Application of a serum metabolite in anti-diabetic sarcopenia

Abstract

The present invention discloses an application of serum metabolites in anti-diabetic sarcopenia, belonging to the field of biomedicine. The present invention first determines through serum metabolomics that 7-ketodeoxycholic acid (7-KDC) is significantly downregulated in T2DM sarcopenia, and finds that 7-KDC can upregulate the differentiation markers Myf5, MyoD, MyoG, and MyHC genes of muscle cells. On this basis, the present invention uses 7-KDC to prevent and / or treat diabetic sarcopenia, providing a new target for the treatment of diabetic sarcopenia.

Description

Technical Field

[0001] The present invention relates to the application of a serum metabolite in anti-diabetic sarcopenia, belonging to the field of biomedicine. Background Art

[0002] Muscle attenuation syndrome (Sarcopenia), referred to as sarcopenia for short, is a progressive and systemic skeletal muscle disease, manifested as the loss of muscle mass and function. With the in-depth study of sarcopenia, it is found that sarcopenia mostly occurs in two groups, one is the elderly group, and the other is the group suffering from chronic and wasting diseases, such as neurodegenerative diseases, type 2 diabetes, etc. Among them, the present invention focuses on the muscle reduction syndrome caused by type 2 diabetes.

[0003] Type 2 diabetes mellitus (T2DM), also known as consumptive thirst, is a chronic metabolic disease. At present, the prevalence of diabetes is increasing year by year and tends to be younger. In recent years, with the in-depth study of diabetes, sarcopenia caused by diabetes has gradually attracted attention. Existing studies have shown that diabetes can cause skeletal muscle injury and atrophy, and inhibit the differentiation ability of its muscle cells. Sarcopenia is mostly age-related and mostly occurs in the elderly group, but with the trend of diabetes towards younger people, diabetic sarcopenia is also becoming more and more obvious in the young group.

[0004] So far, although it has been found that certain substances can relieve diabetic sarcopenia, including metformin, androgen, thiazolidinediones, etc., the above drugs have many side effects that cannot be ignored, such as nausea, vomiting, diarrhea, fatigue, dizziness, hepatocyte injury, endocrine disorders, etc.

[0005] Therefore, there is an urgent need to research and develop safe and effective drugs for preventing or treating diabetic sarcopenia. Summary of the Invention

[0006] To solve the above problems, the present invention first determines through serum untargeted metabolomics that 7-ketodeoxycholic acid, namely 7-keto-3α,12α-dihydroxy cholic acid (7-KDC), is significantly down-regulated in T2DM sarcopenia, and finds that 7-KDC can up-regulate the gene expression levels of the muscle cell differentiation markers Myf5, MyoD, MyoG, and MyHC. On this basis, the present invention uses 7-KDC to prevent and / or treat diabetic sarcopenia, providing a new target for the treatment of diabetic sarcopenia.

[0007] The first object of the present invention is to provide the application of 7-keto-3α,12α-dihydroxy cholic acid or a composition containing 7-keto-3α,12α-dihydroxy cholic acid in the preparation of a drug for preventing and / or treating diabetic sarcopenia.

[0008] In one embodiment, the CAS number of 7-keto-3α,12α-dihydroxy cholic acid (7-KDC) is: 911-40-0; its structural formula is as Figure 6 shown.

[0009] In one embodiment, diabetic sarcopenia is sarcopenia caused by type 2 diabetes, and the symptoms are chronic muscle atrophy and gradual apoptosis of muscle cells.

[0010] In one embodiment, the drug is in any pharmaceutically acceptable dosage form.

[0011] In one embodiment, the dosage forms include: tablets, inhalation preparations, oral liquids, injections, topical preparations.

[0012] In one embodiment, the drug contains pharmaceutically acceptable excipients.

[0013] In one embodiment, the excipients include: one or more of probiotics, lactose, maltose, microcrystalline cellulose, carboxymethyl cellulose, hydroxypropyl methylcellulose, starch, sucrose, glucose, aspartame, water, glycerol, whey protein powder, chitosan oligosaccharide.

[0014] The second object of the present invention is to provide a pharmaceutical composition for preventing and / or treating diabetic sarcopenia, and 7-keto-3α,12α-dihydroxy cholic acid is the main active ingredient in the pharmaceutical composition.

[0015] In one embodiment, the pharmaceutical composition further includes: one or more of metformin, acarbose, glibenclamide, canagliflozin, empagliflozin, dapagliflozin, probiotics.

[0016] In one embodiment, the probiotics include common edible probiotics, including but not limited to Bifidobacterium, Lactobacillus, Bacillus subtilis, etc.

[0017] In one embodiment, the pharmaceutical composition has at least one of the following functions:

[0018] (1) Improve the ability to regulate blood sugar;

[0019] (2) Improve insulin resistance;

[0020] (3) Improve muscle differentiation indicators;

[0021] (4) Alleviate gastrocnemius atrophy.

[0022] The beneficial effects of the present invention:

[0023] The present invention first determines through serum untargeted metabolomics that 7-KDC is significantly downregulated in T2DM sarcopenia, and discovers that 7-KDC can upregulate the differentiation markers Myf5, MyoD, MyoG, and MyHC genes of muscle cells. The present invention uses 7-KDC to prevent and / or treat diabetic sarcopenia, providing a new target for the treatment of diabetic sarcopenia.

[0024] Specifically:

[0025] (1) The 7-KDC of the present invention improves the blood glucose regulation ability of T2DM sarcopenia mice. The blood glucose of mice in the T2DM + 10 mg / kg 7-KDC and T2DM + 30 mg / kg 7-KDC groups is significantly decreased at each time point. The AUC (area under the blood glucose curve) values of the two groups of mice are decreased by 25.66% and 31.17% respectively compared with the model group (T2DM group);

[0026] (2) The 7-KDC of the present invention improves the insulin resistance ability of T2DM sarcopenia mice, and the blood glucose of mice in the T2DM + 10 mg / kg 7-KDC and T2DM + 30 mg / kg 7-KDC groups is decreased at each time point. The AUC values of the two groups of mice are decreased by 36.53% and 38.13% respectively compared with the model group (T2DM group);

[0027] (3) The 7-KDC of the present invention improves the muscle differentiation index of T2DM sarcopenia mice. The size and weight of the gastrocnemius muscle of mice in the T2DM + 10 mg / kg 7-KDC group are significantly increased by 23.21% and 22.66% respectively compared with the model group (T2DM group). The size and weight of the gastrocnemius muscle of mice in the T2DM + 30 mg / kg 7-KDC group are significantly increased by 23.21% and 22.66% respectively compared with the model group (T2DM group);

[0028] (4) The 7-KDC of the present invention upregulates the muscle differentiation index in C2C12 cells and gastrocnemius muscle. The gene expression levels of Myf5, MyoD, MyoG, and MyHC in the gastrocnemius muscle of mice in the T2DM + 10 mg / kg 7-KDC group and the T2DM + 30 mg / kg 7-KDC group are significantly increased. Description of the Drawings

[0029] Figure 1 It is a diagram showing the metabolite changes in the sera of the control group and type 2 diabetes mellitus (T2DM) sarcopenia mice in serum untargeted metabolomics analysis; wherein, Figure 1 A is a heat map showing the expression patterns of different metabolites in the sera of mice; Figure 1 B is a correlation analysis diagram of differential metabolites; Figure 1 C is a bubble diagram of metabolic pathway enrichment; Figure 1D represents the expression of total bacteria; Figure 1 E represents the expression of Lactobacillus; Figure 1 F represents the expression of Bifidobacterium;

[0030] Figure 2 Upregulates the gene expression levels of Myf5, MyoD, MyoG, and MyHC in C2C12 cells under high glucose conditions;

[0031] Figure 3 Improves glucose metabolism and muscle atrophy in T2DM mice; among them, Figure 3 A is the oral glucose tolerance test (OGTT) for each group of mice; Figure 3 B is the insulin tolerance test (ITT) for each group of mice; Figure 3 C-D are the apparent phenomena and quantitative statistics of the gastrocnemius muscle length in each group of mice; Figure 3 E is the weight of the gastrocnemius muscle in each group of mice;

[0032] Figure 4 Promotes the gene expression levels of Myf5, MyoD, MyoG, and MyHC in the muscles of T2DM mice;

[0033] Figure 5 Schematic diagram for constructing a T2DM sarcopenia mouse model;

[0034] Figure 6 Is the chemical structural formula of 7-KDC. Detailed implementation methods

[0035] The following describes the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0036] Raw materials used in the embodiments:

[0037] 7-KDC was purchased from: MedChemExpress (MCE);

[0038] Streptozotocin (STZ) was purchased from: Sigma-Aldrich;

[0039] C2C12 cells were purchased from: Wuhan Boster Biological Engineering Co., Ltd.;

[0040] Mice were purchased from: 42 male C57BL / 6 mice aged 6-8 weeks were purchased from Beijing Sparf Bioscience Co., Ltd.;

[0041] High-fat diet formula: Wuxi Fanbo Biotechnology Co., Ltd. (21.8 kJ / g, 60% fat);

[0042] The basal diet was purchased from: Wuxi Fanbo Biotechnology Co., Ltd. (14.7 kJ / g, 13% fat).

[0043] Experimental methods:

[0044] 1. Construction of T2DM sarcopenia mouse model:

[0045] The schematic diagram for the construction of the T2DM sarcopenia mouse model is as Figure 5 shown. After adaptively feeding according to the SPF-grade maintenance diet standard for 7 days, high-fat diet was fed from day 8 to day 35, and from day 36 to day 40, high-fat diet combined with intraperitoneal injection of streptozotocin (STZ) at 30 mg / kg (10 mM citric acid buffer solution) was used to construct the T2DM mouse model. After continuing to feed the high-fat diet for 7 days, blood glucose was measured. The mice were fasted but allowed to drink water for 10 - 12 hours, and their fasting blood glucose (FBG) was measured using a Roche blood glucose meter. If the FBG (fasting blood glucose) was ≥ 11.1 mmol / L for two consecutive times, the model was considered successfully established.

[0046] The control group of mice was fed with the basal diet, and 10 mM citric acid buffer solution was gavaged according to the body weight of the mice from day 36 to day 40.

[0047] 2. Liquid chromatography-mass spectrometry was used to detect the serum non-target metabolome of mice:

[0048] (1) Serum pretreatment:

[0049] 1 mL of mouse eyeball blood was taken into a centrifuge tube and left to stand in the dark at room temperature until complete coagulation. It was centrifuged at a low speed of 3000 rpm at 4℃ for 5 min, and 450 μL of the upper-layer serum was aspirated into a 1.5 mL centrifuge tube, concentrated and dried; 500 μL of methanol solution was added and mixed evenly; it was centrifuged at 12000 rpm at 4℃ for 10 min, and all the supernatant was taken, concentrated and dried: 150 μL of 2-chloro-L-phenylalanine solution prepared with 80% methanol-water was added for reconstitution, and the supernatant was filtered through a 0.22 μm membrane. The filtrate was subjected to LC-MS detection, and the serum metabolome of mice was analyzed by liquid chromatography-mass spectrometry;

[0050] (2) Liquid chromatography-mass spectrometry:

[0051] Chromatographic conditions: Thermo Vanquish (Thermo Fisher Scientific, USA) ultra-high performance liquid system, using ACQUITY HSS T3 (2.1×100 mm, 1.8 μm) (Waters, Milford, MA, USA) chromatographic column, flow rate of 0.3 mL / min, column temperature of 40 °C, injection volume of 2 μL. Positive ion mode, mobile phase: 0.1% formic acid in acetonitrile (B1) and 0.1% formic acid in water (A1), gradient elution program: 0 - 1 min, 8% B1; 1 - 8 min, 8% - 98% B1; 8 - 10 min, 98% B1; 10 - 10.1 min, 98% - 8% B1; 10.1 - 12 min, 8% B1. Negative ion mode, mobile phase: acetonitrile (B2) and 5 mM ammonium formate in water (A2), gradient elution program: 0 - 1 min, 8% B2; 1 - 8 min, 8% - 98% B2; 8 - 10 min, 98% B2; 10 - 10.1 min, 98% - 8% B2; 10.1 - 12 min, 8% B2.

[0052] Mass spectrometry conditions: Thermo Q Exactive mass spectrometer detector (Thermo Fisher Scientific, USA), electrospray ionization source (ESI), data acquisition in positive and negative ion modes respectively. Positive ion spray voltage is 3.50 kV, negative ion spray voltage is -2.50 kV, sheath gas 40 arb, auxiliary gas 10 arb. Capillary temperature 325 °C, full-scan of the first level is performed at a resolution of 0000, the first-level ion scan range is m / z 100 - 1000, and HCD is used for second-level fragmentation, the collision energy is 30 eV, the second-level resolution is 17500, the first 10 ions of the collected signal are fragmented, and at the same time, dynamic exclusion is used to remove unnecessary MS / MS information.

[0053] Using Metabo Analyst and Cluster 3.0 tools, analyze the serum metabolic profile characteristics of T2DM sarcopenia mice compared with control group mice.

[0054] 3. Detection of the contents of Lactobacillus and Bifidobacterium in mouse feces:

[0055] At the end of the experiment, 2-3 feces of mice were collected under sterile conditions. Sterilized EP tubes and forceps were prepared. Each mouse was placed separately in a clean and sterile container and waited for natural defecation. The feces were collected into sterile EP tubes, placed on ice, and the collected feces were transferred to a -80°C refrigerator for storage within 2 h. Fecal genomic DNA in the control and model groups was extracted according to the instructions of the fecal genomic DNA extraction kit, and the DNA concentration was detected using a microplate reader. Equal amounts of DNA were added for real-time quantitative polymerase chain reaction (RT-PCR) to detect the expression of total bacteria, Lactobacillus, and Bifidobacterium in the feces of the two groups of mice. The primer sequences are shown in Table 1.

[0056] Table 1 Primer sequences

[0057]

[0058] 4. RT-PCR:

[0059] Collect 2×10 6 C2C12 cells and lyse them with trizol free at 4°C. Add chloroform, shake, centrifuge, and take the supernatant after standing. Add an equal amount of isopropanol to precipitate, and then wash the precipitate with 75% ethanol to obtain relatively pure RNA. Add an appropriate amount of enzyme-free water to dissolve the precipitate, measure its absorbance at 260 / 280, and dilute the concentration to between 300 - 500. According to the different concentrations of each group, take 3 μg of RNA and make up the volume to 12 μL. Add 3 μL of 5x buffer, mix well and incubate at 42°C for 2 min. Then add 5 μL of 4x buffer, mix well and perform RT-PCR to obtain cDNA. Take 4 μL of cDNA, add 10 μL of mix, 0.8 μL of forward and reverse primers, and 5.2 μL of water into a 96-well plate for RT-PCR detection. The primer sequences are shown in Table 2. Calculate the relative expression level of the target gene using the 2 -ΔΔCt method and normalize it to β-actin. All experimental steps were carried out according to the instructions.

[0060] Table 2 RT-PCR primer sequences

[0061]

[0062] 5. Mouse blood glucose detection method:

[0063] Mice were fasted overnight, fasted but allowed to drink water for 10 - 12 hours, and blood was collected the next day. The operation was gentle to avoid stress response in mice. The tail tip of the mouse was disinfected with alcohol, and blood was collected with a blood collection needle. Discard the first drop of blood, and take the second drop to measure the blood glucose of the mouse using a Roche blood glucose meter and test strips.

[0064] 6. Oral Glucose Tolerance Test (OGTT):

[0065] On the second day after the gavage of mice, an oral glucose tolerance test (OGTT) was conducted. The mice were fasted for 12 h without water deprivation, and then gavaged with a corresponding volume of glucose solution (2 g / kg, i.g.) according to their body weight. Blood glucose was measured at 15 min, 30 min, 60 min, 90 min, and 120 min after the initial glucose load, and the area under the curve (AUC) at 120 min was calculated.

[0066] 7. Insulin Tolerance Test (ITT):

[0067] One day after the OGTT, the insulin tolerance test (ITT) was performed. The mice were fasted for 6 h without water deprivation and then intraperitoneally injected with insulin (0.75 U / kg) according to their body weight. Timing started from the moment of injection, and blood glucose was measured at 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min, and the area under the curve (AUC) at 120 min was calculated.

[0068] Example 1: Serum non-target metabolomics analysis of mice in the T2DM sarcopenia group

[0069] Twelve mice were randomly divided into two groups (6 mice in each group), namely the control group (Ctrl group) and the T2DM sarcopenia model group (T2DM sarcopenia group).

[0070] Six weeks after T2DM modeling, the fasting blood glucose of the mice was measured, and the results are shown in Table 3.

[0071] Table 3 Fasting blood glucose of mice

[0072] Grouping Fasting blood glucose (6 weeks after modeling) Ctrl group 5.18±0.26 T2DM sarcopenia group 26.32±1.58

[0073] The results showed that the fasting blood glucose of the mice in the T2DM sarcopenia group was 26.32 ± 1.58 six weeks after modeling, indicating successful model construction. Blood was taken from the eyeballs of the mice in the Ctrl group and the T2DM sarcopenia group, and the non-target metabolome of the mouse serum was detected and analyzed by liquid chromatography-mass spectrometry.

[0074] The MetaboAnalyst and Cluster 3.0 tools were used for analysis to compare the serum metabolic profile characteristics of T2DM sarcopenia mice with those of Ctrl group mice. Figure 1 A The results showed that compared with the Ctrl group, 7-ketodeoxycholic acid (7-KDC) was significantly downregulated in the T2DM group; Figure 1B The results showed that 7-KDC was highly correlated with other metabolites. Figure 1 C The results showed that pathway enrichment analysis found significant differences in bile acid pathways.

[0075] 7-KDC can be produced by Lactobacillus and Bifidobacterium under specific conditions. RT-PCR was performed on the feces of the two groups of mice. Figure 1 D. Figure 1 E. Figure 1 The results showed that the expression of total bacteria, lactobacillus and bifidobacterium in the T2DM group was significantly lower than that in the control group.

[0076] Example 2: 7-KDC upregulates muscle differentiation markers in C2C12 cells

[0077] The frozen C2C12 cells were thawed and revived, and cultured in a 60 mm culture dish using DMEM low-glucose (5.56 mmol / L glucose) medium. The cells in the logarithmic growth phase were digested with trypsin, passaged, and inoculated in a 6-well plate.

[0078] There were Ctrl group (5.56mmol / L glucose), HG group (30mmol / L glucose), HG+3μM 7-KDC group, HG+10μM 7-KDC group and HG+30μM 7-KDC group respectively.

[0079] Six replicate wells were set up in each group. After adding different concentrations of 7-KDC for 30 minutes, the cells were incubated with 5.56 or 30 mmol / L glucose for another 24 hours. The cells in each group were collected and the expression levels of Myf5, MyoD, MyoG, and MyHC genes in the cells were detected by RT-PCR.

[0080] The results are as follows Figure 2As shown, compared with the Ctrl group, the mRNA expression levels of muscle differentiation markers Myf5 (0.58 ± 0.03), MyoD (0.44 ± 0.13), MyoG (0.42 ± 0.03), and MyHC (0.40 ± 0.06) genes in the HG group were significantly decreased; compared with the HG group, the effects of HG + 3 μM 7-KDC on Myf5, MyoD, MyoG, and MyHC were not obvious, and HG + 10 μM 7-KDC could restore the decrease in the expression of Myf5 (1.04 ± 0.03), MyoD (1.09 ± 0.08), MyoG (0.96 ± 0.06), and MyHC (1.10 ± 0.12) induced by high glucose; HG + 30 μM 7-KDC could also significantly restore the decrease in the expression of Myf5 (1.13 ± 0.11), MyoD (1.30 ± 0.10), MyoG (1.02 ± 0.15), and MyHC (1.00 ± 0.12) induced by high glucose.

[0081] Example 3: 7-KDC Improves Sarcopenia in T2DM Mice

[0082] Thirty mice were taken and a type 2 diabetes mouse model was constructed by high-fat feeding combined with STZ. After the successful construction of the model, 7-KDC was continuously intragastrically administered for 6 weeks for intervention, and 7-KDC was intragastrically administered every two days. According to the different amounts of 7-KDC intragastrically administered, they were divided into five groups: Ctrl, T2DM sarcopenia group, T2DM + 3 mg / kg 7-KDC, T2DM + 10 mg / kg 7-KDC, and T2DM + 30 mg / kg 7-KDC group. Finally, various indexes of the mice were detected.

[0083] Ctrl group: 1 μg of co-solvent sodium carboxymethylcellulose (CMC-Na) was added to every 200 μL of PBS and intragastrically administered according to 6.7 μL / g of body weight, and intragastrically administered every two days.

[0084] T2DM sarcopenia group: 1 μg of co-solvent sodium carboxymethylcellulose (CMC-Na) was added to every 200 μL of PBS and intragastrically administered according to 6.7 μL / g of body weight, and intragastrically administered every two days.

[0085] T2DM + 3 mg / kg 7-KDC group: 0.09 mg of 7-KDC was added to every 200 μL of PBS, and 1 μg of sodium carboxymethylcellulose (CMC-Na) was added, and intragastrically administered according to 6.7 μL / g of body weight, and intragastrically administered every two days.

[0086] T2DM + 10 mg / kg 7-KDC group: 0.3 mg of 7-KDC was added to every 200 μL of PBS, and 1 μg of sodium carboxymethylcellulose (CMC-Na) was added, and intragastrically administered according to 6.7 μL / g of body weight, and intragastrically administered every two days.

[0087] T2DM + 30 mg / kg 7-KDC group: Add 0.9 mg of 7-KDC to every 200 μL of PBS, and add 1 μg of the solubilizer sodium carboxymethyl cellulose (CMC-Na). Perform gavage according to 6.7 μL / g of body weight, once every two days.

[0088] Model verification results:

[0089] After 6 weeks of model establishment, the fasting blood glucose of the mice was detected. The results are shown in Table 4. The results in Table 4 indicate that the T2DM model was successfully established.

[0090] Table 4 Fasting blood glucose of mice

[0091]

[0092]

[0093] (1) Blood glucose regulation ability

[0094] After the intervention, the mice were fasted overnight without water restriction, and gavaged with an aqueous solution of 20% w / v anhydrous glucose at 2 g / kg. Blood samples were taken from the mouse tail tips at 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min to measure their blood glucose levels, and an oral glucose tolerance test (OGTT) was performed.

[0095] The results are as Figure 3 shown in Figure A and Table 5. Compared with the Ctrl group, the blood glucose levels of the mice in the T2DM sarcopenia group increased at each time point after oral glucose administration, and the AUC value increased significantly by 144.05%; compared with the T2DM sarcopenia group, the blood glucose changes of the mice in the T2DM + 3 mg / kg 7-KDC group were not obvious at each time point, and the AUC value only decreased by 9.23%. However, the blood glucose levels of the mice in the T2DM + 10 mg / kg 7-KDC and T2DM + 30 mg / kg 7-KDC groups decreased significantly at each time point, and the AUC values of the two groups of mice decreased by 25.66% and 31.17% respectively.

[0096] Table 5 Effects of 7-KDC on OGTT in type 2 diabetic mice

[0097]

[0098] (2) Insulin resistance ability

[0099] After the intervention, the mice were fasted overnight without water restriction, and intraperitoneally injected with 0.75 U / kg of insulin solution. Blood samples were taken from the mouse tail tips at 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min to measure their blood glucose levels, and an insulin tolerance test (ITT) was performed.

[0100] The results are as Figure 3 shown in Figure B and Table 6. Compared with the Ctrl group, the blood glucose levels of mice in the T2DM sarcopenia group increased at each time point after intraperitoneal injection of insulin, and the AUC value increased significantly by 193.32%; compared with the T2DM sarcopenia group, the blood glucose levels of mice in the T2DM + 3 mg / kg 7-KDC group remained basically unchanged at each time point, and the AUC value only decreased by 9.41%. In the T2DM + 10 mg / kg 7-KDC and T2DM + 30 mg / kg 7-KDC groups, the blood glucose levels decreased at each time point, and the AUC values of the two groups of mice decreased by 36.53% and 38.13% respectively compared with the T2DM sarcopenia group.

[0101] Table 6 Effects of 7-KDC on ITT in type 2 diabetic mice

[0102]

[0103] Combined with Figure 3 Figures A, 3B, Table 5 and Table 6, it can be seen that as the dosage of 7-KDC increases, the diabetic symptoms of mice are effectively alleviated.

[0104] (3) Gastrocnemius muscle detection

[0105] The mice were sacrificed, and the size and weight of the gastrocnemius muscle were compared. The results are as Figure 3 shown in Figures C to 3E and Table 7. The results show that as the dosage of 7-KDC increases, according to the apparent phenomena and quantitative statistics, the muscle length of mice is significantly improved, and the muscle size and weight are statistically analyzed. Compared with the Ctrl group, the gastrocnemius muscle length and weight of mice in the T2DM sarcopenia group decreased by 23.29% and 18.21% respectively. In the T2DM + 3 mg / kg 7-KDC group, the changes in the size and weight of the gastrocnemius muscle were not obvious. In the T2DM + 10 mg / kg 7-KDC group, the size and weight of the gastrocnemius muscle increased significantly, increasing by 23.21% and 22.66% respectively compared with the T2DM sarcopenia group. In the T2DM + 30 mg / kg 7-KDC group, the size and weight of the gastrocnemius muscle increased significantly, increasing by 21.43% and 23.80% respectively compared with the T2DM group.

[0106] Table 7 Effects of 7-KDC on the gastrocnemius muscle of type 2 diabetic mice

[0107] Group Gastrocnemius length (cm) Gastrocnemius weight / body weight (mg / g) Ctrl group 1.22±0.03 7.59±0.18 T2DM group 0.93±0.03 6.21±0.23 T2DM + 3mg / kg 7-KDC group 1.00±0.03 6.49±0.26 T2DM + 10mg / kg 7-KDC group 1.15±0.03 7.61±0.22 T2DM + 30mg / kg 7-KDC group 1.13±0.03 7.68±0.15

[0108] (4) mRNA detection of Myf5, MyoD, MyoG, and MyHC genes

[0109] RT-PCR was used to detect the mRNA expression of muscle differentiation indexes Myf5, MyoD, MyoG, and MyHC genes in the gastrocnemius muscle. The results are asFigure 4 As shown, compared with the Ctrl group, the mRNA expression levels of muscle differentiation markers Myf5 (0.46±0.08), MyoD (0.37±0.05), MyoG (0.44±0.05), and MyHC (0.48±0.03) genes were significantly decreased in the T2DM sarcopenia group. Compared with the T2DM group, the improvement effect of the expression levels of Myf5, MyoD, MyoG, and MyHC in the mice of the T2DM+3mg / kg 7-KDC group was not obvious compared with the T2DM group. In the mice of the T2DM+10mg / kg 7-KDC group, the expression levels of Myf5 (0.98±0.14), MyoD (0.93±0.06), MyoG (0.94±0.09), and MyHC (1.04±0.05) were significantly increased compared with the T2DM group. In the mice of the T2DM+30mg / kg 7-KDC group, the expression levels of Myf5 (0.98±0.14), MyoD (1.00±0.12), MyoG (1.07±0.15), and MyHC (0.98±0.06) were significantly increased compared with the T2DM sarcopenia group. It can be seen that administering 10mg / kg and 30mg / kg of 7-KDC can restore the decrease in the expression levels of muscle differentiation markers caused by T2DM.

[0110] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. Use of 7-keto-3ɑ,12ɑ-dihydroxycholic acid or a composition containing 7-keto-3ɑ,12ɑ-dihydroxycholic acid in the preparation of a drug for simultaneously preventing and / or treating type 2 diabetes and diabetic sarcopenia; The diabetic sarcopenia is sarcopenia caused by type 2 diabetes, and the symptoms are chronic muscle atrophy and gradual apoptosis of muscle cells; The structural formula of the 7-keto-3ɑ,12ɑ-dihydroxycholic acid is shown below: 。 2. The use according to claim 1, characterized in that: The drug is in any pharmaceutically acceptable dosage form.

3. The use according to claim 2, characterized in that: The dosage forms include: tablets, inhalation preparations, oral liquids, injections, and external preparations.

4. The use according to claim 1, characterized in that: The composition further contains auxiliary materials.

5. The use according to claim 4, characterized in that: The excipients are pharmaceutically acceptable excipients; the excipients include: one or more of probiotics, lactose, maltose, microcrystalline cellulose, carboxymethyl cellulose, hydroxypropyl methyl cellulose, starch, sucrose, glucose, aspartame, water, glycerol, whey protein powder, and chitosan oligosaccharide.

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