Construction of a model of damp-heat syndrome of non-alcoholic fatty liver disease and detection method

Abstract

The application discloses a kind of non-alcoholic fatty liver disease damp-heat syndrome model construction and detection method.The application uses high-fat high-sugar feed to feed experimental animal, while high temperature and high humidity environment is intervened in two stages, and non-alcoholic fatty liver disease damp-heat syndrome animal model is obtained.The model is successfully constructed by observing macroscopic index, pathological tissue, sugar and fat metabolism index, inflammation index, intestinal flora, specific gene, immune index and oxidative stress index of model animal.The application combines non-alcoholic fatty liver disease animal model and traditional Chinese medicine damp-heat syndrome animal model together, the model has high repeatability, is closer to clinical manifestation, and provides ideal animal model for screening new treatment drugs and traditional Chinese medicine mechanism research.

Description

Technical Field

[0001] This invention relates to experimental techniques for medical animal models, and in particular to a method for constructing and evaluating a non-alcoholic fatty liver disease model with damp-heat syndrome. Background Technology

[0002] Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide, characterized by lipid accumulation in the liver and closely associated with obesity, cardiovascular disease, and metabolic syndrome. Globally, NAFLD is on the rise, affecting a quarter of the world's population and posing a serious threat to health. Furthermore, the worsening incidence of NAFLD is increasing the global burden of chronic liver disease. In traditional Chinese medicine (TCM) theory, damp-heat syndrome is closely related to NAFLD and is one of the most common pathogenic syndrome types of NAFLD. Damp-heat syndrome in NAFLD is a chronic and complex condition characterized by persistent fatigue, fever, bitter taste in the mouth, thirst, dark urine, and sticky stools. Currently, the diagnosis of TCM syndrome types mainly relies on subjective observation. Therefore, it is necessary to use a quantitative indicator when evaluating TCM syndrome types, which can also avoid subjective errors by practitioners and further reveal the biological basis of TCM syndromes.

[0003] Animal models can help us better understand diseases and play an important role in the research of therapeutic drugs. Literature review shows that although significant progress has been made in studying the mechanisms of NAFLD over the past few decades, research on animal models of NAFLD with damp-heat syndrome is very limited, lacking unified modeling and evaluation standards. This limitation hinders research into the pathogenesis of NAFLD with damp-heat syndrome and the development of new drugs. Therefore, this study aims to establish a stable and reproducible animal model of NAFLD with damp-heat syndrome. Simultaneously, it is necessary to establish an objective and effective evaluation system for animal models to build evidence-based medicine for the treatment of NAFLD with damp-heat syndrome. Summary of the Invention

[0004] To address the aforementioned issues, this invention aims to provide a method for constructing and evaluating a mouse model of NAFLD damp-heat syndrome, thereby resolving the problem of the lack of unified modeling and evaluation standards for existing animal models of NAFLD damp-heat syndrome.

[0005] The technical solution of this invention is as follows:

[0006] A method for constructing and detecting a damp-heat syndrome model of non-alcoholic fatty liver disease, characterized by the following steps:

[0007] (1) Experimental animals were subjected to a combination of high-fat, high-sugar diet and high-temperature, high-humidity environment;

[0008] (2) evaluating and detecting the non-alcoholic fatty liver disease model of damp-heat syndrome; the evaluation and detection of the model animals include macroscopic indexes, pathological tissues, sugar and lipid metabolism indexes, inflammation indexes, changes in intestinal flora and expression of specific genes.

[0009] The evaluation and detection of the model animals also include immune indexes and oxidative stress indexes.

[0010] Preferably, the high-temperature and high-humidity environment of step (1) refers to placing the model animals into a small animal incubator, setting the temperature to 35℃ and the humidity to 95%, and intervening for 8 hours per day; the model animals are mice.

[0011] The macroscopic indexes include the body weight, food intake, water intake, grip strength, anal temperature, oily hair and open field test of the model animals.

[0012] The pathological tissue indexes include HE staining and oil red O staining to observe the changes in liver tissue.

[0013] The sugar and lipid metabolism indexes include fasting blood glucose, random blood glucose, serum blood glucose, glucose tolerance, insulin tolerance, serum total cholesterol, triglyceride, high-density lipoprotein and low-density lipoprotein.

[0014] The inflammation indexes include IL1β, IL6, IL10 and TNF-α of the liver tissue.

[0015] The changes in intestinal flora refer to the increase in the abundance of Erysipelotrichia, Erysipelotrichales, Erysipelotrichaceae and Faecalibaculum flora.

[0016] The specific genes include cyp4a10, Irf7, cptlb and zbp1.

[0017] The immune indexes include M1 and M2 type macrophages of the liver tissue; the oxidative stress indexes include superoxide dismutase, reduced glutathione, nitric oxide and malondialdehyde of the liver tissue.

[0018] The beneficial effects of the present application are:

[0019] (1) The present application builds the internal cause of the formation of NAFLD damp-heat syndrome by high-fat and high-sugar diet, and builds the external cause of the formation of NAFLD damp-heat syndrome by high-temperature and high-humidity environment intervention, organically combines the animal model of TCM damp-heat syndrome and the animal model of NAFLD, and optimizes the modeling method. No chemical reagent intervention during modeling, the animal model meets the clinical manifestations of NAFLD damp-heat syndrome, simulates the occurrence and development of the disease, and is very close to the pathogenesis of human beings. The results show that the animal model has the characteristics of good stability, high repeatability, easy operation, no chemical toxic side effects and low mortality.

[0020] (2) The evaluation index of the traditional animal model is subjective, while the evaluation index of the present application is specific, including screening the characteristic genes of NAFLD damp-heat syndrome, and finding the dominant flora of NAFLD damp-heat syndrome, which is more scientific and standardized. The present application provides a scientific method for evaluating the NAFLD damp-heat syndrome model.

[0021] (3) The TCM treatment of non-alcoholic fatty liver damp-heat syndrome has definite curative effect and small toxic side effect, and has unique advantages compared with western medicine. Dialectical treatment is the guiding ideology of TCM, and the existing animal model cannot reflect the TCM syndrome type. The animal model built in the present method has the characteristics of non-alcoholic fatty liver disease and damp-heat syndrome, and can be used for research on TCM efficacy evaluation and mechanism of action, and lays a foundation for the pathogenesis and new drug research of non-alcoholic fatty liver TCM syndrome type.

[0022] (4) The present application first proposes to use specific genes to evaluate the damp-heat syndrome model, and finds four genes cyp4a10, Irf7, cpt1b and zbp1 that affect the damp-heat syndrome model animals. The experimental results show that the expression of Cpt1b, Cyp4a10, Zbp1 and Irf7 mRNA in the model animal group is significantly increased; and the Western Blot results confirm that compared with the normal group, the protein expression of Cyp4a10, Zbp1 and Iri7 in the model animal group is increased. This gives a more accurate evaluation method and standard for the establishment of the damp-heat syndrome animal model at the gene level. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The operation schematic diagram of the NAFLD damp-heat syndrome mouse model of the present application;

[0024] Figure 2Figures of macroscopic indicators and pathological tissue changes of mice in each group; Figure A is the change of body weight of mice; Figure B is the body weight of mice after modeling; Figure C is the water intake of mice; Figure D is the food intake of mice; Figure E is the change of anal temperature of mice; Figure F is the change of grip strength of mice; Figure G is the appearance of mice; Figure H is the behavioral changes of mice in open field test; Figure I is the appearance of liver tissue of mice, HE staining and oil red O staining; Figure J is the white adipose tissue and brown adipose tissue index, liver weight and serum glutamic-oxalacetic transaminase content of mice; *P<0.05, **P<0.01, ***P<0.001 compared with the normal group, indicating that the difference is statistically significant;

[0025] Figure 3 Figures of glycolipid metabolism indicators and inflammation indicators of mice in each group; A is the random blood glucose content of mice after modeling; B is the serum blood glucose content of mice after modeling; C and D are oral glucose tolerance test (OGTT) and area under the curve; E and F are insulin tolerance test (ITT) and area under the curve; G is the content of serum triglyceride, total cholesterol, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol; H is the mRNA expression of inflammatory factors in liver tissue detected by qPCR; *P<0.05, **P<0.01, ***P<0.001 compared with the normal group, indicating that the difference is statistically significant;

[0026] Figure 4 Figures of immune indicators and oxidative stress indicators of mice in each group; A is the proportion of M1 and M2 type macrophages in liver tissue of mice detected by flow cytometry; B is the change of oxidative stress related indicators in serum; *P<0.05, **P<0.01, ***P<0.001 compared with the normal group, indicating that the difference is statistically significant;

[0027] Figure 5 Figure of intestinal flora changes of mice in each group; A is the β diversity analysis of intestinal flora of mice; B and C are the α diversity analysis of intestinal flora of mice; D is the species distribution change of intestinal flora of mice at the level of door, class, genus and species; E is LEfSe analysis; F is functional prediction analysis of intestinal flora;

[0028] Figure 6Figure of the results of transcriptomics and specific gene expression of liver tissues of the double-factor group and the normal group; A is a volcano plot of differential gene expression between groups; B and C are GO and KEGG analysis; D is GSEA enrichment analysis; E is differential expression of genes in lipid metabolism, inflammation, oxidative stress and immune pathways; F is GSEA enrichment analysis of the correlation of cyp4a10, Irf7, cpt1b and zbp1 with lipid metabolism, inflammation, immunity and oxidative stress; G is the expression of differential genes cyp4a10, Irf7, cpt1b and zbp1 detected by qPCR and Western blot; compared with the normal group, *P<0.05, **P<0.01, ***P<0.001, indicating that the difference is statistically significant. DETAILED DESCRIPTION

[0029] In order to make the research scheme and advantages of the present application clearer, the present application will be described in detail in conjunction with the drawings. The following description is only used to explain the present application and does not limit the scope of the present application.

[0030] The C57BL / 6J mice are used as model animals in the embodiments of the present application, and the model animals are not limited to mice, but can also be rats, guinea pigs, rabbits and other model animals.

[0031] In the embodiments, SPF level C57BL / 6J male mice with a body weight of 19-21 g and an age of 6-7 weeks are selected. During modeling, the mice are fed with high-fat and high-sugar diet all the time, and high-temperature and high-humidity environment intervention is performed in the 5th-8th week and the 11th-12th week of modeling.

[0032] Embodiment 1: NAFLD mice model with damp-heat syndrome is constructed, and macroscopic indexes, pathological tissues, glycolipid metabolism and inflammation indexes are detected

[0033] SPF level C57BL / 6J male mice are selected (Beijing Vital River Laboratory Animal Technology Co., Ltd., license number: SCXK (Jing) 2021-0006, body weight 19-21 g, age 42-48 weeks. As shown in Figure 1 As shown in the table, the mice are divided into a normal group, an internal factor group, an external factor group and a double-factor group, with 10 mice in each group. The growth environment of the mice in the normal group is a temperature of (20±2) ℃ and a humidity of (45±5) %, and the mice are free to eat and drink, with 12 h of day-night alternation. The mice in the internal factor group are continuously fed with 45% fat energy high-fat and high-sugar feed for 12 weeks in an ordinary environment. The mice in the external factor group are continuously fed with ordinary diet for 12 weeks, wherein the mice are intervened in an ordinary environment for 8 h per day in the 1st-4th week and the 9th-10th week, and intervened in an external damp-heat environment for 8 h per day in the 5th-8th week and the 11th-12th week (temperature 35 ℃, humidity 95%). The mice in the double-factor group are fed with 45% fat energy high-fat and high-sugar feed for 12 weeks, and the external damp-heat environment intervention measures are the same as those of the external factor group.

[0034] Detection index:

[0035] Macroscopic index: The body weight, food intake and water intake of mice were recorded daily during the experiment, and the anal temperature and grip strength of mice were monitored every four weeks.

[0036] Open field test analysis: Before the experiment, first confirm that the experimental box is clean. Set the corresponding parameters in the software, record the grouping and numbering information of the mice. Take the mice out of the feeding cage (be careful not to face the experimenter), quickly place them in the central area of the experimental box, and immediately leave. Turn on the analysis software, the system will automatically record the free activity trajectory of the mice in the box. The experimental time is recorded as 5 minutes. After the experiment, put the mice into the feeding cage prepared in advance, spray with alcohol to remove odor and wipe with paper towel.

[0037] Random blood glucose of mice: At the 4th, 8th and 12th weeks of experimental intervention, the fasting blood glucose of mice was detected (12h fasting at night). At the end of 12 weeks, the tail vein blood of mice was collected to detect the random blood glucose of mice.

[0038] Oral glucose tolerance test (OGTT): At the end of 12 weeks of experimental intervention, the mice were fasted for 12 hours overnight, and the fasting blood glucose of mice was first detected by tail vein blood collection. A 40% glucose solution was administered intragastrically at a dose of 5mL / kg. At 30, 60, 90 and 120 minutes after treatment, tail vein blood was collected to detect blood glucose, and OGTT curve was drawn, and area under the curve (AUC) was calculated.

[0039] Insulin tolerance test (ITT): At the end of 12 weeks of experimental intervention, the mice were fasted for 12 hours overnight, and the fasting blood glucose of mice was first detected by tail vein blood collection. Insulin was injected intraperitoneally at a dose of 5ml / kg. At 30, 60, 90, 120 minutes after treatment, tail vein blood was collected to detect blood glucose, and ITT curve was drawn, and area under the curve (AUC) was calculated.

[0040] Lipid metabolism index detection: Mice were taken blood from the inner canthus, and the blood was allowed to stand at room temperature for 3 hours, then centrifuged at 4℃, 3000rpm / min for 10 minutes. The collected serum was detected for blood lipid level changes according to triglyceride (TG), cholesterol (TC), high-density lipoprotein (HDL-C) and low-density lipoprotein (LDL-C) kits.

[0041] Mouse liver tissue HE staining: (1) The mouse liver tissue was taken and fixed with 4% paraformaldehyde; (2) dehydration and transparency were carried out in a fume hood: the fixed tissue block was taken out, trimmed and flattened, and then placed in an embedding box, and the embedding box with a mark was immersed in 75% ethanol (time unlimited)-95% ethanol (overnight treatment)-100% ethanol I for 1 h-100% ethanol II for 4 h-xylene I for 10 min-xylene II for 50 min. Wax immersion: soft wax for 30 min-hard wax for 1 h; (3) embedding: the melted wax was added to the embedding box, the tissue was placed in the center of the embedding box, and the frozen table at-20°C was cooled to solidify the paraffin; (4) sectioning: the paraffin sectioning machine was used for sectioning, the thickness was 5 um, and the section was flattened in a 47°C sectioning machine, and then the section was taken out with a glass slide; (5) baking: the temperature was set to 65°C, and the section was baked for 6 h; (6) deparaffinization to water: the paraffin tissue section was placed in xylene I for 10 min-xylene II for 10 min-100% ethanol I for 5 min-100% ethanol II for 5 min-95% ethanol for 5 min-75% ethanol for 5 min-tap water for 5 min; (7) hematoxylin staining of cell nucleus: immersion for 7 min, 1% hydrochloric acid alcohol solution differentiation for 3 s, and tap water washing for 5 min to wash away excess dye; (8) eosin staining of cytoplasm: immersion for 1 min, and tap water washing for 30 s to wash away excess dye; (9) dehydration and transparency: 75% ethanol for 3 s-95% ethanol for 3 s-100% ethanol I for 2 min-100% ethanol II for 2 min-xylene I for 5 min-xylene II for 5 min; (10) mounting, microscopic observation: the section was mounted with neutral resin, and the pathological tissue changes were observed under a microscope.

[0042] Mouse liver tissue oil red O staining: (1) The animal tissue block was taken and fixed with 4% paraformaldehyde; (2) dehydration: the fixed tissue was dehydrated in 15% sucrose solution, and then transferred to 30% sucrose solution for further dehydration until the tissue sank to the bottom; (3) sectioning: OCT embedding agent was added to the sample holder of the freezing table for freezing treatment, the trimmed tissue was placed on the frozen OCT embedding agent, and the tissue was wrapped and frozen again after adding OCT embedding agent, and the sectioning was started when the embedding agent turned milky white, and the section thickness was selected to be 10 um; (4) the section was immersed in oil red O working solution for 15 min, washed with tap water twice, washed with 60% isopropanol to remove excess dye, and finally washed with tap water for 3 times; (5) hematoxylin staining of cell nucleus for 15 min, washed with tap water twice, differentiated with 1% hydrochloric acid alcohol solution for 3 s, and then washed with tap water to reverse blue; (6) the section was mounted with water-soluble mounting medium, and observed under a microscope.

[0043] Visceral index: The liver tissue, white adipose tissue and brown adipose tissue of the mice were taken out, washed with normal saline and then dried with filter paper. The visceral index = visceral mass / body weight*100%.

[0044] Liver and kidney function of mice: The mice were taken blood from the inner canthus, and then left to stand at room temperature for 3 hours, centrifuged at 3000 rpm / min for 10 minutes at 4°C, and the collected serum was detected for alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), creatinine (CRE) and blood urea nitrogen (BUN) by an automatic biochemical analyzer.

[0045] Inflammation index detection: qPCR was used to detect the expression of inflammatory factors in the liver tissue of mice. 20 mg of mouse liver tissue was weighed, and RNA was extracted by Trizol method, and RNA quantification was performed by Nanodrop instrument. The corresponding reagents were added according to Table 1 for reverse transcription treatment, and then reacted at 37°C for 15 minutes and at 85°C for 5 seconds.

[0046] Table 1 PCR reverse transcription system

[0047]

[0048] The cDNA was diluted by an appropriate number of times (usually diluted by 2-10 times), and the required reagents for the PCR reaction system were added as shown in Table 2. The primer sequences are shown in Table 3. Then, the machine was detected, and the relative expression amount of the gene was calculated according to the 2- ΔΔCT method.

[0049] Table 2 PCR amplification system

[0050]

[0051] Table 3 Primer sequences

[0052]

[0053] Experimental results

[0054] Macroscopic index change results: From the day of modeling, the body weight, food intake, water intake, anal temperature, grip strength, hair change, movement and liver appearance change of the mice in each group were observed, and the results are shown in Figure 2 (A-F). During the modeling period, the body weight of the mice in each group increased with time. Compared with the normal group, the body weight of the mice in the endogenous group and the double-factor group increased significantly. Compared with the normal group, the mice in the double-factor group showed a decrease in food intake and water intake. In addition, the anal temperature of the mice in the double-factor group was higher than that of the normal group, and the grip strength decreased. As Figure 2(G) shows the changes of the mice's hair: the hair of the normal group mice is smooth and lustrous; the hair of the endogenous group mice is oily; the hair of the exogenous group mice is smooth, and the hair on the face and neck is brownish red; the fur of the double factor group mice is oily, and the color of the face and neck changes to brownish red. As shown in Figure 2 (H) shows that the trajectory graph and the heat map both indicate that the movement trajectory of the normal group mice is chaotic, while the movement trajectory of the endogenous group, the exogenous group and the double factor group mice is simple, and mainly moves along the four sides of the box. In addition, compared with the normal group, the total distance and the average speed of the double factor group mice are significantly reduced, and the static time is significantly increased. Figure 2 (I) shows that the liver tissue of the normal group and the exogenous group mice is dark red and normal in structure. The liver tissue of the endogenous group mice is yellowish white, the liver is enlarged, the capsule is tense, and the surface feels oily. The liver tissue of the double factor group mice is significantly enlarged, the color is yellowish white, the edge is blunt, and the capsule is tense.

[0055] Pathological tissue detection results: after the modeling was completed, the animals in each group were dissected and subjected to HE staining and oil red O staining for pathological histological analysis. The results are shown in Figure 2 (I) shows that the HE staining results show that the liver cells of the normal group mice are normal, the liver cords are arranged in order, and the structure of the liver lobule is clear. The liver cell structure of the exogenous group mice is not obviously abnormal, and part of the inflammatory cells around the central vein are infiltrated. The liver cells of the endogenous group are swollen, with fat vacuoles of different sizes. The liver sinus of the double factor group is significantly narrowed, the liver cord is arranged abnormally, the liver cell division is not clear, and a small amount of inflammatory cell infiltration is accompanied. The oil red O staining results show that the cell nucleus of the normal group and the exogenous group mice is blue, and there is no red lipid droplet accumulation in the cells. There is obvious red lipid droplet accumulation in the liver cells of the endogenous group and the double factor group. As shown in Figure 2 (J) shows that compared with the normal group, the white adipose tissue, liver weight and serum glutathione transaminase content of the double factor group are significantly increased.

[0056] Glycolipid metabolism index detection results: as shown in Figure 3 (A-G) shows that compared with the normal group, the fasting blood glucose, random blood glucose, glucose tolerance, insulin tolerance, TG, TC, HDL-C and LDL-C, GLU of the endogenous group and the double factor group are significantly increased.

[0057] Inflammatory index detection results: as shown in Figure 3 (H) shows that compared with the normal group, the expression of pro-inflammatory factors IL1β and IL6 of the double factor group is significantly increased, TNFα has a trend of increase, and the expression of anti-inflammatory factor IL10 is significantly decreased.

[0058] Example 2: Immune index and oxidative stress index detection

[0059] Immune index detection: Ml and M2 type macrophages in liver tissue were detected by flow cytometry. The liver tissue after sampling was placed in 5% fetal bovine serum DMEM medium. It was gently ground on a 200 mesh screen, and the ground filtrate was transferred to a 15 mL EP tube. After blowing, 100 μL of the suspension was taken into a 1.5 mL centrifuge tube. 4°C, 3000 rpm / min centrifugation for 30s, the excess supernatant was sucked with a syringe, 100 μL of antibody was added (according to the reagent instruction), and the antibody was incubated for 30 min in the dark. Then centrifuge at 4°C, 3000 rpm / min for 30s, discard the supernatant. Add 1 mL of PBS solution and vortex. After washing, centrifuge at 4°C, 3000 rpm / min for 30s, discard the supernatant, and add 500 μL of PBS, ready for machine detection.

[0060] Oxidative stress index detection: The mouse liver tissue was weighed, and the liver homogenate was prepared. According to the biochemical kit instructions, the contents of catalase (CAT), nitric oxide synthase (NOS), reduced glutathione (GSH), lipid oxidation (MDA), nitric oxide (NO), and superoxide dismutase (SOD) were detected.

[0061] Experimental results

[0062] Immune and oxidative stress index detection results: as shown in Figure 4 (A), compared with the normal group, the expression of Ml type macrophages in the double factor group was significantly increased, and the expression of M2 type macrophages was significantly decreased. As shown in Figure 4 (B), compared with the normal group, the SOD and GSH in the double factor group were decreased, and the NO was significantly increased, and the MDA had an increasing trend.

[0063] Example 3: Analysis of the structural characteristics of intestinal flora by 16S rRNA high-throughput sequencing technology

[0064] Experimental steps: (1) Fecal collection: The cecum and its contents were taken when the mouse was taken, and put into a pre-sterilized cryogenic tube. The cryogenic tube was quickly put into liquid nitrogen, and then transferred to a-80°C refrigerator to avoid repeated freezing. (2) Sample DNA extraction and amplification: The DNA of the sample genome was extracted with the corresponding DNA kit, and PCR amplification was performed using specific primers containing barcode and TaKaRa Premix Version 2.0 (TaKaRa Biotechnology Co., Dalian, China). Ultra TM II DNA Library Prep Kit for Library construction was completed using the standard protocol of NEBNext® UltraTM II Directional RNA Library Prep for Illumina® (New England Biolabs, USA). The constructed amplicon library was sequenced using the Illumina Nova 6000 operation platform. (4) Sequencing data processing: In view of the possible interference data of the sequencing results, the following operations were mainly performed: Paired-end Raw Reads data filtering, Paired-end Clean Reads splicing and Raw Tags sequence quality filtering.

[0065] Experimental results: The changes of intestinal flora indexes are shown in Figure 5 (A-D) The microbial community abundance of the double-factor and internal group was significantly lower than that of the normal group, and the distribution of bacteria at each level was severely damaged. As shown in Figure 5 (E), Erysipelotrichia, Erysipelotrichales, Erysipelotrichaceae and Faecalibaculum played an important role in the model of NAFLD with damp-heat syndrome. The dominant species of the normal group were Bacteroidetes, Bacteroidia, Bacteroidales and Muribaculaceae. As shown in Figure 5 (F), KEGG functional enrichment was related to the pathways of Carbohydrate metabolism, Amino acid metabolism, Metabolism of terpenoids and polyketides, Metabolism of cofactors and vitamins, Metabolism of other amino acids, Lipid metabolism, Replication and repair, Energy metabolism, Xenobiotics biodegradation and metabolism, Glycan biosynthesis and metabolism, etc.

[0066] Example 4: Detection of the expression of differential genes and verification of specific genes by liver tissue transcriptome sequencing of mice

[0067] Experimental steps: (1) Extract mouse liver tissue RNA, use NanoDrop 2000 spectrophotometer to detect the purity and concentration of Total RNA, Agient2100 / LabChip GX to detect the integrity of Total RNA; (2) Library construction: after the sample detection is qualified, the library construction is carried out, first use the magnetic bead with Oligo(dT) to enrich the mRNA of eukaryote, Fragmentation Buffer to randomly break the mRNA into fragments, use mRNA as template, reverse transcription into single-strand cDNA and double-strand cDNA, and carry out cDNA purification. Purify the double-strand cDNA, and select the fragment size with AMPure XP beads, finally obtain the cDNA library through PCR enrichment; (3) Library detection: after the library is built, use Qubit 3.0 fluorescence quantifier to quantify, the concentration must reach 1 ng / μL or more, then use Qsep400 high-throughput analysis system to detect the insert fragment of the library, if the insert fragment meets the requirements, then use Q-PCR method to accurately quantify the effective concentration of the library (library effective concentration > 2nM), so as to ensure the quality of the library; (4) Machine detection: after the library detection is qualified, use Illumina NovaSeq6000 sequencing platform to carry out PE150 mode sequencing; (5) Data processing: after the sequencing data is downloaded, get CleanData, compare with the corresponding mouse reference gene, obtain Mapped Data, carry out differential expression analysis, gene function annotation and function enrichment, etc.

[0068] Western Blot detection of differential gene protein expression: take mouse liver tissue, use RIPA lysis buffer to extract total protein, use BCA method to determine protein concentration, determine the loading amount. The sample is separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane. After blocking with 5% skim milk powder for 2h, incubate the primary antibody overnight, wash with TBST for 3 times, incubate the secondary antibody for 1h, wash with TBST for 3 times, develop with ECL luminescent substrate.

[0069] Experimental results: the differential genes of liver tissue are shown in Figure 6 (A-E) According to the screening standard of absolute value of differential fold (Fold Change) ≥1.5 and false discovery rate (FDR) <0.05, the differential expression genes are obtained. Compared with the normal group, there are 846 up-regulated genes and 727 down-regulated genes in the double-factor group. By comparing the biological functions in GO, KEGG and GSEA databases, the differential expression genes of the double-factor group are mainly enriched in glycolipid metabolism, inflammatory response, immune response and oxidative stress related pathways, and the genes with significant difference in each related pathway are screened out. As shown in Figure 6(F-G) showed that RNA-seq screened out characteristic genes Cpt1b, Cyp4a10, Zbp1 and Irf7 of NAFLD of damp-heat syndrome type. Among them, Cpt1b and Cyp4a10 are involved in fatty acid oxidation process. Cyp4a10, Zbp1 and Irf7 are involved in the regulation of inflammatory response, immune balance, etc. In the present application, single gene GSEA analysis found that Cpt1b and Cyp4a10 were closely related to lipid metabolism pathway, and Zbp1 and Irf7 were closely related to immune pathway. The qPCR results confirmed that compared with the normal group, the expression of Cpt1b, Cyp4a10, Zbp1, Irf7 gene mRNA was significantly increased; and the Western Blot results confirmed that compared with the normal group, the protein expression of Cyp4a10, Zbp1 and Irf7 was increased.

[0070] Conclusion: By feeding high-fat and high-sugar diet for 12 weeks combined with high temperature and high humidity environment intervention for 6 weeks, the modeling method can ensure that the model is closer to the clinical manifestations of NAFLD of damp-heat syndrome, which is reflected in macroscopic indicators and pathological changes (increased body weight, decreased food intake, decreased water intake, increased anal temperature, decreased grip strength, decreased exercise, sticky hair, liver pathological tissue with a large number of fat vacuoles and red lipid droplets), changes in sugar and lipid metabolism indicators (increased fasting blood glucose, random blood glucose, serum blood glucose, glucose tolerance, insulin tolerance, total cholesterol, triglyceride, high-density lipoprotein, low-density lipoprotein), changes in inflammatory factors (increased IL1β, IL6, TNF-α, and decreased IL10), changes in immune indicators (increased M1 macrophages and decreased M2 macrophages), changes in oxidative stress indicators (decreased SOD and GSH, increased NO, and increased MDA), changes in intestinal flora biodiversity, Cpt1b, Cyp4a10, Zbp1 and Irf7 as potential marker genes of the disease model. The present application lays a foundation for the basic research and clinical new drug development of NAFLD of damp-heat syndrome.

[0071] The above embodiments are embodiments of the present patent, and appropriate modifications can be made without departing from the technical principles of the present patent. The modifications shall be within the scope of protection of the present patent.

Claims

1. A method for constructing and detecting a non-alcoholic fatty liver disease damp-heat syndrome model, characterized in that, The method comprises the following steps: (1) using high-fat and high-sugar diet and high temperature and high humidity environment to intervene the experimental animals, the high temperature and high humidity environment refers to placing the model animals into a small animal incubator, setting the temperature to 35℃ and the humidity to 95%, and intervening for 8 hours per day; the model animals are mice; (2) evaluating and detecting the non-alcoholic fatty liver disease damp-heat syndrome model; the evaluation and detection of the model animals include macroscopic indexes, pathological tissues, sugar and lipid metabolism indexes, inflammation indexes, intestinal flora changes and expression of specific genes, the specific genes include cyp4a10, Irf7, cpt1b and zbp1.

2. The method for constructing and detecting the non-alcoholic fatty liver disease damp-heat syndrome model according to claim 1, wherein the evaluation and detection of the model animals further include immune indexes and oxidative stress indexes.

3. The method according to claim 1, wherein the method for constructing and detecting the non-alcoholic fatty liver disease with damp-heat syndrome model is characterized by: The macroscopic indexes include the body weight, food intake, water intake, grip strength, anal temperature, oily hair and open field test of the model animals.

4. The method according to claim 1, wherein the method for constructing and detecting the non-alcoholic fatty liver disease with damp-heat syndrome model is characterized by: The pathological tissue indexes include HE staining and oil red O staining to observe the liver tissue changes.

5. The method according to claim 1, wherein the method is characterized by: The sugar and lipid metabolism indexes include fasting blood glucose, random blood glucose, serum blood glucose, glucose tolerance, insulin tolerance, serum total cholesterol, triglyceride, high-density lipoprotein and low-density lipoprotein.

6. The method according to claim 1, wherein the method is characterized by: The inflammation indexes include IL1β, IL6, IL10 and TNF-α of the liver tissue.

7. The method according to claim 2, wherein the method is characterized by: The immune indexes include M1 and M2 type macrophages of the liver tissue; the oxidative stress indexes include superoxide dismutase, reduced glutathione, nitric oxide and malondialdehyde of the liver tissue.

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