Method for constructing arteriosclerosis mouse model by using humanized flora

Abstract

The invention belongs to the technical field of experimental animal model construction, and particularly relates to a method for constructing an arteriosclerosis mouse model by using humanized flora. The technical problem to be solved by the invention is to establish an arteriosclerosis phenotype humanized mouse model taking intestinal flora of a patient with coronary artery disease as a donor source. According to the technical scheme, the method for constructing the arteriosclerosis mouse model by using the humanized flora comprises the following steps: collecting an excrement sample of a patient diagnosed as arteriosclerosis, and preparing a flora suspension; the sterile mouse is subjected to gavage, gavage is conducted once every other day, and gavage is conducted five times; lavage is carried out once a week; and the whole process lasts for 10-13 weeks from the beginning of gavage. The mouse model constructed by the method can systematically evaluate key pathological phenotypes of hypercholesteremia, vascular dysfunction, immune activation and the like of the mouse.

Description

Technical Field

[0001] This invention belongs to the field of experimental animal model construction technology, specifically relating to a method for constructing an arteriosclerosis mouse model using humanized microbiota. Background Technology

[0002] Coronary artery disease (CAD) is a group of chronic, progressive vascular diseases characterized by abnormal lipid deposition in the vessel wall, chronic inflammation, abnormal proliferation of smooth muscle cells, and fibrotic remodeling. It primarily manifests as arteriosclerosis and is the common pathological basis for many major cardiovascular and cerebrovascular diseases, including coronary heart disease, stroke, and peripheral vascular disease. With population aging and changes in lifestyle, the incidence and mortality rates of arteriosclerosis-related diseases continue to rise, becoming a significant public health problem that seriously threatens human health.

[0003] Currently, animal models of arteriosclerosis mainly include high-fat diet-induced models, genetically modified models, and drug-induced models. While these models can simulate the pathological process of arteriosclerosis to some extent, they cannot accurately reflect the overall composition of the gut microbiota in human patients and its pathogenic or proliferative effects in disease development. Increasing evidence suggests that gut microbiota dysbiosis is closely related to the development of cardiovascular disease, and that there are differences in bacterial composition between healthy controls and patients with coronary artery disease. Furthermore, many existing microbiota transplantation models employ antibiotic-based methods to eliminate the gut microbiota before transplantation, which suffers from incomplete elimination, insufficient post-transplantation stability, and poor intergenerational consistency, limiting their application in mechanistic studies and long-term intervention research.

[0004] Therefore, establishing a humanized mouse model that uses the gut microbiota of patients with coronary artery disease as the donor source, germ-free mice as the recipient, has a clear construction process, stable microbiota colonization ability, and can reproducibly present the atherosclerotic phenotype is of great scientific significance and application value for elucidating the role mechanism of gut microbiota in atherosclerosis, screening potential intervention targets, and evaluating relevant prevention and treatment strategies. Summary of the Invention

[0005] The technical problem to be solved by this invention is to establish a humanized mouse model of arteriosclerosis phenotype using the gut microbiota of patients with coronary artery disease as a donor source.

[0006] The technical solution of the present invention is a method for constructing a mouse model of arteriosclerosis using humanized microbiota, comprising the following steps: collecting fecal samples from patients diagnosed with arteriosclerosis and preparing a microbiota suspension; administering gavage to sterile mice once every other day for 5 times; thereafter, gavage once a week; the entire process lasts 10 to 13 weeks from the start of gavage.

[0007] Preferably, the entire process lasts 12 weeks from the start of gavage.

[0008] Specifically, the preparation of the bacterial suspension is carried out as follows: the bacterial suspension is diluted with sterile PBS solution at a weight:volume ratio of 1:5, mixed, centrifuged, and the supernatant is collected to obtain the bacterial suspension.

[0009] Specifically, colony counting was performed on the supernatant to ensure an absorbance value of 0.92 at a wavelength of 620 nm.

[0010] Preferably, the centrifugation is performed at 4°C with 300g for 5 minutes.

[0011] Each mouse was given 200 μL of bacterial suspension by gavage each time.

[0012] Furthermore, the surfaces of the items used in the entire process of the method are sterilized with peracetic acid.

[0013] Preferably, the concentration of the peracetic acid working solution is ≥3%.

[0014] Beneficial effects of the present invention: The present invention discloses a method for constructing a mouse model of arteriosclerosis using humanized microbiota. Compared with traditional modeling methods, the method of the present invention has the following advantages: (1) Patients clinically diagnosed with arteriosclerosis were selected, and their fecal samples were collected. Through human microbiota transplantation, a causal relationship between microbiota dysbiosis and cardiovascular pathological phenotypes was directly established. No anesthesia was required, eliminating the influence of anesthetic drugs on the mice themselves and the entire experiment.

[0015] (2) No open wounds, avoiding other infection situations, and the modeling results are more stable and reliable; (3) This invention does not require microscopes or other experimental equipment. It is implanted into germ-free mice by oral gavage, saving costs.

[0016] (4) The use of standardized sterile mice and microbial transplantation procedures ensured the consistency of experimental conditions and the reproducibility of results.

[0017] (5) Mice that have completed microbial transplantation should be fed and bred in a standardized manner, and the microbial environment should be maintained by regularly supplementing the bacterial solution.

[0018] (6) The constructed mouse model was confirmed and evaluated through microbial sequencing analysis and arteriosclerosis phenotype detection.

[0019] (7) Integrating vascular, immune and microbiome analysis can comprehensively and systematically evaluate the stability and reliability of the model.

[0020] (8) This model is not only applicable to the study of coronary artery disease, but can also be extended to the study of other metabolic diseases.

[0021] This invention utilizes a non-anesthesia, non-invasive oral gavage transplantation technique to directly colonize the gut microbiota of clinical arteriosclerosis patients into germ-free mice. This not only avoids the interference of anesthesia and surgical trauma, ensuring the stability and reliability of the model, but also establishes a direct causal relationship between gut microbiota dysbiosis and cardiovascular phenotypes through a standardized process and low cost. The mouse model constructed using this method can systematically evaluate key pathological phenotypes in mice, such as hypercholesterolemia, vascular dysfunction, and immune activation. This invention provides a stable, reliable, and highly mimicking animal model of human disease for studying the pathogenesis of cardiovascular diseases, developing novel treatment strategies, and evaluating the intervention effects of probiotics or prebiotics. Attached Figure Description

[0022] Figure 1 The figures show the results of physiological indicators and vascular status detection in germ-free mice after intestinal microbiota transplantation from different sources. The microbiota from healthy donors serves as the control group (Con), while the microbiota from patients with coronary artery disease serves as the experimental group (CAD). Figure 1 Figure a shows the results of the detection of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) levels in the serum of mice in the control group and experimental group. Figure 1 b is a graph showing the weight changes of mice in the control and experimental groups during the feeding period. Figure 1 Figure c shows the measurement results of the pulse wave velocity in the left common carotid artery of mice in the control group and the experimental group, which is used to reflect the arterial stiffness.

[0023] Figure 2 The figures show the results of physiological indicators and vascular status detection in germ-free mice after intestinal microbiota transplantation from different sources. The microbiota from healthy donors serves as the control group (Con), while the microbiota from patients with coronary artery disease serves as the experimental group (CAD). Figure 2 Image d shows microscopic images of aortic tissue sections from the control and experimental groups after Masson's trichrome staining, used to show the distribution of collagen fibers in the blood vessel wall. Figure 2 e shows the fluorescence images and quantitative analysis results of the aortic tissue of mice in the control and experimental groups after dihydroethidium (DHE) staining, which are used to reflect the level of reactive oxygen species-related fluorescence signals in the tissue.

[0024] Figure 3 This figure shows the changes in gut microbiota composition and its correlation with bile acid metabolism in recipient mice after fecal microbiota transplantation. The control group (Con) consisted of mice with gut microbiota transplanted from healthy donors, while the experimental group (CAD) consisted of mice with gut microbiota transplanted from patients with coronary artery disease. Figure 3Figure 'a' shows the results of principal coordinate analysis (PCoA) based on the Bray-Curtis distance matrix, used to display the clustering distribution of the gut microbiota community structure in human donor samples and recipient mouse samples. Figure 3 b is a composition distribution diagram of human donor samples and recipient mouse samples at the phylum level, representing the average proportion of each phylum in the overall community. Figure 3 c is a heatmap showing the relative abundance of KEGG orthologs (KOs) related to secondary bile acid metabolism between the control group and the experimental group, and also shows the distribution of representative KO genes in the "secondary bile acid metabolism" pathway module of the KEGG pathway.

[0025] Figure 4 This figure shows the changes in gut microbiota composition and its correlation with bile acid metabolism in recipient mice after fecal microbiota transplantation. The control group (Con) consisted of mice with gut microbiota transplanted from healthy donors, while the experimental group (CAD) consisted of mice with gut microbiota transplanted from patients with coronary artery disease. Figure 4 Figure d shows the Spearman correlation analysis results between the relative abundance of related genes in the gut microbiota and the level of bile acids in fecal samples. Figure 4 e is a graph showing the comparison of the relative abundance of species involved in secondary bile acid transformation between the control group and the experimental group, where the box plot is used to represent the distribution of sample data.

[0026] Figure 5 The figures show the distribution of immune-related cells and the detection results of tight junction proteins in the spleen and small intestine of mice after transplantation of gut microbiota from different sources. The control group consisted of healthy control mice (Con) with gut microbiota transplantation, while the experimental group consisted of mice with gut microbiota transplantation from patients with coronary artery disease (CAD). Figure 5 Figure a shows the results of detecting the proportion of RORγt+T cells in the CD4+T cell population in the spleen of mice in the control group and experimental group. Figure 5 b is a schematic diagram (left) showing the distribution of regulatory T cells (CD4+CD8a⁻CD45+Foxp4+) in the spleen of mice in the control group and experimental group, and a statistical result of their proportion in the spleen (right). Figure 5 Figure c shows the results of detecting the proportion of Th17 cells (CD4+CD8a⁻IL-17a+) in the CD4+ T cell population of the small intestinal lamina propria lymphocytes in the control group and experimental group mice. Figure 5 Figure d shows the distribution of regulatory T cells in the lamina propria of the small intestine of mice in the control and experimental groups, and the statistical results of their proportions. Figure 5 e is a schematic diagram of the distribution of dendritic cells (CD11c+CD103+) in the lamina propria lymphocytes of the small intestine of mice in the control group and experimental group, and a statistical result of their proportion. Figure 5f is a graph showing the statistical results of the ratio of Th17 cells to regulatory T cells in the lamina propria of the small intestine of mice in the control group and the experimental group. Figure 5 Figure g shows the results of detecting the expression of tight junction-related proteins Claudin-1 and ZO-1 in the ileum tissue of mice in the control and experimental groups. Detailed Implementation

[0027] This invention discloses a method for constructing a mouse disease model using humanized gut microbiota. The method first involves isolating feces from a group of patients with coronary artery disease. Subsequently, the obtained microbial suspension is orally injected into germ-free mice, allowing the mice to acquire a microbial community similar to that of humans. By comprehensively utilizing molecular biology, histology, and immunology techniques, key pathological phenotypes in mice, such as hypercholesterolemia, vascular dysfunction, and immune activation, can be systematically evaluated. This invention provides a stable, reliable, and highly mimicking animal model of cardiovascular disease for studying its pathogenesis, developing novel treatment strategies, and evaluating the intervention effects of probiotics or prebiotics.

[0028] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0029] The main reagents and instruments used in the following examples were sourced from the following sources: sterile PBS buffer (Yuanpei Company); peracetic acid (Sinopharm Chemical Reagent Co., Ltd.); fecal DNA extraction kit (QIAGEN); Illumina HiSeq X Ten sequencing platform (Illumina); flow cytometer (BD Biosciences); Western blotting reagents (ThermoFisher Scientific); clean bench (ESCO); high-speed refrigerated centrifuge (Eppendorf); microplate reader (Ausun Company); chemiluminescence imaging system (Tianneng); lipid assay kits (TC, TG, LDL, HDL) (Beijing Jiancheng Bioengineering Institute); Oil Red O staining reagent (Sigma-Aldrich); flow cytometry antibody (BD Biosciences).

[0030] Example 1: Transplantation of fecal microbiota from CAD patients into germ-free mice led to lipid dysregulation and vascular atherosclerosis in the germ-free mice. Patients clinically diagnosed with arteriosclerosis were selected, and fresh fecal and blood samples were collected. Sterile sampling containers were used during collection, and samples were immediately flash-frozen in liquid nitrogen and stored at -80°C for long-term storage. Fecal suspensions were prepared and transplanted into sterile mice via gavage every other day for 5 cycles, then weekly. The entire process lasted 10–13 weeks from the start of gavage. Throughout the procedure, aseptic treatment of peracetic acid was maintained on all surfaces, and the working solution concentration was ≥3%. Serum total lipoprotein, high-density lipoprotein, and low-density lipoprotein levels were assessed in mice; oil red staining of blood vessels revealed plaques, indicating symptoms of arteriosclerosis; and reactive oxygen species (ROS) levels were measured.

[0031] Inclusion criteria for donors: Patients with a clinically confirmed diagnosis of coronary artery disease (CAD) and myocardial infarction were selected as donors in the experimental group, while healthy volunteers matched for age and sex were selected as donors in the control group.

[0032] Sample collection: Fresh fecal samples were collected from the donor, aliquoted into sterile containers, and immediately flash-frozen in liquid nitrogen. The samples were then transferred to an ultra-low temperature freezer at -80°C for long-term storage, or not frozen and immediately used for subsequent experiments. Simultaneously, fasting venous blood was collected from the donor, serum was separated, and cryopreserved for later use.

[0033] Preparation of bacterial suspension and sterile mouse transplantation: Frozen fecal samples from patients were retrieved and processed in a laminar flow hood. The fecal samples were thoroughly ground using two sterilized glass slides and transferred to sterile centrifuge tubes for weighing. Sterile PBS solution was added at a weight:volume ratio of 1:5, and the mixture was thoroughly vortexed for 10 seconds, repeated three times. The mixture was then centrifuged at 300g for 5 minutes at 4°C, and the supernatant was used for subsequent experiments. Colony counting was performed on the supernatant, and its absorbance at 620nm was measured. The supernatant was aliquoted according to a standardized bacterial count, 200μL per tube, for later use. The OD value at 620nm was measured to be 0.92 (1 OD unit = 0.8 × 10⁻⁶). 9 CFU), at 200 μL per tube (approximately 0.74 × 10⁻⁶ CFU). 9 CFU (Cellular Fuel Intake) was aliquoted, labeled, and stored at -80℃ for later use. Each mouse was administered 200 μL of the bacterial suspension or PBS via gavage. The transplantation cycle was as follows: gavage every other day for 5 consecutive days (days 1, 3, 5, 7, and 9); thereafter, gavage was administered weekly until the end of the experiment to ensure long-term stable colonization of the bacterial flora (approximately once a week for 12 weeks). In animal model construction, functional and phenotypic indicators are typically used as experimental endpoints. Specifically, the model construction was considered complete and the experiment terminated when the recipient mice met at least one of the following conditions: (1) The gut microbiota of the recipient mice, as analyzed by 16S rDNA sequencing, showed a high degree of consistency with the donor microbiota in terms of community structure; (2) Recipient mice exhibit metabolic or pathological phenotypes consistent with donor diseases, such as lipid deposition or dyslipidemia; (3) The presence of immune activation or intestinal barrier damage associated with donor disease.

[0034] Serum lipid analysis: At the experimental endpoint, mouse blood was collected and serum was separated. Serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) levels were measured using commercially available kits.

[0035] Vascular function and structural assessment: a. Pulse wave velocity measurement: The pulse wave velocity of the left common carotid artery in mice was non-invasively measured using a high-resolution ultrasound imaging system to assess arterial stiffness. b. Aortic histological analysis: The aortic arch to thoracic aorta segment from mice was paraffin-embedded and sectioned. Collagen deposition was observed using Masson's trichrome staining method. c. Detection of reactive oxygen species (ROS): Since the generation of ROS can trigger endothelial cell apoptosis and alter vasomotor function, the level of superoxide in frozen sections of the aorta was detected using a dihydroethidium fluorescent probe.

[0036] Experimental results ( Figure 1 and Figure 2 Mice transplanted with CAD-associated bacteria exhibited increased vascular stiffness. Collagen deposition levels were elevated in the aorta of CAD-associated mice. Overall superoxide production was increased in the aorta of CAD-associated mice. These results suggest that CAD-associated bacteria promote atherosclerosis through intravascular ROS and vascular fibrosis.

[0037] Example 2: Fecal microbiota transplantation from CAD patients into germ-free mice resulted in disordered gut microbiota composition and bile acid metabolism in the germ-free mice, similar to the donor composition. Fecal samples were collected from mice at the experimental endpoint, and metagenomic shotgun sequencing was performed on the feces to investigate changes in gut microbiota structure and metabolic function. The expression of key genes involved in bile acid synthesis and regulation in the ileum and liver was detected using qRT-PCR.

[0038] Metagenomic Sequencing and Microbial Analysis: Mouse feces were collected, and microbial DNA was extracted using a fecal DNA extraction kit. After passing quality checks, a DNA library was constructed, and shotgun metagenomic sequencing was performed on the Illumina HiSeq XTen platform. Low-quality reads and host genome contamination were removed to obtain high-quality data. De novo assembly of high-quality reads was performed using IDBA_UD software. Gene prediction results were compared with the NCBI NR and KEGG databases for taxonomic and functional gene (KO) annotation. Based on the gene abundance table, the Shannon index was calculated to assess α-diversity, and Bray-Curtis distance was used for principal coordinate analysis to assess β-diversity. The Wilcoxon rank-sum test was used to compare the abundance differences of species, genes, or pathways between groups.

[0039] "Disorders of gut microbiota composition and bile acid metabolism" refer to abnormal changes in the species structure and metabolic functions of the gut microbiota, specifically manifested as: (1) abnormal enrichment of genera with bile acid conversion functions (such as Clostridium, Eggerthella, etc.); (2) enhanced conversion of primary bile acids to secondary bile acids (such as lithocholic acid, deoxycholic acid); and (3) imbalance of host bile acid signaling pathways, which in turn affect lipid metabolism and inflammatory responses. Numerous clinical and animal studies have confirmed that the above-mentioned abnormalities in gut microbiota and bile acid metabolism are common in patients with coronary artery disease.

[0040] Figure 3 and Figure 4The results showed that transplanting gut microbiota from CAD patients not only successfully altered the gut microbiota structure of recipient mice, but more importantly, the enriched specific functional bacteria (such as *Clostridium symbiosum* and *Eggerthella*) disrupted the host's bile acid metabolic homeostasis by enhancing the biosynthesis of secondary bile acids (especially lithocholic acid and deoxycholic acid). Principal coordinate analysis indicated that the gut microbiota structure of the recipient mice clustered with their respective human donors, successfully achieving colonization of human-derived microbiota. At the phylum level, the experimental group mice showed relatively low abundance of Bacteroidetes and Firmicutes, while Proteobacteria were more abundant. In the experimental group microbiota, key genes (such as baiB) responsible for the 7α-dehydroxylation pathway, which converts primary bile acids to secondary bile acids (such as lithocholic acid), and hydroxysteroid dehydrogenase genes were significantly enriched. The abundance of these genes was strongly positively correlated with the levels of lithocholic acid and 7-ketolithocholic acid in feces. Species-level analysis revealed that the intestines of the experimental group mice were rich in bacteria with bile acid conversion functions, such as Clostridium symbiosum, Eggerthella, and Bacteroides intestinalis. Among them, the abundance of Clostridium symbiosum was significantly positively correlated with the host's serum cholesterol level.

[0041] Example 3: Transplantation of fecal microbiota from CAD patients into germ-free mice resulted in immune activation and intestinal disruption in the germ-free mice. Spleens and small intestines were collected from mice at the experimental endpoint. Splenic mononuclear cells and small intestinal lamina propria immune cell samples were extracted and analyzed by flow cytometry. The following immune cell subsets were specifically examined and compared between the control and experimental groups: the proportion of RORγt⁺ cells in spleen CD4⁺ T cells; the proportion of regulatory T cells (phenotype CD4⁺CD8a⁻CD25⁺Foxp3⁺) in spleen; the proportion of Th17 cells (phenotype CD4⁺CD8a⁻IL-17a⁺) in small intestinal lamina propria lymphocytes; the proportion of regulatory T cells in small intestinal lamina propria lymphocytes; and the proportion of dendritic cells (phenotype CD11c⁺CD103⁺) in small intestinal lamina propria lymphocytes. The ratio of Th17 cells to regulatory T cells in small intestinal lamina propria lymphocytes was calculated. In addition, total protein was extracted from the ileum tissue of mice in each group, and the expression levels of tight junction proteins Claudin-1 and ZO-1 were detected by Western blotting.

[0042] Flow cytometry: Lymphocytes from the spleen and lamina propria of the small intestine of mice were isolated, collected, stained with antibodies at 4°C in the dark for 30 min, washed, resuspended in 200 μL PBS, and detected by flow cytometer. Changes were analyzed using Flowjo.

[0043] Western blotting: Cells were lysed using RIPA lysis buffer, and total protein was extracted. Protein separation was performed by SDS-PAGE electrophoresis, followed by transfer to an NC membrane, blocking with 5% skim milk, and immunoassay with tight junction-related proteins Claudin-1 and ZO-1 (primary antibodies), followed by incubation overnight at 4°C. HRP-labeled secondary antibody was added and incubated at room temperature for 1 h. Finally, electrochemiluminescence was used for detection, and the gel was visualized using a chemiluminescent gel imaging system. All data were statistically analyzed and plotted using GraphPad Prism.

[0044] Figure 5 Experimental Results: Immunological activation and intestinal disruption in germ-free mice. The proportion of RORγt⁺ cells in spleen CD4⁺ T cells was increased; the proportion of regulatory T cells (phenotype CD4⁺CD8a⁻CD25⁺Foxp3⁺) in the spleen was decreased; the proportion of Th17 cells (phenotype CD4⁺CD8a⁻IL-17a⁺) in small intestinal lamina propria lymphocytes was increased; the proportion of regulatory T cells in small intestinal lamina propria lymphocytes was decreased; the proportion of dendritic cells (phenotype CD11c⁺CD103⁺) in small intestinal lamina propria lymphocytes was increased; and the ratio of Th17 cells to regulatory T cells in small intestinal lamina propria lymphocytes was increased. The expression levels of tight junction proteins Claudin-1 and ZO-1 were decreased.

[0045] The results of this embodiment demonstrate that the mouse model constructed by transplanting gut microbiota derived from CAD patients exhibits a high degree of consistency with known pathological characteristics of coronary artery disease patients, not only in terms of metabolism and vascular phenotype, but also in terms of immune activation and intestinal barrier disruption. These results further prove, from an immunological and intestinal barrier function perspective, that the mouse model constructed in this invention has good mimicry and biological relevance to human disease states.

[0046] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for constructing a mouse model of arteriosclerosis using humanized microbiota, characterized in that: The procedure includes the following steps: collecting fecal samples from patients diagnosed with arteriosclerosis and preparing a bacterial suspension; administering the suspension to sterile mice by gavage once every other day for 5 times; then switching to once a week thereafter; the entire process lasts 10 to 13 weeks from the start of the gavage.

2. The method according to claim 1, characterized in that: The specific steps for preparing the bacterial suspension are as follows: dilute with sterile PBS solution at a weight:volume ratio of 1:5, mix well, centrifuge, collect the supernatant, and obtain the bacterial suspension.

3. The method according to claim 2, characterized in that: Colony counting was performed on the supernatant to ensure an absorbance value of 0.92 at a wavelength of 620 nm.

4. The method according to claim 2, characterized in that: The centrifugation was performed at 300g for 5 minutes at 4°C.

5. The method according to claim 4, characterized in that: Each mouse was given 200 μL of bacterial suspension by gavage each time.

6. The method according to claim 1, characterized in that: The method involves sterilizing the surfaces of all items used in the entire process with peracetic acid.

7. The method according to claim 6, characterized in that: The concentration of the peracetic acid working solution is ≥3%.

8. The method according to claim 1, characterized in that: The entire process, starting from the start of gavage, lasts 12 weeks.

Images