CN113521123A

CN113521123A - Method and system for treating type 2 diabetes by using astragalus flavone extract

Info

Publication number: CN113521123A
Application number: CN202010353156.3A
Authority: CN
Inventors: 肖敏
Original assignee: Wenzhou Medical University
Current assignee: Wenzhou Medical University
Priority date: 2020-04-20
Filing date: 2020-04-20
Publication date: 2021-10-22

Abstract

Different from other previous researches, the research discusses the action mechanism of the astragalus flavone extract on the treatment of type 2 diabetes mellitus by applying a biological method. The skeletal muscle cells are taken as an experimental object, an experimental group for blocking a P13/Akt signal pathway is constructed, then the astragalus flavone extract is given, the expression of the related carbohydrate genes of the liver cells is analyzed, and the cell mechanism of the astragalus flavone extract on the treatment of type 2 diabetes is discussed. At present, the research is not reported at home and abroad. Therefore, the astragalus flavone extract provides a novel treatment method. The method can save much expenses for purchasing db/db mice and double-gene knockout mice. The research detects the influence of the astragalus flavone extract on the insulin sensitivity of a mouse myoblast cell strain (C2C12) by measuring the protein expression level of an insulin receptor substrate P-IRS-1, and provides a theoretical basis for the treatment of type 2 diabetes.

Description

Method and system for treating type 2 diabetes by using astragalus flavone extract

Technical Field

The field of treatment of obesity-related diseases (such as type 2 diabetes) by astragalus flavone extract, in particular to verification of feasibility of astragalus flavone extract in treatment of type 2 diabetes.

Background

Research has shown that diabetes has become a serious endocrine-metabolic disease threatening human health. Type II diabetes accounts for more than 95% of diabetic patients. The risk of the II type diabetes patients to generate malignant tumors such as colorectal cancer, liver cancer, breast cancer and the like is obviously higher than that of the general population. Insulin resistance is a common cause of obesity and susceptibility to diseases in individuals such as type II diabetes and cardiovascular disease.

Since patients with type II diabetes need to take medicines for a long time to control blood sugar, this puts higher demands on the safety of anti-type II diabetes medicines. Although many anti-type II diabetes drugs are currently approved for marketing, most drugs lower blood glucose and are associated with hypoglycemic side effects. In addition, some drugs can cause gastrointestinal discomfort, edema, increased risk of fracture, and other side effects. Therefore, development of novel anti-type II diabetes drugs with high safety and effective oral administration has been the direction of efforts of researchers.

The extensive biological action and miraculous clinical efficacy of astragalus root have been the focus of all the time, and the components of astragalus root polysaccharide (APS), Astragaloside (AS) and the like play different degrees of roles in resisting inflammation, resisting virus, improving the immune function of the body, resisting tumor, resisting aging and the like. In recent years, researches show that the other component of the astragalus, namely flavonoid (AF), has important pharmacological action in various aspects of removing free radicals, regulating immunity, resisting viruses, resisting inflammation, resisting aging and the like.

The radix astragali flavone extract has effects of reducing blood sugar of diabetic mice. This is probably due to the association of polysaccharides and flavonoids in astragalus, but the mechanism of action still needs to be further studied. By measuring the protein expression level of an insulin receptor substrate P-IRS-1, the influence of the astragalus flavone extract on the insulin sensitivity of a mouse myoblast cell strain (C2C12) is detected, and a theoretical basis is provided for the treatment of type 2 diabetes.

At present, the effect and action mechanism of the astragalus flavone extract are proved at home and abroad by adopting wild type, Adipor1-/-, Adipor2-/-, Adipor1-/-Adipor 2-/-double knockout mice and db/db mice as control experiments, and the improvement effect of the black tartary buckwheat stem and leaf extract on type 2 diabetes is verified by detecting the activity of each conduction path and related gene detection. The astragalus flavone extract obviously reduces the content of triglyceride and oxidative stress, and reduces the expression level of genes encoding proinflammatory cytokines. The source of the Adipor1-/-, the Adipor2-/-, the Adipor1-/-Adipor 2-/-double knockout mice and db/db mice is less, the price is high, and the method is not suitable for being popularized in a laboratory. The same effect is achieved by detecting the influence of the astragalus flavone extract on the AMPK-mediated signal path by using a cheap C57BL/6 mouse, and the method is suitable for popularization in a laboratory.

Disclosure of Invention

In order to overcome the problem that animal models of an Adipor1-/-, an Adipor2-/-, an Adipor1-/-Adipor 2-/-double knockout mouse and a db/db mouse are difficult to realize, the invention constructs a type 2 diabetes mouse model in advance and further detects various indexes of the mouse. Histology level, comparing and observing the tissue characteristics of a common mouse control group (NC), a type II diabetes mouse group (DM), a low-dose astragalus flavone extract mouse group (DM + L) and a high-dose astragalus flavone extract mouse group (DM + H); at the cellular level, the astragalus flavone extract improves the insulin sensitivity of C2C12 cells; performing biochemical detection on related indexes in serum of each group of mice at a molecular level, monitoring the content of each corresponding index in liver in a focused manner, and measuring the blood insulin content of each group of mice; gene level, detection of glucose metabolism related enzyme PEPCK mRNA in liver, measurement of GLUT4mRNA expression in C2C12 cell; comprehensively detecting the influence of the astragalus flavone extract on a PI3K/Akt signal channel.

The invention overcomes the problem of high cost of beta cell gene knockout mice by constructing a type 2 diabetes mouse model, and simplifies the experiment for verifying the treatment effect of the astragalus flavone extract on obesity-related diseases (such as type 2 diabetes). Provides a novel, simple and convenient method and system for the verification experiment of the curative effect of the astragalus flavone extract, promotes the development and clinical treatment progress of the astragalus flavone extract to a certain extent, and has good popularization and feasibility.

Detailed Description

Preparation and group processing of type 1.2 diabetic mouse model and histological examination

40 male C57BL/6 mice (purchased from Shanghai laboratory animal center of Chinese academy of sciences) with 12 weeks old (40 SPF grade male C57BL/6 mice (adaptively fed for 1 week, randomly divided into 10 normal control NC groups and 30 experimental groups, the NC groups were fed with conventional feeds, the experimental groups were fed with diets high-sugar high-fat (MD12031 (gm%/kacl%) protein 19.2/20 fat 4.30/10 carbohydrate 67.3/70 trace element 9.20/0), after 5 weeks, after fasting for 12 hours, the diabetic models were induced by intraperitoneal injection of 1% streptozotocin 40mg/kg, fasting for 4 hours after 72 hours, tail-cutting blood-off blood-taking measurement FPG, blood sugar concentration > 11.8mmol/L, and the occurrence of polydipsia, polyphagia, polyuria and polyuria symptoms suggested that the diabetic mice models were induced to be successfully administered Model + high dose (DM + H) group, normal control NC group. Dissolving radix astragali flavone (concentration of 0.04g/ml) with physiological saline water, intragastrically administering 0.21g/kg.d-1 radix astragali flavone for DM + L group mice, intragastrically administering 0.42g/kg.d-1 radix astragali flavone for DM + H group mice, and intragastrically administering equal amount of physiological saline for NC and DM groups. After 14 days, fasting the mice for 6 hours, weighing the mice, cutting off the tail, taking blood to measure Fasting Blood Glucose (FBG), taking 0.6 ml blood sample from the eyeball, centrifuging, taking the supernatant into a clean centrifugal tube, respectively detecting fasting serum glucose (FBG), taking liver samples of each group, and numbering. Fixing a part of liver tissue with 4% paraformaldehyde, embedding for 24-48h, dehydrating with gradient alcohol, clearing with xylene, soaking in wax, embedding with paraffin, cutting into 4 μm slices, subjecting the sliced liver tissue to HE staining, and observing and photographing the morphological change of each group of liver tissue under a mirror. And subpackaging the rest liver tissue specimens, quickly freezing by using liquid nitrogen, and storing in a refrigerator at the temperature of-80 ℃ for freezing and storing.

2. Detecting related indexes by using a full-automatic biochemical analyzer:

(1) indices of alanine Aminotransferase (ALT), aspartate Aminotransferase (AST) and alkaline phosphatase (ALP) in each group in serum.

(2) Changes of Triglyceride (TG) and blood Glucose (GLU) of each group in serum are detected, and the contents of Insulin (INS) and Free Fatty Acid (FFA) are detected by an ELISA kit.

3. Determination of contents of glucose metabolism related enzyme G6PC and PEPCK mRNA in liver

The determination of the content of glucose-6 phosphatase (G6PC) is carried out by using an ELISA kit, total RNA of mouse liver tissue is extracted by adopting a Trizol one-step method, cDNA is synthesized by reverse transcription, the real-time fluorescent quantitative PCR is used for determining the mRNA expression of the phosphoenolpyruvate carboxykinase (PEPCK) according to the instruction of the kit, and beta-actin is used as an internal reference. PEPCK upstream primer: 5'-TGA AAG GCC GCA CCA TGT AT-3', respectively; PEPCK downstream primer: 5'-GCA CAG ATA TGC CCA TCC GA-3' are provided. Beta-actin upstream primer: 5'-AAC AGT CCG CCT AGA AGC AC-3', respectively; beta-actin downstream primer: 5'-CGT TGA CAT CCG TAA AGA CC-3' are provided. Reaction system 20. mu.l: SYBR Green (2X) 10. mu.l, 10. mu. mol/L upstream and downstream primers 0.4. mu.l each, ddH2O7.2. mu.l, template 0.2. mu.l. Reaction conditions are as follows: denaturation at 95 ℃ for 30 s; and (4) circulating for 40 times: denaturation at 95 ℃ for 5s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 34 s; melting: 95 ℃ for 15s, 60 ℃ for 1min and 95 ℃ for 15 s; and (3) cooling: 60 ℃ for 15 s.

4. Determination of improvement of C2C12 cell insulin sensitivity by astragalus flavone extract

(1) Culture of C2C 12: adding a DMEM high-sugar culture medium containing 10% fetal calf serum, placing the DMEM high-sugar culture medium in an incubator containing 5% CO2 at 37 ℃, culturing, changing the culture medium every other day, and carrying out passage according to the ratio of 1: 3 when the cells grow to 70% -80% of fusion.

(2) C2C12 induced differentiation: when about 80% of C2C12 cells are fused, subculturing the cells in a six-hole plate, and when the cells grow to 80% -90% of fused cells, inducing and differentiating the cells for 4d by using a DMEM medium containing 2% of horse serum, and changing the liquid once every 24 hours; more than 90% became mature skeletal muscle cells after 4 days for the experiment.

(3) Glucose oxidase method for detecting glucose level in cell culture medium

After cell differentiation, 2% HS DMEM medium containing 50ug/ml of astragalus flavone extract, 20ug/ml of insulin, 20ug/ml of astragalus flavone extract +20ug/ml PI3K and 20ug/ml of insulin +20ug/ml PI3K was added to the administration group, and a blank group and a cell-free DMEM group were set. After 12h incubation, the medium was removed and the sugar content of the medium was measured by the glucose oxidase method. The sugar content of each remaining well, i.e., the glucose consumption of the cells of each well for 12h, was subtracted from the average of the sugar content of the DMEM group, and the cells were collected to extract total RNA.

(4) RT-PCR method for detecting GLUT4mRNA expression

Extracting total RNA of each group of cells by a Trizol one-step method, taking 1 mu l of total RNA as a template for reverse transcription, and synthesizing GLUT4 and beta-actin single nucleic acid primers by Shanghai. Upstream of GLUT 4: 5'-GCCCGAAAGAG-3', downstream: 5'-ACTAAGAGCACCGAGACCAA-3', the length of the amplified product is 312 bp. Beta-actin upstream: 5'-CGTGCGTGAGATTAAAGAG-3', downstream: 5'-CTGGAAGGTGGACAGTGAG-3', the length of the amplified product is 435 bp. Amplification conditions: pre-denaturation at 95 ℃ for 5min, followed by 35 cycles of 95 ℃ for 45s, 58 ℃ for 45s, and 72 ℃ for 45s, followed by extension at 72 ℃ for 10min and then at 72 ℃ for 10 min. After the reaction, 1. mu.l of PCR product, 10ul of SYBR Green, 0.8ul of each of the upstream and downstream primers, 7.4ul of ddH2O, was taken and detected by a fluorescence quantitative analyzer.

Description of the drawings: FIG. 1 is a route chart for in vitro experiments

FIG. 2 is a circuit diagram of a type 2 diabetic mouse model constructed in advance and used for monitoring various corresponding indexes in the liver.

Claims

1. A novel mouse experiment mode overcomes the problem that an Adipor1-/-, an Adipor2-/-, an Adipor1-/-Adipor 2-/-double knockout mouse and a db/db mouse animal model are difficult to realize.

2. The novel mouse experimental mode as claimed in claim 1, wherein in the experiment for confirming the effect and action mechanism of the astragalus flavone extract, a type 2 diabetes mouse (using C57BL/6) model is constructed in advance, and then each index of the mouse is further detected. The astragalus flavone extract improves the insulin sensitivity of C2C12 cells and measures the expression of GLUT4mRNA in C2C12 cells; comprehensively detecting the influence of the astragalus flavone extract on a PI3K/Akt signal channel.

3. The novel mouse assay of claim 1, wherein the assay is performed at the histological level, the molecular level, and the cellular level.

4. The novel mouse experimental mode of claim 1, which avoids the problem of high cost of β cell knockout mice and simplifies the experiment for verifying the therapeutic effect of astragalus flavone extract on obesity-related diseases (such as type 2 diabetes).

5. The novel mouse experimental mode of claim 1, which provides a novel, simple and convenient method and system for the verification experiment of the curative effect of the astragalus flavone extract, and has good popularization and feasibility.