Application of a reagent for detecting retinol metabolites in preparing a tool for diagnosing and / or treating pneumoconiosis

Abstract

The present invention provides an application of a reagent for detecting retinol metabolites in the preparation of a tool for diagnosing and / or treating pneumoconiosis, and relates to the field of biomedical technology. The present invention verifies that retinol metabolism disorders and all-trans retinoic acid deficiency are closely related to the occurrence of pneumoconiosis, and therefore proposes to use retinol and all-trans retinoic acid as biomarkers for screening pneumoconiosis and apply them to the preparation of related products for diagnosing pneumoconiosis; at the same time, all-trans retinoic acid intervention for pneumoconiosis can effectively slow down the progression of pneumoconiosis, and therefore proposes for the first time to use all-trans retinoic acid as a drug for treating pneumoconiosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the use of a reagent for detecting retinol metabolites in the preparation of a tool for diagnosing and / or treating pneumoconiosis. Background Art

[0002] Pneumoconiosis is an occupational disease caused by long-term inhalation of pathogenic dust, characterized by diffuse fibrosis of lung tissue. Clinical manifestations include cough, sputum, chest tightness, chest pain, and dyspnea. Pathological changes are characterized by lung inflammation and diffuse nodular fibrosis. Even if you are out of the dusty environment, the disease process cannot be stopped, and it may eventually develop into respiratory failure or even death. Pneumoconiosis often occurs in workers who are exposed to cutting, polishing, and grinding stone, including quarrying, mining, coal mining, sandblasting, pottery manufacturing, masonry, road construction, rock drilling, tunnel operations, jewelry polishing, etc.

[0003] Pneumoconiosis is one of the most important occupational diseases in the world. So far, there is no effective treatment due to the unclear mechanism of pneumoconiosis. Lung transplantation is still the final choice for patients with advanced pneumoconiosis and respiratory failure. At present, there is no treatment plan to improve the prognosis and lung function of pneumoconiosis. Only symptomatic treatment measures such as cough and phlegm, improvement of shortness of breath and dyspnea are taken, and anti-inflammatory treatment is performed when necessary.

[0004] All-trans retinoic acid (ATRA) is an active metabolite of vitamin A. ATRA can regulate genes related to cell growth, proliferation, differentiation, apoptosis, etc. It is the first differentiation inducer used in clinical practice. It is believed to induce abnormally accumulated promyelocytes to differentiate into mature granulocytes, thereby inducing cell death and inhibiting proliferation. It is currently the first choice for the treatment of acute promyelocytic leukemia. However, the therapeutic effect of ATRA in pneumoconiosis fibrosis is still unclear. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a reagent for detecting retinol metabolites for use in the preparation of tools for detecting and / or treating pneumoconiosis, using retinol and ATRA as biomarkers for screening pneumoconiosis and ATRA as a drug for treating pneumoconiosis, with significant therapeutic effects.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides an application of a reagent for detecting retinol metabolites in preparing a pneumoconiosis diagnosis tool.

[0008] Preferably, the retinol metabolites include one or more of all-trans retinoic acid, precursors of all-trans retinoic acid, metabolites of all-trans retinoic acid, and modified products of all-trans retinoic acid.

[0009] Preferably, the detection method of retinol metabolites comprises metabolomics or targeted HPLC-MS / MS.

[0010] Preferably, the detection sample of the retinol metabolites includes plasma and / or lung tissue.

[0011] The present invention also provides the use of one or more combinations of all-trans retinoic acid, all-trans retinoic acid precursors, all-trans retinoic acid metabolites and all-trans retinoic acid modified products in the preparation of drugs for treating pneumoconiosis.

[0012] Preferably, the pneumoconiosis includes silicosis, coal worker's pneumoconiosis, graphite pneumoconiosis, carbon black pneumoconiosis, asbestosis pneumoconiosis, talc pneumoconiosis, cement pneumoconiosis, mica pneumoconiosis, potter's pneumoconiosis, aluminum pneumoconiosis, welder's pneumoconiosis and foundry worker's pneumoconiosis.

[0013] Beneficial effects: The present invention provides an application of a reagent for detecting retinol metabolites in the preparation of a pneumoconiosis diagnostic tool. In the examples, it is found that retinol metabolic disorders and ATRA deficiency are closely related to the occurrence of pneumoconiosis. Therefore, it is proposed to use retinol and ATRA as biomarkers for screening pneumoconiosis and apply them to the preparation of related products for diagnosing pneumoconiosis. At the same time, ATRA intervention in pneumoconiosis can effectively slow down the progression of pneumoconiosis. Therefore, it is the first time to propose ATRA as a drug for treating pneumoconiosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The relationship between the disorder of retinol metabolism pathway and ATRA deficiency in pneumoconiosis tissues, including AC: volcano map, unsupervised clustering heat map and KEGG enrichment pathway of differential metabolites between PBS group and Silica group; D: HPLC-MS / MS detection of ATRA content in lung tissue and serum of mice in PBS and Silica groups; ***P<0.001;

[0015] Figure 2 The results of the effect of ATRA on pneumoconiosis, where A: ATRA treatment experiment schematic diagram; B and C: mouse lung function, IC is the inspiratory volume, Cst is the quasi-static compliance; D: mouse right heart function, RVSP is the right ventricular systolic pressure; E: mouse HE staining; F and G: IL-1β and IL-6 concentrations in mouse bronchoalveolar lavage fluid, BALF is bronchoalveolar lavage fluid; H: mouse Masson staining; I and J: translation and transcription levels of Fn-1 in mouse lung tissue; K: transcription level of Col-Ⅰ in mouse lung tissue; **P<0.01, ***P<0.001;

[0016] Figure 3 The figures show the effects of vitamin A deficiency (VAD) on pneumoconiosis, where A: a schematic diagram for evaluating the effects of ATRA deficiency caused by VAD diet on pneumoconiosis mice; B and C: lung function of mice; D: right heart function of mice; E: HE staining of mice; F and G: concentrations of IL-1β and IL-6 in bronchoalveolar lavage fluid of mice; H: Masson staining of mice; I and J: translation and transcription levels of Fn-1 in mouse lung tissue; K: transcription level of Col-Ⅰ in mouse lung tissue; **P<0.01, ***P<0.001. DETAILED DESCRIPTION

[0017] The present invention provides an application of a reagent for detecting retinol metabolites in preparing a pneumoconiosis diagnosis tool.

[0018] The retinol metabolites of the present invention preferably include all-trans retinoic acid, precursors of all-trans retinoic acid, metabolites of all-trans retinoic acid, and modified products of all-trans retinoic acid. The detection samples of the retinol metabolites of the present invention preferably include plasma and lung tissue. The detection method of the retinol metabolites of the present invention preferably includes metabolomics or targeted HPLC-MS / MS.

[0019] In the embodiment of the present invention, the experiment was conducted using pneumoconiosis mice. Compared with the control mice, the lung metabolism of pneumoconiosis mice was abnormal, and a large number of metabolites increased or decreased significantly. The pathways of abnormal metabolism in pneumoconiosis mainly include tryptophan metabolism, steroid hormone metabolism and retinol metabolism. The active product of retinol metabolism ATRA in the lung tissue and serum of pneumoconiosis mice was significantly lower than that of control mice. Then the pneumoconiosis mice were intervened with VAD diet to consume ATRA in the body and aggravate the progression of pneumoconiosis; compared with the normal diet VAS group, the cardiopulmonary function of pneumoconiosis mice in the VAD group was aggravated to varying degrees. Pathological staining also showed that ATRA deficiency aggravated the inflammation and fibrosis of pneumoconiosis mice. The inflammatory factors IL-1β and IL-6, fibrosis factors FN-1 and Col-Ⅰ of pneumoconiosis mice also increased due to ATRA deficiency. This confirms that retinol metabolites, especially ATRA, can be used as a detection marker for pneumoconiosis.

[0020] The present invention also provides the use of one or more combinations of all-trans retinoic acid, all-trans retinoic acid precursors, all-trans retinoic acid metabolites and all-trans retinoic acid modified products in the preparation of drugs for treating pneumoconiosis.

[0021] The pneumoconiosis described in the present invention preferably includes the types of pneumoconiosis that can be diagnosed according to the "diagnostic criteria for pneumoconiosis" and the "pathological diagnostic criteria for pneumoconiosis", and more preferably includes silicosis, coal worker pneumoconiosis, graphite pneumoconiosis, carbon black pneumoconiosis, asbestosis pneumoconiosis, talc pneumoconiosis, cement pneumoconiosis, mica pneumoconiosis, potter's pneumoconiosis, aluminum pneumoconiosis, welder's pneumoconiosis and foundry worker's pneumoconiosis, etc. In an embodiment of the present invention, ATRA treatment of pneumoconiosis mice can significantly improve the cardiopulmonary function, inflammation and fibrosis of pneumoconiosis mice. Compared with the control group mice, the inspiratory volume (IC) and quasi-static compliance (Cst) of the pneumoconiosis mice group treated with ATRA have improved to a certain extent, and the right ventricular systolic pressure (RVSP) has also decreased to a certain extent. Pathological staining shows that ATRA can reduce the degree of inflammation and fibrosis in pneumoconiosis mice. ATRA can also reduce the concentrations of inflammatory factors IL-1β and IL-6 in the bronchoalveolar lavage fluid (BALF) of pneumoconiosis mice, and can also reduce the levels of fibrosis factors FN-1 and Col-Ⅰ. Therefore, the present invention proposes for the first time to use ATRA as a drug for treating pneumoconiosis.

[0022] The following is a detailed description of the use of a reagent for detecting retinol metabolites provided by the present invention in preparing a tool for diagnosing and / or treating pneumoconiosis in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0023] Example 1

[0024] ATRA is effective in treating pneumoconiosis.

[0025] 1.1 Construction of pneumoconiosis mouse model and ATRA administration

[0026] Male C57BL / 6J mice (8-10 weeks old, 25-30g) were raised in an SPF animal room, and pneumoconiosis model was established by one-time tracheal instillation of silica. The mice were divided into 4 groups (n=9), namely: ①PBS+Vehicle group: 40μL sterile PBS was instilled into the trachea, and corn oil was gavaged 2 weeks later, 3 times a week for 4 weeks; ②PBS+ATRA group: 40μL sterile PBS was instilled into the trachea, and ATRA 10mg / kg was gavaged 2 weeks later, 3 times a week for 4 weeks; ③Si+Vehicle group: Silica suspension (300mg / mL, 40μL) was instilled into the trachea, and corn oil was gavaged 2 weeks later, 3 times a week for 4 weeks; ④Si+ATRA group: Silica suspension (300mg / mL, 40μL) was instilled into the trachea, and ATRA was gavaged 2 weeks later. 10 mg / kg was administered orally, 3 times a week, for 4 weeks. All mice were killed after 6 weeks, and the corresponding samples were collected for testing.

[0027] 1.2 Cardiopulmonary function test

[0028] The anesthetized mice were fixed on the experimental table, and an 8-gauge needle connected to a high-fidelity pressure sensor was inserted into the right ventricle to directly measure the invasive right ventricular systolic pressure (RVSP). RVSP was recorded and analyzed by a PowerLab data acquisition and analysis system (PL 3504, AD Instruments, Australia). The lung function of mice was detected using a pulmonary function testing system (DSIBuxco, USA). Before the experiment, the mice were anesthetized by intraperitoneal injection of 0.4 mL / 100 g 2% pentobarbital, and then the trachea was cut, the endotracheal tube was inserted, and the ventilator was connected. Next, the PFT system automatically detected the FRC test, PV test, FV test, and RC test. Indicators closely related to changes in lung function in pneumoconiosis were selected for statistical analysis, including inspiratory capacity (IC) and static compliance (Cst).

[0029] 1.3 Pathological staining

[0030] The left lung was fixed in 4% paraformaldehyde for 24 hours, dehydrated, embedded in paraffin, and sliced ​​(5 μm) for HE staining and Masson staining. HE staining was used to score inflammation according to Szapiel's score; Masson staining was used to score fibrosis according to Aschroft score. The slices were scanned, filmed, and counted using a 3D HISTECH digital biopsy scanner.

[0031] 1.4 ELISA experiment

[0032] ELISA kit was used to detect the concentration of IL-1β and IL-6 inflammatory factors in mouse BALF

[0033] 1.5 Western blot experiment

[0034] Extract all mouse lung tissue proteins. Prepare 10% polyacrylamide gel, load 10μg protein, and then perform electrophoresis at 80V for 30min; adjust the voltage to 120V and continue electrophoresis until the end. Use PVDF membrane for transfer, block with 5% skim milk powder for 1h, incubate with primary antibody FN-1 (abcam, 1:1000) at 4℃ overnight, secondary antibody at room temperature for 1h, and finally develop using chemiluminescence. Then, incubate the band with β-actin primary antibody, and the remaining steps are the same as above.

[0035] 1.6 QPCR experiments

[0036] RNA was extracted from all mouse lung tissues, and cDNA was obtained using a reverse transcription kit (KR103, Tiangen Biotechnology, Beijing, China). QPCR experiments were performed using a SYBR Green Ⅰ Q-PCR kit (TransGen Biotech, Beijing, China), and data collection and analysis were performed using the Bio-Rad IQ5 system.

[0037] Table 1 Primer sequences (5' to 3')

[0038]

[0039] 1.7 Results Analysis

[0040] Giving ATRA to pneumoconiosis mice can significantly improve cardiopulmonary function, inflammation and fibrosis ( Figure 2 (A). Compared with the control group, the inspiratory volume (IC) of the pneumoconiosis mice treated with ATRA ( Figure 2 B), quasi-static compliance (Cst) ( Figure 2 C) showed a certain degree of improvement, and right ventricular systolic pressure (RVSP) also decreased to a certain extent ( Figure 2 Middle D). Pathological staining showed that ATRA could reduce the degree of inflammation and fibrosis in pneumoconiosis mice ( Figure 2 ATRA can also reduce the concentrations of inflammatory factors IL-1β and IL-6 in the bronchoalveolar lavage fluid (BALF) of pneumoconiosis mice ( Figure 2 F, G), and can also reduce the levels of fibrosis factors FN-1 and Col-Ⅰ ( Figure 2 (Medium IK).

[0041] Example 2

[0042] VAD diet exacerbates pneumoconiosis.

[0043] 2.1 Construction of pneumoconiosis mouse model and corresponding special dietary treatment.

[0044] Male C57BL / 6J mice (8-10 weeks old, 25-30g) were selected and raised in an SPF-grade experimental animal room. The pneumoconiosis model was established by a one-time tracheal instillation of silica. The mice were divided into 4 groups (n=9), namely: ①PBS+VAS group: VAS diet, vitamin A sufficient diet. After 8 weeks of VAS diet, 40μL sterile PBS was instilled into the trachea; ②PBS+VAD group: VAD diet, vitamin A deficient diet. After 8 weeks of VAD diet, 40μL sterile PBS was instilled into the trachea; ③Si+VAS group: After 8 weeks of VAS diet, silica suspension (300mg / mL, 40μL) was instilled into the trachea; ④Si+VAD group: After 8 weeks of VAD diet, silica suspension (300mg / mL, 40μL) was instilled into the trachea. All mice were killed 6 weeks after modeling, and the corresponding samples were collected for detection.

[0045] The rest of the experimental steps are the same as 1.2, 1.3, 1.4, 1.5, 1.6

[0046] 2.2 Results Analysis

[0047] Pneumoconiosis mice were given a VAD diet to consume ATRA in the body, which aggravated the progression of pneumoconiosis ( Figure 3 Compared with the normal diet VAS group, the cardiopulmonary function of the pneumoconiosis mice in the VAD group was aggravated to varying degrees ( Figure 3 BD). Pathological staining also showed that ATRA deficiency aggravated the inflammation and fibrosis of pneumoconiosis mice ( Figure 3 Inflammatory factors IL-1β and IL-6 in pneumoconiosis mice ( Figure 3 F, G); fibrosis factor FN-1 and Col-Ⅰ ( Figure 3 IK) also increases due to ATRA deficiency.

[0048] Example 3

[0049] Pneumoconiosis causes retinol metabolism disorder leading to ATRA deficiency.

[0050] 3.1 Construction of pneumoconiosis mouse model and metabolomics detection.

[0051] Male C57BL / 6J mice (8-10 weeks old, 25-30g) were selected and raised in an SPF-grade experimental animal room. The mice were randomly divided into two groups: ①PBS group (n=10) ②Silica group (n=15). The pneumoconiosis model was established by a one-time tracheal instillation of silica, and the control group was given an equal amount of PBS solution. After 6 weeks of modeling, the mouse lung tissues were collected for non-targeted metabolomics detection based on HPLC-MS / MS. According to the results of non-targeted metabolomics analysis, the pneumoconiosis mouse model was constructed again, and the ATRA content in the lung tissues and serum of pneumoconiosis mice and control mice was detected by targeted quantitative HPLC-MS / MS.

[0052] 3.2 Interpretation of results

[0053] Compared with control mice, pneumoconiosis mice showed abnormal lung metabolism, with a significant increase or decrease in a large number of metabolites ( Figure 1 A, B in the middle). The abnormal metabolic pathways in pneumoconiosis mainly include tryptophan metabolism, steroid hormone metabolism, and retinol metabolism ( Figure 1 C). The levels of retinol metabolite ATRA in lung tissue and serum of pneumoconiosis mice were significantly lower than those in control mice ( Figure 1 (middle D).

[0054] 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of all-trans retinoic acid in the preparation of a drug for treating pneumoconiosis, characterized in that: The pneumoconiosis includes silicosis, coal worker's pneumoconiosis, graphite pneumoconiosis, carbon black pneumoconiosis, asbestosis pneumoconiosis, talc pneumoconiosis, cement pneumoconiosis, mica pneumoconiosis, potter's pneumoconiosis, aluminum pneumoconiosis, welder's pneumoconiosis and foundry worker's pneumoconiosis.

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