Application of branched-chain amino acids in the diagnosis and treatment of diabetic neuropathic pain

Abstract

The present invention provides the use of branched-chain amino acids in the preparation of drugs and diagnostic tools for alleviating diabetic lesions. The connection between branched-chain amino acids and diabetic neuropathy has been discovered, which is of great significance for the diagnosis and treatment of diabetic neuropathy: it provides a new, effective and reliable screening method for the early diagnosis of diabetic neuropathy, and also proposes a new possible treatment method for treating diabetic nerve pain and alleviating the suffering of patients.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular, to the application of branched-chain amino acids in the diagnosis and treatment of diabetic neuropathic pain. Background Art

[0002] Diabetes is the most common complex and comprehensive metabolic disease. The current incidence of diabetes in adults is as high as 12.8%, and this incidence is still rising year by year.

[0003] At the same time, long-term high blood sugar levels damage large and small blood vessels, further harming different tissues throughout the body to varying degrees. Therefore, diabetes is a disease with the most complications, with more than 100 types of complications. [Feldman EL, Callaghan BC, Pop-Busui R, Zochodne DW, Wright DE, Bennett DL, Bril V, Russell JW, Viswanathan V. Diabetic neuropathy. Nat Rev Dis Primers. 2019 Jun 13; 5(1): 42.]

[0004] Among them, diabetic polyneuropathy (DPN) is the most common complication of diabetes. About 50% of diabetic patients will eventually suffer from neuropathy, with symptoms such as numbness, chills, pain in the limbs, gastrointestinal dysfunction, etc., which seriously affect the quality of life of patients.

[0005] The latest "Expert Consensus on Diagnosis and Treatment of Diabetic Neuropathy (2021 Edition)" [Chinese Medical Association Diabetes Branch Neurological Complications Group. Expert Consensus on Diagnosis and Treatment of Diabetic Neuropathy (2021 Edition) [J]. Chinese Journal of Diabetes, 2021, 13(06): 540-557.] points out that the pathogenesis of diabetic neuropathy is still unclear, early diagnosis is difficult, and mainly relies on clinical symptoms. There is no special treatment method. The specific problems are as follows:

[0006] 1) The etiology and pathogenesis of diabetic neuropathy have not been fully elucidated. It is currently believed that it is mainly related to a series of pathological and physiological changes caused by hyperglycemia, lipid metabolism disorders and abnormal insulin signaling pathways. However, a large amount of clinical experience and research have shown that the occurrence of diabetic neuropathy is not strictly positively correlated with the course of diabetes. At the same time, for many patients with type 2 diabetes, good control of blood sugar cannot effectively slow down diabetic neuropathy. These phenomena show that blood sugar is not the key factor affecting diabetic neuropathy, and there are other ways to regulate the occurrence of diabetic neuropathy.

[0007] 2) In addition, the diagnosis of diabetic neuropathy is mainly based on clinical symptoms and signs. There are no diagnostic markers for diabetic neuropathy clinically. At the same time, the early clinical manifestations are often hidden and easy to be ignored. By the time a clinical diagnosis is made, it is often in an irreversible stage.

[0008] 3) Diabetic neuropathy is extremely harmful, but there is currently no special treatment for diabetic nerve damage. It mainly relies on improving lifestyle, controlling blood sugar, nourishing nerves, and managing pain with medication. Moreover, there is currently no strong evidence to support that blood sugar control and lifestyle intervention can improve diabetic neuropathy. A large number of diabetic patients suffer from pain, and severe neuralgia affects the quality of life of diabetic patients, including limited activity, depression, and impaired social function. Therefore, it is extremely important and urgent to find effective diagnosis and treatment methods! Summary of the invention

[0009] The present invention aims to overcome the above-mentioned defects. Starting from the perspective of metabolites, the metabolomics technology is used to detect the serum metabolite spectrum of diabetic patients with or without neuropathy in clinical practice, to find metabolites that can serve as risk factors to indicate the occurrence of diabetic neuropathy, and further propose means of intervention treatment.

[0010] The present invention provides an application of a small molecule compound, characterized in that:

[0011] The above-mentioned small molecule compound is selected from branched-chain amino acids;

[0012] The above application is: used for preparing medicine for alleviating diabetic lesions.

[0013] Furthermore, the application of a small molecule compound provided by the present invention is also characterized in that:

[0014] The above-mentioned diabetic lesions are symptoms of diabetic neuropathy.

[0015] Furthermore, the application of a small molecule compound provided by the present invention is also characterized in that:

[0016] The branched-chain amino acid is selected from at least one of leucine, isoleucine and valine.

[0017] Furthermore, the application of a small molecule compound provided by the present invention is also characterized in that:

[0018] The composition of branched-chain amino acids contained in the above-mentioned drugs is as follows:

[0019] Leucine:isoleucine:valine=2:0.5-2:0.5-2.

[0020] Furthermore, the application of a small molecule compound provided by the present invention is also characterized in that:

[0021] The above-mentioned application also includes: being used to make a screening tool, a prediction tool, or a diagnostic tool for diagnosing whether diabetic neuropathy occurs or not.

[0022] Furthermore, the application of a small molecule compound provided by the present invention is also characterized in that:

[0023] The above-mentioned screening tools, or prediction tools, or diagnostic tools may be test strips, test kits, diagnostic software, testing platforms, or testing analyzers.

[0024] Furthermore, the application of a small molecule compound provided by the present invention is also characterized in that:

[0025] The above diagnostic tool comprises a reagent for detecting the content of leucine, and / or isoleucine, and / or valine;

[0026] The diagnostic strategy of the above diagnostic tool is: when the test result shows that leucine, and / or isoleucine, and / or valine are down-regulated, the probability of diabetic neuropathy is determined to be high.

[0027] The quantitative indicator for the reduction is: not less than 20%.

[0028] Furthermore, the application of a small molecule compound provided by the present invention is also characterized in that:

[0029] The test object of the above test is the patient's ex vivo serum sample.

[0030] In addition, the present invention also provides a method for obtaining metabolic markers of diabetic neuropathy patients, characterized in that:

[0031] Ex vivo serum samples from patients with diabetes and diabetic neuropathy were screened for differential metabolites after non-targeted detection by nuclear magnetic resonance spectroscopy.

[0032] Functions and effects of the present invention:

[0033] In the study of the present invention, it was found that compared with diabetic patients, the serum levels of three branched-chain amino acids, namely leucine, isoleucine and valine, were significantly downregulated in patients with diabetic neuropathy (P<0.0001).

[0034] Subsequently, in the research of the present invention, through mouse experiments, it was further proved that supplementing branched-chain amino acids in feed can effectively alleviate the symptoms of diabetic neuropathy.

[0035] Therefore, the present invention not only discovered for the first time the correlation between branched-chain amino acids and diabetic neuropathy, providing a new, effective and reliable screening method for the early diagnosis of diabetic neuropathy, but also proposed a new treatment method for treating diabetic neuropathy and alleviating the suffering of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 , use nuclear magnetic resonance spectroscopy technology to non-targeted detect the metabolite spectrum of patient serum samples collected clinically.

[0037] They were divided into two groups: Diabetic: diabetic patients without neuropathy, n = 73; DPN: patients with diabetic neuropathy, clinical symptoms manifested as pain, n = 56.

[0038] Figure 2 , to detect the effect of supplementing branched-chain amino acids (BCAA) on alleviating diabetic neuropathy in mice.

[0039] Naive: wild-type C57 / BL6 mice fed a normal diet; DPN: mice fed a high-fat diet to induce diabetic neuropathic pain; DPN+BCAA: DPN mice treated with BCAA. Figure 2 A) and 0.4g( Figure 2 B) Stimulate mice and measure the frequency of mechanical pain withdrawal. DETAILED DESCRIPTION

[0040] 1. Research plan

[0041] 1.1 Subjects

[0042] According to clinical symptoms, the patients were divided into two groups: diabetic group (diabetic, n = 73) and diabetic neuropathy group (DPN, n = 56). The two groups of patients could exclude other factors such as age, gender, BMI, medication history, etc. The collection of samples was in accordance with ethical review and informed consent.

[0043] 1.2 Metabolomics analysis of clinical samples

[0044] Collect patient serum samples, add 800 μL of pre-cooled methanol to every 200 μL serum sample, and let stand overnight at -80°C. Repeat freezing and thawing with liquid nitrogen for 3 times, centrifuge at 4°C, 12000 rpm for 15 minutes, collect the supernatant, add 500 μL of pre-cooled 80% methanol to the precipitate, vortex for 1 minute, then centrifuge at 4°C, 12000 rpm for 15 minutes, collect the supernatant and mix it with the supernatant in the previous step; repeat once.

[0045] The collected metabolite extracts were initially concentrated and dried using an Eppendorf Concentrator plus vacuum concentrator, and then the samples were dried using Labconco The samples were dried thoroughly in a freeze dryer. The processed samples were analyzed by Bruker AVANCE III 600 MHz NMR spectrometer for metabolomics analysis. The data were processed by TOPSPIN (V3.6.0, Bruker Biospin, Germany) and AMIX software package (V3.8.3, Bruker Biospin).

[0046] 1.3 Mouse experiments

[0047] All C57 / BL6 mice were healthy, immunocompetent, with similar pain sensitivity, and had not received other drugs and tests, and met the ethical review of animal experiments. The mice were housed in individually ventilated cages without special microorganisms and provided with sufficient standard food and water. The food and water consumption of the animals was monitored regularly. Six-week-old male mice were fed a high-fat diet (HFD, 60% fat calories), and the control group was fed a control diet (10% fat calories) for 9 weeks. High-fat feeding induced type 2 diabetes in mice as determined by random blood glucose (>16.7mMol / L), intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT). The changes in mechanical pain behavior of the mice were regularly tested to determine the induction of diabetic neuropathic pain phenotype.

[0048] The diabetic neuropathy mice were divided into two groups, one group continued to be fed with a high-fat diet, and the other group was fed with a branched-chain amino acid mixture (leucine: isoleucine: valine = 2:1:1). Mechanical pain behavior tests were continued to observe the therapeutic effect of branched-chain amino acids.

[0049] Mechanical pain paw withdrawal frequency (PWF) determination: The mice were fasted for 12 hours in advance, and a single mouse was placed in a transparent acrylic box separated from other mice and fixed on a metal grid. After the mice adapted to be quiet for about 1 hour, 0.07g of Von Frey fiber was first used to vertically stimulate the central part of the sole of the mouse through the metal grid hole, and pressure was applied to make the fiber bend into an "S" shape for about 1.5 seconds. The stimulation was performed 10 times in total, with an interval of no less than 5 minutes each time, and the number of times the mouse withdrew its leg was recorded. Repeat the above steps with 0.4g of Von Frey fiber and record it. 0.07g represents low-intensity stimulation (allodynia, abnormal pain), and 0.4g represents the mechanical stimulation paw withdrawal frequency under high-intensity stimulation (hyperalgesia, hyperalgesia).

[0050] 2. Results

[0051] 2.1 Clinical patients with diabetic neuropathy have significantly downregulated serum branched-chain amino acids

[0052] The serum samples of 73 diabetic patients (Diabetic) and 56 diabetic neuropathy patients (DPN) were tested by NMR spectrometer in a non-targeted manner, and a total of 16 metabolites were screened, such as Figure 1 As shown, it was found that the three branched-chain amino acids were significantly downregulated in the serum of patients with diabetic neuropathy (P<0.0001).

[0053] 2.2 Branched-chain amino acid supplementation can effectively relieve pain in diabetic neuropathy mice

[0054] like Figure 2 As shown, mice with diabetic neuropathy were supplemented with branched-chain amino acids, and it was found that the symptoms of diabetic neuropathy in mice were significantly alleviated after 5-7 days of supplementation. After 9 days, the mice's response to mechanical pain stimulation almost returned to normal levels.

[0055] Figure 2 :To examine the effect of supplementation of branched-chain amino acids (BCAA) on the relief of diabetic neuropathy in mice. Naive: wild-type C57 / BL6 mice fed a normal diet; DPN: mice induced with diabetic neuropathy phenotype by high-fat diet feeding; DPN+BCAA: DPN mice treated with BCAA. Figure 2 A) and 0.4g( Figure 2 B) Stimulate mice and measure the frequency of mechanical pain withdrawal.

[0056] Functions and effects of this embodiment:

[0057] Diabetic neuropathy is the most common chronic complication of diabetes, and its pathogenesis and etiology have not yet been fully elucidated. Current research focuses on a series of pathological changes caused by disorders of sugar and lipid metabolism and abnormal insulin signaling pathways, as well as further large and small fiber lesions. We should clearly recognize the difference between diabetic complications and diabetes, as the pathogenic mechanisms of the two are not exactly the same.

[0058] As for diabetic neuropathy, a lot of clinical experience and research have shown that many patients with type 2 diabetes cannot effectively slow down diabetic neuropathy even if they control blood sugar well. There is no strong evidence to support that blood sugar control and lifestyle intervention can improve diabetic neuropathy. All these indicate that blood sugar is not the key factor affecting diabetic neuropathy, and there are other metabolic pathways that regulate the occurrence of diabetic neuropathy.

[0059] The present invention starts from the perspective of metabolites, collects serum samples from clinical diabetic patients (n=73) and diabetic neuropathy patients (n=56), uses nuclear magnetic resonance spectroscopy technology to non-targetedly detect the changes in different types of metabolites in blood samples, and screens out that three branched-chain amino acids (leucine, isoleucine, and valine) are significantly downregulated in the serum of diabetic neuropathy patients. Experiments on mice further show that supplementing branched-chain amino acids can indeed effectively alleviate the symptoms of diabetic neuropathy.

[0060] Therefore, the present invention provides clues to reveal the pathogenic mechanism of diabetic neuropathy. It is well known that branched-chain amino acids cause insulin resistance. Therefore, the content of the present invention may break the traditional understanding of branched-chain amino acids. Metabolites are not absolutely "good" or "bad". The roles and functions played by metabolites at different stages of the development of the disease are different.

[0061] For diabetic patients, perhaps high levels of branched-chain amino acids establish a new steady state, and unknown factors cause a decrease in branched-chain amino acid levels, which in turn leads to the occurrence of neuropathy.

[0062] Therefore, the present invention discovered the connection between branched-chain amino acids and diabetic neuropathy, and this discovery is of great significance for the diagnosis and treatment of diabetic neuropathy: clinical serum testing is relatively convenient, fast and reliable, and branched-chain amino acids may be used for clinical testing and early diagnosis in the future. This discovery fills the gap in this regard; it also provides a possible solution to the treatment problem of diabetic neuropathy.

Claims

1. An application of a small molecule compound, characterized in that: The small molecule compound is selected from branched-chain amino acids; The application is: for preparing a drug for relieving diabetic neuropathy; the composition of the branched-chain amino acids contained in the drug is as follows: Leucine:isoleucine:valine = 2:1:1.

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