CN113749050A - Method and system for treating type 2 diabetes mellitus by astaxanthin extract - Google Patents
Method and system for treating type 2 diabetes mellitus by astaxanthin extract Download PDFInfo
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Abstract
Different from other previous researches, the research discusses the action mechanism of the astaxanthin extract on the treatment of type 2 diabetes mellitus by applying a biological method. Constructing an experimental group for blocking a P13/Akt signal pathway by taking skeletal muscle cells as an experimental object, then, administering an astaxanthin extract, analyzing the expression of related carbohydrate genes of the liver cells, and discussing the cell mechanism of the astaxanthin extract on the treatment of type 2 diabetes. At present, the research is not reported at home and abroad. Astaxanthin extract therefore provides a novel therapeutic approach. The method can save much expenses for purchasing db/db mice and double-gene knockout mice. The research detects the influence of the astaxanthin 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
Technical Field
The field of astaxanthin extract treatment on obesity-related diseases (such as type 2 diabetes), and particularly relates to verification of feasibility of the astaxanthin extract on 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. Type II diabetes is mainly caused by the interaction of peripheral insulin resistance and a decline in B cell secretory function. The mere presence of insulin resistance does not necessarily lead to type II diabetes. Therefore, the ideal treatment means for type II diabetes should have both of the above two pathogenesis mechanisms.
Astaxanthin has the chemical name of 3, 3 ' -dihydroxy-4, 4 ' -diketone-beta, beta ' -carotene, is a terpene unsaturated compound, is an antioxidant which is found to be the strongest in the natural world by people so far, has various physiological effects, has the capacity of promoting cell regeneration, has particularly obvious effect of reducing blood sugar, has no obvious side effect, has low adverse reaction and good tolerance, and plays an important role in treating diabetes.
The astaxanthin extract has the function of remarkably reducing the blood sugar of diabetic mice. Probably related to the efficient elimination of free radicals in human bodies, but the action mechanism of the astaxanthin peptide is still required to be further researched, and the influence of the astaxanthin extract 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 astaxanthin 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 astaxanthin extract on type 2 diabetes is verified by detecting the activity of each conduction path and related gene detection. The astaxanthin extract obviously reduces triglyceride content 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 method adopts cheap C57BL/6 mice, achieves the same effect by detecting the influence of the astaxanthin extract on the AMPK mediated signal pathway, and is suitable for popularization in laboratories.
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 astaxanthin extract-given mouse group (DM + L) and a high-dose astaxanthin extract-given mouse group (DM + H); at the cellular level, astaxanthin extract improved C2C12 cell insulin sensitivity; 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, determination of GLUT4 mRNA expression in C2C12 cells; comprehensively detecting the influence of the astaxanthin 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 mellitus mouse model, and simplifies the experiment for verifying the treatment effect of the astaxanthin extract on obesity-related diseases (such as type 2 diabetes mellitus). Provides a novel, simple and convenient method and system for the verification experiment of the curative effect of the astaxanthin extract, promotes the development of the astaxanthin extract and the clinical treatment 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 given conventional feeds, the experimental groups were given high-sugar high-fat (MD12031 (gm%/kacl%) protein 19.2/20 fat 4.30/10 carbohydrate 67.3/70 trace element 9.20/0) diet feeding, after 5 weeks, 12h were fasted, the diabetes model was induced by intraperitoneal injection of 1% streptozotocin 40mg/kg, after 72h, the mice were fasted for 4h, and after tail-cutting blood-taking, the blood glucose concentration was measured to be more than 11.8mmol/L, and the occurrence of polydipsia, polyphagia polyuria and polyuria symptoms suggested that the diabetes model was induced successfully by astaxanthin administration to the control (DM) group, the group + low dose group (DM + L) group, Model + high dose (DM + H) group, normal control NC group. Dissolving astaxanthin (concentration of 0.04g/ml) with physiological saline, intragastrically administering 0.21g/kg.d-1 astaxanthin to DM + L group mice, intragastrically administering 0.42g/kg.d-1 astaxanthin to DM + H group mice, and intragastrically administering equal amount of physiological saline to 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 0.6ml 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 astaxanthin 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
The administration group was prepared by adding 2% HS DMEM medium containing 50ug/ml astaxanthin extract, 20ug/ml insulin, and 20ug/ml astaxanthin extract +20ug/ml PI3K and 20ug/ml insulin +20ug/ml PI3K, respectively, after cell differentiation, while setting a blank group and a cell-free DMEM group. 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 GLUT4 mRNA 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 ddH20, and a fluorescence quantitative analyzer were used for detection.
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 (5)
1. A method and a system for treating type 2 diabetes by an astaxanthin extract, namely a method and a system for treating type 2 diabetes mice by the astaxanthin extract, a novel mouse experimental mode, and solves 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.
2. The novel mouse experimental mode as claimed in claim 1, wherein in the experiment for confirming the effect and action mechanism of astaxanthin extract, a type 2 diabetes mouse (using C57BL/6) model is constructed in advance, and each index of the mouse is further tested. Astaxanthin extract improves insulin sensitivity in C2C12 cells, measurement of GLUT4 mRNA expression in C2C12 cells; comprehensively detecting the influence of the astaxanthin 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 beta cell knockout mice and simplifies the experiment for verifying the therapeutic effect of astaxanthin extract on obesity-related diseases (such as type 2 diabetes).
5. The novel mouse experimental mode of claim 1, which provides a novel and simple method and system for the efficacy verification experiment of astaxanthin extract, and has good popularization and feasibility.
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