Use of soybean extract in prevention or treatment of uranium-induced kidney injury

Abstract

A use of a soybean extract in the preparation of a drug. The drug has one or more of the following uses: a) reducing the kidney coefficient of an individual exposed to uranium; b) improving the uranium excretion capacity of an individual exposed to uranium; and c) preventing or treating uranium-induced kidney injury. The soybean extract is prepared from soybeans by means of a microwave-assisted extraction method.

Description

Application of soybean extract in prevention or treatment of uranium-induced kidney injury TECHNICAL FIELD

[0001] The application belongs to the technical field of kidney injury treatment, and relates to application of soybean extract in prevention or treatment of uranium-induced kidney injury. BACKGROUND

[0002] Uranium has both radioactivity and chemical toxicity, and mainly exists in the form of hexavalent uranyl ion (UO2 2+ ) in the human body. Uranium can enter the human body through the gastrointestinal tract, respiratory tract and skin, and is mainly deposited in the kidney, liver and bone and is difficult to be discharged. Long-term internal irradiation of the nuclide and its chemical toxicity cause serious organ damage, such as kidney failure, liver dysfunction or osteosarcoma, and even death. In living organisms, hexavalent uranyl ion (UO2 2+ ) can be effectively complexed with bicarbonate, citrate and protein. Uranium in the blood reaches the kidney in the form of a low-molecular-weight complex, is filtered through the glomerular membrane together with water, and is then concentrated in the cortex and juxtaglomerular region and combined to the anion site of the brush border membrane of the proximal tubule. Uranium is taken up by the renal tubular epithelial cells through endocytosis and type IIa sodium-phosphorus co-transporter (Napi-IIa), and the uranium not taken up is discharged from the body through urine. Therefore, the kidney is the most sensitive target organ of uranium. Animal experiment studies have shown that a single dose of uranium exposure >2 mg / kg has nephrotoxicity, which is sufficient to change the biochemical parameters of kidney function (blood urea nitrogen, serum creatinine, N-acetylglucosaminidase and alkaline phosphatase).

[0003] At present, the prevention and treatment of uranium poisoning is still a difficult point of research at home and abroad. Uranium chelators and antioxidants are important means for preventing and treating uranium poisoning and alleviating toxicity. The accumulation of uranium in the whole body tissues and organs has long-term harmfulness, and the accumulation of uranium in the body can be reduced by using chelators to increase the elimination of uranium. The formation of soluble chelators can reduce the deposition of uranium in organs and accelerate its discharge from kidney tissues. At present, the main uranium chelators include sodium bicarbonate, ethylenediaminetetraacetic acid (EDTA), DTPA, catechols (CAM), hydroxypyridinones (HOPO), organic phosphates, calixarenes and the like. In addition to chelating uranium, reducing the damage of uranium to tissues and cells is also an important treatment method for detoxification of uranium. At present, it has been found that some drugs can protect the body from uranium invasion. Studies have shown that Zn, metallothionein and hydrogen sulfide may be beneficial to the prevention and treatment of uranium-induced kidney toxicity, but more research is needed before clinical application.

[0004] Qualified uranium chelators should be able to compete with proteins and other competitive binders in vivo to bind uranium, form stable excretable complexes with uranium, and have the characteristics of low toxicity and small side effects. At present, a variety of uranium chelates have been synthesized, but most of them have poor tissue specificity and high toxicity, and many are still in the experimental research stage. Therefore, it is urgent to seek a uranium antidote with biological activities such as non-toxicity, anti-inflammatory, and anti-oxidation. SUMMARY

[0005] In order to overcome the above technical problems, the present application provides an application of a soybean extract in preventing or treating uranium-induced kidney damage, and through an animal model, it is proved that the soybean extract as a natural low-toxicity bioactive substance has the ability to chelate 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.

[0006] In a first aspect, the present application provides an application of a soybean extract in preparing a drug, which has one or more of the following purposes: a) reducing the kidney coefficient of uranium exposed persons; b) improving the uranium excretion capacity of uranium exposed persons; c) preventing or treating uranium-induced kidney damage.

[0007] In the present application, the protective effect of soybean extract on a rat model of uranium-induced kidney damage is analyzed, and the research results show that the soybean extract can reduce the kidney coefficient of uranium exposed rats, improve the histopathological changes caused by uranium in the kidney tissue, and at the same time reduce the expression levels of serum creatinine, urea, total protein in urine, KIM-1, and NGAL, and also has a significant uranium excretion capacity. The above results show that the soybean extract has a certain protective effect on uranium-induced kidney damage.

[0008] In certain embodiments, the soybean extract is prepared from soybeans by a microwave-assisted extraction method. The microwave-assisted extraction method specifically comprises placing water-soaked soybeans in a microwave reaction kettle for treatment, and condensing and recovering the gasified gaseous plasma, to obtain the soybean extract.

[0009] In certain embodiments, the treatment frequency of the microwave reaction kettle is 2000-2100 MHz, the treatment time is 2-3 min, preferably the treatment frequency is 2040-2060 MHz, and more preferably the treatment frequency is 2045 MHz.

[0010] In the present application, the active ingredients in soybeans are extracted by the above-mentioned method. In the extraction step, the soybeans are soaked to have a water content of about 60%, and then are transferred into a microwave plasma effect reaction kettle (i.e., a microwave reaction kettle) for effective separation of active ingredients, and the soybean active ingredient extract (i.e., the soybean extract) is obtained by condensation and recovery.

[0011] In certain embodiments, the uranium exposed person has not developed or has developed uranium-induced kidney damage.

[0012] In a second aspect, the present application also provides a pharmaceutical composition having one or more uses, wherein the active ingredient of the pharmaceutical composition is a soybean extract, and the uses are a), reducing the kidney coefficient of a uranium exposed subject; b), improving the uranium excretion capacity of a uranium exposed subject; c), preventing or treating uranium induced kidney injury.

[0013] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0014] In some embodiments, the soybean extract is prepared from soybean by a microwave-assisted extraction method.

[0015] In some embodiments, the microwave-assisted extraction method specifically comprises placing water-soaked soybean into a microwave reactor for treatment, and condensing and recovering the gasified gaseous plasma to obtain the soybean extract.

[0016] 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.

[0017] In a third aspect, the present application also provides a use of a marker in evaluating the efficacy of a drug for treating uranium induced kidney injury or in screening a drug for treating uranium induced kidney injury, wherein the marker is selected from one or more of a target gene or a urine sample metabolite, and the target gene is C3, Itgb2, Cx3cr1, Ackr3, Col4a3, Lama1, Aldh3b1, Mthfd2 or Kcnq1, and the urine sample metabolite is 5-hydroxylysine, alanylglutamine, aspartate, glycylglycine or phosphoserine.

[0018] In the present application, the potential protective mechanism of soybean extract on uranium induced kidney injury is explored by analyzing the rat kidney transcriptomic and urine metabolomic data. The results show that nine significantly changed genes are screened in the kidney, and five differential metabolites are significantly regulated in the urine. Among them, the C3, Itgb2, Cx3cr1, Ackr3 genes and alanylglutamine are involved in inflammation and immune regulation related pathways such as complement and coagulation cascade, leukocyte transendothelial migration, chemokine signaling pathway; Col4a3, Lama1 genes and 5-hydroxylysine are involved in fibrosis related pathways such as ECM-receptor interaction, relaxin signaling pathway; Aldh3b1, Mthfd2 genes, aspartate, glycylglycine and phosphoserine are involved in oxidative stress related metabolic pathways. In summary, soybean extract may exert its protective effect on the kidney by changing the expression of key genes involved in inflammation, oxidative stress, fibrosis and immune regulation related pathways.

[0019] Compared with the prior art, the application has the following technical effects,

[0020] 1) The application provides a uranium antidote with biological activities such as non-toxicity, anti-inflammatory and anti-oxidation, i.e., soybean extract, and the protective effect of the soybean extract on a rat model of uranium-induced kidney injury is analyzed through research, and the research results show that the soybean extract can reduce the kidney coefficient of uranium-exposed rats, improve the histopathological changes of kidney tissue caused by uranium, and reduce the expression levels of serum creatinine, urea, total protein in urine, KIM-1 and NGAL, and also has significant uranium excretion capacity, thereby proving that the soybean extract has a certain protective effect on uranium-induced kidney injury.

[0021] 2) The application analyzes the rat kidney transcriptome and urine metabolome data to explore the potential protective mechanism of the soybean extract on uranium-induced kidney injury, and provides a marker composition related to the protective effect of the soybean extract on the kidney, including C3, Itgb2, Cx3cr1, Ackr3, Col4a3, Lama1, Aldh3b1, Mthfd2 or Kcnq1 genes, and urine metabolites: 5-hydroxylysine, alanyl glutamine, aspartic acid, glycyl glycine and phosphoserine; the marker composition can be used to effectively evaluate the curative effect of a drug for treating uranium-induced kidney injury and improve the efficiency of screening a drug for treating uranium-induced kidney injury. BRIEF DESCRIPTION OF DRAWINGS

[0022] FIG. 1 is a schematic diagram of experimental grouping and treatment.

[0023] FIG. 2 is the effect of soybean extract on the body weight and kidney coefficient of uranium-exposed rats. (a) Changes in the body weight of rats in each experimental group at different times; (b) Changes in the kidney coefficient of rats in each experimental group.

[0024] FIG. 3 is the pathological changes in the kidney of rats after uranium exposure and soybean extract treatment.

[0025] FIG. 4 is the change level of serum Crea and Urea of rats after uranium exposure and soybean extract treatment. (a) Serum Crea level; (b) Serum Urea level.

[0026] FIG. 5 is the expression level of KIM-1, NGAL and total protein in the urine of rats after uranium exposure and soybean extract treatment. (a) Expression level of KIM-1 protein in urine; (b) Expression level of NGAL protein in urine; (c) Expression level of total protein in urine.

[0027] FIG. 6 is the change level of kidney uranium, urine uranium and blood uranium of rats after uranium exposure and soybean extract treatment. (a) Kidney uranium level; (b) Urine uranium level; (c) Blood uranium level, *P<0.05, ****P<0.0001.

[0028] Figure 7 is a principal component analysis (PCA) plot.

[0029] Figure 8 is a differential expression gene screening plot of each experimental group. (a) volcano plot of differential genes between U group and CON group; (b) volcano plot of differential genes between U+SE group and U group; (c) heat map of expression level of co-expressed differential genes in CON group, U group and U+SE group.

[0030] Figure 9 is a GO functional annotation plot of differential expression genes.

[0031] Figure 10 is a KEGG pathway enrichment analysis plot of differential expression genes.

[0032] Figure 11 is a PLS-DA model plot between different experimental groups.

[0033] Figure 12 is an OPLS-DA model and model permutation test plot between different experimental groups. (a) OPLS-DA model plot between CON group and U group; (b) OPLS-DA model permutation test plot between CON group and U group; (c) OPLS-DA model plot between U group and U+SE group; (d) OPLS-DA model permutation test plot between U group and U+SE group.

[0034] Figure 13 is a volcano plot of differential metabolites between different experimental groups. (a) volcano plot of differential metabolites between CON group and U group; (b) volcano plot of differential metabolites between U group and U+SE group.

[0035] Figure 14 is a common differential metabolite between U / CON group and U+SE / U group. (a) common differential metabolite Wayne plot; (b) common differential metabolite fold change.

[0036] Figure 15 is a KEGG pathway enrichment plot of differential metabolites between different experimental groups. (a) pathway enrichment bubble plot of differential metabolites between U group and CON group; (b) pathway enrichment bubble plot of differential metabolites between U+SE group and U group.

[0037] Figure 16 is a heat map of correlation analysis of co-expressed differential genes and differential metabolites between different experimental groups. DETAILED DESCRIPTION

[0038] The present application will be further described with reference to the following examples. It should be understood that these examples are intended to illustrate the application and are not intended to limit the scope of the application. Unless otherwise indicated, the experimental procedures in the following examples were carried out in accordance with conventional conditions, conditions described in laboratory manuals, or conditions recommended by the manufacturer.

[0039] Preparation Example 1

[0040] The soybean extract was prepared by a microwave-assisted method, and the preparation method was as follows,

[0041] (1) Pretreatment of materials: Screen the soybeans to remove impurities such as dust, wash and drain them, sort them to the same size, and soak them in water until the water content is about 60%.

[0042] (2) Put the prepared materials into the microwave cracking reactor, and treat them at a microwave frequency of 2045 MHz for 2 - 3 minutes. Condense and recover the gaseous plasma vaporized, which is the soybean extract.

[0043] Substances in soybeans are distilled or vaporized 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 O - NMR detection shows that the size of the small - molecule clusters in the essence liquid is 38.81 HZ. The broad - targeted metabolome detection report of the soybean extract shows that it contains 656 small - molecule compounds, all of which are small - molecule compounds below 1000 Da, rich in small - molecule compounds of various secondary metabolites in soybeans. Among them, there are 90 phenolic acids, 90 amino acids and their derivatives, 90 alkaloids and terpenoids, 82 organic acids, 80 flavonoids, 70 lipids, 67 sugars and alcohols and other types, 51 nucleotides and their derivatives, 23 lignins, and 13 vitamins.

[0044] Establishment of the mouse model in Example 1

[0045] Select 24 male SD rats at 10 weeks of age, with a body weight of 440 - 520 g, provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. [SCXK(Beijing)2021 - 0011]. Adaptively raise them for 1 week before the experiment, with 6 rats in each cage. During the experiment, food and water are freely available.

[0046] Take 15 g of uranyl nitrate powder, add 100 mL of double - distilled water, heat slightly until completely dissolved, prepare a solution of 150 mg / mL, adjust the pH = 3 after preparation, and store it at 4°C for later use. In this experiment, an NE - C25S compressed atomizing inhaler is used for exposure.

[0047] Experimental grouping: Randomly divide the 24 SD rats into 4 groups, namely the blank control group (CON), the control group of soybean extract (prepared in Example 1) (SE), the uranium poisoning group (U), and the treatment group of soybean extract (prepared in Example 1) (U + SE).

[0048] As shown in Figure 1, the administration methods of each group are as follows: the rats in the SE group and the U+SE group are given soybean extract (5 mL / kg, 2 times / day) by gavage, and the rats in the U group are given the same volume of double distilled water by gavage. On the 7th day of the experiment, the rats in the U group and the U+SE group are exposed to uranium by inhaling a solution of uranyl nitrate (20 mg / kg), and the rats in the SE group inhale the same volume of double distilled water. Subsequently, the gavage administration is continued every day. The rats in the CON group are given the same volume of double distilled water by the same method at the corresponding time points. The endpoint of the study is 12 days after the intervention.

[0049] Example 2 Determination of the kidney coefficient

[0050] The body weight of the rats in each group is recorded on the 1st, 4th, 8th, and 12th days of the experiment.

[0051] After the rats are sacrificed under sodium pentobarbital anesthesia, all macroscopic changes are recorded, the rat kidneys are dissected and weighed. A portion is fixed in 4% paraformaldehyde and stored at room temperature; another 50 mg of the rat kidney is taken and placed in a cryovial, and 5-10 times the volume of RNAlater solution is poured in, which is stored at 4°C overnight (to allow the solution to fully penetrate the tissue) and stored at -80°C; the remaining kidney is placed in a cryovial and stored at -80°C. The kidney coefficient = kidney weight ÷ rat body weight x 100.

[0052] The body weight is determined and the kidney coefficient is calculated, and the results are shown in Figure 2. The body weight change trend of the SE group is consistent with that of the CON group, indicating that soybean extract has no effect on the body weight of rats. After 7 days of exposure, the body weight of the rats in the U group and the U+SE group decreases, and the body weight decrease trend of the rats in the U+SE group is slightly slower than that of the rats in the U group, but there is no significant difference.

[0053] The kidney coefficient of the rats in the U group is significantly higher than that of the CON group, with a significant difference (P<0.0001). Compared with the U group, the kidney coefficient of the rats in the U+SE group is significantly reduced (P<0.05).

[0054] The results of the determination show that soybean extract can reduce the kidney coefficient of rats exposed to uranium.

[0055] Example 3 Kidney pathological examination (HE staining)

[0056] The rat kidney tissue is fixed with 4% paraformaldehyde. After dehydration, the tissue is embedded in paraffin. A 5 mm section obtained by a microtome is placed on a glass slide coated with paraffin, and is deparaffinized with xylene, ethanol, and water, and then stained with hematoxylin and eosin, and finally mounted with neutral resin. The morphology of the stained tissue is observed under a microscope.

[0057] The rat kidney histopathology examination as shown in Figure 3, the CON group of rats kidney tissue structure is normal, no obvious lesions. U group of rats kidney can see the skin medulla of renal tubular epithelial cell necrosis, exfoliation, interstitial inflammatory cell infiltration, tube cavity can see homogeneous eosinophilic material. U+SE group of kidney injury degree is reduced. Kidney injury score shows that the soybean extract intervention treatment significantly improved the rat uranium-induced kidney injury.

[0058] Example 4 Rat serum Crea and Urea detection results

[0059] In this experiment, the abdominal aorta blood was collected, and the rats were anesthetized with sodium pentobarbital and the abdominal aorta blood was collected.

[0060] Serum collection: 5mL of blood was collected with a yellow head tube containing separation glue / coagulant, and was vertically placed at room temperature for 10-20 minutes. After the blood was completely coagulated, it was placed in a centrifuge at 4°C, 3000r / min for 15min. After centrifugation, the supernatant was collected and stored at-80°C.

[0061] Whole blood collection: 2mL of blood was collected with an EDTA anticoagulant purple head tube and stored at-20°C.

[0062] 2mL of serum was used to determine the content of serum Crea and Urea by biochemical analysis.

[0063] As shown in Figure 4, after exposure to uranium, compared with the CON group, the serum Crea and Urea levels of the U group of rats were significantly higher (P<0.0001). Compared with the U group, the serum Crea and Urea levels of the U+SE group of rats were significantly reduced (P<0.05).

[0064] Example 5 Rat urinary kidney injury marker KIM-1, NGAL and total protein expression levels

[0065] On the 11th day of the experiment, 24-hour urine samples of all rats were collected, and 6mL of urine samples were added with 120μL of concentrated nitric acid and stored at 4°C. The rest of the urine samples were centrifuged at 4°C, 3000r / min for 15min, and the supernatant was collected and stored at-80°C after centrifugation.

[0066] The levels of KIM-1, NGAL and total protein in urine were detected by enzyme-linked immunosorbent assay (ELISA). The test steps were operated according to the reagent instruction: the capture antibody was coated on the enzyme-labeled plate, the target protein in the sample and standard was captured, the biotinylated detection antibody was combined with the target protein, the SABC complex was combined with the biotinylated detection antibody to form an immune complex, and after adding TMB color developing solution, if there is target protein in the reaction well, it will show blue color, and after adding stop solution, it will turn yellow. During the detection process, the free components are all washed away, and the OD value is measured at 450 nm by an enzyme-labeled instrument.

[0067] The detection results are shown in Figure 5. Compared with the U group, the levels of KIM-1, NGAL and TP in the urine of the U+SE group rats were significantly reduced.

[0068] Example 6: Determination of kidney uranium content

[0069] The inductively coupled plasma-mass spectrometry (ICP-MS) was used to detect the uranium content. <0.5 g of organ tissue was weighed into a digestion tank, 6 mL of concentrated nitric acid was added, and pre-digestion was performed on the acid chasing plate, and the acid was chased at 130℃ for 20 min. Then the digestion tank was tightened, and after being sealed, it was placed in a microwave digestion instrument for digestion (Table 1). After digestion was completed, the digestion tank was loosened, and the acid was chased on the acid chasing plate at 160℃ until the remaining acid in the digestion tank was less than 1 mL, and then distilled water was used to make up to 10 mL. Finally, ICP-MS was used to determine the uranium content.

[0070] Table 1: Working program of microwave digestion instrument

[0071] After acute exposure to uranium, a high concentration of uranium accumulated in the kidney. The determination results are shown in Figure 6. Under normal circumstances, the kidney contains only a trace amount of uranium, but after acute uranium exposure, the kidney uranium level will increase significantly. Compared with the U group, the kidney uranium level of the U+SE group rats was significantly reduced (P<0.05); the urine uranium and blood uranium contents showed a downward trend, but there was no significant difference.

[0072] Example 7: Transcriptomic analysis

[0073] The CON group, U group and U+SE group were selected for the experiment. The kidneys of 3 rats randomly selected from each of the above groups were used for transcriptome sequencing; and the urine samples of 5 rats randomly selected were used for metabolome detection.

[0074] 1. Extraction of total RNA

[0075] (1) Tissue sample crushing and lysis

[0076] Liquid nitrogen grinding and crushing: Take an appropriate amount of tissue sample under the protection of liquid nitrogen to grind the tissue sample into powder, and transfer it into an EP tube containing 1.5 mL of TRIzol lysis solution. Place it quietly for 5 min to allow the tissue cells to be fully lysed.

[0077] (2) Sample extraction and purification

[0078] ① Centrifuge the ground and crushed tissue sample at 4°C and 12000g for 5 min. Transfer the supernatant to an EP tube containing 300 μL of chloroform / isoamyl alcohol (24:1). After vigorous shaking, 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).

[0079] 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 in a -20°C refrigerator for more than 2 h.

[0080] ③ After standing, centrifuge at 4°C and 17500g for 25 min. Discard the supernatant and wash the precipitate with 0.9 mL of 75% ethanol. Invert the tube to suspend the precipitate, centrifuge at 4°C and 17500g for 3 min. If necessary, wash again with 75% ethanol, centrifuge at 4°C and 17500g for 3 min.

[0081] ④ Discard the supernatant, briefly centrifuge and absorb the residual liquid, air dry for 3-5 min, and dissolve the precipitate with 20-200 μL of DEPC water or RNase-free water.

[0082] (3) Use a full-automatic capillary electrophoresis instrument to detect the concentration, purity (28S / 18S), and integrity (RIN value) to determine whether the sample quality meets the requirements for library construction and sequencing.

[0083] 2. mRNA library preparation

[0084] (1) mRNA isolation

[0085] Take a certain amount of the qualified Total RNA sample for DNase I digestion. Digest the Total RNA sample with Oligo(dT) magnetic beads to enrich mRNA.

[0086] (2) mRNA fragmentation

[0087] Add fragmentation reagent to the enriched mRNA, react at an appropriate temperature for a certain time, and fragment the mRNA.

[0088] (3) cDNA synthesis

[0089] Preparation of a single-strand synthesis reaction system, synthesis of a single-strand cDNA (fragmented mRNA is added to random primers for cDNA single-strand synthesis), preparation of a double-strand synthesis reaction system, synthesis of a double-strand cDNA (use dUTP instead of dTTP), and then terminal repair and "A" addition and linker ligation are performed on the amplified cDNA.

[0090] (4) PCR reaction

[0091] PCR reaction is performed on the ligation product, and UDG enzyme is used to digest the U-labeled second strand template, and then PCR amplification is performed.

[0092] (5) Library detection

[0093] According to the product requirements, the corresponding detection method is selected for quality inspection of the library.

[0094] (6) PCR product circularization

[0095] After denaturation of the PCR product into a single strand, circularization is performed, and a single-stranded circular DNA library is obtained by circularization. After digestion of the linear DNA molecules that have not been circularized, the final library is obtained.

[0096] 3. Sequencing

[0097] Single-stranded circular DNA molecules form a DNA nanoball (DNB) containing multiple copies through rolling circle replication. The obtained DNBs are added to the mesh-like small holes on the chip using high-density DNA nanochip technology, and sequencing is performed through combined probe anchor polymerization technology (cPAS).

[0098] 4. Data quality assessment

[0099] After obtaining the raw reads of the sequencing data, the filtering software SOAPnuke independently developed by Huada is used for filtering, removing low-quality reads (reads with a proportion of bases with a quality value less than 15 accounting for more than 20% of the total number of bases), adapter contamination, and reads with too high content of unknown bases N (> 5%), obtaining clean reads after quality control, and calculating Q20 and Q30. 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 clean reads with a unique alignment position on the reference genome), and evaluate the quality of the alignment results of this sequencing.

[0100] After obtaining reliable clean reads by RNA-seq sequencing quality assessment, the filtered high-throughput sequencing data was aligned to the reference gene sequence using Bowtie2 to obtain the number of reads aligned to each gene (reads count value), and RSEM software was used to standardize it to TPM expression. By calculating the Pearson correlation coefficient of all gene expressions between each two samples, the correlation of gene expression between samples was reflected. The reliability of the transcriptome sequencing results was ensured by RNA-seq sequencing quality assessment.

[0101] Principal component analysis (PCA) is a clustering analysis based on gene expression of samples, 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.

[0102] 5、Differential gene screening: The results of principal component analysis (PCA) are shown in Figure 7. The data of CON group, U group and U+SE group are obviously separated, indicating that the gene expression of the three experimental groups has changed significantly. The differential genes were screened with the criteria of |FoldChange|>1.5, P.adj<0.05. DESeq2 software was used to analyze the differential genes between different samples. Compared with the CON group, 6216 differentially expressed genes were screened in the U group, including 3187 up-regulated genes and 3029 down-regulated genes (a in Figure 8); compared with the U group, 96 differentially expressed genes were screened in the U+SE group, including 46 up-regulated genes and 50 down-regulated genes (b in Figure 8). To further clarify the main target genes of soybean extract for protecting the kidney of rats from uranium damage, 51 common differentially expressed genes were screened in the three experimental groups (c in Figure 8).

[0103] 6、GO function annotation and enrichment analysis of differentially expressed genes: GO function annotation and enrichment mainly analyze the biological process (BP) in which the gene is involved, the cell component (CC) in which the gene is located, and the molecular function (MF) executed by the gene.

[0104] The GO function annotation analysis of the differentially expressed genes of the CON group, the U group and the U+SE group was performed, and the results are shown in Figure 9. The biological processes (BP) of the differentially expressed genes were mainly related to cell surface receptor signaling pathway, cell adhesion, cell-cell adhesion, transmembrane transport, positive regulation of angiogenesis, positive regulation of cytosolic calcium ion concentration, positive regulation of protein kinase B signaling, response to estradiol, negative regulation of endopeptidase activity, glutathione biosynthetic process, etc.; the cell components (CC) were mainly related to apical plasma membrane, membrane raft, basolateral plasma membrane, integral component of plasma membrane, cell-cell junction, etc.; and the molecular functions (MF) were mainly related to glucose:sodium symporter activity, glutamate-cysteine ligase activity, etc.

[0105] 7、KEGG pathway enrichment analysis of differentially expressed genes: KEGG pathway enrichment analysis was performed on the differentially expressed genes of the CON group, the U group and the U+SE group, and the results are shown in Figure 10. The results showed that the differential genes of the CON group and the U group were enriched in 172 KEGG pathways, and the differential genes of the U group and the U+SE group were enriched in 49 pathways. The intersection of the KEGG pathways of the two comparison groups obtained 27 commonly enriched pathways (P-value < 0.05), mainly involving metabolic pathways, cysteine and methionine metabolism, glutathione metabolism, bile secretion, ECM-receptor interaction, complement and coagulation cascades, protein digestion and absorption, ferroptosis, sphingolipid metabolism, leukocyte transendothelial migration, chemokine signaling pathway, Fc gamma R-mediated phagocytosis, relaxin signaling pathway, Rap1 signaling pathway, etc.

[0106] Example 8 Metabolomics detection

[0107] 1、Extraction of metabolites

[0108] (1) Take 20 μL of sample and 20 μL of standard, add 120 μL of sample release agent, shake at 1200 rpm for 30 min, centrifuge at 18000 g and 4°C for 30 min;

[0109] (2) After centrifugation, transfer 30 μL of supernatant to a 96-well plate, add 20 μL of derivatization reagent and 20 μL of EDC working solution;

[0110] (3) Cover with aluminum film, place the 96-well plate in a constant temperature shaker, react at 40°C, 1200 rpm for 60 min;

[0111] (4) After the oscillation is completed, 4000g, 4℃ centrifugation for 5min, take 30μL and add to a new 96-well plate, add 90μL sample diluent to each well, mix at 600rpm for 10min;

[0112] (5) 4000g, 4℃ centrifugation for 30min, seal the film, and prepare for machine.

[0113] 2. Ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) analysis

[0114] In this experiment, Waters ACQUITY UPLC I-Class Plus (Waters, USA) was used in series with QTRAP6500 Plus high-sensitivity mass spectrometer (SCIEX, USA) to separate and quantitatively detect metabolites.

[0115] Chromatographic conditions: The chromatographic column used was BEH C18 (2.1mm x 10cm, 1.7um, waters), and the column temperature was 40℃. The mobile phase was 0.1% formic acid water (A liquid) and 30% isopropanol acetonitrile (B liquid). The following gradient was used for elution: 0-1.00min, 5%B liquid; 1.00-5.00min, 5%-30%B liquid; 5.00-9.00min, 30%-50%B liquid; 9.00-11.00min, 50%-78%B liquid, 11.00-13.50min, 78%-95%B liquid, 13.5-14.00min, 95%-100%B liquid, above flow rate 0.400mL / min; 14.00-16.00min, 100%B liquid, flow rate 0.600mL / min; 16.00-18.00min, 5%B liquid, flow rate 0.400mL / min.

[0116] Mass spectrometry conditions: For QTRAP 6500 Plus equipped with ESI Turbo ion spray interface, the ion source parameters are set as follows: ion source temperature: 400℃; ion spray voltage (IS): 4500V (positive mode) and -4500V (negative mode); ion source gas I (GS1), gas II (GS2) and curtain gas (CUR) are set to 60, 60 and 35psi respectively. In MRM mode, set the MRM method, which contains the MRM parent-daughter ion pair information of the target metabolite, collision energy (CE) and declustering voltage (DP) and retention time.

[0117] 3. Sample quality control analysis

[0118] During the actual sample mass spectrometry process, a certain number of quality control (QC) samples will be interspersed. The repeatability of QC sample detection is used to evaluate the data quality. In this study, the extracted ion chromatogram (XIC) of the QC sample and the correlation of the QC sample were mainly selected for sample quality control analysis. The extracted ion chromatogram is a graph obtained by continuously depicting the intensity of the target ion in the mass spectrum at each time point. The QC samples in the data summary table were selected, and the Spearman correlation coefficient was calculated according to the quantitative value of the QC sample. The higher the correlation of the QC sample (R2 is closer to 1), the smaller the system error, the better the repeatability of the experiment, and the higher the data quality.

[0119] The original mass spectrometry data was extracted and identified by the quantitative software skyline (version: v.21.1.0.146) to obtain the peak area and identification results of the metabolites. The meta X software was used for further preprocessing of the exported results to obtain compounds and quantitative values that can be used for formal analysis. The identification results of the metabolites were annotated by KEGG and HMDB databases, including KEGG ID, HMDB ID, category, and participation in KEGG metabolic pathways.

[0120] 4. PLS-DA and OPLS-DA analysis:

[0121] Partial least squares discriminant analysis (PLS-DA) is a multivariate statistical analysis method for discriminant analysis, which can establish a relationship model between metabolite expression and sample category to realize the prediction of sample category. In this study, PLS-DA was used to analyze the metabolic profiles of CON group, U group and U+SE group rat urine samples. From the 2D scatter plot of PLS-DA (Figure 11), it can be seen that the samples of each group are within the 95% confidence interval range, indicating that the sample accumulation degree of each group is high and the sample is stable. After uranium exposure and soybean extract treatment, the metabolic profiles of the three experimental groups were obviously separated, indicating that the rat urine metabolites changed significantly after uranium exposure and soybean extract treatment.

[0122] In order to further analyze the changes of rat urine metabolites after uranium exposure and soybean extract treatment, and better distinguish the differences between groups, this study conducted orthogonal partial least squares discriminant analysis (OPLS-DA) on the CON group, U group and U+SE group. The OPLS-DA model and model permutation test between different experimental groups are shown in Table 2 and Figure 12.

[0123] Table 2 OPLS-DA model parameter table

[0124] Note: ① Model parameter R2Y (cum): the explanatory rate of the classification matrix (Y matrix); ② Model parameter Q2 (cum): the predictive ability of the model; ③ The closer R2Y and Q2 are to 1, the more stable and reliable the model is. Generally, Q2 is higher than 0.5, indicating that the prediction effect of the model is better. ④ Model validation: The intercept of R2 and the intercept of Q2 are the intercepts of the regression straight line Y axis when the response ranking test is performed. Generally, the intercept of Q2 is less than 0.

[0125] In the OPLS-DA model, the metabolic profiles of the CON group and the U group, and the U group and the U+SE group were significantly separated, and the model parameters R2Y were all greater than 0.95, and Q2 were all greater than 0.5, indicating that the model was stable and had good prediction effect. In the OPLS-DA model validation, the Q2 intercepts obtained by the CON group and the U group, and the U+SE group and the U group in the permutation test were less than the R2 intercepts of the model, and a red dotted line inclined upward could be seen in the figure, and the Q2 intercepts were all less than 0, indicating that the OPLS-DA model was good and had no overfitting, and the established model was stable and effective.

[0126] 5. Screening of differential metabolites:

[0127] In this study, the screening of differential metabolites was performed by |log2FoldChange|>0.585, P<0.05 and important predictive variables (VIP)>1, and a volcano plot of differential metabolites was drawn (Figure 13). The results showed that compared with the CON group, 240 differential metabolites were screened in the U group, of which 110 were up-regulated and 130 were down-regulated; compared with the U group, 45 differential metabolites were screened in the U+SE group, of which 35 were up-regulated and 10 were down-regulated. According to Figure 14(a), the intersection of the differential metabolites of the U group / CON group and the U+SE group / U group was obtained, and 19 common differential metabolites were obtained, and the difference was shown in Figure 14(b).

[0128] 6. KEGG pathway enrichment analysis of differential metabolites:

[0129] KEGG pathway enrichment analysis of the differential metabolites in rat urine samples after uranium exposure and soybean extract treatment was performed, as shown in Figure 15. According to the -log10(P) value and Pathway Impact value, the U / CON group and the U+SE / U group were significantly enriched in 3 metabolic pathways, including Alanine, aspartate and glutamate metabolism, Glyoxylate and dicarboxylate metabolism, and Cysteine and methionine metabolism. These pathways mainly focused on the synthesis and metabolism of amino acids, carbohydrate metabolism.

[0130] Example 9 Joint analysis of transcriptomics and metabolomics

[0131] In this study, the correlation between the differential genes of transcriptomics and the differential metabolites of metabolomics was analyzed using the Pearson correlation coefficient, and a heat map was drawn, as shown in Figure 16. The vertical coordinate represents the co-expression differential gene name, and the horizontal coordinate represents the co-expression differential metabolite name. Red squares represent positive correlation between differential genes and differential metabolites, blue squares represent negative correlation, and the color intensity represents the strength of correlation. The results showed that under the screening conditions of a correlation coefficient (rho) ≥ 0.7 and a P-value < 0.5, 9 differential genes and 5 metabolites had a high correlation, as shown in Table 3. These genes and metabolites were mainly involved in Metabolic pathways, Complement and coagulation cascades, Cysteine and methionine metabolism, ECM-receptor interaction, Leukocyte transendothelial migration, Chemokine signaling pathway, Relaxin signaling pathway, and Rap1 signaling pathway.

[0132] Table 3 Correlation analysis table of differential genes and differential metabolites

[0133] The embodiments are only illustrative of the present application, and are not intended to limit 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 the modifications are within the scope of the claims of the present application, they are 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) reducing the kidney coefficient of a uranium exposed person; b) improving the uranium excretion capacity of a uranium exposed person; c) preventing or treating uranium induced kidney damage.

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

3. Use according to claim 2, characterized in that, The microwave assisted extraction method specifically comprises placing water soaked soybean 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. 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) reducing the kidney coefficient of a uranium exposed person; b) improving the uranium excretion capacity of a uranium exposed person; c) preventing or treating uranium induced kidney damage.

6. The pharmaceutical composition of claim 5, wherein, The pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

7. The pharmaceutical composition of claim 6, wherein, The soybean extract is prepared from soybean by a microwave assisted extraction method.

8. The pharmaceutical composition of claim 7, wherein, The microwave assisted extraction method specifically comprises placing water soaked soybean in a microwave reaction kettle for treatment, and condensing and recovering the gasified gaseous plasma, to obtain the soybean extract.

9. The pharmaceutical composition of claim 8, wherein, The microwave reaction kettle has a treatment frequency of 2000-2100 MHz and a treatment time of 2-3 min.

10. Use of a marker in the evaluation of the efficacy of a drug for the treatment of kidney damage caused by uranium or in the screening of a drug for the treatment of kidney damage caused by uranium, characterized in that, The marker is selected from one or more of a target gene or a urine sample metabolite, the target gene being C3, Itgb2, Cx3cr1, Ackr3, Col4a3, Lama1, Aldh3b1, Mthfd2 or Kcnq1, and the urine sample metabolite being 5-hydroxylysine, alanylglutamine, aspartate, glycyglycine or phosphoserine.

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