Application of FGF10 (fibroblast growth factor 10) in preparation of medicine for treating obesity

Abstract

The invention provides an application of FGF10 (fibroblast growth factor 10) in preparation of a medicine for treating obesity. Experiments prove that the FGF10, especially the adeno-associated virus carrying the FGF10, can improve abnormal glucose tolerance, relieve energy and glucose metabolism disorders, leptin and insulin resistance and relieve pathological changes of obese patients, and a new strategy is provided for treatment of obesity or obesity-related diseases, especially gene therapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of FGF10 in the preparation of drugs for treating obesity. Background Technology

[0002] In recent years, the global prevalence of obesity has continued to rise, posing a serious threat to public health and the global economy.

[0003] Obesity refers to a significant degree of overweight and excessive fat accumulation, a condition caused by excessive buildup of body fat, especially triglycerides, and is a chronic metabolic disease. Obesity can cause many metabolic-related diseases, such as obesity, metabolic syndrome, and non-alcoholic fatty liver disease, and can lead to insulin resistance, impaired glucose tolerance, dyslipidemia, hepatic steatosis, and impaired liver function.

[0004] Currently, bariatric surgeries, such as Roux-en-Y gastric bypass and vertical sleeve gastrectomy, are among the most effective surgical treatments for obesity. However, these surgeries carry high risks and are expensive, and there are currently few drugs available for treating obesity and related metabolic diseases. Therefore, finding a safe and effective drug is crucial. Summary of the Invention

[0005] In view of this, in order to overcome the shortcomings of the prior art, the present invention is proposed.

[0006] The first aspect of this invention provides the use of FGF10 promoters in the preparation of medicaments for treating and / or preventing obesity or obesity-related diseases.

[0007] In this invention, the FGF10 is fibroblast growth factor 10 (FGF10). The FGF10 promoter can be a human FGF10 promoter or a non-human mammalian FGF10 promoter. Non-human mammals include, but are not limited to, cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.

[0008] In some embodiments, the FGF10 promoter includes one or more of the following: FGF10 itself, substances that upregulate FGF10 expression, substances that enhance the stability of the FGF10 gene or its expression product, and substances that promote FGF10 gene transcription.

[0009] In some implementations, the substances that upregulate FGF10 expression include, but are not limited to, expression constructs that can express FGF10.

[0010] In some implementations, the expression constructs that can express FGF10 include, but are not limited to, plasmids or viruses that can express FGF10.

[0011] In some implementations, the expression constructs capable of expressing FGF10 include, but are not limited to, expression cassettes containing a strong promoter and the FGF10 gene used in homologous recombination or gene editing.

[0012] In some embodiments, the substances that enhance the stability of the FGF10 gene or its expression product include, but are not limited to, protease inhibitors.

[0013] In some implementations, the substances that promote FGF10 gene transcription include, but are not limited to, transcription activators and enhancers.

[0014] In some implementations, the transcription activation complex is recruited to the promoter by using a catalytically inactivated Cas9 protein fused with a transcription activator via a CRISPR-Cas system, and by designing guide RNAs that target the promoter region of the target gene, thereby specifically enhancing the transcription of FGF10.

[0015] In some embodiments, the FGF10 promoter is selected from FGF10 itself or substances that upregulate the expression of FGF10.

[0016] In some embodiments, the expression construct capable of expressing FGF10 comprises a recombinant adeno-associated virus vector with a DIO element and an FGF10 gene expression cassette.

[0017] In this invention, the adeno-associated virus (AAV) consists of a protein capsid and a single-stranded DNA genome.

[0018] The protein capsid of AAV consists of 60 subunits, exhibiting an icosahedral symmetry structure with a diameter of approximately 22 nm, and lacks an envelope. These subunits contain three proteins: VP1, VP2, and VP3, which play crucial roles in viral infection and genome protection. VP1 plays a vital role in viral entry into cells; its unique N-terminal region's phospholipase A (PLA) activity is essential for the transport of the viral genome from endosomal regions to the nucleus and for initiating gene expression. VP2 and VP3 are primarily responsible for maintaining the structural stability of the capsid. The protein capsid is crucial for AAV, protecting the viral genome and mediating viral-host cell interactions. Specific receptor-binding sites on the capsid allow for specific binding to receptors on the host cell surface, determining the infection specificity and host range of AAV. Different serotypes of AAV exhibit differences in the amino acid sequences of their capsid proteins, resulting in varying affinities for different tissues and cells.

[0019] The AAV genome is a linear single-stranded DNA, approximately 4.7 kb in length. At both ends are 145-nucleotide-long inverted terminal repeats (ITRs), which possess a unique secondary structure that forms a T-shaped hairpin structure. This structure is crucial in the AAV life cycle, containing cis-acting elements required for viral replication, packaging, and integration. During viral replication, the ITR provides an initiation site for DNA polymerase, initiating viral genome replication; during packaging, the ITR guides the viral genome to be accurately inserted into the capsid. Two open reading frames (ORFs) in the middle of the genome, the Rep gene and the Cap gene, encode the Rep protein and Cap protein, respectively. The Rep protein participates in viral replication, transcription, and genome integration, while the Cap protein is a major component of the viral capsid.

[0020] In some implementations, the AAV Rep gene is derived from any AAV serotype (including but not limited to AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13 and any variant thereof).

[0021] In some embodiments, the AAV Cap gene may encode a protein capsid from any AAV serotype (including but not limited to AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13 and any variant thereof).

[0022] In this invention, the DIO element is a component that works in conjunction with Cre recombinase to control the expression of the target gene. The DIO element introduces two pairs of lox sites, which, after two rounds of recombination, reach a stable state. Without Cre recombinase, the target gene is not expressed; with Cre recombinase, it induces a reversible rapid flipping of the sequence between the two lox sites. Then, Cre recombinase immediately and irreversibly cleaves the flipped lox sites in the same direction, leaving only the flipped gene and the lox sites, thereby controlling gene expression.

[0023] In some embodiments, the recombinant adeno-associated virus vector with DIO element and FGF10 gene expression cassette includes a promoter.

[0024] In this invention, the term "promoter" generally refers to a deoxyribonucleic acid (DNA) sequence that enables the transcription of a specific gene. A promoter can be recognized by RNA polymerase, which initiates transcription to synthesize RNA. During RNA synthesis, the promoter can interact with transcription factors that regulate gene transcription, controlling the initiation time and extent of gene expression (transcription).

[0025] In some embodiments, the promoters include, but are not limited to, the CAG promoter (hybrid CMV early enhancer, also known as the CAGGS promoter, CB promoter, or CBA promoter), β-actin promoter, small CBA (smCBA) promoter, CBS promoter or CBh promoter, elongation factor 1a short (EFS) promoter, elongation factor 1a (EF1a) promoter, CMV promoter, PGK promoter, UBC promoter, GUSB promoter, UCOE promoter, VMD2 (also known as BEST1) promoter, OPEFS promoter, CYP4V2 autopromoter, RPE65 promoter, or hybrids or derivatives thereof.

[0026] In some implementations, the promoter is derived from a human or a non-human mammal. For example, the promoter may be derived from, but is not limited to, cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.

[0027] In some implementations, the promoter is the human EF1a promoter.

[0028] In some embodiments, the recombinant adeno-associated virus vector with DIO element and FGF10 gene expression cassette also includes a tag protein.

[0029] In some implementations, the tag protein includes HA tag protein, GFP tag protein, and RFP tag protein.

[0030] In some implementations, the tag protein is selected from HA tag proteins.

[0031] In some implementations, the tag protein also includes an enhanced fluorescent protein.

[0032] In some implementations, the enhanced fluorescent protein includes EGFP.

[0033] In one specific embodiment, the recombinant adeno-associated virus vector with a DIO element and an FGF10 gene expression cassette comprises AAV2 / 1x2-hEF1a-DIO-FGF10-3*HA-IRES-EGFP-WPRE-pA. The AAV2 / 1x2-hEF1a-DIO-FGF10-3*HA-IRES-EGFP-WPRE-pA includes AAV2 / 1x2, an hEF1a promoter, a DIO element, a nucleotide sequence encoding FGF10, three HA tag proteins, an internal ribosome entry site IRES, enhanced green fluorescent protein EGFP, a post-transcriptional regulatory element WPRE, and a polyadenylation signal pA.

[0034] In this invention, AAV2 / 1x2 represents a viral vector using an AAV2 genomic backbone and a mixture of AAV1 and AAV2 capsids packaged in a 1:1 ratio.

[0035] In this invention, the nucleotide sequence encoding FGF10 is the nucleotide series shown in Gene ID: 14165, NM_008002.4 CDS, totaling 630 bp.

[0036] In some embodiments, the expression construct capable of expressing FGF10 further includes a recombinant adeno-associated virus vector with Cre elements.

[0037] In some implementations, the recombinant adeno-associated virus vector with Cre elements includes a promoter.

[0038] In some embodiments, the promoters include, but are not limited to, the CAG promoter (hybrid CMV early enhancer, also known as the CAGGS promoter, CB promoter, or CBA promoter), β-actin promoter, small CBA (smCBA) promoter, CBS promoter or CBh promoter, elongation factor 1a short (EFS) promoter, elongation factor 1a (EF1a) promoter, CMV promoter, PGK promoter, UBC promoter, GUSB promoter, UCOE promoter, VMD2 (also known as BEST1) promoter, OPEFS promoter, CYP4V2 autopromoter, RPE65 promoter, Rax promoter, or hybrids or derivatives thereof.

[0039] In some implementations, the promoter is the Rax promoter.

[0040] In some embodiments, the recombinant adeno-associated virus vector with Cre elements also includes a tag protein.

[0041] In some implementations, the tag protein includes HA tag protein, GFP tag protein, and RFP tag protein.

[0042] In some implementations, the tag protein is selected from GFP tag proteins.

[0043] In one specific embodiment, the recombinant adeno-associated virus vector with the Cre element comprises AAV2 / 1x2-Rax-Cre-GFP. The AAV2 / 1x2-Rax-Cre-GFP comprises AAV2 / 1x2, a Rax promoter, a Cre element, and a GFP tag.

[0044] In some embodiments, the titer ratio of the recombinant adeno-associated virus vector with DIO element and FGF10 gene expression cassette to the recombinant adeno-associated virus vector with Cre element is (15-30):(3-10).

[0045] In one specific implementation, the titer ratio of the recombinant adeno-associated virus vector with the DIO element and the FGF10 gene expression cassette to the recombinant adeno-associated virus vector with the Cre element is 20:7.

[0046] In some implementations, the obesity-related diseases include hepatic steatosis, elevated liver function indicators, elevated total cholesterol, elevated triglycerides, adipose tissue accumulation, leptin resistance, insulin resistance, impaired glucose tolerance, fatty liver, and diabetes.

[0047] In one specific implementation, the obesity-related disease is selected from leptin resistance, insulin resistance, and impaired glucose tolerance.

[0048] In some embodiments, the drug comprises a therapeutically and / or preventively effective amount of an FGF10 promoter.

[0049] In this invention, the effective dose for treatment and / or prevention refers to the range of FGF10 promoter doses used to produce the desired effect in order to treat or prevent the occurrence of a disease.

[0050] In some implementations, the drug is administered via injection, inhalation, cavity, mucosa, or skin.

[0051] In one specific implementation, the drug is administered by injection.

[0052] In one specific implementation, the injection site is within the ventricle.

[0053] In one specific implementation, the injection site is the third ventricle.

[0054] In this invention, the term "treatment" generally refers to a clinical intervention used to alter the natural processes of a subject in a clinicopathological process. This may include improving or alleviating a pathological condition, eliminating lesions, or improving prognosis.

[0055] In this invention, the term "prevention" generally refers to the preventive administration of a drug to a healthy subject to prevent the occurrence of a disease or condition. "Prevention" generally does not require the complete elimination of the possibility of a disease or condition occurring; in other words, "prevention" generally means that the probability of a disease or condition occurring is reduced in the presence of the administered drug. In some cases, "prevention" may also mean the complete elimination of the possibility of a disease or condition occurring.

[0056] In some implementations, the subjects may include humans and non-human mammals. For example, the subjects may include, but are not limited to, cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.

[0057] A second aspect of the present invention provides a recombinant adeno-associated virus vector, the recombinant adeno-associated virus vector comprising a recombinant adeno-associated virus vector with a DIO element and an FGF10 gene expression cassette and a recombinant adeno-associated virus vector with a Cre element.

[0058] In some embodiments, the recombinant adeno-associated virus vector with DIO element and FGF10 gene expression cassette includes a promoter.

[0059] In some embodiments, the promoters include, but are not limited to, the CAG promoter (hybrid CMV early enhancer, also known as the CAGGS promoter, CB promoter, or CBA promoter), β-actin promoter, small CBA (smCBA) promoter, CBS promoter or CBh promoter, elongation factor 1a short (EFS) promoter, elongation factor 1a (EF1a) promoter, CMV promoter, PGK promoter, UBC promoter, GUSB promoter, UCOE promoter, VMD2 (also known as BEST1) promoter, OPEFS promoter, CYP4V2 autopromoter, RPE65 promoter, or hybrids or derivatives thereof.

[0060] In some implementations, the promoter is derived from a human or a non-human mammal. For example, the promoter may be derived from, but is not limited to, cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.

[0061] In some implementations, the promoter is the human EF1a promoter.

[0062] In some embodiments, the recombinant adeno-associated virus vector with DIO element and FGF10 gene expression cassette also includes a tag protein.

[0063] In some implementations, the tag protein includes HA tag protein, GFP tag protein, and RFP tag protein.

[0064] In some implementations, the tag protein is selected from HA tag proteins.

[0065] In some implementations, the tag protein also includes an enhanced fluorescent protein.

[0066] In some implementations, the enhanced fluorescent protein includes EGFP.

[0067] In one specific embodiment, the recombinant adeno-associated virus vector with a DIO element and an FGF10 gene expression cassette comprises AAV2 / 1x2-hEF1a-DIO-FGF10-3*HA-IRES-EGFP-WPRE-pA. The AAV2 / 1x2-hEF1a-DIO-FGF10-3*HA-IRES-EGFP-WPRE-pA includes AAV2 / 1x2, an hEF1a promoter, a DIO element, a nucleotide sequence encoding FGF10, three HA tag proteins, an internal ribosome entry site IRES, enhanced green fluorescent protein EGFP, a post-transcriptional regulatory element WPRE, and a polyadenylation signal pA.

[0068] In this invention, AAV2 / 1x2 represents a viral vector using an AAV2 genomic backbone and a mixture of AAV1 and AAV2 capsids packaged in a 1:1 ratio.

[0069] In this invention, the nucleotide sequence encoding FGF10 is the nucleotide series shown in Gene ID: 14165, NM_008002.4 CDS, totaling 630 bp.

[0070] In some implementations, the recombinant adeno-associated virus vector with Cre elements includes a promoter.

[0071] In some embodiments, the promoters include, but are not limited to, the CAG promoter (hybrid CMV early enhancer, also known as the CAGGS promoter, CB promoter, or CBA promoter), β-actin promoter, small CBA (smCBA) promoter, CBS promoter or CBh promoter, elongation factor 1a short (EFS) promoter, elongation factor 1a (EF1a) promoter, CMV promoter, PGK promoter, UBC promoter, GUSB promoter, UCOE promoter, VMD2 (also known as BEST1) promoter, OPEFS promoter, CYP4V2 autopromoter, RPE65 promoter, Rax promoter, or hybrids or derivatives thereof.

[0072] In some implementations, the promoter is the Rax promoter.

[0073] In some embodiments, the recombinant adeno-associated virus vector with Cre elements also includes a tag protein.

[0074] In some implementations, the tag protein includes HA tag protein, GFP tag protein, and RFP tag protein.

[0075] In some implementations, the tag protein is selected from GFP tag proteins.

[0076] In one specific embodiment, the recombinant adeno-associated virus vector with the Cre element comprises AAV2 / 1x2-Rax-Cre-GFP. The AAV2 / 1x2-Rax-Cre-GFP comprises AAV2 / 1x2, a Rax promoter, a Cre element, and a GFP tag.

[0077] In some embodiments, the titer ratio of the recombinant adeno-associated virus vector with DIO element and FGF10 gene expression cassette to the recombinant adeno-associated virus vector with Cre element is (15-30):(3-10).

[0078] In some embodiments, the titer ratio of the recombinant adeno-associated virus vector with the DIO element and the FGF10 gene expression cassette to the recombinant adeno-associated virus vector with the Cre element is 20:7.

[0079] In some embodiments, the titer of the recombinant adeno-associated virus vector with the DIO element and the FGF10 gene expression cassette is (15-30) × 10⁻⁶. 11 vg / mL.

[0080] In some embodiments, the titer of the recombinant adeno-associated virus vector containing the DIO element and the FGF10 gene expression cassette is 20 × 10⁻⁶. 11 vg / mL.

[0081] In some embodiments, the titer of the recombinant adeno-associated virus vector with the Cre element is (3-10) × 10⁻⁶. 11 vg / mL.

[0082] In some embodiments, the titer of the recombinant adeno-associated virus vector with the Cre element is 7 × 10⁻⁶. 11 vg / mL.

[0083] A third aspect of the present invention provides a cell comprising the recombinant adeno-associated virus vector described in the second aspect of the present invention.

[0084] In this application, the term "cell" includes a single cell, a cell line or cell culture, or its cell progeny. Progeny may not necessarily be identical to the original parent cell due to natural, accidental, or intentional mutations. Cells may include cells transfected in vitro using the vector described in this application. Cells may be bacterial cells (e.g., *Escherichia coli*), yeast cells, or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, and NSO cells.

[0085] A fourth aspect of the present invention provides a pharmaceutical composition for treating and / or preventing obesity or obesity-related diseases, the pharmaceutical composition comprising the recombinant adeno-associated virus vector described in the second aspect of the present invention or the cells described in the third aspect of the present invention.

[0086] In some embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipients.

[0087] In some embodiments, the pharmaceutically acceptable carrier and / or excipients include, but are not limited to, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or preservatives.

[0088] In some embodiments, the acceptable carriers and / or excipients of the pharmaceutical composition are non-toxic to the subject at the doses and concentrations used. In some cases, the pharmaceutical composition includes, but is not limited to, liquid, freeze-dried, or lyophilized compositions.

[0089] In some embodiments, the pharmaceutical composition is administered by injection, inhalation, cavity, mucosa, or skin.

[0090] In one specific implementation, the drug is administered by injection.

[0091] In one specific implementation, the injection site is within the ventricle.

[0092] In one specific implementation, the injection site is the third ventricle.

[0093] The advantages and beneficial effects of this invention are as follows:

[0094] This invention provides the application of FGF10 in the preparation of drugs for treating obesity. Through experiments, this invention demonstrates that FGF10, especially adeno-associated viruses carrying FGF10, can improve glucose tolerance, alleviate energy and glucose metabolism disorders, leptin and insulin resistance, and reduce pathological changes in obese patients, providing a new strategy for the treatment of obesity or obesity-related diseases, particularly gene therapy. Attached Figure Description

[0095] Figure 1This section describes the biological characteristics of metabolic dysfunction in mice after high-fat diet-induced obesity. A: Weight gain over 12 weeks in the normal diet group and the high-fat diet group; B: Comparison of average daily energy intake between the normal diet group and the high-fat diet group; C: Glucose tolerance curves for the normal diet group and the high-fat diet group; DE: Oxygen consumption curves (D) and average oxygen consumption (E) at different time points for the normal diet group and the high-fat diet group; FG: Carbon dioxide production curves (F) and average carbon dioxide production (G) at different time points for the normal diet group and the high-fat diet group; HI: Energy consumption curves (H) and average energy consumption (I) at different time points for the normal diet group and the high-fat diet group. Normal diet group: blue; High-fat diet group: red. *: P < 0.05; **: P < 0.01;

[0096] Figure 2 This graph shows the results of leptin and insulin resistance in mice after obesity induced by a high-fat diet. A: Quantitative analysis of serum leptin in the normal diet group and the high-fat diet group; B: Quantitative analysis of serum insulin in the normal diet group and the high-fat diet group. Normal diet group: blue; High-fat diet group: red. *: P < 0.05; **: P < 0.01;

[0097] Figure 3 This image shows the systemic pathological changes in mice after obesity induced by a high-fat diet. A: HE staining of the liver in the normal diet group and the high-fat diet group; B: Quantitative analysis of the grade of steatosis in liver cells in the normal diet group and the high-fat diet group; C: HE staining of white adipose tissue cells in the normal diet group and the high-fat diet group; D: Quantitative analysis of the area of ​​white adipose tissue cells in the normal diet group and the high-fat diet group; E: Quantitative analysis of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in mice in the normal diet group and the high-fat diet group; F: Quantitative analysis of serum triglycerides and cholesterol in mice in the normal diet group and the high-fat diet group. Normal diet group: blue; High-fat diet group: red. *: P < 0.05; **: P < 0.01;

[0098] Figure 4 This image shows the results of decreased FGF10 expression in the hypothalamus caused by a high-fat diet. A: Western blotting images of FGF10 expression levels in the hypothalamus of mice in the normal diet and high-fat diet groups; B: Statistical analysis of FGF10 expression levels in the hypothalamus of mice in the normal diet and high-fat diet groups. Normal diet group: blue; High-fat diet group: red. *: P < 0.05; **: P < 0.01;

[0099] Figure 5This graph shows the results of a single intraventricular injection of FGF10 significantly improving glucose tolerance induced by a high-fat diet. A: Glucose tolerance curves of mice in the high-fat diet control group and the FGF10 intraventricular injection group after 3 days. Control group: red; FGF10 intraventricular injection group: blue; B: Glucose tolerance curves of mice in the high-fat diet control group and the FGF10 intraventricular injection group after 1 week; C: Glucose tolerance curves of mice in the high-fat diet control group and the FGF10 intraventricular injection group after 2 weeks; D: Glucose tolerance curves of mice in the high-fat diet control group and the FGF10 intraventricular injection group after 4 weeks; E: Glucose tolerance curves of mice in the normal diet control group and the FGF10 intraventricular injection group after 3 days; F: Glucose tolerance curves of mice in different intraventricular doses of FGF10 and the high-fat diet control group after 3 days after 3 days; G: Area under the curve (AUC) of the glucose tolerance curves of mice in different intraventricular doses of FGF10 and the high-fat diet control group after 3 days after 3 days. Control group: blue; Intraventricular injection of FGF10 1μg group: red; Intraventricular injection of FGF10 0.5μg group: black; Intraventricular injection of FGF10 0.1μg group: yellow. *: P<0.05; **: P<0.01;

[0100] Figure 6 This is a fluorescence analysis diagram of FGF10 overexpression in elongated cells;

[0101] Figure 7 The biological characteristics of metabolic dysfunction induced by high-fat diet in mice were alleviated by overexpression of FGF10 in elongated cells. A: Glucose tolerance curves of mice in the high-fat diet control group and the elongated cell overexpression of FGF10 group; B: Body weight gain over 12 weeks in the high-fat diet control group and the elongated cell overexpression of FGF10 group; CD: Oxygen consumption curves (C) and average oxygen consumption at different time points (D) in the high-fat diet control group and the elongated cell overexpression of FGF10 group; EF: Carbon dioxide production curves (E) and average carbon dioxide production at different time points (F) in the high-fat diet control group and the elongated cell overexpression of FGF10 group; GH: Energy consumption curves (G) and average energy consumption at different time points (H) in the high-fat diet control group and the elongated cell overexpression of FGF10 group. High-fat diet control group: red; Elongated cell overexpression of FGF10 group: blue. *: P < 0.05; **: P < 0.01;

[0102] Figure 8This is a graph showing the results of FGF10 overexpression in elongated cells alleviating leptin and insulin resistance in mice with high-fat diet-induced obesity. A: Serum leptin quantitative analysis in the high-fat diet control group and the FGF10 overexpression group; B: Serum insulin quantitative analysis in the high-fat diet control group and the FGF10 overexpression group. High-fat diet control group: red; Elongated cell FGF10 overexpression group: blue. *: P < 0.05; **: P < 0.01;

[0103] Figure 9 This image shows the results of how overexpression of FGF10 in elongated cells alleviated the systemic pathological changes in mice after a high-fat diet induced obesity. A: HE staining of the liver in the high-fat diet control group and the FGF10-overexpressing elongated cell group; B: Quantitative analysis of the grade of steatosis in liver cells in the high-fat diet control group and the FGF10-overexpressing elongated cell group; C: HE staining of white adipose tissue cells in the high-fat diet control group and the FGF10-overexpressing elongated cell group; D: Quantitative analysis of the area of ​​white adipose tissue cells in the high-fat diet control group and the FGF10-overexpressing elongated cell group; E: Quantitative analysis of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in mice in the high-fat diet control group and the FGF10-overexpressing elongated cell group; F: Quantitative analysis of serum triglycerides and cholesterol in mice in the high-fat diet control group and the FGF10-overexpressing elongated cell group. High-fat diet control group: red; Elongated cell group: blue. *: P < 0.05; **: P < 0.01. Detailed Implementation

[0104] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0105] Example

[0106] I. Materials and Methods:

[0107] 1. Laboratory animals

[0108] Male SPF-grade 6-8 week old C57BL / 6 mice were purchased from the Animal Experiment Center of Southern Medical University. Under normal conditions, they were provided with unlimited food and water and housed in the SPF-grade standard laboratory animal facility of Southern Medical University's Southern Hospital (environmental humidity maintained at approximately 45-60%, ambient temperature maintained at approximately 22-24℃, daily light and dark cycles each maintained at 12 hours, background noise at 30-45 dB, and circulating purified air). The mice were placed in the experimental environment to acclimatize 3 days prior to the start of the experiment. The conditions for the use and maintenance of the experimental animals complied with the management regulations and guidelines of the Animal Center of Southern Medical University, and all experimental procedures and operations were reviewed by the Ethics Committee of Southern Medical University.

[0109] 2. Monitoring mouse body weight and food intake

[0110] Weigh the food in each cage daily from 3:00 PM to 5:00 PM. Before weighing, zero the electronic scale, pour the food out of the cage and place it on the scale. After the scale reading stabilizes, read the value and record the remaining amount. The difference between the remaining food from the previous day and the remaining food from the current day is the amount of food consumed by each cage of mice. Repeat the measurement three times for each cage and take the average value.

[0111] 3. Analysis of mouse metabolic cages

[0112] The principle of mouse metabolic cage analysis: The metabolic cage system calculates the oxygen consumption and carbon dioxide production of mice during a specific period by detecting the volume of air entering and leaving the cage and the ratio of oxygen to carbon dioxide. Energy consumption is then calculated using a formula. The system continuously monitors the mice's food intake using a gravity sensor in the feeding trough, and monitors their activity level using an infrared system. Experimental procedure: The experimental animal energy metabolism monitoring system is turned on, ensuring all circuits are connected and there is sufficient gas. The system temperature is adjusted to the experimental temperature. The bedding in each channel is changed to ensure adequate feed and water supply. Mice are placed in the energy metabolism monitoring system, one per channel. Mice are numbered according to their channel number and their weight are recorded. Monitoring begins. Mice are monitored in the energy metabolism monitoring system for 48 hours. The first 24 hours are an adaptation phase. Oxygen consumption, carbon dioxide production, food intake, and activity are recorded for the following 24 hours for subsequent analysis. All mice are kept within a normal 12h / 12h light / dark cycle in the energy metabolism monitoring system, with free movement, feeding, and drinking.

[0113] 4. Glucose tolerance test

[0114] The bedding was changed at 6 PM the day before the experiment, and the mice were fasted for 16 hours, with water provided during this period. On the day of the experiment, the experimental environment was kept quiet, and the weight of each mouse was recorded. After calibrating the blood glucose meter, a small incision was made at the tip of the tail of each mouse with sterile scissors, and a suitable amount of blood was gently squeezed out. After removing the first drop of blood with sterile gauze, the fasting blood glucose of the mice was monitored using the blood glucose meter. After all mice had their fasting blood glucose measured, 10% glucose was injected intraperitoneally at a dose of 1 g / kg body weight. Blood glucose was measured at 15, 30, 45, 60, 90, and 120 minutes after intraperitoneal injection using the same blood glucose meter. The data were recorded and compared.

[0115] 5. Mouse perfusion fixation and brain harvesting

[0116] Mice underwent perfusion, fixation, and brain tissue extraction. The specific steps were as follows: Mice were deeply anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution (80 mg / kg). The mice were placed in a supine position with limbs fixed and the abdominal skin maintained at moderate tension. The heart was exposed: the skin and peritoneum were incised along the midline of the abdomen, and the ribs were carefully cut to fully expose the thoracic cavity and heart, avoiding lung damage. Blood was collected: a 1 ml syringe was used to draw a blood sample from the left ventricle for subsequent detection of leptin and insulin in the serum. Perfusion preparation: a purple scalp vein needle was inserted into and fixed in the left ventricle, and a small amount of pre-cooled 4°C saline was injected to inflate the left ventricle. The right atrial appendage was incised, and pre-cooled 4°C saline was perfused into the left ventricle using an automated microinfusion pump until the liver turned white, indicating complete removal of peripheral blood. Fixation was then performed using pre-cooled 4°C paraformaldehyde. Brain tissue extraction: After perfusion, brain tissue was extracted using standard methods for subsequent sectioning.

[0117] 6. Mouse serum leptin / insulin ELISA detection

[0118] According to the kit instructions, serum leptin levels were detected using the leptin ELISA kit, and serum insulin levels were detected using the insulin ELISA kit. The results were then read using a multi-functional microplate reader. The specific steps are as follows: All experimental groups were assigned and equilibrated at room temperature for 2 hours. Calibration wells, sample diluent wells, blank wells, and sample wells were set up. 50 μL of calibrator at different concentrations were added to each calibrator well. 50 μL of sample diluent was added to each sample diluent well. No diluent was added to the blank wells. 50 μL of the sample sample was added to each sample well. Except for the blank wells, 100 μL of biotin antibody was added to each well. The reaction plate was covered with sealing film and incubated at 37°C in a water bath or incubator in the dark for 60 min. The plate was washed 5 times. Except for the blank wells, 100 μL of HRP-labeled avidin was added to each well. The reaction plate was covered with sealing film and incubated at 37°C in a water bath or incubator in the dark for 20 min. The plate was washed 5 times. 100 μL of TMB chromogenic solution was added to all wells. Cover the reaction plate with sealing film and incubate at 37°C in a water bath or incubator in the dark for 15 min. Add 50 μL of stop solution to each well and read the absorbance (OD value) of each well on a 450 nm wavelength microplate reader.

[0119] 7. Immunohistochemistry and immunofluorescence staining of tissue sections

[0120] HE Staining: Dewaxing: Place paraffin sections in a 60°C oven for 2 hours to completely melt the paraffin. Then transfer the sections to a fume hood and treat with xylene twice, 30 minutes each time, to remove the paraffin. Hydration: Immerse the sections sequentially in high- to low-concentration alcohol solutions for hydration, treating each concentration for 5 minutes. Finally, wash the sections three times with PBS buffer, 5 minutes each time, to complete the hydration process. Hematoxylin Staining and Bluing: Immerse the sections in hematoxylin staining solution for 5 minutes, then rinse with tap water. Immerse the sections in hydrochloric acid-alcohol differentiation solution for differentiation, then rinse with tap water. Gradient Dehydration and Eosin Staining: Immerse the sections in eosin staining solution for 10 seconds. Washing and Mounting: Wash the sections three times with PBS buffer, 5 minutes each time, and mount with neutral resin. Observation and Analysis: Observe and photograph under a microscope, and perform quantitative analysis using ImageJ software.

[0121] Immunofluorescence staining: Section washing: Select frozen sections of the appropriate slice, place them in a 6-well plate, and wash three times with PBS phosphate buffer, 5-10 minutes each time. Non-specific antigen blocking: Place the sections in a humidified chamber containing 5% BSA / 0.2% PBS-T buffer and block in a 37°C oven for 1 hour. Primary antibody incubation: Prepare the primary antibody solution, add it to the antibody incubation plate, transfer the sections to the primary antibody, and incubate overnight at 4°C. Secondary antibody incubation: The next day, transfer the sections to 0.2% PBS-T buffer, wash three times, add the corresponding species' secondary antibody, and incubate at 37°C in the dark for 1 hour. DAPI staining and mounting: After incubation, wash three times with 0.2% PBS-T buffer, 5-10 minutes each time. Incubate the slides with DAPI at room temperature in the dark for 5 minutes, wash them, transfer them to a glass slide, wipe off any excess liquid, mount them with an anti-quenching agent, cover them with a coverslip, and finally take images under a laser confocal microscope (Carl Zeiss).

[0122] 8. Tissue protein extraction

[0123] After the postoperative observation period, the experimental animals were deeply anesthetized. After perfusion, brain tissue was removed from the skull and placed in a glass dish containing a small amount of pre-cooled physiological saline. Under a stereomicroscope, the meningeal tissue was carefully separated using the optic chiasm, third ventricle, and pituitary stalk as anatomical markers. The paraventricular nucleus, supraoptic nucleus, part of the hypothalamus, and part of the hippocampus were grasped with microforceps and placed into pre-labeled centrifuge tubes. Brain tissue washing and lysis: The separated brain tissue was washed with PBS buffer three times to remove residual blood. 500 μL of RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors (formulation: 50 mM Tris-HCl, pH 8.0; 1 mM EDTA, pH 8.0; 5 mM DTT; 2% SDS) was added to each centrifuge tube. The ratio of inhibitor to lysis buffer was 1:100. The brain tissue was thoroughly homogenized using a cryo-homogenizer and lysed at 4°C for 30 minutes. The lysed sample was centrifuged at 12,000 rpm for 10 minutes at 4°C. Protein extraction and processing: After centrifugation, the supernatant was carefully aspirated, aliquoted into new centrifuge tubes, and labeled. The protein concentration was determined according to the BCA kit (Beyotime Inc., China) instructions, and diluted to the appropriate concentration with PBS. 5× Loading Buffer was added at a 4:1 ratio and mixed thoroughly. The mixture was heated in a 100°C metal bath for denaturation for 5 minutes, then immediately cooled in an ice-water mixture, centrifuged, aliquoted, and stored at -20°C.

[0124] 9. Protein electrophoresis

[0125] SDS-PAGE (Solid State Discharge-Polypropylene Gel) electrophoresis is used to separate and detect protein samples. SDS-PAGE gels are prepared by adding tissue protein samples to the sample loading wells of the stacking gel, and simultaneously adding protein markers as molecular weight references to appropriate channels. Loading buffer is added to the empty channels on both sides to prevent edge effects. The gel is then mounted in the electrophoresis tank, and electrophoresis buffer (Tris-glycine-SDS buffer) is added. Electrophoresis is performed at a constant voltage of 80 V until the indicator (bromophenol blue) exceeds the stacking gel level. The voltage is then adjusted to 120 V, and electrophoresis continues until the target protein bands are clearly separated. After electrophoresis, the gel is carefully removed, and the gel portion corresponding to the target band is cut off using a gel cutter. A polyvinylidene fluoride (PVDF) membrane (Millipore, USA) is activated with methanol and placed in the transfer apparatus along with the gel. After transfer, the PVDF membrane is blocked at room temperature with freshly prepared 5% fetal bovine serum (BSA) for 1 hour to block non-specific binding. After blocking, the PVDF membrane was incubated overnight with the corresponding primary antibody at 4°C. The next day, the PVDF membrane was washed three times with TBST (Tris-buffered saline containing 0.1% Tween-20), 5 minutes each time. The PVDF membrane was then incubated with a secondary antibody linked to horseradish peroxidase (HRP) for 1 hour at room temperature. After incubation, the PVDF membrane was washed three more times with TBST, 5 minutes each time. The PVDF membrane was stored at 4°C and developed using ECL chemiluminescence.

[0126] 10. Stereotactic microinjection

[0127] Three days prior to the surgery (microinjection), C57 mice were placed in the experimental environment to acclimatize. Glass electrode preparation: One end of the glass electrode was drawn into a thin filament, ensuring a suitable length for accurate delivery to the target brain region without bending. The other end was fixed to the tip of a microinjector, and the syringe was sealed with paraffin oil to prevent leakage. The microinjector with the glass electrode was then mounted on a stereotactic holder, ensuring its stability. The C57 mice were placed in the induction chamber of the small animal anesthesia system and anesthesia was induced using a mixture of 5% isoflurane and air, typically taking 2-3 minutes. When the mice's breathing was stable and they showed no significant response to stimulation, their weight was recorded. The mice's heads were shaved, extending from the line connecting their eyes to behind the occipital protuberance. The mice were placed prone on a stereotactic apparatus, and the anesthetic gas concentration was adjusted to a 1.5-2% isoflurane / air mixture, delivered through a stereotactic mask at a flow rate of 0.5 L / min. The mouse head was stabilized using bilateral ear rods, ensuring consistent ear rod readings, symmetrical and centered head midline, and balanced without lateral deviation. The mouse teeth were stabilized using a mask rod, ensuring anterior and posterior alignment and head stability without slippage. The surgical area was disinfected with 75% alcohol. An incision of approximately 1.5-2 cm was made along the midsagittal line of the skull. Subcutaneous tissue was bluntly dissected to expose the parietal bone. The periosteum was dissected and blood was removed, exposing the bone window area. Under a microscope, the glass electrode tip was moved to the anterior and posterior fontanelles, and coordinates were recorded. The rod height was adjusted to ensure the Z-axis coordinate error of the anterior and posterior fontanelles was within 0.05 mm. Using the anterior fontanelle as the midpoint, 1.5 mm incisions were made to the left and right, recording bone surface coordinates and adjusting the ear rod height to ensure the Z-axis coordinate error was within 0.05 mm. Under a microscope, the glass electrode was moved to the anterior fontanelle and zeroed. According to the Paxinos & Watson mouse stereotactic brain atlas, the relative coordinates of the third ventricle in 7-week-old male C57 mice were (-1.8 mm, 0 mm, -6.0 mm), i.e., 1.8 mm posterior to the anterior fontanelle, 0 mm lateral to it, and 6.0 mm deep. A bone window was created using a miniature handheld cranial drill to expose the brain tissue and superior sagittal sinus. The superior sagittal sinus was used as the Y-axis zeroing point, and the dura mater was gently pried open with a suture needle to avoid obstruction during needle insertion. A glass electrode was positioned in the third ventricle for microinjection. After injection on each side, the needle was left in place for 5 minutes to prevent reflux. After the procedure, the glass electrode was slowly withdrawn, and hemostasis was achieved with sterile cotton balls. Finally, the scalp was sutured. After injection, the injection site was gently pressed to prevent extravasation, and the mouse's response was observed to ensure its normal condition.

[0128] The injection doses of FGF10 protein were 1 μg, 0.5 μg, and 0.1 μg per mouse, with a titer of AAV2 / 1x2-hEF1a-DIO-FGF10-3*HA-IRES-EGFP-WPRE-pA of 20 × 10⁻⁶. 11 The titer of AAV2 / 1x2-Rax-Cre-GFP was 7 × 10⁻⁶ vg / mL. 11 vg / mL, the amount of each of the two carriers was 200 nL.

[0129] II. Analysis of Experimental Results

[0130] 1. Relevant biological characteristics of metabolic dysfunction caused by high-fat diet

[0131] The experimental results for normal diet (CTD) and high-fat diet (HFD) are as follows: Figure 1 As shown, after induction with a high-fat diet, mice exhibited typical metabolic disorder characteristics: a significant increase in energy intake and a significant decrease in energy expenditure. This imbalance in energy directly led to a significant increase in body weight and severe impairment of glucose tolerance.

[0132] 2. Hormonal metabolic disorders and systemic pathological changes following obesity induced by a high-fat diet.

[0133] Biological characteristics of leptin and insulin resistance following a high-fat diet ( Figure 2 It also led to systemic pathological changes ( Figure 3 The study revealed significant fatty degeneration in hepatocytes, accompanied by abnormally elevated serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. Lipid profile analysis showed a significant increase in total cholesterol and triglycerides. Excessive accumulation of adipose tissue was also observed. These findings suggest systemic metabolic pathological changes caused by a high-fat diet.

[0134] 3. High-fat diet-induced obesity leads to a decrease in FGF10 expression in the hypothalamus.

[0135] Experimental results are as follows Figure 4 As shown, a high-fat diet-induced obesity leads to a decrease in FGF10 expression in the hypothalamus.

[0136] 4. A single intraventricular injection of FGF10 can significantly improve glucose intolerance caused by a high-fat diet.

[0137] Mice were injected intracerebroventricularly with FGF10, and their glucose tolerance was assessed at three, one, two, and four weeks post-injection. The results are as follows: Figure 5 A- Figure 5 As shown in E, a single intraventricular (ICV) injection of 3 μg of FGF10 significantly improved glucose intolerance induced by a high-fat diet in mice, and this improvement lasted for at least two weeks.

[0138] The therapeutic effects of different doses of FGF10 on glucose intolerance induced by a high-fat diet were also studied. The mice were divided into three groups: 1 μg, 0.5 μg, and 0.1 μg, with a total weight of approximately 33g. Results ( Figure 5 F- Figure 5 G) indicates that intraventricular injection of 1 μg FGF10 significantly improves glucose intolerance induced by a high-fat diet in mice. This finding suggests that FGF10 has a long-lasting metabolic regulatory effect in the central nervous system, providing important experimental evidence for developing FGF10-based therapeutic strategies for metabolic diseases.

[0139] 5. Overexpression of FGF10 in hypothalamic elongated cells alleviated energy and glucose metabolism abnormalities induced by a high-fat diet.

[0140] After overexpressing FGF10 in hypothalamic elongated cells using AAV2 / 1x2-hEF1a-DIO-FGF10-3*HA-IRES-EGFP-WPRE-pA and AAV2 / 1x2-Rax-Cre-GFP, Figure 6 The mice showed significantly increased energy expenditure, slower weight gain, and improved glucose tolerance, suggesting that overexpression of FGF10 in elongated cells partially reversed the metabolic disorders induced by a high-fat diet by enhancing energy expenditure and improving glucose metabolism. The therapeutic effect of FGF10 overexpression in elongated cells can last for more than 12 weeks, far exceeding the 2-week duration of FGF10 injection. Figure 7 Overexpression of FGF10 in elongated cells improved leptin and insulin resistance induced by a high-fat diet. Figure 8 Overexpression of FGF10 in elongated cells improved systemic pathological changes induced by a high-fat diet, including hepatocellular steatosis, abnormal liver function, elevated blood lipids, and fat accumulation. Figure 9 ).

[0141] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. The use of FGF10 promoters in the preparation of drugs for the treatment and / or prevention of obesity or obesity-related diseases.

2. The application according to claim 1, characterized in that, The FGF10 promoter includes one or more of the following: FGF10 itself, substances that upregulate FGF10 expression, substances that improve the stability of the FGF10 gene or its expression product, and substances that promote FGF10 gene transcription. Preferably, the FGF10 promoter is selected from FGF10 itself or substances that upregulate the expression of FGF10.

3. The application according to claim 2, characterized in that, The substances that upregulate FGF10 expression include expression constructs that can express FGF10; Preferably, the expression construct capable of expressing FGF10 comprises a recombinant adeno-associated virus vector with a DIO element and an FGF10 gene expression cassette; Preferably, the recombinant adeno-associated virus vector with the DIO element and the FGF10 gene expression cassette comprises AAV2 / 1x2-hEF1a-DIO-FGF10-3*HA-IRES-EGFP-WPRE-pA; Preferably, the expression construct capable of expressing FGF10 further includes a recombinant adeno-associated virus vector with Cre elements; Preferably, the recombinant adeno-associated virus vector with Cre element comprises AAV2 / 1x2-Rax-Cre-GFP; Preferably, the titer ratio of the recombinant adeno-associated virus vector with DIO element and FGF10 gene expression cassette to the recombinant adeno-associated virus vector with Cre element is (15-30):(3-10). Preferably, the titer ratio of the recombinant adeno-associated virus vector with DIO element and FGF10 gene expression cassette to the recombinant adeno-associated virus vector with Cre element is 20:

7.

4. The application according to any one of claims 1-3, characterized in that, The obesity-related diseases include hepatic steatosis, elevated liver function indicators, elevated total cholesterol, elevated triglycerides, adipose tissue accumulation, leptin resistance, insulin resistance, impaired glucose tolerance, fatty liver, and diabetes. Preferably, the obesity-related diseases are selected from leptin resistance, insulin resistance, and impaired glucose tolerance.

5. The application according to any one of claims 1-3, characterized in that, The drug contains a therapeutic and / or preventative amount of an FGF10 promoter.

6. The application according to any one of claims 1-3, characterized in that, The drug can be administered via injection, inhalation, cavity, mucosa, or skin. Preferably, the drug is administered by injection; Preferably, the injection site is within the ventricle.

7. A recombinant adeno-associated virus vector, characterized in that, The recombinant adeno-associated virus vectors include recombinant adeno-associated virus vectors with DIO elements and FGF10 gene expression cassettes, and recombinant adeno-associated virus vectors with Cre elements. Preferably, the recombinant adeno-associated virus vector with the DIO element and the FGF10 gene expression cassette comprises AAV2 / 1x2-hEF1a-DIO-FGF10-3*HA-IRES-EGFP-WPRE-pA; Preferably, the recombinant adeno-associated virus vector with Cre element comprises AAV2 / 1x2-Rax-Cre-GFP; Preferably, the titer ratio of the recombinant adeno-associated virus vector with DIO element and FGF10 gene expression cassette to the recombinant adeno-associated virus vector with Cre element is (15-30):(3-10). Preferably, the titer ratio of the recombinant adeno-associated virus vector with DIO element and FGF10 gene expression cassette to the recombinant adeno-associated virus vector with Cre element is 20:

7. Preferably, the titer of the recombinant adeno-associated virus vector containing the DIO element and the FGF10 gene expression cassette is (15-30) × 10⁻⁶. 11 vg / mL; Preferably, the titer of the recombinant adeno-associated virus vector containing the DIO element and the FGF10 gene expression cassette is 20 × 10⁻⁶. 11 vg / mL; Preferably, the titer of the recombinant adeno-associated virus vector with Cre element is (3-10) × 10⁻⁶. 11 vg / mL; Preferably, the titer of the recombinant adeno-associated virus vector with the Cre element is 7 × 10⁻⁶. 11 vg / mL.

8. A cell, characterized in that, The cell contains the recombinant adeno-associated virus vector as described in claim 7.

9. A pharmaceutical composition for treating and / or preventing obesity or obesity-related diseases, characterized in that, The pharmaceutical composition comprises the recombinant adeno-associated virus vector of claim 7 or the cells of claim 8.

10. The pharmaceutical composition according to claim 9, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier and / or excipients.

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