Method for establishing cadmium-induced silkworm hepatotoxicity model

Abstract

The present application relates to a kind of methods for establishing cadmium-induced silkworm liver injury model, comprising: according to the proportion of 10 g mulberry leaf and 1 ml concentration 125 mg / L cadmium chloride solution corresponding to every 25 g silkworm body weight, cadmium chloride solution is evenly applied to the back of mulberry leaf and air-dried, and mulberry leaf is treated with clean water as control;Select five age first day silkworm and divide into model group and blank group, under the condition of controlling temperature and humidity, respectively feed after processing mulberry leaf, calculate the amount of mulberry leaf and solution according to the real-time body weight of silkworm before each feeding, every 12 hours once and continuously feed for 5 days;After feeding, fasting for 12 hours, the weight gain rate of two groups of silkworms, AST and ALT activity in hemolymph are determined, and fat body is observed by histological section;When the weight gain rate of model group is significantly lower than blank group, AST and ALT activity is significantly higher than blank group and the pathological characteristics of karyopyknosis and loose tissue appear in fat body, it is determined that model is established successfully.

Description

Technical Field

[0001] This invention belongs to the field of biological technology, specifically relating to a method for establishing a cadmium-induced liver injury model in silkworms. Background Technology

[0002] Cadmium (Cd), a widespread heavy metal environmental pollutant, can accumulate in organisms through the food chain, causing damage to multiple organ systems, with the liver being one of its main target organs. Accumulation of cadmium in hepatocytes can induce oxidative stress, inflammatory responses, and apoptosis, leading to liver dysfunction and even irreversible damage. Currently, research on the mechanisms of cadmium toxicity and the screening of antidotes heavily rely on animal models. Traditionally, mammalian models such as mice and rats have been widely used, but they have limitations such as long experimental cycles, high breeding costs, high operational technical requirements, and strict ethical reviews, making them particularly unsuitable for large-scale, multi-round preliminary screening and mechanism exploration under conditions of limited funding and personnel.

[0003] In recent years, establishing low-cost, high-throughput toxicity models using model organisms has become an important direction in toxicology research. Silkworm (… Bombyx mori Due to its short lifespan, low breeding costs, high reproductive capacity, and lack of strict ethical constraints, the silkworm shows potential to replace traditional mammalian models. More importantly, the silkworm shares high genetic homology with humans; its fat body is analogous to the liver in mammals in terms of metabolism and detoxification, while its midgut resembles the intestine. This makes the silkworm suitable for studies on the metabolism of exogenous toxins and hepatotoxicity. Existing studies have successfully used silkworms to construct models of diabetes, gout, and drug-induced liver injury, validating its feasibility as a toxicological research tool.

[0004] However, existing methods for constructing cadmium-induced liver injury models using silkworms still face several technical bottlenecks and a lack of standardization. First, the successful construction of the model is highly dependent on the accuracy and stability of the dosage. Using a fixed amount of cadmium solution to treat a fixed mass of mulberry leaves for feeding cannot adapt to the actual situation of continuous weight gain in silkworms during growth, resulting in inconsistent dosage concentrations throughout the experimental period and affecting the model's reproducibility and the reliability of the dose-response relationship. Second, the determination of model success lacks a systematic and objective standardized indicator system. Often, it relies only on elevated individual biochemical indicators (such as serum lymphocyte transaminase activity) or sporadic behavioral observations, lacking a unified standard for verifying multiple correlations between growth inhibition (such as weight gain rate), dynamic changes in key liver function enzyme indicators (such as AST and ALT), and typical histopathological features (such as fat body cell nuclear pyknosis and tissue loosening). Furthermore, the lack of standardized procedures in the preparation of experimental materials and operational processes, such as the pretreatment and disinfection of mulberry leaves, the uniform application of cadmium solution to mulberry leaves, and the controlled drying, may introduce microbial contamination or cause uneven toxicity, affecting the stability and consistency of experimental results.

[0005] Therefore, there is an urgent need in this field to establish a low-cost, easy-to-operate, and reproducible method for constructing a cadmium-induced liver injury model in silkworms. This method needs to address the problem of dynamic and precise control of drug concentration, integrate multi-dimensional and quantitative evaluation criteria, and standardize key experimental procedures, thereby providing a reliable and efficient technical platform for research on the cadmium toxicity mechanism and screening of hepatoprotective drugs based on this model. Summary of the Invention

[0006] The object of the present invention is to overcome at least the aforementioned defects and to provide advantages that will be described later.

[0007] Another objective of this invention is to address the technical problems in the prior art, such as the lack of cadmium-induced liver injury models for silkworms, unclear target organs for liver injury, single model judgment criteria, and non-standard induction and feeding procedures, which lead to poor model stability, low reproducibility, and inability to accurately characterize liver injury.

[0008] To achieve these and other advantages of the present invention, a method for establishing a cadmium-induced liver injury model in silkworms is provided, comprising the following steps: Select mulberry leaves and remove the petioles.

[0009] Mulberry leaves were randomly divided into two groups. In the first group, the mulberry leaves were laid out with the back facing up. The cadmium chloride solution was evenly applied to the back of the mulberry leaves at a ratio of 10 g of mulberry leaves to 1 ml of cadmium chloride solution. The mulberry leaves were then dried until the surface moisture content was ≤5%, resulting in cadmium chloride-treated mulberry leaves. The concentration of the cadmium chloride solution was 125 mg / L. In the second group, the cadmium chloride solution was replaced with an equal volume of water for simultaneous treatment, resulting in water-treated mulberry leaves.

[0010] Silkworms that had completed their fourth molt and were on the first day of the fifth instar were selected and randomly divided into a model group and a control group, with 30 silkworms in each group; they were raised at 18±2℃ and 70% humidity.

[0011] Before each feeding, the total weight of the silkworms in the model group and the control group was weighed. The silkworms in the model group were fed the corresponding amount of cadmium chloride-treated mulberry leaves every 12 hours, according to a feeding ratio of 10g of cadmium chloride-treated mulberry leaves for every 25g of silkworm weight. The silkworms in the control group were fed the corresponding amount of water-treated mulberry leaves every 12 hours, according to a feeding ratio of 10g of water-treated mulberry leaves for every 25g of silkworm weight.

[0012] After 5 days of continuous feeding, all silkworms were fasted for 12 hours, and the following measurements were performed: the weight of silkworms in the model group and the control group was measured, and the weight growth rate was calculated; the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the hemolymph of silkworms in the model group and the control group were measured; fat body sections of silkworms in the model group and the control group were prepared and observed under a microscope; when the average weight growth rate of silkworms in the model group was significantly lower than that in the control group, the activities of liver function indicators aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the hemolymph of silkworms in the model group were significantly higher than those in the control group, and the fat bodies of silkworms in the model group showed pathological features of nuclear pyknosis and tissue loosening, the cadmium-induced silkworm liver injury model was considered to have been successfully established.

[0013] In the above scheme, the concentration of the cadmium chloride solution applied was 125 mg / L. This concentration was determined through multi-gradient preliminary experiments at 75 mg / L, 100 mg / L, 125 mg / L, and 150 mg / L, and it can stably induce liver injury in silkworms, ensuring significant pathological features without the risk of acute mortality, and exhibiting the best model reproducibility. The silkworm variety used was Liangguang No. 2.

[0014] Preferably, the cadmium chloride solution is prepared by water bath heating, specifically including: taking 12.5 mg of anhydrous cadmium chloride and 100 ml of pure water, placing them in a container under water bath conditions and stirring continuously until the solution becomes transparent, thus obtaining the cadmium chloride solution; wherein, the water bath temperature is 50℃~60℃; the stirring rate is 300 r / min, and the stirring time is 15±2 minutes.

[0015] Preferably, the weight of the silkworm is 1g to 1.1g. The silkworm variety is Liangguang No. 2.

[0016] Preferably, the determination of the liver function indicators aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities in hemolymph includes: treating silkworms by abdominal foot puncture and collecting their hemolymph, with hemolymph from every 5 silkworms collected as a single sample, and a single collection volume ≥200 μL; the clarified supernatant is then analyzed using a fully automated biochemical analyzer, employing pre-validated micro-detection conditions and calibration methods suitable for silkworm hemolymph matrix to determine the enzyme activity units of AST and ALT; the reaction temperature is 37°C, the reaction time is 10 minutes, and the calibration curve range is 5~500 U / L.

[0017] Specifically, hemolymph collection was performed as follows: the surface of the third pair of abdominal legs of the silkworm was disinfected by wiping with cotton balls soaked in 75% ethanol. After the ethanol evaporated, the dorsal blood sinuses at the base of the abdominal legs were punctured with a sterile injection needle. The abdominal legs were gently squeezed to allow the hemolymph to flow out naturally. The transparent yellow hemolymph was aspirated with a sterile capillary tube and collected into a pre-cooled centrifuge tube. The hemolymph from every 5 silkworms was mixed into one sample for collection, with a single collection volume of ≥200μL.

[0018] Preferably, the pathological features of nuclear pyknosis and loose tissue in the fat body of silkworms are assessed through the following steps: Live silkworms, after fasting, are rapidly frozen to rigor mortis on dry ice. Immediately after dissection, a fat body sample containing skin is obtained from the dorsal side of the third abdominal segment and placed in 4% paraformaldehyde fixative at 4°C for 24 hours to obtain fixed tissue. The tissue is then dehydrated using a gradient of ethanol, followed by clearing with xylene, and then impregnated with molten paraffin for at least 2 hours to form a paraffin block suitable for ultrathin sections. The paraffin block is cut into continuous thin layers of 5–8 micrometers, which are then spread, attached, and dried. After dewaxing with xylene and rehydration using a gradient of ethanol, the sections are stained using the conventional hematoxylin-eosin staining method. After dehydration and clearing, the sections are then treated with medium... The tissue was sealed with a resin to create permanent sections. Under an optical microscope, adipocytes in the subcutaneous adipose tissue of the third abdominal segment were observed. When the proportion of pyknotted cells was ≥30% and the average width of the interstitial space was ≥20μm, it was determined to meet the pathological characteristics of liver-like injury. The freezing time was ≤3 minutes. Gradient ethanol dehydration included: 70% ethanol for 30 minutes, 80% ethanol for 30 minutes, 95% ethanol for 20 minutes, and anhydrous ethanol for 20 minutes. Xylene clearing treatment of the tissue included xylene I treatment for 10 minutes and xylene II treatment for 10 minutes. Gradient ethanol rehydration included: anhydrous ethanol for 2 minutes, 95% ethanol for 2 minutes, 80% ethanol for 2 minutes, 70% ethanol for 2 minutes, and distilled water for 5 minutes.

[0019] In the above scheme, the freezing time is ≤3 minutes. This duration can quickly stiffen the silkworm and avoid anatomical damage. Moreover, short-term dry ice freezing does not cause ice crystals to damage the cell structure and does not affect pathological observation.

[0020] Preferably, after selecting mulberry leaves and removing petioles, the process includes a pretreatment step: placing the mulberry leaves with the petioles removed, back side up, in a single layer on a breathable stainless steel support mesh, and irradiating them under a UV lamp with a wavelength of 250-260 nm for 15-30 minutes at a distance of 30-50 cm; transferring the UV-sterilized mulberry leaves to a clean air circulation device and circulating them at 15-25℃ and 40-60% relative humidity for 120±10 minutes; and then processing the equilibrated mulberry leaves using one of the following methods: Method 1, completing the subsequent solution application within 10 minutes; Method 2, sealing them in a sterile container, storing them at 4℃, and using them within 24 hours.

[0021] Preferably, the process of uniformly applying cadmium chloride solution or water to the back of mulberry leaves adopts a step-by-step, intermittent application and directional drying method: the pre-treated mulberry leaves are first coated with 50% to 60% of the total amount of cadmium chloride solution or water; the mulberry leaves after the first coating are left to stand for 8 to 12 minutes under laminar flow conditions at a temperature of 20 to 25°C and an air velocity of 0.8 to 1.2 m / s; then the remaining solution is applied a second time, and the mulberry leaves are placed under the same laminar flow conditions until they are dried until the surface moisture content of the leaves is ≤5%.

[0022] Advantages of this invention: 1. The above-mentioned scheme, by clearly defining the silkworm fat body as the target organ for liver-like injury, establishes a multi-dimensional judgment system based on "weight gain rate + hemolymph AST / ALT activity + fat body pathological characteristics," standardizes mulberry leaf treatment, silkworm rearing, and induction parameters, and solves the problems of model deficiency, ambiguous judgment, and non-standardized procedures in existing technologies. It achieves high model stability, strong reproducibility, and accurate characterization of cadmium-induced liver-like injury, providing a low-cost, large-scale applicable silkworm model for related mechanism research and drug screening, filling a gap in the field.

[0023] 2. The above method solves the problem of uneven solution caused by ambiguous parameters in existing preparation methods by limiting the water bath temperature, stirring rate, and time for cadmium chloride solution preparation. It achieves complete dissolution of cadmium chloride and precise consistency of solution concentration, ensuring the stability of the cadmium inducer, providing uniform induction conditions for model establishment, reducing experimental errors caused by differences in inducers, and improving the success rate of model establishment.

[0024] 3. The above scheme, by specifying the silkworm weight as 1g~1.1g, solves the problem of large differences in the physiological state of individual silkworms in existing technologies. It achieves the effect of uniform physiological state of the selected silkworms, consistent tolerance and reactivity to cadmium, providing a standardized experimental baseline for model establishment, reducing the interference of individual differences on experimental results, and further improving the stability and reliability of the model.

[0025] 4. The above scheme, by standardizing the hemolymph collection volume, centrifugation parameters, and adaptation parameters such as detection temperature and time, solves the problem of inaccurate results caused by the incompatibility of existing detection methods with silkworm hemolymph. It achieves accurate and reliable AST and ALT activity detection results, enabling biochemical indicators to truly reflect the degree of liver-like injury, providing a scientific biochemical basis for model assessment, and improving the accuracy of model assessment.

[0026] 5. The above-mentioned scheme, by clearly defining the parameters of each step, such as dehydration and clearing, and the quantitative standards for pathological characteristics, solves the problems of non-standardized pathological assessment procedures and subjective results in the existing system. It achieves high-quality slides, clear pathological observation, and objective and unified judgment standards, accurately verifying the presence of fat-body-like liver injury, providing reliable histological evidence for model establishment, and enhancing the authority of model judgment.

[0027] 6. The above scheme, through ultraviolet disinfection and humidity balancing pretreatment of mulberry leaves, solves the problem of uneven cadmium adsorption caused by existing microbial contamination and uneven leaf conditions. It achieves clean, uncontaminated mulberry leaves with stable humidity, ensuring that the subsequent cadmium solution can be uniformly adsorbed on the leaf surface, guaranteeing the consistency of cadmium dosage ingested by silkworms, reducing experimental interference, and improving the stability of the model's induction effect.

[0028] 7. The above scheme, by employing a step-by-step, spaced-out application and directional drying method, solves the problem of uneven cadmium solution distribution caused by a single application. This achieves the effect of uniform spreading and sufficient adsorption of the cadmium solution on the mulberry leaf surface, further ensuring the consistency of cadmium intake dose among individual silkworms, avoiding excessively high or low local doses, and making the induced liver-like injury degree uniform, thus improving the homogeneity of the model. Attached Figure Description

[0029] Figure 1 This is a comparison chart of the weight gain rate of silkworms with cadmium-induced liver damage according to the present invention; Figure 2 This is a comparative chart of blood, lymph, and liver function indicators in silkworms with cadmium-induced liver injury, as presented in this invention. Figure 3 This is a cross-section of subcutaneous fat in silkworms that caused cadmium-induced liver injury according to the present invention. Detailed Implementation

[0030] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0031] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0032] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0033] Example 1 A method for establishing a cadmium-induced liver injury model in silkworms includes the following steps: Step 1: Select mulberry leaves and remove the petioles; randomly divide the mulberry leaves into two groups. In the first group, lay the mulberry leaves with the back facing up, and apply the cadmium chloride solution evenly to the back of the mulberry leaves at a ratio of 10g of mulberry leaves to 1 ml of cadmium chloride solution. Then, dry the coated mulberry leaves until the surface moisture content is ≤5%, thus obtaining cadmium chloride-treated mulberry leaves. The concentration of the cadmium chloride solution is 125mg / L. In the second group, replace the cadmium chloride solution with an equal volume of water and treat simultaneously, thus obtaining water-treated mulberry leaves.

[0034] Step Two: Select silkworms of the Liangguang No. 2 variety, which have completed their fourth molt and are on the first day of the fifth instar, weighing 1g-1.1g. Randomly divide them into a model group and a control group, with 30 silkworms in each group. Keep them at 18±2℃ and 70% humidity. Before each feeding, weigh the total weight of the silkworms in the model group. Feed them the corresponding amount of cadmium chloride-treated mulberry leaves every 12 hours, according to a feeding ratio of 10g of cadmium chloride-treated mulberry leaves for every 25g of silkworm body weight. Before each feeding, weigh the total weight of the silkworms in the control group. Feed them the corresponding amount of water-treated mulberry leaves every 12 hours, according to a feeding ratio of 10g of water-treated mulberry leaves for every 25g of silkworm body weight. Both the model group and the control group are fed continuously for 5 days.

[0035] Step 3: After feeding for 5 consecutive days, all silkworms were fasted for 12 hours, and then the following measurements were performed: Weight growth rate measurement: The weight of silkworms in the model group and the blank group was measured, and the weight growth rate was calculated.

[0036] Determination of the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in hemolymph: The activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the hemolymph of silkworms in the model group and the blank group were measured.

[0037] Pathological observation of fat bodies: Fat body sections of silkworms in the model group and blank group were prepared and observed under a microscope.

[0038] Model determination: When the average weight gain rate of silkworms in the model group is significantly lower than that in the control group, the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the hemolymph of silkworms in the model group are significantly higher than those in the control group, and the fat bodies of silkworms in the model group show pathological features of nuclear condensation and tissue loosening, the cadmium-induced liver injury model of silkworms is determined to be successfully established.

[0039] Example 2 A method for establishing a cadmium-induced liver injury model in silkworms includes the following steps: 1. Mulberry leaf pretreatment: Select fresh mulberry leaves and remove the petioles. Lay the mulberry leaves in a single layer with the back facing up on a breathable stainless steel support mesh. Place them under a UV lamp with a wavelength of 250~260 nm for 15~30 minutes, with an irradiation distance of 30~50 cm. After UV disinfection, transfer them to a clean air circulation device and circulate them for equilibration at 15~25℃ and 40~60% relative humidity for 120±10 minutes. Complete the solution application within 10 minutes after equilibration.

[0040] 2. Preparation of cadmium chloride solution: The cadmium chloride solution is prepared by water bath heating, specifically by taking 12.5 mg of anhydrous cadmium chloride and 100 mL of pure water, placing them in a container in a water bath at 50℃~60℃, and stirring continuously at a rate of 300 r / min for 15±2 minutes until the solution becomes transparent, thus obtaining a cadmium chloride solution with a concentration of 125 mg / L.

[0041] 3. Selection and grouping of silkworms: Silkworms of the Liangguang No. 2 variety, which are on the first day of the fifth instar after completing the fourth molt and weighing 1g~1.1g, were selected and randomly divided into a model group and a control group, with 30 silkworms in each group; they were raised at 18±2℃ and 70% humidity.

[0042] 4. Mulberry leaf application and directional drying: (1) Model group mulberry leaves: Apply 1 ml of cadmium chloride working solution to 10 g of mulberry leaves in a step-by-step manner: First, apply 50%~60% of the total amount and let stand for 8~12 minutes under laminar flow conditions of 20~25℃ and air velocity of 0.8~1.2 m / s; Second, apply the remaining solution and let stand for 2.5±0.5 minutes under the same laminar flow conditions of 20~25℃ and air velocity of 0.8~1.2 m / s; Then, transfer the mulberry leaves back to laminar flow conditions and air dry until the surface moisture content of the leaves is ≤5%. (2) Blank group mulberry leaves: Replace the cadmium chloride working solution with an equal volume of water and operate according to the same step-by-step application and drying process as the model group, and air dry until the surface moisture content of the leaves is ≤5%.

[0043] 5. Dynamic feeding management: Weigh the total weight of the two groups of silkworms before each feeding, and feed them every 12 hours according to the ratio of 10 g of treated mulberry leaves for every 25 g of silkworm weight, for 5 consecutive days.

[0044] 6. Sample processing, indicator detection and judgment: After 5 consecutive days of feeding, all silkworms were fasted for 12 hours, and the following tests were conducted: (1) Weight growth rate: Measure the weight before and after fasting and calculate the weight growth rate.

[0045] (2) Assay of hemolymph AST / ALT activity: The surface of the third pair of abdominal feet was disinfected with 75% ethanol. After the ethanol evaporated, the dorsal blood sinuses at the base of the abdominal feet were punctured with a sterile injection needle. The abdominal feet were gently squeezed to allow the hemolymph to flow out naturally. The transparent yellow hemolymph was collected with a sterile capillary tube and collected into a pre-cooled centrifuge tube. The hemolymph from every 5 silkworms was mixed into one sample, and the single collection volume was ≥200 μL. The collected hemolymph sample was immediately placed in an environment of 4℃ and centrifuged at 8000~10000 r / min for 8~12 minutes to quickly separate and obtain a clear supernatant. The supernatant was placed in a fully automated biochemical analyzer, and calibration curves of 5, 10, 50, 100, 200, and 500 U / L were prepared using human AST / ALT standards. IFCC was selected. The clinical biochemical test reagent was verified to have no matrix interference by a blank blood lymphocyte spike recovery test. The reaction temperature was set at 37℃ and the reaction time was 10 minutes. The absorbance value was read at 340nm wavelength using the rate method. The enzyme activity units were calculated based on the standard curve established in advance using standards of known concentrations. The calibration curve range was 5~500 U / L. The enzyme activity units of AST and ALT were measured.

[0046] (3) Pathological assessment of fat body: Live silkworms that have been fasted are rapidly frozen on dry ice for 3 minutes (this time can quickly make the silkworms stiff and avoid damage to the fat body caused by the movement of the silkworms during dissection, and short-term dry ice freezing will not form ice crystals that damage the fat body cell structure and will not affect pathological observation) until stiff. Immediately dissect to obtain fat body samples including skin on the back of the third abdominal segment, and put them into 4% paraformaldehyde fixation solution and fix at 4°C for 24 hours to obtain fixed tissue. Fixation with 4% paraformaldehyde at 4℃ for 24 hours; followed by gradient dehydration with ethanol (70% ethanol for 30 minutes, 80% ethanol for 30 minutes, 95% ethanol for 20 minutes and anhydrous ethanol for 20 minutes), clearing with xylene (xylene I treatment for 10 minutes and xylene II treatment for 10 minutes), and paraffin embedding for 2 hours to prepare 5-8 μm sections, which were then stained with hematoxylin and eosin (HE); adipocytes in the subcutaneous adipose tissue of the third abdominal segment were observed under an optical microscope, and five 400x magnification fields were randomly selected to count cells and measure interstitial spaces, with the average value calculated; after verification by three parallel experiments, when the proportion of pyknotted cells was ≥30% and the average width of interstitial spaces was ≥20 μm, it was determined to meet the pathological characteristics of liver-like injury.

[0047] Experiment and Analysis Experiment 1: Model Establishment and Validation Experiment I. Materials 1. Silkworms: Healthy silkworms of the Liangguang No. 2 variety were selected and raised until the first day of the fifth instar after the fourth molt. Individuals weighing between 1g and 1.1g were selected, totaling 60 individuals, and randomly divided into a model group and a control group, with 30 individuals in each group.

[0048] 2. Reagents: Anhydrous cadmium chloride (analytical grade), 4% paraformaldehyde fixative, 0.1 mol / L hydrochloric acid, gradient ethanol (70%, 80%, 95%, anhydrous ethanol), xylene, molten paraffin, hematoxylin-eosin staining solution, neutral resin. Cadmium chloride working solution concentration of 125 mg / L: After preliminary screening with four dose groups of 75 mg / L, 100 mg / L, 125 mg / L, and 150 mg / L, the optimal concentration for modeling silkworms was found to be 125 mg / L, exhibiting 0% mortality, significantly reduced weight gain rate, significantly increased AST / ALT activity, and typical fat body pathological characteristics.

[0049] 3. Instruments: UV lamp (wavelength 250~260nm, used for disinfection), clean air circulation equipment, laminar flow drying device, fully automated biochemical analyzer, high-speed refrigerated centrifuge, optical microscope, microtome, constant temperature water bath, electronic balance, sterile capillary tubes, pre-cooled centrifuge tubes.

[0050] II. Methods Step 1: Place the mulberry leaves (with petioles removed) in a single layer on a breathable stainless steel support mesh with the back side facing up. Place them under a UV lamp with a wavelength of 250~260 nm at a distance of 30 cm for 20 minutes. Then transfer them to a clean air circulation device and circulate them for equilibration at 20℃ and 50% relative humidity for 120 minutes. Apply the solution within 10 minutes after equilibration.

[0051] Step 2: Take 12.5 mg of anhydrous cadmium chloride and 100 mL of pure water, place them in a 55℃ water bath, and stir at 300 r / min for 15 minutes until the solution becomes transparent, to obtain cadmium chloride with a concentration of 125 mg / L.

[0052] Step 3: Place the model group and the control group of silkworms in a rearing environment of 18±2℃ and 70% humidity, respectively. Before each feeding, weigh the total weight of the model group and the control group of silkworms. Calculate the required amount of mulberry leaves for each feeding, based on the ratio of 10g of cadmium chloride-treated mulberry leaves or 10g of water-treated mulberry leaves for every 25g of silkworm weight. Based on the required amount of mulberry leaves, calculate the required amount of cadmium chloride solution and water for each feeding, using a ratio of 10g of mulberry leaves to 1ml of cadmium chloride solution or 1ml of water. Perform step-by-step application and directional drying on the mulberry leaves for each feeding: First, apply 50%~60% solution and let stand under laminar flow for 8~12 minutes; second, apply the remaining solution, let stand for 2.5 minutes, and then transfer the mulberry leaves back to laminar flow conditions to air dry until the surface moisture content of the leaves is ≤5%. The model group silkworms were fed cadmium chloride-treated mulberry leaves every 12 hours as described above, while the control group silkworms were fed mulberry leaves treated with clean water every 12 hours. The feeding was carried out for 5 consecutive days. During this period, the silkworms' feeding and activity were observed daily, and the residual leaves and feces in the feeding box were cleaned up in a timely manner.

[0053] Step 4: After feeding for 5 consecutive days, stop feeding and fast both groups of silkworms for 12 hours.

[0054] Step 5: Measure the weight of the two groups of silkworms after fasting, and calculate the weight growth rate = (weight after fasting - weight before feeding) × 100% / weight before feeding, where the weight before feeding is the initial weight on Day 1.

[0055] Disinfect the surface of the third pair of abdominal legs of silkworms by wiping with cotton balls soaked in 75% ethanol. After the ethanol evaporates, puncture the dorsal blood sinuses at the base of the abdominal legs with a sterile injection needle, gently squeeze the legs to allow the hemolymph to flow out naturally, and collect the transparent yellow hemolymph with a sterile capillary tube into a pre-cooled centrifuge tube, ensuring a single collection volume ≥200 μL. Immediately place the collected hemolymph sample in a 4°C environment and centrifuge at 9000 r / min for 10 minutes to obtain a clear supernatant. Place the supernatant in a fully automated biochemical analyzer, and establish a calibration curve of 5~500 U / L using human AST / ALT standards (concentration points: 5, 10, 50, 100, 200, 500 U / L). Use IFCC detection reagents, verified by matrix effect to be suitable for silkworm hemolymph, set the reaction temperature to 37°C, the reaction time to 10 minutes, and the calibration curve range to 5~500 U / L, and determine the enzyme activity units of AST and ALT. If the sample test value exceeds the upper limit of the calibration curve, it should be diluted with a blank hemolymph matrix and measured again to ensure that the diluted reading falls within the range of the calibration curve.

[0056] After fasting, the silkworms were placed on dry ice and frozen for 3 minutes (this time can quickly stiffen the silkworms without damaging the cells with ice crystals) until stiff. Immediately after stiffening, the fat body samples containing the skin on the back of the third abdominal segment were dissected and obtained. The samples were then placed in 4% paraformaldehyde fixative and fixed at 4°C for 24 hours. The fixed tissue was dehydrated by a gradient of ethanol (70% ethanol for 30 minutes, 80% ethanol for 30 minutes, 95% ethanol for 20 minutes, and anhydrous ethanol for 20 minutes), then cleared by treatment with xylene I and II for 10 minutes each, and finally impregnated with molten paraffin for 2.5 hours to prepare a paraffin block. The paraffin block was cut into continuous thin layers of 6 micrometers, spread, attached, and dried, then dewaxed with xylene, rehydrated by a gradient of ethanol (anhydrous ethanol for 2 minutes, 95% ethanol for 2 minutes, 80% ethanol for 2 minutes, 70% ethanol for 2 minutes, and distilled water for 5 minutes), stained with hematoxylin and eosin, dehydrated and cleared, and then mounted with neutral resin to prepare permanent sections; the adiposome cells were observed under an optical microscope.

[0057] Step 6: Use SPSS 22.0 statistical software to perform independent samples t tests on the weight gain rate, AST and ALT activity data. P<0.05 indicates that the difference is statistically significant.

[0058] III. Results 1. Weight Growth Rate: The daily average weight changes of silkworms in the control group and the model group are shown in Table 1. The weight growth rate was calculated based on the weight changes, and the results are as follows: Figure 1 .

[0059] Table 1: Daily mean body weight changes in silkworms with cadmium-induced liver injury (Mean±SD, n=30) Statistical analysis showed no significant difference in the initial weight (Day 1) of the two groups of silkworms (p>0.05), indicating they were comparable.

[0060] According to Table 1 and Figure 1 The data shows that the average weight gain rate of silkworms in the model group was (101.94±4.99)%, while that in the blank group was (137.04±4.93)%. The model group was significantly lower than the blank group (p<0.0001), indicating that cadmium intake inhibited the growth of silkworms.

[0061] 2. Results of blood lymphocyte AST and ALT activity assays are as follows: Figure 2 When liver cells are damaged, ALT and AST are released into the bloodstream, causing an upregulation of these levels. Silkworms do not have blood; their body fluids are stored in hemolymph. According to... Figure 2 It was found that, compared with the blank group (NC), the activities of ALT and AST in the hemolymph of silkworms in the model group (MC) were significantly increased (p<0.05, p<0.01), indicating that cadmium-induced liver damage can occur in silkworms.

[0062] 3. Pathological characteristics of fat bodies, as follows: Figure 3 The silkworm's fat body functions similarly to the liver of mammals, serving as a primary organ for detoxification of exogenous toxins. Therefore, longitudinal sections of the silkworm's skin allow observation of cellular changes in its subcutaneous fat body. Based on... Figure 3 It was found that the subcutaneous fat structure on the dorsal side of the third abdominal segment in the control group (NC) was intact and relatively firm, with a tight connection between the skin and subcutaneous fat without separation. The adipocytes were regularly arranged, plump, with uniform nuclei and normal staining, presenting an overall healthy tissue state. The average width of the interstitial spaces was 5-10 μm. In contrast, the subcutaneous fat structure on the dorsal side of the third abdominal segment in the model group (MC) showed obvious tissue damage. The skin and subcutaneous fat were clearly separated, the subcutaneous fat tissue became loose and lost its original firmness, and some adipocytes exhibited typical nuclear pyknosis. This indicates that cadmium intake led to liver damage in silkworms.

[0063] IV. Model Judgment Results The results of comprehensive analysis of weight gain rate, blood and lymph biochemical indicators, and pathological characteristics of fat bodies showed that the average percentage of pyknoid cells in the model group was 42.3±5.6% (≥30%), and the average width of interstitial spaces was 28.5±4.2μm (≥20μm). Both indicators met the criteria, and the model group met all the criteria, indicating that the cadmium-induced liver injury model in silkworms was successfully established.

[0064] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Further modifications can be readily implemented by those skilled in the art.

Claims

1. A method for establishing a cadmium-induced liver injury model in silkworms, characterized in that, Includes the following steps: Select mulberry leaves and remove the petioles; Lay mulberry leaves with the back side facing up. Apply cadmium chloride solution evenly to the back of the mulberry leaves at a ratio of 10 g of mulberry leaves to 1 ml of cadmium chloride solution. Then, air dry the mulberry leaves until the surface moisture content is ≤5%, thus obtaining cadmium chloride-treated mulberry leaves. The concentration of the cadmium chloride solution used for application is 125 mg / L. Take another mulberry leaf and replace the cadmium chloride solution with an equal volume of water for simultaneous treatment, thus obtaining water-treated mulberry leaves. Silkworms that had completed their fourth molt and were on the first day of the fifth instar were selected and randomly divided into a model group and a control group, with 30 silkworms in each group; they were raised at 18±2℃ and 70% humidity. Before each feeding, the total weight of the silkworms in the model group and the control group was weighed. The silkworms in the model group were fed the corresponding amount of cadmium chloride-treated mulberry leaves every 12 hours, according to a feeding ratio of 10g of cadmium chloride-treated mulberry leaves for every 25g of silkworm weight. The silkworms in the control group were fed the corresponding amount of water-treated mulberry leaves every 12 hours, according to a feeding ratio of 10g of water-treated mulberry leaves for every 25g of silkworm weight. After 5 days of continuous feeding, all silkworms were fasted for 12 hours, and then the following measurements were performed: the weight of silkworms in the model group and the control group was measured and the weight growth rate was calculated; the activities of aspartate aminotransferase and alanine aminotransferase in the hemolymph of silkworms in the model group and the control group were measured; fat body sections of silkworms in the model group and the control group were prepared and observed under a microscope. The model silkworm's average weight gain rate was statistically significantly lower than that of the control group, the activities of aspartate aminotransferase and alanine aminotransferase in the hemolymph of the model silkworm were statistically significantly higher than those of the control group, and the model silkworm's fat body showed pathological characteristics of nuclear pyknosis and tissue loosening. Therefore, the cadmium-induced liver injury model of silkworm was considered to have been successfully established.

2. The method for establishing a cadmium-induced liver injury model in silkworms as described in claim 1, characterized in that, Cadmium chloride solution is prepared by water bath heating, specifically including: Take 12.5 mg of anhydrous cadmium chloride and 100 ml of pure water, place them in a container in a water bath and stir continuously until the solution becomes transparent to obtain a cadmium chloride solution. The water bath temperature is 50℃~60℃; the stirring rate is 300 r / min; and the stirring time is 15±2 minutes.

3. The method for establishing a cadmium-induced liver injury model in silkworms as described in claim 1, characterized in that, The weight of a silkworm is 1g to 1.1g.

4. The method for establishing a cadmium-induced liver injury model in silkworms as described in claim 1, characterized in that, The determination of liver function indicators such as aspartate aminotransferase and alanine aminotransferase activities in blood and lymphocytes includes: Silkworms were treated by abdominal foot puncture and their hemolymph was collected. The hemolymph from every 5 silkworms was mixed together to form a single sample, with a single collection volume of ≥200μL. The clarified supernatant was analyzed using a fully automated biochemical analyzer. The analysis employed pre-validated micro-detection conditions and calibration methods suitable for silkworm hemolymph matrix to determine the enzyme activity units of aspartate aminotransferase and alanine aminotransferase. The reaction temperature was 37°C, the reaction time was 10 minutes, and the calibration curve range was 5~500 U / L.

5. The method for establishing a cadmium-induced liver injury model in silkworms as described in claim 1, characterized in that, The pathological features of nuclear pyknosis and loose tissue in the fat body of silkworms are assessed through the following steps: Live silkworms that have been fasted are rapidly frozen on dry ice until stiff. Immediately afterward, they are dissected to obtain a fat body sample containing skin on the back of the third abdominal segment. The sample is then placed in 4% paraformaldehyde fixative and fixed at 4°C for 24 hours to obtain fixed tissue. The sample was dehydrated in an ethanol gradient, then cleared with xylene, and then impregnated with molten paraffin for at least 2 hours to form a wax block suitable for ultrathin sectioning. The wax block was cut into continuous thin layers of 5-8 micrometers. After being spread, attached and dried, the sections were dewaxed with xylene and rehydrated with ethanol gradient. They were then stained with conventional hematoxylin-eosin staining method, dehydrated and cleared, and then fixed with neutral resin to make permanent sections. Under an optical microscope, fat body cells in the subcutaneous adipose tissue of the third abdominal segment were observed. When the proportion of pyknotized cells was ≥30% and the average width of the interstitial space was ≥20μm, it was determined to be consistent with the pathological characteristics of liver-like injury. Freezing time ≤ 3 minutes; Gradient ethanol dehydration includes: 70% ethanol for 30 minutes, 80% ethanol for 30 minutes, 95% ethanol for 20 minutes, and anhydrous ethanol for 20 minutes; Xylene-based tissue clearing treatment includes treatment with xylene I for 10 minutes and treatment with xylene II for 10 minutes. Ethanol gradient rehydration includes: 2 minutes with anhydrous ethanol, 2 minutes with 95% ethanol, 2 minutes with 80% ethanol, 2 minutes with 70% ethanol, and 5 minutes with distilled water.

6. The method for establishing a cadmium-induced liver injury model in silkworms as described in claim 1, characterized in that, After selecting mulberry leaves and removing the petioles, the process also includes a pretreatment step for the mulberry leaves: Place the mulberry leaves with the petioles removed, back side up, in a single layer on a breathable stainless steel support mesh, and irradiate them under a UV lamp with a wavelength of 250-260 nm for 15-30 minutes at a distance of 30-50 cm. The mulberry leaves that have been disinfected by ultraviolet light are transferred to a clean air circulation device and circulated for 120±10 minutes at 15~25℃ and 40~60% relative humidity. After balancing, treat the mulberry leaves using one of the following methods: Method 1: Complete the subsequent solution application within 10 minutes; Method 2: Seal in a sterile container, store at 4°C, and use within 24 hours.

7. The method for establishing a cadmium-induced liver injury model in silkworms as described in claim 6, characterized in that, The process of applying cadmium chloride solution or water evenly to the back of mulberry leaves involves a step-by-step, intermittent application and directional drying method. Apply the pre-treated mulberry leaves for the first time, using 50% to 60% of the total volume of cadmium chloride solution or water. After the first application of the solution, let the mulberry leaves stand for 8 to 12 minutes under laminar flow conditions at a temperature of 20 to 25°C and an air velocity of 0.8 to 1.2 m / s. Then apply the remaining solution a second time, placing the mulberry leaves under the same laminar flow conditions until they are dried until the surface moisture content of the leaves is ≤5%.

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