Application of HERP gene knockout mouse model in obesity and metabolic disorders

Abstract

This invention discloses the application of the HERP gene knockout mouse model in obesity and metabolic disorders. The invention found that, compared to the wild-type high-fat diet group, HERP gene knockout leads to earlier and more severe obesity in mice fed a high-fat diet, with increased fat percentage, aggravated adipose tissue inflammation, and phenotypes such as dyslipidemia and glucose metabolism disorders. Furthermore, HERP gene knockout results in increased adipocyte differentiation. This indicates that HERP gene knockout mice are more sensitive to nutrition, develop obesity more quickly on a high-fat diet, and exhibit more severe symptoms such as adipose amplification, adipose tissue inflammation, and glucose and lipid metabolism disorders. This model can be used for molecular mechanism research and drug screening of obesity, adipose tissue disorders, and glucose and lipid metabolism disorders. The invention further found that cilostazol can significantly alleviate obesity and lipid metabolism disorders caused by HERP gene deficiency in mice, but has no significant weight loss effect in mice with normal genotypes, providing a new strategy for precision treatment.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of HERP gene knockout mouse models in obesity and metabolic disorders. Background Technology

[0002] With changing lifestyles, obesity has become one of the greatest threats to human health. Obesity is closely related to hypertension, diabetes, cardiovascular disease, and cancer. Given the prevalence and harmful effects of obesity, it not only poses a serious threat to public health but also imposes a heavy burden on society.

[0003] Animal models play a crucial role in the study of obesity and metabolic disorders, particularly in molecular mechanism research, drug screening, and treatment strategy development. Currently, commonly used animal models for obesity and metabolic disorders include diet-induced models and genetic obesity models. Diet-induced models induce obesity and metabolic disorders through a high-fat (HFD) or high-carbohydrate diet. Their advantage is that they mimic human diet-related metabolic diseases. However, their disadvantages include the fact that the induced obesity effect is closely related to the animal strain and the composition of carbohydrates and fats in the diet, making it difficult to reproduce experimental results across different laboratories, and the modeling time is relatively long (at least 12 weeks). Compared to diet-induced obesity models, genetic obesity models exhibit higher phenotypic stability and are more valuable for studying the molecular mechanisms of the disease and intervention methods. However, the selection of animal models for genetic obesity is currently limited; the most common are the Ob / ob mouse model (leptin gene mutation, manifesting as obesity, insulin resistance, and diabetes) and the db / db mouse model (leptin receptor gene mutation, manifesting as obesity, insulin resistance, and diabetes). Limited genetic obesity models fail to reflect the complex etiology of obesity and restrict research into the molecular mechanisms of obesity and metabolic disorders, as well as the development of obesity treatment strategies and the screening of anti-obesity drugs. Therefore, developing and discovering new animal models of obesity and metabolic disorders remains an urgent need for research on the mechanisms and interventions of obesity and metabolic disorders.

[0004] HERP (encoding herpud1) is an endoplasmic reticulum membrane protein that plays a crucial role in endoplasmic reticulum stress and the degradation pathway of endoplasmic reticulum-related proteins. Literature reports that herpud1 knockout improves fatty liver induced by high-fat diets in mice. GWAS results indicate that herpud1 SNP sites are significantly correlated with blood glucose and lipid levels. However, the function of the herpud1 gene is not fully understood, and its role in obesity remains unexplored.

[0005] Cilostazol is a selective phosphodiesterase III inhibitor, primarily used clinically to treat ischemic symptoms such as ulcers, limb pain, coldness, and intermittent claudication caused by chronic arterial occlusive disease, as well as for the prevention of cerebral infarction. Recent studies have found that cilostazol can inhibit the synthesis of hepatic triglycerides, promote the breakdown of hepatic triglycerides, and reduce the accumulation of lipid droplets in the liver. Cilostazol can also reduce serum triglyceride levels in patients with atherosclerosis. However, the effects of cilostazol on symptoms related to herpud1 gene deficiency have not been reported. Summary of the Invention

[0006] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for constructing a mouse model.

[0007] Another object of the present invention is to provide an application of the mouse model obtained by the above construction method.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A method for constructing a mouse model involves knocking out the HERP gene in mice using sgRNA, feeding the resulting HERP gene knockout mice a high-fat diet, and obtaining a mouse model with any one or more of the following characteristics: I. obesity; II. disordered glucose and lipid metabolism; III. fatty liver.

[0010] Furthermore, the sgRNA is an sgRNA that targets exons 2-4 of the HERP gene.

[0011] Furthermore, the sgRNA includes any one or more of sgRNA1 and sgRNA2; the sequences of sgRNA1 and sgRNA2 are shown below:

[0012] sgRNA1: 5'-CTCCGTGTCCTTAAAGTCACTGG-3'

[0013] sgRNA2: 5'-AGCGTCGTCACAGCTATGAAGGG-3'.

[0014] Furthermore, the high-fat feeding refers to feeding rats with a high-fat rat diet containing 60 kcal of fat calories.

[0015] Furthermore, the high-fat feeding period is at least 5 weeks.

[0016] Furthermore, the high-fat feeding period is 5 to 12 weeks.

[0017] Furthermore, the method of knocking out the HERP gene in mice using sgRNA includes the following steps:

[0018] S1. Design sgRNA1 and sgRNA2 targeting the HERP gene;

[0019] S2. sgRNA1, sgRNA2 and Cas9 enzyme were co-transferred into mouse zygotes;

[0020] S3. The fertilized egg is transplanted into a surrogate mouse to produce F0 generation mice, from which positive F0 generation mice with HERP gene knockout are selected.

[0021] S4. Cross the positive F0 generation mice with wild-type mice to produce F1 generation mice, and select positive F1 generation mice (i.e., heterozygous mice with HERP gene knockout) from them.

[0022] S5. Based on the cross-pairing of positive F1 generation mice, homozygous offspring with HERP gene knockout are screened to obtain homozygous mice with HERP gene knockout.

[0023] Furthermore, the specific steps of step S2 are as follows:

[0024] S21. Synthesize sgRNA1 and sgRNA2. Prepare an sgRNA mixture by mixing sgRNA1 and sgRNA2 in a 1:1 molar ratio. Then, mix Cas9 enzyme and sgRNA mixture in a 1:1 molar ratio and add Opti-MEM culture medium by volume to prepare a gene editing solution.

[0025] S22. Gene editing solution was added to C57BL / 6N mouse zygotes 0.5 days after fertilization, and sgRNA and Cas9 enzyme were transferred into C57BL / 6N mouse zygotes by electrotransfection.

[0026] A mouse model was obtained using the method described above.

[0027] The mouse models obtained by the above construction method can be used to study the mechanisms of any one or more of the following diseases: I. Obesity; II. Metabolic disorders; III. Fatty liver.

[0028] The mouse models obtained by the above construction method can be used to screen drugs that have therapeutic effects on any one or more of the following diseases: I. Obesity; II. Metabolic disorders; III. Fatty liver.

[0029] Application of HERP gene in regulating differentiation of mouse preadipocyte line 3T3-L1.

[0030] Furthermore, the application is as follows: promoting the differentiation of mouse preadipocyte line 3T3-L1 into mature adipocytes by knocking out the HERP gene.

[0031] Furthermore, the knockout is achieved via sgRNA.

[0032] Furthermore, the sgRNA includes any one or more of sgRNA1' and sgRNA2'; the sequences of sgRNA1' and sgRNA2' are shown below:

[0033] sgRNA1': 5'-CTCCGTGTCCTTAAAGTCACTGG-3'

[0034] sgRNA2': 5'-AGCGTCGTCACAGCTATGAAGGG-3'.

[0035] Furthermore, the application includes the following steps:

[0036] T1. First, Cas9 enzyme was transferred into 3T3-L1 cells via liposomes, and then selected with antibiotics to obtain 3T3-L1 cells that stably express Cas9 enzyme.

[0037] T2. Synthesize sgRNA1' and sgRNA2', and transfect sgRNA1' and sgRNA22' into 3T3-L1 cells that stably express Cas9 enzyme at a molar ratio of 1:1 via liposomes. After antibiotic screening, obtain the HERP knockout 3T3-L1 cell line.

[0038] T3. After the obtained HERP knockout 3T3-L1 cell line was grown to 100% confluence, it was induced with a triple inducer and then cultured in a medium containing insulin.

[0039] Furthermore, the triple inducer consists of: 2 μg / ml insulin, 100 ng / ml dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine.

[0040] Furthermore, the insulin-containing culture medium is a culture medium containing 2 μg / ml insulin.

[0041] Furthermore, the induction time is 3 days, and the culture time is 3 days.

[0042] The use of cilostazol or a pharmaceutically acceptable salt thereof in the preparation of drugs for improving, treating and / or preventing HERP gene defect-related obesity and lipid metabolism disorders.

[0043] Furthermore, in the aforementioned applications, the knockout of the HERP gene promotes obesity and lipid metabolism disorders.

[0044] The use of cilostazol or a pharmaceutically acceptable salt thereof in the preparation of an inhibitor of HERP gene defect-associated adipocyte differentiation.

[0045] Furthermore, the differentiation mentioned refers to the differentiation of precursor adipocytes into mature adipocytes.

[0046] Furthermore, in the aforementioned applications, the knockout of the HERP gene promotes adipocyte differentiation.

[0047] Furthermore, the pharmaceutically acceptable salt has an anion that is either inorganic or organic; the inorganic anion is chloride, bromide, iodide, sulfate, nitrate, nitrite, phosphate, or hydrogen phosphate, etc.; the organic anion is acetate, propionate, cinnamate, benzosulfonate, citrate, lactate, or gluconate, etc.

[0048] Furthermore, the drug also includes any one or more of a carrier and an adjuvant.

[0049] Furthermore, the carrier is selected from one or more of lactose, starch, gelatin, sodium carboxymethyl cellulose, methyl cellulose, polyvinylpyrrolidone, and water.

[0050] Furthermore, the additive is selected from one or more of disintegrants, lubricants, and solubilizers.

[0051] The disintegrant is preferably microcrystalline cellulose; the lubricant is preferably one or more of talc, colloidal silica gel, glyceryl stearate, calcium stearate, and magnesium powder; the solubilizer is preferably one or more of methanesulfonic acid, fumaric acid, mannitol, sorbitan monolaurate, monostearate, and monooleate.

[0052] Furthermore, the drug can be prepared into tablets, granules, capsules, oral liquids, or injections.

[0053] The present invention has the following advantages and effects compared with the prior art:

[0054] This invention reveals that, compared to the wild-type high-fat diet group, knocking out the HERP gene leads to earlier and more severe obesity in mice on a high-fat diet, with increased fat percentage, aggravated adipose tissue inflammation, and phenotypes such as dyslipidemia and glucose metabolism disorders. Furthermore, HERP gene knockout results in increased adipocyte differentiation. This indicates that HERP gene knockout mice are more responsive to nutrition, develop obesity more quickly on a high-fat diet, and exhibit more severe symptoms such as adipose amplification, adipose tissue inflammation, and glucose and lipid metabolism disorders. This finding can be used for molecular mechanism research on obesity, adipose tissue disorders, and glucose and lipid metabolism disorders, as well as for drug screening.

[0055] This invention further reveals that cilostazol can significantly alleviate obesity and lipid metabolism disorders caused by HERP gene deficiency in mice, but has no significant weight loss effect in mice with normal genotypes. Based on this finding, this invention provides the application of cilostazol in the preparation of drugs to improve, treat, and / or prevent HERP gene deficiency-related obesity and lipid metabolism disorders, providing a new strategy for precision medicine. Attached Figure Description

[0056] Figure 1 This document presents the construction strategy and gene identification diagram of HERP gene knockout mice; A. Construction strategy; B. Identification band diagram of mice of various genotypes; C. Sanger sequencing diagram;

[0057] Figure 2 This is a graph showing the expression levels of Herp mRNA in the epididymal fat, inguinal fat, and liver of mice; each group consists of 5 mice, with wild-type C57BL / 6N strain mice as controls and β-Actin as an internal control;

[0058] Figure 3 The graphs show mouse weight and body shape; A. Weight gain curve; B. Weight at weeks 5 and 9 of high-fat diet; C. Body shape of mice at week 9 of normal diet and high-fat diet; Number of mice per group = 5;

[0059] Figure 4 The images show the appearance of mouse fat, the percentage of fat in body weight, and inflammation of adipose tissue; A. The appearance of epididymal fat in mice fed a normal diet for 9 weeks; B. The percentage of epididymal fat in mice fed a normal diet for 9 weeks; N=5; C. The appearance of epididymal fat in mice fed a high-fat diet for 9 weeks; D. The percentage of epididymal fat in mice fed a high-fat diet for 9 weeks; E. F4 / 80 staining results of epididymal fat in mice fed a normal diet and a high-fat diet for 9 weeks; F. F4 / 80 staining results of epididymal fat in mice fed a high-fat diet and a high-fat diet for 9 weeks.

[0060] Figure 5 This is a graph showing the blood lipid results of mice; AC. Serum levels of free fatty acids, low-density lipoprotein, and total cholesterol in mice fed a normal diet at week 9; DF. Serum levels of free fatty acids, low-density lipoprotein, and total cholesterol in mice fed a high-fat diet at week 9; FFA, free fatty acids; LDL, low-density lipoprotein; T-CHO, total cholesterol; Number of mice per group = 5;

[0061] Figure 6 This is a fasting blood glucose graph of mice; the number of mice in each group is 5.

[0062] Figure 7 This is an image of a mouse liver section stained with Oil Red O.

[0063] Figure 8These are diagrams showing the differentiation results of mouse primary adipocytes and HERP knockout 3T3-L1 preadipocytes; A. Mouse primary adipocytes; B. HERP knockout 3T3-L1 preadipocytes;

[0064] Figure 9 This is a graph showing the effect of cilostazol on body weight; A. Body weight gain curve; B. Mouse body size; Number of mice in each group = 5;

[0065] Figure 10 These are images showing the effect of cilostazol on white adipose tissue; A. Appearance of epididymal adipose tissue; B. Fat percentage of body weight; C. F4 / 80 staining.

[0066] Figure 11 This is a graph showing the effect of cilostazol on blood lipids; FFA, free fatty acids; LDL, low-density lipoprotein; T-CHO, total cholesterol; number of mice per group = 5;

[0067] Figure 12 This is a diagram showing the effect of cilostazol on the differentiation of primary adipocytes.

[0068] Wherein: WT: wild type; KO: HERP knockout homozygous; WT+LF: wild type mice fed with normal diet; KO+LF: homozygous mice fed with normal diet; WT+HF: wild type mice fed with high-fat diet; KO+HF: homozygous mice fed with high-fat diet; *P<0.05; **P<0.01, ***P<0.001. Detailed Implementation

[0069] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0070] As used herein, the term "gene knockout" or "knockout" refers to editing a gene in a cell (e.g., modifying it by insertion, substitution, and / or deletion) to cause the gene to lose its original function (e.g., to be unable to express a functional protein). Various known molecular biology techniques (e.g., gene editing using zinc finger nucleases, TALEN gene editing, and CRISPR / Cas (e.g., CRISPR / Cas9) gene editing) can be used to edit genes in the cellular genome. Gene knockout is not limited to the complete deletion or removal of an entire gene, but only to the point that the gene loses its original function. For example, gene knockout can be achieved by inserting a foreign DNA fragment into the gene, preventing it from expressing a functional protein, or by inserting or deleting one or more bases into the gene, causing a frameshift mutation. For example, the gene knockout in the following embodiments of this application uses CRISPR / Cas9 gene editing technology.

[0071] Example 1. Construction of HERP gene knockout mice

[0072] This embodiment constructs a HERP gene knockout mouse model. The mouse model is a mouse in which the HERP gene has been knocked out. CRISPR / Cas9 gene editing technology is a mature commercial technology for knocking out gene fragments; for example, Cyagen (Nanjing) Biomedical Technology Co., Ltd. can be commissioned to perform CRISPR / Cas9 gene editing. The gene knockout strategy for this mouse model is as follows: Figure 1 As shown in Figure A. The mice are of the C57BL / 6N strain, with the knockout gene named herpud1 (Gene ID: 64209), and the knockout exons being exons 2-4. The specific construction method includes the following steps:

[0073] 1. Design sgRNA1 and sgRNA2 targeting exons 2-4 of herpud1, with the following specific sequences:

[0074] sgRNA1: 5'-CTCCGTGTCCTTAAAGTCACTGG-3'

[0075] sgRNA2: 5'-AGCGTCGTCACAGCTATGAAGGG-3';

[0076] 2. sgRNA and Cas9 enzyme were co-transferred into C57BL / 6N mouse zygotes; the specific method was as follows:

[0077] (1) Synthesize sgRNA1 and sgRNA2, prepare sgRNA mixture by mixing sgRNA1 and sgRNA2 in a molar ratio of 1:1, then mix Cas9 enzyme and sgRNA mixture in a molar ratio of 1:1 and add Opti-MEM culture medium by volume to prepare gene editing solution;

[0078] (2) Gene editing solution was added to the fertilized eggs of C57BL / 6N mice 0.5 days after fertilization, and sgRNA and Cas9 enzyme were transferred into the fertilized eggs of C57BL / 6N mice by electrotransfection.

[0079] 3. The fertilized eggs were transferred into surrogate C57BL / 6N mice to produce F0 generation mice, from which positive F0 generation mice were selected. Positive F0 generation mice were mated with wild-type mice to produce F1 generation mice, from which positive F1 generation mice (i.e., HERP knockout heterozygotes) were selected. Based on the mating of F1 generation mice, homozygous offspring with HERP gene knockout were selected to obtain homozygous HERP gene knockout mice. The mouse gene identification method is as follows:

[0080] (1) Two weeks after the mice were born, their tails were clipped and genomic DNA was extracted from the tails. High-purity genomic DNA was obtained using the Tiangen Blood / Cell / Tissue Genomic DNA Extraction Kit (catalog number DP304-03). The extraction method was in accordance with the kit instructions.

[0081] (2) PCR identification: Mouse genotypes were identified using two primer pairs (primer 1: F1 / R1; primer 2: F1 / R2), and the amplified products were subjected to gel electrophoresis. The amplification product of PCR primer 1 was 525 bp, and the amplification product of PCR primer 2 was 752 bp. Mice showing only a 525 bp band were homozygous for HERP knockout, mice showing only a 752 bp band were wild-type (WT) mice, and mice showing both 525 bp and 752 bp bands were heterozygous mice. The bands for each genotype are shown below. Figure 1 As shown in Figure B. The PCR primer information used is as follows:

[0082] F1: 5'-AGTTACCCTTGTGCTGAGAGTC-3'

[0083] R1: 5'-CCTTCTTTCCGTTCTAACCAGAG-3'

[0084] R2: 5'-AAGCAAATCTTGGAGACACTGG-3'.

[0085] (3) To ensure that exons 2-4 were knocked out, PCR identification was performed using PCR primer 1, followed by gel extraction of the 525bp sample and verification using Sanger sequencing (sent to an external testing company). Figure 1 As shown in Figure C, the knockout fragment is 3921 bp, indicating that exons 2-4 of the HERP gene were successfully knocked out.

[0086] Example 2. Gene expression in HERP gene knockout mice

[0087] Two groups of mice were set up, with 5 mice in each group. One group consisted of homozygous (KO) male mice constructed according to the method in Example 1; the other group consisted of wild-type (WT) male mice of the C57BL / 6N strain. All mice were breastfed for 4 weeks and then fed a normal diet until 19 weeks of age.

[0088] Inguinal fat, epididymal fat, and liver were collected from mice. RNA was extracted from the tissues using an RNA extraction kit (Axygen, AP-MD-MS-RNA), and then quantitative PCR was performed on HERP mRNA from the inguinal fat, epididymal fat, and liver using a real-time quantitative PCR detection kit (Vazyme, Q226). Specific procedures are detailed in the kit instructions. The primers used are as follows:

[0089] b-actin-F: 5′-GCCATGTACGTAGCCATCCA-3′

[0090] b-actin-R: 5′-ATGTCACGCACGATTTCCCT-3′

[0091] HERP-F: 5′-GCCAGAAACCAGCACAAAGG-3′

[0092] HERP-R: 5′-GTGTAGCCAGAGAAGCCAGG-3′.

[0093] The results are as follows Figure 2 As shown, compared with the WT group, KO mice showed almost no expression of HERP mRNA in epididymal fat, inguinal fat, and liver. These data confirm the successful knockout of the HERP gene.

[0094] Example 3. Effects of HERP gene knockout on mouse body weight and adipose tissue

[0095] Four groups of mice were set up, with five mice in each group. Two groups were homozygous male mice constructed according to the method in Example 1, and the other two groups were wild-type male mice of the C57BL / 6N strain. One group of KO mice and WT mice were fed a normal diet for 9 weeks starting at week 7, while the remaining group of KO mice and WT mice were fed a high-fat diet (Dietz Company, HF60) for 9 weeks starting at week 7. Sufficient food was provided, and the mice were allowed free access to food. Mouse weight was recorded weekly, and food intake was recorded every three days (food consumed = amount of food given - amount of food remaining). Before the end of the experiment, the mice were fasted for 12 hours, and epididymal adipose tissue, inguinal adipose tissue, and liver were collected and weighed using a 0.01% balance. Immunohistochemistry was used to stain the epididymal adipose tissue with F4 / 80.

[0096] 1. Effects on body weight

[0097] From the weight curve ( Figure 3 As shown in Figure A), when fed a normal diet, the weight gain of HERP knockout mice was comparable to that of WT mice. However, when fed a high-fat diet, compared to the WT high-fat group, the average weight of KO mice was significantly higher in the first week of high-fat feeding, and by week 9 (the end of the experiment), the average weight of KO mice was 23.4% higher than that of the WT high-fat group. Physically, the HERP knockout mice in the high-fat feeding group were significantly larger than the wild-type high-fat feeding group. Figure 3 (C). These findings indicate that HERP knockout mice are more sensitive to nutrition and exhibit more severe obesity symptoms.

[0098] Furthermore, high-fat induction is a method for creating obesity models. It is generally believed that in obese animal models, the body weight of the model group is more than 20% greater than that of the control group. Figure 3 As shown in Figure B, for HERP knockout mice, their body weight was already 27% higher than the normal diet group at week 5 of high-fat feeding, while there was no significant difference between the high-fat feeding group and the normal diet group in WT mice at this time. This indicates that obesity occurs earlier after HERP knockout. At week 9 of high-fat feeding, the HERP knockout mice in the high-fat feeding group gained 52% more body weight than the normal diet group, while the WT mice in the high-fat feeding group only gained 16% more body weight than the normal diet group, not yet reaching the standard for obesity. This suggests that HERP knockout mice are easier to model and have more stable model characteristics.

[0099] 2. Effects on adipose tissue

[0100] A characteristic of obesity is the expansion of adipose tissue. Disorders of adipose tissue function, such as inflammation and excessive differentiation of adipocytes, further exacerbate obesity and peripheral metabolic disorders. Figure 4 As observed in the AD study, regardless of whether the diet was normal or high-fat, HERP knockout led to an expansion of white adipose tissue and an increased fat-to-body-weight ratio in mice. F4 / 80 is an indicator of inflammation. From... Figure 4 The EF analysis showed that, compared with WT mice, HERP knockout mice fed a normal diet exhibited mild F4 / 80 positivity, indicating mild inflammation in their adipose tissue. However, when fed a high-fat diet, the inflammation in the adipose tissue of HERP knockout mice was more severe compared to the WT group. This suggests that inflammation in the adipose tissue of HERP knockout mice further exacerbates the development of obesity.

[0101] Example 4. Effects of HERP gene knockout on glucose and lipid metabolism in mice

[0102] Four groups of mice were set up, with five mice in each group. Two groups were homozygous male mice constructed according to the method in Example 1, and the other two groups were wild-type male mice of the C57BL / 6N strain. One group of KO mice and WT mice were fed a normal diet (Dietz, LF10C) for 12 weeks starting at week 7, while the remaining group of KO mice and WT mice were fed a high-fat diet (Dietz, HF60) for 12 weeks starting at week 7. Fasting blood glucose was measured in the mice at week 10. Blood was collected from the mice at week 12, and the supernatant was obtained by centrifugation for lipid analysis.

[0103] 1. Blood lipids

[0104] Serum samples were collected from the four groups of mice in Example 4, and the levels of free fatty acids (Solepro, BC0595), total cholesterol (Nanjing Jiancheng, A111), and low-density lipoprotein (Nanjing Jiancheng, A113) in the serum were detected using the corresponding kits. Specific detection protocols were followed according to the kit instructions.

[0105] Experimental results are as follows Figure 5 As shown, when fed a normal diet, compared with the WT group, HERP knockout mice had higher levels of free fatty acids and low-density lipoprotein, but no significant difference in total cholesterol levels. However, when fed a high-fat diet, compared with the WT group, HERP knockout mice showed significantly increased levels of free fatty acids, low-density lipoprotein, and total cholesterol. These results indicate that HERP knockout mice are nutritionally sensitive and exhibit lipid metabolism disorders.

[0106] 2. Fasting blood glucose

[0107] For the four groups of mice in Example 4, blood glucose levels were measured at week 10 of either a normal diet or a high-fat diet. The specific protocol was as follows: mice were fasted for 12 hours, and blood glucose (fasting blood glucose) from tail tip blood was measured using a blood glucose meter (Roche).

[0108] Experimental results are as follows Figure 6 As shown, when fed a normal diet, HERP knockout had no significant effect on fasting blood glucose levels compared to the WT group. However, when fed a high-fat diet, HERP knockout led to elevated fasting blood glucose levels in mice compared to the WT group. These results indicate that HERP knockout mice are nutritionally sensitive and exhibit disordered glucose metabolism.

[0109] Implementation Case 5. Effects of HERP gene knockout on mouse liver

[0110] For the four groups of mice in Example 4, their livers were collected, photographed, and then frozen sections and stained with Oil Red O.

[0111] Experimental results are as follows Figure 7 As shown, when fed a normal diet, HERP knockout resulted in mild fat accumulation in mice compared to the WT group. However, when fed a high-fat diet, HERP knockout resulted in more severe fat accumulation in mice compared to the WT group. These results indicate that HERP KO mice have severe fatty liver and can be used as a mouse model of fatty liver.

[0112] Example 6. Effects of HERP gene knockout on adipocyte differentiation

[0113] Adipocyte differentiation is a crucial step in the development and progression of obesity. Abnormal adipocyte differentiation leads to obesity. We isolated and cultured primary adipocytes from the epididymal fat of WT and KO mice. We found that HERP knockout promoted primary adipocyte differentiation. Figure 8(A)

[0114] Furthermore, we constructed HERP-knockout 3T3-L1 cells using CRISPR / Cas9 and then induced them to differentiate into mature adipocytes. We found that HERP knockout promoted preadipocyte differentiation. Figure 8 (B) The specific operation is as follows:

[0115] 1. Design sgRNA1' and sgRNA2', with the following specific sequences:

[0116] sgRNA1': 5'-CTCCGTGTCCTTAAAGTCACTGG-3'

[0117] sgRNA2': 5'-AGCGTCGTCACAGCTATGAAGGG-3'.

[0118] 2. Transform sgRNA and Cas9 enzyme together into 3T3-L1 cells; the specific method is as follows:

[0119] First, the Cas9 enzyme was transfected into 3T3-L1 cells using lipo 3000 (Invitrogen), and then selected with antibiotics for two weeks to obtain 3T3-L1 cells that stably express the Cas9 enzyme.

[0120] sgRNA1 and sgRNA22 were then transfected into 3T3-L1 cells stably expressing Cas9 enzyme via lipo 3000 (Invitrogen) at a molar ratio of 1:1, followed by selection with antibiotics for two weeks. HERP knockout 3T3-L1 cell lines were obtained.

[0121] 3. Inducing differentiation into mature adipocytes; the specific method is as follows:

[0122] After the 3T3-L1 cells reached 100% confluence, they were induced for three days with a triple induction agent (2 μg / ml insulin, 100 ng / ml dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine), and then cultured for another three days in a medium containing 2 μg / ml insulin.

[0123] The above results preliminarily suggest that the reason why HERP knockout leads to obesity in mice may be that it promotes the differentiation of adipocytes.

[0124] Example 7: Construction of a mouse model of obesity and metabolic disorder

[0125] 1. Design sgRNA1 and sgRNA2 targeting exons 2-4 of the HERP gene;

[0126] sgRNA1: 5'-CTCCGTGTCCTTAAAGTCACTGG-3'

[0127] sgRNA2: 5'-AGCGTCGTCACAGCTATGAAGGG-3'

[0128] 2. sgRNA and Cas9 enzyme were co-transferred into C57BL / 6N mouse zygotes;

[0129] 3. The fertilized eggs were transferred into surrogate C57BL / 6N mice to produce F0 generation mice, from which positive F0 generation mice were selected. Positive F0 generation mice were mated with wild-type mice to produce F1 generation mice, from which positive F1 generation mice (i.e., HERP knockout heterozygotes) were selected. Based on the mating of F1 generation mice, homozygous offspring with HERP gene knockout were selected to obtain homozygous HERP gene knockout mice.

[0130] 4. Homozygous HERP gene knockout mice were breastfed for 4 weeks, followed by a normal diet for 3 weeks. From 7 weeks of age, they were fed a high-fat diet (Dietz Company, HF60) for 5 weeks. Sufficient food was provided, allowing mice free access to food. Mouse weight was recorded weekly, and food intake was recorded every three days (food consumed = amount of food given - amount of food remaining). Before the end of the experiment, the mice were fasted for 12 hours, and epididymal adipose tissue, inguinal adipose tissue, and liver were collected and weighed using a 0.01% balance. Immunohistochemistry was used to stain the epididymal adipose tissue with F4 / 80. Results showed that at week 5 of the high-fat diet, the body weight was 27% higher than that of the normal diet group. Further experiments showed that the adipose tissue exhibited phenotypes such as inflammation, dyslipidemia, and glucose metabolism disorders. The mouse obesity and metabolic disorder model was successfully established, taking a total of 12 weeks.

[0131] Example 8: Therapeutic effect of cilostazol on obesity in HERP gene-deficient mice

[0132] Experimental animals: 28 mice with HERP gene deletion (KO) (refer to Example 1) and normal genotype (WT) mice were randomly divided into WT solvent group (WT+Veh), WT drug administration group (WT+Cilo), KO solvent group (KO+Veh), and KO drug administration group (KO+Cilo), with 5 mice in each group.

[0133] Administration method: All mice were fed a high-fat diet at 7 weeks of age (refer to Example 3) until the end of the experiment. Mice in the treatment group were given cilostazol (30 mg / kg) intraperitoneally starting from the third week of high-fat feeding (i.e., when the mice were 10 weeks old), while mice in the control group were given an equal volume of solvent (corn oil). The administration was twice a week for 7 consecutive weeks, and then the experiment ended.

[0134] Observation indicators: Mouse body weight was recorded weekly, and food intake was recorded every three days (food consumed = amount of food fed - amount of food remaining). Before the end of the experiment, mice were fasted for 12 hours, and epididymal adipose tissue, inguinal adipose tissue, and liver were collected and weighed using a 0.01% balance. Serum was collected for lipid analysis. Immunohistochemistry was used to stain the epididymal adipose tissue with F4 / 80.

[0135] 1. Effects on body weight

[0136] Figure 9 The results showed that cilostazol had no significant effect on weight gain in wild-type mice, but it delayed weight gain in HERP-deficient mice. At week 3 of administration, the body weight of the KO-treated group was significantly lower than that of the KO-treated group. At week 7 of administration, the body weight of the KO-treated group was reduced by 13.8% compared to the KO-treated group.

[0137] 2. Improvement of adipose tissue function

[0138] One characteristic of obesity is the expansion of adipose tissue. Disorders of adipose tissue function, such as inflammation and excessive differentiation of adipocytes, can further exacerbate obesity and peripheral metabolic disorders. Figure 10 The results showed that cilostazol had no significant effect on the expansion of white adipose tissue in wild-type mice, but it could inhibit the expansion of white adipose tissue (epididymal fat) in HERP knockout mice and reduce the fat-to-body weight ratio.

[0139] F4 / 80 is an indicator of inflammation. Figure 10 As shown in Figure B, cilostazol had no significant effect on adipose tissue inflammation in wild-type mice, but it could alleviate adipose tissue inflammation in HERP knockout mice.

[0140] The above results indicate that cilostazol can improve adipose amplification and adipose tissue inflammation caused by HERP deficiency.

[0141] Example 9: Therapeutic effect of cilostazol on lipid dysregulation in HERP gene-deficient mice

[0142] Serum samples were collected from the four groups of mice in Example 8, and the levels of free fatty acids (Solepro, BC0595), total cholesterol (Nanjing Jiancheng, A111), and low-density lipoprotein (Nanjing Jiancheng, A113) in the serum were detected using the corresponding kits. Specific detection protocols were followed according to the kit instructions.

[0143] Experimental results are as follows Figure 11 As shown, cilostazol can simultaneously reduce free fatty acid, low-density lipoprotein, and total cholesterol levels in both wild-type mice and HERP gene knockout mice. This indicates that cilostazol can improve lipid metabolism disorders caused by HERP gene deletion.

[0144] Example 10: Cilostazol inhibits adipocyte differentiation in HERP gene-deficient mice

[0145] For the four groups of mice in Example 8, after collecting their epididymal adipose tissue, the activation of adipocyte differentiation-related pathways was detected by Western blot.

[0146] The results are as follows Figure 12 As shown, cilostazol can inhibit the expression of key proteins PPARγ, adiponectin, and ACC in the adipose tissue of HERP knockout mice, but has no significant effect on the protein levels of PPARγ, adiponectin, and ACC in normal genotype mice. This indicates that cilostazol can inhibit adipocyte differentiation in HERP-deficient mice.

[0147] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for constructing a mouse model, characterized in that: The HERP gene in mice was knocked out using sgRNA. The resulting HERP knockout mice were fed a high-fat diet to obtain mouse models with any one or more of the following characteristics: I. obesity; II. disordered glucose and lipid metabolism; III. fatty liver. The sgRNA mentioned is an sgRNA that targets exons 2-4 of the HERP gene.

2. The method for constructing a mouse model according to claim 1, characterized in that: The sgRNAs mentioned include sgRNA1 and sgRNA2; the sequences of sgRNA1 and sgRNA2 are shown below: sgRNA1: 5'-CTCCGTGTCCTTAAAGTCACTGG-3' sgRNA2: 5'-AGCGTCGTCACAGCTATGAAGGG-3'; The high-fat feeding refers to feeding rats with a high-fat diet containing 60 kcal of fat calories. The high-fat feeding period is at least 5 weeks.

3. The method for constructing a mouse model according to claim 1 or 2, characterized in that: The method of knocking out the HERP gene in mice using sgRNA includes the following steps: S1. Design sgRNA1 and sgRNA2 targeting the HERP gene; S2. sgRNA1, sgRNA2 and Cas9 enzyme were co-transferred into mouse zygotes; S3. The fertilized egg is transplanted into a surrogate mouse to produce F0 generation mice, from which positive F0 generation mice with HERP gene knockout are selected. S4. Cross the positive F0 generation mice with wild-type mice to produce F1 generation mice, and select positive F1 generation mice from them. S5. Based on the cross-pairing of positive F1 generation mice, homozygous offspring with HERP gene knockout are screened to obtain homozygous mice with HERP gene knockout.

4. The use of the mouse model obtained by the construction method according to any one of claims 1-3 in screening drugs that have therapeutic effects on any one or more of the following diseases: I. Obesity; II. Fatty liver.

5. The use of cilostazol or a pharmaceutically acceptable salt thereof in the preparation of a drug for reducing the weight of obese mice with HERP gene deficiency.

6. The application according to claim 5, characterized in that: The pharmaceutically acceptable salt has an anion that is either inorganic or organic; the inorganic anion is chloride, bromide, iodide, sulfate, nitrate, nitrite, phosphate, or hydrogen phosphate; the organic anion is acetate, propionate, cinnamate, benzosulfonate, citrate, lactate, or gluconate. The drug also includes any one or more of a carrier and an adjuvant.

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