Method for rapidly constructing hepatic fibrosis animal model

Abstract

The invention belongs to the technical field of biology, and particularly discloses a method for rapidly constructing a hepatic fibrosis animal model, and a mouse hepatic fibrosis model is established by jointly using CCl4 and an MEK1 / 2 kinase inhibitor trametinib. The trametinib can be used for inhibiting an ERK1 / 2 pathway by inhibiting MEK1 / 2 kinase; compared with the traditional CCl4 modeling means, the modeling method provided by the invention has the advantages that the modeling speed is high, and obvious hepatic fibrosis phenotypes can be obtained after administration for 2 weeks; after administration for 4 weeks, the hepatic fibrosis degree of a CCl4 + trametinib group is obviously higher than that of a CCl4 group, and meanwhile, the liver injury of a mouse is also obviously aggravated, but the renal function is not obviously influenced. In a word, the hepatic fibrosis model provided by the invention has the advantages of high specificity, high modeling speed, obvious phenotype and simple and safe operation means, and is a modeling means superior to the traditional CCl4.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a method for rapidly constructing an animal model of liver fibrosis. Background Art

[0002] Liver fibrosis is the pathological basis of many chronic liver diseases and an important pathological process for the development of chronic liver damage into diseases such as cirrhosis, liver cancer, and liver failure. Therefore, establishing an efficient animal model of liver fibrosis is of great significance for exploring its pathogenesis, preventing and treating diseases such as cirrhosis.

[0003] There are several common methods for modeling liver fibrosis. ① Carbon tetrachloride (CCl 4 ) is a commonly used drug for constructing animal models of liver fibrosis. 4 It causes liver damage through oxidative stress response, etc. Due to its diverse administration methods, good repeatability, and relatively stable model, it is widely used in the etiology and histology of liver fibrosis, as well as the evaluation of drug effects. 4 The animal model constructed by CCl can well simulate many characteristics of human liver fibrosis and is one of the most commonly used modeling methods. 4 It takes a long time to construct a mouse / rat liver fibrosis model, which requires at least 4-8 weeks. ② Bile duct ligation model: This model mainly simulates cholestatic liver fibrosis and has a relatively limited scope of application. The principle is to construct extrahepatic bile duct obstruction, induce bile duct dilatation, cholestasis and increased intra-biliary pressure, leading to damage of intrahepatic blood vessels, ischemia and necrosis of hepatocytes, and thus causing fibrosis. This method has the characteristics of fast modeling and low spontaneous reversal rate, but it requires high surgical skills of the operator. ③ Thioacetamide (TAA) model: TAA is a weak carcinogen that has toxic effects on hepatocyte DNA, RNA and protein synthase, and can cause hepatocyte necrosis and liver fibrosis. The TAA-induced mouse liver fibrosis model is similar to human liver fibrosis in terms of hemodynamics, morphological characteristics and biochemical metabolic disorders. However, TAA modeling is more complicated than CCl 4 Modeling takes longer, and TAA is volatile and highly toxic, requiring higher levels of personal protection.

[0004] Among the patents published in this field, patent application number CN202210011928.4 discloses that CCl 4 On this basis, by adding special feed to cause vitamin A deficiency in mice, a mouse liver fibrosis model was constructed. However, the advantages of this modeling method are good model stability, no phenotype reversal, and a high modeling success rate, but the modeling time is still relatively long, generally requiring 8-12 weeks. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for rapidly constructing an animal model of liver fibrosis.

[0006] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0007] A method for rapidly constructing an animal model of liver fibrosis comprises the following steps:

[0008] S1. CCl was performed on 5-6 week old C57BL / 6J mice. 4 Administered concurrently with trametinib for 2-4 weeks: CCl 4 The dosage of is 0.5 μL / g, 3 times a week, once every 1-2 days, for 2-4 weeks; the dosage of trametinib is 1.5 mg / kg, 3 times a week, once every 1-2 days, for 2-4 weeks;

[0009] S2, CCL 4 After the withdrawal of trametinib, mice with liver fibrosis were obtained.

[0010] Furthermore, the CCl 4 The drug is administered by intraperitoneal injection, and the trametinib is administered by oral gavage, intraperitoneal injection or intravenous administration.

[0011] Furthermore, the CCl 4 The drug was dissolved in corn oil at a volume ratio of 15% before administration.

[0012] Furthermore, the trametinib is prepared into a storage solution with dimethyl sulfoxide, and then dissolved with PEG300, tween80 and sterile water before administration.

[0013] Compared with the prior art, the present invention combines the use of CCl 4 The mouse liver fibrosis model was established by using the MEK1 / 2 kinase inhibitor trametinib. Trametinib can inhibit the ERK1 / 2 pathway by inhibiting MEK1 / 2 kinases. 4 Modeling means, the modeling method provided by the present invention has a fast modeling speed, and obvious liver fibrosis phenotype is observed after 2 weeks of administration; after 4 weeks of administration, "CCl 4 The degree of liver fibrosis in the "+trametinib" group was significantly stronger than that in the CCl 4 In summary, the liver fibrosis model provided by the present invention has high specificity, fast modeling speed, obvious phenotype, simple and safe operation, and is superior to traditional CCl 4 Modeling means. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Trametinib combined with CCl 4 It can more effectively induce liver fibrosis in mice: A shows the drug administration method and sampling time for mice; B shows HE, Masson's and Sirius red staining after 4 weeks of continuous drug administration; C shows the quantitative analysis of the positive staining areas of Masson's and Sirius red.

[0015] Figure 2 Trametinib combined with CCl 4 Increased ferroptosis of mouse liver cells: A is a representative staining image of mice in each group; B is a quantitative analysis of the Prussian blue staining results; C is a quantitative analysis of the TUNEL staining results.

[0016] Figure 3 Trametinib combined with CCl 4 Aggravate liver function damage in mice: A is the liver function test index; B is the kidney function test BUN index; C is the kidney function test CRE index.

[0017] Figure 4 Trametinib alone cannot induce liver fibrosis in mice: A is a representative staining picture; B is the percentage of positive area stained with Masson, Sirius red and Prussian blue.

[0018] Figure 5 "CCl4+trametinib" administration can cause obvious liver fibrosis after two weeks of administration: A is a representative staining picture; B is the percentage of positive area stained with Masson, picrosirius red and Prussian blue.

[0019] Figure 6 In Prxl2a knockout mice, trametinib+CCl4 can significantly enhance the degree of liver fibrosis: A is a representative picture of various stainings, and B is a quantitative analysis of Masson's, Sirius red and Prussian blue staining. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0021] Unless otherwise specified, the raw materials and reagents used in the following examples were all commercially available.

[0022] CCl 4 Carbon tetrachloride is a traditional liver fibrosis modeling agent, which is dissolved in corn oil at a volume ratio of 15% and then administered.

[0023] Trametinib (Tra) is a MEK1 / 2 kinase inhibitor. It is prepared into a storage solution with dimethyl sulfoxide (DMSO) and then dissolved with PEG300, tween80 and sterile water for administration.

[0024] Example 1

[0025] Wild-type C57BL / 6J mice (purchased from Guangdong Yaokang Biotechnology Co., Ltd.) were used for modeling, aged 5-6 weeks. 4 Administered concurrently with trametinib for 4 weeks: CCl 4 The dosage of 0.5 μL / g was intraperitoneally injected on the first, third, and fifth days of each week (D1, D3, and D5); the dosage of trametinib was 1.5 mg / kg, administered by gavage on the first, third, and fifth days of each week (D1, D3, and D5). After continuous administration for 2 or 4 weeks, mice with liver fibrosis were obtained and recorded as CCl 4 +Tra(CLT) group; samples were collected from mice for testing, and the process was as follows Figure 1 As shown in A.

[0026] Comparative Example 1

[0027] The difference from Example 1 is that only CCl 4 Single administration, recorded as CCl 4 (CL) group.

[0028] Comparative Example 2

[0029] The difference from Example 1 is that only solvents, namely corn oil and DMSO+PEG300+tween80+sterile water, were administered alone, which was recorded as solvent (NC) group.

[0030] After 4 weeks of continuous administration, the mice were killed for related tests. The results were as follows Figure 1 As shown, B in Figure 1 is HE, Masson and Sirius red staining after continuous administration for 4 weeks. HE staining is used to observe tissue morphology, and the latter two stainings are used to observe the degree of liver fibrosis; C in Figure 1 is the quantitative analysis of the positive staining area of ​​Masson and Sirius red, CL is CCl 4 group, CLT is "CCl 4 + trametinib" group. Figure 1 The scale bar for Masson staining is 200 μm, and the scale bars for other stainings are 100 μm. Figure 1 Data are mean ± SD (n = 5), ** represents P < 0.01. Figure 1 B and Figure 1 The results of Masson and Sirius red staining of liver tissue showed that the degree of liver fibrosis in the "CCl4+trametinib" group was significantly higher than that in the group given CCl alone.4 , indicating that trametinib combined with CCl4 can more effectively induce liver fibrosis in mice.

[0031] Four weeks later, liver tissues of mice were collected and stained with Prussian blue and TUNEL to evaluate cell ferroptosis and apoptosis. Figure 2 shown. Figure 2 In the equation, CL is CCl 4 group, CLT is CCl 4 + trametinib group, and NC was the solvent control group. Figure 2 A in the figure is a representative staining image of mice in each group; Figure 2 B in Figure 2 C in the figure is the quantitative analysis of Prussian blue and TUNEL staining results. Figure 2 The scale bar is 100 μm, and the data are mean ± standard deviation (n = 5). ** represents P < 0.01, and NS represents no significant difference (P > 0.05). Figure 2 It can be seen that Prussian blue staining indicated that the cell ferroptosis in the "CCl4+trametinib" group was significantly increased, while TUNL staining showed that the changes in cell apoptosis were not obvious, indicating that trametinib combined with CCl4 increased the ferroptosis of mouse liver cells.

[0032] Liver and kidney function tests are performed by sampling blood from the eye sockets. Figure 3 As shown, Figure 3 A in it is the liver function test index; Figure 3 B in Figure 3 C in the figure is the BUN and CRE index of renal function test. * represents P<0.05, ** represents P<0.01, NS represents no statistically significant difference (P>0.05), and the data in the figure are mean ± standard deviation (n=5). Figure 3 It can be seen that compared with the CCl4 group, the liver function of mice in the "CCl4+trametinib" group further deteriorated, while there was no significant abnormality in renal function, indicating that trametinib combined with CCl4 aggravated liver function damage in mice.

[0033] Trametinib (Tra) was administered by intraperitoneal injection, with the same dosing schedule as Figure 1 A, NC is solvent control, HE staining was used to observe tissue morphology, Masson and Sirius red staining was used to observe liver fibrosis, and Prussian blue staining was used to evaluate cell ferroptosis. Figure 4 As shown, Figure 4 A in the figure is a representative staining picture, and the scale bar in the figure is 100 μm; Figure 4 B in the figure is the percentage of positive area stained by Masson's, Sirius red and Prussian blue. Figure 4 The data are mean ± standard deviation (n = 3), NS (not significant) means there is no statistically significant difference (P>0.05). Figure 4It can be seen that the administration of trametinib alone has no obvious effect on liver fibrosis and liver function in mice, indicating that the administration of trametinib alone cannot induce liver fibrosis in mice.

[0034] Furthermore, after two weeks of continuous administration, liver tissues of mice were taken for testing. The test results were as follows: Figure 5 As shown, Figure 5 A in the figure is a representative staining picture, and the scale bar in the figure is 100 μm; Figure 5 B in the figure is the percentage of positive area stained with Masson's, Sirius red and Prussian blue. * represents P < 0.05, ** represents P < 0.01, and the data in the figure are mean ± standard deviation (n = 5). Figure 5 It can be seen that the degree of liver fibrosis in the "CCl4+trametinib" group was significantly stronger than that in the group given CCl4 alone.

[0035] The above results suggest that "CCl4+trametinib" can accelerate and aggravate the occurrence of liver fibrosis in mice, and the mechanism may be related to the induction of liver cell ferroptosis. "CCl4+trametinib" can cause obvious liver fibrosis after two weeks of administration.

[0036] Example 2

[0037] The wild-type C57BL / 6N and Prxl2a knockout (C57BL / 6N background) mice were used to establish the model. The mice were 5-6 weeks old. The mice were divided into 4 groups: (1) wild-type mice were given solvent, i.e., WT-NC group; (2) wild-type mice were given CCl 4 , namely WT-CL group; (3) Prxl2a knockout mice were given CCl 4 , i.e. KO-CL group; (4) Prxl2a knockout mice were given trametinib + CCl 4 Each group contained 5 mice and was treated with CCl 4 , trametinib or vehicle were used for intervention, 3 times a week for 4 consecutive weeks. 4 The dosage was 0.5 μL / g body weight, injected intraperitoneally; the dosage of trametinib was 1.5 mg / kg body weight, administered intragastricly.

[0038] Four weeks later, liver tissues of mice were taken for testing. The test results were as follows: Figure 6 As shown, Figure 6 A in the figure is a representative picture of various stainings. Figure 6 B in the figure is the quantitative analysis of Masson's staining, Sirius red and Prussian blue staining. The scale bar of Masson's staining is 200 μm, and the scale bar of other staining is 100 μm. The data are mean ± standard deviation (n = 5). Figure 6 It can be seen that compared with the administration of CCl 4In wild-type mice (WT-CL), Prxl2a gene knockout (KO-CL) can inhibit liver fibrosis to a certain extent, but the difference is not significant. 4 + trametinib (i.e., KO-CLT) can significantly enhance liver fibrosis, and Prussian blue staining is enhanced, indicating that liver fibrosis is related to cell ferroptosis ( Figure 6 ). This indicates that in Prxl2a knockout mice, "CCl 4 + trametinib" is also a more effective liver fibrosis modeling method, suggesting that this modeling method has a wider applicability. It can be seen that in Prxl2a knockout mice, trametinib + CCl 4 Can significantly enhance the degree of liver fibrosis.

[0039] In summary, compared with traditional CCl 4 Modeling means, within the same intervention period, "CCl 4 + trametinib" induced a more significant liver fibrosis phenotype and occurred faster, without significant effects on renal function.

[0040] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A method for rapidly constructing an animal model of liver fibrosis, characterized in that: The following steps are involved: S1. CCl4 and trametinib were administered to 5-6 week old C57BL / 6J mice simultaneously for 2-4 weeks, wherein: the dosage of CCl4 was 0.5 μL / g, 3 times a week, once every 1-2 days, for 2-4 weeks; the dosage of trametinib was 1.5 mg / kg, 3 times a week, once every 1-2 days, for 2-4 weeks; After withdrawal of S2, CCL4 and trametinib, mice with liver fibrosis were obtained.

2. The method for rapidly constructing an animal model of liver fibrosis according to claim 1, characterized in that: The CCl4 is administered by intraperitoneal injection, and the trametinib is administered by oral gavage, intraperitoneal injection or intravenous administration.

3. The method for rapidly constructing an animal model of liver fibrosis according to claim 1, characterized in that: The CCl4 was dissolved in corn oil at a volume ratio of 15% and then administered.

4. The method for rapidly constructing an animal model of liver fibrosis according to claim 1, characterized in that: The trametinib is prepared into a storage solution with dimethyl sulfoxide, and then dissolved with PEG300, tween80 and sterile water before administration.

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