Biomarker for diagnosing obesity and complications thereof and application of biomarker

Abstract

The invention discloses a biomarker for diagnosing obesity and complications thereof and application of the biomarker. The biomarker is one or a combination of more of ADAM8, FASN and ELOVL6. The invention also provides a kit for diagnosing obesity and complications thereof. The kit comprises a reagent for detecting the expression level of one or more proteins of ADAM8, FASN and ELOVL6. According to the present invention, the expression levels of ADAM8, FASN and ELOVL6 in serum are detected by using the ELISA kit, such that the sensitivity and the specificity are high. The method is easy and convenient to operate and accurate in result, early diagnosis and intervention of obesity-related hyperlipemia are facilitated, and the burden of patients and the society is relieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to biomarkers for diagnosing obesity and its complications and applications thereof. Background Art

[0002] Obesity and its associated complications, such as hyperlipidemia, have become a global public health problem. Traditional diagnostic methods rely primarily on measurements of body mass index (BMI) and lipid levels, but these methods have limitations in sensitivity and specificity. Therefore, identifying new, more sensitive, and specific biomarkers is crucial for the early diagnosis and intervention of obesity-related hyperlipidemia. Summary of the Invention

[0003] The purpose of the present invention is to provide a biomarker for the diagnosis of obesity and its complications and its application, to explore and discover key molecules of white fat for the diagnosis and treatment of obesity and related complications, to detect key genes in the plasma of clinical patients, and to optimize the detection reagents to verify its diagnostic application for obesity.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] In a first aspect, the present invention provides a biomarker for diagnosing obesity and its complications.

[0006] Biomarkers for diagnosing obesity and its complications, wherein the biomarkers are a combination of one or more of ADAM8, FASN, and ELOVL6.

[0007] Preferably, the biomarker is a combination of ADAM8, FASN, and ELOVL6.

[0008] In a second aspect, the present invention provides a kit for diagnosing obesity and its complications.

[0009] A kit for diagnosing obesity and its complications comprises reagents for detecting the expression levels of one or more proteins of ADAM8, FASN, and ELOVL6.

[0010] Preferably, the reagent is an ELISA reagent, a Western Blot reagent or an immunohistochemistry reagent.

[0011] Preferably, the ELISA reagent includes specific antibodies against ADAM8, FASN, and ELOVL6, and the specific antibodies can recognize and bind to ADAM8, FASN, and ELOVL6 proteins in biological samples, and generate detectable signals through enzymatic reactions.

[0012] Preferably, the biological sample is one or more of serum, plasma, blood, sputum, and cerebrospinal fluid.

[0013] Preferably, the kit includes ELISA reagents, and related buffers, substrates and standards for ELISA detection.

[0014] In a third aspect, the present invention provides the use of a reagent for detecting the expression level of one or more proteins of ADAM8, FASN, and ELOVL6 in the preparation of a product for diagnosing obesity and its complications.

[0015] Preferably, the product distinguishes obese from non-obese people and predicts or diagnoses the risk of obesity-related complications by detecting the expression levels of one or more proteins of ADAM8, FASN and ELOVL6 in biological samples.

[0016] Preferably, the biological sample is one or more of serum, plasma, blood, sputum, and cerebrospinal fluid.

[0017] Preferably, when the ADAM8 expression level is detected to be higher than the reference value, and / or the FASN expression level is detected to be lower than the reference value, and / or the ELOVL6 expression level is detected to be lower than the reference value, it indicates that the subject is at risk of obesity or related complications.

[0018] More preferably, when the ADAM8 expression level is detected to be higher than the reference value, and the FASN expression level is lower than the reference value, and the ELOVL6 expression level is lower than the reference value, it indicates that the subject is at risk of obesity or related complications.

[0019] Preferably, the reference value is the average value of the expression levels of ADAM8, FASN and ELOVL6 proteins in serum or plasma of healthy people.

[0020] Preferably, the complication includes obesity-related hyperlipidemia.

[0021] In this application, ADAM8, FASN, and ELOVL6 correspond to the abbreviations of disintegrin metalloproteinase 8, fatty acid synthase, and long-chain fatty acid elongase 6, respectively. For specific sequences, please refer to databases such as Uniprot / Genbank, and various variants therein are within the scope of this application.

[0022] Based on the knowledge of the sequences and properties of ADAM8, FASN, and ELOVL6, those skilled in the art can routinely obtain reagents for detecting ADAM8, FASN, and ELOVL6 levels, such as ELISA reagents using monoclonal or polyclonal antibodies, Western Blot reagents, or immunohistochemistry reagents. These reagents can be homemade or purchased commercially.

[0023] Beneficial Effects: This invention provides a biomarker for diagnosing obesity and its complications, and its application. The ELISA kit detects the expression levels of ADAM8, FASN, and ELOVL6 in serum with high sensitivity and specificity. This method is simple to use and provides accurate results, facilitating early diagnosis and intervention for obesity-related hyperlipidemia, and reducing the burden on patients and society. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the expression level of serum ADAM8, FASN, and ELOVL6 in obese and normal subjects;

[0025] Figure 2 This is a scatter plot of the correlation between serum ADAM8, FASN, ELOVL6 and BMI in obese and normal people;

[0026] Figure 3 This is the ROC curve of serum ADAM8, FASN, and ELOVL6 in people with increased BMI and normal people. DETAILED DESCRIPTION

[0027] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the embodiments and drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0028] Example 1

[0029] Screening and validation of biomarkers: The present invention first performed bioinformatics analysis using two sequencing datasets related to gonadal white fat in hyperlipidemic mice, GSE38337 and GSE76812, and found that ADAM8 was the only biomarker whose expression was significantly increased in WAT after high fat diet. 24 genes, including Rbp4, Gsta3, Pcx, Immp2l, Cldn22, Gbe1, Enpep, Acss2, Pex11a, Fasn, Acsm3, Oscp1, Gpd2, Pygl, Slc25a1, Tkt, Thrsp, Acaca, Acacb, Nat8l, Pmvk, Elovl6, Tmem120b, and Acly, were genes with decreased expression. Among these genes, only Fasn and Elovl6 had logFC values ​​> 2 in both datasets. In this application, clinical sample validation was performed for the only elevated ADAM8 and the two genes with the largest decreases, FASN and ELOVL6.

[0030] ADAM8, P78325, standard sequence is:

[0031] MRGLGLWLLGAMMLPAIAPSRPWALMEQYEVVLPWRLPGPRVRRALPSHLGLHPERVSYVLGATGHNFTLHLRKNRDLLGSGYTETYTAANGSEVTEQPRGQDHCFYQGHVEGYPDSAASLSTCAGLRGFFQVGSDLHLIEPLDEGGEGGRHAVYQAEHLLQTAGTCGVSDDSLGSLLGPRTAAVFRPRPGDSLPSRETRYVELYVVVDNAEFQMLGSEAAVRHRVLEVVNHVDKLYQKLNFRVVLVGLEIWNSQDRFHVSPDPSVTLENLLTWQARQRTRRHLHDNVQLITGVDFTGTTVGFARVSAMCSHSSGAVNQDHSKNPVGVACTMAHEMGHNLGMDHDENVQGCRCQERFEAGRCIMAGSIGSSFPRMFSDCSQAYLESFLERPQSVCLANAPDLSHLVGGPVCGNLFVERGEQCDCGPPEDCRNRCCNSTTCQLAEGAQCAHGTCCQECKVKPAGELCRPKKDMCDLEEFCDGRHPECPEDAFQENGTPCSGGYCYNGACPTLAQQCQAFWGPGGQAAEESCFSYDILPGCKASRYRADMCGVLQCKGGQQPLGRAICIVDVCHALTTEDGTAYEPVPEGTRCGPEKVCWKGRCQDLHVYRSSNCSAQCHNHGVCNHKQECHCHAGWAPPHCAKLLTEVHAASGSLPVFVVVVLVLLAVVLVTLAGIIVYRKARSRILSRNVAPKTTMGRSNPLFHQAASRVPAKGGAPAPSRGPQELVPTTHPGQPARHPASSVALKRPPPAPPVTVSSPPFPVPVYTRQAPKQVIKPTFAPPVPPVKPGAGAANPGPAEGAVGPKVALKPPIQRKQGAGAPTAP

[0032] FASN, P49327, the standard sequence is:

[0033]

[0034] ELOVL6, Q9H5J4, the standard sequence is:

[0035] MNMSVLTLQEYEFEKQFNENEAIQWMQENWKKSFLFSALYAAFIFGGRHLMNKRAKFELRKPLVLWSLTLAVFSIFGALRTGAYMVYILMTKGLKQSVCDQGFYNGPVSKFWAYAFVLSKAPELGDTIFIIL RKQKLIFLHWYHHITVLLYSWYSYKDMVAGGGWFMTMNYGVHAVMYSYYALRAAGFRVSRKFAMFITLSQITQMLMGCVVNYLVFCWMQHDQCHSHFQNIFWSSLMYLSYLVLFCHFFFEAYIGKMRKTTKAE

[0036] Example 2

[0037] Validation of diagnostic efficacy in real populations

[0038] This example included 78 CAD patients who were screened in the Department of Cardiology, Zhongda Hospital, Southeast University between March 2019 and December 2020. This study was approved by the Ethics Committee of Zhongda Hospital, Southeast University (2018ZDSYLL134-P01). Inclusion criteria were: (1) male or non-pregnant female aged 18-85 years; (2) willingness to sign a written informed consent. Exclusion criteria were: (1) acute myocardial infarction; (2) history of coronary artery bypass grafting surgery; (3) acute ischemic stroke or bleeding; (4) severe liver or kidney disease; (5) uncontrolled severe hypertension; (6) severe dyspnea; (7) recent history of surgery; (8) suffering from other serious diseases with a life expectancy of less than one year; (9) any other condition that the researcher deemed unsuitable for participation in this study. Classification according to BMI further verified that the expression level of ADAM8 in the serum of obese people was significantly higher than that of non-obese people. Serum samples of the above two groups of subjects were collected and stored in a -80℃ refrigerator. Each serum sample was centrifuged at 3000 rpm for 20 minutes at room temperature, repeated twice to completely remove cellular debris, and the supernatant was collected and stored in a -80°C freezer. Based on the patient's BMI calculation results, all patients were divided into three groups: BMI <24 (n=31); 24≤BMI<28 (n=28); and BMI ≥28 (n=19).

[0039] Specific detection steps: First, the applicant designs a reagent step for in vitro quantitative detection of ADAM8, FASN, and ELOVL6 content in human serum, plasma, and related liquid samples. A double-antibody sandwich enzyme-linked immunosorbent assay is used to achieve high-sensitivity and high-specificity detection of the target protein. A 96-well microtiter plate is pre-coated with capture antibodies specific for ADAM8, FASN, and ELOVL6. Lyophilized standards of ADAM8, FASN, and ELOVL6 are prepared, each containing 6 concentration gradients (for example: 2000pg / mL, 1000pg / mL, 500pg / mL, 250pg / mL, 125pg / mL, 62.5pg / mL) for establishing a standard curve. Biotin-labeled anti-ADAM8, FASN, and ELOVL6 detection antibodies are prepared. Horseradish peroxidase-labeled avidin is prepared for binding to the biotin-labeled detection antibody. Sample diluent is used to dilute samples such as serum and plasma to adjust to the appropriate detection concentration range. Wash buffer is used to wash the microplate to remove unbound antibody and impurities. TMB (3,3',5,5'-tetramethylbenzidine) substrate solution is used for the colorimetric reaction. First, dilute the concentrated wash buffer with distilled water at a ratio of 1:25. Simultaneously, reconstitute the standard with sample diluent and serially dilute it to a concentration gradient of 4000, 2000, 1000, 500, 250, 125, 62.5, and 0 pg / mL. Detection antibody-HRP working solution should be diluted 1:100 before use. During the operation, set the standard wells, blank wells and sample wells respectively, add the corresponding liquids in sequence, seal the plate, and incubate at 37°C for 90 minutes; then discard the liquid, wash 5 times, add the detection antibody-HRP working solution, seal the plate again, incubate for 30 minutes and wash; then add 50μL of substrate A and substrate B to each well, incubate at 37°C in the dark for 15 minutes, and then add 50μL of stop solution to terminate the reaction; finally, measure the OD value of each well at a wavelength of 450nm within 15 minutes.

[0040] Compared with the BMI<24 group, the ADAM8 expression level in the 24≤BMI<28 and BMI≥28 groups gradually increased, while the FASN and ELOVL6 expression levels decreased, and both were statistically significant ( Figure 1 Pearson correlation analysis showed that ADAM8 was positively correlated with BMI (r=0.802, P<0.001), triglyceride (TG) (r=0.286, P=0.011), and high-sensitivity C-reactive protein (CRP) (r=0.454, P=0.009); FASN (r=-0.635, P<0.001) and ELOVL6 (r=-0.601, P<0.001) were negatively correlated with BMI ( Figure 2 ).

[0041] ROC curves showed that when comparing the groups with normal BMI and those with elevated BMI, the AUC of ADAM8 was 0.967, P < 0.001; the AUC of FASN was 0.978, P < 0.001; the AUC of ELOVL6 was 0.938, P < 0.001 ( Figure 3 Generally, an AUG of 0.5-0.7 indicates low diagnostic accuracy; an AUG of 0.7-0.9 indicates moderate diagnostic accuracy; and an AUG > 0.9 indicates high diagnostic accuracy. Linear regression suggests that after adjusting for BMI, ADAM8 is an independent and effective factor in predicting TG (P = 0.008). Logistic regression suggests that ADAM8 is an independent and effective factor in predicting TG (P = 0.011).

[0042] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A biomarker for the diagnosis of obesity and its complications, characterized by: The biomarker is one or more combinations of ADAM8, FASN, and ELOVL6.

2. A kit for diagnosing obesity and its complications, characterized by: Contains reagents for detecting the expression level of one or more proteins of ADAM8, FASN, and ELOVL6.

3. The kit according to claim 2, wherein: The reagent is an ELISA reagent, a Western Blot reagent or an immunohistochemistry reagent.

4. The kit according to claim 3, wherein: The ELISA reagent includes specific antibodies against ADAM8, FASN, and ELOVL6. The specific antibodies can recognize and bind to ADAM8, FASN, and ELOVL6 proteins in biological samples and generate detectable signals through enzymatic reactions.

5. The kit according to claim 4, wherein: The kit includes ELISA reagents, and related buffers, substrates and standards for ELISA detection.

6. Use of reagents for detecting the expression levels of one or more proteins of ADAM8, FASN, and ELOVL6 in the preparation of products for diagnosing obesity and its complications.

7. The use according to claim 6, characterized in that: The product distinguishes obese from non-obese people and predicts or diagnoses the risk of obesity-related complications by detecting the expression levels of one or more proteins, ADAM8, FASN and ELOVL6, in biological samples.

8. The use according to claim 7, characterized in that: The biological sample is one or more of serum, plasma, blood, sputum, and cerebrospinal fluid.

9. The use according to claim 6, characterized in that: When the ADAM8 expression level is higher than the reference value, and / or FASN expression level is lower than the reference value, and / or When the ELOVL6 expression level is lower than the reference value, Indicates that the subject is at risk for obesity or related complications.

10. The use according to claim 9, characterized in that: The reference value is the average value of the expression levels of ADAM8, FASN and ELOVL6 proteins in serum or plasma of healthy people.

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