It can be seen that the grip strength / body weight ratio of the fatigue model group test mice is larger than that of the quiet control group, and it can be seen that the periodic training can enhance the grip strength of the mice. The grip strength / body weight ratio of the glycyrrhizin administration group test mice is larger than that of the fatigue model group, and it can be seen that the glycyrrhizin solution can further increase the forelimb and hindlimb grip strength of the mice on the basis of periodic training, therefore, it is shown that glycyrrhizin can improve the endurance of mice, increase the grip strength of mice, and thus enhance the exercise capacity of mice.
[0045] III. ELISA kit detection of inflammatory factors IL-1β, IL-6 and TNF-α
[0046] In long-term strenuous exercise, energy metabolism disorder and oxidative stress damage will occur. Therefore, acute exercise-induced fatigue will cause mechanical damage to muscles. Mechanical tissue damage will cause pro-inflammatory response. Endurance exercise will lead to the systemic release of interleukin 6 and 8 and tumor necrosis factor-a (TNF-a), thereby initiating the recruitment of neutrophils and monocytes.
[0047] 1. Sample processing
[0048] The sample was taken out from the-80℃ refrigerator, thawed on ice, and then the tissue sample was rinsed with PBS to remove residual blood, and the water was wiped dry with gauze. The mouse skeletal muscle tissue and ileum tissue were weighed respectively. The tissue sample was cut into small pieces with surgical scissors and placed in a pre-cooled grinding rod. According to the ratio of 1g tissue to 9ml, the corresponding PBS was added to the grinding rod, and the grinding was carried out on ice. The grinding liquid was transferred to a centrifuge tube, and the supernatant was obtained by centrifugation at 2500xg for 15min.
[0049] 3. Kit detection
[0050] According to the ELISA kit instructions, the absorbance was detected by enzyme-labeled instrument, the standard curve was drawn, and the concentration of IL-1β, IL-6 and TNF-α was calculated.
[0051] 4. Expression level of IL-1β, IL-6 and TNF-α
[0052] After strenuous exercise, the body will be damaged, causing inflammatory infiltration of cells. TNF-α is a pro-inflammatory factor, which is highly expressed when the tissue is damaged, leading to inflammatory cell aggregation and inflammation. From Figure 2 、 Figure 3 and Figure 4 It can be concluded that compared with the quiet control group, the concentration of inflammatory factors IL-1β, IL-6 and TNF-α in the peripheral serum, ileum and skeletal muscle tissue of the fatigue model group is significantly increased, which indicates that the fatigue model group is successfully modeled.
[0053] Compared with the fatigue model group, the concentration of inflammatory factors IL-1β, IL-6 and TNF-α in peripheral serum, ileum and skeletal muscle tissue of glycyrrhizin administration group was significantly reduced; it is proved that glycyrrhizin can improve the increase of systemic and local inflammatory factors caused by fatigue state, thereby having the effect of reducing muscle damage.
[0054] 5. Skeletal muscle HE staining
[0055] The skeletal muscle tissue fixed with 4% paraformaldehyde was embedded with conventional paraffin, sectioned and HE stained, and the pathological changes of the skeletal muscle tissue were observed under an optical microscope.
[0056] The HE results are shown in Figure 5 As shown in the table, the skeletal muscle of the mice in group C appeared different degrees of damage, and the muscle fiber arrangement was destroyed. Compared with group C, the skeletal muscle damage of group D was obviously improved, and the fiber arrangement was uniform. It can be concluded that glycyrrhizin can reduce muscle damage caused by exercise fatigue.
[0057] Four, skeletal muscle immunofluorescence staining
[0058] PI3K / Akt can regulate the NF-κB pathway / transcription factor family of inflammation, cell stress and innate and adaptive immune response. After NF-κB is activated, it can transcribe hundreds of genes containing κB binding sites, which are involved in inflammation, immune and cell survival regulation. In addition to inhibiting the expression of inflammatory factors such as IL-1β, IL-6 and TNF-α, it also participates in the expression of pro-apoptotic gene Bax to cause apoptosis. In apoptosis, Bax and anti-apoptotic gene Bcl-2 regulate the expression of key apoptosis molecules Caspase-3 by inhibiting or promoting the release of mitochondrial cytochrome C, thereby determining cell survival.
[0059] 1. Experimental operation
[0060] The skeletal muscle specimen section (paraffin section) was microwaved in potassium citrate antigen repair solution for 10 min at 98℃, and then cooled to room temperature and soaked in 0.5% Tween-20 for 15 min, and PBS was washed for 3 times, 5 min each time. A hydrophobic pen was used to draw a circle around the tissue, and an appropriate amount of skimmed milk was added to the circle for blocking, and incubated at room temperature for 30 min. The primary antibody (rabbit) was incubated at 4℃ overnight or at room temperature for 1 h, and TBST was washed for 3 times, 5 min each time. Under light-proof conditions, FITC-labeled fluorescent secondary antibody (anti-rabbit Ig G) was added and incubated at room temperature for 1 hour, and TBST was washed for 3 times, 5 min each time, and then distilled water was washed once. Under light-proof conditions, DAH was stained for 5 min, 90% glycerol was used for mounting, and the fluorescence images were collected using a fluorescence microscope, and the fluorescence intensity of PI3K, Akt and Caspase3 was analyzed using Image J, and the results are shown in Figure 6As shown.
[0061] Figure 6 It is shown that glycyrrhizin can up-regulate the ratio of PI3K / Akt protein, inhibit the expression of Caspase 3, so as to inhibit the apoptosis of skeletal muscle cells, reduce inflammatory factors, and reduce muscle damage caused by exercise fatigue.
[0062] In summary, glycyrrhizin reduces muscle damage caused by exercise fatigue from two aspects. On the one hand, glycyrrhizin can reduce inflammatory factor expression by up-regulating the ratio of PI3K / Akt protein, thereby reducing muscle damage caused by exercise fatigue. On the other hand, glycyrrhizin can inhibit muscle cell apoptosis by up-regulating the ratio of PI3K / Akt protein and inhibiting the expression of Caspase 3. In mice, glycyrrhizin can further prolong the exhaustive exercise time of mice, enhance endurance, and improve the exercise capacity of mice on the basis of improving their own endurance through exercise.
Claims
1. Application of glycyrrhizin in the preparation of drugs to alleviate muscle damage caused by exercise fatigue.
2. The application of glycyrrhizin according to claim 1 in the preparation of a drug for alleviating muscle damage caused by exercise fatigue, characterized in that: Glycyrrhizin reduces muscle damage caused by exercise fatigue by inhibiting the expression of inflammatory factors.
3. The application of glycyrrhizin in alleviating exercise-induced muscle damage according to claim 2, characterized in that, The inflammatory factor is at least one of IL-1β, IL-6, and TNF-α.
4. The application of glycyrrhizin according to claim 2 in alleviating exercise-induced muscle damage, characterized in that, Glycyrrhizin inhibits the expression of inflammatory factors by affecting the expression of PI3K / Akt proteins.
5. The application of glycyrrhizin according to claim 4 in alleviating exercise-induced muscle damage, characterized in that, Glycyrrhizin upregulates PI3K / Akt protein expression.
6. The application of glycyrrhizin according to claim 1 in alleviating exercise-induced muscle damage, characterized in that, Glycyrrhizin slows down muscle damage caused by exercise fatigue by inhibiting muscle cell apoptosis.
7. The application of glycyrrhizin according to claim 6 in alleviating exercise-induced muscle damage, characterized in that, Glycyrrhizin inhibits muscle cell apoptosis by affecting PI3K / Akt protein expression and inhibiting Caspase 3 expression.
8. The application of glycyrrhizin according to claim 7 in alleviating exercise-induced muscle damage, characterized in that, Glycyrrhizin upregulates PI3K / Akt protein expression.