Evaluation method of CCl4-induced cirrhosis model and application thereof

Abstract

The invention relates to the technical field of animal model construction and evaluation, in particular to an evaluation method of a CCl4-induced cirrhosis model and application of the evaluation method. Comprising the following steps: S1, establishing a rat liver cirrhosis model by adopting a CCl4 induction mode; s2, performing ultrasonic imaging on the liver cirrhosis model rat to obtain a gray value of the liver of the liver cirrhosis model rat; s3, evaluating the liver injury degree of the liver cirrhosis model rat according to the gray value of the liver of the liver cirrhosis model rat. The invention provides a method for accurately and comprehensively evaluating the liver injury degree without preparing pathological tissue slices or detecting liver functions or additionally adding experimental animals, and the method has important significance on screening of animal models and development of related animal experiments.

Description

Technical Field

[0001] The present invention relates to the technical field of animal model construction and evaluation, and in particular to an evaluation method for a CCl4-induced liver cirrhosis model and its application. Background Art

[0002] In the evaluation of traditional cirrhosis models, two main methods are routinely used to assess the effectiveness of modeling. The first is histopathological assessment. This involves randomly selecting a certain number of experimental rats after the modeling process is complete, sampling their liver tissue, and then conducting a histopathological examination. The second is serological index detection. Similarly, after the modeling is completed, peripheral blood is collected from the experimental rats and serological indicators related to liver function are tested to determine the specific extent of liver damage after modeling. Although these two methods can intuitively determine the extent of liver damage in the selected model rats, the random sampling method used to select experimental subjects inevitably leads to the evaluation results being affected by random factors, making it difficult to fully and accurately reflect the liver damage status of the entire experimental animal population. In addition, to reduce the error caused by randomness and ensure the reliability of the evaluation results, it is usually necessary to increase the number of experimental animals. This not only increases the experimental cost but also violates the "3R" principle of animal experiment protection (replacement, reduction, and optimization).

[0003] Chinese invention patent application CN116837085A discloses a method for simulating the activation and inhibition of the TLR5 signaling pathway in carbon tetrachloride-induced liver injury. By using CBLB502 protein as a TLR5 agonist, it inhibits acute liver injury induced by carbon tetrachloride, solving the problem that the TLR5 signaling has no obvious effect in acute liver injury, significantly reducing liver damage and improving survival rate, providing a simulation method for the activation and inhibition of the TLR5 signaling pathway, and providing a theoretical basis for the prevention and treatment of liver diseases. Chinese invention patent application CN120192910A discloses a method for constructing and evaluating an organoid model of liver injury. By constructing and propagating mouse liver cells in a three-dimensional culture system to simulate the pathological environment of liver injury, it solves the problems of high cost, long cycle and ethical controversy in the construction of liver injury models in the existing technology. However, these existing technologies all rely on pathological tissue sections or tissue cell detection when evaluating the degree of liver injury, and none of them solve the above-mentioned technical problems.

[0004] In this context, it is imperative to provide a method that can accurately and comprehensively assess the degree of liver damage in experimental rats without preparing pathological tissue sections or collecting blood for liver function testing. Summary of the Invention

[0005] The technical problem to be solved by the present invention is that, while traditional liver cirrhosis assessment methods (histopathology or liver function tests) can intuitively determine the extent of liver damage in selected model mice, their use of random sampling leads to significant uncertainty in the evaluation results, making it difficult to fully and accurately reflect the actual extent of liver damage in the entire experimental animal population. Furthermore, this method requires an additional number of experimental animals. The present invention provides a method for accurately and comprehensively assessing the extent of liver damage using a small number of experimental animals, without the need for preparing pathological tissue sections or testing liver function. This method addresses the aforementioned technical problems and is of great significance for the development of animal experiments in the field of liver damage.

[0006] The first aspect of the present invention provides a method for evaluating a CCl4-induced liver cirrhosis model, comprising:

[0007] S1. Establishment of rat liver cirrhosis model by CCl4 induction;

[0008] S2. performing ultrasound imaging on the cirrhosis model rat to obtain a grayscale value of the liver of the cirrhosis model rat;

[0009] S3. Evaluate the degree of liver damage in the liver of the cirrhosis model rat according to the grayscale value of the liver of the cirrhosis model rat.

[0010] The evaluation criteria in step S3 are: grayscale value < X indicates mild liver damage, X ≤ grayscale value ≤ Y indicates moderate liver damage, and grayscale value > Y indicates severe liver damage;

[0011] 0<X<Y.

[0012] In some embodiments, X and Y are determined by fitting the collagen area of ​​liver pathological tissue sections of cirrhosis model rats and the gray value data of the liver of cirrhosis model rats.

[0013] Optionally, the method for determining the evaluation criteria includes: preparing liver pathological tissue sections of cirrhosis model rats and performing Masson staining to obtain the collagen area of ​​the liver pathological tissue sections of the cirrhosis model rats, fitting the statistically obtained collagen area and the grayscale value data of the liver of the cirrhosis model rats to determine the values ​​of X and Y, and then determining the evaluation criteria.

[0014] Optional, 80<X<95, 100<Y<130.

[0015] Further optionally, 85<X<92, 105<Y<120.

[0016] Further optionally, X=90, Y=110.

[0017] In some embodiments, the S1 step includes: injecting a CCl4 olive oil mixed solution into experimental rats to prepare a rat cirrhosis model; the injection frequency is twice a week for a total of 8 to 12 weeks.

[0018] Optionally, the single injection volume of the CCl4 olive oil mixed solution is 1 to 5 mg / kg.

[0019] Optionally, the CCl4 olive oil mixed solution is injected into the peritoneal cavity.

[0020] Optionally, the volume ratio of CCl4 to olive oil in the CCl4 olive oil mixed solution is 1:(1.2-2); further optionally 1:1.5.

[0021] Examples of imaging methods in this field include ultrasound imaging, computed tomography (CT), magnetic resonance imaging (MRI), optical coherence tomography (OCT), infrared thermal imaging, and the like.

[0022] In some embodiments, the step S2 preferably uses ultrasound imaging to obtain the grayscale value of the liver of the cirrhosis model rat.

[0023] The existing technology attempts to use ultrasonic backscatter integrated (IBS) to evaluate the degree of liver fibrosis and cirrhosis in rats. However, IBS relies on the original radio frequency signals collected by ultrasound equipment. These original signals need to be complexly calculated and analyzed during the data processing process, which not only increases the difficulty and time cost of the operation, but also places high demands on the operators. At the same time, IBS analysis requires special equipment, which to a certain extent limits its promotion and application; at the same time, the original radio frequency signals are more sensitive to noise and are easily interfered with during the signal acquisition and processing process, thereby affecting the accuracy of the evaluation results. The present invention specifically selects ultrasound imaging to obtain the grayscale value of the liver of rats with cirrhosis model, and uses grayscale value as a key measurement indicator to evaluate the degree of liver damage. Grayscale value analysis is directly based on the brightness information of B-ultrasound images. Its operation process is simple and clear, and there is no need for complex data processing and conversion of ultrasound signals. The evaluation results can be quickly obtained and real-time evaluation can be achieved. Moreover, this analysis method is suitable for conventional ultrasound equipment and does not require the purchase of additional special equipment, which greatly reduces the use threshold and cost. The present invention utilizes grayscale value analysis of ultrasonic echo signals, which has outstanding advantages in terms of operational convenience, real-time performance, and wide applicability in preclinical research. It has been pioneered in the evaluation of liver damage in a rat cirrhosis model induced by CCl4, achieving accurate and comprehensive evaluation results.

[0024] In some embodiments, the Masson staining step comprises:

[0025] The pathological tissue section is prepared from the liver of a rat, and the section is soaked and incubated in a Boin's liquid, and then dyed by using iron hematoxylin dyeing liquid, acid magenta dyeing liquid and aniline blue dyeing liquid in sequence; then dehydrated, mounted, and observed under a microscope to obtain the dyeing result, and the collagen area is counted.

[0026] Optionally, before being dyed by the aniline blue dyeing liquid, the section is treated by using a phosphomolybdic acid aqueous solution to enhance the contrast of the dyeing; and further optionally, the concentration of the phosphomolybdic acid aqueous solution is 0.1-2%.

[0027] Optionally, the soaking and incubation condition is 50-60 DEG C for 10-30 minutes.

[0028] The second aspect of the present application provides an application of an evaluation method of a CCl4-induced liver cirrhosis model.

[0029] The present application initiatively establishes a non-invasive evaluation method, which does not need to perform pathological histological analysis on the experimental animals after liver sampling, or to perform serological detection of liver function after peripheral blood sampling, but only needs to perform liver ultrasonic imaging examination on the experimental objects, so that the liver damage degree can be accurately evaluated. The evaluation standard is established by exploring the corresponding relationship between the gray value of the liver echo signal of each group of experimental rats and the statistical value of the Masson dyeing collagen area of the liver section, so that the liver damage degree of the liver cirrhosis model can be evaluated by ultrasonic imaging. This method can not only reduce the number of experimental animals, but also more comprehensively evaluate the liver damage degree of each experimental rat after modeling, avoid experimental errors caused by random selection of animals for evaluation, and efficiently and accurately evaluate the liver damage degree of the experimental objects of the liver cirrhosis model. Further, this method can efficiently and accurately screen the liver cirrhosis model animals, and provide strong support for the development of animal experiments such as disease mechanism research, drug research and development, clinical transformation research, disease prevention and early intervention in this field, and has important application value. At the same time, this method is simple and easy to operate, and has strong operability.

[0030] Beneficial effects:

[0031] The present application provides an evaluation method and application of a CCl4-induced liver cirrhosis model, which has the following advantages:

[0032] (1) The liver damage degree of the liver cirrhosis model is evaluated by performing liver ultrasonic imaging examination on the experimental objects, which avoids pathological histological analysis on the experimental animals after liver sampling, or serological detection of liver function after peripheral blood sampling, avoids the trauma of invasive means to the experimental objects, and conforms to the "3R" principle of animal experiment protection.

[0033] (2) By exploring the correspondence between the grayscale value of the liver echo signal of each group of experimental rats and the statistical value of the Masson-stained collagen area of ​​their liver sections, an evaluation standard was established, which can further improve the accuracy of the evaluation results.

[0034] (3) By fitting the grayscale value of the liver of rats with cirrhosis model and the collagen area of ​​the liver pathological tissue sections of rats with cirrhosis model, the evaluation standard was determined as grayscale value <90 for mild liver damage, 90≤grayscale value≤110 for moderate liver damage, and grayscale value >110 for severe liver damage. This evaluation standard can accurately evaluate the degree of liver damage in CCl4-induced cirrhosis model.

[0035] (4) The evaluation results were verified by gross observation of the liver, liver function tests and pathological histological analysis. The results showed that the test results of the three groups of experimental rats with low grayscale values ​​(<90), medium grayscale values ​​(90-110) and high grayscale values ​​(>110) showed significant differences. This verified the feasibility of the method of determining the liver injury evaluation standard by fitting the grayscale value of the liver echo signal with the collagen area of ​​the liver tissue section, and further proved that the evaluation results were accurate and reliable.

[0036] (5) The present invention can replace the traditional method of making pathological tissue sections after dissection or taking peripheral blood to detect liver function. It can not only reduce the number of experimental animals, but also more comprehensively evaluate the degree of liver damage of each experimental rat after modeling. It can efficiently and accurately screen out cirrhosis model animals, which is conducive to the development of animal experiments in related fields.

[0037] (6) The evaluation method of the present invention is simple and easy to use, more convenient to operate, and highly operable; it provides strong support for the development of animal experiments such as disease mechanism research, drug development, clinical translational research, disease prevention and early intervention in this field, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 . The fitting curve of the gray value of the experimental rat liver echo signal and the collagen area of ​​the liver pathological tissue section in the embodiment;

[0039] Figure 2 Gross observation results of the livers of experimental rats in each group;

[0040] Figure 3 .The results of liver pathological histological staining of experimental rats in each group. Figure 3A is the HE staining result of the healthy group; B is the HE staining result of the low gray value model group; C is the HE staining result of the medium gray value model group; D is the HE staining result of the high gray value model group; E is the Masson staining result of the healthy group; F is the Masson staining result of the low gray value model group; G is the Masson staining result of the medium gray value model group; H is the Masson staining result of the high gray value model group; the image scale is 1000 μm;

[0041] Figure 4 .Statistical results of collagen area in liver pathological tissue sections of experimental rats in each group; Figure 4 In the table, * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, and **** represents P < 0.0001. DETAILED DESCRIPTION

[0042] The sources of some consumables used in the specific embodiments of the present invention are as follows.

[0043] CCl4 was sourced from Adamas brand of Shanghai Titan Technology Co., Ltd., with the product number being 65805J.

[0044] Isoflurane was obtained from Tianjin Ruipu Biotechnology Co., Ltd., with the product number of veterinary drug 020037015.

[0045] The medical ultrasonic coupling agent comes from Tianjin Jinya Technology Development Co., Ltd., and the product number is TM-100.

[0046] The multi-mode small animal ultrasound system was from Fujifilm VisualSonics Co., Ltd., model VEVO LAZR-X.

[0047] The linear array probe was from Fujifilm Visual Sonics Co., Ltd., model MX 250S.

[0048] The Masson staining kit was obtained from Shanghai Ruibaohe Biotechnology Co., Ltd., with the catalog number RB0003.

[0049] Neutral gum was obtained from Sinopharm Chemical Reagent Co., Ltd. with the product number 10004160.

[0050] The fully automatic biochemical analyzer was from Shenzhen Raydu Life Science Co., Ltd., model Chemray 240.

[0051] Experimental rats (male Sprague-Dawley rats) were obtained from Shanghai Slake Laboratory Animal Co., Ltd.

[0052] Unless otherwise specified, the reagents, devices and other consumables used in the present application are commercially available; the concentration of the solution is mass concentration.

[0053] Embodiment

[0054] The present embodiment provides an evaluation method of a CCl4-induced cirrhosis model, comprising:

[0055] S1. A CCl4-induced method is used to establish a rat cirrhosis model, and the specific operation method comprises:

[0056] 1) Test grouping: 14 experimental rats are randomly divided into 2 groups (3 healthy rats, 11 model rats), and the experimental rats are male Sprague-Dawley rats, and the weight of a single experimental rat is 200±20g.

[0057] 2) Modeling: the healthy group is not treated (as a control); the model group is injected with a CCl4olive oil mixed solution into the experimental rats by intraperitoneal injection, and 40vol% of the CCl4olive oil mixed solution is injected intraperitoneally once a week on the first day and the fourth day, and the injection dose is 2.5mL / kg of body weight, and a total of 10 weeks of injection is completed, and the preparation of the rat cirrhosis model is completed.

[0058] S2. Ultrasound imaging is performed on the cirrhosis model rats to obtain the gray value of the liver of the cirrhosis model rats, and the specific operation method comprises:

[0059] 1) Hair removal treatment: on the day before the ultrasound detection is carried out, the experimental rats are placed in an anesthesia room, and they are anesthetized by inhaling isoflurane; after the anesthesia takes effect, the hair removal cream is evenly applied to the upper abdomen of the experimental rats, and after 5-10 minutes, the removed rat hair is wiped off with a wet sterile gauze, and if there is a small amount of scattered unshaved hair left, a razor is used to further remove it; complete the hair removal.

[0060] 2) Ultrasound imaging: a multi-mode small animal ultrasound system is used to collect liver section images with a linear array probe with a center frequency of 21MHz; specifically, the anesthetized experimental rats are fixed on the ultrasound imaging operation table, and the upper abdomen is fully exposed; the coupling agent is evenly applied to the detection site, and then the linear array probe is placed in full contact with the skin in the liver detection area of the experimental rats; adjust the angle of the linear array probe to make the image optimal, start automatic scanning, and save the scanning results after scanning is completed.

[0061] 3) Data processing: use Vevo LAB 5.8.2 software to process the scanning results, select the image with the maximum cross-sectional area of the liver in the ultrasound image as the analysis object by Area (region), and calculate the average echo signal gray value (unit: [a.u.] / mm 2); Three different areas were selected for measurement on each transverse plane, and then the average value was calculated and used as the grayscale value of the liver of the cirrhosis model rat.

[0062] The results showed that the grayscale value of liver echo signals in healthy rats was 34.36±4.76[au] / mm 2 , while the grayscale value of the liver of the cirrhosis model mice in the experimental group was 100.73±15.31[au] / mm 2 That is, after 10 consecutive weeks of intraperitoneal injection of CCl4 olive oil mixed solution to induce liver cirrhosis, the grayscale value of the liver of experimental rats increased by 193% compared with healthy rats (P<0.001).

[0063] S3. Evaluate the degree of liver damage in the liver of the cirrhosis model rat according to the grayscale value of the liver of the cirrhosis model rat.

[0064] The evaluation criteria in step S3 are: grayscale value < X indicates mild liver damage, X ≤ grayscale value ≤ Y indicates moderate liver damage, grayscale value > Y indicates severe liver damage; 0 < X ​​< Y.

[0065] The method for determining the evaluation criteria includes: preparing liver pathological tissue sections of cirrhosis model rats and performing Masson staining to obtain the collagen area of ​​the liver pathological tissue sections of the cirrhosis model rats, and fitting the statistically obtained collagen area and the grayscale value data of the cirrhosis model rats to determine X and Y; the specific operation method includes:

[0066] 1) Preparation of tissue sections:

[0067] The experimental rats in each group were anesthetized and then killed by cervical dislocation. The liver tissues were collected. The livers were fixed with 10 times the volume of 10% formalin solution for 3 days and then made into paraffin sections for later use.

[0068] 2) Masson staining:

[0069] Stain using the Masson staining kit as follows:

[0070] 2.1 Dewax the paraffin sections before staining (immerse the sections in xylene I for 20 minutes, xylene II for 20 minutes, anhydrous ethanol I for 10 minutes, anhydrous ethanol II for 10 minutes, then in 95%, 90%, 80%, and 70% alcohol for 5 minutes each, and finally rinse in distilled water for 5 minutes). Soak the dewaxed tissue sections in Bonn's solution at 56°C for 15 minutes, then rinse gently with running water for 3 minutes.

[0071] 2.2 Place the sections in a 0.7% iron hematoxylin staining solution for 5 minutes, then rinse with running water for 3 minutes, and then rinse with deionized water for 1 minute;

[0072] 2.3 Place the sections in a 1% Ponceau acid fuchsin staining solution for 5 minutes and rinse with deionized water for 1 minute.

[0073] 2.4 Place the sections in a 1% phosphomolybdic acid solution for 5 minutes, then directly place them in a 2% aniline blue solution for 5 minutes.

[0074] 2.5 After staining, use filter paper to absorb the aniline blue staining solution and transfer the tissue sections to a 1% acetic acid aqueous solution and soak for 2 minutes;

[0075] 2.6 Rinse the soaked tissue sections with deionized water for 1 minute, then dehydrate with 95% ethanol solution for 2-3 seconds, and then with anhydrous ethanol for 5 seconds, repeating three times;

[0076] 2.7 Place the dehydrated tissue sections in xylene for 1 minute (to make the sections transparent), repeat 3 times, and then use neutral gum to seal the sections;

[0077] 2.8 Observe the staining of tissue sections under a microscope and capture images.

[0078] 3) Calculate collagen area:

[0079] Five randomly selected images from each rat's tissue section were used for statistical analysis of collagen area using ImagePro Plus 6.0 software. The results showed that the collagen area of ​​the healthy rats was 2381.47±786.20, while the collagen fibers of the model rats increased to varying degrees 10 weeks after modeling, reaching 62621.11±35006.99, a 26.30-fold increase (P<0.001).

[0080] 4) Draw the fitting curve:

[0081] The grayscale value of the echo signal of the liver of the rats in each experimental group was used as the horizontal axis, and the collagen area statistical value after Masson staining of the liver tissue section was used as the vertical axis. The corresponding relationship between the two was drawn and the curve fitting was performed (n=14). The fitting results are shown in Figure 1 .

[0082] 5) Determine the evaluation criteria:

[0083] like Figure 1 As shown in the figure, the collagen area of ​​liver tissue sections shows an exponential growth trend with the increase of liver echo signal gray value. 2and 110[au] / mm 2 When the collagen area of ​​the liver section changes significantly and grows more rapidly, the X value is 90 and the Y value is 110; that is, the evaluation standard in step S3 is: grayscale value <90 indicates mild liver damage, 90≤grayscale value≤110 indicates moderate liver damage, and grayscale value >110 indicates severe liver damage.

[0084] Performance Testing

[0085] The accuracy of the evaluation method for the CCl4-induced cirrhosis model provided in the present invention was further verified by gross liver observation, liver function tests, and histopathological examinations. According to the evaluation criteria, rats with a grayscale value <90 were designated as the low grayscale value model group, rats with a grayscale value 90 ≤ ≤ 110 were designated as the medium grayscale value model group, and rats with a grayscale value >110 were designated as the high grayscale value model group.

[0086] 1. Gross Observation of the Liver

[0087] According to the evaluation criteria determined in the example, the model group was further divided into three groups, and the liver of the experimental rats in each group was observed. Figure 2 Among them, the low gray value model group corresponds to samples numbered 29 and 39; the medium gray value model group corresponds to samples numbered 1, 35, and 48; the high gray value model group corresponds to samples numbered 20, 24, and 32; and the healthy group corresponds to samples numbered 75, 77, and 80.

[0088] like Figure 2 As shown in the figure, the livers of the untreated healthy group were brownish-red, soft, smooth, and stretched. However, after 10 weeks of intraperitoneal injection of a CCl4 olive oil mixture, the livers of the model group rats showed varying degrees of change. Among them, the livers of the experimental rats in the low gray value model group were brownish-red, slightly hard, still glossy, and stretched, and overall appeared healthier. In contrast, the livers of the experimental rats in the medium gray value model group became tough and dull in color. A granular liver surface was observed, and adhesions were observed between the liver lobules. The livers of the experimental rats in the high gray value model group were even darker in color, significantly harder in texture, and appeared atrophic and bulging, with blunted edges, an increase in granular nodules on the surface, and obvious adhesions between the liver lobules.

[0089] Figure 2Gross liver observations revealed significant differences in the livers of experimental rats within different liver echo signal grayscale value ranges. In general, as the liver ultrasound grayscale value increased, the livers of the experimental animals gradually became harder, darker, and less lustrous. The number of nodules on the liver surface and adhesions between liver lobules increased significantly, and the degree of liver damage worsened. This result demonstrates that the three grayscale value ranges established by the present invention can characterize different degrees of liver damage. Higher liver ultrasound echo signal grayscale values ​​indicate more severe liver function impairment.

[0090] 2. Liver function tests

[0091] In order to further evaluate the consistency between the degree of liver damage in cirrhosis model mice and the results of liver ultrasound imaging, the present invention simultaneously tested the liver function-related serological indicators of the experimental rats.

[0092] Test Method: Blood was collected from the rats' orbits after anesthesia. The collected blood samples were centrifuged at 3000 rpm for 15 minutes at 2-8°C. Serum samples were collected for liver function testing (samples were stored at -80°C). An automated biochemical analyzer was used to automatically measure various liver function indicators. The results are shown in Table 1.

[0093] In Table 1, model group A is experimental rats with grayscale values ​​less than 90; model group B is experimental rats with grayscale values ​​between 90 and 110; and model group C is experimental rats with grayscale values ​​greater than 110.

[0094] Table 1

[0095]

[0096] Note: Healthy group vs. model group: #: P < 0.05, ##: P < 0.01, ###: P < 0.001; Model group A vs. model group B and model group C: *: P < 0.05, **: P < 0.01.

[0097] The liver function test results in Table 1 show that compared with the healthy control group, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and bile acid (TBA) in the three model groups increased to varying degrees, indicating that the model groups had already developed significant hepatocellular damage and cholestasis 10 weeks after modeling. The ALT, AST, and TBA values ​​in the high grayscale value (>110) model group were 1.82 times, 1.75 times, and 1.17 times, respectively, those in the medium grayscale value (90-110) model group, and 4.11 times (P < 0.01), 6.29 times (P < 0.05), and 2.55 times (P < 0.05), respectively, those in the low grayscale value (<90) model group. These data indicate that higher grayscale values ​​in the rat liver ultrasound echo signals indicate more severe liver damage. In addition, the albumin (ALB) levels of the experimental rats in the medium gray value model group and the high gray value model group decreased by 6.26% and 10.26% respectively compared with the experimental rats in the healthy group, indicating that the experimental rats in the model group after 10 weeks of modeling had already shown a decrease in ALB synthesis, suggesting that the liver function of the rats in the medium and high gray value model groups was severely damaged, indicating that the higher the gray value of the liver ultrasound echo signal, the worse the ALB synthesis ability.

[0098] Liver function test data showed that cirrhosis model mice treated with a CCl4 olive oil solution for 10 weeks had severe liver damage, and significant differences were observed between the liver function index results of the three groups of model mice with different grayscale values. This result demonstrates that the three grayscale value intervals established by the present invention can characterize different degrees of liver damage. The higher the grayscale value of the liver ultrasound echo signal, the more severe the liver damage.

[0099] 3. Histopathological examination

[0100] 3.1 Preparation of tissue sections:

[0101] The experimental rats in each group were anesthetized and then killed by cervical dislocation, and the liver tissues were collected; the livers were fixed with 10 times the volume of tissue fixative for 3 days, and then made into paraffin sections for later use.

[0102] 3.2 Hematoxylin-eosin (HE) treatment:

[0103] 3.2.1 Dewaxing: Dewax the paraffin sections by immersing them in xylene for 5 minutes, repeat twice; then immerse the tissue sections in 100%, 95%, 90%, 80%, and 70% alcohol in gradient concentrations for 30 seconds each, and rinse with tap water after immersion;

[0104] 3.2.2 Hematoxylin staining: Soak the tissue sections in a hematoxylin staining solution for 10 minutes;

[0105] 3.2.3 Eosin staining: Soak the tissue sections in 0.5% eosin staining solution for 3 minutes;

[0106] 3.2.4 Dehydration and mounting: Dehydrate the tissue sections in 70%, 80%, 95% and 100% alcohol, each treatment for 30 seconds. Then, place the tissue sections in xylene for 5 minutes. After removing the sections, remove the remaining xylene around the sections and quickly add neutral gum to the sections. Cover with a coverslip (avoiding air bubbles) to complete the mounting process.

[0107] 3.2.5 Observe the HE staining of tissue sections under a microscope and collect images.

[0108] Note: Alcohol concentration is by volume.

[0109] 3.3Masson staining

[0110] The treatment method is the same as the Masson staining treatment method in the embodiment.

[0111] 3.4 Calculation of collagen area

[0112] The statistical method is the same as the method for counting collagen area in the examples.

[0113] Figure 3 A to D in the figure are the HE staining results of each tissue section. Figure 3 As shown in A, the liver structure of the healthy group rats was clear, with distinct hepatic sinusoids, tightly arranged hepatocytes, centrally centered nuclei, abundant cytoplasm, and no obvious inflammatory cell infiltration. However, the liver structure of the model group rats, which had been continuously injected intraperitoneally with CCl4 olive oil mixed solution for 10 weeks, was damaged to varying degrees, with collagen fibers deposited. Figure 3 As shown in B, the liver structure of the low gray value model group was not obviously damaged, the liver sinusoids were still clearly visible, some cells were necrotic, and a small amount of collagen fibers were deposited; in comparison, Figure 3 C shows that the liver structure of the rats in the medium gray value model group was significantly damaged, with a large amount of collagen fiber deposition in the portal area. The extensive proliferation of fibrous tissue divided and wrapped the original liver lobules into circular structures of varying sizes, forming pseudolobules. The number of necrotic hepatocytes increased significantly, accompanied by inflammatory cell infiltration. Figure 3 As shown in D, the high gray value model group showed more pseudolobules, the hepatocytes in the tissue were disordered, the cell bodies and nuclei became larger and darker in color, and some hepatocytes also showed vesicular and fatty degeneration, indicating more severe hepatocyte damage.

[0114] HE staining results confirmed that liver ultrasound imaging was used to assess the degree of liver damage in rats with cirrhosis, and the results were consistent with the HE staining results. As the grayscale value of the liver echo signal increased across the three model groups, the degree of liver structural damage increased significantly, with significant increases in hepatocyte necrosis and inflammatory cell infiltration. Therefore, this grayscale value range can be used to characterize the different degrees of liver damage.

[0115] Figure 3 Figures E to H are the Masson staining results of each tissue section. Figure 3 As shown in Figure E, only a small amount of collagen fibers were observed around the blood vessels in the healthy control rats, while the model group rats after continuous intraperitoneal injection of CCl4 olive oil mixed solution for 10 weeks showed varying degrees of collagen fiber deposition stained blue. Figure 3 F shows that a small amount of collagen deposition appeared in the low gray value model group. Compared with the low gray value model group, the medium gray value model group ( Figure 3 G) and high gray value model group ( Figure 3 The collagen fibers in the H) increased significantly and interwoven to form a reticular structure, dividing the liver tissue into multiple pseudolobules of varying sizes. The latter showed more pronounced collagen deposition and a greater number of pseudolobules. The Masson staining results confirm that the evaluation criteria, determined by fitting a curve between the grayscale value of the liver echo signal and the collagen area in liver tissue sections, can accurately assess the degree of liver damage.

[0116] The collagen fiber area of ​​the liver tissue sections of each group of experimental rats was further statistically analyzed. Figure 4 As shown in the figure, 10 weeks after modeling, the collagen fibers of the model groups increased to varying degrees. The high gray value model group had the largest collagen area, measuring 110,987.27 ± 10,735.34; the medium gray value model group had the second largest collagen area, measuring 62,754.13 ± 10,716.06; and the low gray value model group had a collagen area of ​​41,261.01 ± 3,399.91. The collagen area in the high gray value model group was 1.77 times (P < 0.001) and 2.69 times (P < 0.0001) that of the medium gray value model group and the low gray value model group, respectively, consistent with the results of liver ultrasound imaging. Furthermore, there was a statistically significant difference between the low gray value and medium gray value model groups (P < 0.05).

[0117] The test results show that as the grayscale values ​​of the three model groups increased, the collagen area also increased accordingly, proving that higher grayscale values ​​of the liver echo signals in the model mice indicate more severe liver damage. Furthermore, significant differences were observed between the grayscale values ​​of the three groups, allowing this grayscale value range to be used to characterize varying degrees of liver damage.

[0118] In summary, the present invention explored the degree of liver damage in three groups of experimental rats with low grayscale value (<90), medium grayscale value (90-110) and high grayscale value (>110) in multiple dimensions through gross liver observation, liver function test and pathological histological test. The test results showed that the gross liver observation, liver function test and pathological histological test results of the three groups of experimental rats showed significant differences, verifying the feasibility of the method of determining the liver damage evaluation standard by fitting the grayscale value of the liver echo signal with the collagen area of ​​the liver tissue section. The obtained evaluation standard can simply and efficiently evaluate the degree of liver damage in rats with CCl4-induced cirrhosis model, and the evaluation results are accurate and reliable.

Claims

1. A method for evaluating a CCl4-induced liver cirrhosis model, characterized in that: include: S1. Establishment of rat liver cirrhosis model by CCl4 induction; S2. performing ultrasound imaging on the cirrhosis model rat to obtain a grayscale value of the liver of the cirrhosis model rat; S3. Evaluating the degree of liver damage in the cirrhosis model rats according to the grayscale value of the liver of the cirrhosis model rats; The evaluation criteria in step S3 are: grayscale value < X indicates mild liver damage, X ≤ grayscale value ≤ Y indicates moderate liver damage, and grayscale value > Y indicates severe liver damage; 0<X<Y; The X and Y are determined by fitting the collagen area of ​​the liver pathological tissue sections of the cirrhosis model rats and the gray value data of the liver of the cirrhosis model rats.

2. The evaluation method according to claim 1, wherein: The method for determining the evaluation criteria includes: preparing liver pathological tissue sections of cirrhosis model rats and performing Masson staining to obtain the collagen area of ​​the liver pathological tissue sections of the cirrhosis model rats, fitting the statistically obtained collagen area and the grayscale value data of the liver of the cirrhosis model rats to determine the values ​​of X and Y, and then determining the evaluation criteria.

3. The evaluation method according to claim 2, wherein: 80<X<95, 100<Y<130.

4. The evaluation method according to claim 3, wherein: 85<X<92, 105<Y<120.

5. The evaluation method according to claim 4, wherein: X=90, Y=110.

6. The evaluation method according to any one of claims 1 to 5, characterized in that: The step S1 comprises: injecting a CCl4 olive oil mixed solution into experimental rats to prepare a rat cirrhosis model; the injection frequency is twice a week for a total of 8 to 12 weeks.

7. The evaluation method according to claim 6, wherein: The single injection amount of the CCl4 olive oil mixed solution is 1-5 mg / kg.

8. The evaluation method according to claim 6, wherein: The CCl4 olive oil mixed solution is injected into the peritoneum.

9. The evaluation method according to claim 6, wherein: The volume ratio of CCl4 to olive oil in the CCl4 olive oil mixed solution is 1:(1.2-2).

10. An application of the method for evaluating a CCl4-induced liver cirrhosis model according to any one of claims 1 to 9, characterized in that: The evaluation method is applied to the screening of animal models.

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