Application of oat-beta-glucan in promoting gallstone formation

Abstract

The invention belongs to the field of medicine, and particularly relates to application of oat-beta-glucan in promoting formation of gallstone, and the oat-beta-glucan can promote formation of gallstone and increase the occurrence rate of gallstone; an intestinal flora detection result shows that the addition of the oat-beta-glucan significantly affects the intestinal flora of a mouse, the abundance change of the desulfurization vibrio is most obvious, the H2S level in serum is significantly increased, the mRNA expression level of FXR and SHP is significantly increased, and the mRNA expression level of CYP7A1 is significantly decreased; after the oat-beta-glucan is added, the FXR level of the liver is obviously increased, and the CYP7A1 is obviously decreased; wB results also prove that after the oat-beta-glucan is added, the FXR level of the liver is remarkably increased, CYP7A1 is remarkably reduced, a model is provided for research of gallstone, and a new thought is also provided for preparation of calculus bovis factitius.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and particularly relates to the application of oat-β-glucan in promoting the formation of gallstones. Background Art

[0002] Gallstones are a common disease of the digestive system, with an incidence rate of up to 10%-15%. Current treatments primarily rely on surgery, resulting in a heavy disease burden and significant health threats. Bile is primarily composed of cholesterol, bile acids, and phospholipids, and supersaturation of cholesterol in bile is a prerequisite for the formation of sterol stones. Under normal physiological conditions, bile acids and phospholipids form mixed micelles to promote the dissolution of hydrophobic cholesterol. However, when cholesterol is over-secreted, bile acids or phospholipids are deficient, or hydrophobic bile acids increase, the homeostasis of cholesterol in bile may be disrupted, leading to supersaturation and precipitation, and the formation of sterol stones.

[0003] Oat-β-glucan is a water-soluble dietary fiber naturally found in oat bran. It is composed of multiple D-glucose molecules connected by β-(1→3) and β-(1→4) glycosidic bonds. Due to its unique structure and health benefits, it is widely used in food, health products and medicine. Existing studies have shown that oat-β-glucan has the function of (1) regulating blood lipids, reducing serum total cholesterol and low-density lipoprotein cholesterol (LDL-C); (2) delaying gastric emptying and glucose absorption, improving postprandial blood sugar response, thereby controlling blood sugar, and is suitable for diabetic patients (see invention patent CN106689959A); (3) activating immune cells such as macrophages and natural killer cells (NK cells), regulating immune responses, and enhancing immunity; (4) as a prebiotic, promoting the proliferation of beneficial bacteria (such as bifidobacteria), inhibiting harmful bacteria, relieving constipation, and improving intestinal health; it can also be used for weight management. However, there are currently no reports on the effect of oat-β-glucan on the formation of gallstones.

[0004] During the research process, the inventors unexpectedly discovered that oat-β-glucan can promote the formation of gallstones and increase the incidence of gallstones; the results of intestinal flora testing showed that the addition of oat-β-glucan significantly affected the intestinal flora of mice, among which the abundance of Desulfovibrio changed most significantly, and the H2S level in the serum increased significantly, providing a model for the study of gallstones and also providing new ideas for the preparation of artificial bezoar. Summary of the Invention

[0005] The primary purpose of the present invention is to provide an application of oat-β-glucan in promoting the formation of gallstones.

[0006] The second object of the present invention is to provide the use of oat-β-glucan in constructing a gallstone experimental animal model.

[0007] The third object of the present invention is to provide the use of oat-β-glucan in the preparation of artificial bezoar.

[0008] The fourth object of the present invention is to provide a method for constructing a gallstone experimental animal model, the method comprising: giving the animal a stone model feed and an aqueous solution containing oat-β-glucan.

[0009] Preferably, the content of oat-β-glucan is 6%.

[0010] The present invention has the following beneficial effects: the present invention provides an application of oat-β-glucan in promoting the formation of gallstones, and the oat-β-glucan can promote the formation of gallstones and increase the incidence of gallstones; intestinal flora detection results show that the addition of oat-β-glucan significantly affects the intestinal flora of mice, among which the abundance of Desulfovibrio changes most significantly, the H2S level in serum significantly increases, the mRNA expression levels of FXR and SHP are significantly upregulated, while the mRNA expression level of CYP7A1 is significantly downregulated; after the addition of oat-β-glucan, the FXR level in the liver is significantly upregulated, and the CYP7A1 is significantly downregulated; WB results also confirm that after the addition of oat-β-glucan, the FXR level in the liver is significantly upregulated, and the CYP7A1 is significantly downregulated, providing a model for the study of gallstones and a new idea for the preparation of artificial bezoar. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Ultrasound test results of mice in the stone model group

[0012] Figure 2 Ultrasound examination results of mice in the oat-β-glucan group

[0013] Figure 3 Anatomical results of mice in the stone model group

[0014] Figure 4 Autopsy results of mice in the oat-β-glucan group

[0015] Figure 5 Intestinal flora 16S test results

[0016] Note: a is the LEFSE branch diagram; b is the quantitative analysis diagram

[0017] Figure 6 Kit for detecting serum H2S content in mice

[0018] Note: LD is the simple stone model group, LDB is the oat-β-glucan group

[0019] Figure 7Comparison of mRNA expression levels of key factors in the liver by real-time fluorescence quantitative PCR Note: a is the mRNA expression level of liver FXR; b is the mRNA expression level of liver SHP; c is the mRNA expression level of liver CYP7A1; NC is the blank control group, LD is the simple stone model group, and LDB is the oat-β-glucan group Figure 8 Western blotting to compare the protein expression levels of key factors in the liver

[0020] Note: a is the protein expression level of liver FXR; b is the protein expression level of liver CYP7A1;

[0021] NC was the blank control group, LD was the simple stone model group, and LDB was the oat-β-glucan group;

[0022] Figure 9 Quantitative analysis of protein WB bands

[0023] Note: a is the protein expression level of liver FXR; b is the protein expression level of liver CYP7A1;

[0024] NC was the blank control group, LD was the simple stone model group, and LDB was the oat-β-glucan group. DETAILED DESCRIPTION

[0025] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0026] It should be noted that, in the following examples, unless otherwise specified, the methods described are conventional methods, and the reagents and consumables used can be purchased from the market.

[0027] Example 1: Animal Experiment

[0028] 1. Main Reagents and Materials

[0029] Oat-β-glucan (purchased from Shaanxi Senfu Biotechnology Co., Ltd.) Mouse stone model feed (purchased from Nantong Trophy Feed Technology Co., Ltd., TP28900)

[0030] 2. Source of experimental animals

[0031] Jiangsu Jicui Yaokang Experimental Animal Technology Co., Ltd., animal species and strain: mouse, C57BL / 6J; sex: male; age: 4 weeks old; weight: 16-20 g.

[0032] 3. Experimental Methods

[0033] Animals were acclimated to feeding for 7 days before the experiment. Healthy animals were selected as test subjects and randomly grouped according to body weight. After grouping, each animal was assigned a unique, permanent animal number. Experimental environment: Animals were housed in an SPF environment with a room temperature of 20-26°C and a relative humidity of 40-70%. The lighting was automated, with a 12-hour light-dark cycle (lights on at 7:00 AM and off at 7:00 PM). The experimental animal housing standards complied with the national standard of the People's Republic of China, GB14925-2010. Drinking water was sterile water obtained through reverse osmosis (RO) membranes. The default stocking density was 4 animals per cage, and bedding was changed weekly.

[0034] Experimental Procedure: After all mice were acclimated for one week, the experiment began. Thirty-six 5-week-old male C57BL / 6J mice (average weight, approximately 18 to 22 g) were randomly divided into three groups of 12 mice each: a blank group (fed a normal growth and reproduction diet and normal drinking water; NC group); a simple stone model group (fed a stone model diet and normal drinking water; LD group); and an oat-β-glucan group (fed a stone model diet and normal drinking water supplemented with a 6% oat-β-glucan solution, with free access; LDM group). The oat-β-glucan solution was replaced daily. From the start of the experiment, mice were weighed every three days, and weight curves were plotted to observe any significant changes or differences between groups. Food and water intake was monitored daily between the experimental and control groups to assess any significant differences. In the fourth week, after abdominal hair removal, B-ultrasound was performed to assess stone formation. Mice were then sacrificed and samples collected for subsequent testing.

[0035] 4. Experimental Results

[0036] The mice in the simple stone model group (LD group) were fed for 4 weeks and then the formation of gallstones was detected by small animal B-ultrasound. The results showed that the final stone incidence rate was 6 / 12 ( Figure 1 ). After 4 weeks of feeding, B-ultrasound showed that the incidence of gallstones in the oat-β-glucan group (LDB group) was 12 / 12 ( Figure 2 The incidence of gallstones was significantly different between the two groups. The mice were killed by overdose of anesthesia and then dissected to further confirm the incidence of gallstones. The dissection results were consistent with the results of small animal B-ultrasound. The incidence of gallstones in the simple stone model group (LD group) was 6 / 12 ( Figure 3 The incidence of gallstones in the oat-β-glucan group was 12 / 12 ( Figure 4 ).

[0037] Example 2: Detection of intestinal flora 16s

[0038] 1. Experimental Methods

[0039] Fresh cecal colon contents were collected from sacrificed mice, and intestinal flora analysis was performed using 16S RNA sequencing with six replicates per experimental group. The samples were placed on dry ice for testing, and the exposure time of the cryopreserved tubes to room temperature was minimized throughout the entire process.

[0040] (1) Extract total genomic DNA from the microbial sample to be tested using a mechanical lysis method;

[0041] (2) Perform PCR amplification using a universal primer pair for the 16S rRNA gene in a reaction solution containing a high-fidelity DNA polymerase, dNTPs, and a buffer system;

[0042] (3) Library construction: Purify the PCR product to remove primer dimers, and use magnetic bead screening to retain the target fragments; add Illumina sequencing adapters and sample-specific index sequences (Index) through secondary PCR or ligation reaction to construct a library that can be mixed and sequenced.

[0043] (4) Library quality control and standardization: Use fluorescence quantification to determine library concentration, and confirm that the fragment size distribution is in the 400-600 bp range by gel electrophoresis or bioanalyzer; mix multiple libraries in equimolar ratios and perform concentration standardization.

[0044] (5) High-throughput sequencing: The mixed library is loaded onto an Illumina sequencing platform, preferably a MiSeq or NovaSeq system, and paired-end sequencing (PE250 or PE300 mode) is performed. A ≥1% PhiX control library is added to correct base recognition errors.

[0045] (6) Data analysis: The raw sequencing data were subjected to quality control (Q≥30 filtering), double-end sequence splicing, and denoising to generate ASV / OTU feature tables, and species classification annotation was completed by comparing with the SILVA or Greengenes database. The sequencing results were clustered into operational taxonomic units (OTUs) based on 97% sequence similarity. OTU rank-abundance curves, rarefaction curves, and Shannon index for α diversity analysis were plotted, and Shannon, Chao1, Simpson, and ACE indices were calculated. A heat map of key OTUs identified by redundancy analysis was generated for β diversity analysis, and principal component analysis, principal coordinate analysis (PCoA), non-metric multidimensional scaling, and unweighted pair group method of arithmetic means (UPGMA) were performed using QIIME. Linear discriminant analysis (LDA) was used to quantitatively evaluate the biomarkers of each group and to draw a LEfSe branch diagram. Non-parametric factorial Kruskal-Wallis sum rank test and (unpaired) Wilcoxon rank sum test were performed to determine the taxa with the greatest abundance differences.

[0046] 2. Experimental Results

[0047] The results of 16S intestinal flora detection showed that the addition of oat-β-glucan significantly affected the intestinal flora of mice. LDA linear discriminant analysis found that the abundance of Desulfovibrionales in the intestine changed most significantly after the addition of oat-β-glucan ( Figure 5 a), further relative abundance quantitative analysis confirmed that the abundance of Desulfovibrio in the oat-β-glucan group was significantly increased compared with the simple stone group ( Figure 5 b) Existing research has demonstrated that Desulfovibrio desulfuricans can degrade sulfur-containing substrates in the intestine into H2S, which acts as an agonist for the liver's FXR receptor. FXR activation can downregulate the liver's bile acid synthase CYP7A1, leading to cholesterol accumulation in the liver bile and the development of gallstones.

[0048] Example 3: Kit for detecting H2S in blood

[0049] 1. Experimental Methods

[0050] Take out fresh mouse serum (within 3 days after sacrifice) and thaw it on ice. Use a kit to detect H2S in the serum. Take out the kit from the 4°C refrigerator 30 minutes before the start of the experiment and equilibrate it to room temperature. Prepare the standard working solution according to the instructions, with concentrations of 1000, 500, 250, 125, 62.5, 31.25, 15.63, and 0 pg / mL respectively. Divide the serum samples into positions on the ELISA plate according to the pre-experimental dilution ratio, add different concentrations of standards and samples, 100uL per well, then add the color developer and iron salt reagent, cover the wells, incubate at 37°C for 15 to 30 minutes, and measure the optical density (OD value) of each well at 680nm using an enzyme-labeled instrument.

[0051] 2. Experimental Results

[0052] The results of blood H2S test confirmed that the H2S level in mouse serum increased significantly after adding oat-β-glucan ( Figure 6 Example 4: Real-time fluorescence quantitative PCR detection of mRNA expression levels of key factors in the liver

[0053] 1. Experimental Methods

[0054] (1) Extract RNA. Take out an appropriate amount of liver sample from the -80℃ freezer and place it in a 5ml EP tube. Add 500μl Trizol to immerse the tissue. Grind the tissue by electric grinding (60s*2) until it is crushed and transfer it to a 1.5ml RNase-free EP tube. Then add 500μl Trizol. Place it on ice for 5 minutes. Then add 200μl chloroform, vortex until it turns milky white and red, and let it stand on ice for 10 minutes. Place it in a pre-cooled centrifuge and centrifuge (4 degrees, 12000rpm, 15 minutes). You can see that there are three obvious layers (the upper colorless and transparent aqueous phase is the RNA layer, the thin milky white layer in the middle is the DNA layer, the lower red layer is the protein layer, and large tissue blocks can be seen at the bottom). Carefully and slowly aspirate the supernatant and place it in another new 1.5ml RNase-free EP tube. Add an equal volume of isopropanol, mix it upside down, and let it stand at room temperature for 15 minutes. Centrifuge (4 degrees, 12000 rpm, 15 minutes) until a white precipitate is visible. Discard the supernatant and add 950 μl of 75% ethanol to each tube. Mix by inverting. Centrifuge (4 degrees, 12000 rpm, 10 minutes) until a clear white precipitate is visible (tissue precipitates are generally larger). After centrifugation, discard the supernatant and centrifuge again for a short spin. Use a 10 ul pipette to remove any remaining liquid. Open the lid and let it air dry (about 5 minutes). Add an appropriate amount of DEPC water (usually 30-50 ul) to each tube according to the size of the precipitate. Dissolve the RNA by pipetting and store in a -80°C refrigerator.

[0055] (2) RNA quality control and concentration determination: Absorbance was measured using a NanoDrop One spectrophotometer to determine RNA concentration (ng / μL) and OD260 / OD280 ratio. The standard OD260 / OD280 ratio is between 1.8 and 2.0; a ratio <1.8 indicates a high level of impurities; a ratio >2.2 indicates significant RNA degradation.

[0056] (3) Reverse transcription reaction: Prepare 10 μL of reverse transcription system reagents on ice according to the TIANGEN kit: Set the PCR instrument reaction conditions (temperature, time) in the following order: 42°C, 15 minutes → 95°C, 3 minutes → 4°Cα, and store the reaction product (cDNA) at -20°C. Prepare the reaction solution on ice according to the instructions, set 3 replicate wells for each sample, and set a blank control reagent for each primer. Set the QuantStudio 3RT-PCR instrument reaction parameters: pre-denaturation: 95°C, 30 seconds, 1 cycle; PCR reaction, a total of 40 cycles: 95°C 10 seconds; 60°C 10 seconds; 72°C 10 seconds. Melting curve analysis: 95°C 5 seconds; 65°C 1 minute; 97°C 0 seconds. After the above program is completed, the CT value of each sample is used to calculate the relative expression of the gene.

[0057] 2. Experimental Results

[0058] qRT-PCR results confirmed that there were significant differences in the mRNA expression levels of FXR, SHP, and CYP7A1 between the simple stone group and the oat-β-glucan group. After the addition of oat-β-glucan, the mRNA expression levels of FXR and SHP in the liver were significantly upregulated, while the mRNA expression level of CYP7A1 was significantly downregulated ( Figure 7 ).

[0059] Example 5: Comparison of protein expression levels of key factors in the liver by Western blotting

[0060] 1. Experimental Methods

[0061] (1) Extract proteins from tissues: Select liver tissue stored in a -80°C refrigerator and cut the target tissue with clean tools. This operation should be performed on ice and completed as quickly as possible to prevent the sample from being degraded by proteases. Place the tissue in a round-bottom centrifuge tube and add 300 μL of pre-cooled lysis buffer to the tube for every 5 mg of tissue. Then, homogenize the tube using an electric homogenizer and centrifuge at 12,000 rpm for 20 minutes at 4°C. Gently remove the centrifuge tube from the centrifuge and place it on ice. Aspirate the supernatant and inject it into a clean tube placed on ice. Store at -80°C and discard the precipitate.

[0062] (2) Prepare separation gel and stacking gel according to the molecular weight of different proteins, add the prepared protein sample to the sample well, perform constant voltage electrophoresis, remove the gel after electrophoresis, process the gel and clamps in the order of black side - cotton pad - gel - PVDF membrane - filter paper - cotton pad - white side, and transfer the membrane at low temperature and constant flow. After the transfer, remove the PVDF membrane, block it with 1% BSA diluted in TBST at room temperature for 1 hour, add Fxr and Cyp7a1 antibodies respectively, and incubate at 4℃ overnight. The next day, wash with TBST 3 times, 5 minutes each time, add secondary antibody and incubate at room temperature for 1 hour, then wash with TBST 3 times, 5 minutes each time. Finally, prepare the luminescent liquid and expose it on the machine.

[0063] (3) Use Image J software for quantitative analysis of WB bands; use the rectangular selection tool to frame the target band area (completely cover the longitudinal range of the band), use the software to generate the grayscale curve corresponding to the band and calculate the area integral optical density value, then measure the band integral optical density of the internal reference target molecule separately, and calculate the relative protein expression (target protein / internal reference protein).

[0064] 2. Experimental Results

[0065] The results of WB bands and quantitative analysis confirmed that there were significant differences in the protein expression levels of FXR and CYP7A1 between the simple stone group and the oat-β-glucan group. The expression level of liver FXR protein was significantly increased after the addition of oat-β-glucan ( Figure 8 a, Figure 9 a), while the protein expression level of liver bile acid synthase CYP7A1 was significantly reduced, which confirms our hypothesis that oat-β-glucan can upregulate the abundance of intestinal Desulfovibrio, thereby activating the liver FXR-SHP signaling pathway and inhibiting the expression level of liver bile acid synthase CYP7A1 by producing H2S, further promoting the accumulation of cholesterol in liver bile and inducing gallstones. ( Figure 8 b, Figure 9 b).

[0066] In summary, the present invention provides a use of oat-β-glucan in promoting the formation of gallstones. The oat-β-glucan can promote the formation of gallstones and increase the incidence of gallstones. The results of intestinal flora detection showed that the addition of oat-β-glucan significantly affected the intestinal flora of mice, among which the abundance of Desulfovibrio changed most significantly, the H2S level in the serum was significantly increased, the mRNA expression levels of FXR and SHP were significantly upregulated, while the mRNA expression level of CYP7A1 was significantly downregulated. After the addition of oat-β-glucan, the level of FXR in the liver was significantly upregulated, and the level of CYP7A1 was significantly downregulated. Western blot results also confirmed that the addition of oat-β-glucan significantly increased the level of FXR in the liver, and significantly downregulated CYP7A1. This provides a model for the study of gallstones and a new idea for the preparation of artificial bezoar.

Claims

1. Application of oat-β-glucan in promoting the formation of gallstones.

2. Application of oat-β-glucan in constructing experimental animal model of gallstones.

3. Application of oat-β-glucan in the preparation of artificial bezoar.

4. A method for constructing a gallstone experimental animal model, characterized in that: The method comprises: administering a stone model feed to an animal and administering an aqueous solution containing oat-β-glucan.

5. The method according to claim 4, wherein The content of the oat-β-glucan is 6%.

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