Biomarkers and their application in chronic nonspecific low back pain

Abstract

The present invention discloses a biomarker for chronic nonspecific low back pain and its application, wherein the biomarker is triacylglycerol. By measuring the level of triacylglycerol in the serum of the sample to be tested and comparing it with the average level of triacylglycerol in the serum of healthy adults, it is determined whether the sample to be tested suffers from chronic nonspecific low back pain. In the present invention, by comparing the lipid groups of healthy adults and adult patients with chronic nonspecific low back pain, it is found that only the content of triacylglycerol has a significant difference between the two groups, indicating that triacylglycerol can be used as a biomarker for chronic nonspecific low back pain, which is helpful for the clinical diagnosis of chronic nonspecific low back pain.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular, relates to a biomarker for chronic nonspecific low back pain and application thereof. Background Art

[0002] The incidence of chronic nonspecific low back pain (cNLBP) is extremely high, accounting for 90% of patients with low back pain. About 80% of adults worldwide will experience cNLBP at different times, with an incidence of 7.6% to 37% and an annual prevalence of 38%. According to the Global Burden of Disease, Injury and Risk Factors Study, the prevalence of cNLBP has been among the top three in the world from 1990 to 2017, and cNLBP ranks among the top five diseases that cause disability and reduce life years. It is the main disease that causes disability and loss of labor force. In my country, cNLBP is the most common disease in rehabilitation medicine, orthopedics and pain medicine, accounting for 1 / 3 of the number of patients seeking medical treatment every day. Its medical expenses have exceeded coronary heart disease, arthritis, diabetes and cerebrovascular disease, bringing a heavy medical economic burden to society. However, due to the lack of clear histopathological changes, cNLBP cannot be diagnosed through clinical examinations such as X-ray, CT and MRI, resulting in difficulties in clinical diagnosis.

[0003] Lipidomics is an emerging discipline that systematically analyzes the entire lipid system. By comparing the changes in lipid metabolic networks under different physiological states, we can understand their interactions and interactions with other biological molecules. Through lipidomics analysis and screening, we can identify key lipid biomarkers in metabolic regulation and ultimately reveal the mechanism of action of lipids in various life activities. In addition, by testing individual lipid levels, we can monitor the progression of many diseases and help improve and provide personalized medical solutions.

[0004] Since the lipid content in serum is relatively low and the components are complex and numerous, how to reduce matrix interference, improve the response of lipid components, and quickly and accurately identify and measure the content of various lipid components from a wide variety of cellular metabolites is the basis for conducting lipid function research. At present, there are no research results on cNLBP biomarkers from the perspective of lipidomics. Therefore, identifying lipid biomarkers of cNLBP is very meaningful to assist in the clinical diagnosis of cNLBP. Summary of the invention

[0005] Based on this, the purpose of the present invention is to provide a biomarker for chronic nonspecific low back pain and its application. The content of the biomarker is significantly different between healthy adults and patients with chronic nonspecific low back pain, and can be used to assist in the diagnosis of chronic nonspecific low back pain.

[0006] The specific technical solutions for achieving the above-mentioned invention objectives include the following.

[0007] In a first aspect, the present invention provides the use of triacylglycerol as a biomarker for chronic nonspecific low back pain.

[0008] In a second aspect, the present invention provides a kit for diagnosing chronic nonspecific low back pain, comprising a reagent for measuring the level of triacylglycerol.

[0009] The third aspect of the present invention provides a method for diagnosing chronic nonspecific low back pain for non-therapeutic purposes, comprising the following steps: using the above-mentioned kit to determine the level of triacylglycerol in the serum of the sample to be tested, and comparing it with a reference value, wherein the reference value is the average level of triacylglycerol in the serum of healthy adults.

[0010] In the present invention, by comparing the lipidomes of healthy adults and adult patients with chronic nonspecific low back pain, it was found that among the 17 lipid classes, only the content of triacylglycerols (TAG) had significant differences between the two groups, and when the specific components of triacylglycerols were analyzed, it was further found that the contents of 6 triacylglycerols - TAG (56:3 / FA18:2), TAG (50:0 / FA16:0), TAG (54:2 / FA18:2), TAG (52:4 / FA18:0), TAG (56:3 / FA18:0) and TAG (54:2 / FA20:0) in patients with chronic nonspecific low back pain were significantly higher than those in healthy people, indicating that triacylglycerols, especially these 6 triacylglycerols, can be used as biomarkers for chronic nonspecific low back pain, which is helpful for the clinical diagnosis of chronic nonspecific low back pain. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The lipidome data of the HV group and cNLBP patients were analyzed by OPLS-DA in Example 1 of the present invention.

[0012] Figure 2 This is a heat map of lipid molecule association in Example 1 of the present invention.

[0013] Figure 3 The content changes of 17 lipid components in the HV group and the cNLBP group in Example 1 of the present invention.

[0014] Figure 4 This is a comparison of the peak areas of 34 triacylglycerol components in the HV group and the cNLBP group in Example 1 of the present invention.

[0015] Figure 5 The content changes of the six triacylglycerol components in the HV group and the cNLBP group in Example 1 of the present invention.

[0016] Figure 6This is an analysis of the sensitivity and specificity of the six triglyceride components as biomarkers for diagnosing cNLBP in Example 2 of the present invention. DETAILED DESCRIPTION

[0017] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. 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.

[0018] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0019] Unless otherwise specified, the examples are based on conventional experimental conditions, such as Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001, or the conditions recommended by the manufacturer's instructions. The raw materials and reagents used in the present invention are all commercially available, and any biological germplasm material can be provided to the outside for scientific research.

[0020] The present invention quantitatively analyzes the serum lipidome of healthy adults and cNLBP adult patients, and identifies a total of 1086 lipids, which are divided into 17 categories. These categories include FA (fatty acids), MAG (monoacylglycerol), DAG (diacylglycerol), TAG (triacylglycerol), CE (cholesterol ester), Cer (ceramide), HexCer (hexosylceramide), SM (sphingomyelin), PA (phosphatidic acid), PC (phosphatidylcholine), PE (phosphatidylethanolamine), PG (phosphatidylglycerol), PI (phosphatidylinositol), PS, LPE (lysophosphatidylethanolamine), LPG (lysophosphatidylglycerol) and LPI (lysophosphatidylinositol). Orthogonal PLS-DA analysis of lipidome data showed that healthy adults and cNLBP patients had different lipidomic characteristics, indicating that there are potential differences in lipid metabolism between cNLBP patients and healthy adults. Further correlation analysis of 17 lipids was performed, and the results showed that among the 17 lipids, only TAG (triacylglycerol) showed obvious differences (significantly increased) between healthy adults and cNLBP patients, while the remaining 16 lipids did not show significant changes. Specifically, TAG (56:3 / FA18:2), TAG (50:0 / FA16:0), TAG (54:2 / FA18:2), TAG (52:4 / FA18:0), TAG (56:3 / FA18:0) and TAG (54:2 / FA20:0) in TAG were significantly higher in cNLBP patients than in healthy adults. Therefore, these 6 TAGs can be used as biomarkers for the evaluation and / or diagnosis of cNLBP.

[0021] In some embodiments of the present invention, the use of triacylglycerol as a biomarker for chronic nonspecific low back pain is disclosed.

[0022] In some embodiments, the triacylglycerol is TAG(56:3 / FA18:2), TAG(50:0 / FA16:0), TAG(54:2 / FA18:2), TAG(52:4 / FA18:0), TAG(56:3 / FA18:0) and TAG(54:2 / FA20:0).

[0023] In other embodiments of the present invention, disclosed is the use of a reagent for determining the level of triacylglycerols in preparing a kit for diagnosing chronic nonspecific low back pain.

[0024] In other embodiments of the present invention, a kit for diagnosing chronic nonspecific low back pain is disclosed, comprising a reagent for measuring the level of triacylglycerol.

[0025] In other embodiments of the present invention, a method for diagnosing chronic nonspecific low back pain for non-therapeutic purposes is disclosed, comprising the following steps: using the above-mentioned kit to determine the level of triacylglycerol in the serum of the sample to be tested, and comparing it with a reference value, wherein the reference value is the average level of serum triacylglycerol in healthy adults.

[0026] In some embodiments, the level of triacylglycerol in the serum of the test sample is determined by liquid chromatography tandem mass spectrometry, and the liquid chromatography conditions include:

[0027] The mobile phase A is an acetonitrile aqueous solution containing 8-12 mM ammonium acetate, wherein the volume ratio of acetonitrile to water is 65-55:35-45; the mobile phase B is an isopropanol acetonitrile solution containing 8-12 mM ammonium acetate, wherein the volume ratio of isopropanol to acetonitrile is 85-95:5-15; the gradient elution program is: 0-1.5 minutes, 68% of the mobile phase A; 1.5-15.5 minutes, 15% of the mobile phase A; 15.5-15.6 minutes, 3% of the mobile phase A; 15.6-18 minutes, 3% of the mobile phase A; 18-18.1 minutes, 68% of the mobile phase A; 18.1-20 minutes, 68% of the mobile phase A.

[0028] In some embodiments, the chromatographic column used in the liquid chromatography is a C18 column.

[0029] In some embodiments, the liquid chromatography conditions also include: injection volume: 4-6 μl; flow rate: 0.25-0.27 mL / min; column temperature: 50-60°C.

[0030] In some of the embodiments, the mass spectrometry conditions include: ion source temperature: 600±10°C; ion source voltage: -4500±50V or 5500±50V; curtain gas: 20±2psi; nebulizer gas and auxiliary gas: both 60±5psi.

[0031] In some of these embodiments, if the level of triglycerides in the serum of the test sample is significantly higher than the reference value, the test sample is diagnosed as chronic nonspecific low back pain.

[0032] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0033] Example 1 Screening of biomarkers for diagnosing chronic nonspecific low back pain in adults

[0034] The following steps are involved:

[0035] 1. Recruitment of healthy volunteers and cNLBP patients

[0036] Twenty-six healthy volunteers (HV group) and 30 cNLBP patients (cNLBP group) were recruited through advertisements. The subjects were between 18 and 65 years old. The cNLBP patients were recruited for pain extending from the lower ribs to the subgluteal fold for more than three months or intermittently for more than six months, and confirmed by two approved physicians according to the diagnostic criteria established by the American Pain Society and the American College of Physicians. In addition, the visual analog scale (VAS) score of cNLBP patients was required to be greater than or equal to 2 points. Other inclusion criteria included right-handedness, no neurological disease or intracranial lesions, and no treatment for back pain in the past three months. Exclusion criteria included: patients with inflammatory spinal disease, vertebral fracture, severe osteoporosis, autoinflammatory arthritis, cancer or unexplained significant weight loss, pregnancy, back pain, postpartum low back pain or menstrual discomfort, cardiovascular and cerebrovascular diseases or endocrine disorders, patients with mental health conditions requiring medication, patients unwilling to sign the study consent form or unwilling to comply with the study protocol, and those with alcohol or substance dependence were excluded from the study. Before the start of the study, pain intensity of all subjects was assessed using VAS, and blood samples were collected for lipidomics analysis. Ethical approval for this study (no.

[2023] 596) was granted by the First Affiliated Hospital of Sun Yat-sen University. All participants provided signed informed consent.

[0037] 2. Extraction of lipid profiles from serum of healthy volunteers and cNLBP patients

[0038] (1) Venous blood was collected from healthy volunteers (HV group) and cNLBP patients (cNLBP group) using heparinized tubes and then centrifuged at 2500 rpm at 4°C to collect serum.

[0039] (2) Take 200 μL of serum sample, add 400 μL of tert-butyl methyl ether (MTBE) and 80 μL of methanol, vortex for 30 seconds, and shake to mix.

[0040] (3) Ultrasonic treatment in an ice-water bath for 10 min, quick freezing in liquid nitrogen for 1 min. Repeat three times. Place at -20°C for 1 h. Centrifuge at 13,000 rpm, 4°C for 15 min. Take the supernatant and blow dry with nitrogen. Add 50 μL of dichloromethane and 50 μL of methanol to redissolve the sample. Oscillate for 30 s, ultrasonic treatment in an ice-water bath for 10 min. Centrifuge at 13,000 rpm, 4°C for 15 min. Take the supernatant, which is the lipidome, and prepare it for detection.

[0041] 3. LC-MS / MS analysis of lipid profiles in healthy volunteers and cNLBP patients

[0042] The lipidomes of healthy volunteers and cNLBP patients were measured using Shimadzu LC-30A (Shimadzu, Japan) coupled with a mass spectrometer (QTRAP 6500, AB SCIEX, USA).

[0043] (1) Liquid chromatography conditions are as follows:

[0044] Column: ACQUITY BEH C18 column (2.1×100mm, 1.7μm, Waters, USA)

[0045] Injection volume: 5 μl

[0046] Flow rate: 0.26mL / min

[0047] Column temperature: 55°C

[0048] Mobile phase A: acetonitrile: H2O (v / v) = 60:40, containing 10 mM ammonium acetate

[0049] Mobile phase B: isopropanol: acetonitrile (v / v) = 90:10, containing 10 mM ammonium acetate

[0050] Gradient elution conditions: 0-1.5 minutes, 68% mobile phase A; 1.5-15.5 minutes, 15% mobile phase A; 15.5-15.6 minutes, 3% mobile phase A; 15.6-18 minutes, 3% mobile phase A; 18-18.1 minutes, 68% mobile phase A; 18.1-20 minutes, 68% mobile phase A.

[0051] (2) Mass spectrometry conditions are as follows:

[0052] Electrospray ionization (ESI) source.

[0053] Ion source temperature: 600°C

[0054] Ion source voltage: -4500V or 5500V

[0055] Curtain gas: 20psi

[0056] Nebulizer gas and auxiliary gas: both at 60psi.

[0057] Multiple reaction monitoring (MRM) was used for scanning.

[0058] 4. Data Analysis

[0059] The abundance of different lipids was quantified based on the peak area. The data were normalized with reference samples and analyzed according to the procedures specified on the website https: / / www.metaboanalyst.ca / . The covariance between the HV group and the cNLBP group was detected by using orthogonal partial least squares discriminant analysis (OPLS-DA). The lipidome data of the HV group and cNLBP patients were analyzed by OPLS-DA. Figure 1As shown, the results showed that the HV group and the cNLBP group had different lipidomic characteristics between individuals, and there were potential differences in lipid metabolism between the HV group and cNLBP patients.

[0060] The 1086 lipids tested were divided into 17 categories, and correlation analysis was performed on these 17 categories of lipids. The lipid molecule correlation heat map shows the correlation between different lipids. The correlation analysis of lipid molecules in different samples can be performed to find lipid molecules with strong correlation in the samples. The results are as follows Figure 2 As shown in the figure, the redder the color, the more positively correlated the two lipids are, and the bluer the color, the more negatively correlated the two lipids are. The results show that TAG has a high correlation with MAG and DAG, indicating that TAG may be converted from these two glycerolipids.

[0061] The changes in the contents of the 17 lipid components measured in the HV group and the cNLBP group were analyzed. The statistical analysis was performed using the Student's t-test (p < 0.05) (data are expressed as mean ± standard error). The results are shown in Figure 3 As shown, from Figure 3 It can be seen that among the 17 lipid components, only TAG showed significant differences (significantly increased) between the HV group and cNLBP patients, while the remaining 16 lipids did not show significant changes. Therefore, TAG can be used as a biomarker for chronic nonspecific low back pain.

[0062] Furthermore, the peak areas of 34 TAG components determined by mass spectrometry were analyzed, and the results were as follows: Figure 4 As shown. Figure 4 It can be seen that the responses of the six triacylglycerols in the cNLBP group - TAG (56:3 / FA18:2), TAG (50:0 / FA16:0), TAG (54:2 / FA18:2), TAG (52:4 / FA18:0), TAG (56:3 / FA18:0) and TAG (54:2 / FA20:0) - in the mass spectrometry were significantly higher than those in the HV group (healthy people, the contents of these six TAGs are shown in Table 1). The six TAG components were accurately quantitatively analyzed based on the internal standard content, and the results are as follows Figure 5 shown.

[0063] Table 1

[0064] Components Content in healthy population (mmol / L) TAG(56:3 / FA18:2) 0.048±0.0005 TAG(50:0 / FA16:0) 0.05±0.04 TAG(54:2 / FA18:2) 0.003±0.004 TAG(52:4 / FA18:0) 0.011±0.013 TAG(56:3 / FA18:0) 0.011±0.009 TAG(54:2 / FA20:0) 0.002±0.003

[0065] In summary, this example identified the differential lipid components between the HV group and the cNLBP group, and clarified from the perspective of lipidomics that six TAGs (TAG(56:3 / FA18:2), TAG(50:0 / FA16:0), TAG(54:2 / FA18:2), TAG(52:4 / FA18:0), TAG(56:3 / FA18:0) and TAG(54:2 / FA20:0)) can be used as biomarkers of cNLBP.

[0066] Example 2 Specificity and sensitivity of the biomarkers for chronic nonspecific low back pain in adults of Example 1

[0067] In order to verify the specificity and sensitivity of the six TAG components screened in Example 1 as biomarkers for chronic nonspecific low back pain in adults, ROC curve analysis was performed on these six TAG components. The results are as follows: Figure 6 shown.

[0068] Figure 6 The results showed that the AUC (ROC curve area) of the six TAG components was 0.7436-0.9013, and the P values ​​were all less than 0.05, which was statistically significant, indicating that the six TAG components were meaningful for the diagnosis of chronic nonspecific low back pain. The larger the AUC, the greater the diagnostic efficacy of the component.

[0069] Example 3 Diagnosis of actual samples using the biomarkers of Example 1

[0070] In this example, the LC-MS / MS detection conditions of Example 1 were used to quantitatively detect the contents of 6 TAG components in the serum lipid groups of 10 samples with known results, and the detection results were analyzed for significant differences with the results in Table 1. The results are shown in Table 2.

[0071] Table 2

[0072]

[0073] The results in Table 2 show that the contents of the six TAGs in the 10 samples were significantly higher than those in the healthy population, and the 10 samples were diagnosed as patients with chronic nonspecific low back pain. The accuracy of using the six TAG components of the present invention as biomarkers to judge chronic nonspecific low back pain was 100%.

[0074] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0075] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Use of triacylglycerol as a biomarker in the preparation of a kit for diagnosing chronic nonspecific low back pain, characterized in that: The triacylglycerols are TAG(56:3 / FA18:2), TAG(50:0 / FA16:0), TAG(54:2 / FA18:2), TAG(52:4 / FA18:0), TAG(56:3 / FA18:0) and TAG(54:2 / FA20:0).

2. Use of a reagent for determining the level of triacylglycerol in preparing a kit for diagnosing chronic nonspecific low back pain, characterized in that: The triacylglycerols are TAG(56:3 / FA18:2), TAG(50:0 / FA16:0), TAG(54:2 / FA18:2), TAG(52:4 / FA18:0), TAG(56:3 / FA18:0) and TAG(54:2 / FA20:0).

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