A combined drug for preventing and treating diabetes

Abstract

The present invention discloses a combination drug for preventing and treating diabetes, belonging to the field of medical technology. The drug comprises shanjiangsu and rosiglitazone, with the mass ratio of shanjiangsu to rosiglitazone being (10-20):1. The present invention combines shanjiangsu and rosiglitazone to more effectively treat diabetes and improve the symptoms of osteoporosis caused by diabetes, providing a new therapeutic drug and approach for the treatment of diabetes.

Description

Technical Field

[0001] The present invention relates to the technical field of medicine, 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 a key characteristic of type 2 diabetes (T2DM), a condition that persists throughout the disease progression from the early stages to the onset. Insulin sensitizers are effective treatments for T2DM. Since their introduction in 1999, thiazolidinediones (TZDs), represented by rosiglitazone, have played a significant role in lowering blood sugar and improving insulin resistance, and are considered the most clinically effective insulin sensitizers. However, these drugs still have drawbacks, including the potential for osteoporosis, cardiovascular risks, and weight gain, limiting their long-term suitability. Therefore, developing an effective, side-effect-free treatment for diabetes is of great clinical significance. 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 shanpinol and rosiglitazone, wherein the mass ratio of shanpinol to rosiglitazone is 10-20:1.

[0007] Thiazolidinediones (TZDs), represented by rosiglitazone (Rosi), are prone to side effects such as osteoporosis in diabetic patients. Furthermore, osteoporosis is a common complication of diabetes. The present invention provides a drug for preventing and treating diabetes. The present invention combines shanjiangsu with rosiglitazone in a specific ratio, which can not only effectively treat diabetes but also improve the symptoms of osteoporosis caused by diabetes.

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

[0009] Preferably, it also includes a pharmaceutically acceptable carrier and / or excipient.

[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 application includes promoting the expression of insulin sensitivity-related pathways, inhibiting the expression levels of osteoclastosis-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 shanjiangsu 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 the 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 results of Micro CT scanning and quantitative analysis of the cancellous bone of T2DM mice treated with drug intervention in Example 3 are shown;

[0019] Figure 5 These are the Micro CT scans and quantitative analysis results of the cortical bones of T2DM mice treated with drug intervention in Example 3. DETAILED DESCRIPTION

[0020] The technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort 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, the English name is: Alpinetin (Apt), CAS No. 36052-37-6, molecular formula C 16 H 14 O4, the chemical structure is as follows:

[0022]

[0023] In the following specific embodiments, the shanjiangsu working solution used is prepared by the following method: 20 mg of purchased shanjiangsu drug powder is taken, and 740 μL of sterile DMSO is added according to the instructions for preparing the mother solution to prepare a shanjiangsu mother solution with a concentration of 100 mM. After the mother solution is fully dissolved, it is divided into EP tubes and sealed, and frozen at -20°C for use. When used, the shanjiangsu mother solution is diluted 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. Culture and intervention of insulin-resistant 3T3-L1 adipocytes

[0027] (1) 3T3-L1 cells were seeded into 48-well plates (1×10 5 After 24 h of contact inhibition, the cells were 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 cells were replaced with adipogenic induction medium B: DMEM culture medium with 10% fetal bovine serum containing 1.5 μg / mL insulin; after 3 days, the cells were 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 at 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 reagents provided by Vazyme. Quantitative PCR (qPCR) experiments were performed on a QuantStudio 6 Flex Real-Time PCR System (Life Technologies, USA). Relative RNA expression levels were quantified using the 2-ΔΔCt method. β-actin was used as an internal reference gene to calculate relative expression abundance. The primer sequences used for amplification are shown in the table below:

[0032]

[0033] qPCR experimental 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 them, the effects of the shanjiangsu + rosiglitazone combined administration group (50 μM Apt + 5 μM Rosi) and the shanjiangsu + rosiglitazone combined administration group (100 μM Apt + 5 μM 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 (normal mice) killed by dislocation in a 50 mL centrifuge tube and soak them in 75% alcohol for 15 minutes. Remove the mice and place them in a sterilized tray in a clean bench. Carefully cut the skin on the back of the mice, peel off the skin of both lower limbs by blunt dissection, separate the lower limbs from the hip joint, cut them at the ankle to free the complete lower limbs, and place them in a 75% alcohol dish. Use sterile ophthalmic scissors and sterile gauze to carefully remove the muscle tissue around the bones. Remove the clean femur and tibia and place them 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 draw α-MEM medium, insert the needle into one end of the femur or tibia, and align it with 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), mix well by pipetting, and seed the cells in a 10 cm culture dish and place it at 37 The cells were cultured in a constant temperature incubator at 4°C and 5% CO2 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). After further culture for 3 days, the supernatant was removed, and the suspended cells were washed with PBS to obtain adherent BMMs.

[0038] BMMs were seeded in 96-well plates at a density of 15,000 cells per well, and in 6-well plates at a density of 250,000 cells per well. They were 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 intervention was 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 intervention was 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 reagents provided by Vazyme. Quantitative PCR (qPCR) experiments were performed on a QuantStudio 6 Flex Real-Time PCR System (Life Technologies, USA). Relative RNA expression levels were quantified using the 2-ΔΔCt method. β-actin was used as an internal reference gene to calculate relative expression abundance. The primer sequences used for amplification are shown in the table below:

[0042]

[0043] qPCR experimental 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 type 2 diabetes (T2DM) mouse models

[0047] Six-week-old male C57BL / 6J mice were acclimated to a normal diet for two days. Ten mice were randomly selected as a control group and fed a normal diet. The remaining mice were fed a high-fat, high-sugar diet (Research diet D12492, 60% kcal). After six weeks of feeding (12 weeks of age), low-dose streptozotocin (STZ, 30 mg / kg, Sigma) was injected intraperitoneally daily for three consecutive days, with fasting for 5 hours before each injection. Two weeks after the first STZ injection (14 weeks of age), fasting blood glucose levels were measured, and mice with blood glucose levels below 190 mg / dL (10.5 mmol / L) were excluded. The experiment was divided into a control group (control), a model group (T2DM), a low-dose shanjiangsu (25 mg / kg) group (equivalent to 2.2 mg / kg in humans), a high-dose shanjiangsu (50 mg / kg) group (equivalent to 4.5 mg / kg in humans), a rosiglitazone (5 mg / kg) group (equivalent to 0.45 mg / kg in humans), a shanjiangsu + rosiglitazone combination group (25 mg / kg + 5 mg / kg), and a shanjiangsu + rosiglitazone combination group (50 mg / kg + 5 mg / kg). The treatment groups received daily oral administration of the corresponding doses of shanjiangsu and rosiglitazone, while the control and model groups received the corresponding volume of solvent (normal saline containing 10% DMSO, 5% Tween 80, and 35% PEG). The mice continued to consume a high-fat, high-sugar diet, and their body weights were recorded weekly until they were sampled and analyzed 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 Sevier Biotechnology Co., Ltd.);

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

[0052] (4) Electrophoresis: Keep the voltage constant 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 minutes, 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 electrophorese at 400 mA for 40 minutes.

[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 three times, 10 min each time; add rabbit anti-p-PI3K, PI3K, p-AKT, AKT, β-TUBLIN monoclonal antibodies (1:1000 TBST dilution), mouse anti-GLUT4 monoclonal antibodies (1:1000 TBST dilution), and incubate at 4°C overnight; wash the PVDF membrane with TBST three times, 5 min each time; add HRP-labeled goat anti-rabbit IgG antibody (BL003A, Biosharp Life Sciences, 1:10000 dilution in TBST), HRP-labeled goat anti-mouse IgG antibody (BL001A, Biosharp Life Sciences, 1:10000 dilution in TBST) and incubate at 37°C for 1 minute. h. Wash the PVDF membrane thoroughly with TBST 3-5 times for 10 min each time. Add ECL chemiluminescent substrate (BL520B, Biosharp LifeSciences) and incubate at room temperature in the dark for 3 min. Observe and record the luminescence 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 mature osteoclasts induced in vitro, 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] A SkyScan 1076 (Bruker, Germany) Micro-CT was used for three-dimensional bone reconstruction and trabecular bone parameter analysis. The mouse femur was fixed in the center of a sample tube and scanned at a resolution of 9 μm / pix. 200 slices below the distal femoral growth plate were selected as region of interest 1 (ROI-1) for analysis of cancellous bone trabecular structure and its parameters, 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 scan results are shown in Figure 1. Figure 4 shown. Figure 4 The results showed that STZ-induced diabetes led to a severe decrease in bone density, and taking the hypoglycemic drug rosiglitazone led to a further decrease in bone density; while the combination of shanjiangsu and rosiglitazone for 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 cortical bone thickness (Ct.Th). 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 sanpin and rosiglitazone increased significantly.

[0059] In summary, the present invention combines shanjiangsu and rosiglitazone in a specific ratio, which can not only treat diabetes more effectively, but also significantly improve the symptoms of osteoporosis caused by diabetes, 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 of the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

Claims

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

1.

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

3. The 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 forms of the drug include but are 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.

Images