Biomarker for progeny cardiac developmental toxicity caused by PM2.5 (Particulate Matter 2.5) and application thereof

Abstract

The invention discloses a biomarker for progeny cardiac developmental toxicity caused by PM2.5 and application of the biomarker. After the parental generation is exposed to PM2.5, the expression quantity of Angptl4 in the heart tissue of the offspring is obviously increased, and the expression quantity of Sirt3 is obviously decreased. According to the application disclosed by the invention, Angptl4 or a combination of Angptl4 and Sirt3 is taken as a marker, the influence of PM2.5 on the filial generation cardiac developmental toxicity is disclosed, and a method is provided for diagnosis and evaluation of the filial generation cardiac developmental toxicity caused by PM2.5 during pregnancy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to 2.5 biomarkers for the impact of PM on the developmental toxicity of the offspring's heart and their applications. Background Art

[0002] Congenital heart disease (CHD) is the most common type of birth defect globally and is an important factor in neonatal and childhood mortality. Therefore, understanding the environmental risk factors and mechanisms causing the incidence and prevalence of CHD has important practical significance for global public health. The Global Burden of Diseases analysis in 2021 showed that PM 2.5 is the leading factor causing the burden of diseases, accounting for 8.0% of the total disability-adjusted life years. A case-control study involving 1,434,998 neonates found that for every 10 μg / m 2.5 increase in the exposure dose of PM during pregnancy, the risk of CHD increases by 2%. Animal experimental studies have found that exposure to PM 3 from gestation to weaning at 3 weeks of age can cause an increase in collagen deposition in the myocardial tissue of the offspring and histopathological changes. In addition, PM 2.5 exposure during pregnancy leads to abnormal expression of mitochondrial fusion / fission genes in the offspring mice, which in turn causes damage to the hearts of the offspring mice. However, so far, the exact underlying mechanism and strong biomarkers for the developmental toxicity of the offspring's heart caused by PM 2.5 exposure during pregnancy still remain a complex problem. 2.5 Angiopoietin-like protein 4 (Angptl4) interacts with CHD-related genes and known pathogenic genes and plays an important role in the regulation of CHD. Sirt3 is a deacetylase mainly located in the mitochondrial matrix. Existing studies have not reported the application of Angptl4 / Sirt3 as biomarkers in the developmental toxicity of the offspring's heart after PM

[0003] exposure during pregnancy. 2.5 exposure during pregnancy. Summary of the Invention

[0004] In order to clarify the impact of PM 2.5 on the developmental toxicity of the heart, and to better diagnose and evaluate the developmental toxicity of the heart, the present invention provides the following technical solutions.

[0005] In a first aspect, the present invention provides a biomarker for the developmental toxicity of the offspring's heart caused by PM 2.5 during pregnancy, and the biomarker is Angptl4 or a combination of Angptl4 and Sirt3.

[0006] Furthermore, by detecting the expression level of Angptl4 mRNA or Angptl4 protein, the developmental toxicity of PM to the offspring's heart can be predicted and evaluated. 2.5 Causing developmental toxicity to the offspring's heart.

[0007] More preferably, the expression level of Angptl4 mRNA or Angptl4 protein is up-regulated.

[0008] Furthermore, while detecting the expression level of Angptl4 mRNA or Angptl4 protein, the expression level of Sirt3 mRNA or Sirt3 protein is additionally detected.

[0009] More preferably, the expression level of Sirt3 mRNA or Sirt3 protein is down-regulated.

[0010] In a second aspect, the present invention provides the use of Angptl4 as a biomarker in the preparation of products for diagnosing, monitoring, evaluating the severity, evaluating the efficacy, evaluating the prognosis, preventing and / or treating the developmental toxicity of PM during pregnancy to the offspring's heart. 2.5 Causing developmental toxicity to the offspring's heart.

[0011] Preferably, the diagnosis, monitoring, evaluation of severity, evaluation of efficacy or prognosis of the developmental toxicity of PM during pregnancy to the offspring's heart includes detecting the expression level of Angptl4. 2.5 Causing developmental toxicity to the offspring's heart.

[0012] Preferably, the product includes at least one of the reagents for detecting the expression level of Angptl4 mRNA, detecting the expression level of Angptl4 protein, down-regulating the expression level of Angptl4 mRNA, and down-regulating the expression level of Angptl4 protein.

[0013] Preferably, the reagents for down-regulating the expression level of Angptl4 mRNA and down-regulating the expression level of Angptl4 protein are selected from at least one of the following:

[0014] 1) A viral vector in which Angptl4 is low-expressed.

[0015] 2) A small molecule drug having the effect of reducing the expression of Angptl4 mRNA and / or Angptl4 protein in heart tissue.

[0016] 3) A small molecule drug or antibody having the effect of reducing the activity of Angptl4.

[0017] In a third aspect, the present invention provides the use of Angptl4 and Sirt3 as combined biomarkers in the preparation of products for diagnosing, monitoring, evaluating the severity, evaluating the efficacy, evaluating the prognosis, preventing and / or treating the developmental toxicity of PM during pregnancy to the offspring's heart. 2.5 Causing developmental toxicity to the offspring's heart.

[0018] Preferably, for the diagnosis, severity assessment, efficacy assessment, or prognosis assessment of PM during pregnancy 2.5 causing offspring cardiac developmental toxicity includes detecting the expression levels of Angptl4 and Sirt3.

[0019] Preferably, the product includes at least one of the reagents for detecting the expression levels of Angptl4 mRNA and Sirt3 mRNA, detecting the expression levels of Angptl4 protein and Sirt3 protein, down-regulating the expression level of Angptl4 mRNA and up-regulating the expression level of Sirt3 mRNA, and down-regulating Angptl4 protein and up-regulating Sirt3 protein expression.

[0020] Preferably, the reagents for down-regulating the expression level of Angptl4 mRNA and up-regulating the expression level of Sirt3 mRNA, and down-regulating Angptl4 protein and up-regulating Sirt3 protein expression are selected from at least one of the following:

[0021] 1) A viral vector that lowly expresses Angptl4 and over-expresses Sirt3;

[0022] 2) A small molecule drug that has the effect of reducing the expression of Angptl4 mRNA and / or Angptl4 protein in cardiac tissue and increasing the expression of Sirt3 mRNA and / or Sirt3 protein in cardiac tissue;

[0023] 3) A small molecule drug or antibody that has the effect of reducing the activity of Angptl4 and increasing the activity of Sirt3.

[0024] Fourthly, the present invention provides a PM 2.5 method for constructing an animal model of offspring cardiac developmental toxicity affected by PM, the method comprising the following steps:

[0025] 1) Exposing the animal to PM 2.5 therein.

[0026] 2) Detecting the expression level of Angptl4 and / or the expression level of Angptl4 mRNA in the cardiac tissue of the offspring animal.

[0027] Preferably, in step 2), the expression level of Sirt3 and / or the expression level of Sirt3 mRNA in the cardiac tissue of the offspring animal is also detected.

[0028] Preferably, the animal is exposed to PM 2.5 for 4 - 8 hours per day, and more preferably 6 hours.

[0029] Preferably, the animal is a rodent.

[0030] More preferably, the rodent is a mouse, and further preferably a C57BL / 6J mouse.

[0031] In a fifth aspect, the present invention provides a PM 2.5 animal model for influencing the developmental toxicity of the offspring's heart, which is constructed according to the method described in the fourth aspect.

[0032] Advantages of the present invention:

[0033] With Angptl4 or a combination of Angptl4 and Sirt3 as a biomarker, the present invention reveals the influence of PM 2.5 on the developmental toxicity of the offspring's heart, provides a method for the diagnosis and evaluation of the developmental toxicity of the offspring's heart caused by PM during pregnancy, and is of great significance for the early risk assessment of the developmental toxicity of the offspring's heart. 2.5 BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Shown is the influence of PM 2.5 on the expression levels of Angptl4 / Sirt3 proteins and mRNAs in the heart tissues of offspring mice. A: Western blot and statistical analysis of mouse heart tissues; B: Results of qRT-PCR;

[0035] Figure 2 Shown is the influence of PM 2.5 on the interaction between Angptl4 and Sirt3 in the heart tissues of offspring mice;

[0036] Figure 3 Shown is the histopathological changes in the heart tissues of mice caused by PM 2.5 exposure.

[0037] Figure 4 Shown is the influence of PM 2.5 exposure on the expression of mitochondrial-related proteins and the ATP content in the heart tissues of mice. A: Western blot and statistical analysis of mouse myocardial tissues; B: ATP content in mouse myocardial tissues.

[0038] Figure 5 Shown is the expression levels of Angptl4 / Sirt3 in umbilical cord serum and the corresponding ROC curves. A: Content of Angptl4 in serum; B: Content of Sirt3 in serum; C: Correlation analysis between PM 2.5 and Angptl4; D: Correlation analysis between PM 2.5 and Sirt3; E: ROC curve of Angptl4; F: ROC curve of Sirt3;

[0039] In the figure, * indicates statistical difference, p < 0.05, ** indicates statistical difference, p < 0.01; # indicates p < 0.05 when comparing Angptl4 knockout mice with wild-type PM 2.5 treatment groups, ## indicates p < 0.01 when comparing Angptl4 knockout mice with wild-type PM 2.5 treatment groups. Detailed implementation manners

[0040] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments. These embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. For the experimental methods without specific conditions indicated in the following embodiments, they are generally carried out according to the conventional conditions in the art or according to the conditions recommended by the manufacturer. Unless otherwise specified, they are all conventional methods. Unless otherwise defined, the professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art.

[0041] "Treatment" as used in the present invention means slowing down, interrupting, preventing, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, disease condition, or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, disease conditions, or disorders.

[0042] "Prognosis assessment" as used in the present invention refers to predicting the possible course and outcome of a disease, including judging the specific consequences of the disease.

[0043] "Therapeutic effect assessment" as used in the present invention refers to evaluating the patient's response to treatment.

[0044] "Monitoring" as used in the present invention refers to observing the occurrence and development dynamics of a disease.

[0045] "Prevention" as used in the present invention refers to an individual taking specific measures to prevent the occurrence of a disease before the disease is diagnosed or has occurred.

[0046] The term "diagnosis" in the present invention refers to ascertaining whether a patient has a disease or disorder in the past, at the time of diagnosis, or in the future, or ascertaining the progression or possible future progression of the disease.

[0047] The sources of some materials and experimental methods involved in this application are as follows:

[0048] SPF-grade wild-type male and female C57BL / 6J mice (8 weeks old), Beijing Vital River Laboratory Animal Technology Co., Ltd. Angptl4 systemic gene knockout mice, Cyagen Biosciences (Suzhou) Inc. Cord serum from healthy controls (n = 40) and children with congenital heart disease (n = 40) was from Beijing Obstetrics and Gynecology Hospital, Capital Medical University. Congenital heart disease was diagnosed using transthoracic color Doppler echocardiography.

[0049] Example 1 Detection of Angptl4 / Sirt3 protein and mRNA expression levels in cardiac tissue of offspring mice

[0050] 1.1 PM 2.5 Establishment of animal model of pregnancy exposure

[0051] After SPF wild-type male and female C57BL / 6J mice (8 weeks old) were acclimated for one week, males and females were mated at night in a ratio of 1:2, and female mice were designated as embryonic day (E) 0 after vaginal plugs were observed. Pregnant mice were divided into three groups: clean air group (FA, 1 μg / m 3 ), Environmental PM 2.5 Group (Ambient PM 2.5 , AP, 30.86 μg / m 3 ), concentrated ambient PM 2.5 Group (Concentrated ambient PM 2.5 , CAP, 367.22 μg / m 3 Mice in the AP and CAP groups were exposed to PM2.5 daily from E6.5 to E18.5 using a small animal whole-body inhalation exposure system. 2.5 6 hours. Pregnant mice in the FA group were housed in individually ventilated cages (IVC) and allowed to move freely and drink water during the exposure period. Mice were euthanized at 1 day after birth and heart tissues were obtained for experiments.

[0052] 1.2 Detection of Angptl4 / Sirt3 protein expression levels in cardiac tissue of 1-day-old mice

[0053] The specific process is as follows:

[0054] (1) Add approximately 0.1 mL of pre-cooled lysis buffer to each tissue and homogenize in a pre-cooled homogenizer. Pipette the homogenate into a pre-cooled 1.5 mL centrifuge tube, centrifuge at 4°C, 12,000 g for 5 min, and transfer the supernatant to a new pre-cooled centrifuge tube.

[0055] (2) The protein concentration of the sample was determined using the BCA protein quantification kit (Dingguo Changsheng, BCA01).

[0056] (3) Proteins were separated by SDS-PAGE and then transferred to nitrocellulose membrane.

[0057] (4) The cells were blocked with skim milk for 1 h at room temperature and then incubated with primary antibodies against Angptl4 (Proteintech, 18374-1-AP) / Sirt3 (CST, D22A3) at 4°C overnight.

[0058] (5) Incubate with anti-rabbit IgG HRP-linked (CST, 7074S) secondary antibody at room temperature for 1 h.

[0059] (6) Visualize protein bands using a ChemiDoc imaging system and analyze their gray values with ImageJ software. The results are shown in Figure 1 .

[0060] 1.3 Detection of the expression levels of Angptl4 / Sirt3 mRNA in the heart tissues of 1-day-old mice

[0061] The specific procedure is as follows:

[0062] (1) Place magnetic beads and heart tissues in a 2.0 mL EP tube and put it into a common homogenizer. Add 0.1 mL of Trizol for homogenization. After homogenization, place the EP tube on ice for 10 min to allow the samples to be fully lysed.

[0063] (2) Add 0.2 volume of chloroform to the Trizol lysate, vortex for 15 s, and place on ice for 5 min. Centrifuge at 12,000 g at 4 °C for 10 min, and then aspirate the upper colorless aqueous phase containing total RNA into a new centrifuge tube.

[0064] (3) Add an equal volume of isopropanol, vortex, and place on ice for 10 min to precipitate RNA. Centrifuge at 12,000 g at 4 °C for 10 min. The RNA precipitate can be seen at the bottom of the tube. Discard the supernatant.

[0065] (4) Add 1 mL of 75% ethanol, vortex. Centrifuge at 12,000 g for 5 min, discard the supernatant. Try to completely aspirate the liquid on the wall. Open the tube mouth and air-dry for 10 min. After the RNA is slightly dry, add 20 μL of DEPC water to dissolve it.

[0066] (5) Use the PrimeScript RT Master Mix (Takara) kit to generate cDNA from the extracted RNA.

[0067] (6) Use the SYBR Premix Ex Taq kit (Takara, Japan) and a real-time fluorescence quantitative PCR detection system to amplify the cDNA. The results are shown in Figure 1 .

[0068] 1.4 Detection of the interaction between Angptl4 and Sirt3 in the heart tissues of 1-day-old mice

[0069] The specific procedure is as follows:

[0070] (1) Add approximately 0.2 mL of pre-cooled lysis buffer to each tissue sample, and homogenize it in a pre-cooled homogenizer. Aspirate the homogenate into a pre-cooled 1.5 mL centrifuge tube, centrifuge at 4°C and 12,000 g for 5 min, and transfer the supernatant to a new pre-cooled centrifuge tube.

[0071] (2) Use a Protein A / G immunoprecipitation kit (Beaver, 22202-20) to detect the interaction between Angptl4 and Sirt3. Take 25 μL of magnetic bead suspension and place it in a 1.5 mL EP tube for magnetic bead pretreatment. Prepare a 200 μL antibody working solution with a final concentration of 25 μg / mL of Angptl4 (Proteintech, 18374-1-AP) antibody using the binding buffer. Resuspend it quickly and gently invert the EP tube on a rotary mixer at room temperature for 15 min, then perform magnetic separation.

[0072] (3) Add 200 μL of the antigen sample prepared in step (1), and gently pipette to evenly disperse the antigen with the magnetic bead-antibody complex. React overnight at 4°C.

[0073] (4) Remove the EP tube from the magnetic separator, add 25 μL of 1×SDS-PAGE Loading Buffer and mix well. Heat at 95°C for 5 min. Then perform magnetic separation to collect the supernatant for SDS-PAGE detection. The results are shown in Figure 2 .

[0074] As Figure 1 shown, compared with the FA group, the protein and mRNA expression levels of Angptl4 in the AP group and CAP group increased, and the protein and mRNA expression levels of Angptl4 increased in a dose-dependent manner; while the protein and mRNA expression levels of Sirt3 decreased, and the protein and mRNA expression levels of Sirt3 decreased in a dose-dependent manner. This indicates that Angptl4 or the combination of Angptl4 and Sirt3 can be used as a biomarker for the developmental toxicity of PM 2.5 to the offspring's heart.

[0075] As Figure 2 shown, there is an interaction between Angptl4 and Sirt3, and PM 2.5 exposure significantly enhanced the binding of Angptl4 and Sirt3. Therefore, by downregulating Angptl4, reducing the binding of Angptl4 and Sirt3, and increasing the content of Sirt3 in the mouse heart tissue, the developmental toxicity of PM 2.5 to the offspring's mouse heart can be alleviated.

[0076] Example 2 Effects of PM 2.5 on the heart development of offspring of Angptl4 knockout pregnant mice

[0077] 2.1 Construction of animal model

[0078] Angptl4 is located on chromosome 17 of mice. CRISPR-AI gene editing technology was used to obtain Angptl4 systemic gene knockout mice (C57BL / 6JCya-Angptl4 em1 / Cya gene knockout mice). At the same time, a wild-type (WT) mouse control group was set up. After the knockout mice and wild-type mice were raised to 8 weeks of age, males and females were mated at night in a ratio of 1:2. Female mice were designated as embryonic day (E) 0 after vaginal plugs were observed. Pregnant mice were divided into two groups: a clean air group (Filtered air, FA, 1 μg / m 3 ) and concentrated ambient PM 2.5 Group (Concentrated ambient PM 2.5 , CAP, 171.95 μg / m 3 Mice in the CAP group were exposed to PM2.5 daily from E6.5 to E18.5 using a small animal whole-body inhalation exposure system. 2.5 6 hours, and the rest of the time was placed in individually ventilated cages (IVC). Pregnant mice in the FA group were housed in the IVC and were free to move and drink water during the exposure period. Mice were euthanized at low temperature on the first day after birth, and heart tissues were obtained for detection.

[0079] 2.2 Hematoxylin-eosin (H&E) staining of heart tissue of 1-day-old mice

[0080] (1) The heart tissue of the offspring mice was fixed with 4% paraformaldehyde and then immersed in 20% sucrose solution at 4°C overnight.

[0081] (2) Prepare wax blocks and paraffin sections.

[0082] (3) Bake the slices at 60℃ for 30 min.

[0083] (4) The paraffin sections were dewaxed by immersion in xylene, dehydrated by gradient concentrations of ethanol, and washed with deionized water for 5 min.

[0084] (5) Incubate with hematoxylin for 5 min and rinse with deionized water for 5 min.

[0085] (6) Differentiation with 1% hydrochloric acid and ethanol for 30 seconds and rinse with deionized water for 5 minutes.

[0086] (7) Stain with eosin solution for 2 min and rinse with deionized water for 5 min.

[0087] (8) After dehydrating the paraffin sections with gradient ethanol, they were cleared with xylene.

[0088] (9) Sealed with neutral balsam.

[0089] (10) Scanned and analyzed with a GT450 fully automatic slide scanning system, and the results are shown in Figure 3 .

[0090] 2.3 Detection of the expression levels of Sirt3, Drp1 and Mfn2 proteins in the heart tissues of 1-day-old mice

[0091] (1) Approximately 0.1 mL of pre-cooled lysis buffer was added to each tissue and homogenized in a pre-cooled homogenizer. The homogenate was aspirated into a pre-cooled 1.5 mL centrifuge tube and centrifuged at 12,000 g for 5 min at 4 °C. The supernatant was transferred to a new pre-cooled centrifuge tube.

[0092] (2) The protein sample concentration was determined using a BCA protein quantification kit (Dingguo Changsheng, BCA01).

[0093] (3) Proteins were separated by SDS-PAGE and then transferred onto a nitrocellulose membrane.

[0094] (4) Blocked with skim milk for 1 h at room temperature, and then incubated overnight at 4 °C with primary antibodies against Sirt3 (CST, D22A3) / Drp1 (CST, 8570S) / Mfn2 (CST, 9482S).

[0095] (5) Incubated with a secondary antibody, anti-rabbit IgG HRP-linked (CST, 7074S), for 1 h at room temperature.

[0096] (6) The protein bands were visualized using a ChemiDoc imaging system, and their gray values were analyzed with ImageJ software. The results are shown in Figure 4 .

[0097] 2.4 Detection of ATP activity in the heart tissues of 1-day-old mice

[0098] (1) Approximately 0.5 mL of pre-cooled lysis buffer was added to each tissue and homogenized in a pre-cooled homogenizer. The homogenate was aspirated into a pre-cooled 1.5 mL centrifuge tube and centrifuged at 12,000 g for 5 min at 4 °C.

[0099] (2) The ATP activity was detected using an ATP content assay kit (Invitrogen, A22066). 10 μL of the supernatant from step 1 and 90 μL of the working solution were added to a 96-well plate.

[0100] (3) Incubated at 37 °C in the dark for 15 min.

[0101] (4) Measure the fluorescence value at 560 nm using a microplate reader to calculate the ATP content. The results are shown in Figure 4 .

[0102] As Figure 3 shown, PM 2.5 exposure significantly induced inflammatory cell infiltration in the heart tissue of the offspring of wild-type mice. Compared with the offspring of wild-type mice treated with PM 2.5 , the inflammatory cell infiltration in the heart tissue of the offspring of Angptl4 - / - mice was not obvious, indicating that after Angptl4 knockout, the inflammatory response of the heart of the offspring mice caused by PM 2.5 could be alleviated.

[0103] As Figure 4 shown, PM 2.5 exposure significantly down-regulated the expression of mitochondrial deacetylase Sirt3, up-regulated the expression of mitochondrial fission protein Drp1, down-regulated the expression of mitochondrial fusion protein Mfn2, and reduced the ATP activity in the heart tissue of the offspring of wild-type mice. Compared with the offspring of wild-type mice treated with PM 2.5 , in the heart tissue of the offspring of Angptl4 - / - mice, the expression of mitochondrial deacetylase Sirt3 was significantly up-regulated, the expression of mitochondrial fission protein Drp1 was significantly down-regulated, the expression of mitochondrial fusion protein Mfn2 was significantly up-regulated, and the ATP activity in the heart tissue was significantly increased. It shows that after Angptl4 knockout, the mitochondrial dynamics disorder and ATP reduction in the heart tissue of the offspring mice caused by PM 2.5 exposure can be alleviated. In addition, the expression level of Sirt3 located downstream of Angptl4 was significantly up-regulated with the knockout of Angptl4, indicating that Angptl4 and Sirt3 are related to the developmental toxicity of the offspring heart caused by PM 2.5 . Angptl4 or the combination of Angptl4 and Sirt3 can be used as biomarkers for the diagnosis and treatment of the developmental toxicity of PM 2.5 to the offspring heart.

[0104] Example 3 Detection of the Contents of Angptl4 and Sirt3 in the Serum of Human Samples and the ROC Curve

[0105] 3.1 Population Study

[0106] Healthy controls (n = 40) and patients with congenital heart disease (CHD) (n = 40) were from Beijing Obstetrics and Gynecology Hospital, Capital Medical University. The diagnosis of CHD was performed using transthoracic color Doppler echocardiography. This study has been reviewed and approved by the Medical Ethics Committee of Beijing Obstetrics and Gynecology Hospital, Capital Medical University, with the approval number 2018-KY-003-02. Written informed consent was obtained from all subjects included in the sampling process. Monthly gridded concentration data of PM2.5 were obtained from the China Ambient Air Pollutant Tracking Dataset (http: / / tapdata.org.cn) and allocated according to the residential and work addresses. The average concentration of PM 2.5 during the exposure window period (12 months or 6 months before pregnancy and the first trimester of pregnancy) was calculated by the weighted average of the monthly average concentrations, with the weights based on the proportion of days covered by each month.

[0107] 3.2 Detection of Angptl4 / Sirt3 protein levels in umbilical cord serum of healthy controls and CHD patients

[0108] (1) Prepare all reagents, sera, and standards according to the instructions using ELISA kits (RayBiotech, ELH-ANGPTL4-1 / ELH-SIRT3-1).

[0109] (2) Add 100 μL of standards or sera to each well and incubate at room temperature for 2.5 h.

[0110] (3) Add 100 μL of the prepared biotinylated antibody to each well and incubate at room temperature for 1 h.

[0111] (4) Add 100 μL of the prepared streptavidin solution to each well and incubate at room temperature for 45 min.

[0112] (5) Add 100 μL of the TMB one-step substrate reagent to each well and incubate at room temperature for 30 min.

[0113] (6) Add 50 μL of the stop solution to each well and immediately read the absorbance at 450 nm.

[0114] As Figure 5 shown, the level of Angptl4 in the serum of CHD patients was significantly upregulated compared with that of healthy controls, while the level of Sirt3 was significantly downregulated. Using R software and the ggplot2 package, the correlation between individual PM 2.5 exposure levels and biomarkers was analyzed. The results showed that PM 2.5The concentration was positively correlated with the Angptl4 content (R = 0.35, p < 0.01) and negatively correlated with the Sirt3 content (R = -0.34, p < 0.01). Using R software and the pROC and ggplot2 packages, the predictive effects of Angptl4 and Sirt3 on PM 2.5 related CHD were analyzed. The results of the ROC curve showed that Angptl4 correctly distinguished 71% and 79% of the patients in the training and test sets, respectively, and Sirt3 correctly distinguished 75% and 78% of the patients in the training and test sets, respectively.

[0115] The above experimental results fully demonstrate that Angptl4 or the combination of Angptl4 and Sirt3 can be used as biomarkers to effectively predict PM 2.5 related CHD.

[0116] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. Use of Angptl4 as a biomarker in the preparation of products for diagnosing, monitoring, assessing severity, evaluating efficacy, assessing prognosis, preventing, and / or treating the developmental toxicity of the offspring's heart caused by PM during pregnancy 2.5 induced by PM during pregnancy.

2. The application according to claim 1, wherein The diagnosis, severity assessment, efficacy assessment, or prognosis assessment of PM during pregnancy 2.5 Inducing offspring cardiac developmental toxicity includes detecting the expression level of Angptl4.

3. The application according to claim 1 or 2, characterized in that, The product described above includes at least one of the reagents for detecting the expression level of Angptl4 mRNA, detecting the expression level of Angptl4 protein, down-regulating the expression level of Angptl4 mRNA, and down-regulating the expression level of Angptl4 protein.

4. The application according to claim 3, characterized in that The reagents for down-regulating the expression level of Angptl4 mRNA and down-regulating the expression level of Angptl4 protein are selected from at least one of the following: 1) A viral vector that has low expression of Angptl4; 2) A small molecule drug that has the effect of reducing the expression of Angptl4, Angptl4 mRNA, and / or Angptl4 protein in cardiac tissue; 3) A small molecule drug or antibody that has the effect of reducing the activity of Angptl4.

5. Use of Angptl4 and Sirt3 as combined biomarkers in the preparation of products for diagnosing, monitoring, assessing severity, evaluating efficacy, predicting prognosis, preventing and / or treating fetal cardiac developmental toxicity caused by PM during pregnancy 2.5 induced by PM during pregnancy 6. The application according to claim 5, characterized in that, The diagnosis, severity assessment monitoring, efficacy assessment, or prognosis assessment of PM during pregnancy 2.5 Inducing offspring cardiac developmental toxicity includes detecting the expression levels of Angptl4 and Sirt3.

7. The application according to claim 5 or 6, characterized in that, The product described above includes at least one of the reagents for detecting the expression levels of Angptl4 mRNA and Sirt3 mRNA, detecting the expression levels of Angptl4 protein and Sirt3 protein, down-regulating the expression level of Angptl4 mRNA and up-regulating the expression level of Sirt3 mRNA, and down-regulating the expression level of Angptl4 protein and up-regulating the expression level of Sirt3 protein.

8. The application according to claim 7, wherein The reagents for down-regulating the expression level of Angptl4 mRNA and up-regulating the expression level of Sirt3 mRNA, and down-regulating the expression level of Angptl4 protein and up-regulating the expression level of Sirt3 protein are selected from at least one of the following: 1) A viral vector that has low expression of Angptl4 and overexpresses Sirt3; 2) A small molecule drug that has the effect of reducing the expression of Angptl4 mRNA and / or Angptl4 protein in cardiac tissue and has the effect of increasing the expression of Sirt3 mRNA and / or Sirt3 protein in cardiac tissue; 3) A small molecule drug or antibody that has the effect of reducing the activity of Angptl4 and has the effect of increasing the activity of Sirt3.

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