Establishment and application of type II diabetes mellitus olfactory disorder / cognitive disorder animal model

Abstract

The invention relates to the technical field of biology, in particular to establishment and application of a type II diabetes mellitus olfactory disorder / cognitive disorder animal model, discloses correlation between GPR88 and T2DM neurodegenerative disease for the first time, and discloses a T2DM mouse model constructed by inhibiting expression of a GPR88 gene. Compared with a common T2DM mouse model which shows more serious olfactory dysfunction and cognitive dysfunction in the T2DM disease course, the animal model constructed by the invention can be used for researching the T2DM pathogenesis through a GPR88 pathway, so that the blank in the prior art is filled, an ideal tool is provided for deeply researching the effect of the target spot in T2DM, and the application prospect is broad. An important foundation is laid for better exploring a new mechanism of GPR88 and diabetes mellitus morbidity and treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a type II diabetes olfactory dysfunction / cognitive dysfunction animal model and its application. BACKGROUND

[0002] Type 2 Diabetes Mellitus (T2DM) is a complex metabolic syndrome caused by the combined action of genetic and environmental factors, characterized by insulin resistance and relative insulin secretion deficiency. Cognitive dysfunction is a common complication of T2DM, and diabetic cognitive dysfunction includes three stages: asymptomatic preclinical stage, mild cognitive impairment (MCI), and dementia. Studies have shown that the prevalence of MCI in T2DM patients is 18.5%-35.8%, and the prevalence of dementia is 10.2%. Olfactory impairment is considered an early predictor of neurodegenerative diseases, and decreased odor recognition ability indicates faster cognitive decline. Studies have found that T2DM patients have olfactory impairment in the preclinical stage of cognitive impairment, and olfactory function and cognitive function are significantly positively correlated. However, there are few studies on olfactory dysfunction in type 2 diabetes.

[0003] GPR88 is an orphan G protein-coupled receptor (GPCR) belonging to the A class of GPCRs, which is classified as an orphan receptor because its endogenous ligand has not been identified, and is highly expressed specifically in the striatum. Studies have shown that GPR88 regulates GABA, glutamate, and dopamine neurotransmitter pathways, affecting behaviors such as motor control, emotion regulation, cognition, and reward learning. Gene knockout studies have shown that GPR88 is closely related to schizophrenia, Parkinson's disease, anxiety, depression, and addiction, and has become a new target for related drug development. However, there is no clear report or recognized conclusion on whether GPR88 is involved in T2DM-like phenotypes. Therefore, it is urgent to cultivate a type II diabetes olfactory dysfunction / cognitive dysfunction animal model to study the pathogenesis and intervention of T2DM. SUMMARY

[0004] In view of the deficiencies of the prior art, the present application provides a type II diabetes olfactory dysfunction / cognitive dysfunction animal model, which provides an ideal tool for in-depth study of the role of this target in T2DM, and better explores the new mechanism of GPR88 and diabetes pathogenesis and treatment.

[0005] To achieve the above-mentioned purpose, the present application is realized by the following scheme: In a first aspect, the present application provides the application of GPR88 gene in constructing a type II diabetes olfactory dysfunction / cognitive dysfunction animal model.

[0006] In the present application, the animal is a non-human mammal; the non-human mammal includes (but is not limited to) mouse, rat, rabbit, monkey, etc., more preferably rat and mouse.

[0007] As used herein, the term "GPR88 gene inactivation" includes the case that one or two GPR88 genes are inactivated, i.e. includes the case that GPR88 gene is inactivated heterozygously and homozygously. For example, the mouse with GPR88 gene inactivation can be a heterozygous or homozygous mouse.

[0008] In the present application, the non-human mammal (e.g. mouse) with GPR88 gene inactivation can be prepared by gene knockout or introduction of foreign gene (or fragment) to inactivate the GPR88 gene, etc. In the art, the technique for inactivating the target gene by gene knockout or introduction of foreign gene is known, and these conventional techniques can be used in the present application.

[0009] In another preferred embodiment of the present application, the inactivation of GPR88 gene is achieved by gene knockout.

[0010] In another preferred embodiment of the present application, the inactivation of GPR88 gene is achieved by insertion of foreign gene (or fragment) into GPR88 gene.

[0011] In a specific example of the present application, a construct containing foreign insertion fragment can be constructed, which contains homologous arms homologous to the flanking sequences on both sides of the insertion site of the target gene, so that the foreign insertion fragment (or gene) can be inserted into the GPR88 genomic sequence (especially the exon region) at a high frequency by homologous recombination, resulting in frame shift, premature termination, or knockout of mouse GPR88 gene, thereby leading to Glce deletion or inactivation.

[0012] The homozygous or heterozygous mouse obtained by the method of the present application is fertile and develops normally. The inactivated GPR88 gene can be inherited to the offspring mouse according to Mendel's law.

[0013] Further, the GPR88 gene is used to construct an animal model of olfactory dysfunction / cognitive dysfunction of type II diabetes, specifically referring to obtaining a GPR88 gene knockout animal model by knocking out the GPR88 gene in the animal or inhibiting the expression of the GPR88 gene in the animal; the GPR88 gene knockout animal model is induced by high-fat diet and STZ injection to construct an animal model of olfactory dysfunction / cognitive dysfunction of type II diabetes.

[0014] It is fully appreciated by those skilled in the art that the GPR88 gene can be knocked out or the expression of the GPR88 gene can be inhibited by using existing gene editing methods. For example, the GPR88 gene can be knocked out or the expression of the GPR88 gene can be reduced by inserting a transposon or a foreign DNA fragment such as a virus into the sequence of the GPR88 gene, or by using a chemical mutagenesis method such as ENU, or a physical mutagenesis method such as X-ray, or a gene targeting method based on embryonic stem cells or CRISPR / Cas9 technology.

[0015] In a second aspect, the present application provides a method for constructing an animal model of type II diabetes mellitus olfactory dysfunction / cognitive dysfunction, comprising the following steps: knocking out the GPR88 gene in an animal or inhibiting the expression of the GPR88 gene in the animal to obtain a GPR88 gene knockout animal model; and inducing the GPR88 gene knockout animal model by a high-fat diet and STZ injection to construct an animal model of type II diabetes mellitus olfactory dysfunction / cognitive dysfunction.

[0016] In particular, the GPR88 gene can be knocked out or the expression of the GPR88 gene can be inhibited by using existing gene editing methods. For example, the GPR88 gene can be knocked out or the expression of the GPR88 gene can be reduced by inserting a transposon or a foreign DNA fragment such as a virus into the sequence of the GPR88 gene, or by using a chemical mutagenesis method such as ENU, or a physical mutagenesis method such as X-ray, or a gene targeting method based on embryonic stem cells or CRISPR / Cas9 technology.

[0017] Further, the animal is a non-human mammal; and more further, the animal is a rat or a mouse.

[0018] In a third aspect, the present application also provides an animal model of type II diabetes mellitus olfactory dysfunction / cognitive dysfunction.

[0019] The following aspects are also within the scope of the present application: (1) Application of the animal model of type II diabetes mellitus olfactory dysfunction / cognitive dysfunction in the functional research of type II diabetes mellitus olfactory dysfunction and cognitive dysfunction.

[0020] (2) Application of the animal model of type II diabetes mellitus olfactory dysfunction / cognitive dysfunction in screening drugs for type II diabetes mellitus olfactory dysfunction and cognitive dysfunction.

[0021] (3) Application of the animal model of type II diabetes mellitus olfactory dysfunction / cognitive dysfunction in the research of the mechanism of action in regulating systemic energy metabolism and glucose homeostasis.

[0022] (4) Application of GPR88 gene as a target in screening drugs or preparations for the treatment of type II diabetes, related neurodegenerative diseases and metabolic diseases, including olfactory dysfunction / cognitive impairment in type II diabetes.

[0023] In this invention, a candidate drug or therapeutic agent refers to a substance known to have a certain pharmacological activity or that is being tested and may have a certain pharmacological activity, including but not limited to nucleic acids, proteins, chemically synthesized small or large molecular compounds, cells, etc. The candidate drug or therapeutic agent can be administered orally, intravenously, intraperitoneally, subcutaneously, via the spinal canal, or directly via intracerebral injection.

[0024] Beneficial effects: This invention reveals for the first time the correlation between GPR88 and T2DM neurodegenerative diseases. The T2DM mouse model constructed by inhibiting the expression of the GPR88 gene showed more severe olfactory and cognitive dysfunction during the course of T2DM compared to the ordinary T2DM mouse model. The animal model constructed by this invention can study the pathogenesis of T2DM through the GPR88 pathway, filling the gap in the existing technology and providing an ideal tool for in-depth research on the role of this target in T2DM, laying an important foundation for better exploring new mechanisms of GPR88 in the pathogenesis and treatment of diabetes. Attached Figure Description

[0025] Figure 1 Comparison of gene identification results for GPR88 mutant homozygous mice; Figure 2 The images show a comparison of body weight between T2DM mice and control mice; 2A shows the body weight comparison of mice in the WT-control group and WT-T2DM group before and after STZ injection; 2B shows the body weight comparison of mice in the GPR88 KO-control group and GPR88 KO-T2DM group before and after STZ injection. Figure 3 The figures show a comparison of food intake between T2DM mice and control mice; 2A shows a comparison of food intake before and after STZ injection in WT-control and WT-T2DM mice; 2B shows a comparison of food intake before and after STZ injection in GPR88 KO-control and GPR88 KO-T2DM mice. Figure 4 The results of glucose tolerance tests in T2DM mice and control mice are shown; among them, 4A is the blood glucose change curve of the GTT test; 4B is the AUC statistical graph of the blood glucose change curve of the GTT test; 4C is the statistical graph of fasting blood glucose in mice. Figure 5The results of insulin tolerance tests (ITT) in T2DM mice and control mice are shown below. 5A shows the percentage change in blood glucose during the ITT test when the insulin injection dose is 0.7 U / kg; 5B shows the AUC statistical graph of the blood glucose change curve during the ITT test when the insulin injection dose is 0.7 U / kg; 5A shows the percentage change in blood glucose during the ITT test when the insulin injection dose is 0.6 U / kg; 5B shows the AUC statistical graph of the blood glucose change curve during the ITT test when the insulin injection dose is 0.6 U / kg. Figure 6 The results of the food burial experiment in T2DM mice are shown below; 6A shows the sniffing time of mice before feeding with a high-fat diet; 6B shows the sniffing time of mice after 6 weeks of T2DM; and 6C shows the sniffing time of mice after 12 weeks of T2DM. Figure 7 The results are from the Y-maze experiment; where 7A represents the total time for mice to enter the new heteroarm; 7B represents the time for mice to enter the new heteroarm. Distance into the new arm / total distance; 7C represents the number of times the mouse entered the new arm; 7D represents the time heatmap of the mouse in the Y maze; Figure 8 The Morris water maze experiment is shown below; 8A represents the escape latency period during the mouse training period; 8B represents the escape distance during the mouse training period; 8C represents the number of times the mouse entered the original platform area during the probe test; 8D represents the distance traveled in the quadrant where the mouse probe test was located / the total distance traveled; 8E represents the time spent in the quadrant where the mouse probe test was located; 8F represents the trajectory of the mouse probe test; and 8G represents the time heatmap of the mouse probe test. Detailed Implementation

[0026] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be described in detail below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or according to the manufacturer's recommendations. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores. Unless otherwise stated, percentages and parts are calculated by weight. Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques are well described in existing literature; see Sambrook et al., *MOLECμLAR CLONING: A BOBRATORY MANUAL*, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., *CURRENT PROTOCOLS IN MOLECμLAR BIOLOGY*, John Wiley & Sons, New York, 1987 and periodic updates; the series *METHODS IN ENZYMOLOGY*, Academic Press, San Diego; Wolffe, *CHROMATIN STRUCTURE AND FUNCTION*, Third edition, Academic Press, San Diego, 1998; *METHODS* IN ENZYMOLOGY, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (PB Becker, ed.), Humana Press, Totowa, 1999, etc. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0027] This invention employs a combined modeling approach of "specific gene defect (GPR88 KO) + environmental stress (high-fat diet) + STZ induction," which can stably and consistently induce typical T2DM phenotypes, including hyperglycemia, insulin resistance, and impaired glucose tolerance. The data reproducibility is excellent, greatly improving experimental reliability. It is an ideal tool for studying T2DM central nervous system complications and their association with metabolic diseases. It can be used to analyze downstream neural circuits and molecular signals, and provides a dedicated, highly relevant preclinical model for drugs targeting downstream pathways, significantly improving the efficiency and success rate of drug screening.

[0028] Example 1: Animal Model of Type II Diabetic Olfactory Dysfunction / Cognitive Impairment Obtaining GPR88 gene (NCBIGeneID:64378) mutant homozygous mice (GPR88 KO): C57BL / 6J GPR88 gene knockout (GPR88+ / -) mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were mated to obtain offspring mice. Genotyping of the offspring mice was performed using polymerase chain reaction (PCR) and DNA sequencing to screen for GPR88- / - homozygous (GPR88 KO) mice. GPR88+ / + wild-type (WT) mice from the same litter were used as controls. The identification results are as follows: Figure 1 As shown.

[0029] Obtaining a type 2 diabetic olfactory / cognitive impairment mouse model (GPR88 KO-T2DM): Experimental grouping: GPR88 KO mice and WT mice of similar weight and about 6 weeks of age were divided into 4 groups: GPR88 KO-T2DM group, WT-T2DM group, GPR88 KO-control group, and WT-control group.

[0030] Mice in the GPR88 KO-T2DM and WT-T2DM groups were fed a purified high-fat diet (60% fat energy) for 8 weeks to induce insulin resistance. After 8 weeks of high-fat diet, mice were fasted for 12 hours for 5 consecutive days and then injected with a small dose of 1% STZ (Streptozotocin, 40 mg / kg) to partially destroy the pancreatic β cells and induce relative insulin deficiency. The mice were then fed a high-fat diet again. Mice in the GPR88 KO-control and WT-control groups were fed a control diet (10% fat energy) for 8 weeks under the same conditions. After fasting for 12 hours for 5 consecutive days, mice were injected with an equal volume of citrate buffer. The mice were then fed a control diet again.

[0031] Seven days after STZ injection, random blood glucose levels at the tail tip of mice were measured. A random blood glucose level >16.7 mmol / L indicated that the T2DM model was essentially established. The GTT (Glucose Tolerance Test), ITT (Insulin Tolerance Test), body weight, and food intake of the model mice were monitored to further validate the T2DM model.

[0032] like Figure 2 As shown, compared with the WT-control group, the body weight of mice in the WT-T2DM group decreased significantly after STZ injection; compared with the GPR88 KO-control group, the body weight of mice in the GPR88 KO-T2DM group decreased significantly after STZ injection. It can be seen that the body weight loss of T2DM mice in both groups is consistent with the characteristics of T2DM disease.

[0033] like Figure 3 As shown, compared with the WT-control group, the food intake of mice in the WT-T2DM group increased significantly after STZ injection; compared with the GPR88 KO-control group, the food intake of mice in the GPR88 KO-T2DM group increased significantly after STZ injection. It can be seen that the increased food intake of the two groups of T2DM mice is consistent with the characteristics of T2DM disease.

[0034] like Figure 4 As shown, the GTT-AUC of the two groups of T2DM mice was significantly larger than that of the Ctrl group, indicating that both groups showed obvious glucose intolerance. Furthermore, the fasting blood glucose of the two groups of T2DM mice was significantly higher than that of the Ctrl group, which is consistent with the characteristics of T2DM mice. In addition, the blood glucose of the GPR88 KO-Ctrl group mice was significantly higher at 15 min and 30 min. Blood glucose was still rising 15 min to 30 min after glucose injection, and began to decrease after 30 min. The decrease in blood glucose was delayed and then gradually returned to normal. This suggests that GPR88 gene deletion may be associated with abnormal insulin secretion or insulin resistance.

[0035] like Figure 5 As shown, the ITT-AUC of the two T2DM mice was significantly larger than that of the Ctrl group, indicating that both groups showed significant insulin resistance. When the insulin injection dose was 0.6 U / kg, the GPR88 KO-T2DM group showed decreased insulin sensitivity compared to the WT-T2DM group, and the GPR88 KO-Ctrl group showed decreased insulin sensitivity compared to the WT-Ctrl group, indicating that GPR88 gene deletion may lead to insulin resistance.

[0036] The above results fully demonstrate that, compared with the pre-STZ injection, T2DM mice showed obvious symptoms of increased appetite and weight loss. Furthermore, the GTT and ITT test results also proved that T2DM mice showed obvious glucose intolerance and insulin resistance, indicating that the T2DM mouse model was successfully established.

[0037] Example 2: Analysis of olfactory function in GPR88 KO-T2DM mouse model After the T2DM mouse model was established, the mice's olfactory function was tested in three stages: 6 weeks of age (before feeding with a high-fat diet), 6 weeks of T2DM disease course, and 12 weeks of T2DM disease course.

[0038] Food burial experiment: Mice were fasted for 12 hours but allowed access to water. The mice were placed in cages containing 3cm of clean bedding to acclimate for 10 minutes. After acclimatization, the mice were removed, the cages were cleaned, and the bedding was replaced with fresh clean bedding. Food was buried in a corner of the cage, ensuring the process was out of the mouse's sight. The mouse was then returned to the cage from the diagonal position where the food was buried, and the time taken from when the mouse returned to the cage until it found the food was recorded. The shorter the time, the better the mouse's sense of smell. After each mouse was tested, the cage was cleaned and the bedding replaced with fresh clean bedding to ensure there were no odors affecting the accuracy of the experimental results.

[0039] like Figure 6 As shown, at 6 and 12 weeks of T2DM disease course, both groups of T2DM mice showed signs of impaired olfactory function. GPR88 KO-T2DM mice had more severe olfactory dysfunction than normal T2DM mice, and the dysfunction gradually worsened as the disease progressed.

[0040] Example 3: Cognitive function analysis of the GPR88 KO-T2DM mouse model Mice were subjected to the Y maze and Morris water maze experiments, respectively, and the results are as follows: Figures 7-8 As shown.

[0041] Y-maze Experiment: The experiment consists of two phases: an adaptation and recognition phase and a testing phase. In the adaptation phase, arm C was closed with a barrier, and the mouse was placed at the distal end of arm A, allowing it to freely explore the areas of arms A and B for 10 minutes. Afterward, the mouse was returned to its cage to rest for one hour, followed by the testing phase. In the testing phase, the barrier closing arm C was removed, and the mouse was placed at the distal end of arm A, facing the central area, allowing it to freely explore the Y-maze for 10 minutes. After the experiment, parameters recorded by Any-maze software were analyzed, including the time, distance traveled, and number of times the mouse entered arm C (the novel arm). Mice naturally tend to explore unknown areas; therefore, the Y-maze experiment can be used to infer the mouse's spatial memory ability. In the testing phase, a higher percentage of the mouse's exploration of arm C suggests a stronger memory of the old arm and better spatial memory.

[0042] like Figure 7 As shown, the time, distance, and number of times GPR88 KO-T2DM mice entered the new heteroarm were significantly lower than those of normal mice, indicating that their short-term spatial working memory was significantly impaired and that they showed short-term memory impairment earlier than normal T2DM mice.

[0043] Morris water maze experiment: The experiment consists of two phases: training and testing. In the training phase, the platform was placed 1.5 cm below the water surface in the center of the SE quadrant for a total of 5 days. Mice were placed into the water maze from four different locations each day for training. During training, mice were placed into the maze sequentially from the same location, and only after all mice had been trained were they placed into the next location. Each mouse was trained for 1 minute per location each day. If a mouse found the platform on its own, training stopped after 3 seconds. Mice that did not find the platform were placed on it for 15 seconds to reinforce their platform location memory. The training phase primarily measured the mice's escape latency (time to find the platform) and escape distance (distance traveled to find the platform). In the testing phase (day 6), the platform was removed, and the mice were placed in the quadrant opposite the platform for 2 minutes. The main data recorded included the number of times the mouse traversed the platform area, the time spent in the target quadrant, and the ratio of the distance traveled in the target quadrant to the total distance. The Morris water maze experiment utilizes the animal's spatial reference memory of the platform's location through distal visual cues. The shorter the escape latency and escape distance (time and distance to find the underwater platform) of mice during the training period, the better their learning and memory abilities. During the testing phase, higher values ​​for the number of times mice entered the original platform area, the distance-to-time ratio of the original platform quadrant, and the time value indicate better long-term spatial reference memory.

[0044] Statistics of water maze training period from day 1 to day 5 (see...) Figure 8 The results (A-8B) showed that mice in the GPR88 KO-T2DM, GPR88 KO-Ctrl, and WT-T2DM groups had longer escape latency and escape distances than normal mice, indicating a decline in their learning and memory abilities. Mice in the GPR88 KO-Ctrl group had longer escape latency and escape distances than normal mice, and mice in the GPR88 KO-T2DM group had longer escape latency and escape distances than those in the WT-T2DM group, suggesting that the absence of GPR88 may lead to a decline in learning ability.

[0045] Statistical results of the probe test (2 min) on day 6 of the water maze (see) Figure 8The results (C-8E) showed that the GPR88 KO-T2DM and WT-T2DM groups entered the original platform region less frequently than the Ctrl group. The GPR88 KO-Ctrl group also entered the original platform region less frequently than normal mice, indicating impaired long-term spatial memory. Furthermore, the GPR88 KO-T2DM group showed significantly reduced distance travel and time to the quadrant containing the original platform compared to other groups. These results suggest that the absence of GPR88 may lead to long-term memory deficits.

[0046] Finally, it should be noted that the above description is only a preferred embodiment of the present invention. Those skilled in the art, under the guidance of the present invention, can make various similar representations without departing from the spirit and claims of the present invention, and such modifications all fall within the protection scope of the present invention.

Claims

1. Application of GPR88 gene in constructing animal models of olfactory / cognitive impairment in type II diabetes.

2. The application according to claim 1, characterized in that, The animal in question is a non-human mammal.

3. The application according to claim 1, characterized in that, GPR88 gene knockout animal models were obtained by knocking out or inhibiting the expression of the GPR88 gene in animals. The GPR88 gene knockout animal models were then induced by a high-fat diet and STZ injection to construct an animal model of olfactory / cognitive impairment in type II diabetes.

4. A method for constructing an animal model of olfactory dysfunction / cognitive impairment in type II diabetes, characterized in that... The procedure includes the following steps: knocking out or inhibiting the expression of the GPR88 gene in animals to obtain a GPR88 gene knockout animal model; and then inducing the GPR88 gene knockout animal model with a high-fat diet and STZ injection to construct a type II diabetic olfactory / cognitive impairment animal model.

5. The method according to claim 4, characterized in that, The animal in question is a non-human mammal.

6. The method according to claim 4 or 5, characterized in that, The animal in question is a rat or a mouse.

7. An animal model of type II diabetes mellitus with olfactory dysfunction / cognitive impairment constructed by any one of claims 4-6.

8. The application of the animal model of olfactory dysfunction / cognitive impairment in type II diabetes as described in claim 7 in the study of olfactory dysfunction / cognitive impairment in type II diabetes.

9. The application of the animal model of olfactory dysfunction / cognitive impairment in type II diabetes as described in claim 7 in screening drugs for the treatment of olfactory dysfunction / cognitive impairment in type II diabetes.

10. Application of GPR88 gene as a target in screening drugs or formulations for the treatment of olfactory / cognitive impairment in type II diabetes.

Images