CN112042592A

CN112042592A - Method and system for treating type 2 diabetes by using black tartary buckwheat stem and leaf extract

Info

Publication number: CN112042592A
Application number: CN201910519710.8A
Authority: CN
Inventors: 肖敏
Original assignee: Wenzhou Medical University
Current assignee: Wenzhou Medical University
Priority date: 2019-06-07
Filing date: 2019-06-07
Publication date: 2020-12-08

Abstract

Different from other previous researches, the research discusses the action mechanism of the black tartary buckwheat stem and leaf extract on the treatment of type 2 diabetes by applying a biological method. The skeletal muscle cells are used as experimental objects to construct an experimental group for blocking a P13/Akt signal pathway, and then the stem and leaf extract of the black tartary buckwheat is given to analyze the expression of the related carbohydrate genes of the liver cells and discuss the cell mechanism of the stem and leaf extract of the black tartary buckwheat for treating the type 2 diabetes. At present, the research is not reported at home and abroad. Therefore, the black tartary buckwheat stem and leaf 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 stem and leaf extract of the black tartary buckwheat 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 black tartary buckwheat stem and leaf extract

Technical Field

The field of treatment of obesity-related diseases (such as type 2 diabetes) by using black tartary buckwheat stem and leaf extracts, and particularly relates to verification of feasibility of treatment of type 2 diabetes by using black tartary buckwheat stem and leaf extracts.

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.

At present, the diabetes treatment mainly depends on medicines, and although the blood sugar can be controlled at a relatively stable low level, the long-term administration of the medicines is bound to harm the health of human bodies. Most institutions concerned believe that effective measures for the management of diabetes are preventive, which can be achieved by altering dietary structure. Therefore, it is urgent to develop natural products and effective ingredients thereof which have hypoglycemic effects and are nontoxic or slightly toxic to human bodies.

The tartary buckwheat belongs to plants in the genus of buckwheat of the family of Polygonaceae and is divided into common tartary buckwheat and black tartary buckwheat, the tartary buckwheat is a unique food used as both medicine and food, modern pharmacological research shows that the common tartary buckwheat has the functions of reducing blood sugar, reducing blood fat, removing free radicals and resisting oxidation and the like, and has the function of adjuvant therapy on cardiovascular and cerebrovascular system diseases, the black tartary buckwheat is a special resource in the western minority regions of Sichuan province, has the reputation of black pearl, and the roots, stems, leaves, flowers and fruits of the black tartary buckwheat all contain various amino acids and trace elements such as selenium and chromium, and the black tartary buckwheat is cool and cold-resistant, has obvious growth advantages in the alpine regions of Sichuan province in China, has rich resources, high biomass of stems and leaves, complete and mature extraction process equipment, is economical and easy to.

The black tartary buckwheat stem and leaf extract has the function of reducing the blood sugar of diabetic mice. The function of damaged beta cells can be improved probably because dietary fibers and flavonoids compounds in the black tartary buckwheat have antioxidation, but the action mechanism of the black tartary buckwheat still needs to be further researched, and the influence of stem and leaf extracts of the black tartary buckwheat on the insulin sensitivity of a mouse myoblast cell line (C2C12) is detected by measuring the protein expression level of an insulin receptor substrate P-IRS-1, so that a theoretical basis is provided for the treatment of type 2 diabetes.

At present, the effect and action mechanism of the stem and leaf extract of the black tartary buckwheat 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 verifying the improvement effect of the stem and leaf extract of the black tartary buckwheat on type 2 diabetes through detecting the activity of each conduction path and related gene detection. The stem and leaf extract of the black tartary buckwheat 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 cheap C57BL/6 mouse is adopted, the same effect is achieved by detecting the influence of the black wasabi stem and leaf extract on an AMPK mediated signal pathway, and the method is suitable for being popularized 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 black wasabi stem and leaf extract mouse group (DM + L) and a high-dose black wasabi stem and leaf extract mouse group (DM + H); at cell level, the black leaf mustard stem and leaf 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 black leaf mustard stem and leaf extract on a PI3K/Akt signal pathway.

The invention overcomes the problem of high cost of a beta cell gene knockout mouse by constructing a type 2 diabetes mouse model, and simplifies the experiment for verifying the treatment effect of the black wasabi stem and leaf 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 black leaf mustard extract, promotes the development and clinical treatment progress of the black leaf mustard 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, fasting for 12 hours, intraperitoneal injection of 1% streptozotocin 40mg/kg induced diabetes model, fasting for 4 hours after 72 hours, tail-cutting blood-off and blood-taking measurement of FPG, blood sugar concentration > 11.8mmol/L, and occurrence of polydipsia, polyphagia polyuria and polyuria symptoms suggested that the diabetes mouse model induced successful administration of the black picea mustard control (DM) stem and leaf fraction, model + low dose (DM + L) group, Model + high dose (DM + H) group, normal control NC group. The normal saline is used for dissolving the stem leaves of the black tartary buckwheat (the concentration is 0.04g/ml), the DM + L group mice are gavaged with 0.21g/kg.d-1 stem leaves of the black tartary buckwheat, the DM + H group mice are gavaged with 0.42g/kg.d-1 stem leaves of the black tartary buckwheat, and the NC and DM groups are gavaged with the same amount of the normal saline. 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 0.6ml 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 insulin sensitivity of C2C12 cells by using black leaf mustard stem and leaf 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 black wasabi stem and leaf extract, 20ug/ml of insulin, 20ug/ml of black wasabi stem and leaf 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 stem and leaf extract of Fagopyrum tataricum, a type 2 diabetes mouse (adopting C57BL/6) model is constructed in advance, and then each index of the mouse is detected. Determination of the expression of GLUT4mRNA in C2C12 cells, and the stem and leaf extracts of black wasabi improve insulin sensitivity in C2C12 cells; comprehensively detecting the influence of the black leaf mustard stem and leaf extract on a PI3K/Akt signal pathway.

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 beta cell gene knockout mice and simplifies the experiment for verifying the treatment effect of the black tartary buckwheat stem and leaf extract on obesity-related diseases (such as type 2 diabetes).

5. The novel mouse experimental mode as claimed in claim 1 provides a novel, simple and convenient method and system for the verification experiment of the curative effect of the stem and leaf extract of tartary buckwheat, and has good popularization and feasibility.