Use of RINL gene and antagonist thereof in treatment of obesity

Abstract

The use of an RINL gene and an antagonist thereof in the treatment of obesity. Specifically provided is the use of an RINL gene or an encoded protein thereof. The RINL gene or the encoded protein thereof is used for one of the following: (1) as a marker for diagnosing, detecting or prognosing obesity; (2) in the preparation of a reagent or kit for diagnosing or detecting obesity; or (3) in the preparation of a drug for treating obesity. RINL-KO significantly improves glucose tolerance and insulin resistance of obese mice, ameliorates the accumulation of subcutaneous fat and visceral fat thereof, alleviates the inflammation of adipose tissue thereof, and greatly improves the health of the mice. Therefore, an RINL antagonist has the potential to efficiently treat obesity. It is proposed for the first time that RINL is used as a new target in the preparation of a drug for treating obesity, which is of great significance for new drug screening, and also provides a new idea for the treatment of obesity.

Description

Use of RINL gene and antagonists thereof in the treatment of obesity

[0001] Cross-reference to Related Applications

[0002] The present disclosure claims priority to Chinese Patent Application No. 2024110399740, filed on July 31, 2024, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present application belongs to the technical field of biological medicine, and specifically relates to the use of RINL gene and antagonists thereof in the treatment of obesity. BACKGROUND

[0004] With the improvement of living standards and changes in lifestyle, obesity has increasingly become a great threat to human health. The main pathological manifestations of obesity are that the percentage of body fat in body weight is too high, and there may also be an increase in fat cells, and it is often accompanied by symptoms of glucose and lipid metabolism imbalance such as hyperglycemia and hyperlipidemia, which can lead to diabetes, etc. At present, the main treatment methods for obesity are drug therapy, surgical operation and life intervention, but all three methods have certain shortcomings. For example, life intervention mainly makes the body's energy expenditure greater than intake through diet control and exercise, but long-term regular life depends on the patient's willpower, and the effect is unstable for many people with weak willpower and is prone to rebound. Surgical treatment mainly reduces the volume of the stomach or fills the stomach with air sacs to enhance satiety, thereby reducing energy intake to achieve therapeutic effect, but it has the problems of high postoperative risk, possible malnutrition, and a small number of people who can accept the treatment. The current drug therapy mainly uses orlistat, chlorocarcinoline and other drugs, which have relatively stable effects, but have large side effects due to lack of good targets, such as orlistat which can cause incontinence. With the development of gene therapy technology and the in-depth understanding of genes related to glucose and lipid metabolism, it is gradually feasible to find new targets at the gene level to treat obesity.

[0005] RINL, which stands for Ras And Rab Interactor Like, is a protein that interacts with Ras and Rab proteins in cells and is involved in various biological processes, particularly in vesicle-mediated transport and regulation of Rab proteins. The protein expressed by RINL can activate the Rab5 subfamily of small GTPases, thereby playing a role in endocytosis. RINL is expressed in various tissues, with higher expression levels in the thymus and spleen. During the experiment, the applicant accidentally found that RINL gene may be related to obesity. Based on this, the present application explores the relationship between RINL gene and obesity and metabolism, and its application potential in obesity drug development. SUMMARY

[0006] In order to solve the problems in the prior art, the purpose of the present disclosure is to provide the use of RINL gene and its antagonist in the treatment of obesity.

[0007] In order to achieve the above-mentioned purpose, the present disclosure adopts the following specific solutions:

[0008] In one aspect, the present disclosure provides the use of RINL gene or its encoded protein, which is: (1) used as a marker for diagnosing, detecting or prognosing obesity; (2) used for preparing a reagent or kit for diagnosing or detecting obesity; or (3) used for preparing a drug for treating obesity.

[0009] In another aspect, the present disclosure provides the use of RINL antagonist in the preparation of a drug for: (1) preventing and / or treating obesity; (2) improving the metabolic capacity of obese patients; (3) preventing and / or reducing the risk of diabetes in obese patients; or (4) improving the immune function of obese patients.

[0010] In another aspect, the present disclosure provides a method for screening a drug for treating and / or preventing obesity, which comprises the following steps: detecting the change of RINL gene expression amount in the body before and after administration.

[0011] In another aspect, the present disclosure provides a RINL antagonist for treating and / or preventing obesity, wherein the RINL antagonist specifically targets the nucleotide sequence shown in SEQ ID NO. 1 or specifically binds to the amino acid sequence shown in SEQ ID NO. 2, and has any one of the following functions:

[0012] (1) inhibiting RINL gene expression;

[0013] (2) deleting or silencing RINL gene;

[0014] (3) knocking out RINL gene;

[0015] (4) antagonizing RINL protein.

[0016] In another aspect, the present disclosure provides a pharmaceutical composition for treating and / or preventing obesity, which comprises a RINL antagonist for inhibiting RINL expression, and optionally a pharmaceutically acceptable carrier or excipient.

[0017] The present disclosure has at least the following advantages:

[0018] The present disclosure establishes high-fat diet-induced mouse obesity, uses gene knockout and gene therapy technology, takes RINL as a target, and reduces the expression of RINL in the whole body to explore the possibility of RINL as a gene therapy target for treating obesity. The results show that RINL-KO can resist obesity caused by high-fat diet, improve the body fat rate of mice, and does not affect the food intake of mice, and can also improve the metabolic capacity of obese mice. In addition, RINL-KO significantly improves the glucose tolerance and insulin resistance of obese mice, improves the accumulation of subcutaneous fat and visceral fat, reduces the inflammation of adipose tissue, and greatly improves the health of mice. Therefore, RINL antagonists have the potential to treat obesity. Therefore, the present application first proposes RINL as a new target for preparing a drug for treating obesity, which has important significance for screening new drugs, and also provides a new idea for the treatment of obesity. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 shows the correlation between the transcription level of human subcutaneous adipose tissue RINL and total cholesterol and triglyceride.

[0020] Figure 2 shows the expression of RINL in an obese mouse model and a mouse adipocyte model.

[0021] Figure 3 shows the effect of siRNA on the expression of RINL in HepG2 cells on cell lipid metabolism.

[0022] Figure 4 shows the body weight changes of male and female mice in different groups within 13 weeks of high-fat feeding.

[0023] Figure 5 shows the metabolic effects of RINL-KO on high-fat diet mice (24 months old).

[0024] Figure 6 shows the OGTT test results of male and female mice in different groups.

[0025] Figure 7 shows the ITT test results of male and female mice in different groups.

[0026] Figure 8 shows the morphologies of various tissues of high-fat diet mice.

[0027] Figure 9 shows the organ weight proportion of male and female mice on high-fat diet.

[0028] Figure 10 shows the HE sections of liver and adipose tissue of male and female mice.

[0029] Figure 11 shows the accumulation of gonadal fat in male and female mice.

[0030] Figure 12 shows the oil red staining section of liver tissue and the fluorescence staining results of adipose tissue.

[0031] Figure 13 shows the results of oxygen consumption rate assay of brown adipose tissue and liver tissue. Pyr + mal, pyruvate and malate, are substrates for mitochondrial complex I, Suc, succinate, is a substrate for mitochondrial complex II, Rot, rotenone, is an inhibitor of mitochondrial complex I, Ant, antimycin, is an inhibitor of mitochondrial complex III, and Asc + TMPD, ascorbic acid and N,N,N',N'-tetramethyl-para-phenylene-diamine, are substrates for mitochondrial complex IV.

[0032] Figure 14 shows the oxygen consumption rate of brown adipose primary cells. Oli, oligomycin, is an inhibitor of ATP synthase, Rot, rotenone, is an inhibitor of mitochondrial complex I, FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, is a mitochondrial uncoupler, Rot, rotenone, is an inhibitor of mitochondrial complex I, and Ant, antimycin, is an inhibitor of mitochondrial complex III.

[0033] Figure 15 shows the transmission electron microscope images of female mouse liver and brown adipose cells (magnification of 4,000x), and the mitochondrial area of brown adipose cells is counted.

[0034] Figure 16 shows the expression levels of heat production and mitochondrial related proteins in brown adipose of female mice detected by WB.

[0035] Figure 17 shows the expression of key heat production protein UCP1 in brown adipose tissue detected by whole tissue immunofluorescence staining of adipose tissue. DETAILED DESCRIPTION

[0036] I. Terminology

[0037] To facilitate the understanding of this disclosure, certain technical and scientific terms are specifically defined below. Unless specifically defined herein, all other technical and scientific terms used in this document concur in meaning with the generally accepted meanings such terms have in the relevant art.

[0038] The articles "a" and "an" as used in this document are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0039] As used herein, "diabetes" refers to a group of disorders that broadly involve the inability to properly regulate the use of glucose or sugar as a result of defects in the production, secretion, or function of the hormone insulin. Abnormal function of insulin leads to abnormal metabolism of carbohydrates, fats, and proteins. Diabetes is divided into two common types. Type 1 or insulin-dependent diabetes mellitus results from an autoimmune reaction that leads to the destruction of the beta islet cells in the pancreas that produce insulin, resulting in a deficiency of insulin throughout the body. Treatment of the disease consists primarily of regular monitoring of blood glucose levels and several injections of insulin per day. Failure to control the insulin dosage can lead to severe hypoglycemia and life-threatening damage to the brain and other functions.

[0040] Type 2 or non-insulin-dependent diabetes mellitus (T2DM) is a more complex disease that typically develops in adults and is associated with glucose-responsive tissues (such as adipose fat tissue, muscle, and liver) developing resistance to the action of insulin. In the early stages of T2DM, the pancreatic islet cells compensate by secreting excess insulin. Without intervention, this leads to dysfunction of the beta islet cells, resulting in decompensation and chronic hyperglycemia. In addition, T2DM can be accompanied by peripheral insulin resistance, in which additional insulin-sensitive cells fail to respond normally. While type 1 diabetes is typically an acute disease that occurs early in life, type 2 diabetes can develop gradually later in life due to a number of factors including genetics and lifestyle. Medications commonly used to treat T2DM fall into several categories: 1) insulin secretagogues that directly stimulate insulin secretion but have the risk of causing hypoglycemia; 2) a dietary insulin secretagogue that enhances glucose-induced insulin secretion but must be taken before each meal; 3) biguanides, including metformin, that reduce glucose produced from digestion; 4) insulin sensitizers, such as the thiazolidinedione derivatives rosiglitazone and pioglitazone, that improve the peripheral response to insulin by modulating the expression of genes of glucose metabolism, but have side effects such as weight gain, edema, and hepatotoxicity; 5) insulin injections, typically required in late T2DM.

[0041] The metabolic nature of type 2 diabetes and the abnormally high blood glucose levels resulting from the condition often lead to the development of symptoms and disorders that affect a wide range of body tissues. Diabetes is associated with a high incidence of obesity, fatty liver disease, hyperlipidemia, fatty liver disease, and GI motility disorders, including gastroparesis and constipation.

[0042] T2DM-associated insulin resistance is often associated with atherosclerosis, obesity, hyperlipidemia, and primary hypertension. This group of abnormal conditions constitutes the "metabolic" or insulin resistance syndrome. In addition, insulin resistance is associated with fatty liver disease, which can lead to chronic inflammation or nonalcoholic steatohepatitis, fibrosis, and cirrhosis. Nonalcoholic fatty liver disease begins with accumulation of triacylglycerol in the liver and is defined as the presence of cytoplasmic lipid droplets in more than 5% of hepatocytes or TAG levels above the 95th percentile of healthy individuals. Both T2DM and fatty liver disease share comorbidities and adversely affect the progression of the respective disease. Type 2 diabetes is a risk factor for progressive liver disease and liver-related death in patients with fatty liver disease, while fatty liver disease can be a marker of cardiovascular risk and mortality in individuals with type 2 diabetes. Nonalcoholic steatohepatitis, a histological subtype of NAFLD characterized by hepatocyte injury and inflammation, is present in approximately 10% of patients with T2DM and is associated with an increased risk of development of cirrhosis and liver-related death.

[0043] Diabetic gastroparesis is a common but serious chronic gastrointestinal disorder defined as the presence of delayed gastric emptying in the absence of a physical obstruction, accompanied by symptoms such as nausea, vomiting, early satiation, bloating, and abdominal pain. Currently, the only FDA-approved drug for the treatment of diabetic gastroparesis is metoclopramide, a dopamine D2 receptor antagonist and 5-HT3 receptor antagonist with weak 5-HT4 agonist activity, which is indicated to reduce symptoms associated with acute and recurrent diabetic gastroparesis for no more than 12 weeks of treatment. However, metoclopramide treatment is associated with significant side effects such as sudden muscle spasms and depression / mood changes.

[0044] As used herein, "RINL" stands for Ras And Rab Interactor Like, a protein involved in various biological processes within the cell, particularly related to vesicle-mediated transport and regulation of Rab proteins. The RINL-expressed protein is capable of activating the Rab5 subfamily of small GTPases, thereby playing a role in endocytosis. RINL is expressed in various tissues, with particularly high levels in the thymus and spleen.

[0045] As used herein, the term "BAT" is Brown Adipose Tissue, referring to a special type of adipose tissue that is primarily responsible for generating heat to maintain body temperature. Unlike White Adipose Tissue (WAT), which is mainly responsible for storing energy, BAT contains a large number of mitochondria and Uncoupling Protein 1 (UCP1), making it play an important role in energy metabolism. BAT is mainly distributed in the interscapular region, neck, axillary region, mediastinum and around the kidney of the human body, especially in infants, while the BAT content in adults is relatively small, accounting for about 1%-2%.

[0046] As used herein, the term "GWAT" is Gonadal White Adipose Tissue, referring to a special type of white adipose tissue that is mainly distributed around the reproductive organs, such as the testes and ovaries. In addition to storing energy, GWAT can secrete some hormones and cytokines, and may also be involved in local immune responses.

[0047] As used herein, the term "Oil Red O staining" is a commonly used chemical detection technique for detecting and quantifying lipids in tissues, especially neutral fats (triglycerides). Oil Red O is a fat-soluble dye that can bind to fatty acid and triglyceride molecules in tissue sections. During the staining process, Oil Red O dye penetrates into cells containing lipids, especially adipocytes, and binds to lipid droplets. Since Oil Red O dye has a red color, when observed under a microscope, cells containing lipids will appear red or pink, while cells without lipids will not be colored.

[0048] The expressions "cell", "cell line", and "cell culture" are used interchangeably and all such designations include the progeny of the original subject cell. Thus, the words "transformant" and "transformed cell" include the primary subject cell and cultures derived therefrom, regardless of the number of transfers. It is also understood that all progeny can not necessarily be identical to the original parent cell, as mutations can occur during proliferation and survival of progeny. Mutated progeny are included so long as they have the same function or biological activity as the original transformed cell for which the screening was performed. Where different designations are intended, this will be clear from the context.

[0049] "Optional" or "optionally" means that the subsequently described event or circumstance can or can not occur, and the description includes situations in which the event or circumstance occurs and situations in which it does not.

[0050] The term "knockout mouse" refers to a mouse that has had a gene inactivated (i.e., "knocked out"). In some embodiments, the gene is inactivated by homologous recombination. In some embodiments, the gene is inactivated by replacement or disruption with an artificial nucleic acid sequence.

[0051] "Administering," "administered," and "administration" when applied to an animal, human, test subject, cell, tissue, organ, or biological fluid means the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, test subject, cell, tissue, organ, or biological fluid. "Administering," "administered," and "administration" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell includes contact of the agent with the cell, as well as contact of the agent with a fluid that is in contact with the cell. "Administering," "administered," and "administration" also mean treatment by an agent, diagnostic, binding composition, or by another cell in vitro and ex vivo, for example. When "treatment" is applied to a human, veterinary, or research subject, it refers to therapeutic treatment, prophylaxis or preventative measures, research, and diagnostic applications.

[0052] "Treatment" means the administration of an internal or external therapeutic agent to a patient having one or more symptoms of a disease, where the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated patient or population, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable extent. The amount of therapeutic agent effective to alleviate any particular symptom of a disease (also referred to as "therapeutically effective amount") can vary according to factors such as the disease state, age, and weight of the patient, and the ability of the drug to elicit a desired effect in the patient. Whether a disease symptom has been alleviated can be assessed by any clinical detection method typically used by a physician or other professional health care provider to assess the severity or progression of the symptom. While embodiments of the disclosure (e.g., therapeutic methods or articles of manufacture) can not be effective in alleviating the target disease symptoms in every patient, it is determined that a statistically significant number of patients should have alleviation of the target disease symptoms according to any statistical test known in the art, such as the Student t-test, the Chi-square test, the U-test according to Mann and Whitney, the Kruskal-Wallis test (H-test), the Jonckheere-Terpstra test, and the Wilcoxon test.

[0053] The term "consisting essentially of or variations thereof, as used throughout this specification and claims, indicates that all the elements or ingredients listed in the composition of matter can be present, and optionally, other elements or ingredients not substantially altering the basic and novel properties of the specified compositions of matter can be present.

[0054] The term "antagonist" refers to an agent that can act at the nucleic acid or protein level to reduce or inhibit the expression or activity of RINL. This includes, but is not limited to, shRNA, siRNA, miRNA, dsRNA, small molecule compounds, stRNA, aptamers, or antibodies or antigen-binding fragments thereof.

[0055] A "vector" is a composition of matter comprising an isolated nucleic acid and is useful for delivering the isolated nucleic acid into a cell. Numerous vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term is also to be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, etc.

[0056] The terms "patient," "subject," "individual," and the like are used interchangeably herein and are intended to include living organisms (e.g., mammals) in which an immune response can be elicited. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.

[0057] The term "kit" is any article of manufacture (e.g., a package or container) comprising at least one reagent (e.g., a therapeutic agent, a probe, a small molecule, etc.) for specifically detecting and / or therapeutically affecting the expression of a marker described herein. The kit can be promoted, distributed, or sold as a whole for performing a method described herein. The kit can comprise one or more reagents necessary for the expression of a composition used in a method described herein. In certain embodiments, the kit can further comprise a reference standard, e.g., a nucleic acid encoding a protein that does not affect or modulate a signal pathway that controls an immune response, cell growth, division, migration, survival, or apoptosis. Those skilled in the art can envision many such control proteins, including but not limited to: common molecular tags (e.g., green fluorescent protein and beta-galactosidase); proteins that are not in a classification that includes any pathway that controls cell growth, division, migration, survival, or apoptosis by reference to Gene Ontology; or ubiquitously present housekeeping proteins. The reagents in the kit can be provided in individual containers, or in a mixture of two or more reagents in a single container. In addition, instructional materials describing the use of the compositions in the kit can be included.

[0058] The term "prognosis" refers to the act of predicting the course and outcome of a disease in advance. More specifically, the course of a disease after treatment can vary depending on the patient's physiological or environmental conditions, and it can be interpreted as all actions that predict the course after treatment in consideration of the patient's overall condition. In terms of the present disclosure, prognosis prediction can be interpreted as predicting the disease-free survival rate of an obese patient by predicting the course and condition after treatment of the obese patient.

[0059] Table 1. Abbreviations

[0060] II. DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

[0061] In one aspect, the present disclosure provides use of a RINL gene or a protein encoded thereby, which: (1) is used as a marker for diagnosing, detecting or prognosing obesity; (2) is used for preparing a reagent or a kit for diagnosing or detecting obesity; or (3) is used for preparing a medicament for treating obesity.

[0062] In another aspect, the present disclosure provides use of a RINL antagonist in the preparation of a medicament for: (1) preventing and / or treating obesity; (2) improving metabolic capacity of an obese patient; (3) preventing and / or reducing the risk of diabetes in an obese patient; or (4) improving immune function of an obese patient.

[0063] In some embodiments, the RINL antagonist specifically targets a nucleotide sequence set forth in SEQ ID NO. 1 or specifically binds to an amino acid sequence set forth in SEQ ID NO. 2, and has any one of the following functions:

[0064] (1) inhibits expression of a RINL gene;

[0065] (2) causes a RINL gene to be deleted or silenced;

[0066] (3) knocks out a RINL gene;

[0067] (4) antagonizes a RINL protein.

[0068] In some embodiments, the prevention and / or reduction of the risk of diabetes is achieved by improving glucose tolerance or improving the degree of insulin resistance.

[0069] In some embodiments, the improvement of immune function of an obese patient is a reduction in inflammation of adipose tissue thereof.

[0070] In some embodiments, the RINL antagonist is selected from the group consisting of shRNA, siRNA, miRNA, dsRNA, stRNA, a small molecule compound, an aptamer, or an antibody or an antigen-binding fragment thereof.

[0071] In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of whole antibody, F(ab)-fragment, F(ab)2-fragment, single chain antibody, chimeric antibody, diabody-construct, synthetic antibody, bispecific single chain antibody, or cross-clonal antibody.

[0072] In some embodiments, the RINL antagonist is an RINL antibody. In some embodiments, the RINL antagonist is an siRNA comprising a sense strand and an antisense strand, the length of which is 15-30 base pairs, the antisense strand comprising a complementary region complementary to mRNA encoding RINL.

[0073] In some embodiments, the nucleotide sequence of the siRNA is selected from any one of the following combinations of sense strand and antisense strand:

[0074] (1) the sense strand having the sequence as shown in SEQ ID NO. 3, and the antisense strand having the sequence as shown in SEQ ID NO. 4; or

[0075] (2) the sense strand having the sequence as shown in SEQ ID NO. 5, and the antisense strand having the sequence as shown in SEQ ID NO. 6; or

[0076] (3) the sense strand having the sequence as shown in SEQ ID NO. 7, and the antisense strand having the sequence as shown in SEQ ID NO. 8.

[0077] In some embodiments, the RINL antagonist is capable of reducing accumulation of subcutaneous fat and visceral fat.

[0078] In some embodiments, the medicament comprises the active ingredient RINL antagonist and a pharmaceutically acceptable carrier or excipient thereof.

[0079] In some embodiments, the content of the RINL antagonist in the medicament is 1% to 99%.

[0080] In another aspect, the present disclosure provides a method for screening a medicament for treating and / or preventing obesity, comprising the step of detecting the change in the expression level of RINL gene in the body before and after administration.

[0081] In another aspect, the present disclosure provides a pharmaceutical composition for treating and / or preventing obesity, comprising a RINL antagonist that inhibits the expression of RINL, and optionally a pharmaceutically acceptable carrier or excipient.

[0082] In some embodiments, the pharmaceutical composition further comprises one or more additional therapeutic components.

[0083] In another aspect, the present disclosure provides a RINL antagonist, wherein the RINL antagonist specifically targets the nucleotide sequence set forth in SEQ ID NO. 1 or specifically binds to the amino acid sequence set forth in SEQ ID NO. 2, and has any one of the following functions:

[0084] (1) inhibits the expression of RINL gene;

[0085] (2) causes the deletion or silencing of RINL gene;

[0086] (3) knocks out RINL gene;

[0087] (4) antagonizes RINL protein.

[0088] In some embodiments, the RINL antagonist is an siRNA, which comprises a sense strand and an antisense strand, and the length of the sense strand and the antisense strand is 15-30 base pairs, and the antisense strand comprises a complementary region complementary to the mRNA encoding RINL.

[0089] In some embodiments, the nucleotide sequence of the siRNA is selected from any one of the following combinations of sense strand and antisense strand:

[0090] (1) the sense strand has the sequence set forth in SEQ ID NO. 3, and the antisense strand has the sequence set forth in SEQ ID NO. 4; or

[0091] (2) the sense strand has the sequence set forth in SEQ ID NO. 5, and the antisense strand has the sequence set forth in SEQ ID NO. 6; or

[0092] (3) the sense strand has the sequence set forth in SEQ ID NO. 7, and the antisense strand has the sequence set forth in SEQ ID NO. 8.

[0093] In another aspect, the present disclosure provides a nucleic acid encoding the aforementioned RINL antagonist.

[0094] In another aspect, the present disclosure provides an expression vector comprising the aforementioned nucleic acid.

[0095] In another aspect, the present disclosure provides a cell comprising the aforementioned nucleic acid and expression vector.

[0096] In another aspect, the present disclosure provides a composition comprising the aforementioned RINL antagonist, and optionally a pharmaceutically acceptable carrier or excipient.

[0097] In another aspect, the present disclosure provides the use of the aforementioned method, the aforementioned RINL antagonist, and / or the aforementioned pharmaceutical composition in the preparation of a medicament for treating and / or preventing obesity in an individual, wherein the medicament is administered to the individual in an effective amount.

[0098] In another aspect, the present disclosure provides a method for treating obesity by administering to a subject a therapeutically effective amount of the aforementioned RINL antagonist and / or the aforementioned pharmaceutical composition.

[0099] Examples

[0100] Further understanding of the present disclosure can be obtained by referring to some specific examples given herein, which are only for illustration of the present disclosure and are not intended to make any limitation on the scope of the present disclosure. Obviously, various modifications and changes can be made to the present disclosure without departing from the essence of the present disclosure, and therefore, such modifications and changes are also within the scope of protection claimed by the present application. The clinical experimental data used in the present disclosure have been obtained with the informed consent of the subjects.

[0101] Example 1: RINL gene is associated with obesity

[0102] Obesity is affected by epigenetic and environmental factors, in order to explore new genetic genes associated with obesity as a therapeutic target for alleviating obesity, the applicant analyzed the transcriptome data of human population in the EGO database, and unexpectedly found that the transcription level of RINL in human subcutaneous adipose tissue was positively correlated with cholesterol and triglyceride in serum. As shown in Figure 1, with the increase of RINL expression, cholesterol and triglyceride have a trend of increasing. Cholesterol and triglyceride as blood lipid indicators reveal that RINL gene may be associated with obesity.

[0103] In order to further demonstrate the relationship between RINL gene and obesity, the applicant used 3T3-L1 adipocyte precursor cell line to induce differentiation of adipocytes, and found that with the increasing degree of lipid accumulation, the expression level of RINL also increased, which proved that the gene was significantly correlated with the degree of obesity. At the same time, C57BL / 6 wild type mice were fed with 60% high-fat feed to construct a high-fat animal model, and a blank control group (i.e. normal diet of mice) was set up, and the expression amount of RINL in epididymal adipose tissue of mice and in vitro adipocyte model was detected, and the results are shown in Figure 2. It was found that compared with the control group, the expression amount of RINL in adipose tissue of obese mice caused by high-fat feeding was significantly increased, which also proved that RINL gene was significantly positively correlated with the degree of obesity and could be used as a target for studying obesity model.

[0104] Example 2: Knocking down the expression of RINL in HepG2 cells using siRNA can alleviate lipid deposition in HepG2 cells

[0105] Considering that the expression level of RINL gene is positively correlated with the degree of obesity, to verify whether knocking down the expression of RINL can alleviate the level of obesity, the applicant first demonstrates in a cell model using HepG2 cells, which are human liver cells and the most common cell model for research on liver diseases. Three groups of siRNAs targeting RINL are designed, and the sequence information is shown in Table 2 below. The siRNA is used to knock down RINL by lipofection method. The HepG2 cells are lysed to collect proteins, and the expression level of RINL in the cells is analyzed by immunoblotting. The results are shown in Figure 3A. The siRNA significantly reduces the expression of RINL in the cells. In addition, after siRINL is used to knock down RINL in HepG2 cells by lipofection method, the cells are subjected to high-fat modeling (treated with 500 μmol / L concentration of oleic acid and 250 μmol / L of palmitic acid for 24 hours), and the triglyceride level in the cells is detected by a triglyceride detection kit (Jizhisheng; AC10179), and the cells are subjected to oil red O staining (Nanjing Jiancheng; D027-1-2) to observe the lipid accumulation. The detection results are shown in Figures 3B and 3C. It can be seen that the treatment of oleic acid and palmitic acid greatly increases the triglyceride content and lipid accumulation in the cells, and knocking down the expression of RINL can alleviate the abnormal increase of triglyceride content and lipid accumulation caused by the treatment of oleic acid and palmitic acid, and has a tendency to return the cells to normal level. That is, the decrease of the expression level of RINL can make HepG2 cells resist the abnormal increase of lipid caused by high-fat modeling, and the low expression of RINL is helpful to alleviate obesity.

[0106] Table 2. Sequence information of siRINL

[0107] Example 3: RINL-KO mice resist obesity caused by high-fat diet

[0108] The conclusion obtained in the cell model needs to be further verified in a mouse model, because the low expression of RINL is associated with resistance to obesity, we constructed a mouse with a whole-body knockout of RINL, and found that the knockout of the gene had no significant effect on the growth and development of the mouse. Eight-week-old female and male mice were selected, and normal female and male mice were selected as 8, and set up as blank control group (WT-NCD), positive control group (WT-HFD) and experimental group (KO-HFD). The positive control group and the experimental group were induced by high-fat diet (i.e. 60% high-fat feed), and the blank group was normally fed. After 13 weeks of high-fat treatment, the body weight change, body fat rate and food intake of the mice in each group were monitored dynamically for 24 hours. The results are shown in Figure 4. After 13 weeks of culture, the body weight of the positive control group of female and male mice increased the fastest, and there was a significant difference with the experimental group of RINL knockout. Similarly, under the condition of basically consistent food intake, the body fat rate of the positive control group of mice was also the highest, especially in female mice, whose body fat rate was nearly 2 times that of the experimental group. The above experiments show that RINL-KO has a significant resistance to the increase in body weight caused by high-fat diet. And it improves the body fat rate of mice, and does not affect the food intake of mice.

[0109] Example 4: RINL-KO improves the metabolic capacity of high-fat diet obese mice

[0110] In order to explore whether RINL knockout can improve the metabolism of mice, this embodiment sets up a positive control group (wild type mice-high fat diet) and an experimental group (RINL gene knockout mice-high fat diet), and uses a 16-channel metabolic monitoring system to monitor the metabolism of mice. The results are shown in Figure 5. Whether it is daytime or nighttime, whether it is female or male, the 24-hour oxygen consumption of RINL knockout mice under high-fat diet is significantly higher than that of wild type mice under high-fat diet. This shows that after RINL gene knockout, the oxygen consumption level of female and male mice is improved, that is, the metabolism of mice is improved, so that the metabolism of mice is more vigorous, and therefore the excessive increase in body weight of mice can be alleviated.

[0111] Example 5: RINL-KO improves the glucose tolerance and insulin resistance of high-fat diet mice

[0112] Sugar metabolism is also an important part of the process of obesity, further, this embodiment explores whether RINL knockout can improve the glucose tolerance of mice. Oral glucose tolerance test (OGTT) is a glucose loading test to understand the function of pancreatic beta cells and the body's ability to regulate blood sugar, which is a commonly used diagnostic test for diagnosing diabetes in clinical practice, and is currently internationally recognized as the gold standard for diagnosing diabetes and glucose regulation abnormalities. On the contrary, if exogenous insulin is given, the blood glucose level is detected within a certain period of time, which is the insulin tolerance test (insulin-induced hypoglycemia test) abbreviated as ITT, and the exogenous insulin is given by intraperitoneal injection; if insulin insensitivity occurs, the blood glucose decreases relatively slowly and rises relatively quickly after insulin injection.

[0113] Female and male mice were selected as blank control group (wild type mice - normal diet), positive control group (wild type mice - high fat diet) and experimental group (RINL knockout mice - high fat diet), and OGTT and ITT tests were performed, respectively, and the results are shown in Figures 6 and 7.

[0114] As shown in Figure 6, compared with the positive control group, RINL-KO significantly improved the glucose tolerance of female mice, although the blood glucose change of RINL-KO male mice was not much different from that of the positive control group, but the area under the blood glucose curve (AUC) showed that RINL knockout improved the overall situation of the change of blood glucose concentration with time.

[0115] As shown in Figure 7, compared with the positive control group, RINL-KO significantly improved the insulin resistance of female and male mice, and similarly, the improvement effect in female mice was better.

[0116] In summary, RINL-KO can improve the glucose tolerance and insulin resistance of mice, that is, it improves the overall health of mice.

[0117] Example 6: RINL-KO improves the accumulation of subcutaneous fat and visceral fat in high-fat diet mice

[0118] After the in vivo experiment of mice, the mice in each group were sacrificed to collect samples, and the molecular indicators of each organ were detected to explore the possible molecular mechanisms. Specifically, after the female and male mice in the blank control group (wild-type mice - normal diet), the positive control group (wild-type mice - high-fat diet) and the experimental group (RINL knockout mice - high-fat diet) were induced by high-fat diet, their individual volume was observed and photographed, then they were sacrificed, and their subcutaneous adipose tissue and organs were taken for analysis of fat content and organ proportion in each group. The results are shown in Figures 8 and 9. The liver, brown adipose tissue and gonadal white adipose tissue of mice in each group were collected for HE section, and the changes in the morphology of each tissue were observed by sectioning and HE staining. The results are shown in Figures 10 and 11.

[0119] As shown in Figure 8, under high-fat diet, the volume of mice in the positive control group was much larger than that of mice in the experimental group, and the lipid accumulation in the subcutaneous inguinal white adipose tissue and the gonadal white adipose tissue was also much higher than that of mice in the experimental group. Similarly, the volume of liver was also larger than that of mice in the experimental group, which may be due to the larger accumulation of visceral fat. The above results show that RINL-KO significantly improves the accumulation of subcutaneous fat and visceral fat in female and male mice.

[0120] Figure 9 shows the weight ratio of each organ in each group of mice, which further shows that RINL-KO significantly improves the proportion of internal organs in female and male mice, i.e. improves the accumulation of visceral fat, and improves the health status.

[0121] Figures 10 and 11 show the pathological observation of each part of the fat of mice in each group, which shows that compared with the control group, the lipid accumulation in the liver, brown fat and white fat of RINL-KO mice under high-fat obesity model is reduced, and the size of fat cells in female mice is reduced more significantly.

[0122] In summary, RINL-KO can improve the accumulation of subcutaneous fat and visceral fat in high-fat diet mice, which helps to maintain the health status of mice.

[0123] Example 7: RINL-KO reduces adipose tissue inflammation caused by obesity in high-fat diet mice

[0124] RINL-KO shows significant improvement in adipose tissue, and we further observed the liver (high-fat diet can induce a significant fatty liver phenotype, and we observed whether RINL-KO can alleviate this phenotype). Oil red O staining (Nanjing Jiancheng; D027-1-2) was performed on the liver of female mice in each experimental group, and F4 / 80 antibody was used to label macrophages in gonadal white adipose tissue for immunofluorescence staining. The results are shown in Figure 12.

[0125] Compared with the positive control group, the lipid ectopic accumulation of the liver of the RINL-KO mouse was reduced, and the liver fat content was close to that of the blank control group. At the same time, the amount of macrophage infiltration in the adipose tissue of the RINL-KO mouse was significantly lower than that of the positive control group mouse, indicating that the knockout of the RINL gene reduced the adipose tissue inflammation caused by obesity in the high-fat diet mouse.

[0126] Example 8: RINL-KO enhances the mitochondrial function of the brown adipose tissue and liver tissue of the high-fat diet mouse

[0127] To further explore the molecular mechanism, we speculate that RINL-KO enhances the metabolism of the mouse, which is closely related to the mitochondrial function of the mouse obesity, so we select the brown adipose tissue and liver tissue of the positive control group (wild-type mouse-high-fat diet) and the experimental group (RINL gene knockout mouse-high-fat diet) to determine the oxygen consumption of each tissue. The oxygen consumption rate of fresh brown adipose tissue and liver tissue homogenate was determined using the mitochondrial function detector Oroboros O2K. By adding the substrates of mitochondrial complexes I and II, the maximum respiratory value was reached, and then the inhibitors of complexes I and III were added to inhibit the mitochondrial respiration, and the difference was the function of the corresponding mitochondrial complex. Finally, the substrate of complex IV was used to directly provide electrons for complex IV to obtain the oxygen consumption of complex IV.

[0128] The experimental results are shown in FIG. 13. The oxygen consumption level of the tissue homogenate of the same weight tissue was determined using O2K. Compared with WT, the BAT and liver part of RINL-KO up-regulated the mitochondrial function, indicating that RINL knockout can enhance the part of the mitochondrial function of the brown adipose tissue and the liver.

[0129] Example 9: RINL-KO enhances the oxygen consumption respiratory rate of the brown adipose tissue primary cells of the high-fat diet mouse

[0130] To further improve the molecular mechanism, we further analyzed the oxygen consumption rate of the brown adipose tissue primary cells of the positive control group (wild-type mouse-high-fat diet) and the experimental group (RINL gene knockout mouse-high-fat diet) at the cellular level using the more detailed cell energy metabolism instrument seahorse XFe24. By adding oligomycin (Oligomycin) to inhibit the ATP synthase activity, and then adding FCCP to inhibit the coupling of ATP synthesis and electron transfer, the oxygen consumption rate reaches the maximum value, and then adding the inhibitors of complexes I and III completely inhibits the overall respiratory function of the mitochondria. The results are shown in FIG. 14. It can be seen that the brown adipose tissue primary cells increase the oxygen consumption respiratory rate after RINL knockout.

[0131] Example 10: RINL-KO increases the mitochondria of the brown adipose tissue cells of the high-fat diet mouse

[0132] In order to more clearly observe the subcellular structure of each lipid metabolism organ, we sent and observed the liver and brown adipose tissue of three groups of female mice, wild type mice-normal diet (NCD-WT), wild type mice-high fat diet (HFD-WT) and RINL KO-high fat diet (HFD-RINL KO) by transmission electron microscopy (the effect of weight loss in female mice is more significant). Through the observation of transmission electron microscopy, the mitochondrial morphology of the liver in the RINL KO group did not change significantly, the mitochondrial density and mitochondrial crista distribution of the brown adipocyte were normal, but the mitochondrial size of the HFD-RINL KO group was significantly increased compared with the HFD-WT group, and the results are shown in Figure 15, and the increase in mitochondrial size is considered to be one of the markers of improving mitochondrial oxygen consumption function.

[0133] Example 11: RINL-KO enhances the expression of brown adipose mitochondrial thermogenic-related proteins in high-fat diet mice

[0134] Based on the above research results, the inventors speculate that knocking out RINL can enhance the mitochondrial thermogenic function of the brown adipose tissue of mice, thereby causing the metabolic rate to increase, so that the RINL knockout mice can resist obesity caused by a high-fat diet. In this embodiment, the mitochondrial-related proteins of the brown adipose tissue are detected for protein content.

[0135] The WB detection results are shown in Figure 16, and it can be seen that the main transcriptional regulator of mitochondrial generation, PGC1a, is significantly up-regulated in the RINL-KO group, the transcription factor PPARa that regulates fatty acid beta-oxidation and the beta-oxidation rate-limiting enzyme CPT1A are significantly increased. At the same time, the expression level of the key gene UCP1 of brown adipose tissue uncoupling thermogenesis is also significantly improved.

[0136] The UCP1 staining of the brown adipose tissue was performed by using the whole tissue immunofluorescence staining method, and the results are shown in Figure 17, and it can be seen that the fluorescence signal of UCP1 in the BAT of the RINL-KO mice is significantly increased.

[0137] In summary, the experimental results show that RINL-KO can resist obesity caused by a high-fat diet, improve the body fat rate of mice, and at the same time, does not affect the food intake of mice, and can also improve the metabolic capacity of obese mice. In addition, RINL-KO significantly improves the glucose tolerance and insulin resistance of obese mice, improves the accumulation of subcutaneous fat and visceral fat, reduces the inflammation of adipose tissue, and greatly improves the health of mice. In addition, RINL-KO enhances the function of mitochondria in brown adipocytes.

[0138] The present application first proposes RINL as a new target for preparing a drug for treating obesity, which has important significance for screening new drugs, and also provides a new idea for the treatment of obesity.

[0139] Any technical features in the above-described embodiments can be combined in any manner, and for the sake of brevity, not all possible combinations are described, however, as long as there is no contradiction, any technical features in the above-described embodiments should be considered to be within the scope of the present disclosure.

[0140] The above-described embodiments only express several implementation manners of the present disclosure, and the description is relatively specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be pointed out that for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present disclosure, and these all belong to the protection scope of the present disclosure. Therefore, the protection scope of the patent of the present disclosure should be subject to the appended claims.

[0141] Table 3. Sequence information

Claims

1. Use of a RINL gene or a protein encoded by the RINL gene, wherein the RINL gene or the protein encoded by the RINL gene: (1) is used as a marker for diagnosing, detecting or prognosing obesity; (2) is used for preparing a reagent or a kit for diagnosing or detecting obesity; or (3) is used for preparing a drug for treating obesity.

2. Use of a RINL antagonist in preparing a drug for: (1) preventing and / or treating obesity; (2) improving metabolic capacity of an obese patient; (3) preventing and / or reducing the risk of diabetes of an obese patient; or (4) improving immune function of an obese patient.

3. Use according to claim 2, wherein, The RINL antagonist specifically targets a nucleotide sequence as shown in SEQ ID NO. 1 or specifically binds to an amino acid sequence as shown in SEQ ID NO. 2, and has any one of the following functions: (1) inhibits expression of the RINL gene; (2) causes deletion or silencing of the RINL gene; (3) knocks out the RINL gene; (4) antagonizes the RINL protein.

4. Use according to claim 2 or 3, wherein, The RINL antagonist is selected from shRNA, siRNA, miRNA, dsRNA, stRNA, a small molecule compound, an aptamer, or an antibody or an antigen-binding fragment thereof.

5. Use according to any one of claims 2-4, wherein, The antibody or the antigen-binding fragment thereof is selected from a whole antibody, a F(ab)-fragment, a F(ab)2-fragment, a single-chain antibody, a chimeric antibody, a diabody-construction, a synthetic antibody, a bispecific single-chain antibody or a cross-clonal antibody.

6. Use according to any one of claims 2-5, wherein, The RINL antagonist reduces accumulation of subcutaneous fat and visceral fat.

7. Use according to any one of claims 2 to 6, wherein, The drug comprises the active ingredient RINL antagonist and a pharmaceutically acceptable carrier or excipient.

8. Use according to any one of claims 2 to 7, wherein, The content of the RINL antagonist in the drug is 1% to 99%.

9. A method for screening a drug for treating and / or preventing obesity, comprising the step of detecting changes in the expression amount of the RINL gene in a body before and after administration.

10. A RINL antagonist for use in the treatment and / or prevention of obesity, wherein, The RINL antagonist specifically targets a nucleotide sequence as shown in SEQ ID NO. 1 or specifically binds to an amino acid sequence as shown in SEQ ID NO. 2, and has any one of the following functions: (1) inhibits expression of the RINL gene; (2) causes deletion or silencing of the RINL gene; (3) knocks out the RINL gene; (4) antagonizes the RINL protein.

11. A pharmaceutical composition for treating and / or preventing obesity, comprising the RINL antagonist of claim 10, and optionally a pharmaceutically acceptable carrier or excipient. Preferably, the pharmaceutical composition further comprises one or more additional therapeutic components.

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