Rapid construction method of hepatic fibrosis disease model based on zebra fish

Abstract

The invention belongs to the technical field of biology, and particularly relates to a rapid construction method of a hepatic fibrosis disease model based on zebra fish. According to the invention, the non-alcoholic fatty liver disease (NAFLD) zebra fish is exposed for 28 days by using chloropolyfluoroalkyl ether potassium sulfonate F-53B with different concentrations (250ng / L, 5 [mu] g / L and 100 [mu] g / L), and the result shows that the hepatic fibrosis zebra fish model can be successfully constructed by low-concentration (250ng / L) exposure. According to the technical scheme, the modeling time is 12 weeks, the modeling time is short, and the stability is high; the hepatic fibrosis model constructed based on zebrafish is low in culture cost, short in experimental period and small in size, so that high-throughput drug screening can be performed, hepatic fibrosis can be induced by the ng / L-level exposure concentration, the exposure concentration is low, and environmental pollution is not easily caused.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for rapidly constructing a liver fibrosis disease model based on zebrafish. Background Art

[0002] The liver is the largest gland in the animal body and has multiple functions such as digestion, metabolism, excretion, detoxification, and immunity. It is the largest metabolic organ in the animal body. In the past few decades, chronic liver diseases have become one of the major diseases and causes of death globally. It is estimated that more than one-fifth of the population in China is affected by some form of liver disease; liver diseases cause nearly 2 million deaths globally every year, posing a serious threat to human health. Liver fibrosis is one of the liver diseases and is a common pathological feature of advanced chronic liver diseases such as viral hepatitis, alcoholic liver disease, metabolic dysfunction-related steatohepatitis, and cholestatic liver disease. After a large number of hepatocytes die due to liver injury, extracellular matrix proteins and fibrous connective tissues will be excessively deposited in the liver, causing fibrosis. Subsequently, the excessive fibers produced by liver fibrosis further change the liver structure, forming scars and regenerative nodules, resulting in abnormal liver function and increased liver blood flow resistance, threatening life. Therefore, it is urgent and necessary to find treatment targets and effective treatment drugs for liver fibrosis.

[0003] The screening of liver fibrosis treatment targets and effective drugs is inseparable from the establishment of liver fibrosis disease models. Rodent models have been widely used in liver fibrosis research, but they have problems such as high breeding costs, long experimental periods, and inability to perform high-throughput drug screening. Even under the condition that rodent liver disease research techniques are mature, there are still many disadvantages that make it difficult to apply them to the determination of liver fibrosis treatment targets and the screening of effective drugs; at the same time, a single animal model cannot cover all aspects of human liver diseases. Combining multiple animal models is more conducive to understanding the development of liver fibrosis diseases, the determination of treatment targets, and drugs.

[0004] Zebrafish is a vertebrate model animal widely used in physiological, toxicological, and molecular genetic research. The zebrafish genome has 87% homology with the human genome, making zebrafish a powerful tool for studying human diseases. Establishing a disease model using zebrafish not only has advantages such as a short experimental period, high model formation efficiency, and low modeling cost, but also has advantages such as high comparability and strong predictability in rodent experiments, and can effectively bridge the huge biological gap between in vivo and in vitro experiments. The zebrafish liver consists of 3 adjacent lobes along the intestine and plays an important role in metabolic homeostasis, including the processing of carbohydrates, proteins, lipids, and vitamins, the synthesis of albumin, fibrinogen, and complement factors, and detoxification functions. 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, diet induction, and gene editing. Among them, diet induction has a long time and lack of reproducibility, resulting in limited application; gene editing methods are also limited in application due to high cost and long gene vector construction time; chemical induction models are very popular among researchers because of their reproducibility and great similarity to human liver fibrosis.

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

[0007] (1) Thioacetamide (TAA). Adult zebrafish are intraperitoneally injected with TAA (300 mg / kg) three times a week for 6 consecutive weeks to show successful liver fibrosis modeling; after treatment of zebrafish larvae with TAA immersion, steatosis and collagen fiber deposition similar to those of intraperitoneal injection of TAA can occur in the liver.

[0008] (2) Diethylnitrosamine (DEN). DEN is a water-soluble substance with stable physical and chemical properties and can be directly administered by water bath. At a concentration of 200 mg / L and intervened for 4 - 6 weeks, a stable zebrafish liver fibrosis model can be obtained.

[0009] (3) Brominated polystyrene (BPS). BPS is similar to DEN and is more likely to cause lipid metabolism disorders and lead to zebrafish liver fibrosis. BPS promotes the accumulation of fat on the inner wall of blood vessels and in the liver. When larvae are exposed to BPS for a long time, the unfolded protein response (UPR) in the liver is activated, resulting in excessive accumulation of triacylglycerol in the liver of male zebrafish and gradually developing into liver fibrosis.

[0010] (4) Drugs. ① Paris polyphylla is a traditional Chinese medicine with antibacterial and anti-inflammatory effects. According to different extractants, the extracts of Paris polyphylla can be divided into many types. Among them, ethyl acetate extract (AcOEtE) can cause liver lipid metabolism disorders and trigger liver fibrosis in larvae. ② After 72 hours of water bath exposure to 0.5 - 2 mmol / L azithromycin, the liver color of larvae deepens and the liver area decreases. As the drug concentration increases, the expression of the liver fibrosis marker tgfb1 gene is up-regulated. ③ Mifepristone can induce an increase in the expression level of the zebrafish Tgf-β1 gene, and significantly increase the expression of laminin and collagen, inducing liver fibrosis.

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

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

[0013] (2) The diet-induced liver fibrosis model takes a long time and lacks reproducibility;

[0014] (3) The cost of constructing a liver fibrosis model by gene editing method is high and the modeling time is long;

[0015] (4) In the construction of a liver fibrosis model based on chemical induction method, the exposure concentration of the compound is extremely high (reaching the mg / L or mg / kg level), which is likely to cause environmental pollution. Summary of the Invention

[0016] In view of the problems existing in the prior art, the present invention provides a method for rapidly constructing a liver fibrosis disease model based on zebrafish, aiming to solve a part of the problems in the prior art or at least alleviate a part of the problems in the prior art. The inventor exposed non-alcoholic fatty liver disease (NAFLD) zebrafish to potassium chlorinated polyfluoroalkyl ether sulfonate F-53B at different concentrations (250 ng / L, 5 μg / L, and 100 μg / L) for 28 days and found that a liver fibrosis zebrafish model can be successfully constructed by exposure to a low concentration (i.e., 250 ng / L). The present invention overcomes the above-mentioned defects and can construct a liver fibrosis model by short-term exposure to a low-concentration compound.

[0017] The present invention is implemented as follows. A method for constructing a liver fibrosis model comprises exposing zebrafish to water containing potassium chlorinated polyfluoroalkyl ether sulfonate.

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

[0019] Further, the concentration of potassium chlorinated polyfluoroalkyl ether sulfonate is above 0.25 μg / L, preferably 0.25 μg / L - 100 μg / L.

[0020] Further, the exposure time is above 28 days.

[0021] Further, a non-alcoholic fatty liver zebrafish model is induced by high-fat diet intervention.

[0022] Further, the zebrafish are 3-month-old adult zebrafish.

[0023] The present invention also provides the application of potassium chlorinated polyfluoroalkyl ether sulfonate in the preparation of reagents for liver fibrosis.

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

[0025] (1) Constructing a liver fibrosis model based on zebrafish has low breeding cost and short experimental period. Its small size enables high-throughput drug screening;

[0026] (2) First, an obese NAFLD zebrafish model was established, mimicking typical pathological characteristics of human obesity.

[0027] (3) The total modeling time was 12 weeks, with a short modeling time and high stability.

[0028] (4) An exposure concentration at the ng / L level can induce liver fibrosis, with a low exposure concentration and not easily causing environmental pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is the construction of the zebrafish NAFLD model; (A) Pictures of fish bodies in the normal diet group and the high-fat diet group; (B) Analysis of liver pathological sections in the normal diet group and the high-fat diet group; (C) Quantitative analysis of body mass index BMI, relative liver weight, hepatic vacuolation density, Oil Red staining optical density, triglyceride, and total cholesterol under different intervention modes. Black arrows indicate hepatic vacuolation. Values = mean ± standard deviation, * , p < 0.05; ** , p < 0.01; *** , p < 0.001.

[0030] Figure 2 It is the construction of the zebrafish liver fibrosis model; (A) Masson staining of liver sections of zebrafish in the normal diet group and the high-fat diet group under different F-53B exposure concentrations; (B) Detection of the content of liver fibrosis biomarkers (α1-type I collagen fiber, prothrombin / coagulation factor II, hyaluronic acid) in the livers of zebrafish in the normal diet group and the high-fat diet group 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 DESCRIPTION OF THE EMBODIMENTS

[0031] In order to make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with embodiments. The equipment and reagents used in each embodiment and test example can be obtained from commercial channels without special instructions. The specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0032] Based on the information contained in this application, various changes to the precise description of the present invention can be easily 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 present invention is not limited to the processes, properties or components defined, as these embodiments and other descriptions are merely for illustrative purposes of specific aspects of the present invention. In fact, all various changes that can be made by those skilled in the art or related fields to the embodiments of the present invention are covered within the scope of the appended claims.

[0033] For a better understanding of the present invention rather than limiting its scope, all numbers representing amounts, percentages, and other numerical values used in this application should be understood as being modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may vary depending on the desired properties to be obtained. Each numerical parameter should at least be regarded as obtained based on the reported significant figures and by the conventional rounding method. In the present invention, "about" means within 10% of a given value or range, preferably within 5%.

[0034] When the temperature is not particularly specified in the following embodiments of the present invention, it is under normal temperature conditions. Normal temperature refers to the natural room temperature conditions in the four seasons without additional cooling or heating treatment. Generally, the normal temperature is controlled at 10 - 30 °C, preferably 15 - 25 °C.

[0035] The present invention discloses a method for rapidly constructing a liver fibrosis disease model based on zebrafish. The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0036] Example 1

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

[0038] Three-month-old wild-type adult zebrafish were cultured at 27±1°C with a light cycle of 14h / 10h in a zebrafish culture system (Tecniplast Zebtec, Tecniplast, Buguggiate, Italy). Among them, 1 / 2 of the fish were intervened with a high-fat diet (HFD) in the first 8 weeks to induce a non-alcoholic fatty liver disease (NAFLD) model. Each fish was fed 0.1 g / d of egg yolk and 0.25 g / d of Artemia nauplii, and was fed 3 times a day. The remaining 1 / 2 of the fish were fed with 0.06 g / d of Artemia shrimp twice a day for 8 weeks as the normal diet group (ND). After 8 weeks of dietary intervention, the fish were photographed, and the body length, body weight, BMI, and relative liver weight were recorded. The liver samples obtained by dissection were used for subsequent hematoxylin / eosin (HE) staining, oil red (ORO) staining, Masson (MASSON) staining, triglyceride (TG) and total cholesterol (T-CHO) quantitative analysis to verify the establishment of the NAFLD model. Among them, BMI = body weight / body length 2 , and the relative liver weight = liver weight / body weight.

[0039] Detection method: 300 μL of PBS (NaCl 8 g / L, KCl 0.2 g / L, Na2HPO4 1.44 g / L, KH2PO4 0.24 g / L, PH = 7.4) was added to the liver samples of each fish in each group for lysis, and then centrifuged at 3,000 g for 10 min at 4°C in a centrifuge to obtain the supernatant. The contents of triglyceride (TG), total cholesterol (T-CHO), α1-type I collagen fiber (Col1A1), prothrombin / coagulation factor II (PT / FII), and hyaluronic acid (HA) in the supernatant were measured. The assay kits for triglyceride (TG), total cholesterol (T-CHO), and α1-type I collagen fiber (Col1A1) were purchased from Nanjing Jiancheng Bioengineering Institute, and the assay kits for prothrombin / coagulation factor II (PT / FII) and hyaluronic acid (HA) were purchased from Jiangsu Enzyme Immunoassay Industry Co., Ltd.

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

[0041] Statistics and analysis: All data were expressed as means ± standard error of the 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 the differences between groups were significant, and p < 0.05 was considered statistically significant.

[0042] Liver fibrosis develops slowly from NAFLD. Therefore, a zebrafish NAFLD model was first constructed by high-fat diet intervention. Specifically, the body mass index of zebrafish induced by high-fat diet increased by 26.92% compared with the normal diet group ( Figure 1 A, C in Figure 1 ), and the relative liver weight increased by 3.09 times ( Figure 1 C in Figure 1 ). Further histopathological section analysis showed that the high-fat diet group significantly induced fatty liver, as evidenced by a 645.95% increase in the vacuole density of the liver after hematoxylin / eosin staining ( Figure 1 B, C in

[0043] 2. Establishment of liver fibrosis model

[0044] The established NAFLD zebrafish model and zebrafish in the ND group were exposed to potassium chloroperfluoroalkyl 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, and zebrafish in the normal diet group at the same exposure concentration were used as controls. After the exposure, liver samples were obtained by dissection and used for Masson staining and determination of the contents of liver fibrosis biomarkers α1-type I collagen fiber (Col1A1), prothrombin / coagulation factor II (PT / FII), and hyaluronic acid (HA).

[0045] The results showed that F-53B induced liver fibrosis in NAFLD zebrafish within the concentration range of 0.25 μg / L - 100 μg / L, while for the normal diet group after exposure, only the 100 μg / L exposure group showed liver fibrosis phenotypes (Figure 2 A). Biochemical identification further verified the pathological section analysis. The liver fibrosis biomarkers, including α1-type I collagen fiber, prothrombin / coagulation factor II, and hyaluronic acid, were all significantly altered in the livers of zebrafish in the high-fat diet group exposed at 0.25 μg / L–100 μg / L. However, for the livers of zebrafish in the normal diet group exposed to F-53B, only the exposure concentration of 100 μg / L significantly altered the contents of α1-type I collagen fiber, prothrombin / coagulation factor II, and hyaluronic acid ( Figure 2 B).

[0046] In summary, the present invention can successfully construct a zebrafish liver fibrosis model in a short time by using a low concentration of F-53B (0.25 μg / L) based on NAFLD zebrafish, overcoming the deficiencies of the prior art.

[0047] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for constructing a liver fibrosis model, characterized in that: Expose zebrafish to water containing potassium chlorinated polyfluoroalkyl ether sulfonate.

2. The method for constructing a liver fibrosis model according to claim 1, characterized in that: The zebrafish are induced to develop non-alcoholic fatty liver, and then exposed to water containing potassium chlorinated polyfluoroalkyl ether sulfonate.

3. The method for constructing a liver fibrosis model according to claim 2, characterized in that: The concentration of the potassium chlorinated polyfluoroalkyl ether sulfonate is above 0.25 μg / L.

4. A method for constructing a liver fibrosis model according to claim 3, characterized in that: The exposure time is above 28 days.

5. The method for constructing a liver fibrosis model according to claim 2, wherein: Induce a non-alcoholic fatty liver zebrafish model through high-fat diet intervention.

6. The method for constructing a liver fibrosis model according to claim 2, characterized in that: The zebrafish are 3-month-old adult zebrafish.

7. Use of potassium chlorinated polyfluoroalkyl ether sulfonate in the preparation of a reagent for liver fibrosis.

Images