Application of LKB1-PD-1 signal axis in preparation of medicine for diagnosing, preventing and treating type 2 diabetes mellitus

Abstract

The invention discloses application of a biomarker LKB1-PD-1 signal axis as a marker for early diagnosis of obesity and type 2 diabetes mellitus and as a type 2 diabetes mellitus treatment target. The invention also discloses a medicine containing the biomarker, a medicine composition, a detection reagent, a kit, an LKB1-PD-1 signal axis regulator, a mouse model R5 / + Stk11f / f with specific deletion of type-2 intrinsic lymphoid cells (ILC2), a mouse model Id2Cre-ERT2Stk11f / f with ILC2 induced knockout of LKB1 and application of the mouse model. The invention also discloses an application of the biomarker LKB1-PD-1 signal axis in preparation of drugs for diagnosis, prevention and treatment of type 2 diabetes mellitus. The invention has a wide application prospect in the field of diagnosis and treatment of obesity and type 2 diabetes mellitus.

Description

Technical Field

[0001] The present invention belongs to the field of medical research technology and relates to the application of the LKB1-PD-1 signaling axis in the preparation of drugs for the diagnosis, prevention and treatment of type 2 diabetes. Background Art

[0002] Obesity and type 2 diabetes. Obesity refers to a chronic metabolic disease in which excessive accumulation and / or abnormal distribution of body fat results in increased body mass due to multiple factors such as genetic genes, environmental factors, lack of exercise and improper diet. In recent years, the incidence of obesity and related metabolic diseases has been increasing year by year, seriously endangering the quality of life and health level of the people, and becoming a risk factor threatening human health (1-2). Obesity-induced chronic inflammation is closely related to a variety of metabolic diseases, such as atherosclerosis, cardiovascular disease, insulin resistance and type 2 diabetes (3-4). The World Health Organization defines BMI ≥ 30 kg / m 2 Obesity is defined as affecting 100% of adults. According to 2016 statistics, over 1 billion people worldwide suffer from obesity, including 650 million adults, 340 million adolescents, and 39 million children. Obesity, particularly central obesity, is the primary cause of insulin resistance in diabetic patients. Insulin resistance (IR) refers to a decrease in the efficiency of insulin in promoting glucose uptake and utilization in the human body due to various factors and is a common feature of obesity and type 2 diabetes. Studies have shown that adipose tissue inflammation is a key factor in obesity-related insulin resistance and metabolic diseases (5-7). Multiple studies have confirmed a positive correlation between proinflammatory cytokine levels and the development of insulin resistance, while levels of anti-inflammatory cytokines such as IL-10 and IL-4 are positively correlated with insulin sensitivity (8-11). Initial evidence for adipose tissue inflammation came from researchers' discovery that levels of proinflammatory cytokines such as TNF-α, IL-6, and IL-1β in adipose tissue are significantly elevated in obese patients and mouse models, and that blocking inflammatory cytokine signaling can improve insulin sensitivity in mice. Inflammation primarily causes insulin resistance by interfering with the insulin signaling pathway. Under physiological conditions, insulin activates the insulin receptor (IR) by binding to the target cell membrane, promoting the phosphorylation of tyrosine residues of the insulin receptor substrate (IRS), activating the downstream PI3K-AKT pathway, and exerting the biological effect of insulin on glucose metabolism; under inflammatory conditions, the serine protein kinases JNK and IKKβ are activated, serine phosphorylating the inhibitory sites of IR and IRS, weakening the signal transduction of insulin (12). Therefore, the high expression of inflammatory factors and over-activated inflammatory pathways in adipose tissue lead to the occurrence of obesity-related insulin resistance by inhibiting the insulin signaling pathway.

[0003] The basic functions of ILC2. Innate lymphoid cells are a group of innate immune cells that have been discovered in recent years and do not express T / B cell surface-specific antigen receptors. Group 2 innate lymphoid cells (ILC2), as a subpopulation of innate lymphoid cells, were first identified in adipose vascular lymphocyte clusters in 2010 (13). They originate from common lymphoid progenitor cell precursors and develop through common auxiliary-like ILC precursors. Their development depends on the transcription factors GATA3 / RORα / Bcl11b / TCF-1 / Gfi1 (14). Their cell marker characteristics are Lin-CD127+CD25+GATA3+ (31). ILC2 migrates from the blood circulation to the tissues in the early stages of the body and, as tissue-resident cells, they are mainly present in various mucosal tissues of the body, including skin / lungs / intestines / liver / fat, etc. (15-18). ILC2s in tissues express multiple surface receptors and can be activated by cytokine signals such as IL-2, IL-7, IL-33, IL-25, and TSLP in the tissue microenvironment. They can also be activated by other signaling substances in the tissue microenvironment, such as neuropeptides (VIP, Neuromedin U, CGRP), leukotrienes, lipids (cysLT, PDG2), and androgens. In addition, ILC2s are also regulated by inhibitory signals such as IFNs, IL-10, TNFα, IL-27, PGE2, PGI2, and catecholamines in the environment (19-20). Activated ILC2s can produce a variety of cytokines, chemokines, and peptides. Among them, type 2 cytokines such as IL-4 / IL-5 / IL-13 / GM-CSF mediate type 2 immune responses in the tissue environment and play a role in tissue inflammation such as respiratory allergic reactions, asthma, and helminth infection (21-22). In addition, ILC2s can also produce amphiregulin Areg (an EGF family cytokine) and IL-9, which are involved in the repair of epithelial tissue after injury (23). Recent studies have shown that ILC2s also play an important role in the occurrence and development of tumors (24-25).

[0004] ILC2 and obesity and type 2 diabetes. Adipose tissue is rich in ILC2, which highly expresses the IL-33 receptor ST2, maintaining the type 2 immune response characteristics of adipose tissue and the immune homeostasis and metabolic homeostasis of adipose tissue (26). Imbalance in the homeostasis of the adipose tissue immune microenvironment is the pathological basis for chronic inflammatory response in tissues (27-29). Under physiological conditions, the adipose tissue immune microenvironment is characterized by type 2 immune response, containing a large number of ILC2, M2 macrophages, eosinophils and Th2, etc., secreting type 2 cytokines IL-4, IL-5, IL-10 and IL-13, manifesting as an anti-inflammatory microenvironment and maintaining an insulin-sensitive state (30-31). In the obese state, adipose tissue mainly contains M1 macrophages, CD8+ T cells, Th1 cells and B cells, etc., which secrete a large amount of inflammatory cytokines such as TNF-α, IL-6, IL-1β, MCP-1 and IFNg, manifesting as a pro-inflammatory microenvironment, inducing adipose tissue inflammation, and manifesting as insulin resistance. Studies have found that ILC2 is a core member of the type 2 immune response network in adipose tissue and plays a key role in regulating chronic inflammatory responses in adipose tissue. ILC2 can recruit eosinophils into adipose tissue and activate them to secrete IL-4 by secreting the cytokine IL-5 (32). In addition, ILC2 is activated and expanded under the stimulation of the alarmin IL-33, expressing IL-5 and IL-13, and promoting the activation of M2 macrophages (33). ILC2 can also interact with other immune cells in adipose tissue. Studies have shown that adipose ILC2 promotes the secretion of type 2 cytokines IL-4 / IL-5 / IL-13 by Th2 cells in adipose tissue and promotes the immunosuppressive function of Treg cells, thereby maintaining the anti-inflammatory state of adipose tissue (34).

[0005] In addition to regulating the inflammatory response of adipose tissue, ILC2 can also directly regulate adipose tissue metabolism. Data from David Artis's research group showed that the IL-33-ILC2 signaling axis can regulate the occurrence and development of obesity and metabolic diseases. The number of ILC2 cells in the adipose tissue of mice lacking IL-33 decreased, which was manifested as weight gain, increased white adipose tissue content, insulin resistance and abnormal glucose metabolism. When IL-33 was injected into mice fed a high-fat diet (HFD), weight gain was prevented and glucose metabolism was improved. Mechanistic studies have found that ILC2 promotes beigeization of white fat by secreting active substances such as methionine-enkephalin, thereby regulating adipose tissue metabolism, inhibiting weight gain and improving glucose metabolism, and preventing the occurrence of obesity and diabetes (26). Studies have also shown that the type 2 cytokine IL-13 secreted by ILC2 can also directly act on adipocyte precursor cells to promote their differentiation into beige adipocytes (35).

[0006] In obese mice or obese people, there is a decrease in the number and function of ILC2 cells, but the mechanism is not yet clear (26, 36-37). Therefore, targeting ILC2 to find drugs that can regulate ILC2 function is an important strategy for treating obesity and its related metabolic diseases. However, the molecular regulatory mechanism of ILC2 functional activation has not yet been elucidated. Clarifying the regulatory mechanism of ILC2 in maintaining immune and metabolic homeostasis in adipose tissue is a prerequisite for revealing these issues. This will also provide theoretical guidance for the study of ILC2 function and its role in disease, and will provide new therapeutic targets and strategies for human obesity and metabolic diseases.

[0007] LKB1 and immune cells. Liver kinase B1 (LKB1), also known as serine / threonine protein kinase 11 (Stk11), activates 13 kinases related to AMPK (a central metabolic sensor) through phosphorylation upon activation. It is a key energy sensor that initiates metabolic pathways and plays a crucial role in regulating cell growth and metabolism. On the one hand, LKB1 can form a heterotrimer with STRAD and MO25, LKB1-STRAD-MO25, which phosphorylates and activates AMPK. AMPK activates the tuberous sclerosis complex (TSC), thereby inhibiting mTORC1 and reducing protein translation and cell growth. On the other hand, AMPK participates in the regulation of FOXO family transcription factors and plays an important role in regulating energy metabolism, cell cycle, and apoptosis (38-39). Studies have found that LKB1 has diverse biological functions in various processes, including energy metabolism, cell cycle, cell proliferation, apoptosis, cell polarization, and cell invasion and metastasis (40-42). In addition, LKB1 is also considered a tumor suppressor. Clinically, germline mutations in the STK11 gene are the main cause of Peutz-Jeghers syndrome (PJS). Germline mutations in LKB1 occur in 80% of PJS patients and are often associated with gastrointestinal malignancies, as well as breast and lung cancer (43). Our previous studies have found that LKB1 can also regulate the growth and metabolism of intestinal epithelial cells, thereby affecting the homeostasis of the intestinal flora (44).

[0008] As a bioenergy sensor, LKB1 not only affects the energy metabolism of various tissue cells, but in recent years, it has also been found that the LKB1 pathway plays an important role in regulating the activation and function of immune cells. First, LKB1 plays an important role in maintaining the homeostasis of hematopoietic stem cells. Knocking out LKB1 can promote the proliferation of hematopoietic stem cells, followed by exhaustion and pancytopenia. Mechanistic studies have found that LKB1 deficiency mainly leads to mitochondrial dysfunction in hematopoietic stem cells (45-47). Secondly, LKB1 is involved in regulating the survival of thymocytes and the positive selection process, and is involved in regulating CD4+ LKB1 regulates T cell glucose metabolism and affects the differentiation of Th1 cells (48-49). Other studies have shown that LKB1 is a negative regulator of DC cell metabolism. Knockout of LKB1 leads to overactivation of DC cells, promotes the expansion of Th17 cells, and causes severe autoimmune diseases in mice (50). In addition, Treg cells can coordinate their cell metabolism and immune balance through LKB1 signaling, and prevent cell apoptosis and functional failure, thereby coordinating the balance between immunity and tolerance (51-52). Recent studies have found that in Th17 cells, the LKB1-mitochondrial axis regulates the cell epigenetic state and affects IL-17A expression (53).

[0009] LKB1 is associated with obesity and type 2 diabetes. Studies have shown that the LKB1-AMPK pathway is functionally abnormal in obesity and type 2 diabetes, and metformin, an AMPK agonist drug targeting this pathway, is currently used to treat type 2 diabetes (54). Previous studies have mainly focused on the regulatory effects of LKB1 on various metabolic tissues such as liver, muscle, and fat. For example, liver-specific knockout of LKB1 leads to the disappearance of AMPK activity, accompanied by increased blood glucose and gluconeogenesis (55). Muscle-specific knockout of LKB1 leads to increased insulin sensitivity (56). Adipose tissue-specific knockout of LKB1 leads to reduced white fat in mice, delayed growth after birth, and death before weaning (57). Since changes in the adipose tissue microenvironment during obesity lead to the infiltration of various inflammatory immune cells, the immune dysregulation state further leads to insulin resistance in the body. Therefore, it is necessary not only to study the effects of LKB1 on the metabolism and function of adipocytes, but also to pay attention to the regulatory effects of LKB1 on immune cells in the tissue microenvironment.

[0010] Current status and challenges in the treatment of obesity and type 2 diabetes. A large number of studies have confirmed that obesity and overweight are risk factors for cardiovascular disease, type 2 diabetes, chronic kidney disease, and malignant tumors. Limited by technical and social factors, the research and development of anti-obesity drugs has always faced huge challenges. There are very few drugs that have truly entered the clinical stage and achieved significant therapeutic effects. The obstacles hindering the development of new drugs are the complexity of the pathways involved in obesity and the limitations of converting preclinical data into human studies. At present, the lipase inhibitor orlistat developed by Roche is the only drug approved by the FDA and EMA for chronic weight management. Orlistat is a highly selective and potent gastrointestinal lipase inhibitor that can slow down the hydrolysis of dietary fat in the gastrointestinal tract, thereby reducing the hydrolysis and absorption of 25-30% of dietary fat, but it has steatorrhea as the most common side effect and other side effects such as hepatotoxicity and acute nephrotoxicity (58).

[0011] Obesity is the main cause of insulin resistance in diabetic patients. In the human body, insulin resistance often includes adipose tissue dysfunction, fat toxicity, oxidative stress, and low-grade inflammation. Epidemiological analysis and pathogenesis research have made it clear that insulin resistance is closely related to a variety of metabolic diseases, cardiovascular diseases, and even neurological diseases. Its combined hyperlipidemia, hypertension, and coronary heart disease also seriously endanger human health. However, there is still a lack of comprehensive understanding of the pathogenesis of insulin resistance in type 2 diabetes, and there is still a lack of effective and specific treatment methods in clinical practice. Many anti-hyperglycemic drugs, such as metformin and glimepiride, can improve blood sugar levels in patients with type 2 diabetes, but their specific mechanisms of action are still controversial (59-60). Thiazolidinediones (such as pioglitazone), as an effective insulin sensitizer, may improve insulin resistance in the liver and peripheral tissues (61), but they can also cause weight gain, edema, and cardiovascular disease, and there are certain risks in terms of safety (62). Considering that inflammation can induce insulin resistance to a certain extent, adipose tissue inflammation is also closely related to obesity and type 2 diabetes, and the use of anti-inflammatory drugs to alleviate insulin resistance in diabetic patients is also a hot topic of current research. Blocking antibodies targeting proinflammatory cytokines, such as infliximab, adalimumab, and etanercept, can inhibit the production of TNF, thereby reducing insulin resistance in inflammatory patients. In a randomized controlled trial of obese patients, researchers found that canakinumab and others can reduce insulin secretion in patients with type 2 diabetes (63); in another placebo-controlled randomized trial, researchers found that tocilizumab can reduce the level of insulin resistance in obese individuals (64). However, the above-mentioned monoclonal antibodies have not yet been tested in phase I clinical trials in obese patients. In addition, energy metabolism regulators such as mitochondrial uncouplers can also improve the level of insulin resistance in rodent models and non-human primates, but their effects in obese patients are still unknown (65). Various adverse reactions of drugs are an important reason for the increasing prevalence of obesity and type 2 diabetes. Therefore, there is an urgent need to develop more effective biomarkers for early diagnosis of obesity and type 2 diabetes and to develop targeted treatment plans for different individuals. Summary of the Invention

[0012] The present invention addresses the shortcomings of the aforementioned prior art and proposes a biomarker, the LKB1-PD-1 signaling axis. In this biomarker, LKB1 is a metabolic kinase that regulates various intracellular metabolic processes, while PD-1 is a transmembrane protein that serves as a co-receptor for the extracellular ligand molecule PD-L1.

[0013] The biomarker LKB1-PD-1 signaling axis of the present invention includes, but is not limited to, human LKB1 gene (STK11) ID: NCBI Gene: 6794, Ensembl: ENSG00000118046, mouse LKB1 gene (Stk11) ID: NCBI Gene: 20869, Ensembl: ENSMUSG00000003068, human PD-1 gene (PDCD1) ID: NCBI Gene: 5133, Ensembl: ENSG00000188389, mouse PD-1 gene (Pdcd1) ID: NCBI Gene: 18566, Ensembl: ENSMUSG00000026285; and / or, the amino acid sequences of the biomarkers LKB1 and PD-1 include, but are not limited to, SEQ ID NO. 1 (human LKB1), SEQ ID NO. 2 (mouse LKB1), SEQ ID NO. 3 (human PD-1), and SEQ ID The sequence shown in NO.4 (mouse PD-1).

[0014] SEQ ID NO.1 (human LKB1):

[0015] MEVVDPQQLGMFTEGELMSVGMDTFIHRIDSTEVIYQPRRKRAKLIGKYLMGDLLGEGSYGKVKEVL

[0016] DSETLCRRAVKILKKKKLRRIPNGEANVKKEIQLLRRLRHKNVIQLVDVLYNEEKQKMYMVMEYCVCGM

[0017] QEMLDSVPEKRFPVCQAHGYFCQLIDGLEYLHSQGIVHKDIKPGNLLLTTGGTLKISDLGVAEALHPFAAD

[0018] DTCRTSQGSPAFQPPEIANGLDTFSGFKVDIWSAGVTLYNITTGLYPFEGDNIYKLFENIGKGSYAIPGDCGP

[0019] PLSDLLKGMLEYEPAKRFSIRQIRQHSWFRKKHPPAEAPVPIPPSPDTKDRWRSMTVVPYLEDLHGADEDE

[0020] DLFDIEDDIIYTQDFTVPGQVPEEEASHNGQRRGLPKAVCMNGTEAAQLSTKSRAEGRAPNPARKACSASS

[0021] KIRRLSACKQQ

[0022] SEQ ID NO.2 (Mouse LKB1):

[0023] MDVADPEPLGLFSEGELMSVGMDTFIHRIDSTEVIYQPRRKRAKLIGKYLMGDLLGEGSYGKVKEVLD

[0024] SETLCRRAVKILKKKKLRRIPNGEANVKKEIQLLRRLRHRNVIQLVDVLYNEEKQKMYMVMEYCVCGMQ

[0025] EMLDSVPEKRFPVCQAHGYFRQLIDGLEYLHSQGIVHKDIKPGNLLLTTNGTLKISDLGVAEALHPFAVDDT <00001​​​​​​​​​​​​​​​​​​YRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKES

[0033] LRAELRVTERRAEVPTAHSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPL

[0034] KEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPED

[0035] GHCSWPL

[0036] SEQ ID NO.4 (mouse PD-1):

[0037] MWVRQVPWSFTWAVLQLSWQSGWLLEVPNGPWRSLTFYPAWLTVSEGANATFTCSLSNWSEDLMLN

[0038] WNRLSPSNQTEKQAAFCNGLSQPVQDARFQIIQLPNRHDFHMNILDTRRNDSGIYLCGAISLHPKAKIEESP

[0039] GAELVVTERILETSTRYPSPSPKPEGRFQGMVIGIMSALVGIPVLLLLAWALAVFCSTSMSEARGAGSKDDT

[0040] LKEEPSAAPVPSVAYEELDFQGREKTPELPTACVHTEYATIVFTEGLGASAMGRRGSADGLQGPRPPRHED

[0041] GHCSWPL

[0042] The present invention innovatively proposes the use of the LKB1-PD-1 signaling axis as a biomarker for the early diagnosis and treatment of obesity and type 2 diabetes. Modulators of the LKB1-PD-1 signaling axis, including but not limited to LKB1 agonists, PD-1 inhibitors, or antagonists (such modulators include small molecule compounds, small interfering RNA, short hairpin RNA, biomacromolecule antibodies, antisense nucleotides, biologically active fragments or homologs of such biomacromolecule antibodies, etc.), can promote type 2 immune responses (secretion of IL-5, IL-13, etc.), thereby inhibiting the occurrence and development of obesity and type 2 diabetes.

[0043] The present invention also proposes that the biomarker LKB1-PD-1 signaling axis is an important target for dysfunctional ILC2s in the adipose tissue of obese mice and humans. LKB1 signaling is downregulated in ILC2s in the adipose tissue of obese mice, while PD-1 expression is significantly increased in ILC2s in the adipose tissue of obese mice. The present invention also proposes that low expression levels of LKB1 signaling in adipose tissue ILC2s are closely associated with insulin resistance in obese individuals. LKB1 expression is not only downregulated in ILC2s in the greater omentum adipose tissue of patients with type 2 diabetes compared to normal ILC2s, but also has decreased phosphorylation levels. PD-1 expression is significantly increased in ILC2s in the greater omentum adipose tissue of patients with type 2 diabetes compared to normal ILC2s.

[0044] The present invention also proposes the use of the biomarker LKB1-PD-1 signaling axis in the preparation of drugs for treating metabolic diseases including obesity and type 2 diabetes. The application uses the biomarker LKB1-PD-1 signaling axis as a target to prepare diagnostic reagents for the occurrence and / or severity of obesity and type 2 diabetes, or to prepare drugs for preventing and treating obesity and type 2 diabetes, or to prepare reagents for predicting the occurrence and / or severity of obesity and type 2 diabetes, or to prepare reagents for evaluating the prognosis of obesity and type 2 diabetes.

[0045] In the application of the present invention, proteins and / or small molecules are prepared by targeting LKB1 or downstream kinases, and antibodies are prepared by immunizing animals with PD-1 (including the extracellular segment, or the full length, or the functional domain) and / or its ligands as immunogens, or antibodies against PD-1 and / or its ligands are synthesized by biological means based on the immunoglobulin sequence of a hybridoma that produces antibodies against PD-1 and / or its ligands.

[0046] In the application of the present invention, the sample for measuring the biomarker LKB1-PD-1 signaling axis is a greater omentum adipose tissue sample obtained from a patient.

[0047] In the application of the present invention, the levels of LKB1 and PD-1 are determined at the protein and nucleic acid levels.

[0048] In the application of the present invention, when the levels of LKB1 and PD-1 are determined at the protein level, the level of LKB1 is determined by flow cytometry, immunohistochemical staining, or immunoblotting to determine its total protein level and / or its phosphorylation level, and the level of PD-1 is determined by flow cytometry, immunohistochemical staining, or immunoblotting.

[0049] In the application of the present invention, when the levels of LKB1 and PD-1 are determined at the nucleic acid level, the levels of LKB1 and PD-1 are determined by quantifying the mRNA encoding LKB1 and PD-1.

[0050] In the application of the present invention, the antibody is used to inhibit the activation of inflammatory immune cells in adipose tissue, promote the secretion of type 2 cytokines (IL-5, IL-13, etc.), inhibit adipose tissue inflammation, and prevent and alleviate obesity and type 2 diabetes.

[0051] In the application of the present invention, the metabolic diseases include type 2 diabetes and insulin resistance caused by obesity.

[0052] The present invention also provides modulators of the LKB1-PD-1 signaling axis, including LKB1 agonists, PD-1 inhibitors, or antagonists. These modulators can activate the expression and activation of the biomarker LKB1, or inhibit or antagonize the expression of the biomarker PD-1 and the binding of PD-1 to its ligand. Preferably, these modulators can actively modulate the biological functions of LKB1 and / or downstream signaling pathways. Preferably, these modulators can inhibit the expression of PD-1 or the binding of PD-1 ligands to the biomarker PD-1, and may or may not inhibit the downstream signaling pathways of LKB1.

[0053] The present invention also proposes the use of the regulator of the biomarker LKB1-PD-1 signaling axis in the preparation of diagnostic reagents for the occurrence and / or severity of obesity and type 2 diabetes, and / or the preparation of drugs for treating obesity and type 2 diabetes, and / or the preparation of reagents for predicting the occurrence and / or severity of obesity and type 2 diabetes, and / or the preparation of reagents for evaluating the prognosis of obesity and type 2 diabetes, and / or the evaluation of the applicability and effectiveness of targeting the LKB1-PD-1 signaling axis to treat obesity and type 2 diabetes.

[0054] The present invention also proposes the use of the regulator of the biomarker LKB1-PD-1 signaling axis in preventing and treating obesity and type 2 diabetes, and in predicting the severity of obesity and insulin resistance in type 2 diabetes.

[0055] The present invention also proposes an inhibitor of the biomarker LKB1-PD-1 signaling axis. The PD-1 antagonist αPD-1 antibody can inhibit the binding of the ligand to PD-1 and may or may not inhibit the downstream signaling pathway of LKB1.

[0056] Furthermore, the antagonist is an antibody, including anti-PD-1 monoclonal antibodies and / or anti-PD-1 ligand monoclonal antibodies, including monoclonal antibodies anti-mouse PD-1 / CD279 antibody (RMP1-14)), anti-mouse PD-1 / CD279 antibody (29F.1A12), anti-human PD-1 / CD279 antibody (J116), anti-mouse PD-L1 (B7-H1) antibody (10F.9G2), anti-human PD-L1 (B7-H1) antibody (29E.2A3), etc.

[0057] Among them, the antagonistic antibodies such as anti-mouse PD-1 / CD279 antibody (RMP1-14), anti-mouse PD-1 / CD279 antibody (29F.1A12), anti-human PD-1 / CD279 antibody (J116), anti-mouse PD-L1 (B7-H1) antibody (10F.9G2), and anti-human PD-L1 (B7-H1) antibody (29E.2A3) all selectively inhibit the binding of PD-1 to its ligand PD-L1.

[0058] The antagonist may also be an anti-PD-1 polyclonal antibody and / or an anti-PD-1 ligand polyclonal antibody.

[0059] The present invention can alleviate the occurrence of obesity-related insulin resistance in mice by injecting anti-PD-1 antibodies and / or anti-PD-1 ligand antibodies.

[0060] The present invention can aggravate obesity-related type 2 diabetes and insulin resistance by knocking out LKB1 gene.

[0061] The present invention also provides a drug / drug composition comprising a regulator of the biomarker LKB1-PD-1 signaling axis as described above, and / or an antibody to the biomarker LKB1-PD-1 signaling axis, wherein the regulator includes but is not limited to an agonist, inhibitor or antagonist.

[0062] The present invention also provides a detection reagent / kit, which contains primers or probes that hybridize to polynucleotides of the biomarker LKB1-PD-1 signaling axis, or contains antibodies that can specifically bind to proteins of the LKB1-PD-1 signaling axis, or contains compounds that can bind to the LKB1-PD-1 signaling axis, including but not limited to small molecule compounds.

[0063] The present invention also proposes uses of the above-mentioned biomarker LKB1-PD-1 signaling axis, regulators of the above-mentioned biomarker LKB1-PD-1 signaling axis, the above-mentioned antibodies, the above-mentioned drugs / drug compositions, and the above-mentioned detection reagents / kits, including: use as a therapeutic target for treating metabolic diseases such as obesity and type 2 diabetes (T2D), use in assessing ILC2 dysfunction, use in early detection of obesity and type 2 diabetes, use in the diagnosis, prevention, and treatment of type 2 diabetes, use in preparing drugs for treating and / or preventing insulin resistance and adipose tissue inflammatory diseases, use in preparing drugs for treating metabolic diseases such as obesity and T2D, use in assessing the impaired effector function of PD-1 upregulation on LKB1-deficient ILC2s, use in assessing the restoration of the number and effector function of adipose ILC2s in mice lacking LKB1 by anti-PD-1 antibody treatment, and / or use in assessing the effectiveness of anti-PD-1 antibody blocking therapy for insulin resistance in the body.

[0064] The modulators, antibodies, drugs / pharmaceutical compositions, and detection reagents / kits described in the present invention can be used alone or in combination, or in combination with hypoglycemic drugs and / or other drugs. The present invention also proposes combining hypoglycemic drugs with antibodies, small molecule compounds, antagonists, and the like that target the LKB1-PD-1 signaling axis, thereby achieving therapeutic effects in preventing and treating insulin resistance caused by obesity and type 2 diabetes.

[0065] The present invention also provides a method for screening candidate drugs for treating obesity and type 2 diabetes and predicting the prognosis of obesity and type 2 diabetes, the method comprising detecting the effect of the candidate drug on the biological effects of the biomarker LKB1-PD-1 signaling axis or its downstream signals (such as type 2 cytokine levels) in a subject or a sample obtained from the subject, wherein, after use of the candidate drug, the biological function of the biomarker LKB1-PD-1 signaling axis is upregulated, indicating that the candidate drug has the effect of treating obesity and type 2 diabetes, alleviating or preventing the occurrence of obesity and type 2 diabetes, and / or improving the prognosis of obesity and type 2 diabetes. The upregulation of the biological function of the biomarker LKB1-PD-1 signaling axis refers to, but is not limited to, an increase in the expression level and / or activation level of the biomarker LKB1, a decrease in the expression level of PD-1, or a weakening of its downstream signaling pathway.

[0066] The present invention also proposes a R5 / +Stk11 f / f The use of a mouse model for predicting obesity and type 2 diabetes by affecting the biomarker LKB1-PD-1 signaling axis, wherein the model is LKB1-specifically deleted in cells expressing or previously expressing IL-5 (R5 / +Stk11 f / fIn mice with either steady-state or diet-induced obesity and type 2 diabetes models, LKB1 expression by adipose ILC2s decreased with increasing insulin resistance, while PD-1 expression by adipose ILC2s increased. LKB1 is lowly expressed and PD-1 is highly expressed in adipose tissue of diabetic patients. Therefore, LKB1 and PD-1 may become effective indicators for assessing the severity of insulin resistance and may become indicators for evaluating the indications for treatment with the LKB1-PD-1 signaling axis. The applications also include: applications in detecting and evaluating the regulatory effects of LKB1 on the homeostatic maintenance and effector function of adipose ILC2s, evaluating the effects of PD-1 in inhibiting the homeostatic maintenance and effector function of adipose ILC2s, and applications of LKB1-PD-1 signaling-related regulators in the preparation of diagnostic reagents for the occurrence and / or severity of obesity and type 2 diabetes, and / or the preparation of therapeutic drugs for obesity and type 2 diabetes, and / or the preparation of prognostic evaluation reagents for obesity and type 2 diabetes, and / or the evaluation of the suitability and effectiveness of treatments targeting LKB1 and PD-1 and / or their ligands for obesity and type 2 diabetes.

[0067] The present invention also provides a method for diagnosing obesity and type 2 diabetes in a subject, or a method for diagnosing obesity and type 2 diabetes in a subject in need, or a method for preventing or treating obesity and type 2 diabetes in a subject in need, or a method for assessing the severity of obesity and type 2 diabetes in a patient, or a method for predicting the risk of obesity and type 2 diabetes in a patient, or a method for predicting or prognosticating whether the patient is suitable for treatment targeting the LKB1-PD-1 signaling axis.

[0068] The method comprises: determining the levels of LKB1 and PD-1 / or their ligands in a sample of abdominal omentum adipose tissue from the patient or subject, wherein the ligand levels are used to prevent or treat obesity and type 2 diabetes in the patient or subject, or to assess the severity of the disease in a patient with obesity and type 2 diabetes, or to predict whether the patient will develop obesity and type 2 diabetes, or to predict or prognose whether the patient is suitable for treatment targeting the biomarker LKB1-PD-1 signaling axis or its ligands.

[0069] The method comprises: using the biomarker LKB1-PD-1 signaling axis and / or its ligand as a target to diagnose the patient suffering from obesity and type 2 diabetes.

[0070] The biomarker LKB1-PD-1 signaling axis and / or its ligand is used as a target to prevent or treat the patient suffering from obesity and type 2 diabetes.

[0071] The severity of the patients suffering from obesity and type 2 diabetes is assessed using the biomarker LKB1-PD-1 signaling axis and / or its ligand as a target.

[0072] The biomarker LKB1-PD-1 signaling axis and / or its ligands are used as targets to predict the occurrence of obesity and type 2 diabetes in the desired subjects.

[0073] The biomarker LKB1-PD-1 signaling axis and / or its ligands are used as targets to predict or evaluate the prognosis of whether the patient is suitable for treatment targeting obesity and type 2 diabetes and / or their ligands.

[0074] In the method of the present invention, the sample for measuring the biomarker LKB1-PD-1 signaling axis is obtained from the greater omentum adipose tissue of the patient.

[0075] In the method of the present invention, the expression levels of LKB1 and PD-1 are determined at the protein level; and / or, the expression levels of LKB1 and PD-1 are determined at the nucleic acid level.

[0076] In the method of the present invention, when the expression levels of LKB1 and PD-1 are determined at the protein level, the expression levels of LKB1 and PD-1 are determined by flow cytometry, immunohistochemical staining, or immunoblotting techniques.

[0077] When the expression levels of LKB1 and PD-1 are determined at the nucleic acid level, the expression levels of LKB1 and PD-1 are determined by quantifying the mRNA levels of genes encoding LKB1 and PD-1.

[0078] The method of the present invention comprises determining the levels of LKB1 and PD-1 and / or their ligands in the sample.

[0079] In the method of the present invention, a panel of combined antibodies is used to determine the expression levels of LKB1 and PD-1 in specific cells in a sample obtained from the patient.

[0080] The levels of LKB1 and PD-1 were quantitatively determined by flow cytometry, immunohistochemical staining, immunoblotting and other methods.

[0081] The levels of LKB1 and PD-1 were quantitatively determined by real-time quantitative PCR.

[0082] In the method of the present invention, the lower the level of LKB1 and the higher the level of PD-1, the more severe the patient's type 2 diabetes and insulin resistance.

[0083] In the method of the present invention, the lower the level of LKB1 and the higher the level of PD-1, the greater the possibility that the patient will develop type 2 diabetes and insulin resistance.

[0084] In the method of the present invention, the lower the level of LKB1 and the higher the level of PD-1, the more suitable the patient is for targeting the LKB1-PD-1 signaling axis and / or its ligands to prevent or treat obesity-related type 2 diabetes and insulin resistance.

[0085] In the method of the present invention, the diagnosis comprises the following steps: i) determining and comparing the expression levels of LKB1 and PD-1 and / or their ligands in the greater omentum adipose tissue obtained from the normal person and the obese patient subject; ii) when the LKB1 level in the adipose tissue determined in step i) is more than 2 times lower than that of the normal group, and the PD-1 expression level is more than 2 times higher than that of the normal group, the likelihood of the individual developing type 2 diabetes and insulin resistance is greater, the degree of insulin resistance is more severe, and the patient is more suitable for a strategy of targeting the LKB1-PD-1 signaling axis to prevent or treat obesity-related type 2 diabetes and insulin resistance.

[0086] The prevention or treatment comprises the following steps: preventing or treating insulin resistance in a subject in need thereof by targeting obesity and type 2 diabetes specific biomarkers, comprising: administering to the subject a pharmaceutical composition comprising a pharmaceutically acceptable carrier, an effective amount of a regulator of obesity and type 2 diabetes specific biomarkers, and optionally an additional therapeutic agent, thereby preventing or treating obesity and type 2 diabetes; wherein the obesity and type 2 diabetes specific biomarkers are selected from the LKB1-PD-1 signaling axis.

[0087] In the present invention, the carrier generally refers to a carrier for administration, which itself does not induce the production of antibodies harmful to the individual receiving the composition and does not have excessive toxicity after administration. These carriers are well known to those skilled in the art. For example, relevant content about pharmaceutically acceptable carriers is disclosed in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991). Specifically, the carrier can include, but is not limited to, one or more combinations of saline, buffer, glucose, water, glycerol, ethanol, adjuvants, etc.

[0088] In the present invention, the pharmaceutical composition includes tablets, capsules, soft capsules, oral liquids, injections, infusions, powder injections or freeze-dried powder injections, etc.

[0089] In the method of the present invention, the regulator is selected from small molecule chemical agents, antisense oligonucleotides, small interfering RNA (siRNA), short hairpin RNA (shRNA), antibodies, and biologically active fragments or homologs of the antibodies.

[0090] The samples referred to in the present invention include adipose tissue samples obtained from patients in any storage method. The LKB1 and PD-1 levels detected include the nucleic acid and protein levels of LKB1 and PD-1 detected by any means (including reagents that can selectively bind to LKB1 and PD-1 and / or their ligands).

[0091] The present invention also proposes that adipose ILC2s in obese and type 2 diabetic humans and mice specifically downregulate LKB1 expression and upregulate PD-1 expression. Simultaneously, using multi-parameter flow cytometry (FACSymphonyA3), the present invention demonstrates that adipose tissue ILC2s in obese individuals express less LKB1 and more PD-1 at the protein level compared to controls. Therefore, the biomarker LKB1-PD-1 signaling axis is a specific target for adipose ILC2 dysfunction in obesity.

[0092] Using a high-fat diet-induced obesity and type 2 diabetes model, the present study suggests that LKB1 expression by adipose ILC2s decreases with increasing insulin resistance, while PD-1 expression by adipose ILC2s increases. Given that adipose tissue in diabetic patients has low LKB1 expression and high PD-1 expression, LKB1 and PD-1 may serve as effective indicators for assessing the severity of insulin resistance and for evaluating treatment indications for the LKB1-PD-1 signaling axis.

[0093] The present invention also proposes that the lack of LKB1 under steady-state conditions through ILC2-specific LKB1 knockout mice significantly inhibits the steady-state maintenance and effector function of adipose ILC2.

[0094] In R5 / +Stk11 f / f High-fat diet was used to induce obesity and type 2 diabetes in mice. f / f LKB1 can be specifically knocked out in ILC2 and induce upregulation of PD-1 expression. f / f The expression of IL-5 and IL-13 by ILC2 in mice decreased significantly, accompanied by aggravated inflammation in adipose tissue and insulin resistance. The present invention uses anti-PD-1 antibodies to treat high-fat-induced obese and type 2 diabetic mice, and proposes that anti-PD-1 antibodies can significantly restore the expression of IL-5 and IL-13 by ILC2, reduce inflammation in adipose tissue, and alleviate insulin resistance. Anti-PD-1 antibodies can also significantly alleviate the Rag1 deficiency of the adaptive immune system. - / -Insulin resistance symptoms were observed in mice, but after ILCs were eliminated with anti-Thy1.2 neutralizing antibodies, anti-PD-1 antibodies were no longer able to effectively inhibit Rag1 - / - The insulin resistance symptoms in mice indicate that ILC2 is a key target cell for the therapeutic effect of PD-1 antibodies.

[0095] In summary, the present invention innovatively proposes that the biomarker LKB1-PD-1 signaling axis is a specific molecular marker for dysfunction of ILC2 in adipose tissue. Under the pathological conditions of obesity and type 2 diabetes, PD-1 expression is increased, and LKB1 expression and phosphorylation levels are decreased. The expression levels of LKB1 and PD-1 in ILC2 in adipose tissue can serve as pathological indicators of obesity-related type 2 diabetes.

[0096] The present invention activates LKB1 or inhibits PD-1 signaling in vivo through methods such as gene knockout and antagonistic antibody drugs, effectively alleviating type 2 diabetes and insulin resistance by activating ILC2s. The LKB1-PD-1 signaling axis specifically regulates the activation of ILC2s in adipose tissue, and therefore serves as a potential target for the diagnosis and treatment of obesity and type 2 diabetes.

[0097] The beneficial effects of the present invention include: the present invention proposes to activate LKB1 or inhibit PD-1 to promote the secretion of type 2 cytokines (IL-5, IL-13, etc.), reduce adipose tissue inflammation, and prevent and alleviate obesity and type 2 diabetes.

[0098] The beneficial effects of the present invention also include: targeting the LKB1-PD-1 signaling axis does not affect glucose metabolism under steady-state conditions, but specifically improves adipose tissue inflammation in the pathological conditions of obesity and type 2 diabetes. Targeting the LKB1-PD-1 signaling axis can specifically target adipose tissue inflammation in diseased conditions while avoiding the side effects of nonspecific effects on other tissues. Targeting the LKB1-PD-1 signaling axis acts on metabolically protective adipose ILC2s, improving the adipose tissue immune microenvironment and achieving more stable therapeutic effects than traditional therapeutic agents that simply target downstream cytokines and related metabolic pathway molecules.

[0099] The present invention also demonstrates that the LKB1-PD-1 signaling axis is a specific biomarker for impaired adipose-derived ILC2s in patients with obesity and T2D. Compared to healthy controls, adipose-derived ILC2s in patients with obesity and type 2 diabetes exhibit lower LKB1 expression and higher PD-1 expression. ILC2-specific LKB1 knockout mice exhibit significantly increased insulin resistance, and treatment with anti-PD-1 antibodies, an antagonist of the LKB1-PD-1 signaling axis, significantly improves insulin resistance in obese and type 2 diabetic mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0100] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without paying any creative work.

[0101] Figure 1 (A) C57BL / 6 wild-type male mice were fed a normal chow (ND) or a high-fat diet (HFD) starting at 6 weeks of age. After 8 weeks, ILC2s in epididymal adipose tissue were isolated by flow cytometry and transcriptome sequencing was performed. Gene expression enrichment analysis of differentially expressed genes in ND / HFD ILC2s was performed (n = 4 / group). (B) Flow cytometry was used to assess mitochondrial mass (MitoTracker) in epididymal adipose tissue ILC2s (n = 3 / group). (C) Ingenuity pathway analysis (IPA) was used to assess upstream metabolic regulators of differentially expressed genes in ND / HFD ILC2s (P value < 0.05, fold change > 1.5). (D) qPCR analysis of Stk11 mRNA expression in adipose ILC2s. (E) Flow cytometry was used to assess phosphorylated LKB1 (P-LKB1) expression in ND / HFD ILC2s (n = 4 / group). All data are presented as mean ± standard error (SD). Data represent at least three independent experiments. (B, DE) Statistical significance was analyzed by two-sided unpaired t-test. *P<0.05, **P<0.01, ***P<0.001.

[0102] Figure 2 (A) qPCR detection of R5 / + and R5 / +Stk11 f / f Stk11 mRNA expression in mouse epididymal fat ILC2s (n=3 / group). (BD) Flow cytometry analysis of Stk11 in R5 / + and R5 / + mice f / f ILC2s (B) and IL-5 in mouse epididymal adipose tissue + ILC2s (C) and IL-13 + The proportion and number of ILC2s (D). The cell gate is based on CD45 + Live cells (B) or CD45 + Lin - Live cells (C, D) (n=7 / group). (E) R5 / + and R5 / +Stk11 f / f Caspase 3 in mice +The proportion of ILC2s (n = 6 / group). (F) Adipose ILC2s were cultured with rmIL-33 (10 ng / mL) and rmIL-7 (10 ng / mL) for 48 hours and then analyzed. Flow cytometry was used to detect early apoptosis (Annexin V + 7-AAD - ) and late apoptosis (Annexin V + 7-AAD + ) ILC2 ratio (n = 3-4 / group). All data are presented as mean ± standard error (SEM) based on at least three independent experiments. (A-F) Statistical significance was analyzed by two-sided unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

[0103] Figure 3 .(AI)R5 / + and R5 / +Stk11 f / f Mice were fed a high-fat diet for 14 weeks starting at 6 weeks of age. Fasting blood glucose (A), fasting insulin (B), intraperitoneal glucose tolerance test (GTT) (C), and insulin tolerance test (ITT) (D) were measured (n=5 / group). (E) Immunoblotting was used to detect phosphorylated Akt (P-Akt, Ser473) and total Akt levels in insulin-sensitive tissues (including epididymal fat, liver, and muscle) (n=3 / group). (FH) Representative images of epididymal fat tissue sections (n=5 / group) stained with hematoxylin and eosin (H&E) (F), Masson's staining (G), and α-smooth muscle actin (α-SMA) (H). Quantitative analysis of coronal structures (CLS) + )(F) and collagen structure (Masson + (G) and α-SMA + (H) Histological density in epididymal adipose tissue. (I) Biolegend LEGENDplex TM Microsphere immunoassay was used to measure the levels of inflammatory factors in epididymal fat (n = 5 / group). All data were obtained from at least three independent experiments and are presented as mean ± standard error. (A, B, F1) Statistical significance was analyzed by two-sided unpaired t-test, and (C, D) by two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.

[0104] Figure 4 (A) Flow cytometry was used to detect the expression of PD-1 in epididymal adipose ILC2s (n=5 / group). (B, C) Flow cytometry was used to detect the expression of IL-5 (B) and IL-13 (C) in epididymal adipose ILC2s (n=5 / group). (DG) Id2 cre- ERT2 Stk11f / f Mice were treated with tamoxifen for 5 consecutive days, and the expression of PD-1 (D), the number of ILC2s (E), and IL-5 in adipose ILC2 were detected on days 2, 5, 7, 9, and 17 after tamoxifen treatment. + ILC2s(F), IL-13 + The proportion of ILC2s (G) (n = 3 / group). All data are presented as mean ± standard error (SEM) based on at least three independent experiments. (A-G) Statistical significance was analyzed by two-sided unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

[0105] Figure 5 .(AD)Id2 cre-ERT2 and Id2 cre-ERT2 Stk11 f / f Mice were treated with anti-PD-1 antibody (200 μg / mouse) for 3 weeks after tamoxifen treatment. (A) Experimental scheme. ILC2s (B), IL-5 + ILC2s (C) or IL-13 + The proportion and number of ILC2s (D) (n = 4-5 / group). All data are presented as mean ± standard error (SEM) based on at least three independent experiments. (B-D) Statistical significance was analyzed by two-sided unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

[0106] Figure 6 (AG) Wild-type mice were treated with anti-PD-1 antibody or isotype control (200 μg / mouse) for 5 weeks after HFD for 3 weeks. Intraperitoneal glucose tolerance test (A), insulin tolerance test (B), total ILC2s number (C), IL-5 + ILC2s ratio and number (D), IL-13 + The proportion and number of ILC2s (E) (n=4 / group). (F,G) Rag1 - / - Mice were given anti-CD90.2 antibodies every 2 days to eliminate ILC2s. After 3 weeks of high-fat feeding, mice were intraperitoneally injected with anti-PD-1 antibodies (200 μg / mouse) every 3 days for a total of 5 weeks. Intraperitoneal glucose tolerance test (F) and insulin tolerance test (G) were measured (n=4 / group). All data included at least three independent experiments, and the data are presented as mean ± standard error. (A, B, F, G) Statistical significance was analyzed by two-way ANOVA, and (CE) statistical significance was analyzed by two-sided unpaired t-test. *P<0.05, **P<0.01, ***P<0.001.

[0107] Figure 7(A) Representative flow cytometry images and cell numbers of ILC2s in the greater omental adipose tissue of healthy controls (LC) and obese patients with type 2 diabetes (T2D) (n=17). Cell gates were obtained from LiveCD45 + Lin - CD127 + Cells. (B, C) Expression levels of IL-5 (B) and IL-13 (C) in adipose ILC2s from patients with LC and T2D (LC = 18, T2D = 8). (D) P-LKB1 levels in adipose ILC2s from patients with LC and T2D (LC = 7, T2D = 9). (E) Correlation between the mean fluorescence intensity of P-LKB1 and adipose insulin resistance index (Adipo-IR), body mass index (BMI), free fatty acids (FFA), and insulin levels in adipose ILC2s from patients with T2D (n = 14). (F, H) Adipose vascular stromal cells from patients with LC and obese T2D were cultured in vitro with rhIL-33 (10 ng / mL) and rhIL-7 (20 ng / mL) for 24 hours. Flow cytometry was used to detect the expression levels of PD-1, IL-5, and IL-13 in ILC2s. (F) Correlation analysis between PD-1 expression levels and IL-5 or IL-13 in ILC2s from LC controls and obese T2D patients (n = 18). (G) Flow cytometry analysis of PD-1 expression levels in human adipose tissue ILC2s (LC = 11, T2D = 7). (H) Adipose vascular stromal cells from obese T2D patients were treated with anti-PD-1 antibodies for 48 hours in the presence of rhIL-33 (10 ng / mL) and rhIL-7 (20 ng / mL). Flow cytometry was used to assess IL-5 production by ILC2s (n = 6 / group). All data were obtained from at least three independent experiments and are presented as mean ± standard error. (AD, G) Statistical significance was analyzed by two-sided unpaired t-test, (E, F) by Spearman correlation test, and (H) by two-sided paired t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

[0108] Figure 8 This is a diagram of the mouse LKB1 protein structure.

[0109] Figure 9 This is a diagram of the structure of human LKB1 protein.

[0110] Figure 10 This is a diagram of the mouse PD-1 protein structure.

[0111] Figure 11 This is a diagram of the structure of human PD-1 protein. DETAILED DESCRIPTION

[0112] The present invention will be further described in detail with reference to the following specific examples and accompanying drawings. The processes, conditions, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art, and the present invention has no particular limitations. The protection content of the present invention is not limited to the following examples. Without departing from the spirit and scope of the inventive concept, variations and advantages that can be imagined by those skilled in the art are included in the present invention and are protected by the appended claims.

[0113] Obviously, the described embodiments are only some of the embodiments of the present invention, rather than all of them. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention, its application, or use. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0114] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not limiting. Therefore, other examples of the exemplary embodiments may have different values.

[0115] Unless otherwise specified, the test materials used in the examples are all conventional biochemical reagents.

[0116] Example 1

[0117] Experimental Methods

[0118] 16.(1.1) High-fat diet-induced obesity / diabetes model in mice

[0119] Obesity / diabetes model was induced in 6-week-old C57BL / 6 wild-type male mice by feeding them with normal diet (ND) / high-fat diet (60% kcal fat) (HFD) for 8 weeks.

[0120] (1.2) Comparison of gene expression differences between obese and control mice ILC2 by gene sequencing

[0121] ILC2s were isolated from epididymal adipose tissue using flow cytometry, and RNA was extracted for transcriptome sequencing. Combining bioinformatics analysis with GO analysis and IPA (Ingenuity Pathway) enrichment analysis, gene expression differences between the ND and HFD groups were compared, and gene sets with specific overexpression or underexpression in HFD ILC2s were enriched.

[0122] (1.3) Flow cytometry verification of impaired mitochondrial homeostasis in HFD-ILC2

[0123] The present invention uses a FACSymphony multi-parameter flow analyzer to perform flow staining analysis on the mitochondrial mass (MitoTracker) and the phosphorylation level of LKB1 in ND and HFD mouse fat ILC2.

[0124] (1.4) qPCR verification of reduced LKB1 expression in HFD-ILC2

[0125] The present invention uses flow cytometry to separate ILC2 from epididymal adipose tissue of ND and HFD mice, extract RNA, and use qPCR to detect the downregulation of LKB1 mRNA level in adipose ILC2 cells of obese mice.

[0126] 2. Experimental results: LKB1 signaling is impaired in adipose ILC2s in obese mice

[0127] Through transcriptome sequencing analysis, the present invention proposes that compared with control mice, adipose ILC2 in obese mice not only has impaired type 2 cytokine secretion function but also has disturbances in cellular energy metabolism and mitochondrial homeostasis ( Figure 1 A). Flow cytometry results also confirmed that the mitochondrial mass of ILC2 in obese mice was significantly reduced ( Figure 1 B) Through IPA pathway enrichment analysis, the present invention found that the expression of LKB1, a key kinase regulating mitochondrial homeostasis, was significantly downregulated in adipose ILC2 of obese mice ( Figure 1 C, D). Flow cytometry results also confirmed that LKB1 activation was impaired in adipose ILC2 of obese mice ( Figure 1 E). These data confirm that LKB1 signaling is impaired in adipose ILC2s in obese mice and suggest that impaired LKB1 signaling may contribute to ILC2 dysfunction in obese mice.

[0128] Example 2

[0129] Experimental Methods

[0130] 18. (1.1) Establishment of an ILC2-specific LKB1-deficient mouse model

[0131] In order to study the effect of LKB1 on adipose ILC2, the ILC2-specific LKB1-deficient mouse model R5 / +Stk11 was established. f / f (R5 / + as WT, R5 / +Stk11 f / f as KO).

[0132] 19.(1.2) qPCR verification of R5 / +Stk11 f / f LKB1 knockout efficiency in mouse adipose ILC2s

[0133] The present invention separates R5 / + and R5 / +Stk11 by flow cytometry. f / fMouse ILC2, LKB1 mRNA levels in both cells were detected by qPCR to verify R5 / +Stk11 f / f LKB1 knockout efficiency in mouse adipose ILC2s.

[0134] 20. (1.3) Flow cytometry verification of changes in ILC2 cell number, cell function, and apoptosis induced by LKB1 deficiency

[0135] The present invention studies the effect of LKB1 knockout on the survival and function of adipose ILC2s, and detects the number, cytokines (IL-5, IL-13), and apoptosis changes of WT and KO ILC2s by flow cytometry.

[0136] 2. Experimental Results: Impaired Adipose ILC2 Activation and Function in Mice with ILC2-Specific LKB1 Deficiency

[0137] Since the cells that produce IL-5 are mainly ILC2, R5 / + mice are widely used to study genes that are selectively deleted in ILC2. f / f Mouse models (R5 / + as WT, R5 / + as Stk11 f / f as KO). From R5 / +Stk11 f / f Mouse ILC2s show efficient deletion of LKB1 ( Figure 2 A) Compared with control mice, R5 / +Stk11 f / f The number of ILC2 and type 2 cytokines IL-5 and IL-13 in mouse fat were significantly reduced ( Figure 2 BD). The present invention also found that R5 / +Stk11 f / f Increased apoptosis of mouse adipose ILC2 cells ( Figure 2 EF). These data demonstrate that LKB1 directly regulates the homeostasis and effector functions of adipose ILC2s.

[0138] Example 3

[0139] Experimental Methods

[0140] 22. (1.1) Feeding R5 / +(WT) and R5 / +Stk11 with a high-fat diet f / f (KO) mouse induced obesity / diabetes model

[0141] The present invention fed 6-week-old WT and KO male mice with normal diet (ND) / high-fat diet (60% kcal fat) (HFD) for 14 weeks, and then tested the mice's fasting blood glucose level, serum insulin level, intraperitoneal glucose tolerance test (GTT), and insulin tolerance test (ITT).

[0142] 23. (1.2) Immunoblotting to detect Akt and p-Akt levels in insulin-sensitive tissues of mice, including epididymal fat, liver, and muscle

[0143] In order to compare the differences in insulin resistance between WT and KO mice, the present invention extracted proteins from epididymal fat, liver, and muscle tissues of WT and KO mice after HFD, and detected the levels of Akt and p-Akt in the insulin-sensitive tissues of mice, epididymal fat, liver, and muscle tissues, by immunoblotting to compare the differences in insulin sensitivity between the two mice.

[0144] 24. (1.3) Histochemical staining to detect inflammatory changes and fibrosis in adipose tissue of WT and KO mice

[0145] In order to compare the differences in adipose tissue inflammation and fibrosis levels between WT and KO mice after HFD, the epididymal adipose tissue of the two mice was stained with HE, Masson and α-SMA.

[0146] 25.(1.4)Biolegend LEGENDplex TM Detection of inflammatory cytokine levels in adipose tissue by microsphere immunoassay

[0147] In order to compare the differences in adipose tissue inflammation between WT and KO mice after HFD, the LEGENDplex TM (Biolegend) cytokine detection kit was used to detect the levels of inflammatory cytokines in epididymal adipose tissue.

[0148] 26.2. Experimental Results - Mice with ILC2-Specific LKB1 Deficiency Develop More Severe Insulin Resistance and Adipose Tissue Inflammation Following High-Fat Diet Feeding

[0149] In the high-fat-induced obesity model, as the disease progressed, the present invention observed that compared with the control group mice, the KO mice had higher fasting blood glucose, impaired glucose tolerance and aggravated insulin resistance ( Figure 3 AD). In the insulin-sensitive tissues of KO mice, the present invention found that activation of the insulin signaling pathway was impaired ( Figure 3 E). Histological analysis showed that KO mice exhibited enhanced adipose tissue inflammation and aggravated adipose tissue fibrosis ( Figure 3 FH). Consistent with the histological data, KO mice showed higher expression of inflammatory cytokines in adipose tissue ( Figure 3 I). These results confirm that obese KO mice develop more severe insulin resistance and adipose tissue inflammation.

[0150] Example 4

[0151] Experimental Methods

[0152] 28. (1.1) Flow cytometry analysis of PD-1 expression in adipose tissue of WT and KO mice and the correlation between cytokine and PD-1 expression

[0153] The present invention compared the PD-1 expression differences between WT and KO mouse adipose ILC2 by flow cytometry and found that the secretion level of ILC2 cytokines was negatively correlated with the expression of PD-1.

[0154] 29. (1.2) Time point experiments to verify the sequential relationship between ILC2 PD-1 expression and downregulation of cell function after LKB1 knockout

[0155] In order to explore the mechanism of downregulation of cell function after the loss of LKB1 in adipose ILC2, we constructed an ILC2-induced LKB1 knockout mouse model, Id2. Cre-ERT2 Stk11 f / f In the mouse model, mice were intraperitoneally injected with tamoxifen to induce LKB1 knockout in ILC2, and the PD-1 expression, ILC2 cell number, and cytokine changes of adipose ILC2 were detected at different time points (2, 5, 7, 9, and 17 days).

[0156] 2. Experimental Results - ILC2-specific LKB1 deficiency leads to PD-1 upregulation, which regulates ILC2 activation and function

[0157] The present invention found that KO mouse adipose ILC2 up-regulated the expression of inhibitory receptor PD-1 ( Figure 4 A) and KO of PD-1 in ILC2 + The secretion of IL-5 and IL-13 by cells was significantly lower than that of PD-1 - cell( Figure 4 BC). To further verify whether PD-1 upregulation leads to impaired effector function of LKB1-deficient ILC2, we performed Id2 Cre-ERT2 Stk11 f / f LKB1 was knocked out by intraperitoneal injection of tamoxifen in mice. We found that upregulation of PD-1 occurred before the impairment of ILC2 effector function, suggesting that PD-1 may regulate the activation function of ILC2 ( Figure 4 DG).

[0158] Example 5

[0159] Experimental Methods

[0160] 31. (1.1) PD-1 antibody blocking experiment

[0161] In order to further prove whether PD-1 regulates the function of adipose ILC2, the present invention Cre-ERT2 Stk11 f / fMice were intraperitoneally injected with tamoxifen to induce ILC2 knockout of LKB1, and at the same time, control and PD-1 antibodies (200 μg / mouse, twice a week) were injected intraperitoneally to block PD-1 signaling.

[0162] 32.(1.2) Flow cytometry

[0163] The present invention is to Id2 Cre-ERT2 Stk11 f / f After PD-1 signaling was blocked in mice, changes in adipose ILC2 cytokines and cell number were detected by flow cytometry.

[0164] 2. Experimental Results: PD-1 Antibody Treatment Restores the Number and Effector Function of Adipose ILC2s in Mice with LKB1 Deficiency

[0165] In order to confirm the effect of PD-1 on ILC2 effector function, the present invention is to investigate the effect of PD-1 on ILC2 Cre-ERT2 Stk11 f / f When mice were intraperitoneally injected with tamoxifen, PD-1 antibodies were used to block the upregulation of PD-1 in LKB1-deficient ILC2s. Figure 5 A). The present invention found that anti-PD-1 antibodies restored the number of LKB1-deficient ILC2s and the levels of IL-5 and IL-13 cytokines to the levels of WT ILC2s ( Figure 5 BD). These results demonstrate that PD-1 antibody treatment can restore the number and effector function of adipose ILC2s in mice with specific LKB1 deficiency.

[0166] Example 6

[0167] Experimental Methods

[0168] (1.1) PD-1 antibody treatment experiment in wild-type mice fed with HFD

[0169] To verify whether PD-1 signaling in adipose ILC2 affects glucose metabolism in mice, the present invention used 6-week-old C57BL / 6 wild-type male mice fed a normal diet (ND) / high-fat diet (60% kcal fat) (HFD) for 3 weeks. The mice were then given intraperitoneal injections of isotype control and anti-PD-1 antibodies (200 μg / mouse, twice a week). Five weeks later, intraperitoneal glucose tolerance tests and insulin tolerance tests were performed on the mice to compare changes in glucose metabolism after treatment.

[0170] (1.2) Flow cytometry analysis of changes in adipose ILC2 function after PD-1 antibody treatment in HFD mice

[0171] The present invention detected changes in the number of adipose ILC2 cells and cytokine levels after PD-1 antibody treatment in HFD mice by flow cytometry.

[0172] 34.(1.3) High-fat fed Rag1 - / - Mouse PD-1 antibody combined with CD90 antibody treatment experiment

[0173] The present invention aims to confirm that the therapeutic effect of PD-1 antibody on obese wild-type mice is dependent on ILC2 and the effect on 8-week-old Rag1 - / - After male mice were fed a high-fat diet (60% kcal fat) (HFD) for 3 weeks, they were intraperitoneally injected with anti-PD-1 antibodies (200 μg / mouse, twice a week) and CD90.2 antibodies (200 μg / mouse, twice a week) to block the PD-1 signal in ILC2 and eliminate ILC cells in the mice, respectively. Intraperitoneal glucose tolerance test and insulin tolerance test were performed on the mice to compare the changes in glucose metabolism after treatment.

[0174] 2. Experimental results - Blocking PD-1 can improve insulin resistance in obese mice

[0175] To confirm the effect of PD-1 signaling in adipose ILC2 on glucose metabolism in mice, the present invention intraperitoneally injected PD-1 antibodies into wild-type mice fed a high-fat diet and found that mice treated with αPD-1 antibodies showed improved blood glucose metabolism and insulin sensitivity compared with control mice ( Figure 6 A, B). At the same time, αPD-1 antibody treatment also restored the number and cytokine levels of mouse adipose ILC2 ( Figure 6 CE). Since PD-1 is also expressed in other cells, we next sought to determine whether the therapeutic effect of PD-1 blockade on insulin resistance depends on ILC2s. We investigated the role of Rag1 in T, B, and NK cells in high-fat-fed rats. - / - The mice were treated with PD-1 antibodies and CD90 antibodies were used to eliminate ILC2 cells in the mice. The present invention found that due to the lack of ILC2s in adipose tissue, anti-CD90.2 antibodies could be used to treat high-fat fed Rag1 - / - Mice have increased glucose intolerance and insulin resistance; when treated with αPD-1 antibodies, Rag1 - / - Mouse Rag1 - / - showed improved glucose tolerance and insulin sensitivity, while the therapeutic effect of PD-1 blockade on insulin was not observed in anti-CD90.2 antibody-treated Rag1 - / - These results indicate that the effectiveness of PD-1 antibody blockade in treating insulin resistance depends on ILC2 ( Figure 6 F, G).

[0176] Example 7

[0177] Experimental Methods

[0178] 36. (1.1) Flow cytometry analysis of differences in ILC2 numbers, cytokines, LKB1 phosphorylation, and PD-1 expression in the greater omental adipose tissue between healthy controls (LC) and obese T2D patients

[0179] The present invention extracted vascular stromal cells (SVF) from the greater omentum adipose tissue of healthy controls and T2D patients, and detected the number of ILC2s, the secretion levels of IL-5 and IL-13, the phosphorylation level of LKB1 and the expression level of PD-1 in the adipose tissue of both groups by flow cytometry.

[0180] 37. (1.2) Spearman correlation analysis was used to examine the correlation between P-LKB1 levels and metabolic parameters in ILC2s of the greater omental adipose tissue of obese patients with T2D, and the correlation between PD-1 expression and IL-5 and IL-13 expression in adipose ILC2s.

[0181] The present invention detects the P-LKB1 level in ILC2 of the greater omental adipose tissue of T2D patients by flow cytometry, and performs correlation analysis with the patients' metabolic parameters Adipo-IR, BMI, FFA or fasting insulin level. The present invention also detects the PD-1 expression level in ILC2 of the greater omental adipose tissue of T2D patients by flow cytometry, and performs correlation analysis with the ILC2 cytokine IL-5 and IL-13 levels.

[0182] 38. (1.3) PD-1 antibody blocking in vitro experiments

[0183] The present invention added anti-PD-1 blocking antibodies to the vascular stromal cell culture medium of obese T2D patients (20 μg / mL). After 48 hours of in vitro culture, it was found that PD-1 blockade restored the IL-5 level produced by adipose ILC2s in obese T2D patients.

[0184] 2. Experimental results: Blocking PD-1 can restore the function of adipose ILC2 in diabetic patients

[0185] To determine whether ILC2s in obese T2D patients have similar functional defects as those in obese mice, the present invention detected the number and cytokines of ILC2s in the greater omentum adipose tissue of the control group and obese T2D patients. It was found that the number of adipose ILC2s and the secretion of IL-5 and IL-13 in obese T2D patients were significantly lower than those in the control group ( Figure 7 AC). These data indicate that the homeostasis and function of adipose ILC2 cells in obese T2D patients are impaired. The present invention further found that, consistent with the mouse data, adipose ILC2 cells in obese T2D patients showed decreased LKB1 activity ( Figure 7D). To determine whether impaired LKB1 signaling in ILC2s is associated with diabetes severity, we performed a correlation analysis between LKB1 phosphorylation levels and metabolic parameters in obese T2D patients. Notably, P-LKB1 was negatively correlated with Adipo-IR, but not with BMI, FFA, or fasting insulin levels ( Figure 7 E). Next, by detecting the expression level of PD-1 on human adipose ILC2s, the present invention found that PD-1 expression was negatively correlated with the expression of IL-5 and IL-13 in adipose ILC2s ( Figure 7 F). In addition, the expression level of PD-1 on ILC2s in obese T2D patients was higher than that in the control group ( Figure 7 G). These data suggest that increased PD-1 expression may be associated with impaired function of adipose ILC2s in patients with T2D. To evaluate the effect of PD-1 on human adipose ILC2 function, in vitro experiments confirmed that PD-1 blockade restored the ability of adipose ILC2s in obese patients with T2D to secrete the cytokine IL-5 ( Figure 7 These data indicate that PD-1 is a negative regulator of adipose-derived ILC2 activation in obese T2D patients, and that LKB1 signaling prevents insulin resistance by inhibiting PD-1 expression and regulating the effector function of ILC2. These findings suggest that the LKB1-PD-1 axis is a new therapeutic target for metabolic diseases such as T2D.

[0186] The correspondence table of the abbreviations used in the present invention to the full names is shown in Table 1 below:

[0187] Table 1

[0188]

[0189]

[0190] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0191] As used in the present invention, the terms "include" and "comprising" are open expressions, that is, including the contents specified in the present invention, but not excluding other aspects.

[0192] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0193] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, any changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the claims.

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Claims

1. A biomarker LKB1-PD-1 signaling axis, characterized in that It includes the metabolic kinase "LKB1" that regulates various intracellular metabolic processes and the transmembrane protein "PD-1"; the "PD-1" is a co-receptor of the extracellular ligand molecule PD-L1; The gene sequence includes one or more of the following sequences: human LKB1 gene STK11 ID: NCBI Gene: 6794, Ensembl: ENSG00000118046, mouse LKB1 gene Stk11 ID: NCBI Gene: 20869, Ensembl: ENSMUSG00000003068, human PD-1 gene PDCD1 ID: NCBI Gene: 5133, Ensembl: ENSG00000188389, mouse PD-1 gene Pdcd1 ID: NCBI Gene: 18566, Ensembl: ENSMUSG00000026285; and / or, the amino acid sequence includes one or more of the following sequences: the human LKB1 sequence shown in SEQ ID NO. 1, the mouse LKB1 sequence shown in SEQ ID NO. 2, the human PD-1 sequence shown in SEQ ID NO. 3, and the mouse PD-1 sequence shown in SEQ ID NO.

4.

2. Use of the biomarker LKB1-PD-1 signaling axis according to claim 1 in the preparation of a drug for treating metabolic diseases including obesity and type 2 diabetes, characterized in that: The application uses the biomarker LKB1-PD-1 signaling axis as a target to prepare diagnostic reagents for the occurrence and / or severity of obesity and type 2 diabetes, or to prepare drugs for preventing and treating obesity and type 2 diabetes, or to prepare reagents for predicting the occurrence and / or severity of obesity and type 2 diabetes, or to prepare reagents for evaluating the prognosis of obesity and type 2 diabetes.

3. The use according to claim 2, characterized in that The sample for measuring the biomarker LKB1-PD-1 signaling axis is a sample obtained from adipose tissue of a patient; and the metabolic disease includes type 2 diabetes and insulin resistance caused by obesity.

4. The use according to claim 2, characterized in that The levels of LKB1 and PD-1 are determined at the protein or nucleic acid level. When the levels of LKB1 and PD-1 are determined at the protein level, the level of LKB1 is determined by flow cytometry, immunohistochemical staining, or immunoblotting to determine its total protein level and / or its phosphorylation level, and the level of PD-1 is determined by flow cytometry, immunohistochemical staining, or immunoblotting. When the levels of LKB1 and PD-1 are determined at the nucleic acid level, the levels of LKB1 and PD-1 are determined by quantifying the mRNA encoding LKB1 and PD-1.

5. A regulator of the biomarker LKB1-PD-1 signaling axis, characterized in that The regulators of the biomarker LKB1-PD-1 signaling axis include agonists of LKB1 and inhibitors or antagonists of PD-1; the regulators include small molecule compounds, small interfering RNA, short hairpin RNA, biological macromolecular antibodies, antisense nucleotides, and biologically active fragments or homologs of biological macromolecular antibodies; the regulators promote type 2 immune response, thereby inhibiting the occurrence and development of obesity and type 2 diabetes.

6. An antibody, characterized in that The antibody is an anti-PD-1 monoclonal antibody or an anti-PD-1 polyclonal antibody; and / or an anti-PD-1 ligand monoclonal antibody or an anti-PD-1 ligand polyclonal antibody; and / or, the antibody includes a monoclonal antibody anti-mouse PD-1 / CD279 antibody (RMP1-14), a monoclonal antibody anti-mouse PD-1 / CD279 antibody (29F.1A12), a monoclonal antibody anti-human PD-1 / CD279 antibody (J116), a monoclonal antibody anti-mouse PD-L1 (B7-H1) antibody (10F.9G2 TM ), monoclonal antibody anti-human PD-L1 (B7-H1) antibody (29E.2A3).

7. A drug / drug composition, characterized in that It comprises the regulator of the biomarker LKB1-PD-1 signaling axis according to claim 5 and / or the antibody according to claim 6.

8. A detection reagent / kit, characterized in that: It includes a primer or probe that hybridizes to the polynucleotide of the biomarker LKB1-PD-1 signaling axis as described in claim 1, or contains an antibody that can specifically bind to the protein of the biomarker LKB1-PD-1 signaling axis, or contains a compound that can bind to the biomarker LKB1-PD-1 signaling axis; the compound includes a small molecule compound.

9. Use of the biomarker LKB1-PD-1 signaling axis according to claim 1, the regulator of the biomarker LKB1-PD-1 signaling axis according to claim 5, the antibody according to claim 6, the drug / drug composition according to claim 7, and the detection reagent / kit according to claim 8, characterized in that: The applications include: use as a therapeutic target for treating obesity and type 2 diabetes, use in evaluating ILC2 dysfunction, use in early diagnosis of obesity and type 2 diabetes, use in diagnosis, prevention and treatment of type 2 diabetes, use in preparing drugs for treating and / or preventing insulin resistance and adipose tissue inflammatory diseases, use in preparing drugs for treating obesity and type 2 diabetes, use in evaluating the effector function of PD-1 upregulation on LKB1-deficient ILC2, use in evaluating the ability of anti-PD-1 antibody treatment to restore the number and effector function of adipose ILC2 in mice lacking LKB1, and / or use in evaluating the effectiveness of anti-PD-1 antibody blocking therapy for insulin resistance and type 2 diabetes in the body.

10. The use according to claim 9, characterized in that The regulator, the antibody, the drug / drug composition, and the detection reagent / kit can be used alone or in combination, and can also be used in combination with hypoglycemic drugs and / or other drugs.

11. ILC2-specific LKB1 deficiency mouse model R5 / +Stk11 f / f ILC2-induced LKB1 knockout mouse model Id2 Cre-ERT2 Stk11 f / f The application is characterized in that The applications include: evaluating the effect of LKB1 on the homeostasis maintenance and effector function regulation of adipose ILC2s, evaluating the role of PD-1 in inhibiting the homeostasis maintenance and effector function of adipose ILC2s, and using the regulator of the biomarker LKB1-PD-1 signaling axis in preparing diagnostic reagents for the occurrence and / or severity of obesity and type 2 diabetes, and / or preparing therapeutic drugs for obesity and type 2 diabetes, and / or preparing prognostic evaluation reagents for obesity and type 2 diabetes, and / or evaluating the applicability and effectiveness of targeting LKB1 and PD-1 and / or their ligands for the treatment of obesity and type 2 diabetes.

12. The use according to claim 11, characterized in that The model is based on the specific deletion of LKB1 in cells expressing or previously expressing IL-5 (R5 / +Stk11 f / f ) mice, in steady-state or diet-induced obesity and type 2 diabetes disease models.

13. A method for screening candidate drugs for treating obesity and type 2 diabetes and evaluating the prognosis of obesity and type 2 diabetes, characterized in that: The method includes detecting the effect of the candidate drug on the biological effects of the biomarker LKB1-PD-1 signaling axis or its downstream signal in the subject or a sample obtained from the subject, wherein, after using the candidate drug, the biological function of the biomarker LKB1-PD-1 signaling axis is upregulated, indicating that the candidate drug has the effect of treating obesity and type 2 diabetes, alleviating or preventing the occurrence of obesity and type 2 diabetes and / or improving the prognosis of obesity and type 2 diabetes.

14. A method for diagnosing obesity and type 2 diabetes in a subject, or a method for diagnosing obesity and type 2 diabetes in a subject in need thereof, or a method for preventing or treating obesity and type 2 diabetes in a subject in need thereof, or a method for assessing the severity of obesity and type 2 diabetes in a patient, or a method for predicting the risk of obesity and type 2 diabetes in a patient, or a method for predicting or prognosticating whether the patient is suitable for treatment targeting the biomarker LKB1-PD-1 signaling axis, characterized in that: The method includes: determining the levels of LKB1 and PD-1 / or their ligands in an adipose tissue sample from the patient or subject, wherein the level of the biomarker LKB1-PD-1 is used to prevent or treat obesity and type 2 diabetes in the patient or subject, or to assess the severity of the disease in a patient with obesity and type 2 diabetes, or to predict whether the patient will develop obesity and type 2 diabetes, or to predict or prognose whether the patient is suitable for treatment targeting the biomarker LKB1-PD-1 signaling axis or its ligand.

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