Use of soy extract for the prevention or treatment of uranium-induced lung damage

Abstract

The present application belongs to the technical field of lung injury treatment and relates to the use of soy extract for the prevention or treatment of uranium-induced lung injury. More specifically, this application relates to the use of soy extract for the manufacture of a medicament with one or more of the following uses: a) improvement of pulmonary fibrosis in a uranium-exposed individual; b) increase of uranium excretion capacity in a uranium-exposed individual; and c) prevention or treatment of uranium-induced lung injury. In this application, the protective effect of soy extract (SE) was investigated in a mouse model of uranium-induced lung injury. The results showed that soy extract can improve pulmonary fibrosis in a uranium-exposed individual.Treatment with soy extract (SE) alleviated uranium-induced pulmonary fibrosis, simultaneously reducing the expression of α-SMA, TGF-β1, and Hyp, and improving uranium-induced pathological changes in lung tissue. Compared to the uranium group, the SE+U group showed a significant reduction in lung uranium concentration of 18.8%. Hematoxylin and eosin staining also revealed a significant reduction in the histological infiltration of inflammatory cells. Soy extract thus possesses a certain protective effect against uranium-induced lung damage.

Description

Technical field

[0001] The present application belongs to the technical field of the treatment of lung damage and concerns the use of soy extract for the prevention or treatment of uranium-induced lung damage. Technical background

[0002] Uranium (U), a strategic resource for national defense and the nuclear energy industry, is widely used in the nuclear industry and military activities, and is also an important material in civilian sectors such as radiation shielding for aircraft. The production, processing, and disposal of nuclear fuel inevitably release uranium into the environment, causing air, soil, and water pollution. In recent years, the rapid development of nuclear energy, the nuclear fuel cycle, and uranium mining in our country has led to an increase in the number of people occupationally exposed to uranium and its compounds. The health consequences, with uranium poisoning being the primary danger, are increasingly coming into focus and directly impact the safe and stable development of our country's nuclear energy industry.

[0003] Most uranium-induced injuries are due to the chemical toxicity of hexavalent uranyl ions (UO2). 2+ ) and not due to radiotoxicity. Inhaled uranium can penetrate deep into the alveoli and cause damage such as emphysema and pulmonary fibrosis, as well as trigger lung cancer. Recent studies have shown that uranium is absorbed into the body via the respiratory tract and causes lung damage through oxidative stress, pro-inflammatory effects, induction of DNA damage, promotion of apoptosis, and autophagy.

[0004] Uranium antidotes and excretion promoters are important tools for preventing and alleviating uranium poisoning. However, uranium antidotes such as DTPA have drawbacks, including high toxicity, low efficacy, and low bioavailability. The use of natural, plant-based agents is among the traditional methods for removing toxic metals from the body. Natural antioxidants found in a normal diet, such as flavonoids, lipoic acid, carotenoids, vitamins C and E, taurine, curcumin, minerals, and amino acids, can increase methylation and scavenge free reactive oxygen species (ROS), and have proven beneficial in mitigating metal toxicity. Uranium antidotes and excretion promoters are important tools for preventing and alleviating uranium poisoning. However, DTPA and other uranium antidotes have drawbacks, including high toxicity, low efficacy, and low bioavailability.

[0005] Therefore, the exploration of new natural uranium antidotes with non-toxic, anti-inflammatory and antioxidant properties remains a challenge. Subject matter of the invention

[0006] To overcome the aforementioned technical problems, this application proposes the use of soy extract for the prevention or treatment of uranium-induced lung damage and demonstrates, using animal models, that soy extract, as a natural, low-toxicity bioactive substance, possesses the ability to chelate uranium and can serve as an effective uranium antidote. This offers a novel preventive or therapeutic approach to the prevention and treatment of uranium poisoning and the mitigation of its toxicity.

[0007] In its first aspect, this application concerns the use of soy extract for the manufacture of a medicinal product: a) Improvement of pulmonary fibrosis in a uranium-exposed subject; b) Increase in uranium excretion capacity in a uranium-exposed object; c) Prevention or treatment of uranium-induced lung damage.

[0008] This application investigated the protective effect of soy extract (SE) in a mouse model of uranium-induced lung injury using scientific research. The results showed that soy extract can improve pulmonary fibrosis in uranium-exposed subjects. Treatment with SE alleviated uranium-induced pulmonary fibrosis and simultaneously reduced the expression of α-SMA, TGF-β1, and Hyp, as well as improving uranium-induced pathological changes in lung tissue. Compared to the uranium group, the SE+U group showed a significant reduction in lung uranium concentration of 18.8%. Hematoxylin and eosin staining revealed a significant reduction in the histological infiltration of inflammatory cells. These results suggest that soy extract possesses some protective effect against uranium-induced lung injury.

[0009] In some embodiments, the soy extract is obtained from soybeans using a microwave-assisted extraction process. This process involves treating water-soaked soybeans in a microwave reactor, condensing the resulting vaporized gas plasma, and recovering it to obtain the soy extract.

[0010] In some embodiments, the microwave reactor has a treatment frequency of 2000-2100 MHz and a treatment time of 2-3 minutes, preferably a treatment frequency of 2040-2060 MHz and more preferably a treatment frequency of 2045 MHz.

[0011] In this application, the active ingredients contained in soybeans are extracted using the method described above. In the extraction step, the soybeans are soaked until their water content is approximately 60%. They are then transferred to a microwave plasma reactor (i.e., a microwave reactor) to separate the active ingredients. The extract containing the active ingredients of the soybeans (i.e., soy extract) is obtained by condensation and recovery.

[0012] In some embodiments, the uranium-exposed object has developed no lung damage or uranium-induced lung damage.

[0013] In the second aspect, this application relates to a pharmaceutical composition with one or more of the uses, wherein the active ingredient of the pharmaceutical composition is a soy extract, and the uses are as follows: a) Improvement of pulmonary fibrosis in a uranium-exposed subject; b) Increase in uranium excretion capacity in a uranium-exposed object; c) Prevention or treatment of uranium-induced lung damage.

[0014] In some embodiments, the pharmaceutical composition also contains a pharmaceutically compatible carrier.

[0015] In some embodiments, the soy extract is obtained from soybeans using a microwave-assisted extraction process.

[0016] In some embodiments, the microwave-assisted extraction process includes treating water-soaked soybeans in a microwave reactor, condensing and recovering the evaporated gas plasma to obtain the soy extract.

[0017] In some embodiments, the microwave reactor has a treatment frequency of 2000-2100 MHz and a treatment time of 2-3 minutes, preferably a treatment frequency of 2040-2060 MHz and more preferably a treatment frequency of 2045 MHz.

[0018] Compared to existing technologies, this application exhibits the following technical effects: This application investigated the protective effect of soy extract (SE) in a mouse model of uranium-induced lung injury using scientific research. The results showed that soy extract can improve pulmonary fibrosis in uranium-exposed subjects. Treatment with SE alleviated uranium-induced pulmonary fibrosis and simultaneously reduced the expression of α-SMA, TGF-β1, and Hyp, as well as improving uranium-induced pathological changes in lung tissue. Compared to the uranium group, the SE+U group showed a significant reduction in lung uranium concentration of 18.8%. Hematoxylin and eosin staining revealed a significant reduction in the histological infiltration of inflammatory cells. Therefore, soy extract possesses a certain protective effect against uranium-induced lung injury. Description of the drawings Fig. Figure 1 shows the model of uranium-induced lung injury and the drug treatment scheme. Fig. Figure 2 shows the mitigating effect of soy extract (SE) on uranium-induced lung damage in rats. (A) U(VI) content in lung tissue (n=5); (B) Representative results of hematoxylin and eosin staining and pathological score of the lungs of the four groups, scale bars: 100 µm; (C) Changes in the lung coefficient of the rats (n=5); (D) Changes in body weight of the rats (n=5). The data represent the mean ± standard error of the mean (SEM) of 5 rats per group. *P < 0.05, ***P < 0.01, ****P < 0.0001, ns means not significant. Fig.Figure 3 shows that treatment with soy extract can improve pulmonary fibrosis in the uranium inhalation model. (A) Masson trichrome staining results and quantitative analysis of the fibrotic area in the uranium inhalation model; scale bar: 100 µm; (B) α-SMA levels in each rat group (n=4); (C) TGF-β1 levels in each rat group (n=4); (D) Hyp levels in each rat group (n=4). Data are given as the mean ± standard error of the mean (SEM) of 5 rats per group. *P < 0.05, **P < 0.01. Fig.Figure 4 shows the possible mechanism for predicting SE treatment of ALI. (A) GO analysis of the entire formulation; the higher the band height, the more significant; (B) Bubble plot of the KEGG enrichment analysis: the larger the bubble, the more targets are affected; the color depth represents the p-value, and the darker the red, the more significant the p-value; (C) Component-target pathway network of the main active ingredient of SE: yellow nodes represent the active compounds, red nodes the target structure, and purple nodes the pathway. Fig. Figure 5 shows the transcriptome analysis of uranium-induced lung injury in rats after SE treatment. (A) PCA score plots of the U group and the U+SE group; (B) Vulkan plot of differentially expressed genes (DEGs); blue indicates downregulation, red upregulation; (C) and (D) Enrichment bubble plots of U+SE and U+SE. Fig.Figure 6 shows the metabolomic analysis of uranium-induced lung injury in rats after SE treatment. (A) PLS-DA score plots of the metabolomic data of the U group and the U+SE group; (B) OPLS-DA score plots of the metabolomic data of the U group and the U+SE group; (C) Validation of the OPLS-DA model; (D) Heatmaps of various metabolites; (E) Volcano plot of differentially expressed genes (DMs); blue indicates downregulation, red upregulation. (F) Bubble diagram of metabolic pathways. Fig.Figure 7 shows the comprehensive multi-omics analysis of uranium-induced lung injury in rats following SE treatment. (A) Shared signaling pathways from network pharmacology and transcriptomics; (B) Bubble plot of the combined transcriptomics and targeted metabolomics analysis based on the MetaboAnalyst database; (C) Heatmap of the correlation between deg and dm; warm and cold colors represent positive and negative relationships, respectively; *P < 0.05, **P < 0.01; (D) Genes and metabolites of important SE compounds acting on drug metabolism—cytochrome P450, complement and coagulation cascades, glutathione metabolism, and IL-17 signaling pathways; yellow nodes represent drugs, red nodes genes, green nodes metabolites, and purple nodes signaling pathways. Fig. Figure 8 shows a mechanism diagram of the effect of SE on uranium-induced lung damage. Detailed description of the embodiments

[0019] The present application is explained in more detail below with reference to specific embodiments. It is understood that these embodiments serve only to illustrate the present application and are not intended to limit the scope of protection of the invention. Unless otherwise stated, the experimental methods in the following examples are generally carried out under standard conditions, the conditions described in the laboratory manual, or the conditions recommended by the manufacturer. Production example:

[0020] Soy extract was produced using a microwave-assisted process as follows: (1) Material pretreatment: Soybeans were sieved to remove dust and other impurities, washed, drained and sorted to a uniform size. They were then soaked in water until the moisture content was approximately 60%; (2) The prepared material was placed in a microwave pyrolysis reactor and treated for 2-3 minutes at a microwave frequency of 2045 MHz. The vaporized gas plasma was condensed and collected; this is the soy extract.

[0021] Soybean components are distilled or vaporized using high-frequency microwave treatment, and the soy extract is obtained by condensation and recovery. 500 g of soy extract can be obtained from 5 kg of soybeans. The pH of the soy extract is approximately 8, indicating a slightly alkaline character. The size of the small molecular clusters in the serum was determined using 17O-NMR analysis determined at 38.81 Hz. The analysis report of the soy extract, based on broad targeted metabolomics, shows that it contains 656 low-molecular-weight compounds, all below 1000 Da, and is a low-molecular-weight compound rich in various secondary metabolites of the soybean, including 90 types of phenolic acids, 90 types of amino acids and their derivatives, 90 types of alkaloids and terpenes, 82 types of organic acids, 80 types of flavonoids, 70 types of lipids, 67 types of sugars, alcohols and other substances, 51 types of nucleotides and their derivatives, 23 types of lignins and 13 types of vitamins. Example 1: Establishing a mouse model

[0022] Twenty-four ten-week-old male SD rats, weighing 440–520 g, were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. [SCXK (Jing)2021-0011]. The rats were acclimatized for one week prior to the start of the experiment, with six rats per cage. Food and water were available ad libitum throughout the experiment.

[0023] Uranyl nitrate hexahydrate (VI) was provided by the China National Nuclear Corporation (Beijing, China). A 150 mg / ml (pH 4) uranyl nitrate stock solution in double-distilled, deionized water was used for all experiments. The solution was stored at 4 °C.

[0024] Experimental group assignment: The 24 rats were randomly assigned to four groups (6 rats per group): one group that received untreated water (CON group), one group that received untreated uranium (U group), one group that received water pretreated with SE (SE group), and one group that received uranium pretreated with SE (U+SE group). The soy extracts (SE) described in the manufacturing example were used in the experiment.

[0025] Rats were anesthetized by intraperitoneal injection of sodium pentobarbital (30 mg / kg). Subsequently, rats in groups SE and U+SE received 10 ml / kg of SE orally daily. On day 7, rats in groups U and U+SE were nebulized with a uranyl nitrate solution (20 mg / kg). The control group received the same amount of purified water. The study endpoint 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 used for uranium content determination and ELISA detection. The remaining tissue was flash-frozen in liquid nitrogen for subsequent experiments. Fig. 1).

[0026] As in Fig.As shown in Figure 1, administration to the individual groups was as follows: Rats in groups SE and U+SE received soy extract (5 ml / kg, twice daily) via gastric tube, while rats in group U received the same volume of double-distilled water daily via gastric tube. On day 7 of the experiment, rats in groups U and U+SE were treated by inhalation of a uranyl nitrate solution containing uranium (20 mg / kg), while rats in group SE received the same volume of double-distilled water nebulized. Subsequently, daily administration via gastric tube was resumed. Rats in group CON received the same volume of double-distilled water at the corresponding time points in the same manner. The study endpoint was 12 days after the intervention. Example 2: Pathological diagnosis of lung tissue

[0027] The left upper lobe of the rat was fixed with 4% paraformaldehyde. After dehydration, the tissue was embedded in paraffin, deparaffinized, rehydrated, and stained with hematoxylin and eosin. The stained tissue sections were examined microscopically (Zeiss, Germany). The severity of lung tissue damage was assessed using the Szapiel score (Szapiel et al., 1979).

[0028] Fresh lower lobe tissue from the left lung was used to determine the uranium content. 0.3 g of lung tissue was digested in 10 ml of PBS using microwave digestion, and the uranium content was determined by inductively coupled plasma mass spectrometry (ICP-MS). Table 1 Operating instructions for the microwave digester Step Maximum microwave power / W Internal temperature limit / °C Temperature control / °C Climbing time / minute Holding time / minute 1 1500 200 120 5 5 2 1500 200 180 5 10

[0029] The diagnostic results are in Fig.Figure 2 shows that in the rat model of uranium inhalation, uranium could be inhaled into the lungs. The uranium content in the lungs was significantly higher in group U than in group CON ( Fig. 2A). Lung damage in group U manifested as interstitial infiltration of inflammatory cells ( Fig. 2B). Compared to group U, the uranium content in the lungs was significantly reduced by 18.8% in group SE+U, and hematoxylin and eosin staining showed a significant reduction in the histological infiltration of inflammatory cells ( Fig. 2A and Fig. 2B). Compared to the CON group, the U group had a lower lung coefficient for lung damage (expressed as relative lung weight) after uranium exposure, and treatment with SE+U showed a significant improvement ( Fig. 2C). Uranium exposure and SE treatment had no effect on the body weight of the rats ( Fig. 2D). Example 3: Masson trichrome staining for the assessment of pulmonary fibrosis

[0030] Masson trichrome staining was performed using a Masson trichrome staining kit (Solarbio, G1346, Beijing). The same lung tissue sections as described above were used. The stained tissue sections were examined microscopically (Zeiss, Germany). The degree of fibrosis was quantified using a numerical scale developed by Ashcroft et al. (1988).

[0031] The α-actin smooth muscle (α-SMA) content in lung tissue was determined using a rat α-SMA EL1SA kit. The lung tissue was homogenized in phosphate-buffered saline (PBS) and centrifuged at 1500 g for 20 minutes to clarify. The supernatant was processed according to the manufacturer's instructions. Absorption was measured at a wavelength of 450 nm. Similar to the α-SMA values, the determination was performed according to the instructions of the transforming growth factor β1 (TGF-β1) and hydroxyproline (Hyp) test kits from Nanjing Jiancheng Pharmaceutical Co., Ltd.

[0032] As in Fig. Figure 3 shows that the results of the Masson trichrome staining showed that the SE treatment significantly reduced the deposition of extracellular matrix compared to the U group ( Fig.3A). Consistent with the Masson staining, the ELISA results showed that the SE-treated rats in the U+SE group exhibited reduced levels of α-SMA, TGF-β1, and Hyp compared to uranium-exposed rats. Fig. 3B to Fig. 3D). SE treatment alleviated uranium-induced pulmonary fibrosis while simultaneously reducing the expression of α-SMA, TGF-β1 and Hyp. Example 4: Network pharmacological analysis

[0033] Screening of soybean extract (SE)-active compounds and targets: A total of 656 metabolites were detected in soybean extract using a UPLC-MS / MS platform. Twenty-two active compounds were identified from SwissADME (http: / / www.swissadme.ch / ) based on criteria such as relative abundance >0.05%, (high) gastrointestinal absorption, and pharmacodynamic activity (at least four "yes" ratings). SE-associated gene targets were determined from the SwissTarget Prediction database (http: / / www.swisstargetprediction.ch / ). Hypothetical targets with a probability >0.1 were selected.

[0034] Screening of target structures for acute respiratory distress syndrome (ALI): Two databases were used to search for therapeutic target structures for ALI: (a) the GeneCards database (https: / / www.genecards.org / ) and (b) the Online Human Mendelian Genetics Database (OMIM, http: / / omim.org / ). An alliance of AI-related gene sets was established.

[0035] Enrichment analysis: Using the KOBAS database (http: / / bioinfo.org / kobas), a gene ontology analysis (GO) and an enrichment of signaling pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed, p < 0.05. Finally, a "SE connection-target / target structure-pathway" network was created using Cytoscape 3.10.1 software.

[0036] The results are in Fig. Figure 4 illustrates this. To better understand the various molecular mechanisms of SE in the treatment of ALI, 18 compounds and 1977 disease targets were extracted from SE-UPLC-MS / MS results and associated databases. A cross-tabulation analysis identified 173 potential SE proteins for ALI treatment. Subsequently, a GO analysis of the mapping target was performed ( Fig.4A). These target structures are involved in biological processes such as the negative regulation of apoptosis, responses to exogenous stimuli, and protein phosphorylation. Cellular components were mainly enriched in membrane rafts and plasma membranes. In terms of molecular function, these target structures are mainly involved in protein kinase activity, enzyme binding, and ATP binding. KEGG results showed that these target structures are associated with the PI3K-Akt pathway, the Rap1 pathway, the T-cell receptor pathway, and the relaxin pathway ( Fig. 4B). Potential mechanism network of SE treatment in ALI ( Fig. 4C). Example 5: Transcriptome analysis

[0037] The experimental groups were CON group, U group, and U+SE group. For transcriptome sequencing, lung samples were randomly selected from three rats in each of the above-mentioned groups; for metabolome analysis, urine samples were randomly selected from five rats. 1. Extraction of total RNA(1) Discharge and lysis of the tissue sample

[0038] Comminution and digestion under liquid nitrogen: A suitable amount of tissue sample was taken and ground into powder under a protective atmosphere with liquid nitrogen, transferred to an Eppendorf tube containing 1.5 ml of TRIzol lysis buffer and left to stand for 5 minutes to ensure complete lysis of the tissue cells. (2) Sample extraction and purification ① The milled and comminuted tissue sample was centrifuged for 5 min at 4 °C and 12,000 g. The supernatant was transferred to an Eppendorf tube containing 300 µL of chloroform / isoamyl alcohol (24:1). After inverting and vigorous mixing, it was centrifuged for 8 min at 4 °C and 12,000 g. If a thick intermediate layer and a cloudy aqueous phase remained, the extraction could be repeated with a volume of chloroform / isoamyl alcohol (24:1), the volume of which corresponded to the volume of the supernatant. ② The supernatant was transferred to a new 1.5 mL centrifuge tube, isopropanol was added to 2 / 3 of the volume of the supernatant, the mixture was carefully inverted and mixed, and then stored in a refrigerator at -20 °C for at least 2 h; ③ After standing, the sample was centrifuged for 25 min at 4 °C and 17,500 g. The supernatant was discarded and the sample was washed with 0.9 ml of 75% ethanol. The sample was inverted, suspended, and precipitated, then centrifuged for 3 min at 4 °C and 17,500 g (depending on the precipitate, it may be washed again with 75% ethanol and centrifuged for 3 min at 4 °C and 17,500 g). ④ The supernatant was discarded, the sample was briefly centrifuged, and the remaining liquid was removed. It was air-dried for 3–5 minutes. The precipitate was dissolved in 20–200 µl of DEPC-treated water or RNase-free water. (3) The fully automated capillary electrophoresis device was used to determine the concentration, purity (28S / 18S) and integrity (RIN value) of the sample and to determine whether the quality of the tested sample meets the requirements for library construction and sequencing. 2. Preparation of the mRNA library(1) mRNA isolation

[0039] A specific amount of qualified total RNA sample was extracted and digested with DNase I. Oligo(dT) magnetic beads were applied to the digested total RNA sample to enrich the mRNA. (2) mRNA fragmentation:

[0040] Fragmentation reagent was added to the enriched mRNA and the reaction was carried out at a suitable temperature for a specific period of time to fragment the mRNA. (3) cDNA synthesis:

[0041] A reaction system for first-strand synthesis was prepared, and the first cDNA strand was synthesized (random primers were added to the fragmented mRNA to synthesize the first cDNA strand). A reaction system for second-strand synthesis was prepared, and the second cDNA strand was synthesized (dUTP was used instead of dTTP). Subsequently, the amplified cDNA was end-repaired and ligated with 'A' and adapters. (4) PCR reaction:

[0042] The ligation product was subjected to a PCR reaction. The uranium-labeled second-strand template was digested with the enzyme UDG. It was subsequently amplified by PCR. (5) Library audit:

[0043] In accordance with the product requirements, suitable testing methods were selected and the library underwent a quality check. (6) Circularization of PCR products:

[0044] After the PCR product was denatured into single strands, it was circularized, and a library of single-stranded circular DNA was obtained through circularization. The non-circularized linear DNA molecules were digested, and the final library was obtained. 3. Sequencing:

[0045] The single-stranded circular DNA molecules were replicated using rolling circle replication, resulting in a multi-copy DNA nanosphere (DNB). The resulting DNBs were then loaded into the mesh wells of the chip using high-density DNA nanochip technology and sequenced by combined probe-anchored polymerization (cPAS). 4. Data quality assessment:

[0046] After obtaining the raw data (raw reads) from the sequencing data, these were filtered using SOAPnuke, filtering software developed by BGI. This process removed low-quality reads (reads where the proportion of bases with a quality score below 15 exceeds 20% of the total number of bases in the reads), adapter-contaminated reads, and reads with an excessively high content (>5%) of unknown base N in the raw data. Quality-controlled clean reads were obtained, and Q20 and Q30 were calculated separately. After obtaining the clean reads, they were aligned to the reference genome sequence (species: Rattus norvegicus; source: NCBI; reference genome version: GCF_015227675.2_mRatBN7.2) using HISAT. The alignment rate (the proportion of clean reads with a unique alignment position in the reference genome) was calculated, and at the same time the quality of the alignment results of this sequencing was evaluated.

[0047] After obtaining reliable clean reads through RNA-Seq sequencing quality assessment, the filtered high-throughput sequencing data were aligned to the reference gene sequence using Bowtie2. The read count for each gene was determined. RSEM software was used to normalize this value to TPM expression levels. The Pearson correlation coefficient for all gene expression levels between any two samples was calculated to determine the correlation of gene expression between the samples. The RNA-Seq sequencing quality assessment ensured the reliability of the transcriptome sequencing results.

[0048] 5. Principal component analysis (PCA) is a cluster analysis of samples based on gene expression levels. It reflects the similarity of gene expression between groups and between samples within groups. In the PCA analysis plot, samples that are closer together show higher similarity. DESeq2 (for repeated samples) was used to identify differentially expressed genes, with p-adjust < 0.05 and a fold change > 1.5 defined as the threshold for a significant difference. GO and KEGG analyses were used for functional annotation and enrichment of signaling pathways associated with differentially expressed genes (DEGs).

[0049] PCA showed significant differences in gene expression between group U and group U+SE, as shown in Fig.Figure 5 shows that, compared to group U, gene expression in the lung tissue of rats in group U+SE showed significant changes ( Fig. 5A). Based on the results of the difference analysis, genes with Padj < 0.05 and (|Fold Change|) > 1.5 were selected as differentially expressed genes (deg). Compared to group U, a total of 67 deg were identified in group U+SE, of which 37 were upregulated and 30 downregulated. A Volcano difference plot was created based on the DEGs ( Fig. 5B). To identify potentially affected signaling pathways, KEGG pathway enrichment bubble diagrams were used ( Fig. 5C) and GO enrichment bubble diagram ( Fig.5D). KEGG pathway enrichment analysis revealed significant enrichment of DEGS in complement and coagulation pathways, metabolic pathways, the IL-17 signaling pathway, and glutathione metabolism. GO analysis showed that, with respect to biological processes (BP), DEGS is primarily associated with the negative regulation of T-cell proliferation, the negative regulation of endopeptidase activity, and apoptosis. Regarding cellular components (CC), DEGS was associated with the extracellular space / region. Molecular function (MF) analysis showed an association with serine endopeptidase inhibitor activity, endopeptidase inhibitor activity, and protease binding. This suggests that SE may influence these pathways and potentially mitigate uranium-induced lung damage in rats. Example 6: Targeted metabolomics analysis 1. Extraction of metabolites

[0050] Tissue samples were milled in 10 µL / mg 80% methanol (v / v) and incubated on ice for 20 minutes. The supernatant was collected after centrifugation at 13,000 rpm and 4 °C for 15 minutes. Finally, 4 µL of supernatant from each group was taken, mixed, and used as a quality control (QC) sample and as a reference for quality control during the analysis. 2. Ultra-high-performance liquid chromatography-mass spectrometry (UPLC-MS) analysis

[0051] For the separation and quantitative determination of the metabolites, a Waters ACQUITY UPLC I-Class Plus (Waters, USA) tandem system with a highly sensitive mass spectrometer QTRAP6500 Plus (SCIEX, USA) was used in the investigation.

[0052] Chromatography conditions: A BEH C18 chromatographic column (2.1 mm × 10 cm, 1.7 µm, Waters) was used. The column temperature was 40 °C. The mobile phase consisted of 0.1% formic acid in water (solution A) and 30% isopropanol in acetonitrile (solution B). Elution was performed with the following gradient: 0–1.00 min, 5% solution B; 1.00–5.00 min, 5–30% solution B; 5.00–9.00 min, 30–50% solution B; 9.00–11.00 min, 50–78% solution B; 11.00–13.50 min, 78–95% solution B. 13.50-14.00 min, 95-100% solution B, each at a flow rate of 0.400 ml / min; 14.00-16.00 min, 100% solution B, at a flow rate of 0.600 ml / min; 16.00-18.00 min, 5% solution B, at a flow rate of 0.400 ml / min.

[0053] Mass spectrometry conditions: For the QTRAP 6500 Plus with ESI turbo ion spray interface, the ion source parameters were set as follows: the ion source temperature was 400 °C; the ion spray voltage (IS) was 4500 V (positive mode) and -4500 V (negative mode); the pressures of the ion source gases I (GS1), II (GS2), and the curtain gas (CUR) were set to 60, 60, and 35 psi, respectively. The MRM method was operated in MRM mode and included information on the MRM parent-daughter ion pairs, the collision energy (CE), the decluster voltage (DP), and the retention time of the target metabolite.

[0054] Mass spectrometry was performed in positive and negative ionization modes, with transitions monitored using multiple reaction monitoring (MRM). MRM conversion and optimization parameters for each metabolite were determined using the G700 Targeted Metabolomics Kit (MetaboProfile, China). Data acquisition and processing were performed using Skyline software (version 21.1).

[0055] Partial Least Squares Discriminant Analysis (PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were performed using MetaboAnalyst 5.0. Differentially expressed metabolites were identified in the research based on |log2FoldChange| > 0.585, p < 0.05, and significant predictor variables (VIP) > 1. The metabolic pathways were then further analyzed using MetaboAnalyst 5.0.

[0056] A comprehensive analysis of the relationships between different omics results was conducted. First, the KEGG co-enrichment pathway was selected based on network pharmacology, transcriptomics, and metabolomics results. Subsequently, the correlation between Deg and DMs was analyzed using Pearson's correlation coefficient. The results were visualized using correlation heatmaps and network diagrams.

[0057] All quantitative data are presented as mean ± standard error (SEM). Statistical analysis was performed using GraphPad Prism 8 (San Diego, CA, USA). A one-way ANOVA was used for group comparisons. The significance level α was set at 0.05.

[0058] Through the identification of metabolites in groups U and U+SE, a total of 393 metabolites were identified in lung tissue, primarily metabolites such as amino acids and peptides, bile acids, lipids, carbohydrates, and vitamins. To investigate changes in metabolites before and after drug administration in lung-damaged rats, PLS-DA and OPLS-DA were used to analyze the data from each group.

[0059] The results are in Fig. 6 shown and demonstrated a good separation in groups U and U+SE ( Fig. 6A - Fig. 6C). A total of 39 differential metabolites (DMs) were identified, of which 9 were downregulated and 30 were upregulated ( Fig. 6D and Fig.6E). MetaboAnalyst was used to analyze metabolic pathways to further investigate the potential effects of these metabolite changes. Based on log(p) and effect sizes, potential pathways for SE therapy in uranium-induced lung injury were identified. The results are presented in the form of a bubble chart ( Fig. 6F). These pathways are mainly associated with vitamin metabolism (folate one-carbon pool, folate biosynthesis, vitamin B6 metabolism) and carbohydrate metabolism (inositol phosphate metabolism, phosphatidylinositol signaling pathway).

[0060] The results are in Fig. Figure 7 shows that the combined analysis of network pharmacology and transcriptomics revealed eight common signaling pathways, including drug metabolism cytochrome P450, the complement and coagulation cascade, glutathione metabolism, and the IL-17 signaling pathway ( Fig.7A). The combined analysis with transcriptomics and targeted metabolomics further enriched pathways such as drug metabolism-cytochrome P450 and glutathione metabolism ( Fig. 7B). Heatmaps of the deg and dm values ​​were created using Pearson correlation coefficient analysis ( Fig. 7C). Genes associated with drug metabolism—cytochrome P450, complement and coagulation cascades, glutathione metabolism, and the IL-17 signaling pathway—including Fgb, Fmo2, Gsta1, and Muc5b, were selected. Finally, a CGMP network diagram was created using Cytoscape 3.10.1 to more clearly illustrate the relationships between key SE components, target genes, and metabolites ( Fig. 7D).

[0061] This application combined network pharmacology, transcriptomics, and metabolomics, investigating the effect of SE on uranium-induced lung injury ( Fig.8) The lung-protective effect of SE was confirmed in a rat model of uranium-induced lung injury. To further explore the potential protective mechanism of SE in the lungs, the results showed that SE may exert its protective effect by inhibiting the expression of key genes associated with inflammatory and immune regulatory processes. Furthermore, SE can upregulate antioxidant enzymes, thereby mitigating uranium-induced oxidative stress. This application elucidates the potential pharmacological mechanism by which SE alleviates uranium-induced lung injury and provides valuable insights for the therapeutic use of SE in lung diseases and related conditions.

[0062] This embodiment serves only to illustrate this application and is not intended to limit the scope of protection of this application. After reading this description, those skilled in the art may, if necessary, make modifications to this embodiment without incurring an inventive step. Provided these modifications are within the scope of the claims of this application, they are protected under patent law. QUOTES INCLUDED IN THE DESCRIPTION

[0000] This list of documents cited by the applicant was automatically generated and is included solely for the reader's convenience. The list is not part of the German patent or utility model application. The DPMA accepts no liability for any errors or omissions. Cited non-patent literature

[0000] Beijing Vital River Laboratory Animal Technology Co., Ltd. [SCXK (Jing)2021-0011

[0022] Szapiel et al., 1979

[0027]

Claims

[1] Use of soy extract for the manufacture of a medicinal product, characterized by that the medicinal product has one or more of the following uses: a) Improvement of pulmonary fibrosis in a uranium-exposed subject; b) Increase in uranium excretion capacity in a uranium-exposed object; c) Prevention or treatment of uranium-induced lung damage. [2] Use according to claim 1, characterized by that the soy extract is obtained from soybeans using a microwave-assisted extraction process. [3] Use according to claim 2, characterized by , that the microwave-assisted extraction process includes treating water-soaked soybeans in a microwave reactor, condensing and recovering the evaporated gas plasma to obtain the soy extract. [4] Use according to claim 3, characterized bythat the treatment frequency of the microwave reactor is 2000-2100 MHz and the treatment time is 2-3 minutes. [5] Use according to claim 1, characterized by that the uranium-exposed object has developed no lung damage or uranium-induced lung damage. [6] Pharmaceutical composition with one or more of the uses, characterized by , that the active ingredient of the pharmaceutical composition is a soy extract, the uses being as follows: a) Improvement of pulmonary fibrosis in a uranium-exposed subject; b) Increase in uranium excretion capacity in a uranium-exposed object; c) Prevention or treatment of uranium-induced lung damage. [7] Pharmaceutical composition according to claim 6, characterized by that the pharmaceutical composition also contains a pharmaceutically compatible carrier. [8] Pharmaceutical composition according to claim 7, characterized bythat the soy extract is obtained from soybeans using a microwave-assisted extraction process. [9] Pharmaceutical composition according to claim 8, characterized by , that the microwave-assisted extraction process includes treating water-soaked soybeans in a microwave reactor, condensing and recovering the evaporated gas plasma to obtain the soy extract. [10] Pharmaceutical composition according to claim 9, characterized by that the treatment frequency of the microwave reactor is 2000-2100 MHz and the treatment time is 2-3 min.

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