Tyrosine-containing marker and application thereof in diagnosis of diabetic peripheral neuralgia

Abstract

The invention belongs to the technical field of diagnostic markers, and particularly relates to a tyrosine-containing marker and application thereof in diagnosis of diabetic peripheral neuralgia. The invention provides a new approach for diagnosing, evaluating, preventing and treating diabetic peripheral neuralgia based on tyrosine, and the tyrosine is found to be abnormally reduced in a DPNP patient and a related animal model and is closely related to occurrence and development of the peripheral neuralgia. Therefore, a new biomarker and a potential intervention means are provided for early recognition, risk assessment and targeted intervention of DPNP.

Description

Technical Field

[0001] This invention belongs to the field of diagnostic biomarker technology, specifically relating to a tyrosine-containing biomarker and its application in the diagnosis of diabetic peripheral neuropathy. Background Technology

[0002] Type 2 diabetes mellitus (T2DM) is a systemic chronic metabolic syndrome. Long-term hyperglycemia and metabolic disorders affect various tissues, leading to a variety of chronic complications. Diabetic peripheral neuropathic pain (DPNP) is one of the most common chronic complications of T2DM, with more than 50% of T2DM patients experiencing DPNP symptoms. The main symptom of DPNP is bilateral symmetrical distal limb pain, severely impacting patients' physical and mental well-being, reducing their quality of life and work, and placing a heavy burden on society and families. Multiple domestic and international DPNP diagnosis and treatment guidelines and expert consensus indicate that DPNP lacks effective early diagnosis and specific treatment methods; its prevention and treatment mainly rely on good lifestyle interventions, intensive glycemic control, and pain medication. Developing biomarkers and preventative measures for DPNP is extremely important and urgent.

[0003] Current treatments for diabetic peripheral neuropathy primarily include symptomatic treatments such as antidepressants, antiepileptic drugs, and opioid analgesics. However, these treatments generally suffer from limited efficacy, significant adverse reactions, large individual variability, and difficulty in halting disease progression. Furthermore, effective early diagnostic indicators and etiological intervention targets are lacking. Therefore, identifying endogenous metabolites closely related to the development and progression of diabetic peripheral neuropathy, and possessing potential diagnostic and therapeutic value, is crucial for the precise diagnosis and treatment of this disease.

[0004] Existing research suggests that the amino acid metabolic profile is significantly altered in diabetes, but the specific characteristics of tyrosine changes in diabetic peripheral neuropathy and its relationship with pain intensity and neurological function impairment remain unclear. Furthermore, whether abnormal tyrosine metabolism directly participates in the development and progression of diabetic peripheral neuropathy in animal experiments, and its feasibility as a potential diagnostic biomarker or intervention target, still lacks systematic research and translational applications. Therefore, conducting systematic research on the characteristics of tyrosine changes in diabetic peripheral neuropathy and its application value is of great significance for promoting the early diagnosis and development of novel treatment strategies for this disease. Summary of the Invention

[0005] To address the above problems, the present invention provides a diagnostic biomarker, including tyrosine.

[0006] The present invention also provides the application of the above-mentioned diagnostic markers in the preparation of products for diagnosing diabetic peripheral neuropathy.

[0007] The present invention has the following beneficial effects:

[0008] This invention provides a novel approach for the diagnosis, assessment, and prevention of diabetic peripheral neuropathy (DPNP) based on tyrosine. By discovering that tyrosine levels are abnormally low in DPNP patients and related animal models and are closely related to the occurrence and development of peripheral neuropathy, this invention provides new biomarkers and potential intervention methods for the early identification, risk assessment, and targeted intervention of DPNP. Attached Figure Description

[0009] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. 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 creative effort.

[0010] Figure 1 A unidimensional test was used to plot a volcano plot of differential metabolites between DPNP patients and T2DM patients.

[0011] Figure 2 The concentrations of tyrosine in peripheral blood of the healthy group (n = 60), the T2DM group (n = 103), and the DPNP group (n = 111) were compared. One-way ANOVA was used, and multiple correction tests were performed using the Bonferroni method. The values ​​in the figure are mean ± standard error (mean ± SEM), and ns (no significance) indicates no significant difference.

[0012] Figure 3 ROC curve of decreased tyrosine levels predicting the risk of DPNP.

[0013] Figure 4 Tyrosine deficiency led to an increased frequency of paw withdrawal in db / db mice stimulated with 0.07 g von Frey filaments, indicating mechanical hyperalgesia; tyrosine supplementation had the opposite effect. Mice in the db / m and db / db groups were fed a standard tyrosine concentration of 4 g / kg, the db / db-low Tyr group received 2 g / kg, and the db / db-high Tyr group received 8 g / kg. One-way ANOVA was performed, and multiple correction tests were conducted using the Bonferroni method. Values ​​in the figure are mean ± standard error (mean ± SEM), with n = 6 mice in each group.

[0014] Figure 5Tyrosine deficiency led to increased paw withdrawal frequency in db / db mice stimulated with 0.4 g von Frey filaments, indicating mechanical hyperalgesia; tyrosine supplementation had the opposite effect. Mice in the db / m and db / db groups were fed a standard tyrosine concentration of 4 g / kg, the db / db-low Tyr group received 2 g / kg, and the db / db-high Tyr group received 8 g / kg. One-way ANOVA was used, and multiple correction tests were performed using the Bonferroni method. Values ​​in the figure are mean ± standard error (mean ± SEM), with n = 6 mice in each group.

[0015] Figure 6 Tyrosine deficiency led to a shortened paw withdrawal latency in db / db mice to noxious thermal stimuli, indicating hyperalgesia of heat pain; tyrosine supplementation had the opposite effect. The tyrosine content in the diets of db / m and db / db mice was the standard concentration of 4 g / kg, the db / db-low Tyr group was 2 g / kg, and the db / db-high Tyr group was 8 g / kg. One-way ANOVA was used, and multiple correction tests were performed using the Bonferroni method. The values ​​in the figure are mean ± standard error (mean ± SEM), and n = 6 mice per group.

[0016] Figure 7 Tyrosine deficiency led to a shortened paw withdrawal latency in db / db mice to noxious cold stimuli, indicating hyperalgesia for cold pain; tyrosine supplementation had the opposite effect. The tyrosine content in the diets of db / m and db / db mice was the standard concentration of 4 g / kg, the db / db-low Tyr group was 2 g / kg, and the db / db-high Tyr group was 8 g / kg. One-way ANOVA was used, and multiple correction tests were performed using the Bonferroni method. The values ​​in the figure are mean ± standard error (mean ± SEM), with n = 6 mice in each group.

[0017] Figure 8 Example image of whole-cell patch-clamp recording of primary DRG neurons after tyrosine deficiency treatment.

[0018] Figure 9 Tyrosine deficiency treatment significantly increased the resting membrane potential of primary DRG neurons in db / db mice. The control group received tyrosine at a standard concentration of 0.39 mM, while the deficiency group received 0.13 mM for 24 h. Unpaired t-tests were used to test for normality and homogeneity of variance. The values ​​in the figure are mean ± standard error (mean ± SEM), n = 15.

[0019] Figure 10Tyrosine deficiency significantly reduced the basal currents of primary DRG neurons in db / db mice. The control group received 0.39 mM tyrosine at the standard concentration, while the deficiency group received 0.13 mM, with a treatment duration of 24 h. Unpaired t-tests were used to test for normality and homogeneity of variance. The values ​​in the figure are mean ± standard error (mean ± SEM), n = 15. Detailed Implementation

[0020] Various exemplary embodiments of the present invention are now described in detail. Unless otherwise specified, the methods used in the embodiments are conventional methods, and the reagents used are commercially available reagents or reagents prepared using conventional methods. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Experimental process

[0026] 1. Collection of serum samples and detection of metabolites from clinical trial patients

[0027] This study included healthy adults, adults with type 2 diabetes mellitus (T2DM) without neuropathic pain, and adults with diabetic dysplasia of the hip (DPNP). Participants with cardiovascular disease, malignant tumors, or abnormal liver or kidney function were excluded. The study protocol was reviewed, approved, and supervised by the Ethics Committee of Xinhua Hospital, and complied with the relevant principles of the Declaration of Helsinki. All participants were fully informed and signed informed consent forms before enrollment.

[0028] The inclusion criteria for the DPNP group were: 1) a clear history of type 2 diabetes mellitus (T2DM); 2) the appearance of neuropathic pain symptoms at or after the clinical diagnosis of diabetes; 3) clinical symptoms of peripheral neuropathic pain, characterized by bilateral distal symmetrical limb pain, initially affecting the distal extremities of both feet, and gradually progressing proximally to the lower legs and hands; supplemented by electromyography to diagnose neuropathy; 4) patients with numbness or chills but no pain were excluded; patients with neuropathic pain caused by other reasons were also excluded.

[0029] The T2DM group included patients with type 2 diabetes mellitus (T2DM) who were matched in terms of age, sex, duration of diabetes, fasting blood glucose, medication use, liver and kidney function, and no neuropathic pain.

[0030] Fasting peripheral blood samples were collected from the aforementioned clinical participants, and a fully quantitative metabolomics analysis was performed using ultra-high performance liquid chromatography-triple quadrupole-tandem mass spectrometry (UPLC-QQQ-MS / MS). The analysis was conducted by MetaBio (Shanghai) Co., Ltd. The screening scope covered a total of 204 metabolites, including amino acids, organic acids, fatty acids, bile acids, carnitine, and indole. Potential biomarkers were screened using multidimensional statistical analysis, unidimensional statistical analysis, pathway enrichment analysis, and modeling prediction.

[0031] 2. Establishment of the mouse model

[0032] The establishment of the mouse DPNP model relies on the T2DM model. This project uses spontaneously induced T2DM mice (db / db) with leptin receptor gene deficiency as the research subjects. All animal experiments in this project were approved by the Animal Welfare and Ethics Committee of Xinhua Hospital affiliated to Shanghai Jiao Tong University School of Medicine and conducted in accordance with the standards set by the committee.

[0033] Tyrosine, an essential amino acid, can be controlled in vivo by adjusting its intake. By customizing the diet, the tyrosine content in a standard L-amino acid diet for mice (containing 4 g / kg tyrosine) was adjusted to half (containing 2 g / kg tyrosine) or twice (containing 8 g / kg tyrosine) to achieve tyrosine deficiency or supplementation treatment.

[0034] 3. Mouse pain behavior test

[0035] Behavioral tests on mice were conducted according to standard methods. Before testing, mice were placed in a temperature-controlled (24 ± 2℃) and noise-free testing room for at least one hour to acclimatize. In this patented procedure, mechanical pain, thermal pain, and cold pain tests were performed on the mice.

[0036] Mechanical pain test in mice: Mice were placed on a rack with a metal mesh and isolated by covering them with a perforated plexiglass box, allowing them to acclimatize for at least half an hour. Once the mice were completely calm, the center of the hind paw was carefully stimulated with calibrated 0.07 g (low) and 0.4 g (medium) von Frey silk, respectively, to simulate touch-induced mechanical pain. The pressure was kept constant during stimulation, causing the von Frey silk to bend into a "C" shape and maintain this position for 1.5 seconds. A positive result was defined as a mouse exhibiting obvious, rapid paw withdrawal or licking of the paw. Each mouse was stimulated 10 times with different mechanical forces of von Frey silk, with an interval of at least 5 minutes between stimulations. The paw withdrawal frequency (PWF) for each mechanical force was calculated using the following formula: (Number of positive responses / Number of stimulations 10) × 100% = PWF.

[0037] To determine the paw withdrawal latency (PWL) of mice to noxious thermal stimuli: Mice were placed on a rack with a metal mesh, and isolated by covering them with a perforated plexiglass box, allowing them to acclimatize for at least half an hour. Once the mice were completely calm, a light beam was shone from the light box onto the center of the sole of the mouse's hind paw. The light beam was turned off when the mouse withdrew its paw or jumped. PWL was defined as the time from the start to the end of the light beam. The experiment was repeated 5 times on each hind paw, with an interval of at least 10 minutes between each stimulus. A 20-second protection time was used to avoid hind paw tissue damage.

[0038] To determine the paw withdrawal latency (PWL) in mice to noxious cold stimuli: Mice were placed in individual plexiglass chambers containing a cold aluminum plate (temperature controlled at 0 °C). PWL was defined as the time from when the mouse was placed in contact with the cold aluminum plate to the first jump or paw withdrawal. The experiment was repeated 5 times per mouse, with an interval of at least 10 minutes between stimuli. A 20-second protection time was provided to avoid hind paw tissue damage.

[0039] 4. Isolation and whole-cell patch-clamp recording of primary DRG neurons in mice

[0040] L3-L5 DRG neurons were isolated from db / db mice approximately 8 weeks old. After euthanasia, the lumbar spinal canal was rapidly dissected and the spinal cord removed. DRG tissue was located near the intervertebral foramina on both sides of the spine, and the L3-L5 DRG was precisely extracted. The DRG was then prepared using Hank's balanced buffer (Ca-free). 2+ and Mg 2+ Rinse twice, then digest and separate the cells at 37 °C for 20-30 min with a mixture of collagenase I (1 mg / mL) and dispersin II (5 mg / mL) prepared in Hank's equilibration buffer, gently shaking once every 10 min. After adding 1 mL of fetal bovine serum to stop digestion, collect the cell suspension, centrifuge at 200 × g for 5 min and discard the supernatant; then add Hank's equilibration buffer again, centrifuge at 200 × g for 5 min and discard the supernatant. Carefully pipette and aspirate the cell pellet into Neurobasal™-A medium containing 15% fetal bovine serum, and seed it onto cell slides coated with laminin (20 μg / mL). Incubate statically in a cell culture incubator at 37 °C and 5% CO2.

[0041] The excitability of primary DRG neurons was recorded using whole-cell patch-clamp recording. Neurons were placed in a constant-temperature chamber (35 °C) of an inverted microscope, and recording was performed using borosilicate glass microelectrodes with a resistance of 3-5 MΩ. The specific method is as follows: 1) Locate the target neuron and carefully adjust the micromanipulator to slowly bring the microelectrode filled with electrode fluid into contact with the target neuron. Liquid junction potential compensation is performed as the neuron approaches, and appropriate suction is carefully applied to induce a high-resistance seal. After the seal is formed, fast capacitive current compensation is performed. Subsequently, the membrane is gently broken with suction to enter whole-cell recording mode, and slow capacitive current compensation is performed to optimize recording quality. 2) Current pulses of different intensities (0-750 pA, 25 pA interval, 200 ms) are applied to the neuron to record parameters such as resting membrane potential, base current, and decay time. 3) All recorded signals are low-pass filtered at 2.9 kHz and digitized at a sampling rate of 20 kHz.

[0042] Results Analysis

[0043] To further elucidate the metabolic causes of diabetic dysplasia of the kidney in patients with type 2 diabetes mellitus (T2DM) complicated with diabetic dysplasia of the kidney, we employed ultra-high performance liquid chromatography-triple quadrupole-tandem mass spectrometry (UHPLC-TMS) for fully quantitative metabolomics analysis to improve detection sensitivity and specificity. We also expanded the coverage of metabolites to include 204 metabolites, such as amino acids, organic acids, fatty acids, bile acids, carnitine, and indole, thus providing a more comprehensive analysis of the metabolic characteristics of DPNP patients. Peripheral blood samples were collected from 60 healthy individuals, 103 T2DM patients (without DPNP symptoms), and 111 DPNP patients (matched for age, sex, duration of diabetes, fasting blood glucose, medication use, and liver and kidney function). The results showed that compared with T2DM patients, DPNP patients had significantly lower peripheral blood tyrosine levels (P < 0.0001), with a decrease of 12.66%. Figure 1 2). This suggests that tyrosine is closely related to DPNP. To investigate whether tyrosine levels can predict the risk of DPNP in patients with T2DM, a Logistic Regression model was used to model and predict the above data ( Figure 3 The results show that the area under the ROC curve (AUC) is 0.849, which can be used as a potential predictor of DPNP.

[0044] Next, using the spontaneous T2DM model db / db mice as the research subject, we investigated the causal relationship between decreased tyrosine levels and the occurrence of DPNP. Tyrosine is an essential amino acid, and its intake level was altered by synthetic diet (1 / 2 or 2 times the standard content), and the aggravation or improvement of DPNP symptoms in db / db mice was examined. Mechanical pain tests showed that after 21 days of tyrosine deficiency treatment, the frequency of paw withdrawal in mice stimulated by 0.07 g and 0.4 g von Frey filaments significantly increased. Figure 4 ,5); Noxious heat and cold stimulation tests showed that the withdrawal latency of mice was significantly shortened, suggesting increased pain sensitivity ( Figure 6 7). Tyrosine supplementation reversed the above phenotype. These results collectively indicate that tyrosine deficiency exacerbates DPNP symptoms in db / db mice, while tyrosine supplementation improves symptoms, and decreased tyrosine levels are an important metabolic trigger for DPNP.

[0045] The dorsal root ganglion (DRG) is a cluster of peripheral sensory neurons, containing primary sensory neurons responsible for the upward transmission of pain. It plays a crucial role in pain perception and is a classic subject of study in the pathogenesis of DPNP. To investigate whether decreased tyrosine levels cause excitability disorders in DRG neurons, primary DRG neurons from db / db mice were isolated, treated with tyrosine deficiency, and their excitability was recorded using whole-cell patch-clamp. The results showed that tyrosine deficiency significantly increased the resting membrane potential and significantly decreased the rheobase current in primary DRG neurons. Figure 8-10 The above results indicate that tyrosine deficiency leads to increased excitability of primary DRG neurons in mice, thereby enhancing pain perception.

[0046] This study clarifies that decreased tyrosine levels are an important metabolic trigger for DPNP, providing a theoretical basis for early biomarker screening and targeted intervention strategies for DPNP, and promoting the improvement of DPNP prevention and treatment strategies centered on blood glucose management.

[0047] Compared with existing technologies, this invention is the first to propose the use of tyrosine in the diagnosis, risk assessment, and prevention of diabetic peripheral neuropathy. This not only provides a new biomarker for the early identification and clinical stratification of this disease but also offers a new potential therapeutic target for targeted intervention. The technical solution of this invention has clear scientific basis and promising clinical translation prospects, and can provide a new technical pathway for the prevention and treatment of diabetic peripheral neuropathy.

[0048] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. 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 creative effort.

Claims

1. A diagnostic biomarker, characterized in that, Including tyrosine.

2. The use of the diagnostic marker as described in claim 1 in the preparation of products for diagnosing diabetic peripheral neuropathy.

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