A method for constructing a liver fibrosis animal model

Abstract

The application belongs to the technical field of biology and particularly relates to a method for constructing a liver fibrosis animal model. The application discloses, for the first time, application of a TLR1 / TLR2 specific agonist in improving the efficiency of a CCL4-induced liver fibrosis animal model. Although the TLR1 / TLR2 specific agonist itself cannot induce the liver fibrosis animal model, it can improve the efficiency of the CCL4-induced liver fibrosis animal model.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for constructing an animal model of liver fibrosis. Background Technology

[0002] Liver fibrosis / cirrhosis is a disease caused by multiple etiologies, prevalent globally, and poses a serious threat to human health. Liver fibrosis is a key pathological stage in the progression of various chronic liver injuries (such as viral hepatitis, alcoholic liver disease, and non-alcoholic fatty liver disease) to cirrhosis and even liver cancer. Its core pathogenesis involves persistent liver injury activating hepatic stellate cells (HSCs), which transform into myofibroblasts, synthesizing and depositing large amounts of extracellular matrix (ECM), thus disrupting the normal liver structure. If the underlying cause persists, fibrosis further progresses to cirrhosis, accompanied by liver failure. Because early-stage human liver fibrosis is asymptomatic and repeated biopsies are difficult, coupled with ethical restrictions, animal models have become essential tools for studying its mechanisms and anti-fibrotic drugs. An ideal model should mimic the etiology, pathology, and reversibility of human fibrosis as closely as possible. Therefore, it is necessary to develop a method for constructing an animal model of liver fibrosis. Summary of the Invention

[0003] The first aspect of the present invention is to provide the application of TLR1 / TLR2 specific agonists.

[0004] A second aspect of the present invention aims to provide a combination of reagents for constructing animal models of liver fibrosis.

[0005] The third aspect of this invention aims to provide the application of the reagent combination of the second aspect of this invention.

[0006] The fourth aspect of this invention aims to provide a method for constructing an animal model of liver fibrosis.

[0007] The fifth aspect of this invention aims to provide an application of the animal model obtained by the construction method of the fourth aspect of this invention.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the invention provides the use of a TLR1 / TLR2 specific agonist in any one of a1)-a2); a1) Improve the efficiency of CCL4-induced liver fibrosis animal models; a2) Prepare products that improve the efficiency of CCL4-induced liver fibrosis animal models.

[0009] In this invention, the TLR1 / TLR2 specific agonist may include, but is not limited to: Pam3CSK4, SMU-C68, SMU-C409, CU-T12-9, Diprovocim-X, or pharmaceutically acceptable salts thereof; further including Pam3CSK4 or pharmaceutically acceptable salts thereof.

[0010] In some embodiments, improving the efficiency of CCL4-induced liver fibrosis animal models includes: shortening the time of CCL4-induced liver fibrosis animal model (e.g., from 4-8 days to 2 days), and / or increasing the degree of liver fibrosis in the animal model (e.g., increasing the Sirius red positive staining area in the liver of the animal model, increasing the content of AST and / or ALT in the serum).

[0011] In some embodiments, the product includes reagents, reagent kits, or reagent packages.

[0012] In this invention, the animal is a non-human animal.

[0013] In this invention, the non-human animals include non-human mammals; further include at least one of non-human primates, rodents, cattle (e.g., cows, bulls, buffalo), deer, pigs, sheep (e.g., sheep, goats), dogs, rabbits, chickens, cats, ferrets, and horses; and further include rodents.

[0014] In this invention, the non-human primates include at least one of orangutans, apes, and monkeys (e.g., marmosets, rhesus monkeys).

[0015] In this invention, the rodent includes at least one of mice, rats, hamsters, and guinea pigs; more specifically, at least one of mice and rats.

[0016] In this invention, the rodents are selected from the rat family; further selected from the families Cricetidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muridae (true mice and rats, gerbils, spiny rats, crested rats), Madagascar Muridae (climbing mice, rock mice, tailed rats, Madagascar rats and mice), Spiny Dormicidae (e.g., spiny dormice), and Mole-like Muridae (e.g., mole rats, bamboo rats, and mole rats).

[0017] In this invention, the rodent is a mouse of the C57BL strain, which is selected from C57BL / a, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL6, C57BL / 10, C57BL10ScSn, C57BL / 10Cr, and C57BL / Ola. In this invention, the mouse is a 129 strain selected from 129 / J, 129 / ReJ, 129 / OlaHsd, 129 / Sv, 129 / SvJ, 129 / Re, 129 / RrJ, and 129 / Sv-ter / +. These mice are described, for example, in Festing et al., Revised nomenclature for strain 129 mice, Mammalian Genome 10: 836 (1999); Auerbach et al., Establishment and Chimera Analysis of 129 / SvEv- and C57BL / 6-DerivedMouse Embryonic Stem Cell Lines (2000), the relevant content of which is incorporated herein by reference in its entirety. In this invention, the mice are a cross between the 129 strain and the C57BL / 6 strain. In this invention, the mice are a cross between the 129 strain or the BL / 6 strain. In this invention, the mice are a BALB strain, such as the BALB / c strain. In this invention, the mice are a cross between the BALB strain and another strain. In this invention, the mice are derived from a hybrid line (e.g., 50% BALB / c - 50% 12954 / Sv; or 50% C57BL / 6 - 50% 129). In this invention, the non-human animal is a rodent. Specifically, the non-human animal is a mouse with the strains BALB / c, BALB / cHeAn, BALB / cJ, BALB / cRl, BALB / cWt, C57BL / 10, C57BL / 10ScSn, C57BL (C57BL / 10Cr and C57BL / Ola), C58, CBA / Br, CBA / Ca, CBA / J, CBA / st, or CBA / H, and NOD, NOD / SCID, or NOD-Prkdc. scid IL-2rg null The mouse in the background.

[0018] In this invention, the pharmaceutically acceptable salt comprises at least one of a metal salt, an ammonium salt, a salt formed with an organic base, a salt formed with an inorganic acid, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid.

[0019] In this invention, the metal salt comprises at least one of alkali metal salts (e.g., sodium salts, potassium salts, etc.), alkaline earth metal salts (e.g., calcium salts, magnesium salts, barium salts, etc.), and aluminum salts.

[0020] In this invention, the salt formed with an organic base includes salts formed with one or more of the following organic bases: trimethylamine, triethylamine, pyridine, methylpyridine, 2,6-dimethylpyridine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine.

[0021] In this invention, the salt formed with the inorganic acid includes salts formed with one or more of the following inorganic acids: hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid.

[0022] In this invention, the salt formed with organic acids includes salts formed with one or more of the following organic acids: formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.

[0023] In this invention, the salt formed with the basic amino acid includes salts formed with one or more of the following basic amino acids: arginine, lysine, ornithine.

[0024] In this invention, the salt formed with acidic amino acids includes salts formed with one or more of the following acidic amino acids: aspartic acid and glutamic acid.

[0025] CCL4-induced liver fibrosis animal models are the most classic and widely used method for establishing liver fibrosis animal models in this field. For example, this method is disclosed in documents such as "Detailed Methods for Establishing Animal Models, Volume 5 | Mouse Liver Fibrosis Model" (https: / / muhn.edu.cn / ecmd / info / 1481 / 20795.htm) and "[Animal Modeling - Drug Efficacy Evaluation] - Carbon Tetrachloride-Induced Liver Fibrosis Animal Model" (https: / / www.zvast-bio.com / newsarticle / 9681.html).

[0026] A second aspect of the invention provides a reagent combination for constructing an animal model of liver fibrosis, comprising: a TLR1 / TLR2 specific agonist and CCL4.

[0027] In some embodiments, the TLR1 / TLR2 specific agonist and CCL4 are present independently.

[0028] In some embodiments, the TLR1 / TLR2 specific agonist and CCL4 are administered separately; the CCL4 and TLR1 / TLR2 specific agonist are administered sequentially and separately.

[0029] In some embodiments, when administered, the TLR1 / TLR2 specific agonist and CCL4 are in a mass-volume ratio (mg:mL) of (1-100):1; further, (5-25):1; and even further, (9-11):1.

[0030] In some embodiments, the reagent combination also includes an instruction manual describing a method for constructing an animal model of liver fibrosis.

[0031] In some embodiments, the method for constructing the animal model of liver fibrosis includes the following steps: administering the TLR1 / TLR2 specific agonist and CCL4 to the animal.

[0032] In some embodiments, the method for constructing the animal model of liver fibrosis includes the following steps: sequentially administering CCL4 and a TLR1 / TLR2 specific agonist to the animal.

[0033] In some embodiments, the method for constructing the animal model of liver fibrosis includes the following steps: 1) Perform the following operations 1-3 times per week (preferably 2 times per week): The animals were sequentially administered CCL4 and TLR1 / TLR2 specific agonists, wherein the interval between administration of CCL4 and TLR1 / TLR2 specific agonists was 0.5-3 days (preferably 0.5-2 days; more preferably 0.8-1.2 days).

[0034] In some implementations, step 1) is performed for 2-10 weeks; preferably for 2-10 weeks; more preferably for 2-6 weeks; and even more preferably for 2-4 weeks.

[0035] In some implementations, when the above operations are performed 2-3 times per week, the interval between the two operations is 0.5-5 days (preferably 0.5-3.5 days; more preferably 0.8-2.2 days; and even more preferably 0.8-1.2 or 1.8-2.2 days).

[0036] In some embodiments, the administration methods of the TLR1 / TLR2 specific agonist and CCL4 are each independently selected from oral and parenteral administration; preferably, the administration methods of the TLR1 / TLR2 specific agonist and CCL4 are each independently selected from oral, intravenous, intramuscular, intraperitoneal, intrathecal, intracranial, and subcutaneous administration.

[0037] In some embodiments, the TLR1 / TLR2 specific agonist and CCL4 are administered via intraperitoneal injection.

[0038] In some embodiments, the dosage of the TLR1 / TLR2 specific agonist is 0.1-10 mg / kg per dose in mice (further, 0.5-3 mg / kg per dose; even further, 0.8-1.2 mg / kg per dose); the dose per unit body weight for different subjects can be calculated using equivalent dose conversion relationships.

[0039] In some embodiments, the dosage of CCL4 administered is 0.01-1 mL / kg·dose in mice (further, 0.05-2 mL / kg·dose; even further, 0.08-0.12 mL / kg·dose); the dose per unit body weight for different subjects can be calculated using equivalent dose conversion relationships.

[0040] A third aspect of the present invention provides the use of the reagent combination of the second aspect of the present invention in constructing an animal model of liver fibrosis or in preparing a product for constructing an animal model of liver fibrosis.

[0041] In some implementations, the product includes a reagent kit or reagent kit.

[0042] A fourth aspect of the present invention provides a method for constructing an animal model of liver fibrosis, comprising the step of using a reagent combination from a second aspect of the present invention.

[0043] In some embodiments, the method for constructing the animal model of liver fibrosis includes the following steps: administering the TLR1 / TLR2 specific agonist and CCL4 to the animal.

[0044] In some embodiments, the method for constructing the animal model of liver fibrosis includes the following steps: sequentially administering CCL4 and a TLR1 / TLR2 specific agonist to the animal.

[0045] In some embodiments, the method for constructing the animal model of liver fibrosis includes the following steps: 1) Perform the following operations 1-3 times per week (preferably 2 times per week): The animals were sequentially administered CCL4 and TLR1 / TLR2 specific agonists, wherein the interval between administration of CCL4 and TLR1 / TLR2 specific agonists was 0.5-3 days (preferably 0.5-2 days; more preferably 0.8-1.2 days).

[0046] In some implementations, step 1) is performed for 2-10 weeks; preferably for 2-10 weeks; more preferably for 2-6 weeks; and even more preferably for 2-4 weeks.

[0047] In some implementations, when the above operations are performed 2-3 times per week, the interval between the two operations is 0.5-5 days (preferably 0.5-3.5 days; more preferably 0.8-2.2 days; and even more preferably 0.8-1.2 or 1.8-2.2 days).

[0048] In some embodiments, the administration methods of the TLR1 / TLR2 specific agonist and CCL4 are each independently selected from oral and parenteral administration; preferably, the administration methods of the TLR1 / TLR2 specific agonist and CCL4 are each independently selected from oral, intravenous, intramuscular, intraperitoneal, intrathecal, intracranial, and subcutaneous administration.

[0049] In some embodiments, the TLR1 / TLR2 specific agonist and CCL4 are administered via intraperitoneal injection.

[0050] In some embodiments, the dosage of the TLR1 / TLR2 specific agonist is 0.1-10 mg / kg per dose in mice (further, 0.5-3 mg / kg per dose; even further, 0.8-1.2 mg / kg per dose); the dose per unit body weight for different subjects can be calculated using equivalent dose conversion relationships.

[0051] In some embodiments, the dosage of CCL4 administered is 0.01-1 mL / kg·dose in mice (further, 0.05-2 mL / kg·dose; even further, 0.08-0.12 mL / kg·dose); the dose per unit body weight for different subjects can be calculated using equivalent dose conversion relationships.

[0052] A fifth aspect of the present invention provides the application of animal models obtained by the construction method of the fourth aspect of the present invention in screening anti-hepatic fibrosis drugs or in liver fibrosis research (e.g., research on the pathogenesis of liver fibrosis).

[0053] The beneficial effects of this invention are: This invention discloses for the first time the application of TLR1 / TLR2 specific agonists in improving the efficiency of CCL4-induced liver fibrosis animal models. Although TLR1 / TLR2 specific agonists themselves cannot induce liver fibrosis animal models, they can improve the efficiency of CCL4-induced liver fibrosis animal models.

[0054] This invention discloses for the first time a reagent combination for constructing an animal model of liver fibrosis, comprising: a TLR1 / TLR2 specific agonist and CCL4. This reagent combination has the following advantages for constructing an animal model of liver fibrosis: 1) Accelerated modeling speed: This invention combines TLR1 / TLR2 agonists with CCL4, allowing for the observation of a significant liver fibrosis phenotype within just 2 weeks of administration, while traditional CCL4 models typically require 4-8 weeks. This significantly shortens the experimental cycle and improves research efficiency.

[0055] 2) Enhanced fibrosis: Under the same administration time (e.g., 4 weeks), the degree of liver fibrosis (e.g. Sirius red positive area) in the combined model group was significantly stronger than that in the CCL4-only group, and the model phenotype was more stable and easier to observe and quantify.

[0056] 3) Simple and safe operation: This invention does not require complicated surgery, but only uses conventional drug administration methods (such as intraperitoneal injection), which is simple to operate and requires low skill from the experimenters.

[0057] 4) No significant effect on renal function: Experimental results showed that while the combined administration group significantly aggravated liver damage, it had no significant adverse effect on renal function (such as blood urea nitrogen and creatinine levels) in mice, indicating that the TLR1 / TLR2 agonist (Pam3CSK4) has high safety and does not increase the risk of toxicity. Attached Figure Description

[0058] Figure 1 The experimental procedures of Examples 1-2 are shown in schematic diagram.

[0059] Figure 2 HE staining, Sirius red staining, and Masson staining results are shown for the normal control group (NC group), the model group (CCL4 group), and the combined modeling group (CCl4+Pam3CSK4) in Example 1.

[0060] Figure 3 The results of detecting liver and kidney function indicators in the serum of mice in the normal control group (NC group), model group (CCL4 group), and combined modeling group (CCl4+Pam3CSK4) in Example 1 are shown.

[0061] Figure 4 The results of Sirius red staining and detection of liver function indicators in mouse serum are shown for the normal control group (NC group) and the Pam3CSK4 model group (Pam3CSK4 group) in Example 1.

[0062] Figure 5 The results of pathological staining (HE, Sirius red staining and Masson staining) and detection of serum liver and kidney function indicators in mice are shown in Example 2 for the normal control group (NC group), model group (CCL4 group), and combined modeling group (CCl4+Pam3CSK4).

[0063] Figure 2-3 and Figure 4 This is based on the results of two batches of experiments. Detailed Implementation

[0064] 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. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention. Such structures and techniques have also been described in many publications.

[0065] definition Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0066] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0067] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0068] Those skilled in the art will understand that when a numerical range is listed herein, it is intended to indicate that any intermediate value or range of values ​​within the listed numerical range is also within the scope of this invention.

[0069] As used herein, the terms “including,” “comprising,” “having,” “containing,” or “involving,” and their other variations thereof, are inclusive or open-ended and do not exclude other unlisted elements or method steps.

[0070] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available. For reagents whose manufacturers are listed, similar products from other manufacturers are substituted.

[0071] The experimental subjects involved in the following examples were 6-8 week old male C57BL / 6J mice; the experimental reagents included: carbon tetrachloride (CCl4) (purchased from MCE, catalog number: HY-Y0298), a classic liver fibrosis modeling reagent, which was dissolved in corn oil at a volume ratio of 20% to obtain a CCl4 solution; Pam3CSK4 (purchased from MCE, catalog number: HY-P1180A) is a TLR1 / TLR2 specific agonist, which was prepared as a storage solution with dimethyl sulfoxide (DMSO) and as a working solution with physiological saline.

[0072] Example 1: Eight-week-old healthy male C57bl / 6J mice were randomly divided into four groups: a normal control group (NC group), a model group (CCL4 group), a Pam3CSK4 model group (Pam3CSK4 group), and a combined model group (CCl4 + Pam3CSK4), with six mice in each group. The overall experimental design is as follows: Figure 1 As shown, the specific steps are as follows: CCl4 solution was injected intraperitoneally on the first and fourth days of each week at a rate of 0.5 μL / g. One day after each CCl4 injection, Pam3CSK4 was injected intraperitoneally at a rate of 1 mg / kg. Each injection was administered at the same time point (e.g., CCl4 solution was injected at 10:00 AM on day 1, Pam3CSK4 at 10:00 AM on day 2, CCl4 solution at 10:00 AM on day 4, and Pam3CSK4 at 10:00 AM on day 5). After four weeks of continuous administration, the mice were sacrificed, and blood was collected from the orbital sinus. Serum was separated to detect liver and kidney function indicators (ALT, AST, BUN, CREA). Liver tissue was collected for pathological staining (HE, Sirius red stain, and Masson stain).

[0073] HE staining, Sirius red staining, and Masson staining results of the normal control group (NC group), model group (CCL4 group), and combined modeling group (CCl4+ Pam3CSK4) are as follows: Figure 2 As shown: At 4 weeks of modeling, both the model group (CCL4) and the combined modeling group (CCl4+Pam3CSK4) showed obvious liver fibrosis characteristics after HE staining, Sirius red staining, and Masson staining. Quantitative analysis of collagen-stained areas in Sirius red staining revealed that the degree of fibrosis in the combined modeling group was significantly higher than that in the model group, indicating that the mice in the combined modeling group had a stronger degree of collagen accumulation and more severe liver fibrosis. The results of detecting liver and kidney function indicators in the serum of mice in the normal control group (NC group), model group (CCL4 group), and combined modeling group (CCl4+Pam3CSK4) are shown below. Figure 3As shown: Compared with the normal control group, the liver damage indicators ALT and AST in the model group and the combined model group were significantly increased, and the combined model group was higher than the model group, indicating that the liver damage in the combined model group was more severe; the kidney damage indicators BUN and CREA did not change significantly, indicating that the combined model group (CCl4+ Pam3CSK4) did not cause significant kidney damage.

[0074] Sirius red staining results and serum liver function indicators in mice from the normal control group (NC group) and the Pam3CSK4 model group (Pam3CSK4 group) are as follows: Figure 4 As shown: there was no significant difference in the Sirius red positive staining area between the normal control group and the Pam3CSK4 model group; in fact, the Sirius red positive staining area in the Pam3CSK4 model group was slightly lower than that in the normal control group. There was no significant difference in ALT between the normal control group and the Pam3CSK4 model group; in fact, the ALT in the Pam3CSK4 model group was slightly lower than that in the normal control group. There was no significant difference in AST between the normal control group and the Pam3CSK4 model group; in fact, the AST in the Pam3CSK4 model group was slightly lower than that in the normal control group. This indicates that Pam3CSK4 alone cannot induce liver fibrosis. Figure 2 The results showed that the Sirius red positive staining area in the combined model group (CCl4+ Pam3CSK4) was significantly higher than that in the normal control group (NC group) than the sum of the individual Sirius red positive staining areas in the model group (CCL4 group) and the Pam3CSK4 model group (Pam3CSK4 group) compared to the normal control group (NC group). Similarly, the ALT level in the combined model group (CCl4+ Pam3CSK4) was significantly higher than that in the normal control group (NC group) than the sum of the individual ALT levels in the model group (CCL4 group) and the Pam3CSK4 model group (Pam3CSK4 group) compared to the normal control group (NC group). The increase in AST of Pam3CSK4 compared to the normal control group (NC group) was much greater than the sum of the increases in AST of the model group (CCL4 group) and the Pam3CSK4 model group compared to the normal control group (NC group). This means that Pam3CSK4 can synergistically accelerate the process of CCL4-induced liver fibrosis, and Pam3CSK4 and CCL4 have a synergistic effect in inducing liver fibrosis.

[0075] Example 2: Eight-week-old male C57bl / 6J mice were randomly divided into three groups: a normal control group (NC group), a model group (CCL4 group), and a combined modeling group (CCl4 + Pam3CSK4), with six mice in each group. The overall experimental design is as follows: Figure 1As shown, the specific steps are as follows: CCl4 solution was injected intraperitoneally on the first and fourth days of each week at a rate of 0.5 μL / g. One day after each CCl4 injection, Pam3CSK4 was injected intraperitoneally at a rate of 1 mg / kg. Each injection was administered at the same time point (e.g., CCl4 solution was injected at 10:00 AM on day 1, Pam3CSK4 at 10:00 AM on day 2, CCl4 solution at 10:00 AM on day 4, and Pam3CSK4 at 10:00 AM on day 5). After two weeks of continuous administration, the mice were sacrificed, blood was collected from the orbital sinus, and serum was separated to detect liver and kidney function indicators (ALT, AST, BUN, CREA). Liver tissue was collected for pathological staining (HE, Sirius red stain, and Masson stain).

[0076] The pathological staining (HE, Sirius red staining, and Masson staining) and serum liver and kidney function indicators of mice in the normal control group (NC group), model group (CCL4 group), and combined modeling group (CCl4+ Pam3CSK4) are as follows: Figure 5 As shown, no obvious liver fibrosis was observed in the model group (CCL4) at 2 weeks of modeling. However, significant liver fibrosis was observed in the combined modeling group (CCl4 + Pam3CSK4) using HE staining, Sirius red staining, and Masson staining. Quantitative analysis of collagen staining areas in Sirius red staining revealed no significant increase in positive staining areas in the model group compared to the normal control group, while the degree of fibrosis was significantly increased in the combined modeling group, indicating successful establishment of liver fibrosis in the combined modeling group. Serum liver function indicators showed significantly elevated ALT and AST levels in the combined modeling group compared to the normal control group, with the combined modeling group exhibiting higher levels than the modeling group, indicating more severe liver damage. These results combined with the CCL4 + Pam3CSK4 modeling approach accelerates the establishment of the liver fibrosis model, with significant liver fibrosis observed at 2 weeks of modeling.

[0077] The technical solutions disclosed herein are not limited to the specific embodiments described above. Any technical modifications made based on the technical solutions disclosed herein shall fall within the protection scope of this disclosure.

Claims

1. Application of TLR1 / TLR2 specific agonists in any of a1)-a2); a1) Improve the efficiency of CCL4-induced liver fibrosis animal models; a2) Prepare products that improve the efficiency of CCL4-induced liver fibrosis animal models.

2. The application according to claim 1, characterized in that, The improvement of the efficiency of CCL4-induced liver fibrosis animal models includes: shortening the time of CCL4-induced liver fibrosis animal model and / or increasing the degree of liver fibrosis in the animal model; Preferably, the product includes reagents, reagent kits, or reagent sets; Preferably, the TLR1 / TLR2 specific agonist includes Pam3CSK4 or a pharmaceutically acceptable salt thereof.

3. A reagent set, comprising: TLR1 / TLR2 specific agonists and CCL4.

4. The reagent combination according to claim 3, characterized in that, The TLR1 / TLR2 specific agonists and CCL4 exist independently in various forms. Preferably, the TLR1 / TLR2 specific agonist comprises Pam3CSK4 or a pharmaceutically acceptable salt thereof; Preferably, the reagent kit also includes an instruction manual that describes a method for constructing an animal model of liver fibrosis.

5. The use of the reagent combination of any one of claims 3 to 4 in constructing an animal model of liver fibrosis or in preparing a product for constructing an animal model of liver fibrosis.

6. A method for constructing an animal model of liver fibrosis, comprising the step of using a combination of reagents according to any one of claims 3 to 4.

7. The reagent combination or construction method according to claim 4 or 6, characterized in that, The method for constructing the animal model of liver fibrosis includes the following steps: administering the TLR1 / TLR2 specific agonist and CCL4 to the animal; Preferably, the method for constructing the animal model of liver fibrosis includes the following steps: sequentially administering CCL4 and a TLR1 / TLR2 specific agonist to the animal; Preferably, the method for constructing the animal model of liver fibrosis includes the following steps: 1) Perform the following operations 1-3 times per week: The animals were administered CCL4 and TLR1 / TLR2 specific agonists sequentially, with an interval of 0.5-3 days between administrations of the CCL4 and TLR1 / TLR2 specific agonists. Preferably, step 1) is performed for 2-10 weeks; Preferably, when the operation is performed 2-3 times per week, the interval between the two operations is 0.5-5 days.

8. The reagent combination or construction method according to claim 7, characterized in that, The TLR1 / TLR2 specific agonist and CCL4 are each administered via oral or parenteral route. Preferably, the administration methods of the TLR1 / TLR2 specific agonist and CCL4 are each independently selected from oral, intravenous, intramuscular, intraperitoneal, intrathecal, intracranial, and subcutaneous injections. Preferably, the dosage of the TLR1 / TLR2 specific agonist is 0.1-10 mg / kg per dose in mice. Preferably, the dosage of CCL4 is 0.01-1 mL / kg per dose for mice.

9. The use of the animal model obtained by the construction method according to any one of claims 6 to 8 in screening anti-liver fibrosis drugs or in liver fibrosis research.

10. The application, reagent combination, or construction method according to any one of claims 1 to 9, characterized in that, The animal in question is a non-human animal; Preferably, the non-human animal includes non-human mammals; further includes rodents; and even further includes mice, rats, or guinea pigs.

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