Application of plumbagin in the preparation of drugs for combating sarcopenia

Abstract

The present invention relates to the use of plumbagin in the preparation of a drug for combating sarcopenia. Experiments have shown that plumbagin can improve the muscle mass and strength of sarcopenia model mice, improve functional disorders, and promote myoblast differentiation and myotube fusion, which provides a new drug for the treatment of sarcopenia.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a new use of plumbagin, and more specifically to an application of plumbagin in the preparation of a drug for combating sarcopenia. Background Art

[0002] Sarcopenia, also known as "skeletal muscle aging" or "sarcopenia" in clinical practice, refers to the decline in skeletal muscle mass and muscle strength caused by aging. As humans enter an aging society, the incidence of aging-related diseases such as sarcopenia is increasing year by year in countries around the world. It is estimated that there are currently about 50 million people suffering from this disease in the world, and it is expected that the number of people suffering from sarcopenia will reach 500 million by 2050. Age-related loss of muscle strength and muscle mass in the elderly is associated with adverse clinical outcomes (such as falls, limited mobility, accidental disability and fractures), ultimately leading to reduced quality of life and increased medical expenses. Therefore, sarcopenia has become an important health issue for humans, and how to prevent and treat sarcopenia has also become an important long-term research topic for humans. There is currently no specific drug for the treatment of sarcopenia, so it is crucial to explore therapeutic drugs and potential targets for sarcopenia.

[0003] Previous studies have shown that plumbagin (hereinafter referred to as PB, also known as lanxelquinone, molecular formula: C 11 H8O3, CAS No.: 481-42-5) is a naphthoquinone substance, mainly found in the whole plant of Plumbago and the root of Plumbago. Its molecular structure is as follows Figure 2 A. Studies have shown that PB can inhibit inflammatory response (see Chinese patent application publication number CN117298079A, which discloses the use of plumbagin in the preparation of drugs for treating atopic dermatitis), promote osteoblast differentiation in vitro, inhibit osteoclast differentiation, and improve bone loss caused by estrogen deficiency, suggesting that PB plays an important role in estrogen deficiency-related diseases. PB can also inhibit ferroptosis of mouse chondrocytes, produce anti-inflammatory effects, and relieve osteoarthritis in mice.

[0004] In addition, PB also has a significant therapeutic effect on myocardial cell damage, improving hemodynamic parameters and alleviating inflammation and apoptosis of cardiac tissue. Zhang Qianrui et al. published a study titled "Study on the Protective Effect of Plumbagin on TBHP-Induced H9c2 Myocardial Cell Damage" in the Journal of Chinese Hospital Pharmacy, Issue 19, 2021, which showed that plumbagin has a protective effect on TBHP-induced myocardial cell damage, which may be related to PLB's inhibition of ROS-mediated oxidative stress, apoptosis and autophagy.

[0005] With the rapid increase of the aging population worldwide, sarcopenia has brought a huge burden to patients, families and the socio-economic situation. However, there is currently no specific drug to treat or improve sarcopenia, so it is urgent to explore the pathogenesis and treatment of sarcopenia. Summary of the invention

[0006] In view of the above-mentioned defects of the prior art, the present invention provides the use of plumbagin in the preparation of a drug for combating sarcopenia.

[0007] The technical objectives of the present invention are achieved through the following technical solutions:

[0008] The invention relates to application of plumbagin in preparing a medicine for treating and / or preventing sarcopenia.

[0009] Furthermore, the drug for treating and / or preventing sarcopenia of the present invention also includes pharmaceutically acceptable excipients and / or drugs compatible with plumbagin.

[0010] Furthermore, the auxiliary materials of the present invention include at least one of a solvent, a disintegrant, a flavoring agent, a colorant, a lubricant, an antioxidant, a preservative, a binder, a filler or a thickener.

[0011] Preferably, the dosage of the drug of the present invention is 9.1-27.3 mg / kg·d.

[0012] Preferably, the administration of the drug of the present invention includes oral administration, intravenous injection or intraperitoneal injection.

[0013] Preferably, the dosage forms of the drug for treating and / or preventing sarcopenia of the present invention include capsules, granules, tablets, oral liquids, pills, injections, ointments, liposome nanoparticles, sustained-release agents, controlled-release agents or dispersible tablets.

[0014] Another technical purpose of the present invention is to provide the use of plumbagin in the preparation of a drug for promoting myogenic differentiation and myotube fusion.

[0015] Another technical objective of the present invention is to provide a pharmaceutical composition for treating and / or preventing sarcopenia, which comprises a pharmaceutically active ingredient and excipients, wherein the pharmaceutically active ingredient is plumbagin.

[0016] Furthermore, the administration of the pharmaceutical composition of the present invention includes oral administration, intravenous injection or intraperitoneal injection; preferably intraperitoneal injection; more preferably, the dosage of the pharmaceutical composition is 9.1-27.3 mg / kg·d, and the administration time is 3 to 4 months.

[0017] Furthermore, the dosage forms of the pharmaceutical composition of the present invention include capsules, granules, tablets, oral liquids, pills, injections, ointments, liposome nanoparticles, sustained-release agents, controlled-release agents or dispersible tablets.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] Studies have shown that plumbagin can improve muscle mass and strength in sarcopenia model mice, improve functional disorders, and promote myoblast differentiation and myotube fusion. Intraperitoneal injection of plumbagin active ingredients at a dose of 1-3 mg / kg·d (9.1-27.3 mg / kg·d in human body) for 3 months can improve muscle mass and strength in sarcopenia model mice, improve functional disorders, and promote myotube fusion and myoblast differentiation in vitro, which will provide new drugs for the treatment and / or prevention of sarcopenia. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Intraperitoneal injection of plumbagin improves muscle mass and strength in sarcopenia model mice Figure: Figure 1 A in the figure indicates female mice injected intraperitoneally with plumbagin, and representative images of HE staining in each group; B. Body weight of mice in each group; C. Tensile force value of mice in each group; D. Quadriceps mass of mice in each group; E. Average cross-sectional area of ​​quadriceps fibers in each group.

[0021] Figure 2 This is a diagram showing that plumbagin promotes the myogenic differentiation of mouse myoblasts (C2C12); A. The molecular structure of plumbagin; B. CCK8 was used to detect the effect of plumbagin on the proliferation of C2C12; C. Giemsa staining was used to detect the effect of plumbagin on the myogenic differentiation of C2C12; D. The myotube fusion rate of each group of cells was evaluated; E. RT-qPCR was used to detect the expression of myogenic differentiation marker genes MuRF1 and Atrogin1 in mouse myoblasts; F. Western blot was used to detect the expression of myogenic differentiation marker proteins MuRF1 and Atrogin1.

[0022] Figure 3 Figure 2 shows that plumbagin promotes myogenic differentiation of human skeletal muscle cells (HsKMC); A. CCK8 was used to detect the effect of plumbagin on the proliferation of HsKMC; B. Giemsa staining was used to detect the effect of plumbagin on the myogenic differentiation of HsKMC; C. The myotube fusion rate of each group of cells was evaluated; D. RT-qPCR was used to detect the expression of myogenic differentiation marker genes MuRF1 and Atrogin1; E. Western blot was used to detect the expression of myogenic differentiation marker protein genes MuRF1 and Atrogin1. DETAILED DESCRIPTION

[0023] In order to better illustrate the technical purpose, technical solution and advantages of the present invention, the present invention is further described in conjunction with the accompanying drawings and specific embodiments.

[0024] The method for calculating the dosage for humans is: the clinical dosage for humans is X mg / kg, which is converted to the dosage for rats:

[0025] Dose for rats = X mg / kg × 70kg × 0.018 / 200g

[0026] =X mg / kg×70kg×0.018 / 0.2kg

[0027] =6.3 X mg / kg

[0028] That is, calculated based on the dose per unit body weight, the equivalent dose for rats is 6.3 times that for humans.

[0029] This method is similar to:

[0030] The dose for mice = X mg / kg × 70 kg × 0.0026 / 20 g = 9.1 X mg / kg.

[0031] Example

[0032] 1. Experimental subject treatment

[0033] The experimental subjects were 12-week-old C57BL / 6 female mice purchased from the Guangdong Medical Experimental Animal Center. All animal experiments were approved by the Ethics Committee of the First Affiliated Hospital of Guangzhou University of Chinese Medicine (approval number GZTCMF1-2023010). The mice were randomly divided into four groups (n = 8), namely, sham operation (12-week-old C57BL / 6 female normal mice) plus 1% DMSO group, model (muscle atrophy caused by 12-week-old C57BL / 6 female mice with ovarian removal) plus 1% DMSO group, model plus 1 mg / kg plumbagin group and model plus 3 mg / kg plumbagin group. During the operation, 1% isoflurane and 99% oxygen were used for anesthesia at a rate of 20 ml / min. After the operation, intervention treatment was carried out according to the group. Intraperitoneal injections were performed 5 times a week for 12 weeks, and the samples were collected immediately after the intervention.

[0034] 2. Mouse tensile test

[0035] The mouse was asked to grasp the horizontal grid connected to the small animal grip dynamometer with its forelimbs. The peak force applied to the grid was recorded each time and measured in Newtons. The average of the five measurements was normalized to the whole body weight. The results are shown in Figure 1 -B, C, and D: B represents the weight of mice in each group; C represents the pulling force of mice in each group; and D represents the mass of quadriceps muscles of mice in each group. Figure 1 -C shows that the skeletal muscle tension of mice increased significantly after plumbagin intervention. Figure 1 -D shows that the quadriceps weight of mice was significantly increased after plumbagin intervention.

[0036] 3. Histological staining and cross-sectional area (CSA) calculation

[0037] The muscle tissue of the mouse excised after the treatment in "1. Experimental Subject Treatment" was fixed with 4% paraformaldehyde and embedded in paraffin. The slice thickness of the tissue sample was 5 μm, and the slice was stained with hematoxylin-eosin (HE). The results are as follows Figure 1 -A. The average cross-sectional area of ​​muscle fibers in each group of mice was determined using Image J software. The results are shown in Figure 1 -E. The test results show that plumbagin intervention can significantly increase the cross-sectional area of ​​quadriceps muscle fibers in mice.

[0038] 4. Myoblast Culture and Induction

[0039] C2C12 myoblasts were purchased from Wuhan Pronocell Life Science Co., Ltd. and cultured in DMEM medium containing 10% fetal bovine serum, 1% penicillin and streptomycin in a cell culture incubator at 37°C and 5% CO2. Human skeletal muscle cells (HsKMC) were purchased from Guangzhou Geneo Biotechnology Co., Ltd. and cultured in HsKMC-specific medium, also in a cell culture incubator at 37°C and 5% CO2. Myogenic induction medium (DMEM, containing 2% horse serum) was used to induce myogenic differentiation and culture C2C12 and HsKMC in vitro. The myogenic induction time was 7 days, and the myogenic induction medium was replaced every 3 days.

[0040] 5. CCK8 assay to detect the effect of plumbagin on cell proliferation

[0041] C2C12 or HsKMC treated in “4. Myoblast cell culture and induction” were cultured at 2×10 3 Cells were seeded into 96-well plates at a ratio of 1 cell / well and treated with different concentrations of plumbagin. Cell proliferation was assessed using CCK8 assay on day 3. Optical density (OD) was measured at 450 nm. Figure 2 -B (CCK8 detection of the effect of plumbagin on C2C12 proliferation) and Figure 3 -A (CCK8 detection of the effect of plumbagin on HsKMC proliferation). Figure 2 -B It can be seen that plumbagin neither promotes nor inhibits the proliferation of C2C12 when the concentration does not exceed 1.75 μmol / L; Figure 3 -A showed that plumbagin neither promoted nor inhibited the proliferation of HsKMC when the concentration did not exceed 2.5 μmol / L.

[0042] 6. Giemsa staining to detect the effect of plumbagin on promoting cell myogenic differentiation

[0043] The steps of Giemsa staining are as follows: After discarding the myogenic induction medium, wash with distilled water 3 times. Use 70% ethanol to fix (muscle cells treated with plumbagin at concentrations of 0μmol / L, 0.5μmol / L, and 1.5μmol / L) for 20 minutes, wash with distilled water 3 times, and stain with the diluted 1× Giemsa staining solution. After staining for 25 minutes, discard the staining solution and add distilled water to wash 3 times. After drying, observe and take pictures under a microscope. The myotube fusion rate (%) was calculated according to the formula = number of myotube nuclei / total number of nuclei × 100%. The results of Giemsa staining to detect the effect of plumbagin on promoting C2C12 myogenic differentiation are shown as follows. Figure 2 -C; The results show that the formation of myotubes increased; the myotube fusion rate of each group of cells (cells treated with 0μmol / L, 1.5μmol / L, and 1.75μmol / L plumbagin) was evaluated as follows Figure 2 -D shows that the myotube fusion rate increased; Giemsa staining was used to detect the effect of plumbagin on promoting myogenic differentiation of human skeletal muscle cells. Figure 3 -B shows that (cell myotube formation increased) and the cell myotube fusion rate of each group (cells treated with 0μmol / L, 0.5μmol / L, 1μmol / L, and 2.5μmol / L plumbagin) was evaluated. Figure 3 -C, the results showed that the myotube fusion rate was increased.

[0044] 7. RT-qPCR detection of the expression of myogenic differentiation marker genes MuRF1 and Atrogin1

[0045] Cells were seeded in 6-well plates. After induction of myogenic differentiation, 1 mL of TRIzol reagent was added to each well to extract total cellular RNA. The concentration and purity of RNA were analyzed using a Thermo full-wavelength microplate reader and a PrimeScript TM cDNA synthesis reaction was performed using the RTMaster Mix reverse transcription kit according to SYBR PremixEx Taq TM II operation steps, using Bio-Rad CFX96 real-time fluorescence quantitative PCR instrument for two-step fluorescence quantitative analysis, the reaction conditions are set at 95℃ for 30s, 95℃ for 5s, 60℃ for 1min extension, 40 cycles, and 3 technical replicates for each sample. The expression results of the myogenic differentiation marker genes MuRF1 and Atrogin1 in mouse myoblasts are shown in Figure 2. Figure 2 -E, it can be seen that the expression of MuRF1 and Atrogin1 was significantly downregulated, and muscle atrophy was inhibited; the expression of myogenic differentiation marker genes MuRF1 and Atrogin1 in human skeletal muscle cells was detected Figure 3 -D, it can be seen that the expression levels of MuRF1 and Atrogin1 were significantly downregulated, and muscle atrophy was inhibited.

[0046] 8. Western blot detection of the expression of myogenic differentiation marker proteins MuRF1 and Atrogin1

[0047] The cells were seeded in a 100 mm culture dish. After inducing myogenic differentiation, 200 μL of RIPA lysis buffer prepared with phosphatase inhibitors and protease inhibitors was added to extract proteins. The final concentration of the sample was 6.0 μg / μl. 5× protein sample buffer was added and denatured in a 95°C metal bath for 5 minutes. Gel preparation was performed according to the molecular weight of the target protein, and conventional sample loading, electrophoresis, and transfer were performed. After transfer, the membrane was completely immersed in 5% BSA blocking solution and incubated on a horizontal shaker at room temperature for 2 hours for blocking. The primary antibody was diluted with TBST and incubated on a horizontal shaker at 4°C overnight. The next day, the membrane was washed 3 times with TBST and incubated with the corresponding secondary antibody at room temperature for 60 minutes. After washing the membrane 3 times with TBST, it was developed and exposed to film. The grayscale value of the protein bands was analyzed using Image J software, and the grayscale value of the target protein / grayscale value of the β-Tubulin band was used to represent the relative protein expression level of the target gene. The expression results of the myogenic differentiation marker proteins MuRF1 and Atrogin1 in mouse myoblasts are shown in the figure. Figure 2 -F, detection of the expression of myogenic differentiation marker proteins MuRF1 and Atrogin1 in human skeletal muscle cells Figure 3 -E, it can be seen that the expression levels of MuRF1 and Atrogin1 were significantly downregulated, and muscle atrophy was inhibited.

[0048] 9. Statistical analysis

[0049] All data were checked for normality and homogeneity of variance. All data were expressed as mean ± standard deviation. All data were normally distributed and had homogeneous variance. One-way analysis of variance was used to compare univariate samples among multiple groups. p<0.05 was considered statistically significant.

[0050] It was found that the relative tensile force of estrogen-deficient mice decreased significantly, and the relative muscle weight was significantly downregulated. The reason for the weight gain of mice after ovariectomy was excluded. It was considered whether ovariectomy would cause atrophy of mouse muscles themselves. Therefore, pathological section analysis of mouse muscles was performed. HE staining showed that the cross-sectional area of ​​muscle fibers in estrogen-deficient mice became smaller and the centralization of cell nuclei increased. The study found that plumbagin has the effect of promoting the differentiation of mouse myoblasts in vitro and has the potential to treat muscle atrophy in vivo. The results of in vivo experiments showed that plumbagin can significantly increase the cross-sectional area of ​​muscle fibers in estrogen-deficient mice and inhibit the centralization of cell nuclei. In vitro cell experiments showed that plumbagin can accelerate the differentiation of myoblasts and fuse them into myotubes. In the case of muscle atrophy, plumbagin can maintain the myogenic potential of muscle cells and maintain the growth of myotubes by adding nuclei from unfused myoblasts. Therefore, it can be used to prevent and treat muscle atrophy. Considering that the previous experiments were conducted on mouse myoblasts and mice, human skeletal muscle cells were also used for partial verification. The results showed that plumbagin can also promote the generation and fusion of myotubes of human skeletal muscle cells.

[0051] The above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the protection scope of the present invention. In addition, the present invention may also be implemented in other ways. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the technical solution of the present invention may be modified or equivalently replaced, but these modifications or replacements are all within the protection scope of the present invention.

Claims

1. Use of plumbagin as an active ingredient for promoting myogenic differentiation and myotube fusion in the preparation of drugs for treating and / or preventing sarcopenia.

2. The use according to claim 1, characterized in that: The drug for treating and / or preventing sarcopenia also includes pharmaceutically acceptable excipients and / or drugs compatible with plumbagin.

3. The use according to claim 2, characterized in that: The auxiliary materials include at least one of solvents, disintegrants, flavoring agents, colorants, lubricants, antioxidants, preservatives, adhesives, fillers or thickeners.

4. The use according to claim 1, characterized in that: The dosage of the drug is 9.1-27.3 mg / kg·d.

5. The use according to claim 1, characterized in that: The administration of the drug includes oral administration, intravenous injection or intraperitoneal injection.

6. The use according to claim 1, characterized in that: The dosage forms of the drug for treating and / or preventing sarcopenia include capsules, granules, tablets, oral liquids, pills, injections, ointments, liposome nanoparticles, sustained-release agents, controlled-release agents or dispersible tablets.

7. Use of plumbagin in the preparation of drugs for promoting myogenic differentiation and myotube fusion.

8. Use of a composition comprising plumbagin and excipients in the preparation of a medicament for treating and / or preventing sarcopenia, characterized in that: The plumbagin is used as an active ingredient for promoting myogenic differentiation and myotube fusion, and the auxiliary materials include at least one of a solvent, a disintegrant, a flavoring agent, a colorant, a lubricant, an antioxidant, a preservative, an adhesive, a filler or a thickener.

9. The use according to claim 8, characterized in that: The administration method of the pharmaceutical composition includes oral administration, intravenous injection or intraperitoneal injection; the dosage of the pharmaceutical composition is 9.1-27.3 mg / kg·d, and the administration time is 3-4 months.

10. The use according to claim 8, characterized in that: The dosage form of the pharmaceutical composition includes capsules, granules, tablets, oral liquids, pills, injections, pastes, liposome nanoparticles, sustained-release agents, controlled-release agents or dispersible tablets.

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