Combined medicine for preventing and treating diabetes

Abstract

The invention discloses a combined medicine for preventing and treating diabetes mellitus, and belongs to the technical field of medicines. The medicine for preventing and treating diabetes mellitus is prepared from alpinetin and rosiglitazone, and the mass ratio of the alpinetin to the rosiglitazone is (10-20): 1. According to the invention, alpinetin and rosiglitazone are combined for use, so that diabetes mellitus can be treated more effectively, osteoporosis symptoms caused by diabetes mellitus can be improved, and a new therapeutic drug and approach are provided for treatment of diabetes mellitus.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a combined medicine for preventing and treating diabetes. Background Art

[0002] Type 2 diabetes mellitus (T2DM) is a common chronic metabolic disease caused by a variety of factors that lead to insufficient insulin secretion or the body's inability to effectively utilize insulin, resulting in persistently high blood sugar levels.

[0003] Insulin resistance is the main feature of T2DM, which runs through the entire disease process from the early stage to the development of T2DM, and insulin sensitizers are effective drugs for the treatment of T2DM. Since its launch in 1999, thiazolidinediones (TZDs), represented by rosiglitazone, have played an important role in lowering blood sugar and improving insulin resistance, and are considered to be the best insulin sensitizers in clinical effect. However, these drugs still have shortcomings, which may cause osteoporosis, cardiovascular risks and weight gain, limiting their applicability for long-term use. Therefore, it is of great clinical significance to develop a drug that can effectively treat diabetes without side effects. Summary of the invention

[0004] In view of the above deficiencies in the prior art, the present invention provides a combined drug for preventing and treating diabetes, which can effectively treat diabetes and improve the symptoms of osteoporosis caused by diabetes.

[0005] To achieve the above purpose, the specific technical solutions of the present invention are as follows:

[0006] In a first aspect, the present invention provides a combined drug for preventing and treating diabetes, comprising shanpin and rosiglitazone, wherein the mass ratio of shanpin to rosiglitazone is 10-20:1.

[0007] Thiazolidinediones (TZDs) drugs represented by rosiglitazone (Rosi) are likely to cause side effects such as osteoporosis in diabetic patients, and osteoporosis is a common complication of diabetes. The present invention provides a drug for preventing and treating diabetes. The present invention combines sansingol with rosiglitazone in a specific ratio, which can effectively treat diabetes and improve the symptoms of osteoporosis caused by diabetes.

[0008] Preferably, the effective amount of shanpin is 4.5 to 9 mg / kg per day; the effective amount of rosiglitazone is 0.45 mg / kg per day.

[0009] Preferably, a pharmaceutically acceptable carrier and / or excipient is also included.

[0010] Preferably, the dosage form of the drug includes but is not limited to tablets, granules, capsules, pills, sustained-release preparations, oral liquid preparations, and injections.

[0011] In a second aspect, the present invention provides use of the drug in preparing a drug for preventing and treating diabetes.

[0012] Preferably, the use includes promoting the expression of insulin sensitivity-related pathways, inhibiting the expression level of osteoclast-related genes, and alleviating the symptoms of diabetic osteoporosis.

[0013] Compared with the prior art, the present invention is beneficial in that:

[0014] The present invention uses shanpinol and rosiglitazone in combination, which can not only effectively treat diabetes, but also improve the osteoporosis symptoms caused by diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The results of drug intervention in Example 1 on the expression levels of insulin sensitivity-related genes Slc2a4, Pparg, and Adipoq;

[0016] Figure 2 The results of drug intervention in Example 2 on the expression levels of osteoclast-related genes Ctsk, Mmp9, and Trap;

[0017] Figure 3 The results of the drug intervention in Example 3 on the expression of insulin sensitivity-related pathway proteins p-PI3K, PI3K, p-AKT, AKT, and GLUT4;

[0018] Figure 4 The Micro CT scan and quantitative analysis results of the drug intervention on cancellous bone of T2DM mice in Example 3;

[0019] Figure 5 Micro CT scanning and quantitative analysis results of the cortical bone of T2DM mice after drug intervention in Example 3. DETAILED DESCRIPTION

[0020] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] In the following specific embodiments, the alpinol can be directly purchased from the market or prepared by conventional methods in the art; the alpinol can be derived from the mature seeds of the ginger plant Alpinia cardamomum, and its English name is: Alpinetin (Apt), CAS No. 36052-37-6, and its molecular formula is C 16 H 14 O4, the chemical structure is shown below:

[0022]

[0023] In the following specific embodiments, the sanpin working solution used is prepared by the following method: take 20 mg of purchased sanpin drug powder, add 740 μL of sterile DMSO according to the instructions for preparing the mother solution to prepare a sanpin mother solution with a concentration of 100 mM, wait until the mother solution is fully dissolved, divide it into EP tubes and seal them, and freeze them at -20°C for use. When using, dilute the sanpin mother solution to the required working concentration (25~100 μM).

[0024] Example 1

[0025] Effects of drugs on the 3T3-L1 cell model of insulin resistance

[0026] 1. Cultivation and intervention of insulin-resistant 3T3-L1 adipocytes

[0027] (1) 3T3-L1 cells were seeded into 48-well plates (1×10 5 / well), after 24 h of contact inhibition, the medium was replaced with adipogenic induction medium A: DMEM culture medium with 10% fetal bovine serum containing 1 μM dexamethasone, 1.5 μg / mL insulin, and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX); after 3 days of induction, the medium was replaced with adipogenic induction medium B: DMEM culture medium with 10% fetal bovine serum containing 1.5 μg / mL insulin; after 3 days, the medium was replaced with normal culture medium;

[0028] (2) TNF-α (final concentration 10 ng / mL) was added to the treatment group and incubated for 24 h;

[0029] (3) Drug treatment groups were added with drug solutions with final concentrations of 25 μM Apt, 50 μM, 100 μM, 5 μM Rosi, 25 μM Apt + 5 μM Rosi, 50 μM Apt + 5 μM Rosi, and 100 μM Apt + 5 μM Rosi, respectively, and incubated for 24 h;

[0030] 2. qPCR assay

[0031] Total RNA was extracted from 3T3-L1 adipose tissue using TRIzol reagent (Takara, Japan) and dissolved in enzyme-free water. The extracted RNA was reverse transcribed into complementary DNA (cDNA) using reverse transcription reagent provided by Vazyme. Quantitative PCR (qPCR) experiments were performed on the QuantStudio 6 Flex Real-Time PCR System (Life Technologies, USA). The 2-ΔΔCt method was used to quantify the relative RNA expression level. β-actin was used as an internal reference gene to calculate the relative expression abundance. The primer sequences used for amplification are shown in the following table:

[0032]

[0033] The qPCR results are as follows Figure 1 As shown. Figure 1 It can be seen that in 3T3-L1 adipocytes, the combination of shanjiangsu and rosiglitazone promoted the expression levels of insulin-sensitive genes Slc2a4, Pparg, and Adipoq, among which the effects of the shanjiangsu + rosiglitazone combined administration group (50 μM Apt + 5μM Rosi) and the shanjiangsu + rosiglitazone combined administration group (100 μM Apt + 5μM Rosi) were particularly significant.

[0034] Example 2

[0035] Effects of drugs on osteoclasts in vitro

[0036] 1. Isolation of bone marrow macrophages (BMMs) and osteoclast induction

[0037] Place the mice (ordinary mice) killed by dislocation in a 50 mL centrifuge tube and soak them in 75% alcohol for 15 minutes. Take out the mice and place them in a sterilized tray in the clean bench. Carefully cut the skin on the back of the mice, peel off the skin of both lower limbs of the mice by blunt separation, separate the lower limbs from the hip joint, cut off the complete lower limbs at the ankles, and place them in a 75% alcohol dish. Carefully remove the muscle tissue around the bones with sterile ophthalmic scissors and sterile gauze, and place the clean femur and tibia in a sterile PBS solution dish to prevent the tissue from drying. Use new sterile ophthalmic scissors to cut off both ends of the femur and tibia, then use a 1 mL syringe to absorb α-MEM culture medium, insert the needle into one end of the femur or tibia, and aim at a 50 mL centrifuge tube to flush the medullary cavity and collect BMMs until the femur or tibia turns white. After flushing, filter the cells through a 40 μm filter, add M-CSF (30 ng / mL), blow and mix, inoculate the cells in a 10 cm culture dish, and place it at 37 ℃, 5% CO2 constant temperature incubator for 16 hours; the supernatant was aspirated into a new 10 cm culture dish, and α-MEM medium was added to 8 mL, supplemented with M-CSF (30 ng / mL), and the supernatant was removed after continuing to culture for 3 days. The suspended cells were washed with PBS to obtain the attached BMMs.

[0038] BMMs were seeded in 96-well plates at 15,000 cells per well, and 250,000 cells per well in 6-well plates, and cultured in α-MEM medium containing 30 ng / mL M-CSF. When the cell density reached 60%-80%, 75 ng / mL RANKL (Receptor Activator of Nuclear Factor-κ B Ligand) was added to the culture medium for induction. The cells were observed under an optical microscope every day. Large, fully fused, differentiated, mature multinucleated giant cells, namely osteoclasts, began to appear on the 5th day of culture.

[0039] Drug interventions were grouped according to the following concentrations: 25 μM Apt, 50 μM, 100 μM, 5 μM Rosi, 25 μM Apt+5 μM Rosi, 50 μM Apt+5 μM Rosi, 100 μM Apt+5 μM Rosi, and drug interventions were carried out simultaneously with RANKL induction.

[0040] 2. qPCR assay

[0041] Total RNA was extracted from osteoclasts using TRIzol reagent (Takara, Japan) and dissolved in enzyme-free water. The extracted RNA was reverse transcribed into complementary DNA (cDNA) using reverse transcription reagent provided by Vazyme. Quantitative PCR (qPCR) experiments were performed on the QuantStudio 6 Flex Real-Time PCR System (Life Technologies, USA). The 2-ΔΔCt method was used to quantify the relative RNA expression level. β-actin was used as an internal reference gene to calculate the relative expression abundance. The primer sequences used for amplification are shown in the following table:

[0042]

[0043] The qPCR results are as follows Figure 2 As shown. Figure 2 It can be seen that in the in vitro induced mature osteoclasts, the combined use of shanjiangsu and rosiglitazone significantly inhibited the expression levels of osteoclast-related genes Ctsk, Mmp9, and Trap compared with the group treated with rosiglitazone alone.

[0044] Example 3

[0045] Effects of drug intervention on T2DM mice

[0046] Construction of type 1 and 2 diabetes mellitus (T2DM) mouse model

[0047] Six-week-old male C57BL / 6J mice were selected and fed with normal feed for 2 days. Ten mice were randomly selected as the normal control group and given normal feed. The rest of the animals were given high-fat and high-sugar feed (Research diet D12492, 60% kcal%). After 6 weeks of feeding (12 weeks of age), low-dose streptozotocin (STZ, 30 mg / kg, Sigma) was injected intraperitoneally every day for 3 consecutive days, and fasted for 5 hours before each injection. Fasting blood glucose levels were measured two weeks after the first injection of STZ (14 weeks of age), and mice with blood glucose levels below 190 mg / dL (10.5 mmol / L) were excluded. The experiment was divided into control group (Control), model group (T2DM), low-dose shanjiangsu administration group (25 mg / kg) (equivalent to 2.2 mg / kg for humans), high-dose shanjiangsu administration group (50 mg / kg) (equivalent to 4.5 mg / kg for humans), rosiglitazone administration group (5 mg / kg) (equivalent to 0.45 mg / kg for humans), shanjiangsu + rosiglitazone combined administration group (25 mg / kg + 5 mg / kg), shanjiangsu + rosiglitazone combined administration group (50 mg / kg + 5 mg / kg). The administration group was gavaged with corresponding doses of shanjiangsu and rosiglitazone every day, and the control group and model group were gavaged with corresponding volumes of solvent (the solvent was saline containing 10% DMSO, 5% Tween 80 and 35% PEG). The mice continued to eat high-fat and high-sugar feed, and the weight of the mice was recorded every week until the mice were sampled for testing at 22 weeks of age.

[0048] 2. Western blot experiment

[0049] (1) Epididymal white fat pad (eWAT) was collected and quickly frozen in liquid nitrogen. Tissue lysis buffer was added and ultrasonically disrupted at 4°C for 20 min.

[0050] (2) Prepare 10% concentration of SDS-PAGE gel (G2043-50T, Wuhan Saiweier Biotechnology Co., Ltd.);

[0051] (3) Determine protein concentration by BCA method to ensure consistent protein loading;

[0052] (4) Electrophoresis separation: Keep constant voltage at 200-250 V for 30 min. Stop electrophoresis when the phenol blue indicator reaches the bottom of the gel. For ease of comparison, use the same volume of each sample during electrophoresis.

[0053] (5) Transfer and antibody reaction: After the electrophoresis is completed, remove the gel and wash it with clean water. At the same time, soak the PVDF membrane and filter paper in ice-free rapid transfer buffer (G2148-1L, Wuhan Sevier Biotechnology Co., Ltd.) for 5 min, lay the gel and NC membrane in the order of cathode-sponge-filter paper-gel-NC membrane-filter paper-sponge-anode, put them into the electrophoresis tank, and electrophoresed at 400 mA for 40 min at low temperature;

[0054] (6) Remove the PVDF membrane and wash it with 1×PBST (PBS with 0.1% Tween-20) for 5 min, add TBST blocking buffer containing 5% skim milk powder, and incubate at 37°C for 2 h; wash the PVDF membrane with TBST for 3 times, 10 min each time; add rabbit anti-p-PI3K, PI3K, p-AKT, AKT, β-TUBLIN monoclonal antibodies (1:1000 TBST dilution) and mouse anti-GLUT4 monoclonal antibodies (1:1000 TBST dilution) and incubate at 4°C overnight; wash the PVDF membrane with TBST for 3 times, 5 min each time; add HRP-labeled goat anti-rabbit IgG antibody (BL003A, Biosharp Life Sciences, 1:10000 dilution in TBST) and HRP-labeled goat anti-mouse IgG antibody (BL001A, Biosharp Life Sciences, 1:10000 dilution in TBST) and incubate at 37°C for 1 h, wash the PVDF membrane thoroughly with TBST 3-5 times, 10 min each time, add ECL chemiluminescent substrate (BL520B, Biosharp LifeSciences) at room temperature and avoid light for 3 min, and observe and record the results in the dark.

[0055] Figure 3 Western blot was used to detect the effect of combined drug use on the expression of proteins related to the insulin signaling pathway. Figure 3 It can be seen that in the in vitro induced mature osteoclasts, the combination of shanjiangsu and rosiglitazone significantly promoted the expression of related proteins p-PI3K, PI3K, p-AKT, AKT, and GLUT4.

[0056] 3. Micro CT Scanning

[0057] SkyScan1076 (Bruker, Germany) Micro-CT was used for bone 3D reconstruction and trabecular parameter detection. The femur of the mouse was fixed in the center of the sample tube and scanned at a resolution of 9 μm / pix. The 200 slices below the distal femoral growth plate were selected as the region of interest 1 (ROI-1) to analyze the trabecular structure and parameters of cancellous bone, including bone mineral density (BMD), cancellous bone volume ratio (BV / TV), cancellous bone trabecular thickness (Tb.Th) and cancellous bone trabecular spacing (Tb.Sp). The scanning results are shown in Figure 4 shown. Figure 4 The results showed that STZ-induced diabetes led to a severe decrease in bone density, which was further reduced after taking the hypoglycemic drug rosiglitazone; while the combination of sansevieriaceae and rosiglitazone in the treatment of diabetes could improve the symptoms of diabetic osteoporosis.

[0058] The 100 slices of the femoral shaft in the middle of the femur were selected as the region of interest 2 (ROI-2) for analyzing the three-dimensional structure of the cortical bone and the thickness of the cortical bone (Ct.Th). The results are shown in Figure 5 As shown. Figure 5 It can be seen that the cortical bone tissue volume (TV) and cortical bone area (Ct. Ar) of the group treated with rosiglitazone alone decreased significantly, while the cortical bone tissue volume (TV) and cortical bone area (Ct. Ar) of the group treated with combined treatment of sansevieriaceae and rosiglitazone increased significantly.

[0059] In summary, the present invention uses shanpinol and rosiglitazone in combination according to a specific ratio, which can not only treat diabetes more effectively, but also significantly improve the osteoporosis symptoms caused by diabetes, thereby providing a new therapeutic drug and approach for the treatment of diabetes.

[0060] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A combined drug for preventing and treating diabetes, characterized in that: It comprises shanpin and rosiglitazone, wherein the mass ratio of shanpin to rosiglitazone is (10-20):

1.

2. A combined drug for preventing and treating diabetes according to claim 1, characterized in that: The effective dosage of the shanjiangsu is 4.5 to 9 mg / kg per day, and the effective dosage of the rosiglitazone is 0.45 mg / kg per day.

3. A combined drug for preventing and treating diabetes according to claim 1, characterized in that: Pharmaceutically acceptable carriers and / or excipients are also included.

4. The combined drug for preventing and treating diabetes according to claim 1, characterized in that: The dosage form of the drug includes but is not limited to tablets, granules, capsules, pills, sustained-release preparations, oral liquid preparations, and injections.

5. Use of the drug according to any one of claims 1 to 4 in the preparation of a drug for preventing and treating diabetes.

6. Use of the drug according to claim 5 in preparing a drug for preventing and treating diabetes, characterized in that: The application includes promoting the expression of insulin sensitivity-related pathways, inhibiting the expression level of osteoclast-related genes, and alleviating the symptoms of diabetic osteoporosis.

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