CN118243921A - A serum marker for assisting diagnosis of mild traumatic brain injury - Google Patents

Abstract

The invention discloses a serum marker for assisting in diagnosing mild traumatic craniocerebral injury. The invention discovers that the concentration of mycophenolate mofetil in the brain tissue and serum of a mouse with mild traumatic brain injury is obviously improved, and the concentration change is changed along with the different time periods after the injury, so that the mycophenolate mofetil can be used as an auxiliary diagnosis marker for the mild traumatic brain injury. The invention is helpful to solve the problem that the light traumatic craniocerebral injury is difficult to diagnose in time and accurately clinically, and also expands a new view for researching the pathological mechanism of the light traumatic craniocerebral injury. Meanwhile, mycophenolate mofetil is suggested to be used as a medicament which is already marketed, and is possible to have the potential of playing a brand new pharmacological action in the aspect of light traumatic craniocerebral injury.

Description

A serum marker for assisting diagnosis of mild traumatic brain injury

Technical Field

The invention belongs to the technical field of craniocerebral injury diagnosis, and specifically relates to a serum marker for auxiliary diagnosis of mild traumatic craniocerebral injury.

Background technique

The high incidence of mild traumatic brain injury (mTBI) has always been a global public health problem. Although mTBI has received increasing public attention, there is still a lack of standardized definitions and precise grading indicators for mTBI in clinical practice. Currently, the Glasgow Coma Scale combined with imaging examinations is used to assess and judge mTBI in clinical practice, but the scale evaluation is too subjective and not friendly to children and the elderly, and imaging examinations are insensitive to mTBI. About 29% of patients have false negative CT diagnostic results when imaging examinations are used as the basis for the diagnosis of mTBI. In contrast, biomarkers are a type of indicator that can be objectively measured and evaluated. Their appearance or concentration changes can truly reflect the patient's pathophysiological state, effectively judge the occurrence, development and prognosis of the disease, and can make up for the shortcomings of traditional diagnostic methods. In recent years, serum markers used to assist in the diagnosis and assessment of TBI severity have become a research hotspot. For example, biomarkers related to neuronal and axonal injury include ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), neuron-specific enolase (NSE), Tau protein, and neurofilament light chain protein (NFL); biomarkers related to astrocyte and cerebrovascular injury include calcium-binding protein B (S100B) and glial fibrillary acid protein (GFAP). A large number of studies have shown that these serum markers can assist in the diagnosis and prognosis of TBI, but as independent detection factors, especially for the assessment of mTBI, there are still deficiencies and they have not been actually applied in clinical practice. Therefore, how to screen better detection markers to improve the accurate diagnosis of mTBI remains a challenge.

We constructed a diffuse closed mild craniocerebral injury mouse model to simulate the occurrence of mTBI in actual scenarios, used metabolomics technology to analyze the differences in metabolic levels between the experimental group and the control group, identified differential metabolites, and used ELISA technology to preliminarily verify the differential metabolites, and found that mycophenolate mofetil can be used as a potential molecular marker for mTBI. Its concentration changes in serum can effectively indicate the occurrence of mTBI, help the accurate diagnosis of mTBI, and provide a new perspective for the study of the pathological mechanism of mTBI.

Summary of the invention

The purpose of the present invention is to provide a molecular marker that can be used to evaluate mTBI in view of the deficiencies in existing mTBI diagnostic technologies.

A serum marker for assisting the diagnosis of mild traumatic brain injury, wherein the marker is mycophenolate mofetil.

Compared with normal people, the content of mycophenolate mofetil is increased in patients with mild traumatic brain injury.

The method for determining the content of mycophenolate mofetil is ELISA detection method, chromatography analysis method or chromatography-mass spectrometry analysis method.

Detecting the expression level of mycophenolate mofetil in serum is used to achieve the following functions:

(1) Assist in the diagnosis of mTBI;

(2) Assist in distinguishing mTBI patients from healthy controls.

A kit for assisting in the diagnosis of mild traumatic brain injury comprises a reagent for determining the content of mycophenolate mofetil in a serum sample.

A system for assisting in the diagnosis of mild traumatic brain injury includes a method for establishing a mathematical model for detecting the concentration of mycophenolate mofetil, wherein the mathematical model is obtained according to the following steps:

(A1) Detect the concentration of mycophenolate mofetil in the serum of n1 type A samples and n2 type B samples respectively;

(A2) Taking the mycophenolate mofetil concentration data of all samples obtained in step (A1), a mathematical model is established based on Youden index analysis to determine the threshold for classification judgment.

The Class A sample and the Class B sample are any of the following:

(B1) samples of mild traumatic brain injury and healthy controls;

(B2) samples with mild traumatic brain injury and samples without traumatic brain injury;

(B3) Samples at different time periods after mild traumatic brain injury.

Beneficial effects of the present invention: The present invention found that the concentration of mycophenolate mofetil in the brain tissue and serum of mice with mild traumatic brain injury was significantly increased, and the concentration changed with different time periods after the injury, indicating that mycophenolate mofetil can be used as an auxiliary diagnostic marker for mild traumatic brain injury. The present invention helps to solve the difficult problem of timely and accurate diagnosis of mild traumatic brain injury in clinical practice, and also expands new perspectives for the study of the pathological mechanism of mild traumatic brain injury. At the same time, it is suggested that mycophenolate mofetil, as a type of drug already on the market, may have the potential to play a new pharmacological role in mild traumatic brain injury.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of mouse modeling.

Figure 2 shows the percentage of identified metabolites in brain tissue.

Figure 3 shows the percentage of identified metabolites in serum.

Figure 4 is a Venn diagram of the results of differential metabolite screening of brain tissue samples.

Figure 5 is a Venn diagram of the results of differential metabolite screening of serum samples.

Figure 6 shows the intersection of metabolites between brain tissue and serum in different comparison groups.

Figure 7 shows the mycophenolate mofetil test results.

Figure 8 is the ROC curve of mycophenolate mofetil detection.

Figure 9 shows the results of repeated validation of mycophenolate mofetil.

Detailed ways

In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

Example 1 Diffuse closed mild craniocerebral injury model in mice

The mice were randomly divided into a sham operation group (Sham group, n=9) and an experimental group (mTBI group, n=9). The experimental group was hit with a cylindrical weight with a mass of 80 g, a diameter of 13 mm, and an injury height of 20 cm.

The mice were fasted and deprived of water for 12 hours before modeling. 1% sodium pentobarbital was prepared with normal saline as an anesthetic, the mice were weighed, and the mice were anesthetized by intraperitoneal injection at 6 μL/g. After the mice were fully anesthetized (the corneal reflex disappeared, the pain reflex disappeared, the respiratory rate decreased, and the muscle tension decreased), the hair on the top of the mouse's skull was shaved, and the mice were fixed in a prone position on a sponge pad on the operating table. The elastic coefficient of the sponge pad was K=2.95 N/cm.

Use a stereotaxic percussion device to fix the position of the mouse. Make the mouse bite the front crossbar of the instrument with its front teeth to fix the front and back position of the mouse and ensure that the mouse's brain is level; push the ear bars on the left and right sides of the instrument horizontally into the lower ear canals of the left and right sides of the mouse (in the depression of the mandible), hold the head, adjust the position so that the ear bars and the percussion pipe are on the same horizontal line, and fix the left and right position of the mouse; align the percussion pipe with the right lateral brain of the mouse, the front edge of the weight is tangent to the tail of the mouse's eye, and the tangent line of the weight is tangent to the coronal axis of the mouse; adjust the position of the percussion pipe downward so that the contact surface is flush with the plane of the skull top. Ensure that the mouse's breathing is normal during the adjustment (Figure 1).

To prevent the weight rebounding in the striking channel from causing secondary damage to the mouse, the weight traction rope was immediately pulled after the weight hit the mouse's head. After the strike, the mouse was immediately removed from the device and placed on a 37°C constant temperature pad. After 30 minutes, it was returned to the cage and waited for recovery. The sham group was operated in the same manner except that no strike was applied.

Example 2 Metabolomics Analysis

2.1 Sample collection

Brain tissue samples and serum samples were collected from the sham group mice and the experimental group mice 3h and 6h after modeling (Table 1) for metabolomics analysis.

Table 1 Sampling groups

Brain tissue collection steps:

(1) Anesthesia. Use 1% sodium pentobarbital prepared in normal saline as an anesthetic, weigh the mouse, and anesthetize it by intraperitoneal injection at a rate of 6 μL/g.

(2) Perfusion. The anesthetized mouse was fixed in a supine position. The chest was opened with surgical scissors. A small leak was cut in the right atrial appendage of the mouse heart. 60 mL of normal saline was perfused through the left ventricle until cool liquid was observed to flow out of the right atrial appendage.

(3) Freezing. After perfusion, the mouse skull was opened quickly, and the brain tissue was removed and frozen in liquid nitrogen.

Serum collection steps:

(1) Blood collection. Blood was collected from the mice by removing their eyeballs. After blood collection, the mice were killed by dislocating their necks.

(2) Separation of serum. Place the centrifuge tube containing the collected blood at room temperature for 2 hours, then centrifuge at 3000 rpm for 10 minutes at 4°C. The upper light yellow clear liquid is the mouse serum.

(3) Freezing. The mouse serum was frozen and stored at -80°C.

2.2 Chromatography-mass spectrometry analysis

After the collected samples were slowly thawed at 4°C, they were added to a pre-cooled methanol/acetonitrile/water mixed solution (volume ratio 2:2:1), vortexed, sonicated at low temperature for 30 minutes, left to stand at -20°C for 10 minutes, centrifuged at 14000g for 20 minutes at 4°C, and the supernatant was vacuum dried. For mass spectrometry analysis, 100 μL of acetonitrile aqueous solution (volume ratio 1:1) was added for re-dissolution, vortexed, centrifuged at 14000g for 15 minutes at 4°C, and the supernatant was sampled for analysis.

Chromatographic conditions:

Column temperature 25 °C, flow rate 0.5 mL/min, injection volume 2 μL;

Mobile phase A: water + 25 mM ammonium acetate + 25 mM ammonia, mobile phase B: acetonitrile;

Gradient elution program: 0-0.5min, 95% mobile phase B elution; 0.5-7min, mobile phase B linearly changes from 95% to 65%; 7-8min, mobile phase B linearly changes from 65% to 40%; 8-9min, mobile phase B maintains 40%; 9-9.1min, mobile phase B linearly changes from 40% to 95%; 9.1-12min, mobile phase B maintains 95%. The samples were placed at 4℃ for automatic injection during the whole process.

Mass spectrometry conditions:

Electrospray ionization (ESI) positive ion and negative ion modes were used for detection, and the ESI source and mass spectrometer setting parameters were as follows:

Nebulizer gas auxiliary heating gas 1 (Gas1): 60, auxiliary heating gas 2 (Gas2): 60, curtain gas (CUR): 30psi, ion source temperature: 600℃, spray voltage (ISVF) ±5500 V (positive and negative modes);

The primary mass-to-charge ratio detection range is 80-1200 Da, the resolution is 60000, and the scanning accumulation time is 100ms. The secondary adopts a segmented acquisition method with a scanning range of 70-1200 Da, a secondary resolution of 30000, a scanning accumulation time of 50ms, and a dynamic exclusion time of 4 s.

2.3 Results Analysis

2.3.1 Number of metabolites identified

For brain tissue samples, a total of 1,325 metabolites were identified after merging the positive and negative ion modes; for serum samples, a total of 1,267 metabolites were identified after merging the positive and negative ion modes.

According to the chemical classification, the proportion of each type of metabolites is shown in Figure 2-3.

2.3.2 Screening of differential metabolites

The variable weight value (Variable Importance for the Projection, VIP) obtained by the OPLS-DA model can be used to measure the influence and explanatory power of each metabolite expression pattern on the classification and discrimination of each group of samples, and to mine differential metabolic molecules with biological significance. Metabolites with VIP>1 are usually considered to have significant contributions in model interpretation.

This experiment strictly used OPLS-DA VIP>1 and P value<0.05 as the screening criteria for significantly different metabolites. The screening results are shown in Table 2-3 and Figure 4-5.

Table 2 Screening results in brain tissue

Note: B1 is the brain tissue sample Sham group, B2 is the brain tissue sample mTBI 3h group, B3 is the brain tissue sample mTBI 6h group.

Table 3 Screening results in serum

Note: S1 is the serum sample Sham group, S2 is the serum sample mTBI 3h group, S3 is the serum sample mTBI 6h group.

Based on the metabolites screened out in the positive and negative ion modes, the differential metabolites between the mTBI 3h group and the Sham group in the brain tissue samples were compared with the differential metabolites between the mTBI 3h group and the Sham group in the serum (Figure 6a), and three differential metabolites were obtained: tamoxifen, cysteine-tryptophan, and phosphatidylglycerol 38:6; the differential metabolites between the mTBI 6h group and the Sham group in the brain tissue samples were compared with the differential metabolites between the mTBI 6h group and the Sham group in the serum (Figure 6b), and two metabolites were obtained: xanthine and phenylethylamine; the differential metabolites between the mTBI 6h group and the mTBI3h group in the brain tissue samples were compared with the differential metabolites between the mTBI 6h group and the mTBI 3h group in the serum (Figure 6c), and one differential metabolite was obtained: mycophenolate mofetil.

Among them, mycophenolate mofetil was present in the mTBI 6h and mTBI 3h groups, and also in the brain tissue samples mTBI 6h and control groups, and serum samples mTBI 3h and control groups. This suggests that mycophenolate mofetil is related to the occurrence of mTBI and may become a potential molecular marker for mTBI.

Example 3 Verification of metabolic differences and establishment of mathematical model

Mice were repeatedly modeled according to the diffuse closed mild craniocerebral injury model, and mouse serum was collected at 1h, 3h, 6h and 8h after mTBI, respectively. A negative control group was set up, and the serum of mice in the negative control group and the experimental group was tested using the mycophenolate mofetil ELISA detection kit. Three sets of replicates were set for each group of serum samples.

The test results are shown in Figure 7. By establishing the ROC curve for Youden index analysis (Figure 8), it was concluded that when the concentration of mycophenolate mofetil was less than 6.198pbb, it was mTBI negative, and when the concentration of mycophenolate mofetil was greater than 6.198pbb, it was mTBI positive. At this time, the test specificity was 100%, the sensitivity was 81.82%, and the Youden index was the largest.

From the concentration trend, the serum level of mycophenolate mofetil rose rapidly and reached a peak 1 hour after mTBI, then slowly decreased, and was still higher than the control group level at 8 hours after mTBI (Figure 9). Its levels at 3 hours and 6 hours were higher than those in the control group, and the 6 hour level was lower than that at 3 hours, which was consistent with the screening intersection.

Potential molecular markers of TBI need to satisfy the requirement that the serum level concentration has good stability over time. Mycophenolate mofetil (MMF) is an ester derivative of mycophenolic acid (MPA) with unique immunosuppressive effects and high biosafety. It has been used as a commercial drug to treat rheumatic immune diseases such as lupus erythematosus. MMF can be rapidly hydrolyzed into active metabolites MPA in the body. MPA reduces the synthesis of guanine nucleotides by inhibiting the key rate-limiting enzymes in the de novo synthesis pathway of purine nucleotides, thereby selectively inhibiting the proliferation and function of T and B lymphocytes. At present, there are no reports on the relationship between MMF and TBI. However, from the perspective of the primary injury and secondary injury mechanism of TBI, MMF may be related to the activation of astrocytes and subsequent metabolic pathways, and suggests potential drug treatment targets for mTBI, which may be related to new drug uses of mycophenolate mofetil.

Claims (7)

1. A serum marker for aiding in the diagnosis of mild traumatic brain injury, wherein the marker is mycophenolate mofetil.

2. The serum marker for aiding diagnosis of mild traumatic brain injury according to claim 1, wherein said mycophenolate mofetil is present in an elevated amount in patients suffering from mild traumatic brain injury compared to normal persons.

3. The serum marker for aiding diagnosis of mild traumatic brain injury according to claim 2, wherein said mycophenolate mofetil content assay is an ELISA assay, a chromatographic assay or a chromatographic-mass spectrometry.

4. The serum marker for aiding diagnosis of mild traumatic brain injury according to claim 1, wherein the expression level of mycophenolate mofetil in serum is detected for achieving the following functions:

(1) Auxiliary diagnosis of mTBI occurrence;

(2) Helping to distinguish mTBI patients from healthy controls.

5. A kit for aiding in the diagnosis of mild traumatic brain injury, comprising a reagent for determining the mycophenolate mofetil content in a serum sample.

6. A system for aiding in the diagnosis of mild traumatic brain injury, comprising a mathematical model building method for detecting mycophenolate mofetil concentration, said mathematical model being obtained by the steps of:

(A1) Respectively detecting the concentration of mycophenolate in serum of n1 class A samples and n2 class B samples;

(A2) Taking the mycophenolate mofetil concentration data of all the samples obtained in the step (A1), establishing a mathematical model according to about dengue index analysis, and determining the threshold value of classification judgment.

7. The system for aiding in the diagnosis of mild traumatic brain injury according to claim 6, wherein said class a sample and said class B sample are any one of the following:

(B1) A sample of mild traumatic craniocerebral injury and a healthy control sample;

(B2) A sample of mild traumatic brain injury and a sample of non-traumatic brain injury;

(B3) Samples in different time periods after mild traumatic craniocerebral injury.