A zebrafish-based liver fibrosis disease model rapid construction method

Abstract

The present application belongs to the technical field of biology, and particularly relates to a method for rapidly constructing a liver fibrosis disease model based on zebrafish. In the present application, non-alcoholic fatty liver disease (NAFLD) zebrafish are exposed to different concentrations (250 ng / L, 5 μg / L and 100 μg / L) of chlorinated polyfluoroalkyl ether potassium sulfonate F-53B for 28 days, and it is found that a low concentration (i.e. 250 ng / L) exposure can successfully construct a liver fibrosis zebrafish model. The technical scheme of the present application has a total modeling time of 12 weeks, has a short modeling time and high stability; the liver fibrosis model based on zebrafish has low breeding cost, a short experimental period, and small individuals, so that high-throughput drug screening can be carried out, and a ng / L-level exposure concentration can induce liver fibrosis, so that the exposure concentration is low and environmental pollution is not easy to cause.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a method for rapidly constructing a zebrafish-based liver fibrosis disease model. Background Technology

[0002] The liver, the largest gland in an animal's body, performs multiple functions including digestion, metabolism, excretion, detoxification, and immunity, making it the largest metabolic organ in the animal. Over the past few decades, chronic liver disease has become one of the leading causes of disease and death worldwide. It is estimated that more than one-fifth of China's population is affected by some form of liver disease; liver disease causes nearly 2 million deaths globally each year, posing a serious threat to human health. Liver fibrosis is a type of liver disease and a common pathological feature of advanced chronic liver diseases such as viral hepatitis, alcoholic liver disease, metabolic dysfunction-related fatty liver disease, and cholestatic liver disease. After liver damage causes massive death of hepatocytes, extracellular matrix proteins and fibrous connective tissue excessively deposit in the liver, causing fibrosis. Subsequently, the excessive fibrosis further alters the liver structure, forming scars and regenerative nodules, leading to abnormal liver function and increased hepatic blood flow resistance, threatening life. Therefore, finding therapeutic targets and effective drugs for liver fibrosis is urgent and necessary.

[0003] The establishment of liver fibrosis disease models is essential for screening therapeutic targets and effective drugs. Rodent models have been widely used in liver fibrosis research, but they suffer from high breeding costs, long experimental cycles, and limitations in high-throughput drug screening. Even with mature rodent liver disease research techniques, several drawbacks hinder their application in identifying therapeutic targets and screening effective drugs for liver fibrosis. Furthermore, a single animal model cannot encompass all aspects of human liver diseases. Combining multiple animal models is more conducive to understanding the development of liver fibrosis and identifying therapeutic targets and drugs.

[0004] Zebrafish are widely used as a vertebrate model organism in physiological, toxicological, and molecular genetic studies. The zebrafish genome shares 87% homology with the human genome, making it a powerful tool for researching human diseases. Using zebrafish to establish disease models offers advantages such as short experimental cycles, high modeling efficiency, and low cost, while also providing high comparability and predictability compared to rodent experiments, effectively bridging the significant biological gap between in vivo and in vitro experiments. The zebrafish liver consists of three adjacent lobes along the intestine and plays a crucial role in metabolic homeostasis, including the processing of carbohydrates, proteins, lipids, and vitamins, the synthesis of albumin, fibrinogen, and complement factors, and detoxification. Four days after fertilization, the zebrafish liver develops all major cell types and begins to function.

[0005] Traditional liver fibrosis models are mainly constructed based on methods such as chemical induction, dietary induction, and gene editing. Among these, dietary induction is time-consuming and lacks reproducibility, limiting its application; gene editing methods are costly and require long gene vector construction times, also limiting their application; chemical induction models are highly popular among researchers due to their reproducibility and strong similarity to human liver fibrosis.

[0006] Currently, the main substances used to construct zebrafish liver fibrosis models based on chemical induction methods are as follows:

[0007] (1) Thioacetamide (TAA). Adult zebrafish were injected intraperitoneally with TAA (300 mg / kg) three times a week for six consecutive weeks, and liver fibrosis modeling was successfully established. After TAA immersion treatment, zebrafish showed fatty degeneration and collagen fiber deposition in the liver similar to those after intraperitoneal injection of TAA.

[0008] (2) Diethylnitrosamine (DEN). DEN is a water-soluble substance with stable physicochemical properties and can be administered directly via water bath. A stable zebrafish liver fibrosis model can be obtained by intervention at a concentration of 200 mg / L for 4–6 weeks.

[0009] (3) Brominated polystyrene (BPS). Similar to DEN, BPS tends to cause lipid metabolism disorders, leading to liver fibrosis in zebrafish. BPS promotes the accumulation of fat in the vascular lining and liver. In juvenile fish exposed to BPS for extended periods, the unfolded protein response (UPR) in the liver is activated, resulting in excessive accumulation of triglycerides in the liver of male zebrafish, which gradually develops into liver fibrosis.

[0010] (4) Drugs. ① Paris polyphylla is a traditional Chinese medicine with antibacterial and anti-inflammatory effects. Depending on the extractant, Paris polyphylla extracts can be classified into many types. Among them, the ethyl acetate extract (AcOEtE) can cause liver lipid metabolism disorders and induce liver fibrosis in juvenile fish. ② After 72 hours of exposure to 0.5–2 mmol / L azithromycin in a water bath, the liver color of juvenile fish darkened, and the liver area decreased. With increasing drug concentration, the expression of the liver fibrosis marker TGF-β1 gene was upregulated. ③ Mifepristone can induce an increase in the expression of the TGF-β1 gene in zebrafish, and significantly increase the expression of laminin and collagen, thus inducing liver fibrosis.

[0011] Based on the above, previous methods for constructing liver fibrosis have the following shortcomings:

[0012] (1) The construction of liver fibrosis models based on rodents has problems such as high breeding costs, long experimental cycles, and inability to conduct high-throughput screening of therapeutic drugs.

[0013] (2) The diet-induced liver fibrosis model is time-consuming and lacks reproducibility;

[0014] (3) Gene editing methods for constructing liver fibrosis models are costly and time-consuming;

[0015] (4) The liver fibrosis model constructed based on chemical induction method has extremely high compound exposure concentrations (reaching mg / L or mg / kg level), which can easily cause environmental pollution. Summary of the Invention

[0016] To address the problems existing in the prior art, this invention provides a rapid method for constructing a zebrafish-based liver fibrosis disease model, aiming to solve or at least alleviate some of the problems in the prior art. The inventors exposed zebrafish with non-alcoholic fatty liver disease (NAFLD) to different concentrations (250 ng / L, 5 μg / L, and 100 μg / L) of potassium chlorofluoroalkyl ether sulfonate F-53B for 28 days, finding that a low concentration (i.e., 250 ng / L) of exposure was sufficient to successfully construct a zebrafish model of liver fibrosis. This invention overcomes the aforementioned deficiencies, enabling the construction of a liver fibrosis model using low-concentration compound exposure for a short period.

[0017] This invention is achieved by a method for constructing a liver fibrosis model, which involves exposing zebrafish to water containing potassium chlorinated polyfluoroalkyl ether sulfonate.

[0018] Furthermore, the zebrafish were induced to develop non-alcoholic fatty liver disease and then exposed to water containing potassium chlorinated polyfluoroalkyl ether sulfonate.

[0019] Furthermore, the concentration of the potassium chloropolyfluoroalkyl ether sulfonate is 0.25 μg / L or higher, preferably 0.25 μg / L-100 μg / L.

[0020] Furthermore, the exposure time was more than 28 days.

[0021] Furthermore, a zebrafish model of non-alcoholic fatty liver was induced through a high-fat diet intervention.

[0022] Furthermore, the zebrafish is a 3-month-old adult zebrafish.

[0023] This invention also provides the application of potassium chloropolyfluoroalkyl ether sulfonate in the preparation of reagents for liver fibrosis.

[0024] In summary, the advantages and positive effects of this invention are as follows:

[0025] (1) The zebrafish liver fibrosis model has low breeding cost, short experimental cycle, and small size, which allows for high-throughput drug screening.

[0026] (2) First, an obese NAFLD zebrafish model was established to mimic the typical pathological characteristics of obesity in humans.

[0027] (3) The total molding time is 12 weeks, which is short and has high stability;

[0028] (4) Exposure concentrations at the ng / L level can induce liver fibrosis. The low exposure concentrations are unlikely to cause environmental pollution. Attached Figure Description

[0029] Figure 1 This document describes the construction of a zebrafish NAFLD model; (A) images of fish from the normal diet group and the high-fat diet group; (B) histopathological analysis of liver sections from the normal diet group and the high-fat diet group; and (C) quantitative analysis of body mass index (BMI), relative liver weight, liver vacuolation density, Oil Red staining density, triglycerides, and total cholesterol under different intervention modes. Black arrows indicate liver vacuolation. Values ​​= mean ± standard deviation. * p<0.05; ** p<0.01; *** ,p<0.001.

[0030] Figure 2 This study describes the construction of a zebrafish liver fibrosis model; (A) Masson staining of zebrafish liver sections from normal and high-fat diet groups under different F-53B exposure concentrations; and (B) detection of liver fibrosis biomarkers (α1-I type collagen fibers, prothrombin / coagulation factor II, and hyaluronic acid) in the livers of zebrafish from normal and high-fat diet groups under different F-53B exposure concentrations. Yellow arrows indicate liver fibrosis. Values ​​= mean ± standard deviation. * p<0.05; ** p<0.01; *** ,p<0.001. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0032] Based on the information contained in this application, various changes to the precise description of the invention can be readily made by those skilled in the art without departing from the spirit and scope of the appended claims. It should be understood that the scope of the invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are merely illustrative of specific aspects of the invention. In fact, various modifications to embodiments of the invention that will be apparent to those skilled in the art or related fields are covered within the scope of the appended claims.

[0033] To better understand the invention and not to limit its scope, all figures indicating amounts, percentages, and other numerical values ​​used in this application should, in all cases, be understood to be modified by the word "about". Therefore, unless specifically stated otherwise, the numerical parameters listed in the specification and appended claims are approximate values ​​and may vary depending on the desired properties being sought. Each numerical parameter should at least be considered as obtained based on reported significant figures and through conventional rounding methods. In this invention, "about" means within 10%, preferably within 5%, of a given value or range.

[0034] Unless otherwise specified, the following embodiments of the present invention are all under normal temperature conditions. Normal temperature refers to the natural room temperature conditions in all four seasons, without additional cooling or heating treatment. Generally, the normal temperature is controlled between 10 and 30°C, preferably between 15 and 25°C.

[0035] This invention discloses a method for rapidly constructing a zebrafish-based liver fibrosis disease model. The technical solution of this invention will be clearly and completely described below with reference to embodiments thereof.

[0036] Example 1

[0037] 1. Induction of a zebrafish model of non-alcoholic fatty liver disease (NAFLD)

[0038] Three-month-old wild-type adult zebrafish were cultured in a zebrafish culture system (Tecniplast Zebtec, Tecniplast, Buguggiate, Italy) at 27±1℃ with a photoperiod of 14h / 10h. Half of the fish were subjected to a high-fat diet (HFD) intervention for the first 8 weeks to induce a non-alcoholic fatty liver disease (NAFLD) model, with each fish fed 0.1 g / day of egg yolk and 0.25 g / day of brine shrimp three times a day. The remaining half of the fish were fed 0.06 g / day of brine shrimp twice a day for 8 weeks, serving as the normal diet group (ND). After 8 weeks of dietary intervention, the fish were photographed to record body length, weight, BMI, and relative liver weight. Liver samples obtained from dissection were used for subsequent hematoxylin / eosin (HE) staining, Oil Red (ORO) staining, Masson's staining, and quantitative analysis of triglycerides (TG) and total cholesterol (T-CHO) to validate the establishment of the NAFLD model. BMI was calculated as body weight / body length. 2 Relative liver weight = liver weight / body weight.

[0039] Detection Method: Liver samples from each fish in each group were lysed in 300 μL PBS (NaCl 8 g / L, KCl 0.2 g / L, Na2HPO4 1.44 g / L, KH2PO4 0.24 g / L, pH 7.4), and then centrifuged at 3,000 g for 10 min at 4°C to obtain the supernatant. The supernatant was analyzed for triglyceride (TG), total cholesterol (T-CHO), α1-I collagen fibers (Col1A1), prothrombin / factor II (PT / FII), and hyaluronic acid (HA) levels. Kits for triglyceride (TG), total cholesterol (T-CHO), and α1-I collagen fibers (Col1A1) were purchased from Nanjing Jiancheng Bioengineering Institute, and kits for prothrombin / factor II (PT / FII) and hyaluronic acid (HA) were purchased from Jiangsu Enzyme Immunoassay Co., Ltd.

[0040] Tissue section staining: Zebrafish liver samples obtained from dissection were fixed overnight in 4% paraformaldehyde, dehydrated, clarified, and then embedded in paraffin. Each embedded liver tissue section was cut into multiple 5μm thick sections and placed on glass slides for hematoxylin / eosin (HE) and MASSON staining. For liver samples requiring oil red staining, frozen sections of 8-10μm thickness were prepared for staining. The stained sections were observed and photographed under an optical microscope (Nikon, Eclipse Ti-U), and the liver vacuolar density and optical density of oil red positive staining areas were statistically analyzed using ImageJ software (https: / / imagej.nih.gov / ij / ).

[0041] Statistical Analysis: All data are expressed as mean ± standard deviation of mean (SEM). Statistical analysis was performed using GraphPad Prism 9.0 software (GraphPad software Inc., La Jolla, CA, USA). Unpaired t-tests and two-way ANOVA were used to determine whether differences between groups were statistically significant; p < 0.05 was considered statistically significant.

[0042] Liver fibrosis develops gradually from NAFLD; therefore, a zebrafish NAFLD model was first established through a high-fat diet intervention. Specifically, the high-fat diet induced a 26.92% increase in body mass index in zebrafish compared to the normal diet group. Figure 1 (A and C in the original text) increased the relative liver weight by 3.09 times. Figure 1 (C in the text). Further histopathological analysis showed that the high-fat diet group significantly induced fatty liver, as evidenced by a 645.95% increase in liver vacuolar density after hematoxylin / eosin staining. Figure 1 In the B and C sections, the optical density of the positive areas stained with Oil Red staining increased by 1.01 times. Figure 1 The results (B and C in the original text) indicate significant lipid accumulation in the liver of the high-fat diet group; simultaneously, the Masson staining experiment confirmed that the high-fat diet did not induce liver fibrosis. Biochemical results further confirmed the success of our NAFLD model: the triglyceride and total cholesterol levels in the liver of the high-fat diet group were significantly higher than those of the normal diet group by 46.08% and 80.78%, respectively. Figure 1 (C in the text). The above data proves that a high-fat diet intervention successfully induced NAFLD in zebrafish.

[0043] 2. Establishment of a liver fibrosis model

[0044] The established NAFLD zebrafish model and ND group zebrafish were exposed to potassium chlorofluoroalkyl ether sulfonate F-53B (purity ≥98%, CAS#73606-19-6, Shanghai Maikun Chemical Reagent Co., Ltd.) at concentrations of 250 ng / L, 5 μg / L, and 100 μg / L for 28 days. Zebrafish from the normal diet group, exposed to the same concentration, served as controls. After exposure, liver samples were obtained by dissection and used for Masson staining and to determine the levels of liver fibrosis biomarkers α1-I type collagen fibers (Col1A1), prothrombin / coagulation factor II (PT / FII), and hyaluronic acid (HA).

[0045] The results showed that F-53B induced liver fibrosis in zebrafish with NAFLD within the concentration range of 0.25 μg / L–100 μg / L, while in the normal diet group after exposure, only the 100 μg / L exposure group showed a liver fibrosis phenotype. Figure 2 A). Biochemical identification further validated the pathological section analysis. Liver fibrosis biomarkers, including α1-I type collagen fibers, prothrombin / coagulation factor II, and hyaluronic acid, were significantly altered in the livers of zebrafish fed a high-fat diet with exposure from 0.25 μg / L to 100 μg / L. In contrast, for zebrafish fed a normal diet with exposure to F-53B, only a 100 μg / L exposure concentration significantly altered the levels of α1-I type collagen fibers, prothrombin / coagulation factor II, and hyaluronic acid. Figure 2 B).

[0046] In summary, this invention successfully constructs a zebrafish liver fibrosis model in a short time using a low concentration of F-53B (0.25 μg / L) based on NAFLD zebrafish, overcoming the shortcomings of previous techniques.

[0047] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing a liver fibrosis model, characterized in that: Three-month-old adult zebrafish were induced to develop non-alcoholic fatty liver disease by a high-fat diet, and then exposed to water containing potassium chlorinated polyfluoroalkyl ether sulfonate at concentrations of 0.25 μg / L to 100 μg / L for more than 28 days.

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