Use of soy extract in preparation of drug for preventing or treating uranium-induced lung injury

Abstract

A use of a soy extract in the preparation of a drug. The drug has one or more of the following uses: a) mitigating pulmonary fibrosis of uranium-exposed individuals; b) improving the uranium excretion capability of the uranium-exposed individuals; and c) preventing or treating uranium-induced lung injury.

Description

Use of soybean extract in preparation of drugs for preventing or treating uranium-induced lung injury TECHNICAL FIELD

[0001] The present application belongs to the technical field of lung injury treatment, and relates to the use of soybean extract in preventing or treating uranium-induced lung injury. BACKGROUND

[0002] As a strategic resource for national defense and nuclear power industry, uranium (U) is widely used in nuclear industry and military activities, and is also an important material in civil fields such as aircraft radiation shielding. In the process of nuclear fuel manufacturing, processing and disposal, uranium is inevitably released into the environment, causing air, soil and water pollution. In recent years, with the rapid development of nuclear power, nuclear fuel cycle and uranium mining and metallurgy in China, the number of people occupationally exposed to uranium and its compounds has increased, and the health effects of uranium poisoning have attracted increasing attention, which directly affects the safe and stable development of China's nuclear power industry.

[0003] Most of the injuries caused by uranium are due to the chemical toxicity of hexavalent uranyl ions (UO2 2+ ) rather than radioactive toxicity. Inhaled uranium can penetrate into the alveoli, causing emphysema, pulmonary fibrosis and other injuries, and may lead to lung cancer. Recent studies have shown that uranium is absorbed by the human body through the respiratory tract and causes lung injury through oxidative stress, promotion of inflammation, induction of DNA damage, promotion of apoptosis and autophagy, etc.

[0004] Uranium antidotes and chelators are important means for preventing and reducing uranium toxicity. Uranium antidotes such as DTPA have the disadvantages of high toxicity, low efficiency and low bioavailability. At the same time, the application of natural plant active ingredients is one of the traditional methods for removing toxic metals from the body. Natural antioxidants found in normal diet, such as flavonoids, thioctic acid, carotenoids, vitamins C and E, taurine, curcumin, minerals, amino acids, etc., have the ability to increase methylation and scavenge free ROS, and show beneficial effects in reducing metal toxicity. Uranium antidotes and chelators are important means for preventing and reducing uranium toxicity. Uranium antidotes such as DTPA have the disadvantages of high toxicity, low efficiency and low bioavailability.

[0005] Therefore, it is still challenging to study new natural uranium antidotes with non-toxic, anti-inflammatory, antioxidant and other biological activities. SUMMARY

[0006] In order to overcome the above technical problems, the present application provides the use of soybean extract in preventing or treating uranium-induced lung injury, and through animal models, it is proved that the soybean extract as a natural low-toxicity bioactive substance has the ability to chelate with uranium and can be used as an effective uranium antidote, providing a new prevention or treatment approach for preventing and treating uranium poisoning and reducing toxicity.

[0007] In a first aspect, the present application provides use of a soybean extract in preparation of a medicament,

[0008] a) improving lung fibrosis of a uranium exposed subject;

[0009] b) improving uranium excretion of a uranium exposed subject;

[0010] c) preventing or treating uranium induced lung injury.

[0011] In the present application, the protective effect of soybean extract (SE) on uranium induced lung injury mouse model was analyzed, and the results showed that soybean extract could improve lung fibrosis of uranium exposed subject, SE treatment could reduce uranium induced lung fibrosis, and reduce the expression of α-SMA, TGF-β1 and Hyp, and improve the pathological changes of lung tissue caused by uranium. Compared with the U group, the lung uranium level of the SE+U group was significantly reduced by 18.8%, and hematoxylin and eosin staining showed that the histological inflammatory cell infiltration was significantly reduced. The above results showed that soybean extract had a certain protective effect on uranium induced lung injury.

[0012] In some embodiments, the soybean extract is prepared from soybean by a microwave assisted extraction method. The microwave assisted extraction method is specifically that the water soaked soybean is placed in a microwave reaction kettle for treatment, and the gasified gaseous plasma is condensed and recovered, to obtain the soybean extract.

[0013] In some embodiments, the treatment frequency of the microwave reaction kettle is 2000-2100 MHz, and the treatment time is 2-3 min, preferably the treatment frequency is 2040-2060 MHz, and more preferably the treatment frequency is 2045 MHz.

[0014] In the present application, the active ingredients in soybean are extracted by the above method, and the water content of the soybean after soaking is about 60% in the extraction step, then the soybean is transferred into a microwave plasma effect reaction kettle (i.e. microwave reaction kettle) for effective separation of active ingredients, and the soybean active ingredient extract (i.e. soybean extract) can be obtained by condensation and recovery.

[0015] In some embodiments, the uranium exposed subject has not developed or has developed uranium induced lung injury.

[0016] In a second aspect, the present application also provides a pharmaceutical composition with one or more uses, wherein the active ingredient of the pharmaceutical composition is a soybean extract, and the uses are,

[0017] a) improving lung fibrosis of a uranium exposed subject;

[0018] b) improving uranium excretion of a uranium exposed subject;

[0019] c) preventing or treating uranium induced lung injury.

[0020] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0021] In some embodiments, the soybean extract is prepared from soybean by a microwave-assisted extraction method.

[0022] In some embodiments, the microwave-assisted extraction method specifically comprises placing water-soaked soybean in a microwave reactor for treatment, and recovering the gasified gaseous plasma by condensation to obtain the soybean extract.

[0023] In some embodiments, the microwave reactor has a treatment frequency of 2000-2100 MHz and a treatment time of 2-3 min, preferably a treatment frequency of 2040-2060 MHz, and more preferably a treatment frequency of 2045 MHz.

[0024] Compared with the prior art, the present application has the following technical effects,

[0025] The present application studies and analyzes the protective effect of soybean extract (SE) on a uranium-induced lung injury mouse model, and the research results show that the soybean extract can improve lung fibrosis of uranium exposure subjects, SE treatment can reduce uranium-induced lung fibrosis, and the expression of α-SMA, TGF-β1 and Hyp is reduced, and the lung histopathology change caused by uranium is improved. Compared with the U group, the lung uranium level of the SE+U group is significantly reduced by 18.8%, and hematoxylin and eosin staining shows that the histological inflammatory cell infiltration is significantly reduced. Therefore, the soybean extract has a certain protective effect on uranium-induced lung injury. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is a uranium-induced lung injury model and a drug treatment scheme.

[0027] Figure 2 is a soybean extract (SE) that reduces uranium-induced lung injury in rats. (A) U(VI) content in lung tissue (n=5); (B) representative hematoxylin and eosin staining results and lung pathology scores of the four groups, the scale bar represents 100 μm; (C) changes in rat lung coefficient (n=5); (D) changes in rat body weight (n=5). The data are the average values of 5 rats in each group ± SEM. *P<0.05, ***P<0.01, ****P<0.0001, ns means not significant.

[0028] Figure 3 Soybean extract treatment can improve lung fibrosis in uranium inhalation model. (A) Masson's trichrome staining results and quantitative analysis of fibrosis area in uranium inhalation model, scale bar represents 100 pm; (B) a-SMA levels in each group of rats (n = 4); (C) TGF-β1 levels in each group of rats (n = 4); (D) Hyp levels in each group of rats (n = 4). Data are the mean ± SEM of 5 rats in each group. *P < 0.05, **P < 0.01.

[0029] Figure 4 Potential mechanisms of SE in treating ALI. (A) GO analysis chart in the whole prescription, the higher the wave band height, the more significant; (B) KEGG enrichment analysis bubble chart, the larger the bubble, the more targets it acts on; color depth represents p value, the darker the red color, the more significant the p value; (C) "Component-Target-Pathway" network of the main active ingredients of SE; yellow nodes represent the main active compounds, red nodes represent the target points, and purple nodes represent the signal pathways.

[0030] Figure 5 Transcriptomic analysis of SE-treated rats with uranium-induced lung injury. (A) PCA score plot of U group and U+SE group; (B) volcano plot of DEGs; blue represents down-regulation, and red represents up-regulation; (C) and (D) enrichment bubble chart of U+SE and U+SE.

[0031] Figure 6 Metabolomic analysis of SE-treated rats with uranium-induced lung injury. (A) PLS-DA score plot of U group and U+SE group metabolomic data; (B) OPLS-DA score plot of U group and U+SE group metabolomic data; (C) Validation of OPLS-DA model; (D) Heat map of different metabolites; (E) Volcano plot of DMs; blue represents down-regulation, and red represents up-regulation; (F) Metabolic pathway bubble chart.

[0032] Figure 7 Multi-omics comprehensive analysis of SE-treated uranium-induced lung injury. (A) Common pathways of network pharmacology and transcriptomics; (B) Bubble chart of joint analysis of transcriptomics and targeted metabolomics based on MetaboAnalyst database; (C) Correlation heat map of deg and dm; warm or cold color represents positive or negative relationship; *P < 0.05, **P < 0.01; (D) Main compounds from SE acting on genes and metabolites of drug metabolism—cytochrome P450, complement and coagulation cascade, glutathione metabolism, and IL-17 signaling pathway; yellow nodes represent active compounds, red nodes represent genes, green nodes represent metabolites, and purple nodes represent signal pathways.

[0033] Figure 8 Mechanism diagram of the effect of SE on uranium-induced lung injury. DETAILED DESCRIPTION

[0034] The present application will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present application and not to limit the scope of the present invention. For the experimental methods without specific conditions noted in the following embodiments, they are usually carried out under conventional conditions, the conditions described in laboratory manuals, or the conditions recommended by the manufacturer.

[0035] Preparation Example

[0036] The soybean extract was prepared by microwave-assisted method, and the preparation method is as follows.

[0037] (1) Pretreatment of materials: The soybeans were screened to remove impurities such as dust, washed and drained, sorted to the same size, and soaked in water until the water content was about 60%.

[0038] (2) The prepared materials were put into a microwave cracking reactor and treated at a microwave frequency of 2045 MHz for 2 - 3 min. The gasified plasma was condensed and recovered to obtain the soybean extract.

[0039] The substances in soybeans are distilled or gasified under the action of high-frequency microwaves, and the soybean extract is obtained by condensation and recovery. 500 g of soybean extract can be collected from 5 kg of soybeans. The pH of the soybean extract is about 8, showing weak alkalinity. Through 17 1H-NMR detected that the small molecular cluster structure size of the essence liquid was 38.81 HZ. The extensive targeted metabolome detection report of the soybean extract showed that it contained 656 small molecular compounds, all of which were small molecular compounds below 1000 Da, rich in small molecular compounds of various secondary metabolites in soybeans. Among them, there were 90 phenolic acids, 90 amino acids and their derivatives, 90 alkaloids and terpenoids, 82 organic acids, 80 flavonoids, 70 lipids, 67 sugars, alcohols and other types, 51 nucleotides and their derivatives, 23 lignins, and 13 vitamins.

[0040] Establishment of the mouse model in Example 1

[0041] Twenty-four 10-week-old male SD rats, weighing 440 - 520 g, were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. [SCXK(Beijing) 2021-0011]. They were adaptively fed for 1 week before the experiment, with 6 rats in each cage. Food and water were freely available during the experiment.

[0042] Uranyl nitrate hexahydrate (VI) was provided by China National Nuclear Corporation (Beijing, China). All experiments were carried out with the original uranyl nitrate solution with a concentration of 150 mg / mL (pH 4) in double-distilled deionized water. The solution was stored at 4 °C for later use.

[0043] Experimental groups: 24 rats were randomly divided into 4 groups (6 rats in each group): no pre-treatment water administration group (CON group), no pre-treatment uranium administration group (U group), water administration and SE pre-treatment group (SE group), uranium administration and SE pre-treatment group (U+SE group). The soy extracts (SE) were prepared according to the preparation example.

[0044] The rats were anesthetized by intraperitoneal injection of sodium pentobarbital (30 mg / kg). Then, the rats in the SE group and the U+SE group were orally administered with SE 10 mL / kg / d. On day 7, the rats in the U group and the U+SE group were inhaled with uranyl nitrate solution (20 mg / kg) by nebulization. The control group was given the same amount of purified water. The endpoint of the study was 12 days after the intervention. The left upper lobe of the lung was fixed with 4% paraformaldehyde for histopathological analysis. The left lower lobe was taken for uranium content determination and ELISA detection, and the remaining tissue was rapidly frozen in liquid nitrogen for subsequent experiments (Figure 1).

[0045] As shown in Figure 1, the administration methods of each group were as follows: the rats in the SE group and the U+SE group were orally administered with soy extracts (5 mL / kg, 2 times / day), and the rats in the U group were orally administered with the same volume of double distilled water. On day 7 of the experiment, the rats in the U group and the U+SE group were inhaled with uranyl nitrate solution (20 mg / kg) by nebulization, and the rats in the SE group were inhaled with the same volume of double distilled water by nebulization. Subsequently, the daily oral administration was continued. The rats in the CON group were given the same volume of double distilled water in the same way at the corresponding time points. The endpoint of the study was 12 days after the intervention.

[0046] Example 2 Histopathological diagnosis of lung tissue

[0047] The left upper lobe of the lung of the rats was fixed with 4% paraformaldehyde. After dehydration of the tissue, it was embedded in paraffin, and then deparaffinized, hydrated, stained with hematoxylin and eosin. The stained tissue sections were observed under a microscope (Zeiss, Germany). The severity of lung tissue damage was evaluated according to the Szapiel scoring system (Szapiel et al., 1979).

[0048] Fresh left lower lung tissue was taken for uranium content determination. 0.3 g of lung tissue was digested into 10 mL of PBS using a microwave digestion instrument, and the uranium content was detected by inductively coupled plasma mass spectrometry (ICP-MS). The ICP-MS was used for uranium content determination.

[0049] Table 1 Working program of microwave digestion instrument

[0050] The results of the diagnosis are shown in Figure 2. In the model of uranium inhalation rats, uranium can be inhaled by the lung, and the lung uranium level of the U group is significantly higher than that of the CON group (Figure 2A). The lung injury of the U group is manifested as interstitial inflammatory cell infiltration (Figure 2B). Compared with the U group, the lung uranium level of the SE+U group is significantly reduced by 18.8%, and hematoxylin and eosin staining shows that the histological inflammatory cell infiltration is significantly reduced (Figures 2A and 2B). Compared with the CON group, the lung coefficient of lung injury expressed by relative lung weight is reduced after uranium exposure, and SE+U treatment is significantly improved (Figure 2C). Uranium exposure and SE treatment have no effect on the body weight of rats (Figure 2D).

[0051] Example 3 Masson's trichrome staining to observe lung fibrosis

[0052] Masson's trichrome staining was performed using a Masson's trichrome staining kit (Solarbio, G1346, Beijing). The same lung tissue sections as described above were used for Masson's staining. The stained tissue sections were observed under a microscope (Zeiss, Germany). The degree of fibrosis was quantified according to the numerical scale of Ashcroft et al. (Ashcroft et al., 1988).

[0053] The lung tissue alpha-smooth muscle actin (a-SMA) level was detected using a rat a-SMA enzyme-linked immunosorbent assay kit. The lung tissue was homogenized with phosphate buffered saline (PBS) and centrifuged at 1500g for 20 minutes. The supernatant was treated according to the manufacturer's procedure. The absorbance at 450 nm wavelength was finally determined. Similar to the a-SMA level, the detection was performed according to the instructions of the transforming growth factor-β1 (TGF-β1) detection kit and the hydroxyproline (Hyp) detection kit of Nanjing Jiancheng Bioengineering Institute.

[0054] As shown in Figure 3, the Masson's trichrome staining results show that SE treatment significantly reduces extracellular matrix deposition compared with the U group (Figure 3A). Consistent with Masson staining, ELISA results show that the a-SMA, TGF-β1, and Hyp levels of the SE-treated U+SE group rats are reduced compared with uranium-exposed rats (Figures 3B-3D). SE treatment can alleviate uranium-induced lung fibrosis while reducing the expression of a-SMA, TGF-β1, and Hyp.

[0055] Example 4 Network pharmacology analysis

[0056] Screening of SE active compounds and targets: A total of 656 metabolites were detected in SE by UPLC-MS / MS platform. According to the conditions of relative abundance > 0.05%, gastrointestinal absorption (high), and drug properties (at least 4 "yes") in SwissADME (http: / / www.swissadme.ch / ), 22 active compounds were screened. SE-related gene targets were collected by SwissTarget Prediction (http: / / www.swisstrgetprediction.ch / ) database. Hypothesized targets with a probability > 0.1 were selected.

[0057] Screening of acute lung injury (ALI) targets: Two databases were used to search for therapeutic targets for ALI: (a) GeneCards database (https: / / www.genecards.org / ), (b) Online Mendelian Inheritance in Man (OMIM, http: / / omim.org / ). A union of al -related gene sets was established.

[0058] Enrichment analysis: Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were performed using the KOBAS database (http: / / bioinfo.org / kobas) with a p-value < 0.05. Finally, the "SE-compound-target-pathway" network was constructed using Cytoscape 3.10.1 software.

[0059] The results are shown in Figure 4. To better understand the multiple molecular mechanisms of SE in treating ALI, 18 compounds and 1977 disease targets were collected from the UPLC-MS / MS results of SE and related databases. Through cross-table analysis, 173 potential SE proteins for ALI treatment were identified. Subsequently, GO analysis was performed on the mapped targets (Figure 4A). These targets were involved in biological processes such as negative regulation of apoptotic process, response to external stimulus, and protein phosphorylation. Cell components were mainly enriched in lipid rafts and plasma membrane. In terms of molecular function, these targets were mainly involved in protein kinase activity, enzyme binding, and ATP binding. KEGG results showed that these targets were related to "PI3K-Akt signaling pathway", "Rap1 signaling pathway", "T cell receptor signaling pathway", and "Relaxin signaling pathway" (Figure 4B). The potential mechanism network of SE in treating ALI (Figure 4C).

[0060] Example 5 Transcriptomic analysis

[0061] The experimental groups are selected as CON group, U group and U+SE group. From each group, 3 rats are randomly selected for lung transcriptome sequencing, and 5 rats are randomly selected for urine sample for metabolome detection.

[0062] 1. Total RNA extraction

[0063] (1) Tissue sample crushing and lysis

[0064] Liquid nitrogen grinding and crushing: Take an appropriate amount of tissue sample under liquid nitrogen protection and grind the tissue sample into powder, and transfer it into an EP tube containing 1.5 mL TRIzol lysis solution. Let it stand for 5 min to fully lyse the tissue cells.

[0065] (2) Sample extraction and purification

[0066] ① Centrifuge the ground and crushed tissue sample at 4°C and 12000g for 5 min, and transfer the supernatant to an EP tube containing 300 μL chloroform / isoamyl alcohol (24:1). After mixing well by inverting and shaking vigorously, centrifuge at 4°C and 12000g for 8 min. If the intermediate layer is thick and the aqueous phase is turbid, extract again with an equal volume of chloroform / isoamyl alcohol (24:1).

[0067] ② Absorb the supernatant into a new 1.5 mL centrifuge tube, add isopropanol at 2 / 3 of the volume of the supernatant, mix gently by inverting, and place it in a -20°C refrigerator for more than 2 h.

[0068] ③ After standing, centrifuge at 4°C and 17500g for 25 min, discard the supernatant, and wash with 0.9 mL of 75% ethanol. Invert and suspend the precipitate, centrifuge at 4°C and 17500g for 3 min (depending on the precipitate, it can be washed again with 75% ethanol at 4°C and 17500g for 3 min).

[0069] ④ Discard the supernatant, centrifuge briefly, and absorb the residual liquid. Dry for 3-5 min, and dissolve the precipitate with 20-200 μL of DEPC water or RNase-free water.

[0070] (3) Use the full-automatic capillary electrophoresis instrument to detect the concentration, purity (28S / 18S), and integrity (RIN value) to determine whether the measured sample quality meets the requirements for library construction and sequencing.

[0071] 2. mRNA library preparation

[0072] (1) mRNA isolation

[0073] Take a certain amount of the qualified Total RNA sample for DNase I digestion. The digested Total RNA sample is used to enrich mRNA using Oligo(dT) magnetic beads.

[0074] (2) mRNA fragmentation

[0075] Add fragmentation reagent to the enriched mRNA, and react at proper temperature for a certain time to fragment the mRNA.

[0076] (3) cDNA synthesis

[0077] Prepare a reaction system for single-strand synthesis, synthesize single-strand cDNA (fragmented mRNA is added with random primers to synthesize single-strand cDNA), prepare a reaction system for double-strand synthesis, synthesize double-strand cDNA (use dUTP instead of dTTP), and then perform end repair and "A" addition and adaptor ligation on the amplified cDNA.

[0078] (4) PCR reaction

[0079] Perform PCR reaction on the ligation product, digest the U-labeled second-strand template with UDG enzyme, and then perform PCR amplification.

[0080] (5) Library detection

[0081] Select corresponding detection methods according to product requirements to perform quality inspection on the library.

[0082] (6) PCR product circularization

[0083] After denaturation of the PCR product into single-strand, perform circularization, obtain single-strand circular DNA library by circularization, digest linear DNA molecules that are not circularized, and obtain the final library.

[0084] 3. Sequencing on machine

[0085] Single-strand circular DNA molecules form a DNA nanoball (DNB) containing multiple copies through rolling circle replication. The obtained DNBs are added to the reticular small holes on the chip using high-density DNA nanochip technology, and sequencing is performed through combined probe anchor and polymerization technology (cPAS).

[0086] 4. Data quality evaluation

[0087] After obtaining the raw reads after sequencing, the filtering software SOAPnuke independently developed by Huada is used for filtering, removing low-quality (reads with a proportion of bases with a quality value less than 15 accounting for more than 20% of the total number of bases in the reads), adapter contamination and reads with too high content of unknown bases N (> 5%) in the raw data raw reads, obtaining the quality-controlled data clean reads, and calculating Q20 and Q30 respectively. After obtaining the clean reads, we use HISAT to align the clean reads to the reference genome sequence (species name: Rattus norvegicus; source: NCBI; reference genome version: GCF_015227675.2_mRatBN7.2), calculate the alignment rate (the proportion of the number of clean reads with unique alignment positions on the reference genome), and at the same time, evaluate the quality of the alignment results of this sequencing.

[0088] After obtaining reliable clean reads through RNA-seq sequencing quality evaluation, the filtered high-throughput sequencing data is aligned to the reference gene sequence using Bowtie2 to obtain the number of reads aligned to each gene (reads count value), and RSEM software is used to standardize it to TPM expression. By calculating the Pearson correlation coefficient of the expression of all genes between each two samples, the correlation of gene expression between samples is reflected. The reliability of the transcriptome sequencing results is ensured through RNA-seq sequencing quality evaluation.

[0089] 5、Principal component analysis (PCA) is a clustering analysis of samples based on gene expression, reflecting the similarity of gene expression between groups and samples. The closer the distance between sample points in the PCA analysis graph, the higher the similarity between them. DESeq2 (repeated samples) is used to screen differential expression genes, p-adjust < 0.05, and fold change > 1.5 as the significant difference threshold. GO and KEGG analysis are used for functional annotation and enrichment pathways related to differential expression genes (DEGs).

[0090] PCA showed that the gene expression of U group and U+SE group was significantly different, as shown in Figure 5. Compared with U group, the gene expression in lung tissue of U+SE group rats changed significantly (Figure 5A). According to the results of differential analysis, genes with Padj<0.05 and |Fold Change|>1.5 were screened as differential expression genes (deg). Compared with U group, 67 degs were identified in U+SE group, of which 37 were up-regulated and 30 were down-regulated. According to DEGs, a volcano plot was drawn (Figure 5B). In order to identify the pathways that may be affected, we obtained the KEGG pathway enrichment bubble chart (Figure 5C) and GO enrichment bubble chart (Figure 5D). KEGG pathway enrichment analysis showed that DEGS were significantly enriched in complement and coagulation cascade pathway, metabolic pathway, IL-17 signaling pathway and glutathione metabolism. GO analysis results showed that in the aspect of biological process (BP), DEGS were mainly related to negative regulation of T cell proliferation, negative regulation of endopeptidase activity, and apoptosis process. Regarding cell component (CC), degs were related to extracellular space / extracellular region. Molecular function (MF) analysis was related to serine-type endopeptidase inhibitor activity, endopeptidase inhibitor activity and protease binding. It is suggested that SE may affect these pathways and alleviate uranium-induced lung injury in rats.

[0091] Example 6 Targeted metabolomics detection

[0092] 1. Extraction of metabolites

[0093] Tissue samples were ground in 80% methanol (v / v) at a concentration of 10 μL / mg, and incubated on ice for 20 minutes. After centrifugation at 13000 rpm, 4°C for 15 minutes, the supernatant was collected. Finally, 4 μL of supernatant from each group was mixed as a quality control (QC) sample, which served as a reference for quality evaluation during analysis.

[0094] 2. Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) analysis

[0095] In this experiment, Waters ACQUITY UPLC I-Class Plus (Waters, USA) coupled with QTRAP6500 Plus high sensitivity mass spectrometer (SCIEX, USA) was used for separation and quantitative detection of metabolites.

[0096] Chromatographic conditions: The chromatographic column used was BEH C18 (2.1 mm x 10 cm, 1.7 um, waters) with a column temperature of 40 °C. The mobile phase was 0.1% formic acid water (A liquid) and 30% isopropanol acetonitrile (B liquid). Elution was performed using the following gradient: 0-1.00 min, 5% B liquid; 1.00-5.00 min, 5%-30% B liquid; 5.00-9.00 min, 30%-50% B liquid; 9.00-11.00 min, 50%-78% B liquid, 11.00-13.50 min, 78%-95% B liquid, 13.5-14.00 min, 95%-100% B liquid, above the flow rate was 0.400 mL / min; 14.00-16.00 min, 100% B liquid, flow rate was 0.600 mL / min; 16.00-18.00 min, 5% B liquid, flow rate was 0.400 mL / min.

[0097] Mass spectrometry conditions: For QTRAP 6500 Plus equipped with ESI Turbo ion spray interface, the ion source parameters were set as follows: ion source temperature: 400 °C; ion spray voltage (IS): 4500 V (positive mode) and -4500 V (negative mode); ion source gas I (GS1), gas II (GS2) and curtain gas (CUR) were set to 60, 60 and 35 psi, respectively. The MRM method was set in MRM mode, which contained the MRM parent-daughter ion pair information of the target metabolites, collision energy (CE) and declustering voltage (DP) and retention time.

[0098] Mass spectrometry detection was performed in positive and negative ionization modes, and multiple reaction monitoring (MRM) transitions. The MRM transition and optimization parameters for each metabolite were from the G700 targeted metabolomics kit (MetaboProfile, China). Skyline software (Version 21.1) was used for data acquisition and processing.

[0099] Partial least squares discriminant analysis (PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were performed using MetaboAnalyst 5.0. In this study, the screening of differential metabolites was performed by |log2FoldChange|>0.585, P<0.05 and important predictive variables (VIP)>1. Metabolic pathways were further analyzed in MetaboAnalyst 5.0.

[0100] The connection between different omics results was comprehensively analyzed. First, the KEGG co-enriched pathways were selected according to the results of network pharmacology, transcriptomics and metabolomics. Then, the correlation between deg and DMs was analyzed using Pearson correlation coefficient. The results were visualized by correlation heat map and network diagram.

[0101] All quantitative data are expressed as mean ± standard error of mean (SEM). Statistical analysis was performed using GraphPad Prism 8 (San Diego, CA, USA). One-way ANOVA was used for intergroup analysis. The alpha criterion was set at 0.05.

[0102] By identifying the metabolites of U and U+SE groups, a total of 393 metabolites were identified in lung tissue, mainly amino acids and polypeptides, bile acids, lipids, carbohydrates, vitamins, and other metabolites. To explore the changes of metabolites before and after administration in lung injury rats, PLS-DA and OPLS-DA were used to analyze the data of each group.

[0103] As shown in Figure 6, the U and U+SE groups were well separated (Figures 6A-6C). A total of 39 differential metabolites (DMs) were screened, of which 9 were down-regulated and 30 were up-regulated (Figures 6D and 6E). MetaboAnalyst was used to analyze the metabolic pathways and further explore the potential impact of metabolite changes. According to the log(p) and effect value, the potential pathways of uranium treatment of SE lung injury were determined. The results were presented in the form of bubble chart (Figure 6F). These pathways were mainly related to vitamin metabolism (one-carbon folate pool, folate biosynthesis, vitamin B6 metabolism) and carbohydrate metabolism (inositol phosphate metabolism, phosphatidylinositol signaling system).

[0104] As shown in Figure 7, the combined analysis of network pharmacology and transcriptomics showed that 8 common pathways, such as drug metabolism-cytochrome P450, complement and coagulation cascade, glutathione metabolism, IL-17 signaling pathway, etc. (Figure 7A). Combined analysis of transcriptomics and targeted metabolomics also enriched pathways such as drug metabolism-cytochrome P450 and glutathione metabolism (Figure 7B). The heatmap of deg and dm was obtained by Pearson correlation coefficient analysis (Figure 7C). The genes related to drug metabolism-cytochrome P450, complement and coagulation cascade, glutathione metabolism and IL-17 signaling pathway were selected, including Fgb, Fmo2, Gsta1 and Muc5b. Finally, Cytoscape 3.10.1 was used to construct the C-G-M-P network diagram, which more directly reflects the relationship between the key components of SE, target genes and metabolites (Figure 7D).

[0105] The present application combines network pharmacology, transcriptomics and metabolomics to study the effect of SE on uranium-induced lung injury (Figure 8). The lung protective effect of SE was verified in a rat model of uranium-induced lung injury. To further explore the potential protective mechanism of SE on the lung, the results showed that SE may exert its protective effect by inhibiting the expression of key genes related to inflammation and immune regulation pathways. In addition, SE can also up-regulate antioxidant enzymes, thereby attenuating uranium-induced oxidative stress. The present application elucidates the potential pharmacological mechanism of SE in reducing uranium-induced lung injury, providing valuable insights for the therapeutic application of SE in the lung and related diseases.

[0106] The specific embodiments are only an explanation of the present application, which is not a limitation to the present application, and those skilled in the art can make modifications to the embodiments without creative contribution after reading the specification, but as long as it is within the scope of the claims of the present application, it is protected by the patent law.

Claims

1. Use of a soy extract for the preparation of a medicament, characterized in that, The medicine has one or more of the following uses, a) improving lung fibrosis of uranium exposed persons; b) improving uranium excretion of uranium exposed persons; c) preventing or treating uranium induced lung injury.

2. Use according to claim 1, characterized in that, The soybean extract is prepared from soybeans by a microwave assisted extraction method.

3. Use according to claim 2, characterized in that, The microwave assisted extraction method specifically comprises the following steps: placing water soaked soybeans in a microwave reaction kettle for treatment, and condensing and recovering the gasified gaseous plasma to obtain the soybean extract.

4. Use according to claim 3, characterized in that, The microwave reaction kettle has a treatment frequency of 2000-2100 MHz and a treatment time of 2-3 min.

5. The use according to claim 1, characterized in that, The uranium exposed person has not developed or has developed uranium induced lung injury.

6. A pharmaceutical composition having one or more of the following uses, characterized in that, The active ingredient of the pharmaceutical composition is the soybean extract, and the uses are, a) improving lung fibrosis of uranium exposed persons; b) improving uranium excretion of uranium exposed persons; c) preventing or treating uranium induced lung injury.

7. The pharmaceutical composition of claim 6, wherein, The pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

8. The pharmaceutical composition of claim 7, wherein, The soybean extract is prepared from soybeans by a microwave assisted extraction method.

9. The pharmaceutical composition of claim 8, wherein, The microwave assisted extraction method specifically comprises the following steps: placing water soaked soybeans in a microwave reaction kettle for treatment, and condensing and recovering the gasified gaseous plasma to obtain the soybean extract.

10. The pharmaceutical composition of claim 9, wherein, The microwave reaction kettle has a treatment frequency of 2000-2100 MHz and a treatment time of 2-3 min.

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