Application of salvianolic acid B in preparation of medicine for treating aging-related muscle hypofunction

Abstract

The invention discloses application of salvianolic acid B in preparation of a medicine for treating aging-related muscle hypofunction. The research finds that the salvianolic acid B has a remarkable effect of improving the aging-related muscle hypofunction. As a natural product, the salvianolic acid B has higher safety and fewer side effects, and is milder and safer compared with certain drugs for treatment. Secondly, compared with long-term physical treatment and nutritional supplement, the treatment mode of the salvianolic acid B is simpler and more convenient, and patients can accept and follow the treatment scheme more easily. Therefore, the invention provides the application of the salvianolic acid B in preparing the medicine for treating the aging-related muscle hypofunction, and the mechanism of the salvianolic acid B is possibly related to inflammatory response reduction and autophagy change promotion. The discovery that salvianolic acid B improves aging-related muscle hypofunction adds a new possibility for the lack of sarcopenia treatment means at present.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular, relates to the application of salvianolic acid B in the preparation of a medicine for treating aging-related muscle dysfunction. Background Art

[0002] Sarcopenia is also clinically referred to as "skeletal muscle aging" or "sarcopenia". Among them, aging-related muscle function decline is an important feature of the occurrence and development of sarcopenia. Therefore, improving aging-related muscle function decline is the key to treating sarcopenia. Among them, aging-related muscle function decline may be caused by multiple factors, including increased inflammatory response and impaired cell autophagy. Senescent cell-associated secretory phenotype (SASP) refers to a series of extracellular factors released by aging cells, including cytokines, growth factors, proteases, extracellular matrix proteins, etc. At present, studies have shown that inhibiting the senescent cell-associated secretory phenotype (SASP) can reduce the inflammatory response of muscle tissue, promote the regeneration and repair of muscle cells, and thus slow down the progression of sarcopenia. In addition, promoting intracellular autophagy function is also considered to be a potential therapeutic strategy. TP53INP2 is an important autophagy regulator in the autophagy process. Studies have found that chronic activation of autophagy by muscle-specific overexpression of TRP53INP2 can prevent sarcopenia and muscle function decline in mice. Therefore, promoting autophagy in senescent cells is also an important therapeutic strategy to improve aging-related muscle function decline. At present, there is a lack of treatment options for sarcopenia, which are mainly physical therapy, nutritional supplements and a small amount of drug therapy. Physical therapy and nutritional supplements require long-term treatment and are difficult to adhere to. Long-term nutritional supplements may increase the burden on the kidneys of the elderly, and drug treatments such as growth hormone may cause adverse side effects. Long-term safety and effectiveness need further study.

[0003] Salvianolic acid B is the main effective water-soluble component of Salvia miltiorrhiza, and has pharmacological properties such as antioxidant, anti-inflammatory and cardiovascular protection. Although people have conducted many studies on the uses and mechanisms of action of salvianolic acid B, no studies have shown that it has the effect of improving aging-related muscle dysfunction. Patent CN115414409A shows that salvianolic acid A can relieve muscle atrophy, myopathy and musculoskeletal complications caused by diabetes, but salvianolic acid A and salvianolic acid B are two active substances with different structures. At the same time, it targets muscle dysfunction caused by diabetes, not muscle dysfunction caused by aging. Muscle dysfunction caused by other factors may be the result of abnormal changes in the body, and the treatment plan is also to treat the specific causes of these changes, rather than directly treating muscle loss itself. Muscle dysfunction related to aging usually occurs gradually with normal aging, and can be improved by increasing nutritional intake and doing moderate exercise. Therefore, treatment methods for other pathological factors may not be suitable for treating muscle dysfunction associated with normal aging. Summary of the invention

[0004] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art and provide the use of salvianolic acid B in the preparation of a drug for treating aging-related muscle dysfunction.

[0005] The above-mentioned object of the present invention is achieved through the following technical solutions: The present invention has found that salvianolic acid B has a series of anti-aging effects, such as reducing the SASP of senescent cells, promoting autophagy of senescent cells, reducing the level of ROS in senescent cells, and increasing the ATP of senescent cells. Salvianolic acid B and its decomposition products can inhibit the expression of genes related to the senescence-associated secretory phenotype (SASP); in subsequent animal experiments, it was also found that salvianolic acid B improves the morphological function of skeletal muscle and the body's motor ability. It has a significant effect on improving aging-related muscle dysfunction, and its mechanism may be related to the fact that salvianolic acid B can reduce the SASP of senescent cells and promote the autophagy of senescent cells. The weakening of autophagic ability is one of the causes of muscle atrophy, and promoting cell autophagy is the key to improving muscle function. The present invention believes that salvianolic acid B not only has the effect of improving the phenotype of cell aging, but also has the effect of directly improving the symptoms of muscle dysfunction. This discovery adds a new possibility to the current lack of treatment methods for sarcopenia, and compared with some drug treatments, the application of salvianolic acid B to treat aging-related muscle dysfunction has higher safety and fewer side effects. Secondly, compared with long-term physical therapy and nutritional supplementation, it is relatively simple and easy, and patients can more easily accept and follow the treatment plan.

[0006] Therefore, the present invention provides the use of salvianolic acid B and / or its active hydrolyzate in the preparation of a drug for treating aging-related muscle dysfunction.

[0007] The present invention also provides the use of salvianolic acid B and / or its active hydrolyzate in the preparation of medicines for treating diseases caused by aging-related muscle dysfunction.

[0008] The above-mentioned treatment refers to intervention after the symptoms or diseases occur to eliminate or control the symptoms or diseases.

[0009] Furthermore, the active hydrolysis product is sodium danshensu (SDSS) and / or rosmarinic acid (RA).

[0010] Furthermore, the disease caused by aging-related muscle dysfunction is sarcopenia.

[0011] Furthermore, the drug treats aging-related muscle dysfunction by improving skeletal muscle morphology, skeletal muscle function and / or body motor ability.

[0012] Furthermore, the drug can treat aging-related muscle dysfunction by activating cell and tissue autophagy activity and reducing the production of inflammatory factors. The drug can upregulate TP53INP2 and promote cell autophagy, thereby directly improving muscle function.

[0013] Furthermore, the effective dose of salvianolic acid B in the medicine is not less than 25 mg / Kg.

[0014] Furthermore, the medicine also contains pharmaceutically acceptable excipients.

[0015] Preferably, the auxiliary material is selected from any one or more of an excipient, a diluent, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent or a preservative.

[0016] Furthermore, the dosage form of the drug is any one of injection, oral solution, capsule, tablet or granule.

[0017] Preferably, the pharmaceutical dosage form is an injection.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides the use of salvianolic acid B and its active hydrolyzate in the preparation of drugs for treating aging-related muscle dysfunction. The present invention has found that salvianolic acid B has the functions of reducing SASP of senescent cells, promoting autophagy of senescent cells, reducing ROS levels in senescent cells and increasing ATP of senescent cells, improving skeletal muscle morphology and function and body movement ability, and has a significant effect on improving aging-related muscle dysfunction. As a natural product, salvianolic acid B has high safety and fewer side effects, and is milder and safer than certain drug treatments. Secondly, the treatment method of salvianolic acid B is relatively simple and easy compared with long-term physical therapy and nutritional supplements, and patients can more easily accept and follow the treatment plan. Therefore, the present invention proposes the use of salvianolic acid B in the preparation of drugs for treating aging-related muscle dysfunction, and its mechanism may be related to reducing inflammatory response and promoting autophagy. The discovery that salvianolic acid B improves aging-related muscle dysfunction adds a new possibility to the current lack of treatment methods for sarcopenia. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the result of in vitro cell experiments to explore the effect of salvianolic acid B on senescent cells; Figure 1 A shows the process of conversion of salvianolic acid B into sodium danshensu and rosmarinic acid, B-D show the effects of different concentrations of salvianolic acid B, sodium danshensu and rosmarinic acid on cell viability, E-G show the effects of different concentrations of salvianolic acid B, sodium danshensu and rosmarinic acid on the expression of SASP-related genes in senescent cells, H-J show the effects of salvianolic acid B treatment on total cellular ROS and ATP content in senescent cells.

[0020] Figure 2 The transcriptome sequencing results of cells treated with salvianolic acid B are shown in Figure 2. Figure 2 A is a volcano plot of gene expression changes in aging cells after treatment with salvianolic acid B, B is the result of GO functional enrichment analysis using significantly changed differentially expressed genes, C is a Venn diagram of the intersection of differentially expressed genes in multiple autophagy-related pathways obtained from Figure B, D is a heat map of the common differentially expressed genes obtained after the intersection of multiple autophagy-related pathways, E is the expression level of TP53INP2, and F to H are the expression levels of autophagy-related proteins p62 and LC3BⅠ / Ⅱ, respectively.

[0021] Figure 3 To explore the effect of salvianolic acid B on aging-related muscle loss using a mouse premature aging model; Figure 3 A is the experimental flow chart, B-D are the effects of different concentrations of salvianolic acid B treatment on the body weight, running endurance and limb grip strength of mice, and E-F are the staining images of the cross-sectional area of ​​mouse muscle fibers and the results of the cross-sectional area of ​​muscle fibers after treatment with different concentrations of salvianolic acid B. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0023] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0024] Example 1 In vitro experiment 1. Experimental methods: (1) Cell acquisition: BJ cell line, derived from human skin fibroblasts, was purchased from the American Type Culture Collection (ATCC).

[0025] (2) Construction of replicative senescent cell model: BJ cells were continuously passaged until they no longer had the ability to proliferate and then processed. The BJ cell line was derived from human skin fibroblasts and purchased from the American Type Culture Collection (ATCC).

[0026] (3) Construction of irradiated replicative senescent cell model: BJ cells were irradiated with 10 Gy, 25 mA X-rays (Rad Source Technologies, USA) and cultured for 1 week before subsequent treatment.

[0027] (4) RNA extraction and quantitative real-time PCR (qPCR): Total RNA was isolated using Trizol (Takara, Japan) according to the manufacturer's protocol. The purity and concentration of RNA were determined by the Nano-Drop system (Thermo, USA). 1 μg of total RNA was reverse transcribed into cDNA using the HiScriptIII RT SuperMix for qPCR Kit (Vazyme, China). Real-time fluorescence quantitative PCR was performed using 2xRealStar Power SYBR qPCR Mix (Genstar, USA). The qPCR operation was as follows: incubation at 50°C for 2 min and at 95°C for 10 min. PCR was incubated at 95°C for 15 s and at 60°C for 1 min for a total of 40 cycles. At the melting curve stage, incubation was performed at 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. Gene expression in each sample was then analyzed using the QuantStudio™6 Pro Real-Time PCR System (Thermo Fisher Scientific, USA). β-actin mRNA was used as the internal reference. The relative expression of each gene was calculated using the 2-ΔΔCT formula. PCR primers are shown in Table 1: Table 1 PCR primers

[0028] (5) Cell viability experiments were performed using a CCK-8 kit (Beyotime, China). 4,000 cells were seeded per well in a 96-well plate and cultured in a cell culture incubator at 37 degrees Celsius for 24 hours. After 24 hours, the corresponding solvent or salvianolic acid B (Selleck, USA) was added to each well and cultured in a cell culture incubator at 37 degrees Celsius for 24 hours. After 24 hours, the cell culture medium in the 96-well plate was aspirated, 100 μL of 10% CCK-8 reagent was added to each well, and the cells were cultured in a cell culture incubator at 37 degrees Celsius for about 1 hour. Subsequently, the absorbance value was measured at a wavelength of 450 nm using a microplate reader (Biotek, USA) to evaluate cell viability. Cell viability (%) = (absorbance value of the experimental group - background absorbance value) / (absorbance value of the control group - background absorbance value) × 100%.

[0029] (6) Western blotting: Samples were lysed in SDS-PAGE protein loading buffer (Beyotime, China). After centrifugation, the supernatant was collected and the protein concentration was determined using the Pierce™ BCA protein assay kit (Thermo Fisher Scientific, USA). Protein samples were separated on SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in 5% bovine serum albumin for 1 h at room temperature and then washed three times with TBST. The membranes were trimmed and incubated with different primary antibodies overnight. After washing three times with TBST, the secondary antibodies were incubated for 1 h at room temperature. After washing three times with TBST, they were exposed to Amersham ImageQuant 600 (GE Healthcare, USA). Western blotting was performed using primary antibodies against p62 (MCE, USA) and LC3B (proteintech, China). Rabbit IgG (CST, USA) secondary antibody was used.

[0030] (7) ROS detection: The ROS detection experiment used a reactive oxygen species detection kit (Beyotime, China). DCFH-DA was diluted with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10 μmol / L. The cell culture medium was removed and 1 mL of the diluted DCFH-DA was added. The cells were incubated in a 37°C cell culture incubator for 20 minutes. The cells were washed twice with serum-free cell culture medium to fully remove the DCFH-DA that had not entered the cells. After digesting the cells and washing the cells twice again with serum-free cell culture medium, 1 μL of DAPI (Sigma Germany) was added and the fluorescence intensity was detected by flow cytometry (BD, USA) under the FITC fluorescence spectrum.

[0031] (8) ATP detection: The ATP detection experiment uses an enhanced ATP detection kit (Beyotime, China). Melt the reagents to be used in an ATP ice bath, and dilute the ATP standard solution (0.5mM) with ATP detection lysis buffer to the concentrations of 0, 0.01, 0.05, 0.1, 0.5, 1, 5, and 10μM. Prepare an appropriate amount of ATP detection working solution (1:9) according to the ratio of 100μL ATP detection working solution for each sample or standard. Aspirate the cell culture medium of the six-well plate, add 200μL lysis buffer to each well, and use a pipette to repeatedly blow or shake the culture plate to allow the lysis buffer to fully contact and lyse the cells. After washing with PBS, centrifuge at 12000g at 4℃ for 5 minutes, take 20μL of the supernatant to measure ATP, and the rest can be used to measure BCA. Add 100μL ATP detection working solution to the detection well. Leave it at room temperature for 3 to 5 minutes to consume all the background ATP, thereby reducing the background. Add 20 μL of sample or standard to each well, quickly mix with a gun (micropipette), and measure the RLU value with a microplate reader (Biotek, USA) after at least 2 seconds. Relative ATP content = RLU value / protein content.

[0032] (9) Transcriptome sequencing: This experiment was commissioned to Beijing Novogene Bioinformatics Technology Co., Ltd.

[0033] (10) Statistical analysis: All data are expressed as mean ± SD. Graph Pad Prism 8.0 software (Graph Pad Prism software, La Jolla, CA, USA) was used to analyze the significant differences between two groups using unpaired t-test, and one-way analysis of variance was used to analyze the significant differences between multiple groups. P < 0.05 was considered statistically significant.

[0034] 2. Experimental results Salvianolic acid B (SAB) can be decomposed into caffeic acid and danshensu, and danshensu can be transformed into danshensu sodium (SDSS) and rosmarinic acid (RA) under certain conditions ( Figure 1 In Figure A), in order to determine the safe concentrations of salvianolic acid B, sodium tanshinone and rosmarinic acid for young BJ cells and replicative senescent BJ cells, the present invention uses CCK8 to explore the safe concentrations. The CCK8 results show that after treating young BJ cells and replicative senescent BJ cells with 200μM and below concentrations of salvianolic acid B, sodium tanshinone and rosmarinic acid for 24 hours, the three drugs have no significant changes in the cell viability of young and replicative senescent cells compared with the group without drug treatment. ( Figure 1 Middle B~D) Real-time fluorescence quantitative PCR detected that the expression of multiple SASP-related genes in senescent cells treated with different concentrations of salvianolic acid B, sodium danshensu, and rosmarinic acid was significantly reduced, among which salvianolic acid B inhibited the most types of SASP-related genes. Figure 1 (middle E~G).

[0035] Treatment of senescent BJ cells with 200 μM salvianolic acid B also reduced total cellular ROS and increased intracellular ATP ( Figure 1 This indicates that salvianolic acid B can improve various aging phenotypes of cells and is a powerful anti-aging substance.

[0036] The results of transcriptome sequencing of cells treated with salvianolic acid B showed that salvianolic acid B can affect the changes in the autophagy pathway of aging cells ( Figure 2 A to D in the middle). Among them, the expression of TP53INP2 in senescent cells treated with salvianolic acid B was upregulated ( Figure 2 E). Previous studies have shown that upregulation of this gene can enhance autophagy in cells and muscle tissue, thereby enhancing the improvement of muscle tissue atrophy. Further cell experiments also observed a decrease in p62 and an increase in the ratio of LC3BⅡ / LC3BI, which further confirmed that salvianolic acid B has the effect of promoting cell autophagy ( Figure 2 Therefore, the cell experiment verified that salvianolic acid B up-regulated TP53INP2 and promoted cell autophagy, that is, salvianolic acid B can activate cell autophagy, and the weakening of autophagy ability is one of the causes of muscle atrophy, and promoting cell autophagy is the key to improving muscle function. The present invention believes that salvianolic acid B not only has the effect of improving the phenotype of cell aging, but also has the effect of directly improving the symptoms of muscle function reduction.

[0037] Example 2 In vivo experiment 1. Experimental methods: (1) Establishment and administration of mouse muscle dysfunction symptom model: 8-week-old male C57 background mice were purchased from Guangdong Yaokang Biotechnology Co., Ltd. Under SPF conditions, 5 mice were placed in each cage and allowed to freely follow a 12-hour light-dark cycle. All animal experiments were conducted in accordance with the institutional guidelines and protocols approved by the Animal Care and Ethics Committee of Jinan University (approval number 20240428-10). In order to construct a mouse muscle dysfunction symptom model, X-ray (Rad Source Technologies, USA) mice were used with a dose of 4.5 Gy, 25 mA, and were fed a normal diet for two months after irradiation. Two months later, salvianolic acid B was injected intraperitoneally once every 2 days for a total of 28 days. The constructed mouse muscle dysfunction symptom model had symptoms of aging and muscle dysfunction.

[0038] (2) Treadmill test: The treadmill (Anhui Zhenghua Biological Instrument Equipment Co., Ltd., China) was set at an angle of 15° to the ground. The mice were subjected to running adaptation training for 3 consecutive days before the formal treadmill test. The running speed and time during the training were as follows: 5rpm 2min, 7rpm 2min, and 9rpm 1min. In the formal experiment, the initial running speed of the mice was 5rpm, and the speed increased by 2rpm every 2min until the mice were exhausted and the running distance of the mice was recorded. Exhaustion means that the mice still stopped running for 3s or more under electrical stimulation. Running work of mice (KJ) = mouse weight (kg) × mouse running distance (m) × g (9.8m / s 2 )×sin(15°) (3) Grip strength test: Place the mouse's limbs on the grid of a grip force meter (Shanghai Xinruan, China), pull the mouse's tail backward until the mouse's limbs are out of the grid, and the grip force meter records the instantaneous grip force (gf) of the mouse out of the grid.

[0039] (4) H&E staining and statistics of muscle fiber cross-sectional area (CSA): Fresh muscle tissue was fixed in 4% paraformaldehyde for 24 hours and then dehydrated with graded alcohol. The tissue was embedded in a wax block for embedding. The embedded tissue was cut into tissue sections with a thickness of 4 µm. After dewaxing of the paraffin sections, hematoxylin was used to stain the nuclei and eosin was used to stain the cytoplasm. After staining, the sections were dehydrated, sealed and dried to obtain image information. The sections were then scanned using a panoramic scanner (3D HISTECH, Hungary) to obtain high-definition images for observation and analysis. The average CSA area was calculated using slide viewer 2.7. Three fields of view were selected for each section, and the areas of 80 to 90 muscle fibers were counted in each field of view.

[0040] (5) Statistical analysis: All data are expressed as mean ± SD. Graph Pad Prism 8.0 software (Graph Pad Prism software, La Jolla, CA, USA) was used to analyze the significant differences between two groups using unpaired t-test, and one-way analysis of variance was used to analyze the significant differences between multiple groups. P < 0.05 was considered statistically significant.

[0041] 2. Experimental results By constructing a mouse model of muscle dysfunction, we observed that this model can lead to muscle loss in mice. Through treadmill experiments and grip strength experiments, we observed that supplementing mice with salvianolic acid B can effectively improve the running endurance and limb grip strength of mice ( Figure 3 A~D in the middle). And through HE staining, we can also observe that after supplementing with salvianolic acid B, the cross-sectional area of ​​mouse muscle fibers increased ( Figure 3E-F in the figure), that is, salvianolic acid B can improve the morphological function of skeletal muscle and the body's motor ability. The above experiments show that injecting salvianolic acid B into mice can improve the muscle loss associated with aging.

Claims

1. Use of salvianolic acid B and / or its active hydrolyzate in the preparation of a drug for treating aging-related muscle dysfunction.

2. Use of salvianolic acid B and / or its active hydrolyzate in the preparation of drugs for treating diseases caused by aging-related muscle dysfunction.

3. The use according to claim 1 or 2, characterized in that: The active hydrolysis product is sodium danshensu and / or rosmarinic acid.

4. The application according to claim 2, characterized in that: The disease is sarcopenia.

5. The use according to claim 1 or 2, characterized in that: The drug treats aging-related muscle dysfunction by improving skeletal muscle morphology, skeletal muscle function and / or body movement capacity.

6. The use according to claim 1 or 2, characterized in that: The drug treats aging-related muscle dysfunction by activating cell and tissue autophagy activity and reducing the production of inflammatory factors.

7. The use according to claim 1, characterized in that: The medicine also contains pharmaceutically acceptable excipients.

8. The use according to claim 7, characterized in that: The auxiliary material is selected from any one or more of an excipient, a diluent, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent or a preservative.

9. The use according to claim 1, characterized in that: The dosage form of the drug is any one of injection, oral solution, capsule, tablet or granule.

10. The use according to claim 9, characterized in that: The drug dosage form is an injection.

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