Application of salvianolic acid B in preparing a drug for treating senescence-related muscle hypofunction

Abstract

The present invention discloses the use of salvianolic acid B in the preparation of a medicament for treating senescence-related muscle hypofunction. The research of the present invention finds that salvianolic acid B has a significant effect on improving senescence-related muscle hypofunction. As a natural product, salvianolic acid B has high safety and few side effects, and is gentler and safer compared to certain drug treatments. Secondly, the treatment method of salvianolic acid B is relatively simple and easy compared with long-term physical therapy and nutritional supplementation, 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 a medicament for treating senescence-related muscle hypofunction, and its mechanism may be related to reducing inflammatory response and promoting autophagy improvement. The discovery that salvianolic acid B improves senescence-related muscle hypofunction adds a new possibility to the lack of current treatment means for sarcopenia.

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 active water-soluble component of Salvia miltiorrhiza, with pharmacological properties such as antioxidant, anti-inflammatory, and cardiovascular protection. Although extensive research has been conducted on the uses and mechanisms of salvianolic acid B, there is currently no study indicating its effect on improving age-related muscle function decline. Patent CN115414409A shows that salvianolic acid A can alleviate muscle atrophy, myopathy, and musculoskeletal complications caused by diabetes. However, salvianolic acid A and salvianolic acid B are two active substances with different structures, and it targets muscle function decline caused by diabetes, rather than that caused by aging. Muscle function decline caused by other factors may be the result of abnormal body changes, and the treatment plan is targeted at the specific causes of these changes, rather than directly treating sarcopenia itself. Age-related muscle function decline usually occurs gradually with normal aging and can be improved by increasing nutritional intake and performing appropriate exercise. Therefore, treatment methods for other pathological factors may not be applicable to treating age-related muscle function decline associated with normal aging. Summary of the Invention

[0004] The object 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 age-related muscle function decline.

[0005] The above object of the present invention is achieved by the following technical solutions:

[0006] The present invention has found through research that salvianolic acid B has a series of anti-aging effects, such as reducing the senescence-associated secretory phenotype (SASP) of senescent cells, promoting autophagy of senescent cells, reducing the intracellular ROS level of senescent cells, and increasing ATP in senescent cells. Salvianolic acid B and its degradation 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 locomotor ability of the body. It has a significant effect on improving age-related muscle function decline, and its mechanism may be related to the ability of salvianolic acid B to reduce the SASP of senescent cells and promote autophagy of senescent cells. 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 senescent cell phenotype, but also has the effect of directly improving the condition of muscle function reduction. This discovery adds a new possibility to the lack of current treatment methods for sarcopenia. Compared with some drug treatments, the application of salvianolic acid B to treat age-related muscle function decline has higher safety and fewer side effects. Secondly, it is relatively simple and easy compared with long-term physical therapy and nutritional supplementation, and patients can more easily accept and follow the treatment plan.

[0007] Therefore, the present invention provides the use of salvianolic acid B and / or its active hydrolysis products in the preparation of a drug for treating age-related muscle function decline disorders.

[0008] The present invention also provides the use of salvianolic acid B and / or its active hydrolysis product in the preparation of a medicament for treating diseases caused by age-related muscle hypofunction.

[0009] The above treatment refers to intervening after the occurrence of a disorder or disease to eliminate or control the disorder or disease.

[0010] Further, the active hydrolysis product is sodium danshensu (SDSS) and / or rosmarinic acid (RA).

[0011] Further, the disease caused by age-related muscle hypofunction is sarcopenia.

[0012] Further, the medicament treats age-related muscle hypofunction by improving skeletal muscle morphology, skeletal muscle function, and / or body motor ability.

[0013] Further, the medicament treats age-related muscle hypofunction by activating cell and tissue autophagy activity and reducing the production of inflammatory factors. The medicament can up-regulate TP53INP2 and promote autophagy, thereby directly improving muscle function.

[0014] Further, the effective dose of salvianolic acid B in the medicament is not less than 25 mg / Kg.

[0015] Further, the medicament also contains pharmaceutically acceptable excipients.

[0016] Preferably, the excipients are selected from any one or more of excipients, diluents, lubricants, wetting agents, sweeteners, flavoring agents, emulsifying agents, suspending agents, or preservatives.

[0017] Further, the dosage form of the medicament is any one of injection, oral liquid, capsule, tablet, or granule.

[0018] Preferably, the dosage form of the medicament is injection.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention provides the use of salvianolic acid B and its active hydrolysis products in the preparation of a medicament for treating senescence-related muscle hypofunction. The present invention has found through research that salvianolic acid B has the functions of reducing the senescence-associated secretory phenotype (SASP) of senescent cells, promoting autophagy of senescent cells, reducing the intracellular reactive oxygen species (ROS) level of senescent cells, and increasing the adenosine triphosphate (ATP) of senescent cells, improving the morphological function of skeletal muscle and the locomotor ability of the body, and having a significant effect on improving senescence-related muscle hypofunction. As a natural product, salvianolic acid B has high safety and few side effects, and is gentler and safer compared to some drug treatments. Secondly, the treatment method of salvianolic acid B is relatively simple and easy compared with long-term physical therapy and nutritional supplementation, 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 a medicament for treating senescence-related muscle hypofunction, and its mechanism may be related to reducing the inflammatory response and promoting autophagy. The discovery that salvianolic acid B improves senescence-related muscle hypofunction adds a new possibility to the lack of current treatment means for sarcopenia. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Results of in vitro cell experiments to explore the effect of salvianolic acid B on senescent cells; among them, Figure 1 A in FIG. is the process of salvianolic acid B being converted into sodium danshensu and rosmarinic acid, and B-D are the results of the effects of different concentrations of salvianolic acid B, sodium danshensu, and rosmarinic acid on cell viability, and E-G are the results of the effects of different concentrations of salvianolic acid B, sodium danshensu, and rosmarinic acid treatments on the expression of SASP-related genes in senescent cells, and H-J are the effects of salvianolic acid B treatment on senescent cells on the total ROS and ATP content of the cells.

[0022] Figure 2 Results of transcriptome sequencing of cells after treatment with salvianolic acid B; among them, Figure 2 A in FIG. is a volcano plot of gene expression changes in senescent cells after treatment with salvianolic acid B, B is the result of GO functional enrichment analysis using significantly changed differential genes, C is a Venn diagram of the intersection of differential genes in multiple autophagy-related pathways obtained from FIG. B, D is an expression heat map of the common differentially expressed genes obtained after intersection in multiple autophagy-related pathways, E is the expression level of TP53INP2, and F-H are the expression levels of autophagy-related proteins p62 and LC3BⅠ / Ⅱ, respectively.

[0023] Figure 3 Results of exploring the effect of salvianolic acid B on senescence-related muscle loss through a premature mouse model; among them, Figure 3 A in FIG. is an experimental flow chart, and 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 maps of the cross-section of muscle fibers and the results of the cross-sectional area of muscle fibers in mice after treatment with different concentrations of salvianolic acid B. DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention will be further described below in conjunction with the specification drawings and specific embodiments, but the embodiments 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 technical field.

[0025] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0026] Example 1 In vitro experiment

[0027] 1. Experimental method:

[0028] (1) Obtaining cells: The BJ cell line, derived from human skin fibroblasts, was purchased from the American Type Culture Collection (ATCC).

[0029] (2) Construction of a replicative senescence cell model: The BJ cells were continuously passaged until the BJ cells no longer had the ability to proliferate and then subsequent treatments were performed. The BJ cell line, derived from human skin fibroblasts, was purchased from the American Type Culture Collection (ATCC).

[0030] (3) Construction of an irradiated replicative senescence cell model: The BJ cells were irradiated with 10 Gy, 25 mA X-rays (Rad Source Technologies, USA), and cultured for 1 week after irradiation before subsequent treatments.

[0031] (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 HiScript III RT SuperMix for qPCR Kit (Vazyme, China). Real-time fluorescence quantitative PCR was performed using the 2xRealStar Power SYBR qPCR Mix (Genstar, USA). The qPCR procedure was as follows: incubated at 50°C for 2 minutes and at 95°C for 10 minutes. PCR was performed at 95°C for 15 seconds and at 60°C for 1 minute for a total of 40 cycles. During the melting curve stage, incubated at 95°C for 15 s, at 60°C for 1 min, and at 95°C for 15 s. Then the gene expression in each sample was analyzed using the QuantStudio™ 6 Pro Real-Time PCR system (Thermo Fisher Scientific, USA). β-actin mRNA was used as an internal reference. The relative expression levels of each gene were calculated using the 2-ΔΔCT formula. The PCR primers are shown in Table 1:

[0032] Table 1 PCR Primers

[0033]

[0034] (5) Cell viability assay was performed using a CCK-8 kit (Beyotime, China). 4000 cells per well were seeded in a 96-well plate and cultured in a cell incubator at 37 °C for 24 h. After 24 h, the corresponding solvent or salvianolic acid B (Selleck, USA) was added to each well and cultured in a cell incubator at 37 °C for 24 h. After 24 h, 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 incubator at 37 °C for about 1 h. 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 - absorbance value of the background) / (absorbance value of the control group - absorbance value of the background) × 100%.

[0035] (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 measured using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, USA). Protein samples were separated on an SDS-PAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% bovine serum albumin at room temperature for 1 h and then washed 3 times with TBST. The membrane was trimmed and incubated with different primary antibodies overnight. After washing 3 times with TBST, the secondary antibody was incubated at room temperature for 1 h. After washing 3 times with TBST, it was exposed to Amersham ImageQuant 600 (GE Healthcare, USA). Primary antibodies against p62 (MCE, USA) and LC3B (proteintech, China) were used for Western blotting. Rabbit IgG (CST, USA) secondary antibody was used.

[0036] (7)ROS detection: The ROS detection experiment used a reactive oxygen species detection kit (Beyotime, China). DCFH-DA was diluted 1:1000 with serum-free culture medium to a final concentration of 10 μmol / L. The cell culture medium was removed, and 1 mL of the diluted DCFH-DA was added. Incubate in a 37 °C cell culture incubator for 20 minutes. Wash the cells twice with serum-free cell culture medium to completely remove the DCFH-DA that did not enter the cells. Digest the cells and wash the cells twice again with serum-free cell culture medium, then add 1 μL of DAPI (Sigma, Germany). Detect the fluorescence intensity by flow cytometry (BD, USA) under the FITC fluorescence spectrum.

[0037] (8)ATP detection: The ATP detection experiment used an enhanced ATP detection kit (Beyotime, China). The reagents for ATP detection were thawed on ice. The ATP standard solution (0.5 mM) was diluted with the ATP detection lysis buffer to concentrations of 0, 0.01, 0.05, 0.1, 0.5, 1, 5, and 10 μM. Prepare an appropriate amount of ATP detection working solution (1:9) according to the ratio of 100 μL of ATP detection working solution per sample or standard. Aspirate the cell culture medium in the six-well plate, add 200 μL of lysis buffer to each well, and use a pipette to repeatedly pipette or shake the culture plate to allow the lysis buffer to fully contact and lyse the cells. After washing with PBS, centrifuge at 12,000 g for 5 minutes at 4 °C. Take 20 μL of the supernatant to measure ATP, and the rest can be used to measure BCA. Add 100 μL of ATP detection working solution to the detection wells. Incubate at room temperature for 3 - 5 minutes to consume all the background ATP, thereby reducing the background. Add 20 μL of the sample or standard to each well, quickly mix with a pipette (micropipette), and at least 2 seconds later, measure the RLU value with an enzyme-linked immunosorbent assay reader (Biotek, USA). Relative ATP content = RLU value / protein content.

[0038] (9)Transcriptome sequencing: This experiment was commissioned to Beijing Novogene Bioinformatics Technology Co., Ltd. to complete.

[0039] (10)Statistical analysis: All data were expressed as mean ± SD. GraphPad Prism 8.0 software (GraphPad Prism software, La Jolla, CA, USA) was used. Unpaired t-tests were used to analyze the significant differences between two groups, and one-way ANOVA was used to analyze the significant differences between multiple groups. P < 0.05 was considered statistically significant.

[0040] 2. Experimental results

[0041] Salvianolic acid B (SAB) can be decomposed into caffeic acid and danshensu, and danshensu can be converted into sodium danshensu (SDSS) and rosmarinic acid (RA) under certain conditions ( Figure 1 In A), to determine the safe concentrations of salvianolic acid B, sodium danshensu and rosmarinic acid on 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 200 μM or lower concentrations of salvianolic acid B, sodium danshensu and rosmarinic acid for 24 h respectively, compared with the drug-free treatment group, the cell viability of the three drugs on young and replicative senescent cells has no significant change. ( Figure 1 In B-D)

[0042] By real-time fluorescence quantitative PCR, it is detected that the expression of various SASP-related genes in senescent cells treated with different concentrations of salvianolic acid B, sodium danshensu and rosmarinic acid has a significant decrease, and salvianolic acid B inhibits the most types of SASP-related genes. ( Figure 1 In E-G).

[0043] Treatment of senescent BJ cells with 200 μM salvianolic acid B also has the effects of reducing the total cellular ROS and increasing the intracellular ATP ( Figure 1 In H-J). This indicates that salvianolic acid B can improve various senescent phenotypes of cells and is a powerful anti-aging substance.

[0044] The transcriptome sequencing results of cells treated with salvianolic acid B show that salvianolic acid B can affect the changes in the autophagy pathway of senescent cells ( Figure 2 In A-D). Among them, the expression of TP53INP2 in senescent cells treated with salvianolic acid B is up-regulated ( Figure 2 In E). According to previous studies, the up-regulation of this gene can enhance autophagy in cells and muscle tissues, thereby enhancing the improvement of muscle tissue atrophy. And in further cell experiments, a decrease in p62 and an increase in the ratio of LC3BⅡ / LC3BⅠ are also observed, which further confirms that salvianolic acid B has the effect of promoting autophagy in cells ( Figure 2 In F-H). Therefore, through cell experiments, it is verified that salvianolic acid B up-regulates TP53INP2 and promotes autophagy in cells, that is, salvianolic acid B can activate autophagy in cells, and the weakening of autophagy ability is one of the causes of muscle atrophy, and promoting autophagy in cells is the key to improving muscle function. The present invention believes that salvianolic acid B not only has the effect of improving cell senescent phenotypes, but also has the effect of directly improving muscle function and reducing diseases.

[0045] Example 2 In vivo experiment

[0046] 1. Experimental method:

[0047] (1)Establishment of a mouse model of muscle function decline and drug administration: Male C57-background mice at 8 weeks of age were purchased from Guangdong Medicilon Biotech Co., Ltd. Under SPF conditions, 5 mice were housed in each cage, and they were freely subjected to a 12-hour light-dark cycle. All animal experiments were conducted in accordance with the institutional guidelines and protocols approved by the Animal Protection and Ethics Committee of Jinan University (approval number: 20240428-10). To construct a mouse model of muscle function decline, X-ray (Rad Source Technologies, USA) was used on the mice at a dose of 4.5 Gy and 25 mA. After irradiation, the mice were fed normal feed for two months. Two months later, salvianolic acid B was injected intraperitoneally once every 2 days for a total of 28 days. The constructed mouse model of muscle function decline exhibited symptoms of aging and muscle function decline.

[0048] (2)Treadmill test: The treadmill (Zhenghua Biological Instrument and Equipment Co., Ltd., Anhui, China) was set at an angle of 15° to the ground. Mice were subjected to running adaptation training for 3 consecutive days before the formal treadmill test. The running speed and time during the training period were as follows: 5 rpm for 2 min, 7 rpm for 2 min, and 9 rpm for 1 min. In the formal experiment, the initial running speed of the mice was 5 rpm, and the speed was increased by 2 rpm every 2 min until the mice were exhausted, and the running distance of the mice was recorded. Exhaustion was defined as the mice stopping running for 3 s or more under electrical stimulation. The work done by the mice running (KJ) = mouse body weight (kg) × mouse running distance (m) × g (9.8 m / s 2 ) × sin(15°)

[0049] (3)Grip strength test: The four limbs of the mice were placed on the grid of a grip strength meter (Shanghai Xinruan, China), and the mouse's tail was pulled backward until the four limbs of the mouse detached from the grid. The grip strength meter recorded the grip strength (gf) at the moment the mouse detached from the grid.

[0050] (4)H&E staining and statistical analysis of the cross-sectional area (CSA) of muscle fibers: Fresh muscle tissue was fixed in 4% paraformaldehyde for 24 hours and then dehydrated with gradient alcohol. The tissue was embedded in paraffin blocks. The embedded tissue was cut into tissue sections with a thickness of 4 µm. After dewaxing the paraffin sections, the cell nuclei were stained with hematoxylin and the cytoplasm was stained with eosin. After staining, the sections were dehydrated, sealed, and dried to obtain image information. Then the sections were scanned with a panoramic scanner (3D HISTECH, Hungary) to obtain high-definition images for observation and analysis. The average CSA area was statistically analyzed using slide viewer 2.7. Three fields of view were selected for each section, and the areas of 80-90 muscle fibers were statistically analyzed in each field of view.

[0051] (5)Statistical analysis: All data were expressed as mean ± SD. GraphPad Prism 8.0 software (GraphPad Prism software, La Jolla, CA, USA) was used. Unpaired t-tests were used to analyze the significant differences between two groups, and one-way ANOVA was used to analyze the significant differences among multiple groups. P < 0.05 was considered statistically significant.

[0052] 2. Experimental results

[0053] By constructing a mouse model of muscle hypofunction symptoms, we observed that this model could lead to muscle loss in mice. Through treadmill experiments and grip strength experiments, we could observe that supplementing salvianolic acid B to mice could effectively improve their running endurance and limb grip strength ( Figure 3 in A–D). Moreover, through HE staining, we could also observe that after supplementing salvianolic acid B, the cross-sectional area of mouse muscle fibers increased ( Figure 3 in E–F), that is, salvianolic acid B could improve the morphological function of skeletal muscle and the locomotor ability of the body. The above experiments showed that injecting salvianolic acid B into mice had the effect of improving age-related muscle loss.

Claims

1. Use of salvianolic acid B and / or its active hydrolysis product as the sole active ingredient in the preparation of a medicament for treating senescence-related muscle hypofunction; the active hydrolysis product is sodium danshensu and / or rosmarinic acid; the senescence-related muscle hypofunction is sarcopenia.

2. The application according to claim 1, characterized in that, The medicament treats senescence-related muscle hypofunction by improving skeletal muscle morphology, skeletal muscle function, and / or body motor ability.

3. The application according to claim 1, characterized in that The medicament treats senescence-related muscle hypofunction by activating cell and tissue autophagy activity and reducing the production of inflammatory factors.

4. The application according to claim 1, wherein The medicament further contains pharmaceutically acceptable excipients.

5. The application according to claim 4, wherein, The excipients are selected from any one or more of excipients, diluents, lubricants, wetting agents, sweeteners, flavoring agents, emulsifying agents, suspending agents, or preservatives.

6. The application according to claim 1, wherein The dosage form of the medicament is any one of injection, oral liquid, capsule, tablet, or granule.

7. The application according to claim 6, wherein The dosage form of the medicament is injection.

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