Application of Endothelin-1 in central nervous injury diagnosis and severity grading evaluation

Abstract

The invention discloses application of Endothelin-1 in central nervous injury diagnosis and severity grading evaluation, belongs to the technical field of laboratory medicine, and particularly discloses application of ET-1 as a molecular marker in preparation of a central nervous injury clinical diagnosis reagent and injury severity evaluation. It is proposed for the first time that ET-1 can be used as a novel plasma marker after spinal cord injury / brain injury, the plasma concentration of ET-1 can be used for reflecting the disease severity of spinal cord injury / brain injury, and research finds that the ET-1 protein content in plasma is positively correlated with the injury severity of spinal cord injury / brain injury. And the severity of central nervous injury and prognosis evaluation can be dynamically monitored by utilizing the ET-1 expression level.

Description

Technical Field

[0001] The present invention belongs to the technical field of laboratory medicine, and particularly relates to the application of Endothelin-1 in the diagnosis and severity grading assessment of central nervous system injury. Background Art

[0002] Traumatic spinal cord injury (SCI) and traumatic brain injury (TBI) are common, high-risk traumatic illnesses with extremely high rates of disability and mortality. The more severe the injury, the worse the functional prognosis and the higher the mortality rate. Therefore, the severity of the injury is often positively correlated with disability and mortality. Currently, clinicians rely solely on initial imaging studies and neurological assessments to assess the severity of SCI / BBI, which are limited and subjective, with issues such as radiation exposure, high device dependency, and subjective error. Crucially, the pathological process of acute SCI / BBI is divided into primary and secondary damage. Secondary damage includes increased bleeding, tissue swelling, neuronal and oligodendrocyte death, gliosis, and cystic cavity formation. Therefore, initial imaging studies and functional assessments cannot accurately and promptly assess the impact of secondary damage on functional prognosis. Frequent follow-up imaging studies are cumbersome and medically unsafe for patients with limb paralysis in the early stages of the disease.

[0003] At present, some diseases can be monitored dynamically using clear hematological molecular markers in clinical practice. For example, the dynamic monitoring blood index for lung cancer is CEA, the dynamic monitoring index for pancreatic cancer is CA199, the dynamic monitoring index for liver cancer is AFP, and the dynamic monitoring index for bacterial infectious diseases is white blood cell count and CRP. However, there is still a lack of reliable dynamic monitoring indicators in peripheral blood for spinal cord injury / brain injury. This phenomenon is closely related to the existence of a natural physical barrier between the central nervous system and peripheral tissues, namely the blood-spinal cord barrier / blood-brain barrier and its special immune regulation mechanism. Markers in peripheral blood for dynamic monitoring of the degree of injury in spinal cord injury / brain injury must have the following characteristics: (1) extremely low expression under normal physiological conditions, but upregulated expression after spinal cord injury / brain injury; (2) correlated with the severity of nerve damage in spinal cord injury / brain injury; (3) able to penetrate the blood-spinal cord barrier after injury and maintain a certain abundance and time window in peripheral blood. Summary of the Invention

[0004] Technical issues solved:

[0005] This application addresses the shortcomings of existing technologies and solves technical problems such as the limitations and subjectivity of existing technologies, radiation exposure, strong equipment dependence, subjective errors, the inability of imaging examinations and functional assessments to accurately and timely judge the impact of secondary injuries on functional prognosis, and the troublesome and medically unsafe nature of frequent follow-up imaging examinations for patients with limb paralysis in the early stages of the disease. It also provides the application of Endothelin-1 in the diagnosis and severity grading assessment of central nervous system injuries.

[0006] Technical solution:

[0007] To achieve the above objectives, this application is implemented through the following technical solutions:

[0008] Endothelin-1 is used in the diagnosis and severity grading assessment of central nervous system injury. Endothelin-1 is used as a molecular marker to prepare an in vitro diagnostic reagent for diagnosing or assessing the severity of central nervous system injury.

[0009] Furthermore, the detection object of the in vitro diagnostic reagent is Endothelin-1 protein or gene in plasma.

[0010] Furthermore, the central nervous system injury is spinal cord injury (SCI) and / or traumatic brain injury (TBI).

[0011] Furthermore, the graded assessment includes a graded assessment of injury severity and a graded assessment of functional recovery.

[0012] Furthermore, the in vitro diagnostic reagent is a reagent for quantitatively detecting Endothelin-1 protein or gene in plasma.

[0013] Furthermore, the in vitro diagnostic reagent is used for diagnosis of central nervous system injury using enzyme-linked immunosorbent assay (ELISA), chemiluminescence or Q-PCR real-time fluorescence quantitative PCR.

[0014] Beneficial effects:

[0015] This application provides the use of Endothelin-1 in the diagnosis and severity grading assessment of central nervous system injury, which has the following beneficial effects compared with the existing technology:

[0016] 1. This study reveals for the first time that ET-1 levels in peripheral plasma are significantly and abnormally upregulated after central nervous system injury, and that this change is positively correlated with the severity of injury.

[0017] 2. This invention establishes for the first time an injury assessment system based on dynamic monitoring of plasma ET-1. Samples are easily accessible. Plasma sampling is convenient, enabling non-invasive and continuous monitoring. This overcomes the invasive risks of traditional cerebrospinal fluid testing and the temporal and spatial limitations of imaging examinations. Therefore, plasma ET-1 testing can elevate the severity detection and assessment of spinal cord injury / brain injury to a new level.

[0018] 3. Widely applicable detection technology: Detection using ELISA, chemiluminescence, or Q-PCR methods breaks through the temporal and spatial limitations of traditional central nervous system injury diagnosis, providing accurate quantitative analysis and economical applicability.

[0019] 4. Improved precision of clinical assessment: Establish a quantitative correspondence model between ET-1 concentration gradient and degree of injury (ASIA classification), providing an objective biomarker standard for severity classification;

[0020] 5. Optimization of diagnosis and treatment processes: For patients and their families, reducing movement during imaging examinations is more convenient. For clinicians, it can provide more objective, accurate, and dynamic data to assess severity and track prognosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a graph showing the correlation between ET-1 expression levels in spinal cord tissue samples and blood samples in the acute phase after spinal cord injury in rats of this application and spinal cord injury, wherein A is a graph showing the expression levels of ET-1 in spinal cord injury tissue at different times; B is a graph showing the expression levels of ET-1 in plasma at different times after injury;

[0022] Figure 2 This is a graph showing the correlation between the degree of spinal cord injury and ET-1 expression in rats of this application, wherein A is a graph showing the expression level of ET-1 in the spinal cord tissue of rats after different degrees of spinal cord injury; B is a graph showing the expression level of ET-1 in the plasma of rats after different degrees of spinal cord injury; and C is a graph showing the correlation analysis between different degrees of spinal cord injury and the expression level of ET-1 in rat plasma.

[0023] Figure 3 The following are correlation diagrams of the degree of spinal cord injury and ET-1 expression levels in patients with spinal cord injury in this application, wherein A is a graph showing the expression of ET-1 protein levels in plasma after injury, B is a graph showing the correlation analysis between functional scores and ET-1 expression levels 6 months after injury, and C is a graph showing the relationship between plasma ET-1 concentration and the degree of spinal cord injury in patients with spinal cord injury.

[0024] Figure 4These are graphs showing the protein expression levels of D-DT, IL-6, IL-1β, and CCL2 in the plasma of patients with vertebral fractures and different degrees of spinal cord injury during the acute phase after injury in this application, wherein A is a graph showing the expression level of D-DT in plasma, B is a graph showing the expression level of IL-6 in plasma, C is a graph showing the expression level of IL-1β in plasma, and D is a graph showing the expression level of CCL2 in plasma. DETAILED DESCRIPTION

[0025] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples are conventional methods generally known to those skilled in the art and do not constitute any limitation to the present invention in any way.

[0026] Endothelin-1 (ET-1) (NCBI (https: / / www.ncbi.nlm.nih.gov / ) Gene ID: 1906; Uniprot (https: / / www.uniprot.org) ID: P05305) in the following examples.

[0027] ET-1 is a vasoactive peptide and a member of the endothelin system. It is expressed in organs and tissues such as the kidneys, lungs, heart, and blood vessels, while its expression is extremely low or absent in organs and organs such as the brain, spinal cord, liver, pancreas, and thyroid. Although ET-1 expression is upregulated in various central nervous system tissues, such as those in multiple sclerosis and cerebral infarction, no studies have demonstrated that abnormally upregulated ET-1 can be detected in the peripheral blood of patients with spinal cord injury or traumatic brain injury.

[0028] Example 1:

[0029] The application of Endothelin-1 in the diagnosis and severity grading assessment of central nervous system injury, wherein Endothelin-1 is used as a molecular marker to prepare an in vitro diagnostic reagent for diagnosing or assessing the severity of central nervous system injury, wherein the central nervous system injury is spinal cord injury (SCI) and / or traumatic brain injury (TBI), and the grading assessment includes a grading assessment of injury severity and a grading assessment of functional recovery. The in vitro diagnostic reagent is a reagent for quantitatively detecting Endothelin-1 protein or gene in plasma. The correlation between ET-1 and spinal cord injury in acute phase blood samples and spinal cord tissue samples of rats with spinal cord injury is as follows:

[0030] Twenty-four adult male Sprague-Dawley rats weighing 180-200g were anesthetized intraperitoneally. The surgical site was thoroughly disinfected with iodine and then decolorized with alcohol. With the rats in the prone position, a 2.5cm longitudinal incision was made along the midline of the back, centered at the T9 spinal vertebra, exposing the white aponeurosis and muscles. A scalpel was used to separate the muscles above and on both sides of the spine, fully exposing the T8-T10 vertebrae.

[0031] The IH-0400 impactor (Precision Systems and Instruments) was used as the injury device, with the impact force set to 150 kDyne. Immediately after the contusion, the impactor was removed. The rats experienced spasmodic tremors or tail-flicking reflexes at the moment of impact, followed by hematoma formation at the spinal cord impact site. The dura mater was intact and purple-red, indicating that the model was successfully established. After pressing to stop bleeding, the rats were sutured and maintained at different time points (0 days,

[0032] Peripheral blood was drawn from the tail vein and stored in an EDTA-containing anticoagulant tube. At the same time, 0.5 cm of tissue above and below the injury center was removed for protein extraction and frozen in a -80°C refrigerator for subsequent use.

[0033] ET-1 ELISA test, the specific process is as follows:

[0034] Step 1, sample preparation: Prepare the sample according to the above extraction method.

[0035] Step 2: Add reagent diluent: Add 150 μL of sample diluent RD1-105 to each well.

[0036] Step 3: Add 75 μL of standard and sample to each well. Cover with sealing film and incubate on a horizontal microplate shaker at room temperature for 1 hour.

[0037] Step 4: Add ET-1 labeled antibody: discard the liquid, spin dry, add 400 μL of washing buffer to each well and wash the plate three times, add 100 μL of HRP labeled antibody to each well, cover with sealing film, and incubate at room temperature for 3 hours on a horizontal microplate shaker.

[0038] Step 5: Add substrate solution: remove the liquid in the wells, spin dry, add 200 μL substrate solution to each well, and incubate in the dark for 30 minutes.

[0039] Step 6. Termination: Add 50 μL of stop solution to each well to terminate the reaction.

[0040] Step 7: Result analysis: Within 30 minutes after the reaction is terminated, the absorbance at a wavelength of 450 nm is measured sequentially using a multifunctional microplate reader. The OD value detected for each sample is inserted into the standard curve to calculate the concentration of ET-1 in plasma.

[0041] The above kits were purchased from R&D Systems.

[0042] The results are as follows Figure 1 As shown, Figure 1 The results in Figure A showed that the ET-1 protein content in the injured tissue was significantly increased at 1 day and 4 days after spinal cord injury in rats. Figure 1 The results in Figure B showed that ET-1 was significantly upregulated in the plasma of rats 1 day after spinal cord injury. The above results indicate that ET-1 expression was significantly upregulated in the spinal cord tissue and plasma during the acute phase of spinal cord injury in rats.

[0043] Example 2:

[0044] Correlation between the degree of spinal cord injury and ET-1 expression level in rats:

[0045] After 24 adult male SD rats weighing 180-200g were anesthetized by intraperitoneal anesthesia, the surgical site was thoroughly disinfected with iodine tincture and then decolorized with alcohol. The rats were placed in a prone position, and a 2.5cm longitudinal incision was made along the midline of the back with the T9 vertebra of the spinal cord as the center to expose the white aponeurosis and muscles. The muscles above and on both sides of the spine were separated with a scalpel to fully expose the T8-T10 vertebrae. The IH-0400 impactor (precision system and instrument) injury device was used, and the impact force was set to 0, 50, 100, 150, 200, and 250kDyne. The impact rod was removed immediately after the contusion ended. At the moment of impact, the rat's body showed spasmodic tremors or tail-wagging reflexes, followed by hematoma formation at the spinal cord impact point, and the dura mater was intact and purple-red, indicating that the model was successfully established. After pressing to stop bleeding, suture and maintain the wound. 24 hours after injury, peripheral blood was drawn from the tail vein and stored in an anticoagulant tube containing EDTA. At the same time, 0.5 cm of tissue above and below the injury center was removed for protein extraction and frozen in a -80°C refrigerator for later use. Spinal cord tissue and plasma ET-1 ELISA testing was performed using the same procedures as in Example 1. Figure 2 As shown, Figure 2 The results in Figure A show that the more severe the spinal cord injury, the higher the expression level of ET-1 in the spinal cord tissue. Figure 2 The results in Figure B indicate that the more severe the spinal cord injury, the higher the expression level of ET-1 in plasma, indicating that the expression level of ET-1 is correlated with the degree of spinal cord injury in rats. Figure 2 The correlation analysis between the degree of spinal cord injury and the expression level of ET-1 in the acute phase was performed in C. The results showed that the more severe the spinal cord injury, the higher the expression level of ET-1, and the two were positively correlated (Spearman, r 2 =0.96,P<0.001).

[0046] Example 3

[0047] Correlation analysis between functional recovery and ET-1 expression level in patients with different degrees of spinal cord injury:

[0048] Peripheral blood was collected from 68 patients with simple vertebral fractures without neurological symptoms and patients with different degrees of spinal cord injury in anticoagulant tubes (containing EDTA) at the Affiliated Hospital of Nantong University. According to the American Spinal Injury Association (ASIA) grading standard, they were rated as A, B, C, and D. Among them, 20 patients with simple vertebral fractures were used as controls (male / female = 13 / 7), ASIA-A: 12 cases (male / female = 8 / 4), ASIA-B: 12 cases (male / female = 9 / 3), ASIA-C: 12 cases (male / female = 9 / 3), and ASIA-D: 12 cases (male / female = 5 / 7). Peripheral blood was centrifuged at 1000g for 5 min and the supernatant was taken. ET-1 ELISA test (detection kit was human ET-1 ELISA kit) was performed with the same steps as in Example 1.

[0049] At the 6-month follow-up assessment, the strength of key upper and lower limb muscles should be systematically assessed, including the elbow flexors, wrist extensors, elbow extensors, flexors of the distal phalanx of the middle finger, abductors of the little finger, hip flexors, knee extensors, ankle dorsiflexors, extensor pollicis longus, and ankle plantar flexors. A six-point scale (5 points: normal strength; 0 points: complete paralysis) is used to calculate the total bilateral muscle group score as an objective indicator of neurological recovery.

[0050] The results are as follows Figure 3 shown. Figure 3 The results in Figure A showed that during the acute phase of injury, the protein expression level of ET-1 in the peripheral blood of the spinal cord injury group was significantly higher than that of the control group, and the degree of injury was significantly positively correlated with the expression of ET-1. Figure 3 In Figure B, the correlation analysis between ET-1 protein expression level and functional score 6 months after injury was performed. The results showed that the acute phase ET-1 level was significantly negatively correlated with the functional score 6 months after injury (Spearman, r 2 =0.92, P <0.001), indicating that elevated ET-1 expression may predict poor long-term functional recovery.

[0051] According to the above Figure 3 Receiver Operating Characteristic analysis (ROC analysis) was performed on the results of A to determine the cut-off value (Cut-off) of plasma ET-1 concentration and its association with prognosis. The corresponding cut-off value is as follows Figure 3As shown in C. The results suggest that: in the acute phase of injury, when the plasma ET-1 value of patients with spinal cord injury is less than 0.85ng / ml, it indicates a good prognosis and no neurological dysfunction (ASIA grading does not meet the injury standard); when the ET-1 value is between 0.85-2.14ng / ml, it corresponds to ASIA grading D; when the ET-1 value is between 2.14-3.59ng / ml, it corresponds to ASIA grading C; when the ET-1 value is between 3.59-4.85ng / ml, it corresponds to ASIA grading B; when the ET-1 value exceeds 4.85ng / ml, it corresponds to ASIA grading A, which indicates a poor prognosis and poor functional recovery. The results of this study indicate that the plasma ET-1 concentration in the acute phase can be used as a biomarker to predict the functional prognosis of patients, and its cut-off value ( Figure 3 Middle C) has potential application value in clinical grading and prognosis assessment.

[0052] Example 4

[0053] In spinal cord injury, overactivated inflammatory response is the key driving factor of secondary spinal cord injury, and the process is mainly mediated by the injury-related pattern molecule D-DT (D-dopachrome tautomerase), inflammatory factors (TNF-α, IL-1β) and chemokine CCL2. It has been demonstrated in previous papers published by the inventors of this application (Bingqiang He, LiNiu, Shaolan Li et al., Sustainable inflammatory activation following spinal cord injury is driven by thrombin-mediated dynamic expression of astrocyticchemokines[J]. Brain Behav Immun, 2024, 116(85-100); Hui Li, Bingqiang He, XingyuanZhang et al., D-dopachrome tautomerase drives astroglial inflammation via NF-kappaB signaling following spinal cord injury[J]. Cell Biosci, 2022, 12(1): 128) that after spinal cord injury in rats, the expression of D-DT, IL-6, IL-1β and CCL2 in the injured tissue increased rapidly. Therefore, it is necessary to detect whether the above four inflammatory indicators in the plasma of injured patients can also indicate the severity of spinal cord injury.

[0054] The specific implementation method is the same as that in Example 3. ELISA kits are used to detect 24 indicators including D-DT, IL-6, IL-1β and CCL. The results are as follows: Figure 4 As shown. The results showed that there was no significant difference in the expression of D-DT, IL-1β, and CCL2 in the plasma of patients with vertebral fractures compared with patients with spinal cord injury, and there was no significant difference in the expression levels of D-DT, IL-1β, and CCL2 in the plasma of patients with different degrees of spinal cord injury. Although ELISA results showed that there was a difference in the expression of IL-6 in the plasma of patients with vertebral fractures compared with patients with spinal cord injury, there was no significant difference in the expression of IL-6 in the plasma of patients with different degrees of spinal cord injury. The above results suggest that although the expression level of IL-6 is correlated with spinal cord injury, it cannot be used as an important marker for the clinical classification of patients with spinal cord injury.

[0055] All the above human specimens have been approved by the Ethics Committee of the Affiliated Hospital of Nantong University, and the animal samples have been approved by the Animal Ethics Committee of Nantong University.

[0056] The above is an exemplary description of the present invention. It should be noted that, without departing from the core of the present invention, any simple deformation, modification or other equivalent replacement that can be made by other skilled in the art without expending creative labor falls within the scope of protection of the present invention.

Claims

1. The application of Endothelin-1 in the diagnosis and severity assessment of central nervous system injury is characterized by: described Endothelin-1 is used as a molecular marker to prepare in vitro diagnostic reagents for diagnosing or evaluating the severity of central nervous system damage.

2. The use of Endothelin-1 in the diagnosis and severity grading assessment of central nervous system injury according to claim 1, characterized in that: The in vitro diagnostic reagent detects Endothelin-1 protein or gene in plasma.

3. The use of Endothelin-1 in the diagnosis and severity grading assessment of central nervous system injury according to claim 1, characterized in that: The central nervous system injury is spinal cord injury (SCI) and / or traumatic brain injury (TBI).

4. The use of Endothelin-1 in the diagnosis and severity grading assessment of central nervous system injury according to claim 1, characterized in that: The graded assessment includes a graded assessment of injury severity and a graded assessment of functional recovery.

5. The use of Endothelin-1 in the diagnosis and severity grading assessment of central nervous system injury according to claim 2, characterized in that: The in vitro diagnostic reagent is a reagent for quantitatively detecting Endothelin-1 protein or gene in plasma.

6. The use of Endothelin-1 in the diagnosis and severity grading assessment of central nervous system injury according to claim 2, characterized in that: The in vitro diagnostic reagent is used for diagnosis of central nervous system damage by enzyme-linked immunosorbent assay (ELISA), chemiluminescence or Q-PCR real-time fluorescence quantitative PCR.

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