Use of igfbp3 protein as intervention target for diabetic amyotrophy and use of NBI-31772 in preparation of drug for ameliorating diabetic amyotrophy

Abstract

Use of an IGFBP3 protein as an intervention target for diabetic amyotrophy and use of NBI-31772 in the preparation of a drug for ameliorating diabetic amyotrophy. In the use of the IGFBP3 protein as the intervention target for diabetic amyotrophy, the functional activity of the IGFBP3 protein is subjected to targeted inhibition, thereby ameliorating symptoms of diabetic amyotrophy and improving muscle strength and function. Furthermore, in another aspect, provided is the use of NBI-31772 in the preparation of a drug for ameliorating diabetic amyotrophy. By reducing the accumulation of the IGFBP3 protein in cells, NBI-31772 can effectively inhibit the occurrence of diabetic amyotrophy and improve muscle strength and function, while additionally improving insulin sensitivity, alleviating hyperlipidemia, and inhibiting fatty liver. The small molecule can be used as a clinical drug for diabetic amyotrophy, and can also be used in combination with clinical diabetic drugs to achieve comprehensive improvement of diabetes and related complications.

Description

Application of IGFBP3 protein as intervention target point of diabetic muscle atrophy and application of NBI-31772 in preparation of drug for improving diabetic muscle atrophy

[0001] Related applications

[0002] The present application claims priority to the Chinese patent application No. 202411091850.7, filed on August 09, 2024, entitled "Application of IGFBP3 protein as intervention target point of diabetic muscle atrophy", and the Chinese patent application No. 202411091849.4, filed on August 09, 2024, entitled "Application of NBI-31772 in preparation of drug for improving diabetic muscle atrophy", the contents of which are hereby incorporated by reference in their entirety. TECHNICAL FIELD

[0003] The present application belongs to the technical field of biological medicine, and particularly relates to the application of IGFBP3 protein as intervention target point of diabetic muscle atrophy and the application of NBI-31772 in preparation of drug for improving diabetic muscle atrophy. BACKGROUND

[0004] Diabetic muscle atrophy is one of the typical complications of diabetes, but unlike traditional cognition, the muscle pathology of diabetic patients is not an advanced complication of diabetes. The muscle of prediabetic patients begins to show mild atrophy, which can also be called muscle degradation or muscle loss, that is, the skeletal muscle index is low, but the muscle activity is normal. With age and other factors becoming more serious, it gradually develops into diabetic skeletal muscle disease, which is manifested as muscle atrophy and accompanied by muscle dysfunction. Since muscle loss and muscle atrophy are typical characteristics of the human aging process, they are often overlooked as complications in diabetic populations in clinical practice. The exact pathophysiological mechanism of muscle loss, atrophy and progressive muscle dysfunction caused by diabetes has not been fully understood, and there is a serious lack of clinical targeted treatment strategies.

[0005] Early studies have shown that the imbalance of protein synthesis and degradation in muscle cells is the key mechanism leading to the occurrence of muscle atrophy, and E3 ubiquitin ligases such as MuRF1 and Atrogin-1 associated with protein degradation are considered to be key effector molecules, and their expression is significantly increased in diabetic and obese patients. Insulin resistance, inflammation and other factors in diabetic patients are considered to be key factors for muscle disease, but this hypothesis has not been effectively supported by clinical studies. The effect of insulin sensitizers such as metformin on diabetic muscle atrophy is not consistent in various studies. Some studies have shown that the grip strength of elderly patients improved after receiving metformin treatment, but in another study, the grip strength of overweight patients with type 2 diabetes and chronic obstructive pulmonary disease decreased significantly after receiving metformin treatment for 24 weeks. Glucagon-like peptide-1 (GLP-1) receptor agonists such as liraglutide also have no consistent effect on skeletal muscle mass index in patients with type 2 diabetes. PPARγ agonists such as pioglitazone have no significant effect on muscle loss in patients with type 2 diabetes, and even in individuals without type 2 diabetes, taking pioglitazone was found to cause loss of thigh muscle in men. The above studies fully indicate that the existing technology has a serious lack of understanding of the pathogenesis of diabetic muscle atrophy, and the treatment of diabetic muscle atrophy lacks clear intervention targets.

[0006] Sherryline Jogie-Brahim et al. in the article of Endocrine reviews, doi: 10.1210 / er.2008-0028 have pointed out that IGFBP3 (Insulin-like growth factor-1 binding protein 3) is a secreted protein, and its main function is believed to be to bind IGF-1 protein (Insulin-like growth factor-1) in the blood circulation, protect IGF-1 from rapid degradation, and enable it to reach the target cells to activate downstream Akt and other signaling pathways through binding IGF-1R (IGF-1 receptor) to regulate protein synthesis, cell proliferation and survival, etc. Recent studies have shown that IGFBP3 may have IGF-1-independent functions in addition to IGF-1-dependent cell functions. During the occurrence of diabetic muscle atrophy, there is a large accumulation of IGFBP3 protein in muscle cells, so whether IGFBP3 protein can be used as an intervention target for diabetic muscle atrophy needs to be studied.

[0007] NBI-31772 (1-[(3,4-dioxocyclohexa-1,5-dien-1-yl)-hydroxymethylidene]-6,7-dihydroxy-2H-isoquinoline-3-carboxylic acid) is a non-selective inhibitor of IGFBP family proteins, CAS No. 374620-70-9, but whether it can be used as a clinical drug for muscle atrophy caused by diabetes has not been studied and reported. SUMMARY

[0008] In order to overcome the defects of the prior art, one aspect of the present application is to provide the application of IGFBP3 protein as a target for intervention of diabetic muscle atrophy, and the inhibition of the activity and cell localization of the protein can effectively improve the state of muscle atrophy. Also provided is a substance based on the application of IGFBP3 protein as a target for intervention of diabetic muscle atrophy, which silences, mutates or knocks out IGFBP3 or reduces the physiological activity of IGFBP3, and is applied in the preparation of a clinical drug for diabetic muscle atrophy. Finally, an animal model for screening of a drug for diabetic muscle atrophy is provided, which has typical symptoms of diabetic muscle atrophy and can be used for screening and verification of a clinical drug. The present application fully proves that the occurrence of diabetic muscle atrophy is closely related to the accumulation of IGFBP3 protein in muscle cells, and IGFBP3 protein can be used as a target for intervention of diabetic muscle atrophy.

[0009] In order to achieve the above-mentioned purpose, the technical solution of the present application is as follows:

[0010] The application of IGFBP3 protein as a target for intervention of diabetic muscle atrophy.

[0011] The IGFBP3 protein targets the functional activity thereof to improve the symptoms of diabetic muscle atrophy and improve muscle strength and function.

[0012] A substance based on the application of IGFBP3 protein as a target for intervention of diabetic muscle atrophy, which is applied in the preparation of a clinical drug for diabetic muscle atrophy, and the substance silences, mutates or knocks out IGFBP3 or reduces the physiological activity of IGFBP3.

[0013] The drug can reduce the expression level of IGFBP3 protein, including any one or several of the following:

[0014] (1) reducing the transcription level of IGFBP3 coding gene; (2) reducing the translation level of IGBFP3 protein; (3) increasing the degradation rate of IGFBP3 protein.

[0015] The drug can reduce or block the physiological function of IGFBP3 protein, or directly bind to IGFBP3 and reduce the binding of IGFBP3 to other proteins;

[0016] The drug reduces or blocks the physiological function of IGFBP3, specifically the drug reduces or blocks the entry of IGFBP3 protein into muscle cells;

[0017] The drug binds to IGFBP3 and reduces the binding of IGFBP3 to other proteins, specifically the binding of IGFBP3 to KPNB1, or the binding of IGFBP3 to FOXO1 and other muscle atrophy regulatory proteins.

[0018] The drug, which can be any one or more of the following types:

[0019] (1) polypeptide; (2) host cell, genetically engineered polypeptide or nucleotide; (3) small molecule compound; (4) polynucleotide encoding polypeptide, or polynucleotide capable of inhibiting the interaction of IGFBP3 with FOXO1, or the interaction of IGFBP3 with KPNB1, blocking the expression and / or function of IGFBP3, including vectors expressing polynucleotides; (5) protein, antibody, antisense oligonucleotide, siRNA, antisense expression vector, recombinant virus, other agents capable of blocking the interaction of IGFBP3 with FOXO1 and IGFB, or blocking the entry of IGFBP3 protein into cells.

[0020] The application also includes the use in animal models of diabetic muscle atrophy, and the use in animal models of diabetic muscle atrophy in which the expression of the SDHAF4 gene in adipose tissue is reduced or deleted, the expression of IGFBP3 in adipose tissue is increased, and the content of IGFBP3 in blood is increased.

[0021] The model can be used to screen candidate drugs that reduce the expression of IGFBP3, or to screen candidate drugs that reduce the symptoms of muscle atrophy, which are used for the treatment of diabetic muscle atrophy.

[0022] The animal is selected from at least one of mice, rats or primates.

[0023] Another aspect of the present application is to provide the use of NBI-31772 in the preparation of a drug for improving diabetic muscle atrophy, which can effectively inhibit the occurrence of diabetic muscle atrophy by reducing the accumulation of IGFBP3 protein in cells, and improve muscle strength and function. The small molecule can be used as a clinical drug for diabetic muscle atrophy, and can also be used in combination with clinical diabetes drugs to achieve comprehensive improvement of diabetes and related complications.

[0024] In order to achieve the above-mentioned purposes, the present application is realized by the following technical solutions:

[0025] Use of NBI-31772 in the preparation of a drug for improving diabetic muscle atrophy, wherein the NBI-31772 is a small molecule inhibitor that targets the activity and functional localization of IGFBP3 protein, reduces the accumulation of IGFBP3 protein in cells, and inhibits the occurrence of diabetic muscle atrophy.

[0026] The NBI-31772 can improve the reduction of muscle strength in a diabetic state.

[0027] The NBI-31772 can improve muscle strength and function in a diabetic state.

[0028] The NBI-31772 can improve the reduction of muscle strength in a diabetic state.

[0029] The NBI-31772 can improve insulin sensitivity and reduce blood sugar.

[0030] The NBI-31772 can improve the reduction of muscle strength in a diabetic state.

[0031] The NBI-31772 can reduce non-alcoholic fatty liver symptoms in a diabetic state.

[0032] Compared with the prior art, the present application has the following beneficial technical effects:

[0033] 1. The present application fully proves through experiments that the occurrence of diabetic muscle atrophy is closely related to the accumulation of IGFBP3 protein in muscle cells. The IGFBP3 protein is overexpressed and secreted by adipocytes during the development of diabetes, and the IGFBP3 protein in the blood further enters muscle cells. IGFBP3 protein binds to KPNB1 and FOXO1 to promote FOXO1 nuclear translocation, and further activates the expression of muscle atrophy-related genes, causing the occurrence and development of muscle atrophy. Inhibiting the function of IGFBP3 protein can effectively inhibit the occurrence of muscle atrophy, and the gene IGFBP3 can be used as a target for the preparation of a drug for treating diabetic muscle atrophy.

[0034] 2. Mice with adipose tissue-specific knockout of SDHAF4 exhibit overexpression and secretion of IGFBP3 by adipose tissue, increased blood IGFBP3 content, increased accumulation of IGFBP3 protein in muscle cells, and progressive pathological changes in muscle atrophy. The mice exhibit consistent pathological manifestations of diabetic muscle atrophy, and are effective models for screening drugs for diabetic muscle atrophy. Therefore, the adipose SDHAF4 knockout mouse is an effective model for screening drugs for diabetic muscle atrophy.

[0035] 3, NBI-31772 of the present application can effectively inhibit the occurrence of diabetic muscle atrophy, improve muscle strength and function by reducing the accumulation of IGFBP3 protein in cells, and can be used as a clinical drug for diabetic muscle atrophy. It can also be used in combination with clinical diabetes drugs to achieve comprehensive improvement of diabetes and related complications. NBI-31772 and modified compounds based thereon have good application prospects.

[0036] 4, The present application first confirms that NBI-31772 targets and inhibits the activity of IGFBP3 protein and reduces the accumulation of IGFBP3 in cells; NBI-31772 has a more specific and single inhibition target, and the small molecule has a short metabolic cycle, is easily excreted through urine, has less accumulation in the body, and has no side effects.

[0037] In summary, NBI-31772 can reduce the accumulation of IGFBP3 protein in cells, improve insulin sensitivity, reduce blood lipids, improve fatty liver symptoms, inhibit the occurrence of muscle atrophy, and improve muscle strength and function. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 is a schematic diagram of significant muscle atrophy in diabetic mice; Figure 1A is the oral glucose tolerance test result of 8-week-old db / db mice and control db / m mice; Figure 1B is the oral insulin tolerance test result of 8-week-old db / db mice and control db / m mice; Figure 1C is a schematic diagram of muscle strength of 4-week-old to 8-week-old db / db mice and control db / m mice detected by mouse grip strength meter; Figure 1D is a schematic diagram of muscle strength of 4-week-old and 8-week-old db / db mice and control db / m mice detected by mouse grip roller experiment; Figure 1E is a schematic diagram showing significant atrophic muscle fibers in the muscles of 8-week-old db / db mice and control db / m mice;

[0039] Figure 2 is a diagram showing that the expression of SDHAF4 in fat is decreased and the expression of IGFBP3 is increased in diabetic mice. Figure 2A is a diagram showing the mRNA level of IGFBP3 gene in white adipose tissue (iWAT) and brown adipose tissue (BAT) of 8-week-old db / db mice. Figure 2B is a diagram showing the protein content of IGFBP3 and SDHAF4 in white adipose tissue (iWAT) of 8-week-old db / db mice detected by Western blot. Figure 2C is a diagram showing the statistical analysis of the results of Figure 2B (n=6, p<0.001). Figure 2D is a diagram showing the protein content of IGFBP3 and SDHAF4 in brown adipose tissue (BAT) of 8-week-old db / db mice detected by Western blot. Figure 2E is a diagram showing the statistical analysis of the results of Figure 2D (n=6, p<0.01). Figure 2F is a diagram showing the immunohistochemical analysis of muscle sections of 8-week-old db / db mice and control db / m mice.

[0040] Figure 3 is a diagram showing that the increase of serum IGFBP3 protein promotes the occurrence of muscle atrophy. Figure 3A is a diagram showing that the grip strength of mice is significantly reduced (n=6, p<0.01) detected by a grip meter. Figure 3B is a diagram showing that the grip strength of mice is significantly reduced (n=6, p<0.01) detected by a rolling wheel experiment. Figure 3C is a diagram showing that IGFBP3 protein has binding ability with FOXO1, a key regulatory protein of muscle atrophy, and KPNB1 protein, which assists the nuclear translocation of proteins, suggesting that IGFBP3 can promote the nuclear translocation of FOXO1 protein through KPNB1. Figure 3D is a diagram showing the occurrence of muscle atrophy after 10 micrograms of IGFBP3 protein is injected into the tail vein of wild-type mice for 2 weeks.

[0041] Figure 4 is a diagram showing that fat knockout SDHAF4 mice exhibit consistent muscle atrophy pathology as diabetic mice. Figure 4A is a diagram showing the verification of the knockout efficiency of fat tissue-specific knockout SDHAF4 mice. Figure 4B is a diagram showing the IGFBP3 mRNA analysis of fat tissue-specific knockout SDHAF4 mice. Figure 4C is a diagram showing that the content of IGFBP3 protein in the serum of fat tissue-specific knockout SDHAF4 mice is significantly increased (n=3) detected by Western blot.

[0042] Figure 4D is a diagram showing that the grip strength of fat tissue-specific knockout SDHAF4 mice is significantly reduced at 10 weeks of age (n=6, p<0.001) detected by a grip meter. Figure 4E is a diagram showing that the grip strength of fat tissue-specific knockout SDHAF4 mice is significantly reduced at 10 weeks of age (n=6, p<0.001) detected by a rolling wheel experiment.

[0043] Figure 5 is a schematic diagram showing that fat knockout IGFBP3 improves the muscle atrophy status of a model mouse; Figure 5A is a schematic diagram showing that the grip strength meter test shows that the muscle strength of the mouse is significantly increased (n = 6, p < 0.001); Figure 5B is a schematic diagram showing that the rolling wheel test shows that the muscle strength of the mouse is significantly increased (n = 6, p < 0.001); Figure 5C is a schematic diagram showing that the real-time fluorescent quantitative PCR detects the expression of muscle atrophy marker genes; Figure 5D is a schematic diagram showing that HE staining of muscle sections shows that atrophic muscle fibers disappear;

[0044] Figure 6 is a structural formula of NBI-31772;

[0045] Figure 7 is a schematic diagram showing that NBI-31772 reduces the accumulation of IGFBP3 protein in muscle cells of diabetic mice; muscle section immunofluorescence analysis shows that the IGFBP3 protein content is significantly reduced after NBI-31772 treatment, wherein the red fluorescence represents IGFBP3 protein;

[0046] Figure 8 is a schematic diagram showing the improvement of NBI-31772 on muscle atrophy in diabetic mice; Figure 8A shows the improvement of muscle fiber structure in diabetic mice after 8 weeks of NBI-31772 treatment; Figure 8B shows that the protein degradation in muscle cells is significantly reduced in diabetic mice after 8 weeks of NBI-31772 treatment; Figure 8C shows that the fibrous protein content in muscle cells is significantly increased in diabetic mice after 8 weeks of NBI-31772 treatment; wherein TA represents the tibialis anterior muscle, GA represents the gastrocnemius muscle, and QC represents the quadriceps muscle;

[0047] Figure 9 is a schematic diagram showing the improvement of NBI-31772 on muscle function in diabetic mice; Figure 9A shows that the rolling wheel test confirms that the muscle strength is increased in diabetic mice after 8 weeks of NBI-31772 treatment; Figure 9B shows that the grip strength meter confirms that the forelimb strength is increased in diabetic mice after 8 weeks of NBI-31772 treatment;

[0048] Figure 10 is a schematic diagram showing that NBI-31772 improves the insulin sensitivity of diabetic mice; Figure 10A is the body weight of diabetic mice (db / db) after 4 weeks of NBI-31772 treatment; Figure 10B is the body weight of diabetic mice (db / db) after 8 weeks of NBI-31772 treatment; Figure 10C is the insulin tolerance test of diabetic mice (db / db) after 4 weeks of NBI-31772 treatment; Figure 10D is the insulin tolerance test of diabetic mice (db / db) after 8 weeks of NBI-31772 treatment;

[0049] Figure 11 is a diagram showing the improvement of blood lipid of diabetic mice by NBI-31772; Figure 11A is the change of total cholesterol content of diabetic mice after 8 weeks of treatment with NBI-31772; Figure 11B is the change of triglyceride content of diabetic mice after 8 weeks of treatment with NBI-31772;

[0050] Figure 12 is a diagram showing the improvement of fatty liver of diabetic mice by NBI-31772; Figure 12A is the change of liver weight of diabetic mice after 8 weeks of treatment with NBI-31772; Figure 12B is the change of HE staining of liver section of diabetic mice after 8 weeks of treatment with NBI-31772; wherein the white vacuoles in the section represent large lipid droplets, and the white dots represent small lipid droplets in the liver cells. DETAILED DESCRIPTION

[0051] The embodiments of the present application will be described in detail below with reference to the examples.

[0052] The temperature parameters in the present application, if not particularly limited, allow both constant temperature treatment and fluctuation within a certain temperature range. It should be understood that the constant temperature treatment allows fluctuation within the accuracy range controlled by the instrument. Fluctuation within the range of, for example, ±5°C, ±4°C, ±3°C, ±2°C, ±1°C is allowed. In various embodiments of the present application, the size of the serial numbers of the processes does not mean the order of execution, and the execution order of the processes should be determined according to their functions and inherent logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0053] Example 1

[0054] 1. Animal breeding and breeding

[0055] Db / db mice were purchased from Janvier Labs. Fat-specific knockout SDHAF4 mice were designed and bred by Cre / loxP system. Sdhaf4f / f mice were made by Beijing Boao Saitu Co., Ltd. using conventional homologous recombination in embryonic stem cells, which designed two sgRNAs to generate a ~3 kb chromosomal deletion (exons 1-2) at the Sdhaf4 site in the mouse genome. PCR and Southern blot methods were used to identify F1 mutants, and allelic cloning and sequencing were performed. All mice were backcrossed with C57BL / 6 mice for at least 7 generations. Fabp4-Cre mice were obtained from Jackson lab (018965). Mice were genotyped using custom TaqMan genotyping assays (GeneStar). Tail DNA was extracted using proteinase K lysis buffer (100 mM Tris-HCl (pH 8.0), 5 mM EDTA (pH 8.0), 200 mM sodium chloride, 0.2% SDS, 0.1 mg / ml proteinase K). The DNA was then analyzed by PCR.

[0056] 2. Rolling experiment

[0057] The motor coordination and balance of the mouse model were evaluated by the rolling experiment. Mice were trained on a rotating rod at a constant speed of 4 revolutions per minute for 3 consecutive days. On the fourth day, they received an accelerated rotating rod test. The test started at 4 revolutions per minute and gradually increased to 40 revolutions per minute over 5 minutes. During the test, if the experimental animal fell off the rotating rod or grabbed the rod twice in a row instead of trying to run on it, the test was terminated, and the time on the rod was recorded. Data were presented as the maximum value of the time the mouse stayed on the rotating rod (from three trials).

[0058] 3. Grip strength test

[0059] The maximum grip strength of the mice was measured using a Bioseb grip strength machine (BIO-GS3, Bioseb, Florida, USA). The mice were placed on the detection grid so that they could hold the grid with their front limbs or both front and hind limbs simultaneously. Then the tail of the mouse was gently pulled, pulling in the direction of the force detected by the device. When the mouse released the grid, the detector recorded its peak grip strength, and the grip strength was expressed in grams (g). Each mouse was tested three times, and the maximum value was recorded as the grip strength of the mouse.

[0060] 4. Real-time quantitative PCR

[0061] Total RNA was extracted from mouse tissues using TriPure Isolation Reagent (Roche, Basel, Switzerland) and reverse transcribed into cDNA using the iScript cDNA Synthesis Kit (BioRad, Hercules, CA, USA). Real-time quantitative PCR reactions were performed using iQ SYBR Green Supermix (BioRad) and data were analyzed using the CFX Connect real-time PCR detection system (BioRad).

[0062] 5. Glucose and insulin tolerance tests

[0063] Glucose tolerance tests (OGTT) and insulin tolerance tests (ITT) were performed according to published standard protocols. For OGTT, mice were fasted overnight and then given glucose (2 g / kg), and blood glucose levels were measured at 0, 15, 30, 45, 60, 90 and 120 minutes. For ITT, mice were fasted for 6 hours and then given insulin (0.7 U / kg for normal diet) intraperitoneally, and blood glucose levels were measured at 0, 15, 30, 45, 60, 90 and 120 minutes.

[0064] 6. Statistical analysis

[0065] Data are presented as mean ± SEM. Data were analyzed using Prism (GraphPad). Two-tailed Student's T test was used for pairwise comparisons. p less than 0.05 was considered significant.

[0066] Example 2

[0067] 1. Experimental materials

[0068] NBI-31772 was purchased from Tocris Bioscience; Cholesterol, triglyceride assay kit was purchased from Nanjing Jiancheng Bioengineering Institute; TRIzol reagent was purchased from Invitrogen; RNA reverse transcription kit, SYBR fluorescent dye were purchased from Dalian Baobio Company. RNA primer sequences were ordered and synthesized from Beijing Aoke Dingsheng Biological Technology Co., Ltd.

[0069] 2. Experimental animal feeding and model establishment

[0070] db / db male mice were purchased from Nanjing Jiqie Pharmaceutical Animal Co., Ltd. Mice were housed in a room with controlled temperature (22 to 28 degrees) and humidity (60%) and light maintained on a 12-h light, 12-h dark cycle. Mice were allowed free access to food and water during the experiment. The experimental mice were divided into two groups, each group of 8 mice. Two groups were: (1) control group, normal diet, intraperitoneal injection of normal saline every 3 days (6 mg / kg / day) (2) drug intervention group, normal diet, intraperitoneal injection of NBI-31772 every 3 days (6 mg / kg / day). The experimental period was 2 months, and insulin tolerance test was performed once at 4 weeks and 8 weeks of drug intervention, and muscle function detection such as rolling wheel test, grip strength test, gait analysis was performed at 8 weeks.

[0071] 3. Experimental methods

[0072] (1) Insulin tolerance test

[0073] Insulin tolerance test (ITT) was performed according to the published standard protocol. Mice were fasted for 6 hours, then intraperitoneally injected with insulin (0.7 U / kg for normal diet), and blood glucose levels were measured at 0, 15, 30, 45, 60, 90 and 120 minutes.

[0074] (2) Rolling wheel test

[0075] The motor coordination and balance of the mouse model were evaluated by the rolling wheel test. Mice were trained on a rotating rod at a constant speed of 4 revolutions per minute for 3 consecutive days. On the fourth day, they received an accelerated rotating rod test. The test started at 4 revolutions per minute and gradually increased to 40 revolutions per minute within 5 minutes. During the test, if the experimental animal fell off the rotating rod or grabbed the rod more than twice without attempting to run on it, the test was terminated, and the time on the rod was recorded. Data were presented as the maximum value of the time the mouse stayed on the rotating rod (from three trials).

[0076] (3) Grip strength test

[0077] The maximum grip strength of mice was measured using a Bioseb grip strength tester (BIO-GS3, Bioseb, Florida, USA). The mice were placed on the detection grid so that they could grab the grid with their front limbs or both front and hind limbs simultaneously. Then the tail of the mouse was gently pulled, pulling in the direction of the force detected by the device. When the mouse released the grid, the detector recorded its peak grip strength, and the grip strength was expressed in grams (g). Each mouse was tested three times, and the maximum value was recorded as the grip strength of the mouse.

[0078] (4) mRNA content detection

[0079] The reverse transcription RNA-real-time fluorescent quantitative PCR method is used for detection. The specific method is as follows:

[0080] RNA extraction: 50 mg of liver tissue was added with 800 μL of TRIzol reagent, and the liver tissue was manually ground and broken. After 5 min of shaking bed at room temperature, 200 μL of chloroform (1 / 5 of the total volume) was added to extract protein, and after 15 s of vigorous mixing, it was placed at room temperature for 15 min, and then centrifuged at 13,000 g at 4°C for 20 min. The upper aqueous phase was transferred to another RNase-Free EP tube, and an equal volume of isopropanol was added. After mixing and placing at room temperature for 15 min, it was centrifuged at 13,000 g at 4°C for 20 min, and the supernatant was discarded. 500 μL of pre-cooled 75% ethanol was added, mixed well, and centrifuged at 13,000 g at 4°C for 10 min. The supernatant was discarded, and the ethanol was completely volatilized. It was dissolved in 10-20 μL of DEPC water. The concentration was determined by ultraviolet spectrophotometry, and it was used for reverse transcription.

[0081] RNA reverse transcription: The transcription volume was 20 μL, 500 ng of RNA was taken out, 4 μL of 5X Master Mix was added, DEPC water was added to 20 μL, and it was incubated at 37°C for 60 min and treated at 80°C for 15 s. It was placed at -20°C for standby.

[0082] Real-time fluorescent quantitative PCR (Real-time PCR): SYBR Green method was used, and the reaction system included 1 μL of cDNA, 5 μL of 2X Premix Ex Taq TM II, 0.5 μL of upstream and downstream primer mixture (10 μM), and sterile water was added to 10 μL. The reaction conditions were as follows: 95°C for 10 min, 40 cycles of PCR (each cycle included 95°C for 30 s, 55°C for 30 s, and 72°C for 20 s), and finally the melting curve was observed (95°C for 15 s, 60°C for 15 s, and 95°C for 15 s).

[0083] (5) Statistical analysis

[0084] The data is expressed as mean ± SEM. The data was analyzed by Prism (GraphPad). Two-tailed Student's T test was used for pairwise comparison. Significant statistical significance is *p<0.05, **p<0.01.

[0085] The relevant results of the experiments of the present application examples 1-2 are given below:

[0086] I. Significant muscle atrophy in diabetic mice

[0087] Db / db mice are a classic mouse model of diabetes mellitus in scientific research, which has been widely used in basic pathology and drug screening studies. Mice show a significant decrease in glucose metabolism and insulin sensitivity at the age of 8 weeks. At the same time, the muscle strength of the mice also shows a progressive change, which is not different from that of normal mice at the age of 4-7 weeks, but the muscle strength of db / db mice at the age of 8 weeks is significantly reduced, and muscle sections show a large number of atrophic muscle fibers, suggesting that diabetic db / db mice at the age of 8 weeks have significant diabetic muscle atrophy. As shown in Figure 1, Figure 1A is the oral glucose tolerance test result of 8-week-old db / db mice and control db / m mice, showing that the glucose metabolism of db / db mice is significantly decreased (n=6, p<0.001). Figure 1B is the oral insulin tolerance test result of 8-week-old db / db mice and control db / m mice, showing that the insulin sensitivity of db / db mice is significantly decreased (n=6, p<0.01); Figure 1C is a schematic diagram of the muscle strength of 4-week-old to 8-week-old db / db mice and control db / m mice detected by mouse grip strength meter, showing that the grip strength of 8-week-old db / db mice is significantly reduced (n=6, p<0.001); Figure 1D is a schematic diagram of the muscle strength of 4-week-old and 8-week-old db / db mice and control db / m mice detected by mouse grip roller experiment, showing that the grip strength of 8-week-old db / db mice is significantly reduced (n=6, p<0.001); Figure 1E is a schematic diagram of muscle sections of 8-week-old db / db mice and control db / m mice showing that db / db mice have significant atrophic muscle fibers.

[0088] II. Diabetic mice secrete IGFBP3 from fat and accumulate in muscle cells.

[0089] In the study of muscle pathology, adipose tissue has been considered as an important peripheral factor in regulating muscle pathology, but it has not been deeply studied in the pathogenesis of diabetic muscle atrophy. The present application identifies a significantly increased secreted protein IGFBP3 in white adipose tissue and brown adipose tissue, and the expression of the protein is increased due to the decreased expression of SDHAF4 gene. IGFBP3 protein is considered to play a basic function in blood circulation, and the present application first discovers that the protein can accumulate in muscle cells of diabetic mice, and the present application speculates that the secretion of IGFBP3 protein from adipocytes and the accumulation in muscle cells may be a key potential mechanism for promoting the onset of diabetic muscle atrophy, as shown in Figure 7, Figure 7A is a schematic diagram of the mRNA level of IGFBP3 gene in white adipose tissue (iWAT) and brown adipose tissue (BAT) of 8-week-old db / db mice, showing that the expression of IGFBP3 in adipose tissue of db / db mice is significantly increased (n = 6, p < 0.01); Figure 7B is a Western blot detection of the protein content of IGFBP3 and SDHAF4 in white adipose tissue (iWAT) of 8-week-old db / db mice, showing that the protein content of IGFBP3 in white adipose tissue of db / db mice is significantly increased, and the protein content of SDHAF4 is significantly decreased; Figure 7C is a statistical analysis of the results of Figure 7B (n = 6, p < 0.001); Figure 7D is a Western blot detection of the protein content of IGFBP3 and SDHAF4 in brown adipose tissue (BAT) of 8-week-old db / db mice, showing that the protein content of IGFBP3 in brown adipose tissue of db / db mice is significantly increased, and the protein content of SDHAF4 is significantly decreased; Figure 7E is a statistical analysis of the results of Figure 7D (n = 6, p < 0.01); Figure 7F is a schematic diagram of immunohistochemical analysis of muscle sections of 8-week-old db / db mice and control db / m mice, showing that the protein content of IGFBP3 in muscle cells of db / db mice is significantly increased.

[0090] III. Elevated serum IGFBP3 protein promotes the occurrence of muscle atrophy.

[0091] To confirm that the increase of IGFBP3 protein can induce muscle atrophy, the present application expresses and purifies mouse IGFBP3, and then injects 10 micrograms of IGFBP3 protein into the tail vein of normal mice. After 2 weeks of injection, the present application finds that the muscle strength of normal mice is significantly reduced, and the expression of the muscle atrophy marker genes Atrogin-1 and MuRF-1 is significantly increased, suggesting that IGFBP3 protein can directly promote the occurrence of muscle atrophy. At the same time, FOXO1 protein is the core factor regulating muscle atrophy, and is also the transcriptional regulator of Atrogin-1 and MuRF-1 genes. The activation of FOXO1 protein is a direct factor for the occurrence of muscle atrophy. The present application first finds that IGFBP3 protein can bind to FOXO1, and the present application speculates that IGFBP3 activates the transcriptional activity of FOXO1 by binding to FOXO1, thereby inducing the occurrence of muscle atrophy. As shown in Figure 8, Figure 8A is a schematic diagram showing that the grip strength of wild-type mice was significantly reduced after injection of 10 micrograms of IGFBP3 protein into the tail vein for 2 weeks (n = 6, p < 0.01); Figure 8B is a schematic diagram showing that the grip strength of wild-type mice was significantly reduced after injection of 10 micrograms of IGFBP3 protein into the tail vein for 2 weeks (n = 6, p < 0.01); Figure 8C is a Pulldown experiment confirming that IGFBP3 protein has binding ability with the key regulatory protein FOXO1 of muscle atrophy and the KPNB1 protein assisting the nuclear transport of FOXO1 protein, suggesting that IGFBP3 can promote the nuclear transfer of FOXO1 protein through KPNB1; Figure 8D is a schematic diagram showing that the expression of the key marker genes Atrogin-1 and MuRF-1 of muscle atrophy in mice was significantly increased (n = 6, p < 0.01) after injection of 10 micrograms of IGFBP3 protein into the tail vein of wild-type mice for 2 weeks, indicating the occurrence of muscle atrophy.

[0092] IV. Fat knockout SDHAF4 mice exhibit muscle atrophy consistent with diabetic mice.

[0093] In the previous study of db / db diabetic mice, the present application found that the decrease of adipose SDHAF4 could promote the expression of IGFBP3, and further verified this phenomenon. The present application constructed a mouse with adipose tissue SDHAF4 knockout, and the results showed that the gene expression of IGFBP3 in SDHAF4 adipose knockout mice was indeed significantly increased, and the protein content of IGFBP3 in the blood was also significantly increased, which suggested that the SDHAF4 adipose mouse increased the secretion of IGFBP3 protein. Subsequently, the present application detected the muscle strength of the mouse and found that the muscle strength of the SDHAF4 adipose knockout mouse was significantly decreased at the age of 10 weeks, suggesting that adipose SDHAF4 presented consistent muscle atrophy pathology with diabetic mice, and the knockout mouse was a good animal model for screening drugs for diabetic muscle atrophy. As shown in Figure 9, Figure 9A is a schematic diagram of the knockout efficiency verification of adipose tissue-specific SDHAF4 knockout mice, showing that the SDHAF4 mRNA of brown adipose tissue and white adipose tissue is significantly reduced (n = 6, p < 0.01); Figure 9B is a schematic diagram of IGFBP3 mRNA analysis of adipose tissue-specific SDHAF4 knockout mice, showing that the mRNA expression of IGFBP3 in white adipose tissue and brown adipose tissue of the knockout mice is significantly increased (n = 6, p < 0.01); Figure 9C is a schematic diagram of Western blot analysis showing that the content of IGFBP3 protein in the serum of adipose tissue-specific SDHAF4 knockout mice is significantly increased (n = 3); Figure 9D is a schematic diagram of grip strength meter detection showing that the grip strength of adipose tissue-specific SDHAF4 knockout mice is significantly decreased at the age of 10 weeks (n = 6, p < 0.001); Figure 9E is a schematic diagram of the rolling wheel experiment showing that the grip strength of adipose tissue-specific SDHAF4 knockout mice is significantly decreased at the age of 10 weeks (n = 6, p < 0.001).

[0094] Five, adipose knockout IGFBP3 improves the muscle atrophy state of model mice.

[0095] To further verify that IGFBP3 is an effective target for intervention and treatment of diabetic muscle atrophy, the present application further knocks out IGFBP3 in SDHAF4 adipose knockout mice to achieve inhibition of IGFBP3 expression. Analysis results show that simultaneous knockout of SDHAF4 and IGFBP3 in adipose tissue can effectively increase the muscle mass of mice, and the expression of muscle atrophy marker genes in the muscle of mice is also significantly decreased, and muscle tissue sections show that atrophic muscle fibers disappear in SDHAF4 and IGFBP3 knockout mice, and these experimental evidences not only further prove that the SDHAF4 adipose knockout mouse is a better diabetic muscle atrophy screening model, but also prove that IGFBP3 is an effective target for treatment of diabetic muscle atrophy, as shown in FIG. 10, A in FIG. 10 is a schematic diagram of simultaneous knockout of IGFBP3 in adipose tissue-specific SDHAF4 knockout mice, grip strength meter detection shows that the muscle strength of mice is significantly increased (n = 6, p < 0.001); B in FIG. 10 is a schematic diagram of simultaneous knockout of IGFBP3 in adipose tissue-specific SDHAF4 knockout mice, the roller experiment shows that the muscle strength of mice is significantly increased (n = 6, p < 0.001); C in FIG. 10 is a schematic diagram of real-time fluorescent quantitative PCR detection of muscle atrophy marker genes in adipose tissue-specific SDHAF4 knockout mice, the results show that the expression of muscle atrophy key genes is significantly decreased (n = 6, p < 0.01); D in FIG. 10 is a schematic diagram of HE staining of muscle sections in adipose tissue-specific SDHAF4 knockout mice, showing that atrophic muscle fibers disappear.

[0096] Six, NBI-31772 can reduce the accumulation of IGFBP3 protein in cells

[0097] IGFBP3 protein mainly exists in blood circulation, and the content of IGFBP3 protein in cells under normal conditions is less. As shown in FIG. 7, immunofluorescence analysis of muscle sections shows that there is significant accumulation of IGFBP3 protein in the tibialis anterior muscle and gastrocnemius muscle cells of diabetic mice (red fluorescence signal represents IGFBP3 protein), and the db / db mice used in the experiment are a mouse model commonly used in basic scientific research and clinical drug research of diabetes, and are a classic model for scientific research of diabetes. After 8 weeks of NBI-31772 treatment, the protein content of IGFBP3 in the muscle is significantly reduced. These data confirm that NBI-31772 can inhibit the functional activity of IGFBP3 entering the cells.

[0098] Seven, NBI-31772 can improve the symptoms of diabetic muscle atrophy

[0099] Muscle atrophy is a typical complication of diabetes, and the protein degradation of muscle cells is increased, and the muscle content is reduced. As shown in Figure 8A, the muscle sections of db / db mice show that there are a large number of atrophic muscle fibers in various muscles, and after 8 weeks of NBI-31772 treatment, the atrophic muscle fibers obviously disappeared, and the muscle structure was normal. As shown in Figure 8B, after 8 weeks of NBI-31772 treatment, the expression of key genes regulating muscle atrophy in muscle cells was significantly decreased. As shown in Figure 8C, the content of key fibrous protein MYHC in muscle cells was significantly increased after 8 weeks of NBI-31772 treatment, suggesting an increase in muscle mass. These data all confirm that NBI-31772 can significantly improve the symptoms of muscle atrophy.

[0100] Eight, NBI-31772 can improve muscle strength in diabetic state

[0101] The main occurrence of diabetic muscle atrophy is accompanied by a decrease in muscle strength, which is a key factor leading to various mechanical injuries and accidents. As shown in Figure 9A, the rolling wheel experiment shows that after 8 weeks of NBI-31772 treatment, the body strength and balance ability of diabetic mice are significantly increased. As shown in Figure 9B, the grip strength test shows that after 8 weeks of NBI-31772 treatment, the muscle strength of the forelimbs of mice is significantly increased. These data all confirm that NBI-31772 can significantly improve muscle strength and body balance after acting.

[0102] The above experimental results prove that NBI-31772 can inhibit the accumulation of IGFBP3 protein in cells, reduce the activity of IGFBP3 protein, improve insulin sensitivity, reduce blood lipids, improve fatty liver, inhibit the progression of diabetic muscle atrophy, improve muscle strength and function, and achieve the relief and improvement of diabetic muscle atrophy.

[0103] Nine, NBI-31772 improves insulin sensitivity in diabetic mice

[0104] db / db mice are a mouse model commonly used in basic scientific research and clinical drug development of diabetes, and are a classic model for studying diabetes in the scientific community. As shown in Figure 10, db / db mice were divided into two groups, one group was a control group, and the other group was a drug intervention group, and the drug intervention was a total of 8 weeks, of which the body weight difference and insulin sensitivity of the mice were detected at the fourth week. As shown in Figure 10, the intervention of the compound had no effect on the body weight of the mice at 4 weeks (Figure 10A) and 8 weeks (Figure 10B). However, the insulin sensitivity of the mice was significantly improved after 4 weeks of drug intervention (Figure 10C), and the insulin sensitivity was also improved after 8 weeks of drug intervention (Figure 10D). This shows that NBI-31772 can quickly and continuously improve insulin sensitivity and has the effect of reducing blood glucose.

[0105] X. Improvement of hyperlipidemia by NBI-31772 in diabetic mice

[0106] A significant indicator of diabetes is the significantly elevated cholesterol and triglyceride levels in the blood. As shown in Figure 11 A, the total cholesterol level in the blood was significantly reduced after 8 weeks of NBI-31772 treatment. Figure 11 B shows that the triglyceride level in the blood was also significantly reduced after 8 weeks of NBI-31772 treatment. These results suggest that NBI-31772 can significantly improve the hyperlipidemia symptoms associated with diabetes.

[0107] XI. NBI-31772 can improve the symptoms of non-alcoholic fatty liver disease

[0108] Obesity and diabetes are also associated with the development of non-alcoholic fatty liver disease. As shown in Figure 12 A, the liver weight was significantly reduced after 8 weeks of NBI-31772 treatment. As shown in Figure 12 B, the HE staining of liver sections showed that db / db mice had excessive lipid droplet accumulation in the liver, while the liver lipid droplets were significantly reduced after 8 weeks of NBI-31772 treatment. These data demonstrate that NBI-31772 can significantly improve the symptoms of fatty liver disease.

Claims

1. Use of IGFBP3 protein as a target for intervention of diabetic muscle atrophy.

2. The use of IGFBP3 protein as an intervention target for diabetic muscle atrophy according to claim 1, characterized in that, The IGFBP3 protein targets the functional activity thereof, improves the symptoms of diabetic muscle atrophy, and enhances muscle strength and function.

3. Substances based on the use of the IGFBP3 protein according to claim 1 as an intervention target for diabetic muscle atrophy, characterized in that, The substance is used in the preparation of a clinical drug for diabetic muscle atrophy, which silences, mutates or knocks out IGFBP3 or reduces the physiological activity of IGFBP3.

4. The substance for use according to claim 3, wherein the IGFBP3 protein is used as a target for intervention in diabetic muscle atrophy. The drug can reduce the expression level of IGFBP3 protein, including any one or several of the following: (1) reducing the transcription level of IGFBP3 coding gene; (2) reducing the translation level of IGBFP3 protein; (3) increasing the degradation rate of IGFBP3 protein.

5. The use of the IGFBP3 protein as a target for intervention in diabetic muscle atrophy according to claim 3, characterized in that, The drug can reduce or block the physiological function of IGFBP3 protein, or directly bind to IGFBP3 and reduce the binding of IGFBP3 to other proteins. The drug reduces or blocks the physiological function of IGFBP3, specifically the drug reduces or blocks the entry of IGFBP3 protein into muscle cells. The drug binds to IGFBP3 and reduces the binding of IGFBP3 to other proteins, specifically the binding of IGFBP3 to KPNB1, or the binding of IGFBP3 to FOXO1 and other muscle atrophy regulatory proteins.

6. The use of the IGFBP3 protein as a target for intervention in diabetic muscle atrophy according to claim 3, characterized in that, The drug, which can be any one or more of the following: (1) polypeptide; (2) host cell, genetically engineered polypeptide or nucleotide; (3) small molecule compound; (4) polynucleotide encoding polypeptide, or polynucleotide capable of inhibiting the interaction of IGFBP3 with FOXO1, or the interaction of IGFBP3 with KPNB1, blocking IGFBP3 expression and / or function, including vectors expressing polynucleotides; (5) protein, antibody, antisense oligonucleotide, siRNA, antisense expression vector, recombinant virus, and other agents capable of blocking the interaction of IGFBP3 with FOXO1 and IGFB, or blocking the entry of IGFBP3 protein into cells.

7. The use of IGFBP3 protein as an intervention target for diabetic muscle atrophy according to claim 1, characterized in that, It also includes the use in animal models of diabetic muscle atrophy, in which the expression of adipose tissue SDHAF4 gene is decreased or deleted, the expression of adipose tissue IGFBP3 is increased, and the content of blood IGFBP3 is increased.

8. The use of IGFBP3 protein as a target for intervention of diabetic muscle atrophy according to claim 7, characterized in that, The model can be used to screen candidate drugs that reduce IGFBP3 expression, or to screen candidate drugs that reduce muscle atrophy symptoms, which are used for the treatment of diabetic muscle atrophy.

9. The use of IGFBP3 protein as an intervention target for diabetic muscle atrophy according to claim 7, characterized in that, The animal is selected from at least one of mice, rats or primates.

10. Use of NBI-31772 for the preparation of a medicament for ameliorating diabetic muscle atrophy, characterized in that, The NBI-31772 is a small molecule inhibitor that targets the activity and function of IGFBP3 protein, reduces the accumulation of IGFBP3 protein in cells, and inhibits the occurrence of diabetic muscle atrophy.

11. Use of NBI-31772 according to claim 10 for the preparation of a medicament for improving diabetic muscle atrophy, characterized in that, The NBI-31772 can improve the decline of muscle strength in diabetic state.

12. Use of NBI-31772 according to claim 10 for the preparation of a medicament for improving diabetic muscle atrophy, characterized in that, The NBI-31772 can enhance muscle strength and function in diabetic state.

13. Use of NBI-31772 according to claim 10 for the preparation of a medicament for improving diabetic muscle atrophy, characterized in that, Compounds based on the structure modification and modification of NBI-31772, as long as they can inhibit the activity and intracellular localization of IGFBP3 protein, can be used to prepare drugs for improving diabetic muscle atrophy.

14. Use of NBI-31772 according to claim 10 for the preparation of a medicament for improving diabetic muscle atrophy, characterized in that, The NBI-31772 can improve insulin sensitivity and reduce blood sugar.

15. Use of NBI-31772 according to claim 10 for the preparation of a medicament for improving diabetic muscle atrophy, characterized in that, The NBI-31772 can improve hyperlipidemia in a diabetic state.

16. Use of NBI-31772 according to claim 10 for the preparation of a medicament for improving diabetic muscle atrophy, characterized in that, The NBI-31772 can reduce non-alcoholic fatty liver symptoms in a diabetic state.

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