Construction method of 3DG-induced senile prediabetic disease animal model

Abstract

The invention provides a construction method of an animal model of senile prediabetic disease, which comprises the following steps: performing intragastric administration induction on an experimental animal by using 3-deoxyglucuronidone to construct and form the animal model of senile prediabetic disease with impaired fasting blood glucose and / or hypoglucose tolerance as a main marker. Researches find that 3-deoxyglucal not only induces glucose and lipid metabolism disorder and peripheral adipose tissue insulin resistance and islet secretion function damage, but also causes'fat-islet axis' dual hormone feedback loop imbalance, and is mainly related to interaction damage of adiponectin / adiponectin receptor pathway and glucose / sweet receptor pathway. The composition accords with physiological and pathological characteristics of senile prediabetic diseases. The model provided by the invention has the advantages of prevention, treatment and reversibility, is simple to operate, economical and applicable, and high in modeling success rate, solves the core problem of complexity of the existing animal model for the senile prediabetic disease, and provides an experimental basis for research and development of related drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of life sciences, and in particular relates to a method for constructing a 3DG-induced elderly prediabetic animal model. Background Art

[0002] The prevalence of diabetes in the elderly has become an important public health issue affecting global human health, bringing huge economic burdens to countries, medical systems, patients and their families. Prediabetes mellitus (Pre-DM) in the elderly is an important risk, main pathogenic factor and prevention node for the development of type 2 diabetes (T2DM) in the elderly. Pre-DM refers to impaired glucose regulation (IGR), elevated fasting plasma glucose (FPG) and / or 2h plasma glucose (2hPG) after oral glucose tolerance test (OGTT) but not reaching the criteria for the formation of diabetes, that is, impaired fasting glucose (IFG), impaired glucose tolerance (IGT), or both. In the prevention and treatment strategy of "focusing on the starting point and balancing the target" in the "Clinical Guidelines for the Prevention and Treatment of T2DM in the Elderly in China (2022 Edition)", primary prevention management recommends lifestyle intervention and the use of hypoglycemic drugs such as non-insulin secretagogues for prevention and treatment, but the long-term safety and effectiveness of fasting blood glucose in multi-heterogeneous elderly patients still need to be verified. Therefore, further searching for effective intervention measures for the core pathological mechanisms of elderly Pre-DM is a key scientific issue that needs to be solved urgently. Animal or cell models play an irreplaceable role in biotechnology and medical research. They are our bridge to understand human physiology and disease mechanisms, and they are also key tools for drug development, etc. At present, the methods for modeling syndrome models of elderly Pre-DM animals can be roughly divided into three categories: (1) feeding with high-energy feed, or combining activity space restriction, low-dose injection of streptozotocin (STZ), etc.; (2) using combined drug modeling; (3) using transgenic mice to create a spontaneous model combined with syndrome modeling factors. The main problems of the above methods are: too many intervention factors and poor reproducibility; or too long a cycle may lead to syndrome transformation and no difference in evaluation indicators; or irreversible damage to animals after the modeling is stopped; or the syndrome manifestation observation indicators should be consistent with the clinical practice, etc. Elderly patients with Pre-DM have distinct characteristics of functional aging, central or ectopic obesity, chronic inflammation and other heterogeneity. Adipocyte aging promotes adipose tissue inflammation and varying degrees of systemic insulin resistance (IR), which is the core pathological change and impaired pancreatic β-cell function, and is the key intrinsic essence that mainly triggers T2DM. The "fat-islet axis" dual hormone feedback loop is one of the important endocrine systems to maintain the body's metabolic balance. Among them, the interaction mechanism between insulin and the fat factor adiponectin (APN) is closely related to the occurrence and development of elderly diabetes. In the imbalance of the "fat-islet axis" in elderly Pre-DM, pancreatic β cells respond to glucose (Glc) stimulation to synthesize and secrete insulin in a biphasic manner. This "triggering pathway" is crucial for "reshaping" the patient's glucose homeostasis. The insulin secretion of pancreatic β cells is also regulated by multiple factors such as calcium, metabolites and secreted peptides / proteins. Among them, in addition to APN / adiponectin receptor 1 (Adiponectin receptor 1, AdipoR1) involved in adipocyte-β cell endocrine signal transduction, sweet taste receptor (STRs) family members T1R2-T1R3 (TAS1R2-TAS1R3), as receptors for nutrients such as Glc, mediate the secretory function of pancreatic cells in a manner similar to a "bypass switch". The latest cutting-edge research has revealed that T1R2-T1R3 participates in the "fat-islet axis" dialogue and synergistically maintains the balance of glucose and lipid metabolism. During the aging process, advanced glycation products (AGEs) accumulate in cells and tissues. 3-deoxyglucosone (3DG) is an important precursor of AGEs and is the main material basis for "rich food" causing Pre-DM. It is closely related to the occurrence and development of aging and diabetes. In the long-term work accumulation, the applicant's research team found that 3DG may become part of the presumptive biomarker for judging prediabetes in the elderly; it proved that the increase in circulating 3DG levels is one of the important pathological mechanisms causing IGT in the elderly. Summary of the invention

[0003] Technical problems to be solved: In order to solve the technical defects of the current technology in establishing the simulated disease characteristics, physiological and pathological indicators, pharmacological effects and repeatability of the elderly Pre-DM animal model, and break through the technical bottleneck of long cycle and complex operation, the present invention discloses a method for constructing a 3DG-induced elderly prediabetes animal model. By gavage 3DG rats, the characteristic model of acute or chronic elderly Pre-DM can be created, and the problems of too many intervention factors, poor reproducibility, or the transformation of syndromes and no difference in evaluation indicators due to too long cycle, or irreversible damage to animals after the modeling is stopped, or the syndrome characterization observation indicators should be consistent with the clinical situation, etc. The 3DG-induced Pre-DM model has multiple characteristics, including rapid onset, easy operation, good reproducibility, etc., which enables researchers to better simulate the endocrinology and pathology characteristics of elderly Pre-DM under certain experimental conditions, and the changes in sweet taste receptors in the fat-islet axis of the model are closely related to the occurrence and development of elderly Pre-DM. The established animal model can be applied to the research of endocrine-related Chinese and Western medicines and research fields such as biology.

[0004] Technical solution: A method for constructing a 3DG-induced prediabetes animal model in the elderly: This method uses 3DG gavage to induce experimental animals to construct a prediabetes animal model in the elderly with impaired fasting blood glucose or impaired glucose tolerance, or both as the main signs. Furthermore, the experimental animals are SD rats or mice. Furthermore, the intragastric administration amount of 3DG is 50 mg / kg, once a day. Furthermore, the intragastric administration also includes supplying water to the experimental animals. Furthermore, the experimental animals were supplied with secondary reverse osmosis water, with a feeding amount of 1 mL / 100 g, once a day. Furthermore, the induction time is 2-10 weeks. Furthermore, the animal model constructed by the construction method is used in the study of the pathogenesis of prediabetes in the elderly. Furthermore, the animal model constructed by the construction method is used in the screening of endocrine-related Chinese and Western medicines. Beneficial effects: 1. The present invention uses 3-deoxyglucosone to intervene to construct an elderly Pre-DM animal model, and verifies it through glucose / insulin tolerance test (OGTT test), serum "fat-islet axis" related biochemical index detection, adipose and pancreatic tissue damage pathological analysis and cell structure changes, and adipose and pancreatic tissue protein immunoblotting detection. The results show that in the elderly Pre-DM disease syndrome combined rat model and cell damage model induced by 3DG, 3DG not only induces glucose and lipid metabolism disorders and peripheral adipose tissue IR and impaired islet secretion function, but also causes an imbalance in the dual hormone feedback loop of the "fat-islet axis" that maintains the body's metabolic balance, which is mainly related to the interactive damage of the APN / AdipoR1 pathway and the Glc / T1R2-T1R3 pathway in the "fat-islet axis", providing a new idea for the study of the pathogenesis of elderly Pre-DM; 2. The method of constructing an elderly Pre-DM animal model of the present invention has the advantages of rapid onset, easy operation, and good replicability. It solves the key problem of the complexity of preparing existing elderly Pre-DM animal models and provides an experimental basis for the research and development of related drugs. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 The data show that intravenous injection of 3DG induced acute glucose intolerance in SD rats, where A is the monitoring of plasma 3DG levels after intravenous injection of 3DG (50 mg / kg); B is the monitoring of plasma blood glucose levels in intravenous glucose tolerance test; C is the monitoring of plasma insulin levels in intravenous glucose tolerance test; D is the result of 2NBD sugar uptake test in adipose tissue of rats isolated after intravenous injection of 3DG; E is the result of glucose-stimulated insulin secretion test (GSIS) in pancreatic islet tissue of rats isolated after intravenous injection of 3DG; F is the result of WB detection of relative protein expression level of p85-PI3K in pancreatic islet tissue of rats isolated after intravenous injection of 3DG. Figure 2 The data are as follows: oral glucose intolerance in SD rats induced by a single oral gavage of 3DG, where A is blood glucose; B is the area under the blood glucose curve; C is plasma insulin; D is the area under the insulin curve; E is plasma glucagon; F is the area under the glucagon curve; G is plasma total GLP-1; and H is plasma total GIP. Figure 3 The results show that oral administration of 3DG for 2 weeks induced increased fasting blood glucose concentration and impaired oral glucose tolerance in SD rats. A is fasting blood glucose; B is the result of oral glucose tolerance test; C is the area under the glucose curve. Figure 4 Figure 2 shows the general conditions of the normal group and the model group and the successful evaluation of the model, where A is blood glucose; B is serum insulin; C is HE staining; D is body weight; E is abdominal fat mass; and F is abdominal fat mass / body weight. Figure 5 Figure 2 is a comparison chart of serum related indexes detected in rats of the normal group and the model group, where A is fasting blood glucose (FBG); B is fasting insulin (FINS); C is insulin resistance index (HOMA-IR); D is serum triglyceride (TG); E is serum total cholesterol (TC); F is serum low-density lipoprotein cholesterol (LDL-C); G is serum high-density lipoprotein cholesterol (HDL-C); H is serum glutamate pyruvate aminotransferase (ALT); I is serum glutamate oxaloacetate aminotransferase (AST); J is superoxide dismutase (SOD); K is glutathione peroxidase (GSH); L is skeletal muscle malondialdehyde (MDA); M is serum adiponectin (APN) content; N is serum leptin content (LP); O is serum tumor necrosis factor (TNF-α) content. Figure 6 The results of the detection of mRNA and protein related to the fat-islet axis in the normal group and the model group rats are shown in Figure 1, where A is the relative expression of AdipoR1, T1R2, T1R3, IRS1, and GLUT4 mRNA; B is the protein immunoblot band; C is the relative protein expression; D is the relative expression of AdipoR1, T1R2, T1R3, IRS2, and GLUT2 mRNA; E is the protein immunoblot band; and F is the relative protein expression. DETAILED DESCRIPTION The present invention is further described below in conjunction with the accompanying drawings and embodiments. The following embodiments are explanations of the present invention and the present invention is not limited to the following embodiments: Preliminary basic experiments (1) SD rats were intravenously injected with 50 mg / kg 3DG With the acute increase of circulating 3DG ( Figure 1 A), impaired intravenous glucose tolerance in normal rats ( Figure 1 B), while plasma insulin levels increase ( Figure 1 C); 3DG-mediated impaired glucose tolerance and reduced insulin-stimulated glucose uptake in adipose tissue ( Figure 1 D) and decreased glucose-stimulated insulin secretion in pancreatic tissue ( Figure 1 E), the phosphorylation level of p85-PI3K in pancreatic tissue was decreased ( Figure 1 F); Figure 1 B, D and E, F also show that aminoguanidine [an inhibitor of terminal carbonyl products (AGEs)] can inhibit impaired glucose tolerance and insulin resistance by regulating the phosphorylation of p85-PI3K in the aged rat Pre-DM animal model induced by 3DG (intravenous injection). (2) A single oral administration of 3DG (20 mg / kg) to normal SD rats Impaired glucose tolerance in rats during the oral glucose tolerance test (OGTT) Figure 2 A), plasma insulin levels increase ( Figure 2 C) No change in glucagon ( Figure 2 E), which may be related to fluctuations in GLP-1 and GIP levels ( Figure 2 G, H). (3) 3DG (50 mg / kg) was administered orally to normal rats for 2 weeks The fasting blood glucose level was significantly higher than that of the control group ( Figure 3 A), 3DG-induced decreased oral glucose tolerance in rats compared with the control group ( Figure 3 B) and the glucose area under the curve (AUC) was significantly higher than that of the control group ( Figure 3 C). Example 1 1. Experimental Animal Information Species and strain: SD; Grade: SPF (Specific pathogen Free); Number and sex: 20, male; Supplier: Zhaoyan (Suzhou) New Drug Research Center Co., Ltd., Animal Production License: SCXK(Su)2018-0006, Animal Use License SYXK(Su)2017-0041. 2. Animal feeding and management After receiving the animals, the veterinarian will conduct a physical examination on all animals and carry out quarantine and adaptation in the early stage of the experiment. The period from the arrival of the animals to the day of model induction is the early stage of the experiment, which is at least 1 week. The animals were kept in a barrier system. The environmental conditions of the animal room were: temperature: 20-26°C, humidity: 40-70%. The keepers recorded the temperature and humidity data once in the morning and afternoon every day. The air in the animal room was exchanged 10 to 20 times per hour. The lighting in the animal room was alternating according to a 12 / 12 hour light and dark cycle, except for necessary interruptions during experimental activities, but all interruptions were recorded. After the group experiment, multiple animals were kept in cages, 5 in each cage. The animal cages were made of polysulfone plastic, and corn cob bedding was laid in the animal cages. Toys for animals to chew or cover were also arranged in the animal cages. A cage card for identifying the group was placed in each cage. The cage card was marked with information such as the test number, group, gender, animal name and number, and the animals were arranged in order according to the group. Normal feed was purchased from Shuangshi Experimental Animal Co., Ltd., and animals were allowed to eat freely. Any additional food addition had to be approved by the subject leader and recorded. The animals are watered by secondary reverse osmosis water provided by automatic drinking devices. 3. Main instruments and reagents Instruments: Low-temperature refrigerated centrifuge (TG-16M): Shanghai Lu Xiangyi Centrifuge Instrument Co., Ltd.; microplate reader (MK3): Regal, Finland; upright microscope (CX41): OLYMPUS, Japan; transmission electron microscope (HT7700), scanning browsing software (CaseViewer2.4) and panoramic slide scanner (PANNORAMICDESK / MIDI / 250 / 1000): Hitachi (Japan Electronics and Electrical Company); Real-time detector (ABI-7300): ABI, USA; electrotransfer machine (TE77XP): HOEFER, USA; electrophoresis apparatus (mini protean 3cell): BIO-RAD, USA; imaging system (5200): Shanghai Tianneng Technology Co., Ltd.; vortex oscillator (K30): Shanghai Qingpu Luxi Instrument Factory; water bath (HI1210): Leica, Germany, etc. Reagents: 3-deoxyglucosone (3DG), synthesized by the Central Laboratory of Suzhou Traditional Chinese Medicine Hospital. 4. Experimental Procedure 1. Experimental grouping and model establishment After the rats had undergone environmental adaptation in the early stage of the experiment, they were divided into a normal group (n=10) and a model group (n=10) according to their body weight. The rats in the normal group and the model group were fed with normal feed, which was renewed every two days, and they were allowed to eat freely for 6 weeks. The rats in the model group were given 3-deoxyglucosone (3DG) by gavage, and the rats in the normal group were given the same volume of normal saline for 6 weeks. 2. General situation and model success evaluation After the model group experiment was completed, that is, 6 weeks after gavage of 3DG induction, the fasting body weight was measured after fasting for 18 hours without water, and then an oral glucose tolerance test (OGTT) was performed (the amount of glucose was configured according to the body weight of each rat at a dose of 2g / kg, the time interval between oral gavage was 1min, and blood glucose was measured at 0min before injection and 30min, 60min, and 120min after injection), and blood was collected and stored under anesthesia. The rats were euthanized in strict accordance with ethics, and the pancreatic islets and adipose tissue were dissected and stored according to the corresponding requirements. The pancreatic tissue was observed by HE staining, and the total abdominal fat mass was measured. Figure 4 The general conditions of rats in the normal group and model group and the results of model identification are given. Figure 4 A and Figure 4 B shows that compared with the rats in the normal group, the blood glucose levels of the rats in the model group at 30min, 60min and 120min in the OGTT test were significantly increased, and the insulin level at 30min in the OGTT test was delayed with improved insulin peak time; Figure 4C shows that compared with the normal group rats, the pancreatic islet tissue of the model group rats showed mild atrophy and damage; Figure 4 D shows that compared with the normal group rats, the body weight of the model group rats is similar, but Figure 4 E and Figure 4 F shows that compared with the normal group rats, the abdominal fat mass and index of the model group rats were significantly increased, which is consistent with the characteristic manifestations of central obesity in the elderly Pre-DM; T-test: ** P<0.01, *** P<0.001vs. normal group. (III) Serum-related index detection After the model group experiment was completed, that is, 6 weeks after oral administration of 3DG induction, the animals in the normal group and the model group were fasted for 18 hours, and peripheral blood was collected from all animals in the normal group and the model group to prepare serum. The fasting blood glucose (FBG) of the rats was measured by ELISA method ( Figure 5 A) Fasting insulin (FINS) Figure 5 B) and calculated the insulin resistance index (Homeostasis Model Assessment-Insulin Resistance, HOMA-IR) ( Figure 5 C); and determination of serum triglyceride (TG) ( Figure 5 D) Total cholesterol (TC) Figure 5 E) Low density lipoprotein cholesterol (LDL-C) Figure 5 F) High-density lipoprotein cholesterol (HDL-C) Figure 5 G), glutamate pyruvate transaminase (ALT) Figure 5 H), glutamate oxaloacetic transaminase (AST) ( Figure 5 I), skeletal muscle malondialdehyde (Malondialdehyde, MDA) Figure 5 L), superoxide dismutase (SOD) Figure 5 J), glutathione peroxidase (Glutathioneperoxidase, GSH) Figure 5K) and serum adiponectin (APN) ( Figure 5 M), serum leptin content (Leptin, LP) ( Figure 5 N), serum tumor necrosis factor-alpha (TNF-α) ( Figure 5 O). Figure 5 The results of serum related index detection of rats in normal group and model group are given. Figure 5 AC shows that compared with the normal group rats, the FBG and HOMA-IR of the model group rats were significantly increased and the FINS was significantly decreased, which is completely consistent with the judgment basis of international organizations for Pre-DM. Figure 5 DI showed that compared with the normal group rats, the relevant biochemical indicators of the model group rats were abnormal, among which TG, LDL-C, HDL-C and AST showed statistical differences; Figure 5 JL It can be seen that compared with the normal group rats, the model group rats had abnormalities in the related oxidative stress indicators, among which SOD and MDA showed statistical differences; Figure 5 MO showed that compared with the normal group rats, the APN of the model group rats was significantly decreased and the LP was significantly increased, while TNF-α was increased but with no statistical difference, which was completely consistent with the judgment basis of Pre-DM by international organizations and the basic physical and chemical manifestations of the Pre-DM model in elderly rats. (IV) Based on the detection of mRNA and protein related to the fat-islet axis After the model group experiment was completed, that is, 6 weeks after gavage of 3DG induction, the animals were fasted but not watered for 18 hours, and peripheral blood was collected from all animals in the normal group and model group after anesthesia. The rats were euthanized in strict accordance with ethics, and the pancreatic islets and adipose tissue were dissected and preserved according to the corresponding requirements. RT-qPCR and Western blot were used to detect the changes in the expression of genes and proteins such as AdipoR1, T1R2-T1R3, IRS, and GLUT in adipose and pancreatic tissues, respectively. Figure 6 The results of the mRNA and protein detection of fat-islet axis related rats in the normal group and model group are given. Figure 6 A shows that compared with the normal group, the relative values ​​of AdipoR1, IRS1 and GLUT4 mRNA in the adipose tissue of rats in the model group were significantly decreased, and T1R2-T1R3 mRNA was significantly increased; Figure 6 As can be seen from B and C, compared with the normal group, the expression of IRS1 and GLUT4 proteins in the adipose tissue of the rats in the model group was significantly downregulated, and the expression of T1R2 protein was significantly upregulated. Figure 6D shows that compared with the normal group, the relative values ​​of AdipoR1, T1R2-T1R3, IRS2, and GLUT2 mRNA in the pancreatic islet tissue of rats in the model group were significantly reduced; Figure 6 As can be seen from E and F, compared with the normal group, the expression of T1R2-T1R3 and GLUT2 proteins in the pancreatic islet tissue of the model group rats was significantly downregulated. The above results show that the 3DG-induced Pre-DM model of aged rats is closely related to the fat-islet axis, among which the impaired APN / AdipoR1 and Glc / STRs pathways are involved in the crosstalk of signal transduction between adipocytes and pancreatic β cells in the Pre-DM model state of aged rats, which fully suggests that this model can be used as one of the potential tools for the research of the "fat-islet axis" AdipoR1 / STRs signaling pathway as the main potential target and effect pathway for the prevention and treatment of elderly Pre-DM. Based on the above test results, it can be seen that a good elderly Pre-DM animal model was obtained by feeding rats with 3-deoxyglucosone for a period of time using the method of the present invention, which provides a new idea for the study of the pathogenesis of elderly Pre-DM. The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the above disclosed methods and technical contents without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of protection of the technical solution of the present invention.

Claims

1. A method for constructing a 3DG-induced prediabetes animal model in the elderly, characterized in that: The method uses 3DG gavage to induce experimental animals, thereby constructing an animal model of elderly prediabetes with impaired fasting blood glucose and / or impaired glucose tolerance as the main signs.

2. The method for constructing a 3DG-induced elderly prediabetes animal model according to claim 1, characterized in that: The experimental animals are SD rats or mice.

3. The method for constructing a 3DG-induced prediabetes animal model in the elderly according to claim 1, characterized in that: The intragastric administration amount of 3DG is 50 mg / kg, once a day.

4. The method for constructing a 3DG-induced elderly prediabetes animal model according to claim 1, characterized in that: The intragastric administration also includes supplying water to the experimental animals.

5. The method for constructing a 3DG-induced elderly prediabetes animal model according to claim 4, characterized in that: The experimental animals were supplied with secondary reverse osmosis water at a feeding rate of 1 mL / 100 g, once a day.

6. The method for constructing a 3DG-induced elderly prediabetes animal model according to claim 1, characterized in that: The induction time is 2-10 weeks.

7. Use of the animal model constructed by the construction method according to any one of claims 1 to 6 in the study of the pathogenesis of prediabetes in the elderly.

8. Application of the animal model constructed by the construction method according to any one of claims 1 to 6 in the research of endocrine-related Chinese and Western medicines.

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