A method for rapidly constructing an animal model of liver fibrosis

Abstract

The application belongs to the technical field of biology, and specifically discloses a method for rapidly constructing a liver fibrosis animal model, which establishes a mouse liver fibrosis model by jointly using CCl4 and a MEK1 / 2 kinase inhibitor, i.e., trametinib. The trametinib can inhibit the MEK1 / 2 kinase and the ERK1 / 2 pathway. Compared with a traditional CCl4 modeling method, the modeling method provided by the application has the advantages of rapid modeling speed, obvious liver fibrosis phenotype after two weeks of administration, and the like. After four weeks of administration, the liver fibrosis degree of the "CCl4+trametinib" group is significantly higher than that of the CCl4 group, and meanwhile, the liver damage of the mouse is also significantly aggravated, but the trametinib has no obvious influence on the kidney function. In general, the liver fibrosis model provided by the application has the advantages of high specificity, rapid modeling speed, obvious phenotype, simple and safe operation, and the like, and is a modeling method superior to the traditional CCl4 modeling method.

Description

Technical Field

[0001] The present 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, the pathological basis of various chronic liver diseases, is a key pathological process in the progression of chronic liver damage to diseases such as cirrhosis, liver cancer, and liver failure. Therefore, establishing an efficient animal model of liver fibrosis is crucial for exploring its pathogenesis and preventing and treating diseases such as cirrhosis.

[0003] Common methods for modeling liver fibrosis include the following. ① Carbon tetrachloride (CCl4) is a commonly used drug for establishing animal models of liver fibrosis. CCl4 causes liver damage through oxidative stress and other mechanisms. Due to its diverse administration methods, good reproducibility, and relatively stable models, it is widely used in studies of the etiology and histology of liver fibrosis, as well as in evaluating drug efficacy. Animal models established using CCl4 closely mimic many features of human liver fibrosis and are one of the most commonly used modeling methods. However, establishing a mouse / rat liver fibrosis model using CCl4 is time-consuming, requiring at least 4-8 weeks. ② Bile duct ligation model: This model primarily simulates cholestatic liver fibrosis and has a relatively limited scope of application. It works by creating extrahepatic bile duct obstruction, inducing bile duct dilation, cholestasis, and elevated intrabiliary pressure. This leads to intrahepatic vascular damage, hepatocyte ischemia and necrosis, and consequently, fibrosis. This method offers rapid modeling and a low rate of spontaneous reversal, but requires high surgical skill on the part of the operator. Thioacetamide (TAA) model: TAA is a weak carcinogen with toxic effects on hepatocyte DNA, RNA, and protein synthases, causing 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 requires longer time than CCl4 modeling, and TAA is volatile and highly toxic, requiring higher personal protective equipment requirements.

[0004] Among the patents disclosed in this field, patent application number CN202210011928.4 discloses that on the basis of administering CCl4, by adding special feed, vitamin A deficiency is caused in mice, thereby constructing a mouse liver fibrosis model. 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 application is implemented according to the following technical scheme:

[0007] A method for quickly constructing a liver fibrosis animal model, comprising the following steps:

[0008] S1, CCl4 and trametinib are simultaneously administered to 5-6 week old C57BL / 6J mice for 2-4 weeks, wherein: the administration dose of CCl4 is 0.5 μL / g, and the administration is performed 3 times a week with 1-2 days interval, and the administration is continuously performed for 2-4 weeks; the administration dose of trametinib is 1.5 mg / kg, and the administration is performed 3 times a week with 1-2 days interval, and the administration is continuously performed for 2-4 weeks;

[0009] S2, after the administration of CCL4 and trametinib is stopped, a liver fibrosis mouse is obtained.

[0010] Further, the CCl4 is administered by intraperitoneal injection, and the trametinib is administered by gavage, intraperitoneal injection or intravenous injection.

[0011] Further, the CCl4 is dissolved in corn oil at a volume ratio of 15% before administration.

[0012] Further, the trametinib is configured into a storage solution by using dimethyl sulfoxide, and then is administered after being assisted by dissolving PEG300, tween80 and sterile water.

[0013] Compared with the prior art, the present application establishes a mouse liver fibrosis model by using CCl4 and MEK1 / 2 kinase inhibitor trametinib in combination. Trametinib can inhibit the ERK1 / 2 pathway by inhibiting MEK1 / 2 kinase. Compared with the traditional CCl4 modeling method, the modeling method provided by the present application has a fast modeling speed, and obvious liver fibrosis phenotype is obtained after 2 weeks of administration. After 4 weeks of administration, the degree of liver fibrosis in the "CCl4+trametinib" group is significantly higher than that in the CCl4 group, and the liver damage of the mouse is also significantly aggravated, but has no obvious effect on the kidney function. In summary, the liver fibrosis model provided by the present application has high specificity, fast modeling speed, obvious phenotype, simple and safe operation method, and is a modeling method superior to the traditional CCl4. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 In order to make trametinib combined with CCl4 more effective in inducing the occurrence of liver fibrosis in mice: A is the administration method of the mouse and the sampling time; B is the HE, Masson and Sirius red staining after 4 weeks of continuous administration; C is the quantitative analysis of the positive staining area of Masson and Sirius red.

[0015] Figure 2Trametinib combined with CCl4 increases ferroptosis in mouse liver cells: A is a representative staining image of mice in each group; B is the quantitative analysis of Prussian blue staining results; C is the quantitative analysis of TUNEL staining results.

[0016] Figure 3 Trametinib combined with CCl4 aggravates liver function damage in mice: A is the liver function test index; B is the BUN index of renal function test; C is the CRE index of renal function test.

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

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

[0019] Figure 6 Figure 3: Trametinib + CCl4 significantly enhanced the degree of liver fibrosis in Prxl2a knockout mice: A is a representative image of various stainings, and B is a quantitative analysis of Masson's, Picrosirius red, and Prussian blue staining. DETAILED DESCRIPTION

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

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

[0022] CCl4 is carbon tetrachloride, a traditional liver fibrosis modeling agent, and is dissolved in corn oil at a volume ratio of 15% before administration.

[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 before administration.

[0024] Example 1

[0025] Wild-type C57BL / 6J mice (purchased from Guangdong Yaokang Biotechnology Co., Ltd.) were used to establish the model, aged 5-6 weeks. CCl4 and trametinib were administered simultaneously for 4 weeks, wherein: the dosage of CCl4 was 0.5 μL / g, administered by intraperitoneal injection, and administered on the first, third, and fifth days of each week (D1, D3, D5); the dosage of trametinib was 1.5 mg / kg, administered by gavage, and administered on the first, third, and fifth days of each week (D1, D3, D5). After continuous administration for 2 or 4 weeks, mice with liver fibrosis were obtained and recorded as the CCl4+Tra (CLT) group; the mice were sampled for testing, and the process is as follows: Figure 1 As shown in A.

[0026] Comparative Example 1

[0027] The difference from Example 1 is that only CCl4 was administered alone, which was recorded as the CCl4 (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 the solvent (NC) group.

[0030] After 4 weeks of continuous administration, the mice were killed for relevant tests. The results were as follows: Figure 1 As shown, B in Figure 1 is HE, Masson's and Sirius red staining after 4 weeks of continuous administration. 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 areas of Masson's and Sirius red, CL is the CCl4 group, and CLT is the "CCl4+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 The data are mean ± standard deviation (n = 5), ** represents P < 0.01. Figure 1 B and Figure 1 As shown in C, Masson and picrosirius red staining of liver tissue revealed that the degree of liver fibrosis in the "CCl4+trametinib" group was significantly higher than that in the case of CCl4 alone, 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 table, CL is the CCl4 group, CLT is the CCl4 + trametinib group, and NC is 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 2C 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 represents BUN and CRE indicators of renal function test. * represents P < 0.05, ** represents P < 0.01, and NS represents no statistically significant difference (P > 0.05). 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 the solvent control. The tissue morphology was observed by HE staining, liver fibrosis was observed by Masson and Sirius red staining, and cell ferroptosis was evaluated by Prussian blue staining. 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 4 It 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 of ​​Masson, Picrosirius red and Prussian blue staining. * represents P < 0.05, ** represents P < 0.01, and the data in the figure are mean ± standard deviation (n = 5). Figure 5It 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. 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] Wild-type C57BL / 6N and Prxl2a knockout (C57BL / 6N background) mice were used for modeling. The mice were 5-6 weeks old. The mice were divided into four groups: (1) wild-type mice were given solvent, i.e., WT-NC group; (2) wild-type mice were given CCl4, i.e., WT-CL group; (3) Prxl2a knockout mice were given CCl4, i.e., KO-CL group; (4) Prxl2a knockout mice were given trametinib + CCl4, i.e., KO-CLT group. Each group contained 5 mice and was treated with CCl4, trametinib, or solvent three times a week for four consecutive weeks. The CCl4 dose was 0.5 μL / g body weight by intraperitoneal injection; the trametinib dose was 1.5 mg / kg body weight by gavage.

[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, Picrosirius red and Prussian blue staining. The scale bar of Masson 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 wild-type mice given CCl4 (i.e., WT-CL), knockout of the Prxl2a gene (i.e., KO-CL) can inhibit liver fibrosis to a certain extent, but the difference is not significant; if CCl4+trametinib is given to Prxl2a knockout mice (i.e., KO-CLT), liver fibrosis can be significantly enhanced, and Prussian blue staining is enhanced, suggesting that liver fibrosis is related to cell ferroptosis ( Figure 6 This indicates that CCl4 + trametinib is also a more potent approach for modeling liver fibrosis in Prxl2a knockout mice, suggesting that this approach has broad applicability. Thus, in Prxl2a knockout mice, trametinib + CCl4 significantly enhances the degree of liver fibrosis.

[0039] In summary, compared with the traditional CCl4 modeling method, within the same intervention time, the liver fibrosis phenotype induced by "CCl4+trametinib" is more significant and occurs faster, while having no obvious effect on renal function.

[0040] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. Any 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 simultaneously to 5-6 week old C57BL / 6J mice for 2-4 weeks: CCl4 was administered at a dose of 0.5 μL / g, three times a week, every 1-2 days, for 2-4 weeks; trametinib was administered at a dose of 1.5 mg / kg, three times a week, 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 was prepared into a storage solution with dimethyl sulfoxide, and then dissolved with PEG300, tween80 and sterile water before administration.

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