Application of timosaponin in preparation of amyotrophy treatment medicine

Abstract

The invention discloses an application of timosaponin in preparation of a medicine for treating amyotrophy. The research finds that on one hand, the timosaponin can enhance the vitality of myoblasts and up-regulate the expression level of GPX4 protein in the myoblasts; when the GPX4 is not knocked out, the timogenin can obviously reduce abnormal expression of cigarette-induced myoblast Atrogin-1 and MuRF-1 proteins, but has no influence after the GPX4 is knocked out; on the other hand, the timogenin can obviously enhance the behavioral movement ability of a cigarette smoke induced amyotrophy model mouse, increase the cross sectional area of muscle fibers and reduce the protein expression of Atrogin-1 and MuRF-1; the experimental results show that the timogenin has a remarkable inhibiting effect on the cigarette smoke induced muscle atrophy, and meanwhile, the timogenin is small in effective dose toxicity, high in activity, low in price and easy to obtain, so that the timogenin is expected to become a potential medicine for clinically treating the muscle atrophy.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to the application of timosaponin in the preparation of a drug for treating muscular atrophy. Background Art

[0002] Skeletal muscle is an important organ responsible for movement and multiple metabolic functions, and plays an important role in maintaining respiration, supporting physical activity, and balancing the homeostasis of glucose, amino acids, and lipids. Skeletal muscle atrophy is the result of protein degradation exceeding protein synthesis due to disease, aging, and lack of physical activity, which manifests as a decrease in the number and size of muscle fibers, resulting in reduced muscle strength.

[0003] At present, there is no FDA-approved drug for the treatment of muscular atrophy, so it is crucial to develop drugs that can improve and treat muscular atrophy. Anemarrhena sapogenin is a biologically active steroidal saponin isolated from the medicinal plant Anemarrhena asphodeloides, which has anti-aging, anti-inflammatory and antioxidant biological activities. At present, there is no report on the use of Anemarrhena sapogenin in the treatment of muscular atrophy. Summary of the invention

[0004] The invention aims to provide the use of timosaponin in the preparation of a drug for treating muscle atrophy, thereby providing an effective way for improving and treating muscle atrophy.

[0005] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0006] Application of sapogenin from Anemarrhena asphodeloides in the preparation of drugs for treating muscular atrophy.

[0007] It is well known that Atrogin-1 and MuRF-1, two E3 ubiquitin ligases, are important regulators of ubiquitin-mediated protein degradation in skeletal muscle. Targeted regulation of their expression may prevent or reverse muscle atrophy. At the same time, glutathione peroxidase 4 (GPX4) increases in expression in highly oxidative muscle fibers and is a key factor in maintaining muscle stability.

[0008] The present invention is the first to study and find that in in vitro cell experiments, timosaponin can enhance the vitality of myoblasts and upregulate the expression level of GPX4 protein in myoblasts; in myoblasts without GPX4 knockout, timosaponin can significantly reduce the abnormal expression of Atrogin-1 and MuRF-1 proteins induced by cigarette smoke, but in myoblasts with GPX4 knockout, timosaponin has no effect on the abnormal expression of Atrogin-1 and MuRF-1 proteins induced by cigarette smoke; and in in vivo experiments, timosaponin can significantly enhance the behavioral motor ability of mice with muscle atrophy model induced by cigarette smoke, increase the cross-sectional area of ​​muscle fibers, and reduce the expression of Atrogin-1 and MuRF-1 proteins; these experimental results all indicate that timosaponin has a significant inhibitory effect on muscle atrophy induced by cigarette smoke, and at the same time, the effective dose of timosaponin has low toxicity, high activity, and is cheap and easy to obtain, making timosaponin a potential drug for the clinical treatment of muscle atrophy.

[0009] In the present invention, the structural formula of the timosaponin is as follows:

[0010]

[0011] Preferably, in the above application, the drug for treating muscle atrophy is a drug for treating skeletal muscle atrophy.

[0012] Compared with the prior art, the technical effects of the present invention are embodied in:

[0013] The present invention studies and finds for the first time that, in an in vitro cell experiment, timosaponin can enhance the activity of myoblasts and upregulate the expression level of GPX4 protein in myoblasts; in myoblasts without GPX4 knockout, timosaponin can significantly reduce the abnormal expression of Atrogin-1 and MuRF-1 proteins induced by cigarette smoke, but in myoblasts with GPX4 knockout, timosaponin has no effect on the abnormal expression of Atrogin-1 and MuRF-1 proteins induced by cigarette smoke; and in an in vivo experiment, timosaponin can significantly enhance the behavioral motor ability of mice with muscle atrophy model induced by cigarette smoke, increase the cross-sectional area of ​​muscle fibers, and reduce the expression of Atrogin-1 and MuRF-1 proteins; these experimental results all indicate that timosaponin has a significant inhibitory effect on muscle atrophy induced by cigarette smoke, and at the same time, timosaponin has low toxicity, high activity, and is cheap and easy to obtain at an effective dose, making timosaponin a potential drug for the clinical treatment of muscle atrophy. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The effect of sapogenin from Anemarrhena asphodeloides on the viability of myoblasts induced by cigarette smoke;

[0015] Wherein, CSE represents cigarette smoke, SAR represents timosaponin, NAC represents N-acetylcysteine, L, M and H represent low, medium and high doses of timosaponin, respectively, Cell viability (% of control) represents cell viability (percentage compared with the control group), the same below;

[0016] Figure 2 The effect of sapogenins on the expression of GPX4 protein in myoblasts induced by cigarette smoke;

[0017] Among them, Relative GPX4 protein expression indicates the relative expression level of GPX4 protein;

[0018] Figure 3 The effect of sapogenins on the expression of Atrogin-1 protein in myoblasts induced by cigarette smoke;

[0019] Among them, Si-GPX4 means that the GPX4 gene was knocked out, Si-NC means that it was not knocked out, and Relative Atrogin-1protein expression means the relative expression level of Atrogin-1 protein; the same below;

[0020] Figure 4 The effect of sapogenins on the expression of MuRF-1 protein in myoblasts induced by cigarette smoke;

[0021] Among them, Relative MuRF-1protein expression indicates the relative expression level of MuRF-1 protein;

[0022] Figure 5 The effect of sapogenins on cigarette smoke-induced grasping force in mice;

[0023] Among them, Relative grip strength refers to relative grip strength;

[0024] Figure 6 The effect of sapogenin from Anemarrhena asphodeloides on the pole climbing ability of mice induced by cigarette smoke;

[0025] Among them, Relative climbing time on pole test indicates the time spent climbing the pole;

[0026] Figure 7 The effect of sapogenins on the balance ability of mice induced by cigarette smoke;

[0027] Among them, Relative hanging endurance means relative hanging endurance;

[0028] Figure 8 The effect of sapogenin from Anemarrhena asphodeloides on the rotarod ability of mice induced by cigarette smoke;

[0029] Among them, Relative time on rotarod represents the relative length of time spent on the rotating rod;

[0030] Fig. 9 The effect of sapogenin on swimming ability of mice induced by cigarette smoke;

[0031] Among them, Relative time of swimming means relative swimming time;

[0032] Fig.10 The effect of sapogenin on the cross-sectional area of ​​muscle fibers in mice induced by cigarette smoke;

[0033] Among them, Muscle fiber CSA represents muscle fiber cross-sectional area, Disturibution represents distribution ratio, CON represents control group, MOD represents model group, L-SAR represents low-dose timosaponin group, H-SAR represents high-dose timosaponin group, and NAC represents N-acetylcysteine ​​group;

[0034] Fig.11 The effect of sapogenin on the expression of GPX4 protein in gastrocnemius muscle of mice induced by cigarette smoke;

[0035] Fig.12 The effect of Anemarrhena sapogenin on the expression of Atrogin-1 protein in gastrocnemius muscle of mice induced by cigarette smoke;

[0036] Fig.13 To investigate the effect of sapogenin from Anemarrhena asphodeloides on the expression level of MuRF-1 protein in the gastrocnemius muscle of mice induced by cigarette smoke. DETAILED DESCRIPTION

[0037] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0038] Example 1 In vitro evaluation of the efficacy of timosaponin in treating muscular atrophy

[0039] In this example, low, medium and high doses of sapogenin (hereinafter referred to as SAR) and N-acetylcysteine ​​(hereinafter referred to as NAC) were used to test myoblasts induced by cigarette smoke extract (hereinafter referred to as CSE) to evaluate the therapeutic effect of SAR on muscle atrophy. The specific operation steps are as follows: (1) Preparation of cigarette smoke extract: burn an unfiltered Marlboro cigarette and allow the smoke to pass through 1 mL PBS. After filtering through a 0.22 μm microporous filter to remove particles and bacteria, the pH was adjusted to 7.00-7.40, which was defined as 100% CSE. (2) Cell grouping and administration: myoblasts were cultured in DMEM culture medium containing 10% fetal bovine serum and incubated in a 37°C incubator. When the cells grew to 60-70% confluence, they were divided into a blank group, a CSE group, a SAR low-dose group, a SAR medium-dose group, a SAR high-dose group and a NAC group and treated for 24 hours, and then the corresponding cell experiments were performed.

[0040] In this embodiment, all experimental processes were repeated 6 times, and the data were statistically analyzed and plotted using GraphPad Prism 8.0.1 software. The difference analysis used Student's T test or one-way ANOVA, and the values ​​were expressed as "mean ± SD". The test result was considered to be statistically significant when P < 0.05.

[0041] 1. Preparation of in vitro models

[0042] C1C12 myoblasts were divided into a blank group (CON), a model group (1% CSE), a low-dose group of timosaponin (1% CSE, SAR 10 -2 μM), medium dose group of timosaponin (1% CSE, SAR 10 -1 μM), high-dose timosaponin group (1% CSE, SAR 1 μM) and N-acetylcysteine ​​group (1% CSE, NAC 1 mM), for a total of 6 groups.

[0043] 2. Cell viability detection

[0044] The viability of myocytes in each group was detected using an MTT kit, and the absorbance was recorded at 490 nm using an enzyme marker. The results are shown in Figure 1 shown.

[0045] Depend on Figure 1 It can be seen that compared with the control group, CSE significantly reduced the viability of myocardial cells in the model group; however, SAR effectively reversed the decrease in cell viability in a dose-dependent manner, and NAC also significantly increased cell viability (*P<0.05, **P<0.01 represent the comparison between each group and the CSE group). #P < 0.05, representing the comparison between SAR-H group and NAC group).

[0046] 3. Immunoblotting to detect the expression levels of GPX4, Atrogin-1 and MuRF-1 proteins

[0047] On the one hand, each group of myocytes was lysed with RIPA buffer, and the protein concentration was determined using a BCA kit. Then, 30 μg of protein samples were dissolved in a 10-12% SDS polyacrylamide gel and electrotransferred to a 0.45 μm PVDF membrane. The membrane was soaked in 5% bovine serum albumin and incubated at room temperature for 1 hour, and then incubated with the primary antibody at 4°C overnight. After washing with TBST, it was incubated with a secondary antibody of goat anti-rabbit IgG HRP (1:5000) for 1 hour. The primary antibodies used are as follows: GPX4 (1:1000). The protein bands were detected by enhanced chemiluminescence ECL and visualized using the ChemiDoc MP imaging system; the results are shown in Figure 2. Figure 2 shown.

[0048] On the other hand, the GPX4 gene knockout experiment was performed on the blank group (CON), model group (1% CSE), and high-dose group of timosaponin (1% CSE, SAR 1 μM), and then the expression levels of Atrogin-1 and MuRF-1 proteins in cells before and after GPX4 gene knockout were detected by the same method as above. The primary antibodies used were as follows: Atrogin-1 (1:1000) and MuRF-1 (1:1000); the results are shown in Figure 2. Figure 3 and Figure 4 shown.

[0049] Figure 2 The results of immunoblotting showed that the expression level of GPX4 protein in the model group cells was lower than that in the control group, while both SAR and NAC significantly increased the decrease in GPX4 expression level induced by CSE exposure (*P<0.05, **P<0.01 represents the comparison between each group and the CSE group).

[0050] Figure 3 and Figure 4 The results of immunoblotting showed that in the cell group without GPX4 knockout, SAR could restore the up-regulation of the expression levels of muscle atrophy-related proteins MuRF-1 and Atrogin-1 induced by CSE exposure; however, after GPX4 knockout, SAR treatment no longer had a restorative effect on the expression of these proteins (*P<0.05, **P<0.01 represents a comparison with the si-NC+CSE group, $ P<0.05, $$ P<0.01 represents the comparison between si-GPX4+ control group and si-NC+ control group, # P<0.05, ##P<0.01 represents the comparison between si-GPX4+CSE+SAR group and si-NC+CSE+SAR group).

[0051] The above results show that SAR can enhance the cell viability of myoblasts and achieve the purpose of treating CSE-induced muscle atrophy by upregulating the level of GPX4 protein. In addition, SAR can restore the levels of muscle atrophy factors MuRF-1 and Atrogin-1 in CSE-induced myoblasts, but knocking out GPX4 can eliminate the above effects. These results show that SAR can improve and treat muscle atrophy, and GPX4 is a key regulator of myogenic differentiation.

[0052] Example 2 In vivo efficacy evaluation of timosaponin in the treatment of muscular atrophy

[0053] In this example, low and high doses of timosaponin were used to conduct experiments on C57BL / 6 male mice induced by cigarette smoke to evaluate the therapeutic effect of timosaponin on muscle atrophy.

[0054] In this embodiment, all experimental processes were repeated at least 3 times, and the data were statistically analyzed and plotted using GraphPad Prism 8.0.1 software. The difference analysis used Student's T test or one-way ANOVA, and the values ​​were expressed as "mean ± SD". The test result was considered to be statistically significant when P < 0.05.

[0055] 1. Preparation of animal model

[0056] C57BL / 6 male mice were randomly divided into five groups: blank group (PBS, nasal drip 50 μL / d), model group (100% CSE, nasal drip 50 μL / d), low-dose timosaponin group (100% CSE, nasal drip 50 μL / d; SAR, intragastric administration 5 mg / kg / d), high-dose timosaponin group (100% CSE, nasal drip 50 μL / d; intragastric administration SAR 10 mg / kg / d) and N-acetylcysteine ​​group (100% CSE, nasal drip 50 μL / d; intragastric administration NAC 100 mg / kg / d), with 8 C57BL / 6 mice in each group, five days a week for six weeks.

[0057] 2. Grip test

[0058] The grip strength test was used to test the grip strength of mice: the mice's forelimbs were placed flat on the grip strength test bar and the mice's tails were lifted. Next, the mice's tails were pulled uniformly until they released the grip strength test bar. The measurement was repeated 3 times and the average value was recorded. The results were as follows: Figure 5 shown.

[0059] 3. Pole climbing test

[0060] The climbing pole experiment was used to test the motor coordination ability of mice: a rough wooden pole (50 cm long, 8 mm in diameter) was fixed vertically on the ground, and then the mouse was placed on the top of the pole. The time it took for the mouse to reach the ground from the top was measured. The measurement was repeated 3 times and the average value was recorded. The results are shown in Table 1. Figure 6 shown.

[0061] 4. Balance test

[0062] The hanging test can test the mouse's motor abilities such as grip, coordination and endurance. Fix the two ends of a metal wire (50 cm long, 2 mm in diameter) on a platform 37 cm above the ground, slowly place the mouse in the middle of the wire, and let the mouse's forelimbs grab the wire. Record the time it takes for the mouse to reach the two platforms within 30 seconds, and record the mouse's score. Figure 7 shown.

[0063] The specific scores are as follows: 0 points: the mouse falls; 1 point: the mouse grabs the wire with both forelimbs; 2 points: the mouse grabs the wire with both forelimbs and struggles upward; 3 points: the mouse grabs the wire with both forelimbs and one or both hind limbs; 4 points: the mouse grabs the wire with all four limbs and the tail; 5 points: the mouse successfully climbs to any platform at either end within 30 seconds.

[0064] 5. Rotarod test

[0065] The rotarod test can be used to assess muscle endurance, coordination, and balance. Before the experiment, mice were first subjected to 60 seconds of adaptive training. During the formal experiment, the rotation speed was accelerated from 4 rpm to 40 rpm for 10 minutes. When the mouse fell, the time of the mouse falling was recorded. If the mouse did not fall from the rotating rod within 10 minutes, the test was stopped and the falling time was recorded as 600 seconds. Each mouse was repeated 3 times and the average value was taken. The results are shown in Figure 8 shown.

[0066] 6. Weighted swimming test

[0067] The weighted swimming test is used to test the exercise endurance and fatigue resistance of mice: prepare a water tank of about 50cm*50cm*40cm, fill it with water at about 28-30℃, tie a weight (7% of the mouse's body weight) to the base of the mouse's tail with tape, put 4 mice into the water tank at the same time, and start timing. When the mouse sinks into the water and does not float to the surface for more than 5s, stop timing, fish out the mouse, and record the mouse's swimming time. The results are as follows: Fig. 9 shown.

[0068] from Figures 5 to 9It can be seen that compared with the control group, the grip strength, pole climbing, hanging endurance, rotation ability and swimming ability of the model group were significantly decreased; compared with the model group, the grip strength of the CSE+SAR-L group was improved, and the grip strength, hanging endurance, pole climbing time, rotation time and swimming time of the CSE+SAR-H and CSE+NAC groups were significantly improved; and the grip strength and pole climbing time of the CSE+SAR-H group were higher than those of the CSE+NAC group (*P<0.05, **P<0.01 represent the comparison between each group and the CSE group, # P<0.05, ## P < 0.01 represents the comparison between CSE + SAR-H group and CSE + NAC group).

[0069] 7. Histochemical analysis of skeletal muscle

[0070] After the mice were sacrificed, gastrocnemius muscle samples were taken and fixed with 4% paraformaldehyde, embedded in paraffin, and stained with hematoxylin and eosin according to standard staining procedures. Images of the stained sections were then captured under a light microscope. The muscle fiber cross-sectional area (CSA) of each mouse was measured using Image J software, and the results are shown in Figure 2. Fig.10 shown.

[0071] Depend on Fig.10 HE staining results of gastrocnemius muscle of mice showed that the cross-sectional area of ​​muscles in CSE group was concentrated in the range of 400-800 μm. 2 The muscle fiber area of ​​the CSE+SAR-L group was 800-1200 μm 2 In addition, the CSE+SAR-H and CSE+NAC groups showed comparable effects to the control group, with muscle fiber area distribution ranging from 1200 to 1600 μm 2 range (*P<0.05, **P<0.01 represent the comparison between each group and CSE group).

[0072] 8. Immunoblotting to detect the expression levels of GPX4, Atrogin-1 and MuRF-1 proteins

[0073] Gastrocnemius muscle was lysed with RIPA buffer, and protein concentration was determined using a BCA kit. Then, 30 μg of protein sample was dissolved in a 10-12% SDS polyacrylamide gel and electrotransferred to a 0.45 μm PVDF membrane. The membrane was soaked in 5% bovine serum albumin and incubated at room temperature for 1 hour, and then incubated with the primary antibody at 4°C overnight. After washing with TBST, it was incubated with a secondary antibody of goat anti-rabbit IgG HRP (1:5000) for 1 hour. The primary antibodies used were as follows: GPX4 (1:1000), Atrogin-1 (1:1000), and MuRF-1 (1:1000). Protein bands were detected by enhanced chemiluminescence ECL and visualized using the ChemiDoc MP imaging system; the results are shown in Figure 2. Fig.11 , Fig.12 and Fig.13 shown.

[0074] Fig.11 Western blotting results showed that compared with the control group, the expression of GPX4 in the CSE group was significantly decreased, while SAR significantly upregulated the expression of this protein (*P<0.05, **P<0.01 represent the comparison between each group and the CSE group).

[0075] like Fig.12 and Fig.13 As shown, the expression of MuRF-1 and Atrogin-1 proteins in the CSE group was significantly increased, while SAR could significantly restore the CSE-induced MuRF-1 and Atrogin-1 protein expressions to normal levels (*P<0.05, **P<0.01 represent the comparison between each group and the CSE group).

[0076] The above experimental results show that SAR can improve the grasping strength, climbing pole, hanging endurance, rotation ability and swimming ability of CSE-induced mice, and can increase muscle fiber area by upregulating the expression of GPX4 and reducing the expression of muscle atrophy factors in CSE-induced muscles, thereby increasing muscle mass and improving muscle atrophy.

Claims

1. Application of Anemarrhena sapogenin in the preparation of drugs for the treatment of muscular atrophy.

2. The use according to claim 1, characterized in that The structural formula of the described timosaponin is as follows:

3. The use according to claim 1, characterized in that The muscle atrophy therapeutic drug is a skeletal muscle atrophy drug.

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