Adipose tissue lipid metabolite for evaluating fat health state and detection kit and application of adipose tissue lipid metabolite

Abstract

The invention relates to adipose tissue lipid metabolites for evaluating the health state of fat and a detection kit and application of the adipose tissue lipid metabolites. Specifically, the lipid metabolites arachidonic acid and docosapentaenoic acid can be used as markers for evaluating the health state of adipose tissue metabolism; the arachidonic acid can be used for improving obesity metabolic disorder; according to the kit, fat tissue lipid metabolites are rapidly detected, so that the fat tissue is directly and rapidly evaluated; according to the detection kit, the technical requirements on operators are reduced, the operation steps are simplified, and the detection kit can be applied on a large scale, so that early detection and prevention and treatment of obesity-related diseases are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection and its related clinical medical application, and in particular to adipose tissue lipid metabolites and detection kits and applications thereof for evaluating the health status of fat. Background Art

[0002] Obesity is a global public health problem. It is not only associated with a variety of chronic diseases, but may also lead to complications such as metabolic inflammation. Biomarkers play an important role in the diagnosis, risk assessment and treatment response monitoring of obesity. At present, conventional obesity biomarkers include: blood tests: abdominal blood glucose, insulin levels and lipid profiles, leptin and adiponectin, high-sensitivity C-reactive protein (CRP); urine tests: creatinine and microalbumin; tissue tests: gene expression analysis of adipose tissue, etc. In recent years, researchers have discovered some new biomarkers, such as: the secretory form of PDIA3 is a reliable marker for the degree of metabolic inflammation in obesity; leptin (LEP), adrenomedullin (ADM), fatty acid binding protein 4 (FABP4), insulin-like growth factor binding protein 1 (IGFBP1) and advanced glycation end product specific receptor (AGER) are key biomarkers for predicting body mass index (BMI); differentially expressed proteins (DEPs) in plasma exosomes between obese and non-obese diabetic patients; MT1-MMP can be used as a potential target for obesity treatment, etc.

[0003] However, the above biomarkers are mainly indirect indicators used to characterize obesity symptoms, and their relationship with the health status of fat is not clear. Adipose tissue, as a key energy storage organ, has a very close and direct relationship with the occurrence and development of obesity. Experimental studies have shown that collecting abdominal wall adipose tissue samples can provide valuable data for studying adipocyte function, changes in inflammatory markers, and gene expression. Unlike traditional indirect detection indicators, direct and rapid detection of adipose tissue can not only provide more intuitive metabolic health status information, but also has great significance for understanding the mechanism of obesity. At present, the metabolic markers for monitoring the health status of adipose tissue mainly include triglycerides, leptin, adiponectin, adenosine, and some inflammatory factors such as C-reactive protein (CRP) and tumor necrosis factor α (TNF-α). The above metabolic markers can effectively evaluate the health status of adipose tissue and its impact on systemic metabolism. By detecting the changes in the levels of these markers, it can provide an important reference for the diagnosis, treatment and prevention of obesity.

[0004] However, the detection methods of the above-mentioned biomarkers have the following shortcomings: (1) Traditional methods of detecting obesity-related prevention and treatment markers, such as blood drawing, genetic screening, and fecal microbial flora analysis, are relatively complicated to operate; moreover, these methods are mainly based on indirect markers or metabolic risk assessments, lack direct assessment and detection of adipose tissue, and are difficult to reflect the patient's obesity-related pathological status indicators; (2) Although there are some studies that have conducted direct detection of adipose tissue, in these studies, the sampling and analysis methods of adipose tissue are usually cumbersome, require high professional skills from operators, are highly invasive to patients, and have many postoperative complications; many corresponding analysis methods are expensive and not worth large-scale clinical application. For example, the currently commonly used mass spectrometry method for detecting lipid metabolites in adipose tissue and the western blot method for detecting protein expression are costly, complex to operate, and require a large amount of samples. They are difficult to use on a large scale in clinical work, which limits their practicality and popularity.

[0005] In addition, although there are studies on the metabolites of adipose tissue, for example, the lipid metabolites creatinine and / or xanthine in adipose tissue can be detected for the diagnosis of osteoarthritis, no studies have reported the role of arachidonic acid and docosapentaenoic acid in assessing the health status of fat, and there is still a lack of a complete set of detection kits for adipose tissue lipid metabolites. Summary of the invention

[0006] In order to overcome at least one problem existing in the prior art, the present invention conducts in-depth research on adipose tissue lipid metabolites and finds that arachidonic acid and docosapentaenoic acid can be used as markers of healthy metabolic adipose tissue. Using arachidonic acid to treat obese mice induced by a high-fat diet can effectively improve their obesity metabolic disorders. The present invention also provides a rapid detection kit for adipose tissue lipid metabolites, which directly uses adipose tissue as a sample, thereby achieving direct and rapid evaluation of adipose tissue.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention is to provide the use of adipose tissue lipid metabolites as markers for assessing fat health status, wherein the adipose tissue lipid metabolites include at least one of arachidonic acid (AA, C20:4) and docosapentaenoic acid (C22:5).

[0009] Furthermore, the adipose tissue lipid metabolites also include at least one of conventional lipid metabolism detection markers, and the conventional lipid metabolism detection markers include: fatty acids (FA), triglycerides (TG), total cholesterol (TC), cholesterol esters (CE), free cholesterol (FC), high-density lipoprotein cholesterol (HDL-c), and low-density lipoprotein cholesterol (LDL-c).

[0010] Further, the adipose tissue lipid metabolites are selected from orbital fat, abdominal subcutaneous fat, and adipose tissue lipid metabolites in serum. Preferably, the adipose tissue lipid metabolites are adipose tissue lipid metabolites in abdominal subcutaneous fat.

[0011] The second aspect of the present invention is to provide the use of adipose tissue lipid metabolites in the preparation of a preparation for treating obesity, wherein the adipose tissue lipid metabolites include at least one of arachidonic acid and docosapentaenoic acid.

[0012] Furthermore, the adipose tissue lipid metabolite is arachidonic acid, which is used to prepare a preparation for improving obesity metabolic disorders.

[0013] Furthermore, the improvement of obesity metabolic disorders includes: reducing fat weight; improving blood lipid and blood sugar metabolism indicators; improving serum inflammation levels and leptin and adiponectin levels; improving fat inflammation and fat cell size; and improving fatty liver and liver fibrosis.

[0014] Furthermore, the preparation contains other components for preventing and treating obesity, and may also contain pharmaceutically acceptable auxiliary ingredients.

[0015] Further, in a specific embodiment, the amount of arachidonic acid used is 2 mg / kg mouse, and the administration period is 7 weeks.

[0016] The third aspect of the present invention is to provide a use of any adipose tissue lipid metabolite described in the first aspect in the preparation of a kit for assessing the metabolic health status of adipose tissue of a subject.

[0017] The fourth aspect of the present invention is to provide a kit for detecting adipose tissue lipid metabolites, wherein the adipose tissue lipid metabolites are any adipose tissue lipid metabolites described in the first aspect.

[0018] Furthermore, the kit includes an antibody pre-coated microplate, a sample homogenization lysis solution, a sample simple homogenizer, a standard diluent, a standard, a labeled antibody biotinylated antibody, a horseradish peroxidase label, a washing solution, a color developer, a stop solution, a plate sealing film, and a sample centrifuge tube. The kit may also include an instruction manual to explain the components of each reagent and the operating steps of the kit.

[0019] Furthermore, the kit also includes a fat tissue collection device, which can be a biopsy needle, anesthesia biopsy needle, etc. conventionally used in the art. The fat tissue used can be directly used for the detection of fat tissue lipid metabolites after sample pretreatment.

[0020] Furthermore, the kit uses an absorbance (OD value) determination method to indirectly determine the content of lipid metabolites in the adipose tissue, specifically, after adding a substrate for color development, the absorbance (OD value) is determined at a specific wavelength using an enzyme marker.

[0021] The fifth aspect of the present invention is to provide an application of any kit described in the fourth aspect, which is selected from at least one of the following applications: application in evaluating the metabolic health status of adipose tissue of a subject; application in screening drugs for improving diseases related to abnormal lipid metabolism; application in evaluating the effects of drugs or treatments on lipid metabolism.

[0022] Specifically, the above-mentioned kit can be used for personalized medicine in clinical medicine. By detecting the levels of specific lipid metabolites, it can provide a scientific basis for formulating personalized nutrition and drug intervention plans, thereby optimizing the treatment effect and preventing related metabolic diseases; in public health and epidemiological research, the above-mentioned kit is used for large-scale population screening, evaluating the healthy status of lipid metabolism, and obtaining epidemiological data on the effects of nutrition, lifestyle and genetic factors on lipid metabolism, thereby promoting a deeper understanding and implementing more effective management and treatment strategies; at the same time, the above-mentioned kit plays an important role in drug research and development and efficacy evaluation, helping to evaluate the effects of emerging drugs or therapeutic interventions on lipid metabolism, and supporting the development of drugs and their application research in humans.

[0023] Compared with the prior art, the present invention adopts the above technical solution to have the following beneficial effects:

[0024] (1) The present invention innovatively proposes that lipid metabolites arachidonic acid and docosapentaenoic acid can be used as markers for assessing the metabolic health of adipose tissue, and further verifies that arachidonic acid can be used for the prevention and treatment of obesity;

[0025] (2) In view of the complexity of the prior art in the detection of obesity-related markers and the lack of assessment of adipose tissue directly related to obesity, the present invention introduces a rapid kit for direct assessment of adipose tissue, which can directly and quickly assess adipose tissue by rapidly detecting adipose tissue lipid metabolites (metabolic composition), thereby generating more accurate individual pathological status data. Compared with the detection of indirect indicators, the detection kit used in the present invention has higher clinical practicality and diagnostic value.

[0026] (3) In view of the problems of complex operation and high cost of direct detection methods of adipose tissue in the prior art, the rapid detection kit designed in the present invention reduces the technical requirements for operators and simplifies the operation steps, so that it can be applied on a larger scale, thereby improving the early detection and management and treatment effects of obesity-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0028] Figure 1 Schematic diagram of immunohistochemical staining of CD68, a marker of adipose tissue macrophages, in orbital fat (OF) and abdominal subcutaneous fat (SF) of obese people in one embodiment of the present invention;

[0029] Figure 2 Schematic diagram of the comparison of the contents of adipose tissue lipid metabolites arachidonic acid and docosapentaenoic acid in orbital fat (OF) and abdominal subcutaneous fat (SF) of obese people in one embodiment of the present invention;

[0030] Figure 3 This is a schematic diagram comparing the content of arachidonic acid in adipose tissue of normal mice and obese mice induced by a high-fat diet in one embodiment of the present invention;

[0031] Figure 4 This is a schematic diagram of the changes in body weight, white fat weight and liver weight of high-fat diet obese mice after arachidonic acid intervention in one embodiment of the present invention;

[0032] Figure 5 This is a schematic diagram of changes in blood lipid and blood glucose metabolism indicators in high-fat diet obese mice after arachidonic acid intervention in one embodiment of the present invention;

[0033] Figure 6 This is a schematic diagram of changes in serum inflammation levels and leptin and adiponectin levels in high-fat diet obese mice after arachidonic acid intervention in one embodiment of the present invention;

[0034] Figure 7 This is a schematic diagram of changes in fat inflammation and adipocyte size in high-fat diet obese mice after arachidonic acid intervention in one embodiment of the present invention;

[0035] Figure 8 This is a schematic diagram of the changes in fatty liver and liver fibrosis in obese mice fed a high-fat diet after arachidonic acid intervention in one embodiment of the present invention. DETAILED DESCRIPTION

[0036] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The experimental methods without specific conditions in the following embodiments are usually measured according to national standards. The experimental materials without the source indicated in the following embodiments are all commercially available raw materials. The equipment used in each step in the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standards, conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise specified, all parts are weight parts and all percentages are mass percentages. Unless otherwise defined or specified, all professional and scientific terms used in the present invention have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equal to the recorded content can be applied to the method of the present invention.

[0037] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention is further described below in conjunction with the drawings and specific embodiments, but is not intended to be a limitation of the present invention.

[0038] Example 1 - Screening of adipose tissue lipid metabolites for assessing adipose health status

[0039] This example analyzes the content of lipid metabolites in adipose tissue and determines that arachidonic acid and docosapentaenoic acid can be used as markers for assessing the health status of fat, specifically including the following steps:

[0040] (1) In adipose tissue in a healthy metabolic state, the levels of arachidonic acid and docosapentaenoic acid are elevated;

[0041] 1) Collection and post-processing steps of orbital fat (OF) and abdominal subcutaneous fat (SF) of obese patients: In clinical practice, excess orbital fat tissue is collected from patients during blepharoplasty, and subcutaneous fat tissue is collected from patients during abdominal wall plastic surgery and liposuction for subsequent experiments. The collection process follows a strict and sterile operating procedure, and the above collection has obtained the informed consent of the patient and the approval of the ethics committee. An appropriate amount of adipose tissue was taken out under a sterile environment, and the sample was gently washed with saline under sterile conditions to remove blood and impurities, and then placed in a sterile clean EP tube. The fat samples used for subsequent immunohistochemical observation were fixed in formalin and stored at room temperature to maintain the cell structure. The samples used for the subsequent lipid metabolite determination step were kept at a low temperature by dry ice, quickly transferred to the laboratory, and kept on ice during transportation. Precautions include maintaining sterile operation, shortening the collection and processing time as much as possible to reduce the risk of sample degradation, and recording each step and any abnormal phenomena observed in detail.

[0042] 2) Immunohistochemical staining of CD68 was used to detect macrophages in adipose tissue and compare the inflammatory conditions of periorbital fat and abdominal fat to establish a research model for metabolic health of periorbital adipose tissue. Antigen retrieval was performed at high temperature using sodium citrate buffer to expose antigen epitopes. Then, the sections were treated with hydrogen peroxide to block endogenous peroxidase activity. Next, normal goat serum was added to the sections to block nonspecific binding sites. Subsequently, the primary antibody for CD68 was applied and incubated under appropriate conditions. After washing, the corresponding biotinylated secondary antibody was added and incubated again. Afterwards, horseradish peroxidase-labeled streptavidin and DAB were used for color development to reveal brown positive signals. Finally, hematoxylin was used for counterstaining to facilitate microscopic observation of cell nuclei, and the sections were dehydrated with gradient alcohol and sealed with neutral gum. The distribution and density of macrophages were subsequently observed and analyzed under a microscope. The above test results are shown in Figure 1 .

[0043] like Figure 1 As shown, compared with abdominal subcutaneous fat (SF), orbital fat (OF) of obese people has smaller adipocytes and mild adipose inflammation, which is a metabolically healthy adipose tissue; while SF has larger adipocytes, significant macrophage infiltration (red arrows), and obvious macrophage crown structures (red triangles), indicating chronic inflammation of adipose tissue. The results show that orbital adipose tissue is a good model for studying healthy fat metabolism.

[0044] 3) Lipid metabolites were determined using conventional chromatography-mass spectrometry targeted metabolite detection methods in the art, and the results were as follows: Figure 2 As shown in the figure, the contents of arachidonic acid and docosapentaenoic acid were found to be significantly higher in OF than in SF.

[0045] These results suggest that arachidonic acid and docosapentaenoic acid are markers of healthy metabolic adipose tissue.

[0046] (2) In the adipose tissue of obese mice, the content of arachidonic acid (AA) is decreased;

[0047] 1) Construction of a high-fat diet-induced obese mouse model: C57BL / 6 mice were used and fed with a high-fat diet for 8 weeks. First, healthy 4-week-old C57BL / 6 mice were selected and fed under normal diet conditions for 1 week to adapt them to the laboratory environment. Then the mice were randomly divided into a normal diet group (control group, NCD group) and a high-fat diet group (experimental group, HFD group). The experimental group began to feed a high-fat diet containing 45%-60% fat, while the control group continued to feed a normal low-fat diet. The feed was sourced from Jiangsu Collaborative Biological Co., Ltd. During this period, the mice were supplied with sufficient fresh feed and water every day, and maintained a 12-hour light / dark cycle, a temperature of 22±2℃, and a humidity of 40%-60%. At the same time, the mice were weighed weekly and weight changes were recorded, and the food intake and daily behavioral health status were observed. After 8 weeks of high-fat diet feeding, the weight and length of the mice were measured, the mice were anesthetized, and tissue sampling and blood index determination were performed to evaluate the obesity effect and metabolic status, thereby successfully constructing a high-fat diet-induced obese mouse model.

[0048] 2) After 8 weeks, the mice were anesthetized and their adipose tissue was collected. The mice were killed by cervical dissection and dissected to expose the abdominal cavity. The target epididymal adipose tissue (eWAT) was carefully separated using sterile scissors and forceps and quickly placed in a pre-cooled sterile centrifuge tube for further analysis of the adipose tissue sample or stored at -80°C to maintain its biological activity and integrity for subsequent molecular biology experiments. The above operations were performed under the premise of ensuring animal welfare and experimental guidelines.

[0049] 3) Use the ELISA method to determine the arachidonic acid content in adipose tissue. First, extract samples from mouse adipose tissue, and properly process and dilute them to meet the requirements of the ELISA kit. Then, prepare the ELISA reaction plate according to the instructions of the kit, and add standards, samples, and capture antibodies specific to arachidonic acid to each well. Next, incubate the plate to allow the antibody to bind to arachidonic acid, and wash to remove unbound substances. Add enzyme-labeled antibodies, incubate and wash again, add substrate solution, and the enzyme reaction will produce a color change. Finally, read the absorbance of each well at a specific wavelength (450nm) using an enzyme-linked immunosorbent assay, and calculate the arachidonic acid content in the adipose tissue by comparing it with the standard curve. Each step of this process must be strictly followed in accordance with the instructions of the kit to ensure the accuracy and reliability of the results. The above-mentioned detection results of arachidonic acid in adipose tissue are shown in Figure 3 .

[0050] like Figure 3 As shown, the arachidonic acid (AA) content in the adipose tissue of high-fat diet-induced obese mice was significantly decreased compared with that of normal diet mice, demonstrating that arachidonic acid is a marker of healthy metabolic adipose tissue.

[0051] Example 2-Arachidonic acid (AA) improves obesity metabolic disorders in mice

[0052] This example further studies the effect of arachidonic acid on the treatment of obesity, and specifically includes the following experimental steps:

[0053] 1) The construction process of high-fat diet-induced obese mice is as described in Example 1. After 8 weeks, the high-fat diet mice group was randomly divided into an arachidonic acid (AA) intervention group (HFD+AA group) and a blank control group (HFD-control group); similarly, the normal diet mice group was randomly divided into an arachidonic acid (AA) intervention group (NCD+AA group) and a blank control group (NCD-control group). AA comes from APExBIO arachidonic acid C4223-50MG. The mice in the AA intervention group were gavaged with a dose of 2 mg / kg per day, and the blank group was gavaged with a solvent without AA per day for 7 weeks. After the intervention, as described above, the weight and height of the mice were measured, and the mice were anesthetized to collect blood samples. After cervical vertebrae were dissected and killed, the white fat (eWAT of epididymal fat, pWAT of perirenal fat, and iWAT of subcutaneous fat in the inguinal area) and liver tissue samples of the mice were collected for subsequent analysis.

[0054] 2) The detection steps of relevant indicators are briefly described as follows: The steps for measuring the body mass and length of mice are as follows: Soothe the mouse to a relaxed state, then place it on a scale (active mice are weighed in a container), and record the displayed mass value. Next, prepare a soft ruler and soothe the mouse to stabilize its emotions, and gently grasp the mouse's neck and tail, place it on a flat surface, and stretch its body naturally. Use a soft ruler to measure the length of the mouse from the tip of its nose to the base of its tail, and record the measurement results.

[0055] The mice were then anesthetized, the eyeballs were removed using sterile forceps, and the blood that flowed out was quickly and carefully collected into a clean EP tube for subsequent separation of serum. The blood sample was allowed to stand at room temperature for 30 minutes, and then the serum was separated by centrifugation at 3000rpm for 10 minutes, and the separated serum was transferred to a new centrifuge tube for further analysis or stored at -80°C. The mice were killed by cervical dissection and dissected to expose the abdominal cavity. The target white adipose tissue and liver tissue were carefully separated using sterile scissors and forceps, and quickly placed in a pre-cooled sterile centrifuge tube.

[0056] The steps for measuring serum glucose metabolism and lipid metabolism indexes are as follows: As mentioned above, the supernatant is obtained by centrifugation as a serum sample; then, the chemical reaction analysis is performed according to the instructions using a fully automatic biochemical analyzer and a kit according to specific indicators such as glucose, insulin, and cholesterol and triglycerides; finally, the results are obtained according to the standard curve or instrument readings, ensuring that the standard operating procedures are followed to ensure the accuracy and reliability of the data. The serum inflammatory indexes and leptin and adiponectin levels of mice were determined by ELISA, and the steps are as follows: the serum is added to the ELISA plate coated with specific antibodies according to the instructions, and appropriate washing steps are performed after incubation; then, the enzyme-labeled secondary antibody is added, incubated and the plate is washed again, and then the color substrate is added, and the optical density (OD value) is measured by an enzyme reader after the reaction; finally, the specific concentrations of inflammatory indexes, leptin and adiponectin in the sample are converted according to the standard curve, ensuring the strictness of each step to ensure the accuracy of the results. HE staining was used to observe the inflammatory status and adipocyte size of mouse adipose tissue, and immunohistochemistry was used to analyze the macrophage-specific marker F4 / 80. The specific steps were as follows: first, the mouse adipose tissue was fixed with formalin, embedded and sliced. HE staining was performed to observe the tissue structure and adipocyte size; then immunohistochemistry was performed, and macrophages were labeled with F4 / 80 antibodies, and then observed after antigen repair, blocking, antibody incubation and color development. HE staining, immunohistochemistry F4 / 80, α-SMA staining and Oil Red O staining were used to observe the inflammation, fibrosis and fatty liver of mouse liver tissue. The detailed steps are as follows: First, the liver tissue was fixed in 10% neutral formalin, dehydrated, transparent and paraffin-embedded, and the slice thickness was 4-5 microns; when performing HE staining, it was first dewaxed to water, then stained with hematoxylin for 5-10 minutes, rinsed with tap water to show blue, then differentiated with hydrochloric acid alcohol, and blued with tap water, then stained with eosin and dehydrated with different concentrations of ethanol, and finally transparentized with xylene and sealed with neutral gum; for immunohistochemistry F4 / 80, the slices needed to be distilled water, and then washed with tap water to show blue. After toluene and ethanol were reduced to water, microwave antigen repair was performed in citric acid buffer, followed by blocking endogenous enzymes with 3% hydrogen peroxide, blocking nonspecific binding with normal serum, and then incubating with F4 / 80 antibody, adding secondary antibody and developing color; when performing α-SMA staining, the basic process was similar, using antibodies against α-SMA to observe liver fibrosis; finally, Oil Red O staining was used for frozen sections, first treated with 30% sucrose solution, so that the tissue sections were stained in Oil Red O dye, and then washed with water and observed to evaluate liver fat deposition; in summary, these staining methods were used to comprehensively evaluate the pathophysiological state of mouse liver. The above test results are shown in Figures 4 to 8 .

[0057] Depend on Figure 4 As shown in Figure 2, after AA intervention, the body weight and fat weight of mice fed a high-fat diet were significantly reduced. Figure 5As shown in Figure 2, after AA intervention, the blood lipid and blood glucose metabolism indicators of mice were significantly improved. Figure 6 As shown in Figure 2, after AA intervention, the serum inflammation level and leptin and adiponectin levels of high-fat diet obese mice were significantly improved. Figure 7 The results showed that after AA intervention, epididymal fat inflammation and adipocyte size in obese mice fed a high-fat diet were significantly improved. Figure 8 The results showed that fatty liver and liver fibrosis in high-fat diet obese mice were significantly improved after AA intervention.

[0058] From the above experimental results, it can be seen that intervention with arachidonic acid (AA) can significantly improve obesity metabolic disorders in mice, and it has a certain preventive and therapeutic effect on obesity.

[0059] Example 3 - Rapid Detection Kit for Adipose Tissue Lipid Metabolites

[0060] This embodiment constructs a rapid detection kit for lipid metabolites in adipose tissue based on the research of embodiment 1 and embodiment 2, which can directly and rapidly detect lipid metabolites (metabolic composition) in adipose tissue; wherein the lipid metabolites include: arachidonic acid AA (C20:4), docosapentaenoic acid (C22:5), fatty acid FA, triglyceride TG, total cholesterol TC, cholesterol ester CE, free cholesterol FC, high-density lipoprotein cholesterol HDL-c, low-density lipoprotein cholesterol LDL-c; the detection results of the above kit will be used to evaluate the metabolic health status of adipose tissue of the subject. The kit mainly indirectly determines the content of the above lipid metabolites by absorbance (OD value) determination method, and after adding substrate for color development, the absorbance (OD value) is determined at a specific wavelength using an enzyme marker.

[0061] The above-mentioned kit mainly includes: 1) antibody pre-coated microplate; 2) sample homogenate lysis solution (PBS solution): the mass volume ratio of tissue to lysis solution is recommended to be 1:5; 3) sample simple homogenizer; 4) standard diluent; 5) standard; 6) labeled antibody biotinylated antibody; 7) horseradish peroxidase label (HRP); 8) washing solution; 9) color developer (TMB solution); 10) stop solution; 11) sealing film; 12) instruction manual; 13) several 1.5mL sample centrifuge tubes; 14) adipose tissue collection device (the adipose tissue obtained through it can be directly used for the detection of adipose tissue lipid metabolites after sample pretreatment).

[0062] The operating steps of the above kit are briefly described as follows:

[0063] (1) Processing of adipose tissue. Wash the tissue in pre-cooled sample homogenization solution (PBS solution) to remove blood, and weigh it for later use. If the tissue block is large, cut it into pieces before homogenization. After homogenization using the matching sample simple homogenizer, centrifuge at 5000×g for 5 minutes, and collect the supernatant for subsequent testing;

[0064] (2) Take the kit out of the refrigerator and equilibrate at room temperature for 20 minutes;

[0065] (3) Add deionized water to the standard to prepare an appropriate amount of standard dilution solution and incubate at room temperature for 15 minutes;

[0066] (4) Perform serial dilution of the standard.

[0067] (5) Setting standard wells and sample wells;

[0068] (6) Add the diluted standard to the pre-coated microplate wells in sequence; add 50uL of the standard of different concentrations to each standard well; add 50uL of the sample to be tested to the sample well; and do not add anything to the blank well;

[0069] (7) Seal the reaction wells with a sealing film and incubate at room temperature for 120 min;

[0070] (8) Wash the plate 5 times and pat dry on thick absorbent paper for the last wash;

[0071] (9) After adding the labeled biotinylated antibody, seal the reaction wells with a sealing film and incubate at room temperature for 60 min;

[0072] (10) Wash the plate 5 times and pat dry on thick absorbent paper for the last wash;

[0073] (11) Except for the blank wells, add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each well of the standard well and sample well; seal the reaction wells with a sealing film and incubate at room temperature for 20 min;

[0074] (12) Discard the liquid, pat dry on absorbent paper, fill each well with washing solution (350uL), let stand for 1 min; shake off the washing solution, pat dry on absorbent paper, and repeat this process 5 times;

[0075] (13) Add 100 uL / well of TMB solution, seal the wells with a sealing film, and incubate at room temperature in the dark for 20 min;

[0076] (14) Add 50uL of stop solution to each well;

[0077] (15) Within 15 min, measure the absorbance (OD value) of each well at a wavelength of 450 nm using a microplate reader;

[0078] (16) Calculation of experimental results: After the test is completed, the concentration of the standard is used as the ordinate and the corresponding absorbance (OD value) is used as the abscissa. The four-parameter logistic curve fitting (4-pl) is used to create the standard curve equation. The absolute concentration value of the detected metabolite in the sample is calculated using the equation based on the absorbance (OD value) of the sample;

[0079] (17) The absolute concentration value of the sample obtained is compared with the reference range provided in the kit (the specific range can be determined by further in-depth research), so as to evaluate the healthy metabolic status of the patient's adipose tissue and provide personalized suggestions for the prevention and treatment of obesity in the patient.

[0080] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. The use of adipose tissue lipid metabolites as markers for assessing fat health status, characterized in that: The adipose tissue lipid metabolites include at least one of arachidonic acid and docosapentaenoic acid.

2. The use according to claim 1, characterized in that: The adipose tissue lipid metabolites also include at least one of conventional lipid metabolism detection markers, and the conventional lipid metabolism detection markers include fatty acids, triglycerides, total cholesterol, cholesterol esters, free cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol.

3. Use of adipose tissue lipid metabolites in the preparation of a preparation for treating obesity, characterized in that: The adipose tissue lipid metabolites include at least one of arachidonic acid and docosapentaenoic acid.

4. The use according to claim 3, characterized in that: The adipose tissue lipid metabolite is arachidonic acid, which is used for preparing a preparation for improving obesity metabolic disorders.

5. The use according to claim 4, characterized in that: The improvement of obesity metabolic disorders includes: reducing fat weight; improving blood lipid and blood sugar metabolism indicators; improving serum inflammation levels and leptin and adiponectin levels; improving fat inflammation and fat cell size; and improving fatty liver and liver fibrosis.

6. A kit for detecting lipid metabolites in adipose tissue, characterized in that: The adipose tissue lipid metabolites include arachidonic acid and docosapentaenoic acid.

7. The kit according to claim 6, characterized in that The adipose tissue lipid metabolites also include fatty acids, triglycerides, total cholesterol, cholesterol esters, free cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol.

8. The kit according to claim 6, characterized in that The kit comprises an antibody pre-coated microplate, a sample homogenization lysis solution, a sample simple homogenizer, a standard diluent, a standard, a labeled antibody biotinylated antibody, a horseradish peroxidase label, a washing solution, a color developer, a stop solution, a plate sealing film, and a sample centrifuge tube.

9. The kit according to claim 6, characterized in that The kit adopts absorbance determination method to indirectly determine the content of lipid metabolites in the adipose tissue.

10. A use of the kit according to any one of claims 6 to 9, characterized in that: The application is selected from at least one of the following applications: application in evaluating the metabolic health status of a subject's adipose tissue; application in screening drugs for improving diseases related to abnormal lipid metabolism; application in evaluating the effects of drugs or treatment methods on lipid metabolism.

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