A method for evaluating the liver damage induced by substances based on the area ratio of zebrafish liver compartments

Abstract

The present invention provides a method for evaluating the liver-damaging effect of a substance based on the area ratio of zebrafish liver partitions. The present invention belongs to the technical field of toxicological detection. The present invention divides the liver shown in a liver fluorescence image in the lateral position of a zebrafish into partitions, and the highest point where a horizontal line intersects the liver is recorded as point a. The intersection point of the zebrafish's melanin stripe and the eye in the lateral position is recorded as point c. A horizontal line is drawn through point c, and the intersection point of the horizontal line with the right side of the liver is recorded as point b. Points a and b are connected by a straight line, and the area below the line a and b is recorded as area I, and the area above the line a and b is recorded as area II. The present invention uses comparison of changes in the liver partition area ratio (area of area II / area of area I) to quickly evaluate the effect of a substance on the zebrafish liver. This overcomes the problem of using the total liver area as an inaccurate indicator for evaluating the liver-damaging effect of a substance on the zebrafish, and also avoids experimental errors caused by different total liver areas due to individual differences in zebrafish.

Description

Technical Field

[0001] The present invention belongs to the technical field of toxicological detection, and in particular relates to a method for evaluating the liver damage caused by a substance based on the area ratio of zebrafish liver partitions. Background Art

[0002] The incidence of chemical-induced liver injury is on the rise due to factors such as drug abuse, excessive alcohol consumption, and environmental pollution. As the body's primary detoxification organ responsible for metabolizing and excreting foreign substances, liver injury caused by exogenous compounds can not only lead to major liver diseases such as fatty liver, cirrhosis, and liver cancer, but in severe cases, can even threaten the patient's life. Therefore, establishing a method that can quantitatively, rapidly, and accurately evaluate compound liver injury is beneficial for more accurate assessment of the liver-damaging effects of exogenous compounds. Traditional models for evaluating liver toxicity, such as in vitro cell models and rodent models, have numerous problems. In vitro cell experiments cannot accurately reflect the effects of compounds in vivo, making it impossible to accurately determine their hepatotoxic effects. In vivo rodent experiments, on the other hand, have high experimental costs, long experimental cycles, and ethical issues, making it difficult to rapidly and efficiently evaluate compounds for liver injury risks.

[0003] Due to their unique biological advantages, zebrafish hold great potential for evaluating the hepatotoxicity of compounds. The zebrafish liver's structure and function closely resemble those of humans, making it an excellent model animal for studying liver function. Zebrafish have a short reproductive cycle, large egg production, rapid development, and a small size, significantly reducing experimental timelines and the amount of test substance used. Furthermore, the transparency of zebrafish embryos facilitates direct microscopic observation and recording of the effects of compounds on various organs. Current methods for studying liver function in zebrafish utilize transgenic zebrafish with green fluorescently labeled livers. Measuring liver area is a commonly used and intuitive indicator for evaluating compound liver damage. When a compound exhibits toxic effects on the liver, pathological changes such as edema and necrosis occur in hepatocytes, and the liver may enlarge or decrease in size. Therefore, measuring the effect of a compound's liver area on zebrafish liver size can quickly assess whether it has liver-damaging effects.

[0004] In experiments, the inventors discovered that some compounds can cause enlargement of the lower half of the zebrafish liver and shrinkage of the upper half. While the researchers observed significant changes in the zebrafish liver morphology under a microscope, their liver area was statistically significant when measured between the compound-treated and blank control groups. Therefore, using total liver area as an indicator of a compound's effect on zebrafish liver damage is inaccurate. Summary of the Invention

[0005] In response to the technical problem discovered by the inventors, the present invention provides a method for evaluating the liver damage caused by substances based on the area ratio of zebrafish liver partitions.

[0006] During the experiment, the inventors found that the liver shown in the liver fluorescence image of the zebrafish in the lateral position can be divided into zones. The highest point where the horizontal line intersects the liver is marked as point a, the intersection point of the zebrafish's melanin stripe and the eye in the lateral position is marked as point c, a horizontal line is drawn through point c, and the intersection point of the horizontal line with the right side of the liver is marked as point b. A straight line is connected between points a and b, and the area below the line a and b is marked as zone I, and the area above the line a and b is marked as zone II (see Figure 1 The present invention uses a comparison of changes in the liver area ratio (area of zone II / area of zone I) to rapidly evaluate the effects of substances on the zebrafish liver. This overcomes the inaccuracy of using total liver area as an indicator of liver damage in zebrafish. It also avoids experimental errors caused by individual differences in total liver area. This method aligns with liver pathological changes, allowing for a more scientific, objective, and accurate assessment of whether a substance has a damaging effect on the liver.

[0007] The technical solutions of the present invention are as follows:

[0008] A method for evaluating the liver damage induced by a substance based on the area ratio of zebrafish liver partitions comprises the following steps:

[0009] (3) 72 hours after fertilization, the zebrafish with normal development were continuously incubated with the test substance, which was recorded as the test substance treatment group; at the same time, a control group was set up by incubating without the test substance under the same conditions, which was recorded as the control group;

[0010] (4) After incubation in the anesthesia step (1), the liver fluorescence images of each zebrafish in the control group and the test substance treatment group were collected. The liver fluorescence images of the zebrafish in the lateral position were divided into zones. The highest point where the horizontal line intersected with the liver was recorded as point a. The intersection point of the zebrafish's lateral position melanin stripe and the eye was recorded as point c. A horizontal line was drawn through point c. The intersection point of the horizontal line with the right side of the liver was recorded as point b. A straight line was drawn to connect points a and b. The area below the line a and b was recorded as zone I, and the area above the line a and b was recorded as zone II. The area of zone I of the zebrafish liver (A) was calculated. Ⅰ ), liver zone II area (A Ⅱ );

[0011] (3) Calculate the liver partition area ratio of each group of zebrafish obtained in step (2) according to the following formula:

[0012] Liver area ratio = (liver area II (A Ⅱ ) / area of liver zone I (A Ⅰ ))×100%

[0013] The significance of the difference in liver area ratio between the control group and the test substance treatment group is analyzed and compared; when there is a significant difference in the liver area ratio of the zebrafish in the test substance treatment group and the control group, it indicates that the test substance has a damaging effect on the liver.

[0014] According to the present invention, preferably, in step (1), the zebrafish is a transgenic zebrafish whose liver cells are labeled with green fluorescent protein.

[0015] Preferably, according to the present invention, in step (1), the test substance is directly dissolved in zebrafish culture water to prepare a test solution; or the test substance is dissolved in a co-solvent to prepare a mother solution, and then diluted with zebrafish culture water to prepare a test solution.

[0016] More preferably, the cosolvent is DMSO, and the final concentration of DMSO in the detection solution is less than 0.5% by volume.

[0017] More preferably, a co-solvent control group is added when a co-solvent is used.

[0018] Preferably, according to the present invention, in step (1), the zebrafish is incubated with the test substance for 1 to 3 days.

[0019] Preferably, according to the present invention, in step (3), the significance of the difference in liver area ratio between the control group and the test substance-treated group is analyzed and compared, and a t-test is used for statistical analysis.

[0020] The beneficial effects of the present invention include at least the following:

[0021] 1. The present invention is the first to discover that the zebrafish liver partition area ratio is used as an evaluation indicator for zebrafish liver damage, which is more scientific and objective, and improves the accuracy of the results. The established zebrafish liver partition area ratio evaluation system and method have the advantages of simple operation, rapidity, stability, reliability and good repeatability.

[0022] 2. The evaluation method provided by the present invention avoids experimental errors caused by differences in total liver area due to individual differences in zebrafish. By comparing changes in liver partition area ratios, the effects of compounds on the zebrafish liver are quickly evaluated to match the pathological changes in the liver. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a schematic diagram of the zebrafish liver divisions;

[0024] In the figure: the blue box is the liver, the fluorescence image of the liver in the side position of the zebrafish, the highest point where the horizontal line intersects the liver is marked as point a, the intersection of the melanin stripe and the eye in the side position of the zebrafish is marked as point c, a horizontal line is drawn through point c, and the point where the horizontal line intersects with the right side of the liver is marked as point b. The area below the line connecting a and b is area I, and the area above the line is area II.

[0025] Figure 2 This is a fluorescence image of the zebrafish liver in the blank control group in the study of hepatotoxicity induced by Psoralea corylifolia aqueous extract in zebrafish;

[0026] In the figure: The blue box represents the liver. This is a fluorescence image of the zebrafish liver in the lateral position. The highest point where the horizontal line intersects the liver is marked as point a. The intersection of the melanin stripe and the eye in the lateral position is marked as point c. A horizontal line is drawn through point c, and the point where it intersects the right side of the liver is marked as point b. Area I is below the line connecting a and b, and area II is above the line.

[0027] Figure 3 This is the fluorescence image of zebrafish liver in the Psoralea corylifolia water extract group;

[0028] In the figure: the blue box is the liver, the fluorescence image of the liver in the side position of the zebrafish, the highest point where the horizontal line intersects the liver is marked as point a, the intersection of the melanin stripe and the eye in the side position of the zebrafish is marked as point c, a horizontal line is drawn through point c, and the point where the horizontal line intersects with the right side of the liver is marked as point b. The area below the line connecting a and b is area I, and the area above the line is area II.

[0029] Figure 4 This is a statistical graph of the total fluorescence area of zebrafish liver by Psoralea corylifolia aqueous extract;

[0030] In the figure: ns: no statistical difference, p>0.05.

[0031] Figure 5 This is a statistical graph of the fluorescence area of zebrafish liver area I by Psoralea corylifolia aqueous extract;

[0032] In the figure: **p<0.01 compared with the control group.

[0033] Figure 6 This is a statistical graph of the fluorescence area of zebrafish liver area II by Psoralea corylifolia aqueous extract;

[0034] In the figure: ***p<0.001 compared with the control group.

[0035] Figure 7 This is a statistical chart of the ratio of the area of the zebrafish liver to the water extract of Psoralea corylifolia.

[0036] In the figure: ***p<0.001 compared with the control group.

[0037] Figure 8 This is a picture of zebrafish liver tissue in the blank control group in the study of psoralea corylifolia-induced zebrafish hepatotoxicity.

[0038] Figure 9 This figure shows the effect of Psoralea corylifolia aqueous extract on zebrafish liver tissue.

[0039] Figure 10This is a fluorescence image of the zebrafish liver in the blank control group in the study of alcohol-induced zebrafish hepatotoxicity;

[0040] In the figure: the blue box is the liver, the fluorescence image of the liver in the side position of the zebrafish, the highest point where the horizontal line intersects the liver is marked as point a, the intersection of the melanin stripe and the eye in the side position of the zebrafish is marked as point c, a horizontal line is drawn through point c, and the point where the horizontal line intersects with the right side of the liver is marked as point b. The area below the line connecting a and b is area I, and the area above the line is area II.

[0041] Figure 11 Fluorescent images of zebrafish liver in the alcohol group;

[0042] The blue box in the figure is the liver, and the liver fluorescence image in the zebrafish lateral position. The highest point where the horizontal line intersects the liver is marked as point a, and the intersection of the melanin stripe and the eye in the zebrafish lateral position is marked as point c. A horizontal line is drawn through point c, and the point where the horizontal line intersects with the right side of the liver is marked as point b. The area below the line connecting a and b is area I, and the area above the line is area II.

[0043] Figure 12 This is a statistical diagram of the total fluorescence area of zebrafish liver affected by alcohol;

[0044] In the figure: ns means no statistical difference, p>0.05.

[0045] Figure 13 This is a statistical diagram of the effect of alcohol on the fluorescence area of zone Ⅰ in zebrafish liver;

[0046] In the figure: ns means no statistical difference, p>0.05.

[0047] Figure 14 This is a statistical diagram of the effect of alcohol on the fluorescence area of zone II in zebrafish liver;

[0048] In the figure: ***p<0.001 compared with the control group.

[0049] Figure 15 This is a statistical diagram of the effect of alcohol on the area ratio of zebrafish liver partitions;

[0050] In the figure: ***p<0.001 compared with the control group.

[0051] Figure 16 This is a diagram of zebrafish liver tissue in the blank control group in the study of alcohol-induced zebrafish hepatotoxicity.

[0052] Figure 17 A diagram showing the effects of alcohol on zebrafish liver tissue.

[0053] Figure 18 This is a fluorescence image of the zebrafish liver in the blank control group in the study of α-naphthyl isothiocyanate-induced zebrafish hepatotoxicity.

[0054] In the figure: the blue box is the liver, the fluorescence image of the liver in the side position of the zebrafish, the highest point where the horizontal line intersects the liver is marked as point a, the intersection of the melanin stripe and the eye in the side position of the zebrafish is marked as point c, a horizontal line is drawn through point c, and the point where the horizontal line intersects with the right side of the liver is marked as point b. The area below the line connecting a and b is area I, and the area above the line is area II.

[0055] Figure 19 This is the fluorescence image of zebrafish liver in the α-naphthyl isothiocyanate group;

[0056] In the figure: The blue box represents the liver. This is a fluorescence image of the zebrafish liver in the lateral position. The highest point where the horizontal line intersects the liver is marked as point a. The intersection of the melanin stripe and the eye in the lateral position is marked as point c. A horizontal line is drawn through point c, and the point where it intersects the right side of the liver is marked as point b. Area I is below the line connecting a and b, and area II is above the line.

[0057] Figure 20 This is a statistical graph of the total fluorescence area of zebrafish liver induced by α-naphthyl isothiocyanate;

[0058] In the figure: ns means no statistical difference, p>0.05.

[0059] Figure 21 This is a statistical graph of the fluorescence area of zebrafish liver area Ⅰ induced by α-naphthyl isothiocyanate;

[0060] In the figure: ***p<0.001 compared with the control group.

[0061] Figure 22 This is a statistical graph of the fluorescence area of zebrafish liver area II induced by α-naphthyl isothiocyanate;

[0062] In the figure: ***p<0.001 compared with the control group.

[0063] Figure 23 This is a statistical diagram of the ratio of α-naphthyl isothiocyanate to the area of zebrafish liver partitions;

[0064] In the figure: ***p<0.001 compared with the control group.

[0065] Figure 24 This is a picture of zebrafish liver tissue in the blank control group in the study of zebrafish hepatotoxicity induced by α-naphthyl isothiocyanate.

[0066] Figure 25 This figure shows the effect of α-naphthyl isothiocyanate on zebrafish liver tissue. DETAILED DESCRIPTION

[0067] The present invention will be further described in detail below with reference to the Examples, but the scope of the present invention is not limited thereto. Where specific conditions are not specified in the Examples, conventional conditions or conditions recommended by the manufacturer were used. Reagents or instruments used without manufacturer's indication are conventional products that can be purchased commercially.

[0068] 1. Methods for evaluating the effects of substances on liver area ratios

[0069] 1.1 Experimental animals: The present invention uses the transgenic zebrafish Tg (l-fabp: EGFP) in which liver cells are labeled with green fluorescent protein as the experimental animal. It can be purchased from the National Zebrafish Resource Center, and ordinary commercial products can be used. Female and male zebrafish are raised separately, with alternating lighting for 14 hours and darkness for 10 hours, and the temperature is controlled at 28°C. Female and male zebrafish are placed in a mating tank with partitions in a male-to-female ratio of 1:2. The partitions are removed at 8:30 the next morning, and the male and female zebrafish are mated. Fertilized eggs are obtained after 1-2 hours. The fertilized eggs are washed with fresh zebrafish culture water and disinfected with methylene blue, and then placed in a constant temperature incubator at 28°C for light control. Culture to 72hpf (hours post fertilization), and healthy fry are selected for subsequent experiments.

[0070] Zebrafish culture water: 5.0 mM NaCl, 0.17 mM KCl, 0.4 mM CaCl2, 0.16 mM MgSO4.

[0071] 1.2 Hepatotoxicity evaluation method: After the substance treatment and the control group treatment, 10-15 healthy zebrafish were taken from each group and anesthetized with an appropriate amount of tricaine. The zebrafish were placed on their sides with their eyes overlapped on a glass slide containing 4% methylcellulose. After adjusting to the lateral position, the zebrafish liver was observed under an inverted fluorescence microscope and photographed. In the fluorescence image of the zebrafish liver in the lateral position, the highest point where the horizontal line intersects the liver is marked as point a. The intersection of the melanin stripe and the eye in the lateral position of the zebrafish is marked as point c. A horizontal line is drawn through point c, and the point where the horizontal line intersects with the right side of the liver is marked as point b. The area below the line connecting a and b is area I, and the area above the line is area II. Figure 1 Image pro plus 6.0 was used to calculate the total liver area (A 总 ) and the area of liver zone I and II (A Ⅰ 、A Ⅱ ), and the liver partition area ratio of each group of zebrafish was calculated according to the following formula:

[0072] Liver area ratio = (liver area II (A Ⅱ ) / area of liver zone I (A Ⅰ ))×100%

[0073] By comparing the coefficient of variation of the total liver area and the liver area ratio of normal zebrafish, it can be shown that the liver area ratio has a higher precision. The smaller the coefficient of variation, the smaller the deviation of the measurement result; the larger the coefficient of variation, the greater the deviation of the measurement result. The coefficient of variation calculation formula is as follows, C·V is the coefficient of variation, SD is the standard deviation, is the mean.

[0074]

[0075] The structure and function of the zebrafish liver are similar to those of humans. The zebrafish liver is essentially formed at 48 hpf after fertilization, and its morphology and function are essentially mature after 72 hpf. The zebrafish liver is very sensitive to the intervention of foreign substances. Treating zebrafish with hepatotoxic substances for 1-3 days can alter the physiological structure of the zebrafish liver and induce liver damage. Therefore, the present invention uses 72 hpf zebrafish and treats them with exogenous substances for 1-3 days to observe the effects of the substances on the zebrafish liver.

[0076] The coefficient of variation of the total liver area and liver area ratio of normal zebrafish at 5 and 6 days post-fertilization (dpf) (5 and 6 dpf) was calculated, as shown in Table 1. The results showed that the coefficient of variation of the liver area ratio for each group of zebrafish was lower than that of the total liver area, indicating that the liver area ratio is more stable, has less dispersion, and is more precise than the total liver area, allowing for more accurate assessment of whether a substance is damaging the liver. Comparing the liver area ratios of zebrafish in the blank control group with those in the substance-treated groups showed a significant increase or decrease in the liver area ratio in the substance-treated groups (p < 0.05), indicating that the substance is damaging the liver.

[0077] Table 1 Coefficient of variation (%) of the ratio of total liver area to liver area of zebrafish at different developmental stages

[0078]

[0079] 2. Case Study 1: Evaluation of the Hepatotoxicity of Psoralea corylifolia to Zebrafish

[0080] 2.1 Research Background on Psoralea corylifolia-induced Hepatotoxicity in Zebrafish

[0081] Psoralea corylifolia (Bu Gu Zhi) has a long history of clinical use in my country and is a traditional and commonly used tonic Chinese medicine. However, reports of adverse reactions to Bu Gu Zhi have been increasing in recent years, with hepatotoxicity garnering widespread attention from drug regulatory authorities and researchers both domestically and internationally. According to statistics, liver damage accounts for as high as 55.95% of adverse reactions to Bu Gu Zhi, with some patients even experiencing liver failure and death.

[0082] Clinical manifestations of psoralea corylifolia liver damage primarily include jaundice of the skin and sclera, dark urine, fatigue, and loss of appetite, accompanied by elevated biochemical markers such as alanine aminotransferase (ALT), total bilirubin (T-Bil), and direct bilirubin (D-Bil). Animal experiments have also shown that aqueous and alcoholic extracts of psoralea corylifolia cause varying degrees of liver damage in mice and rats.

[0083] In this study, transgenic zebrafish with green fluorescent protein-labeled livers were used to evaluate the hepatotoxicity of Psoralea corylifolia aqueous extract in zebrafish using the total liver area, liver zone I area, liver zone II area, and liver zone area ratio as evaluation indicators. Combined with alanine aminotransferase (ALT), a gold marker of liver function, and liver tissue sections, the hepatotoxicity of Psoralea corylifolia aqueous extract in zebrafish was evaluated.

[0084] 2.2 Experimental methods

[0085] 2.2.1 Drugs and reagents

[0086] Psoralea corylifolia (Batch No. 20102401-1) was purchased from Xinjiang Renyu Chinese Medicine Pieces Co., Ltd., tricaine (Batch No. A5040) was purchased from Sigma-Aldrich, USA, CMC-Na (Batch No. 419281) was purchased from Sigma-Aldrich, USA, and an alanine aminotransferase (ALT) assay kit (Batch No. 20221130) was purchased from Shenzhen Raydu Life Sciences Co., Ltd. Zebrafish culture water (5.0 mM NaCl, 0.17 mM KCl, 0.4 mM CaCl2, 0.16 mM MgSO4) was used.

[0087] 2.2.2 Animals and breeding conditions

[0088] Transgenic zebrafish Tg(l-fabp:EGFP) with green fluorescent protein labeling of liver cells were used as experimental animals. These can be purchased from the National Zebrafish Resource Center or other commercially available products. Male and female zebrafish were housed separately, with alternating lighting cycles of 14 h / darkness of 10 h, and the temperature was controlled at 28°C. Male and female zebrafish were placed in a mating tank with a partition at a ratio of 1:2. The partition was removed at 8:30 the next morning, allowing the male and female zebrafish to mate. Fertilized eggs were obtained 1-2 hours later. The fertilized eggs were washed with fresh zebrafish culture water and disinfected with methylene blue, then placed in a 28°C constant temperature incubator with controlled light. The juveniles were cultured to 72 hpf, and healthy fry were selected for subsequent experiments.

[0089] 2.2.3 Experimental Grouping

[0090] Normally developing zebrafish (72 hpf) were selected and transferred to a 24-well sterile culture plate, with 20 per well. A blank control group was treated with zebrafish culture water, while a psoralea corylifolia extract (WEFP) group was treated with 500 μg / mL WEFP. Three replicate wells were used for each group. Each well was filled with zebrafish culture water to a total volume of 2 mL. The zebrafish were then placed in a lighted incubator (28°C) to continue developing. Mortality was recorded for three consecutive days in each group.

[0091] 2.2.4 Liver area statistics

[0092] 72 hours after drug treatment, 10-15 healthy zebrafish from each group were anesthetized with tricaine. The fish were placed in lateral recumbency with their eyes closed on a glass slide containing 4% methylcellulose. After adjusting the fish to a lateral position, the fluorescent area of the zebrafish liver was observed and photographed using an inverted fluorescence microscope. Image Pro Plus 6.0 was used to calculate the total liver area and the areas of zones I and II. The liver area ratios of each group were calculated. GraphPad Prism 9.5.0 software was then used to plot the data into bar graphs for visual comparison.

[0093] 2.2.5 Liver tissue sections

[0094] Five to ten zebrafish per group were anesthetized with tricaine and fixed in 4% paraformaldehyde. After 96 hours, the zebrafish were dehydrated and fixed using a gradient of ethanol (75% for 3 hours, 85% for 2 hours, 90% for 2 hours, 95% for 1 hour, and 100% for 40 minutes twice), followed by benzene for 5 minutes, xylene for 5 minutes twice, and paraffin for 1 hour three times. After dehydration, the zebrafish were embedded in paraffin and sectioned at 4 μm in an embedding machine. The sections were deparaffinized to water and then rinsed with purified water for hematoxylin and eosin staining. The sections were then washed and placed in a gradient of ethanol (95% for 10 minutes twice, 100% for 10 minutes twice). The sections were then dehydrated in xylene until transparent, air-dried, and mounted with neutral gum. Histopathological changes in the livers of zebrafish in the blank control group and the psoralea corylifolia aqueous extract group were observed under a microscope to assess whether the psoralea corylifolia aqueous extract induces pathological changes in liver tissue.

[0095] 2.2.6 Detection of the effect of Psoralea corylifolia aqueous extract on zebrafish ALT

[0096] After drug administration according to the experimental grouping method in 2.2.3, 30 healthy zebrafish from each group that had been administered for 72 h were placed in a 1.5 mL enzyme-free sterilized EP tube. The fish were rinsed twice with purified water to remove moisture, and 200 μL of normal saline was added. The fish were shaken and disrupted for 1 min, and centrifuged at 3000 rpm at 2-8°C for 15 min. The supernatant was collected and loaded onto the sample. The corresponding parameters were set for automatic determination on the fully automatic biochemical analyzer, and the ALT content was detected by the fully automatic biochemical analyzer.

[0097] 2.2.7 Statistics

[0098] SPSS 28.0 software was used to perform statistical analysis on the experimental data. The differences between the two groups were compared using the t-test and plotted as a bar graph using GraphPad Prism 9.5.0 software. *p<0.05, **p<0.01, ***p<0.001, ns: no statistical difference compared with the blank control group (p>0.05).

[0099] 2.3 Experimental Results

[0100] 2.3.1 Effect of Psoralea corylifolia on zebrafish liver area

[0101] Zebrafish were treated with 500 μg / mL of Psoralea corylifolia aqueous extract. The results showed that compared with the blank control group, the Psoralea corylifolia aqueous extract caused significant changes in the morphology of the zebrafish liver. The lower half of the liver was significantly swollen, and the upper half of the liver was significantly atrophied. Figure 2 、 Figure 3 The liver area was calculated using Image pro plus 6.0 and then a histogram was drawn using GraphPad Prism 9.5.0 software. It was found that there was no statistically significant difference in the total liver area between the zebrafish in the Psoralea corylifolia water extract group and the blank control group (p>0.05). Figure 4 .

[0102] After statistically analyzing the liver area of the two groups of zebrafish and calculating the liver area ratio, it was found that compared with the blank control group, the area of zone I in the psoralea corylifolia water extract group was significantly increased (p<0.01), the area of zone II was significantly decreased (p<0.001), and the liver area ratio was significantly decreased (p<0.001). There was a statistical difference. Figure 5 、 Figure 6 、 Figure 7 .

[0103] Table 2 shows the statistical means and p-values for the ratios of total liver area, liver area I, liver area II, and liver subarea area in each zebrafish group. The results showed that the liver subarea ratio had the smallest SD value, indicating that using the liver subarea ratio as an indicator of the liver damage effect of Psoralea corylifolia aqueous extract has a low error rate. The coefficient of variation of the liver subarea ratio in each group was lower than that of the total liver area, indicating that the liver subarea ratio is more stable, has a smaller dispersion, and is more precise than the total liver area test, allowing for more accurate assessment of whether the Psoralea corylifolia aqueous extract has a liver damage effect.

[0104] 2.3.2 Effects of Psoralea corylifolia on zebrafish liver histopathological sections

[0105] By staining liver tissue sections, it can be determined whether there is damage to the liver tissue structure. Figure 8As shown in Figure 2, cells in normal zebrafish liver tissue are densely arranged. Figure 9 As shown in the results, compared with the blank control group, the aqueous extract of Psoralea corylifolia caused obvious vacuoles between zebrafish liver cells, severe defects in liver structure, blurred boundaries of liver cells, and severe damage to cell structure. This indicates that the aqueous extract of Psoralea corylifolia caused damage to the zebrafish liver.

[0106] 2.3.3 Effects of Psoralea corylifolia on zebrafish ALT and comprehensive analysis of various evaluation indicators

[0107] Alanine aminotransferase (ALT) is one of the most commonly used sensitive indicators of liver damage, with elevated ALT levels reflecting liver dysfunction. As shown in Table 2, ALT levels in zebrafish significantly increased under the influence of Psoralea corylifolia. This, combined with liver morphology and liver tissue pathology analysis, suggests that Psoralea corylifolia aqueous extract causes severe liver damage in zebrafish.

[0108] Comprehensive analysis of various evaluation indicators revealed that the aqueous extract of Psoralea corylifolia caused significant structural damage to zebrafish liver tissue, with a significant increase in ALT, a sensitive marker of liver function. However, there was no statistically significant difference in the total liver area between the Psoralea corylifolia aqueous extract group and the blank control group. Statistical analysis of liver area revealed a significant increase in liver zone I and a significant decrease in liver zone II in the Psoralea corylifolia aqueous extract group compared with the blank control group, demonstrating statistically significant differences consistent with changes in liver morphology, histopathology, and ALT. Calculation of the liver zone area ratio revealed that the liver zone area ratio had the smallest SD compared to the total liver area, indicating that using the liver zone area ratio as an indicator of liver damage by the Psoralea corylifolia aqueous extract has a low error rate. The coefficient of variation of the liver zone area ratio across all groups was lower than that of the total liver area, indicating that the liver zone area ratio is more stable, has less variability, and is more precise than the total liver area measurement, making it a more accurate indicator of liver damage by the Psoralea corylifolia aqueous extract.

[0109] The experimental results show that when using zebrafish to evaluate the hepatotoxicity of substances, the total liver area as a detection indicator is not sensitive and accurate. It is more sensitive and accurate to evaluate the liver damage caused by substances by calculating the liver partition area ratio.

[0110] Table 2 Effects of Psoralea corylifolia on liver area and ALT in zebrafish larvae ( n=30)

[0111]

[0112] Note: Compared with the blank control group, p>0.05 means no significant difference, p<0.05 means significant difference, p<0.01 means a significant difference, and p<0.001 means an extremely significant difference.

[0113] 2.4 Conclusion

[0114] Psoralea corylifolia aqueous extract caused significant changes in zebrafish liver morphology and pathological changes, including a significant increase in alanine aminotransferase (ALT), a sensitive marker of liver function. However, the total liver area of zebrafish treated with the Psoralea corylifolia aqueous extract was not statistically different from that of the blank control group. The liver was divided into two zones and the changes in liver area and the liver area ratio were compared. The results showed that compared with the blank control group, the liver area of zone I increased significantly, the area of zone II decreased significantly, and the liver area ratio decreased significantly. These differences were statistically significant, consistent with the changes in liver morphology, liver pathology, and ALT. Furthermore, the coefficient of variation of the liver area ratio in each group was lower than that of the total liver area, indicating that the liver area ratio is more stable, has less variability, and is more precise than the total liver area, making it more accurate for assessing the liver damage caused by the Psoralea corylifolia aqueous extract. These results suggest that using total liver area as a metric for evaluating the hepatotoxicity of compounds in zebrafish is not sensitive or accurate. Comparing changes in liver area in different zones is a more sensitive and accurate method for assessing the liver damage of compounds.

[0115] III. Example 2: Evaluation of the Effect of Alcohol on Zebrafish Hepatotoxicity

[0116] 3.1 Research Background on Alcohol-Induced Zebrafish Hepatotoxicity

[0117] Alcoholic liver disease (ALD) is liver damage caused by long-term, excessive alcohol consumption. Initially, it often presents as alcoholic fatty liver disease (AFLD) and alcoholic hepatitis, which can progress to liver fibrosis, cirrhosis, and even liver failure. AFLD is induced by excessive alcohol consumption, causing intracellular lipid accumulation exceeding 5% of liver wet mass, or fatty degeneration of more than one-third of liver tissue per unit area, representing the early stages of ALD. Studies have shown that 90% of heavy drinkers develop fatty liver, 30% of whom develop liver fibrosis, and 8%-20% progress to cirrhosis. ALD is the leading cause of liver disease-related morbidity and mortality. Acute alcohol intoxication is more common than chronic alcoholism. Animal studies have also shown that alcohol can cause liver damage in mice, rats, and zebrafish.

[0118] This study used transgenic zebrafish with green fluorescent protein-labeled livers. The total liver area, liver zone I area, liver zone II area, and liver zone area ratio were used as evaluation indicators, combined with liver tissue pathological sections to evaluate the hepatotoxicity of alcohol in zebrafish.

[0119] 3.2 Experimental methods

[0120] 3.2.1 Drugs and reagents

[0121] Alcohol was purchased from Nanjing Chemical Reagent Co., Ltd., tricaine (product batch number: A5040) was purchased from Sigma-Aldrich, USA, and fish fixative CMC-Na (product batch number: 419281) was purchased from Sigma-Aldrich, USA. Zebrafish culture water (5.0 mM NaCl, 0.17 mM KCl, 0.4 mM CaCl2, 0.16 mM MgSO4) was used.

[0122] 3.2.2 Animals and breeding conditions

[0123] Transgenic zebrafish Tg(l-fabp:EGFP) with green fluorescent protein labeling of liver cells were used as experimental animals. These can be purchased from the National Zebrafish Resource Center or other commercially available products. Male and female zebrafish were housed separately, with alternating lighting cycles of 14 h / darkness of 10 h, and the temperature was controlled at 28°C. Male and female zebrafish were placed in a mating tank with a partition at a ratio of 1:2. The partition was removed at 8:30 the next morning, allowing the male and female zebrafish to mate. Fertilized eggs were obtained 1-2 hours later. The fertilized eggs were washed with fresh zebrafish culture water and disinfected with methylene blue, then placed in a 28°C constant temperature incubator with controlled light. The juveniles were cultured to 72 hpf, and healthy fry were selected for subsequent experiments.

[0124] 3.2.3 Experimental Grouping

[0125] Select normally developing zebrafish at 72 hpf and transfer them to a 24-well sterile culture plate, 20 per well. A blank control group was treated with zebrafish culture water, while an alcohol (EtOH) group was treated with 1.5% EtOH. Three replicate wells were used per group. Each well was filled with zebrafish culture water to a total volume of 2 mL. The zebrafish were then placed in a lighted incubator (28°C) to continue developing. Mortality was recorded for three consecutive days in each group.

[0126] 3.2.4 Liver area statistics

[0127] After 72 hours of treatment with 1.5% EtOH, 10-15 healthy zebrafish from each group were anesthetized with an appropriate amount of tricaine. The fish were placed in lateral recumbency with their eyes closed on a glass slide containing 4% methylcellulose. After adjusting the fish to a lateral position, the fluorescent area of the zebrafish liver was observed and photographed using an inverted fluorescence microscope. Image Pro Plus 6.0 was used to calculate the total liver area and the areas of zones I and II, and the liver zone area ratio was calculated for each group. GraphPad Prism 9.5.0 software was then used to plot the data into bar graphs for visual comparison.

[0128] 3.2.5 Liver tissue sections

[0129] After 72 hours of treatment with 1.5% EtOH, 5-10 zebrafish per group were anesthetized with tricaine and fixed in 4% paraformaldehyde. After 96 hours, the zebrafish were dehydrated and fixed using a gradient of ethanol (75% for 3 hours, 85% for 2 hours, 90% for 2 hours, 95% for 1 hour, and 100% for 40 minutes twice), followed by benzene for 5 minutes, xylene for 5 minutes twice, and paraffin for 1 hour three times. After dehydration, the zebrafish were embedded in paraffin and sectioned at 4 μm in an embedding machine. After dewaxing to water, the sections were rinsed with purified water and stained with hematoxylin and eosin. The sections were then washed and placed in a gradient of ethanol (95% for 10 minutes twice, 100% for 10 minutes twice). The sections were then dehydrated in xylene until transparent, air-dried, and mounted with neutral gum. Histopathological changes in the livers of the control and alcohol-treated zebrafish were observed under a microscope to assess whether alcohol-induced pathological changes in liver tissue occurred.

[0130] 3.2.6 Data Statistics

[0131] SPSS 28.0 software was used to perform statistical analysis on the experimental data. The differences between the two groups were compared using the t-test and plotted as a bar graph using GraphPad Prism 9.5.0 software. Compared with the blank control group, *p<0.05, **p<0.01, ***p<0.001, ns, showed no statistically significant difference.

[0132] 3.3 Experimental Results

[0133] 3.3.1 Effects of alcohol on zebrafish liver area

[0134] Zebrafish were treated with 1.5% EtOH. The results showed that compared with the blank control group, 1.5% EtOH caused significant changes in the morphology of the zebrafish liver. The lower half of the liver was obviously swollen, the upper half of the liver was atrophied, and the swim bladder completely disappeared. Figure 10 、 Figure 11 The liver area was calculated using Image pro plus 6.0 and then a bar graph was drawn using GraphPad Prism 9.5.0 software. It was found that there was no statistical difference in the total liver area between the alcohol group and the blank control group (p>0.05). Figure 12 .

[0135] After statistically analyzing the liver area of the two groups of zebrafish and calculating the liver area ratio, it was found that the liver area of the zebrafish in the alcohol group had no significant difference in area I (p>0.05), the area of area II was significantly reduced (p<0.001), and the liver area ratio was significantly reduced (p<0.001). There was a statistical difference, such as Figure 13 、 Figure 14 、 Figure 15 .

[0136] Table 3 shows the statistical means and p-values for the ratio of total liver area to liver area, zone I, zone II, and liver subarea area in each zebrafish group. The results showed that the liver subarea ratio had the smallest SD value, indicating that using the liver subarea ratio as an indicator of alcohol-induced liver damage has a low error rate. The liver subarea ratio also had the smallest p-value, indicating that the liver subarea ratio has a high sensitivity for assessing liver damage and exhibits the most significant variation. The coefficient of variation for the liver subarea ratio in each group was lower than that for the total liver area, indicating that the liver subarea ratio is more stable, has a smaller dispersion, and is more precise than the total liver area, making it a more accurate indicator of alcohol-induced liver damage.

[0137] By staining liver tissue sections, it can be determined whether there is damage to the liver tissue structure. Figure 16 As shown in Figure 2, cells in normal zebrafish liver tissue are densely arranged. Figure 17 As shown in the figure, compared with the blank control group, 1.5% EtOH caused severe vacuolation between cells in the zebrafish liver, severe defects in liver structure, blurred boundaries of liver cells, and irregular arrangement of liver cells, indicating that alcohol causes damage to the zebrafish liver.

[0138] 3.3.3 Comprehensive analysis of various evaluation indicators of the effects of alcohol on zebrafish liver

[0139] Comprehensive analysis of various evaluation indicators, as shown in Table 3, revealed that alcohol causes significant structural damage to zebrafish liver tissue. However, there was no statistically significant difference in total liver area between the alcohol-treated zebrafish and the blank control group. Analyzing liver area by zonal area revealed a significant decrease in Zone II area in the alcohol-treated zebrafish compared with the blank control group, consistent with changes in liver morphology and histopathology. Calculation of liver area ratios revealed that the SD value for the liver area ratio was the smallest compared with the total liver area, indicating that using the liver area ratio as an indicator of alcohol-induced liver damage has a low error rate. The liver area ratio also had the lowest p-value, indicating that the liver area ratio has high sensitivity and the most significant variation in assessing liver damage. The coefficient of variation for the liver area ratio in each group was lower than that for the total liver area, indicating that the liver area ratio is more stable, has less variability, and is more precise than the total liver area, making it a more accurate indicator of alcohol-induced liver damage.

[0140] The experimental results show that when using zebrafish to evaluate the hepatotoxicity of compounds, the total liver area as a detection indicator is not sensitive and accurate. Evaluating the liver damage effect of compounds by calculating the liver partition area ratio is more sensitive and accurate.

[0141] Table 3 Effects of alcohol on liver area of zebrafish larvae ( n=30)

[0142]

[0143]

[0144] Note: Compared with the blank control group, p>0.05 indicates no significant difference, and p<0.001 indicates a very significant difference.

[0145] 3.4 Conclusion

[0146] Alcohol causes significant changes in zebrafish liver morphology and pathological changes in liver tissue. However, the total liver area of zebrafish in the alcohol group was not statistically different from that in the blank control group. The liver was divided into two zones and the changes in liver area and the liver area ratio were compared. Results showed that compared with the blank control group, the liver area of Zone I in the alcohol group was not significantly different, while the area of Zone II was significantly reduced, and the liver area ratio was significantly reduced. These differences were statistically significant, consistent with changes in liver morphology, liver pathology, and ALT. Furthermore, the coefficient of variation of the liver area ratio in each group was lower than that of the total liver area, indicating that the liver area ratio is more stable, has less variability, and is more precise than the total liver area, making it more accurate for assessing alcohol-induced liver damage. These results suggest that using total liver area as a metric for evaluating the hepatotoxicity of compounds in zebrafish is not sensitive or accurate. Comparing changes in liver area in different zones is a more sensitive and accurate method for assessing the liver damage of compounds.

[0147] Example 3: Evaluation of the effect of α-naphthyl isothiocyanate (ANIT) on zebrafish hepatotoxicity

[0148] 4.1 Introduction to the research background of α-naphthyl isothiocyanate (ANIT)-induced hepatotoxicity in zebrafish

[0149] α-Naphthyl isothiocyanate (ANIT) is a commonly used cholestasis modeling agent that causes inflammatory damage to the small bile ducts of the liver and induces intrahepatic cholestatic liver disease (CLD). A multicenter epidemiological study of chronic liver disease in Shanghai showed that the prevalence of cholestasis was 10.26% among 4660 patients with chronic liver disease. CLD has an insidious onset, with no obvious clinical symptoms in the early stages. In the advanced stages, a series of hyperbilirubinemia-related manifestations such as jaundice, dark urine, and itchy skin may appear, and it may further develop into liver fibrosis, cirrhosis, and even terminal events such as liver failure. Histopathology shows damage to intrahepatic bile duct epithelial cells and apoptosis, necrosis, hypertrophy, and fibrosis of periportal hepatocytes, with unclear hepatocyte boundaries. Plasma biochemical analysis found that after ANIT treatment, the levels of liver enzymes related to cholestasis, such as ALT and AST, were significantly increased.

[0150] In this study, transgenic zebrafish with green fluorescent protein-labeled livers were used to evaluate the hepatotoxicity of α-naphthylisothiocyanate (ANIT) in zebrafish using liver tissue sections, total liver area, liver zone I area, liver zone II area, and liver zone area ratio as evaluation indicators.

[0151] 4.2 Experimental methods

[0152] 4.2.1 Drugs and reagents

[0153] α-Naphthyl isothiocyanate (ANIT) was purchased from Beijing Beihai Liuhuan Technology Development Co., Ltd., tricaine (product batch number: A5040) was purchased from Sigma-Aldrich, USA, and the fish fixative CMC-Na (product batch number: 419281) used for photography was purchased from Sigma-Aldrich, USA. Zebrafish culture water (5.0 mM NaCl, 0.17 mM KCl, 0.4 mM CaCl2, 0.16 mM MgSO4) was used.

[0154] 4.2.2 Animals and breeding conditions

[0155] Transgenic zebrafish Tg(l-fabp:EGFP) with green fluorescent protein labeling of liver cells were used as experimental animals. These can be purchased from the National Zebrafish Resource Center or other commercially available products. Male and female zebrafish were housed separately, with alternating lighting cycles of 14 h / darkness of 10 h, and the temperature was controlled at 28°C. Male and female zebrafish were placed in a mating tank with a partition at a ratio of 1:2. The partition was removed at 8:30 the next morning, allowing the male and female zebrafish to mate. Fertilized eggs were obtained 1-2 hours later. The fertilized eggs were washed with fresh zebrafish culture water and disinfected with methylene blue, then placed in a 28°C constant temperature incubator with controlled light. The juveniles were cultured to 72 hpf, and healthy fry were selected for subsequent experiments.

[0156] 4.2.3 Experimental Grouping

[0157] Normally developing zebrafish (72 hpf) were selected and transferred to a 24-well sterile culture plate, with 20 per well. A blank control group was treated with zebrafish culture water, while the α-naphthyl isothiocyanate group was treated with 5 μM ANIT. Three replicate wells were used for each group. Each well was filled with zebrafish culture water to a total volume of 2 mL. The zebrafish were then placed in a lighted incubator (28°C) to continue developing. Mortality was recorded for three consecutive days in each group.

[0158] 4.2.4 Liver area statistics

[0159] After 72 hours of treatment with 5μM α-naphthyl isothiocyanate, 10-15 healthy zebrafish from each group were anesthetized with an appropriate amount of tricaine. The fish were placed in lateral recumbency with their eyes closed on a glass slide containing 4% methylcellulose. After adjusting the fish to a lateral position, the fluorescent area of the zebrafish liver was observed and photographed using an inverted fluorescence microscope. Image Pro Plus 6.0 was used to calculate the total liver area and the areas of zones I and II, and the liver zone area ratio was calculated for each group. GraphPad Prism 9.5.0 software was then used to plot the data into bar graphs for visual comparison.

[0160] 4.2.5 Liver tissue sections

[0161] After 72 hours of treatment with 5μM α-naphthyl isothiocyanate, 5-10 zebrafish per group were anesthetized with tricaine and fixed in 4% paraformaldehyde. After 96 hours, the zebrafish were dehydrated and fixed using a gradient of ethanol (75% for 3 hours, 85% for 2 hours, 90% for 2 hours, 95% for 1 hour, and 100% for 40 minutes twice), followed by benzene for 5 minutes, xylene for 5 minutes twice, and paraffin for 1 hour three times. After dehydration, the zebrafish were embedded in paraffin and sectioned at 4μm in an embedding machine. After deparaffinization to water, the zebrafish were rinsed with purified water and stained with hematoxylin and eosin. The sections were then washed and placed in a gradient of ethanol (95% for 10 minutes twice, 100% for 10 minutes twice). The sections were then dehydrated in xylene until transparent, air-dried, and sealed with neutral gum. The pathological changes in the liver tissue of zebrafish in the blank control group and α-naphthyl isothiocyanate group were observed under a microscope to evaluate whether α-naphthyl isothiocyanate caused pathological changes in liver tissue.

[0162] 4.2.6 Data Statistics

[0163] SPSS 28.0 software was used to perform statistical analysis on the experimental data. The differences between the two groups were compared using the t-test and plotted as a bar graph using GraphPad Prism 9.5.0 software. Compared with the blank control group, *p<0.05, **p<0.01, ***p<0.001, ns, showed no statistically significant difference.

[0164] 4.3 Experimental Results

[0165] 4.3.1 Effects of α-naphthyl isothiocyanate on zebrafish liver area

[0166] Zebrafish were treated with 5μM α-naphthyl isothiocyanate. The results showed that compared with the blank control group, 5μM α-naphthyl isothiocyanate caused significant changes in the liver morphology of zebrafish. The liver edge was obviously blackened, the lower half of the liver was obviously swollen, the upper half of the liver was obviously atrophied, and the swim bladder completely disappeared. Figure 18 、 Figure 19The liver area was calculated using Image pro plus 6.0 and then a bar graph was drawn using GraphPad Prism 9.5.0 software. It was found that there was no statistically significant difference in the total liver area between the zebrafish in the α-naphthyl isothiocyanate group and the blank control group (p>0.05). Figure 20 .

[0167] After statistically analyzing the liver area of the two groups of zebrafish, it was found that compared with the blank control group, the area of zone I in the zebrafish treated with α-naphthyl isothiocyanate was significantly increased (p<0.001), the area of zone II was significantly decreased (p<0.001), and the liver area ratio was significantly decreased (p<0.001). There was a statistical difference, such as Figure 21 、 Figure 22 、 Figure 23 .

[0168] Table 4 shows the statistical means and p-values for the total liver area and liver areas I and II in each zebrafish group. The results showed that the liver area ratio had the smallest SD value, indicating that using the liver area ratio as an indicator of liver damage by α-naphthalene isothiocyanate has a small error rate. The liver area ratio also had the smallest p-value, indicating that the liver area ratio has a high sensitivity for liver damage assessment and the most significant variation. The coefficient of variation for the liver area ratio in each group was lower than that for the total liver area, indicating that the liver area ratio is more stable, has a smaller dispersion, and is more precise than the total liver area, allowing for more accurate assessment of liver damage by α-naphthalene isothiocyanate.

[0169] 4.3.2 Effects of α-naphthyl isothiocyanate on zebrafish liver histopathological sections

[0170] By staining liver tissue sections, it can be determined whether there is damage to the liver tissue structure. Figure 24 As shown in Figure 2, cells in normal zebrafish liver tissue are densely arranged. Figure 25 As shown in the figure, compared with the blank control group, 5μM α-naphthyl isothiocyanate caused severe vacuolation between zebrafish liver cells, severe defects in liver structure, blurred boundaries of liver cells, and irregular arrangement of liver cells, indicating that 5μM α-naphthyl isothiocyanate caused damage to the zebrafish liver.

[0171] 4.3.3 Comprehensive Analysis of Evaluation Indicators of the Effects of α-naphthylisothiocyanate on Zebrafish Liver

[0172] Comprehensive analysis of various evaluation indicators, as shown in Table 4, revealed that 5 μM α-naphthalene isothiocyanate (α-NITC) caused significant structural damage to zebrafish liver tissue. However, the total liver area of zebrafish treated with α-NITC was not statistically different from that of the blank control group. Statistical analysis of liver area revealed a significant increase in Zone I and a significant decrease in Zone II in the α-NITC group compared with the blank control group, consistent with changes in liver morphology and histopathology. Calculation of the liver area ratio revealed that the SD value of the liver area ratio was the smallest compared with the total liver area, indicating that the liver area ratio has a low error rate when used as an indicator of α-NITC-induced liver damage. The liver area ratio also had the lowest p-value, indicating that the liver area ratio has high sensitivity and the most significant variation for assessing liver damage. The coefficient of variation of the liver area ratio in each group was lower than that of the total liver area, indicating that the liver area ratio is more stable, has less variability, and is more precise than the total liver area, making it a more accurate indicator of liver damage.

[0173] The experimental results show that when using zebrafish to evaluate the hepatotoxicity of compounds, the total liver area as a detection indicator is not sensitive and accurate. Evaluating the liver damage effect of compounds by calculating the liver partition area ratio is more sensitive and accurate.

[0174] Table 4 Effects of α-naphthyl isothiocyanate on the liver area of zebrafish larvae ( n=30)

[0175]

[0176] Note: Compared with the blank control group, p>0.05 indicates no significant difference, and p<0.001 indicates a very significant difference.

[0177] 4.4 Conclusion

[0178] α-Naphthyl isothiocyanate (α-NITC) caused significant changes in zebrafish liver morphology and pathological changes in liver tissue. However, the total liver area of zebrafish treated with α-NITC was not statistically different from that of the blank control group. The liver was divided into two zones and the changes in liver area and liver zone area ratio were compared. The results showed that compared with the blank control group, the area of zone I increased significantly, the area of zone II decreased significantly, and the liver zone area ratio decreased significantly. These differences were statistically significant, consistent with the changes in liver morphology, liver pathology, and ALT. The coefficient of variation of the liver zone area ratio in each group was lower than that of the total liver area, indicating that the liver zone area ratio is more stable, has less variability, and is more precise than the total liver area, making it a more accurate indicator of liver damage caused by α-NITC. These results suggest that using total liver area as a metric for evaluating the hepatotoxicity of compounds in zebrafish is not sensitive or accurate. Comparing changes in liver zone area is a more sensitive and accurate method for assessing the liver damage of compounds.

[0179] The present invention uses the zebrafish liver partition area ratio as an evaluation indicator for zebrafish liver damage for the first time, overcoming the problem of using the total liver area as an inaccurate indicator for evaluating the effect of a substance on zebrafish liver damage. It also avoids experimental errors caused by different total liver areas due to individual differences in zebrafish. It is more scientific and objective, improves the accuracy of the evaluation results, and has the advantages of simple, rapid, stable, reliable and reproducible operation.

Claims

1. A method for evaluating the liver damage induced by a substance based on the area ratio of the zebrafish liver compartment, comprising the following steps: (1) 72 hours after fertilization, normal zebrafish were continuously incubated with the test substance, which was recorded as the test substance treatment group; at the same time, a control group was set up by incubating without the test substance under the same conditions, which was recorded as the control group; (2) After incubation in the anesthesia step (1), collect the liver fluorescence image of each zebrafish in the control group and the test substance treatment group, divide the liver fluorescence image of the zebrafish in the lateral position into zones, mark the highest point where the horizontal line intersects with the liver as point a, mark the intersection point of the zebrafish's lateral position melanin stripe and the eye as point c, draw a horizontal line through point c, mark the intersection point of the horizontal line with the right side of the liver as point b, connect points a and b with a straight line, mark the area below the line connecting a and b as zone I, and mark the area above the line connecting a and b as zone II, and calculate the area of zone I and zone II of the zebrafish liver; (3) Calculate the liver partition area ratio of each group of zebrafish obtained in step (2) according to the following formula: Liver zone area ratio = (liver zone II area / liver zone I area) × 100% Analyze and compare the significance of the difference in liver area ratio between the control group and the test substance treatment group; When there is a significant difference between the liver area ratio of the zebrafish in the test substance treatment group and the liver area ratio of the zebrafish in the control group, it indicates that the test substance has a damaging effect on the liver.

2. The method according to claim 1, wherein In step (1), the zebrafish is a transgenic zebrafish whose liver cells are labeled with green fluorescent protein.

3. The method according to claim 1, wherein In step (1), the test substance is directly dissolved in zebrafish culture water to prepare a test solution; or the test substance is dissolved in a co-solvent to prepare a mother solution, and then diluted in zebrafish culture water to prepare a test solution.

4. The method according to claim 3, wherein The cosolvent was DMSO.

5. The method according to claim 4, wherein The final concentration of DMSO in the detection solution is less than 0.5% by volume.

6. The method according to claim 4, wherein When using a co-solvent, a co-solvent control group should be added.

7. The method according to claim 1, wherein In step (1), the zebrafish are incubated with the test substance for 1 to 3 days.

8. The method according to claim 1, wherein In step (3), the significance of the difference in liver area ratio between the control group and the test substance-treated group was analyzed and compared, and the t-test was used for statistical analysis.

Images