Application of Xinmaitong capsule in preparation of medicine for treating type 2 diabetes mellitus

Abstract

The invention discloses an application of Xinmaitong capsules in preparation of a medicine for treating type 2 diabetes mellitus, which is characterized in that a luciferase reporter gene plasmid is constructed by targeting a glucagon-like peptide-1 receptor (GLP-1R), which is one of the most effective targets for treating the type 2 diabetes mellitus at the present stage, and HEK-293T is used as a tool cell in vitro, so that the medicine for treating the type 2 diabetes mellitus is obtained. GLP-1R luciferase reporter gene plasmids are transfected into the Xinmaitong capsules, luciferase reporter gene experiments find that the Xinmaitong capsules can significantly up-regulate the activity of GLP-1R promoters, and further in-vivo experiments verify that the Xinmaitong capsules can significantly improve the activity of the GLP-1R promoters. Results show that the Xinmaitong capsule has a remarkable improvement effect on glycolipid metabolism of mice with the type 2 diabetes by influencing a GLP-1R signal channel, so that the Xinmaitong capsule can be used as a preparation for treating the type 2 diabetes and can be prepared into oral liquid, injection, tablets, capsules, granules, dripping pills and the like which are permissible in pharmacy.

Description

Technical Field

[0001] The invention belongs to the technical field of new uses of traditional Chinese medicine compound, and particularly relates to the use of Xinmaitong capsule in preparing a medicine for treating type 2 diabetes. Background Art

[0002] Xinmaitong Capsules, manufactured by Guizhou Yibai Pharmaceutical Co., Ltd., are capsules with the following functions: promoting blood circulation and removing blood stasis, promoting blood circulation and nourishing the heart, lowering blood pressure and lipids. They are used to treat hypertension, hyperlipidemia, etc. The main ingredients include Chinese Angelica, Cassia Seed, Uncaria, Achyranthes, Salvia miltiorrhiza, Pueraria lobata, Sophora japonica, Ilex pubescens, Prunella vulgaris, and Panax notoginseng. The contents are brown to brown powder; the taste is slightly bitter and astringent.

[0003] Type 2 diabetes is a metabolic disease characterized by persistent hyperglycemia, which usually evolves from insufficient insulin secretion or insulin resistance. In recent years, its prevalence and mortality caused by complications caused by hyperglycemia have continued to increase. A series of health problems caused by type 2 diabetes have become the focus of attention in the development of metabolic disease drugs. At present, hypoglycemic drugs are widely used in clinical practice, including different types of hypoglycemic drugs such as insulin preparations, insulin secretagogues and insulin sensitizers. However, they all have adverse reactions such as hypoglycemia and gastrointestinal discomfort, and these drugs have no protective effect on damaged pancreatic β cells. They only focus on insulin itself and its corresponding targets and ignore the functional protection of pancreatic β cells. A large number of studies have shown that in pancreatic β cells, GLP-1R has the functions of promoting pancreatic cell proliferation, inhibiting pancreatic cell apoptosis, and promoting insulin release. It has a good pancreatic function protection effect. After being activated, it releases cAMP as a second messenger to activate downstream signaling pathways and thus play an anti-type 2 diabetes role. Therefore, GLP-1R is one of the most effective targets for the treatment of type 2 diabetes and has important research significance.

[0004] Traditional Chinese medicine has multiple components, multiple targets, and overall regulatory functions. It has been widely used in China for thousands of years and has shown great potential in improving health and preventing and treating various diseases. Its adverse reactions are relatively small in patients treated with traditional Chinese medicine. Therefore, it is a feasible way to explore the treatment of type 2 diabetes with traditional Chinese medicine compound preparations to find drugs that have therapeutic effects on type 2 diabetes and further develop high-efficiency and low-toxic drugs. Summary of the invention

[0005] The purpose of the present invention is to find a new drug with significant effect in treating type 2 diabetes in view of the shortcomings of the current traditional Chinese medicine compound preparations in treating type 2 diabetes.

[0006] To achieve the above object, the present invention provides the use of Xinmaitong Capsule in preparing a medicine for treating type 2 diabetes.

[0007] The present invention takes GLP-1R as the entry point, and through the construction of GLP-1R luciferase reporter gene plasmid, screens out Xinmaitong Capsule, which can promote the increase of GLP-1R promoter activity for transcription, and has potential anti-type 2 diabetes effects. Further in vivo experiments have verified that Xinmaitong Capsule can reduce the amount of water consumed, urine volume and fasting blood sugar levels in mice, and significantly improve insulin resistance, and can be used as a drug for the treatment of type 2 diabetes.

[0008] The present invention directly prepares Xinmaitong Capsules into a preparation for treating type 2 diabetes or mixes them with other drugs for treating type 2 diabetes to prepare a compound preparation for treating type 2 diabetes, wherein the preparation can be any one of oral liquid, injection, tablet, capsule, granule, and pill allowed in pharmacy.

[0009] The beneficial effects of the present invention are as follows:

[0010] 1. Compared with the existing uses of Xinmaitong Capsules, the present invention finds that Xinmaitong Capsules have a good in vivo therapeutic effect on type 2 diabetes and discovers a new use of this drug.

[0011] 2. Xinmaitong Capsule is a compound Chinese medicine preparation. As a drug for the treatment of type 2 diabetes, it has little toxicity and side effects. The GLP-1R hypoglycemic effect is a glucose-dependent mechanism, and patients will not experience adverse reactions such as hypoglycemia.

[0012] 3. Xinmaitong Capsule is a drug that has been on the market for many years and is used by a large population. It can be directly applied to the human body without the need for clinical safety evaluation. It has a good application prospect and can quickly increase the market share of this product. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is a schematic diagram of the construction of pLG3-Basic-GLP-1R plasmid and the drug screening process.

[0014] Figure 2 The MTT method was used to screen the concentrations of the two compound drugs on HEK-293T cells. Figure 2 A) and luciferase reporter gene assay to screen drugs that promote upregulation of luciferin expression in HEK-293T cells transfected with pLG3-Basic-GLP-1R plasmid ( Figure 2 B).

[0015] Figure 3 This is the effect of Xinmaitong Capsule on water intake (A), body weight (B), and urine volume (C) in type 2 diabetic mice.

[0016] Figure 4The figure shows the effect of Xinmaitong capsule on biochemical indexes and insulin resistance in mice with type 2 diabetes. A and B are the random postprandial blood glucose and random fasting blood glucose levels of mice in each group, respectively; C is the glucose level in serum; D and E are the results of OGTT experiment and AUC statistics, respectively; F, G, H, and I are the effects of Xinmaitong capsule on HDL-C, LDL-C, TC, and TG in mice with type 2 diabetes, respectively; J is the level of serum insulin; and K is the improvement effect of Xinmaitong capsule on insulin resistance.

[0017] Figure 5 H&E staining was used to observe the effects of Xinmaitong Capsule on the liver (A) and pancreatic tissue (B) of type 2 diabetic mice.

[0018] Figure 6 The effect of Xinmaitong capsule on the fluorescence of pancreatic tissue-related proteins in type 2 diabetic mice was detected by immunofluorescence (IF). A is the result of immunofluorescence staining of pancreatic tissue of mice in each group, and B and C are statistical analyses of GLP-1R and Insulin, respectively.

[0019] Figure 7 Western blot was used to detect the effect of Xinmaitong Capsule on GLP-1R signaling pathway-related proteins in pancreatic tissue of type 2 diabetic mice. A is a protein map, B and C are the expression levels of GLP-1R and PDX-1 relative to the internal reference protein β-actin. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments, but the protection scope of the present invention is not limited to these embodiments.

[0021] Example 1

[0022] Application of Xinmaitong capsule in the preparation of drugs for treating type 2 diabetes

[0023] 1. Experimental Materials

[0024] Drug sources: Xinmaitong Capsules were purchased from Guizhou Yibai Pharmaceutical Co., Ltd., and Xingpi Yanger Granules were purchased from Guizhou Jianxing Pharmaceutical Co., Ltd.

[0025] Preparation of sample solutions: Xinmaitong Capsules and Xingpiyanger Granules were dissolved in 60% ethanol aqueous solution at a concentration of 2g / 40mL, and the ethanol was removed by a rotary evaporator. The powdered drugs were lyophilized in a freeze dryer and stored at -20°C. Sample solutions with Chinese medicine compound concentrations of 10mg / mL, 1mg / mL, 0.1mg / mL, 0.001mg / mL, and 0.0001mg / mL were prepared with normal saline before use.

[0026] Preparation of 5 mg / mL MTT solution: weigh 0.5 g of MTT powder, add 100 mL of sterilized PBS, dissolve in a 60°C water bath overnight, filter through a 0.22 μm microporous filter to sterilize, and store at 4°C.

[0027] Cell line: HEK-293T cells were stored by Guizhou Natural Product Research Center; the cells were cultured in a 37°C 5% CO2 incubator using DMEM medium containing 5% FBS.

[0028] Animal source: Healthy SPF male c57 mice were purchased from Guizhou Huiyu Biotechnology Co., Ltd.

[0029] The above-mentioned DMEM culture medium and FBS (fetal bovine serum) were purchased from Gibco (USA), and MTT (tetramethylthiazolyl salt) was purchased from Beijing Solebow Technology Co., Ltd.

[0030] 2. Experimental content

[0031] (1) Plasmid construction and drug screening methods

[0032] Design and synthesize oligonucleotides: First, design an oligonucleotide sequence containing the GLP-1R promoter region. This sequence contains approximately 2000 base pairs (bp). The synthesized oligonucleotide sequence is double-digested with restriction enzymes so that it can be inserted into the pGL3-Basic vector. After connecting the oligonucleotide sequence to the pGL3-Basic vector, the ligation product is transformed into Escherichia coli DH5α to screen positive clones. PCR and sequencing methods are used to identify positive clones to ensure that the inserted sequence is correct. See the plasmid map and drug screening process diagram for details. Figure 1 .

[0033] 5×10 3 HEK-293T cells in the exponential proliferation phase were inoculated in a 96-well plate at a density of cells / well, with 90 μL of DMEM medium containing 5% FBS in each well. After overnight culture, 10 μL of sample solution containing different concentrations of Chinese herbal compound was added to each well until the cells adhered to the wall, and 5 replicate wells were set up for each concentration. A blank control group was also set up. After the cells were cultured in a 37°C incubator for 24 hours, 10 μL of 5 mg / mL MTT solution was added to each well. After continuing to culture for 4 hours, the cell supernatant was removed, 160 μL DMSO was added, and the plate was placed on a flat shaker in the dark and shaken for 15 minutes. The absorbance of each well was measured using an enzyme reader with a test wavelength of 490 nm, and the cell viability was calculated. The results are shown in the table. Figure 2 .

[0034] Figure 2The results of A showed that Xinmaitong Capsule (XMT) and Xingpi Yanger Granule (XPYR) had low toxicity to HEK-293T cells at a certain concentration. Therefore, normal saline was used to prepare sample solutions of the Chinese medicine compound at concentrations of 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0.125 mg / mL for luciferase reporter gene experiments. Among them, Xinmaitong Capsule could significantly upregulate the fluorescence expression value of GLP-1R, indicating that Xinmaitong Capsule can promote the expression activity of GLP-1R promoter (see Figure 2 B), has potential anti-type 2 diabetes effects.

[0035] (2) Establishment of animal model and effects of Xinmaitong capsule on water intake, urine volume, and body weight of type 2 diabetic mice

[0036] Healthy SPF male c57 mice (4 weeks old, 12-15 g) were raised under SPF conditions. After 1 week of adaptive feeding, the mice were randomly divided into a normal group (n=7) and a model group (n=35). The mice in the model group were fed a high-fat, high-sugar diet (HFD) with 60% fat energy for 4 weeks. In the 5th week, 50 mg / kg of freshly prepared streptozotocin (STZ) in citric acid buffer (0.1 mol / L, pH 4.5) was injected intraperitoneally for 5 days. The normal group was fed ordinary feed and injected with an equal volume of citric acid buffer. Blood glucose levels in samples obtained from the tail vein were measured on two random days 5 days after the end of STZ injection. Mice with blood glucose concentrations >11.1 mmol / L were considered to have successfully established the T2DM model.

[0037] The mice with successful modeling were randomly divided into 5 groups according to the random number allocation method: model group, Xinmaitong capsule low-dose group (0.655g / kg / d), medium-dose group (1.966g / kg / d), high-dose group (3.275g / kg / d) and metformin group (250mg / kg / d, MET). The mice in the normal group and model group were given an equal volume of drinking pure water. Body weight and fasting blood sugar were measured once a week, and the amount of water consumed by the mice was measured every day for 8 weeks. The urine volume of the mouse cages in each group was weighed before and after the experiment.

[0038] Figure 3 The results showed that the type 2 diabetes mouse model was successfully established, and Xinmaitong Capsule was able to reduce the amount of water consumed and urine produced by type 2 diabetic mice and restore weight loss in the type 2 diabetic state.

[0039] (3) Detection of the effects of Xinmaitong capsule on biochemical indexes and insulin in type 2 diabetic mice

[0040] The fasting blood glucose level of mice was tested at a fixed time every week. At 22:00 on the night before the test, mice in each group were fasted but not watered. At the 8th week, an oral glucose tolerance test (OGTT) was performed. After fasting but not watering for 10 h, all mice were orally fed with 2 g / kg of glucose, and then the tail end was cut off at 0, 30, 60, 90 and 120 min to measure blood glucose. The integral area under the curve (AUC) was used to compare the glucose tolerance level between the Xinmaitong capsule treatment group and the control group. Serum high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), triglyceride (TG), and fasting blood glucose (GLU) levels were measured according to the instructions of the kit (manufacturer Nanjing Jiancheng). Serum insulin concentration was measured using an ELISA kit. The homeostasis model assessment of insulin resistance (HOMA-IR) was used to estimate the level of insulin resistance. HOMA-IR was calculated as follows: HOMA-IR = GLU × FINS / 22.5, where FINS is the fasting insulin level.

[0041] Figure 4 The results of blood glucose and biochemical index tests showed that the postprandial blood glucose and random fasting blood glucose levels in the model group were significantly higher than those in the normal group. After treatment with Xinmaitong capsules, the blood glucose levels of each group were significantly lower than those in the model group ( Figure 4 A, B), the serum glucose content was further detected, which was consistent with the blood sugar level. After treatment with Xinmaitong capsule, the serum glucose content of each group was significantly lower than that of the model group ( Figure 4 C). In order to evaluate its glucose tolerance, we conducted an OGTT experiment. The results showed that Xinmaitong Capsule can effectively improve the glucose tolerance of type 2 diabetic mice, and the area under the curve ( Figure 4 D, E), Xinmaitong capsule also reduced LDL-C, TC, TG, and increased HDL-C in type 2 diabetic mice ( Figure 4 F, G, H, I), reduce insulin secretion levels in type 2 diabetes and improve insulin resistance ( Figure 4 J, K).

[0042] (4) Pathological sections of pancreas and liver of mice with type 2 diabetes treated with Xinmaitong capsule

[0043] After the experiment, the pancreatic tissue of the killed mice was immediately separated, and the liver and pancreas of appropriate size were cut and fixed in 4% formaldehyde. After fixation for 24 hours, they were embedded in paraffin and made into slices. H&E staining was used to observe the effect of Xinmaitong Capsule on the physiological structure of mouse liver and pancreatic tissue. All slices were scanned using Pannoramic 250Flash III slide scanner, and images were collected using CaseViewer software.

[0044] Figure 5The results of H&E staining of the liver and pancreas of mice showed that compared with the normal group, the liver cells of the model group mice were significantly enlarged with macrovesicular fatty degeneration, the pancreatic islet cells showed fatty vacuolar degeneration, some acinar cells were loose and edematous, some acinar cells showed eagle-eye giant cell lesions, and the structure of the pancreatic islet cell group was destroyed. Compared with the model group, the fatty degeneration of hepatocytes and the structure of pancreatic islet cells, as well as the pathological state of the liver and pancreas were significantly improved after treatment with Xinmaitong Capsule and metformin.

[0045] (5) Immunofluorescence (IF) of Xinmaitong Capsule on GLP-1R and Insulin in pancreatic tissue of type 2 diabetic mice

[0046] The pancreatic tissue sections were placed in dewaxing solution and anhydrous ethanol for repair in sequence. During the repair process, excessive evaporation of the buffer solution should be prevented to prevent dry sections. After the repair was completed, the sections were rinsed with distilled water and cooled naturally. Then the sections were placed in PBS (pH 7.4) and washed on a decolorizing shaker for 3 times, each time for 5 minutes. After the sections were slightly dried, circles were drawn around the tissue with a tissue pen, and 3% BSA was added for blocking for 30 minutes. GLP-1R antibody (1:1000) and Insulin antibody (1:500) were added, and the sections were placed in an incubation box and incubated overnight at 4°C. Then the sections were placed in PBS (pH 7.4) and washed on a shaker for 3 times, each time for 5 minutes. CY3-labeled goat anti-mouse IgG and Alexa Fluor 488-labeled goat anti-rabbit IgG were added, and incubated at room temperature in the dark for 50 minutes. Then the sections were placed in PBS (pH 7.4) and washed on a decolorizing shaker for 3 times, each time for 5 minutes. DAPI staining solution was added and incubated at room temperature in the dark for 10 minutes. Then the sections were placed in PBS (pH 7.4) Wash the sections on a decolorizing shaker three times for 5 min each time, add autofluorescence quencher solution for 5 min, rinse with running water for 10 min, add anti-fluorescence quenching sealing agent to seal the sections, and observe and collect images under a fluorescence microscope.

[0047] Figure 6 The IF results showed that the fluorescence values ​​of GLP-1R and Insulin were significantly upregulated in type 2 diabetic mice treated with Xinmaitong Capsule.

[0048] (6) Effect of Xinmaitong Capsule on pancreatic tissue protein levels in type 2 diabetic mice

[0049] Take pancreatic tissue of each group of animals to extract protein samples, prepare separation gel and compression gel of appropriate concentration according to the molecular weight of the target protein; prepare protein loading, with a loading volume of 5-10 μL per well (total protein 50 μg), and a protein marker loading volume of 4 μL; turn on the power to start electrophoresis, 80V electrophoresis for 30 minutes, 120V electrophoresis for 90 minutes, and stop electrophoresis when the blue indicator buffer swims out of the gel; cut a PVDF membrane of 8 cm × 5 cm in size, and soak it in methanol for 30 seconds to activate it; cut the separation gel and put it into the pre-cooled transfer solution, and place the PVDF membrane in the form of a "sandwich" from top to bottom in sequence. Place sponges, filter paper, PVDF membrane, separation gel, filter paper, and sponges, assemble the wet transfer system, and perform wet transfer at 220 mA on ice for 120 min. After the transfer, remove the PVDF membrane and block it with 1×TBS containing 5% skimmed milk powder for 1 h. Recover the blocking solution, wash the membrane 3 times with 1×TBST for 5 min each time, pour in the primary antibody that covers the PVDF membrane, and incubate overnight on a shaker at 4°C. Recover the primary antibody, wash the membrane 3 times with 1×TBST for 5 min each time, pour in the secondary antibody, and incubate at room temperature in the dark for 2 h. Recover the secondary antibody, wash the membrane 3 times with 1×TBST for 5 min each time, and then develop the PVDF membrane using an infrared fluorescence imaging instrument.

[0050] Figure 7 The WB results showed that the expression levels of GLP-1R and PDX-1 proteins in pancreatic tissue of type 2 diabetic mice treated with Xinmaitong Capsule were significantly upregulated.

[0051] From the above results, it can be seen that Xinmaitong Capsule has potential value in the preparation of drugs for the treatment of type 2 diabetes.

Claims

1. Use of Xinmaitong Capsule in preparing medicine for treating type 2 diabetes.

2. Use of the Xinmaitong capsule according to claim 1 in preparing a drug for treating type 2 diabetes, characterized in that: The Xinmaitong capsule can be directly prepared into a preparation for treating type 2 diabetes or can be compounded with other drugs for treating type 2 diabetes to prepare a compound preparation for treating type 2 diabetes.

3. Use of the Xinmaitong capsule according to claim 2 in preparing a drug for treating type 2 diabetes, characterized in that: The preparation is any one of oral liquid, injection, tablet, capsule, granule and pill.

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