Use of podophyllotoxin in inducing hypothyroid animal models and cell models and model establishment method

Abstract

The application provides an application of a podophyllotoxin in inducing a hypothyroidism animal model and a cell model and a model establishment method, and belongs to the field of basic medicine. A method for establishing a podophyllotoxin-induced hypothyroidism animal model comprises the following steps: (1) providing rats; (2) dissolving the podophyllotoxin in a solvent to prepare a podophyllotoxin preparation; (3) performing gavage administration of the podophyllotoxin preparation to the rats, and the administration dose is 10-20 mg / kg of body weight per day, and the administration is continuously performed for 3-5 days; and (4) obtaining a hypothyroidism rat model. The present application aims at the defects of a long modeling cycle, complex operation, single mechanism and the like of the existing method for establishing a hypothyroidism model, and the lack of a stable and repeatable hypothyroidism model, and establishes a podophyllotoxin-induced hypothyroidism animal model and a podophyllotoxin-induced hypothyroidism cell model.

Description

Technical Field

[0001] This invention relates to the field of basic medicine, and in particular to the application of podophyllotoxin in inducing animal and cell models of hypothyroidism and methods for establishing such models. Background Technology

[0002] Podophyllotoxin (PPT) belongs to the aryltetrahydronaphthalene lignan class and is mainly derived from plants in the genera *Polygonum*, *Diphylleia*, and *Pholiota*. Currently, it is primarily used clinically for its antiviral effects in treating diseases such as genital warts and herpes. It also possesses antibacterial, antiviral, and antioxidant pharmacological effects. Podophyllotoxin also inhibits cell division, and its derivatives VP-16 and VM-26 are representative antitumor drugs. However, the high toxicity of podophyllotoxin limits its clinical application. Existing studies have reported that podophyllotoxin can cause nephrotoxicity, hepatotoxicity, cardiotoxicity, neurotoxicity, and reproductive toxicity. However, whether the thyroid gland is a target organ for its toxicity is not currently reported in the literature.

[0003] Hypothyroidism is an endocrine disorder caused by insufficient synthesis, secretion, or biological effects of thyroid hormones. Stable and reliable animal and cell models of hypothyroidism are indispensable research tools in basic medical research and drug screening. Currently, the main methods for establishing hypothyroidism animal models in existing technologies include:

[0004] (1) Propylthiouracil (PTU) induction method: PTU is mixed into the feed at a ratio of 0.05%-0.15% and fed to animals, or a 0.05%-0.1% PTU aqueous solution is prepared and allowed to be drunk freely by the animals. The modeling period is usually 4-10 weeks. This method reduces the synthesis of thyroid hormones by inhibiting the activity of thyroid peroxidase (TPO), blocking the oxidation of iodine and the iodination of tyrosine.

[0005] (2) Methimazole (MMI) induction method: Administered by gavage at a dose of 5-10 mg / (kg·d), the modeling period is relatively long. MMI also reduces the synthesis of triiodothyronine (T3) and tetraiodothyronine (T4) by inhibiting TPO activity.

[0006] (3) Thyroidectomy: The thyroid tissue of rats is surgically removed to induce permanent hypothyroidism. This method is complex and invasive.

[0007] While the aforementioned methods can induce hypothyroidism to some extent, they still have the following limitations: PTU feeding or drinking water methods typically require 4-10 weeks to establish a stable hypothyroidism model, and MMI gavage also requires several weeks to months. The long modeling period increases experimental and time costs, and may also lead to animal death during the experiment; the modeling effect may also vary. Existing models all reduce thyroid hormone synthesis by inhibiting TPO activity, which is a single-target chemical intervention. This single intervention mode is difficult to simulate the complex pathogenesis of hypothyroidism in clinical practice, and cannot reflect the damage to the thyroid gland caused by drugs or other factors through non-TPO pathways. Summary of the Invention

[0008] The purpose of this invention is to provide an application and model establishment method of podophyllotoxin in inducing hypothyroidism in animal and cell models. The technical problem to be solved is that existing methods for establishing hypothyroidism models have shortcomings such as long modeling cycle, complex operation, and single mechanism of action, and lack stable and reproducible hypothyroidism models. The invention aims to establish an animal model and a cell model of hypothyroidism induced by podophyllotoxin.

[0009] To address the aforementioned technical problems, the application of podophyllotoxin in inducing hypothyroidism in animal and cell models, and the method for establishing these models, provided by this invention, are implemented as follows:

[0010] Application of podophyllotoxin in the preparation of animal models of hypothyroidism.

[0011] This invention reveals that podophyllotoxin is toxic to the thyroid gland, causing thyroid tissue damage and a decrease in thyroid hormone levels. This discovery not only expands the research field of podophyllotoxin but also provides a novel approach for establishing hypothyroidism models.

[0012] Optionally, the animal is a rodent.

[0013] Optionally, the method for establishing the hypothyroid animal model includes: administering podophyllotoxin into the animal to induce thyroid tissue damage.

[0014] Optionally, the administration method is by gavage; the dosage of the podophyllotoxin is 10-20 mg / kg body weight / day, and the administration cycle is 3-5 days.

[0015] A method for establishing an animal model of podophyllotoxin-induced hypothyroidism includes the following steps:

[0016] (1) Provide rats;

[0017] (2) Dissolve podophyllotoxin in a solvent to prepare a podophyllotoxin preparation;

[0018] (3) The podophyllotoxin preparation was administered to rats by gavage at a dose of 10-20 mg / kg body weight / day for 3-5 consecutive days;

[0019] (4) Obtain a rat model of hypothyroidism.

[0020] The animal model establishment method of this invention only requires daily gavage administration for 3-5 consecutive days to complete the modeling process. It is simple to operate, requires no special equipment, and is suitable for large-scale modeling. Compared to existing thyroidectomy surgery, this invention is simpler to operate; compared to the simple PTU and MMI induction methods, this invention has a shorter administration cycle.

[0021] Optionally, the rats are SPF-grade male SD rats aged 6-8 weeks with a weight of 190-230 g; the solvent in step (2) is a mixed solution of 2% DMSO and 0.5% sodium carboxymethyl cellulose.

[0022] Application of podophyllotoxin in the preparation of hypothyroid cell models.

[0023] This invention has found that podophyllotoxin directly damages thyroid follicular epithelial cells, leading to a decrease in the synthesis and secretion of thyroid hormones, and thus inducing hypothyroidism.

[0024] Optionally, the method for establishing the hypothyroid cell model includes: diluting podophyllotoxin to a working concentration with cell culture medium and adding it to thyroid follicular epithelial cells to induce cell damage.

[0025] A method for establishing a podophyllotoxin-induced hypothyroidism cell model includes the following steps:

[0026] (1) Provide rat thyroid follicular epithelial cells;

[0027] (2) Dilute podophyllotoxin to the working concentration using cell culture medium, wherein the working concentration of podophyllotoxin is 5-10 nM; add it to thyroid follicular epithelial cells and culture for 20-28 hours;

[0028] (3) Obtain a hypothyroid cell model.

[0029] The method for establishing the cell model of this invention is simple to operate. It only requires diluting podophyllotoxin to the working concentration with cell culture medium and then treating the cells. The cell model can be obtained by treating for only 20-28 hours, providing an efficient and convenient research tool for in vitro research.

[0030] Optionally, the rat thyroid follicular epithelial cells are rat thyroid epithelial cells PR2036. Attached Figure Description

[0031] Figure 1 This is a graph showing the change in rat body weight during the drug administration process in Example 1 of the present invention;

[0032] Figure 2 This is a graph showing the changes in rat body temperature during drug administration in Example 1 of the present invention;

[0033] Figure 3 This is a graph showing the changes in serum oxidative stress indicators in rats 4 days after administration of drug in Example 1 of the present invention; where A is MDA level and B is SOD activity;

[0034] Figure 4 This is a graph showing the changes in serum thyroid hormone levels in rats 4 days after administration of drug in Example 1 of the present invention; where A is TT3, B is TTT4, and C is TSH.

[0035] Figure 5 This is the pathological examination result of rat thyroid tissue 4 days after administration in Example 1 of the present invention. Note: Scale bar: 100 μm;

[0036] Figure 6 This is a diagram showing the CCK8 results of podophyllotoxin acting on rat thyroid epithelial cells in Example 2 of this invention;

[0037] Figure 7 This is a diagram showing the changes in the activity of rat thyroid epithelial cells after podophyllotoxin modeling in Example 2 of this invention.

[0038] Figure 8 This is Example 2 of the present invention, showing the changes in thyroid hormone levels in rat thyroid epithelial cells after podophyllotoxin modeling;

[0039] Figure 9 This is a diagram showing the apoptosis of rat thyroid epithelial cells after podophyllotoxin modeling in Example 2 of this invention. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the following embodiments provide a more detailed description of the invention. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0042] Example 1: Establishment of an animal model of hypothyroidism induced by podophyllotoxin

[0043] 1. Laboratory animals

[0044] SPF-grade male SD rats aged 6-8 weeks, weighing 190-230 g, were selected. The animals were housed in a 12-hour alternating light and dark environment with an ambient temperature of 21-25℃ and an ambient humidity of 30-40%. Food and water were not restricted, and the animals were given an acclimatization diet for 3 days.

[0045] 2. Animal grouping

[0046] Sixty SD rats were randomly divided into four groups (n=15 per group):

[0047] Blank control group (CON): Administered an equal volume of solvent via gavage.

[0048] PPT low-dose group (PPT-L): 10 mg / kg / day podophyllotoxin administered orally.

[0049] High-dose PPT group (PPT-H): 20 mg / kg / day podophyllotoxin administered orally.

[0050] 3. Preparation of modeling drugs

[0051] Accurately weigh podophyllotoxin powder, dissolve it in 2% DMSO, and make up to volume with 0.5% sodium carboxymethyl cellulose to prepare podophyllotoxin preparation.

[0052] 4. Model Building Methods

[0053] Healthy SPF-grade male SD rats aged 6-8 weeks were administered podophyllotoxin via gavage at a dose of 10-20 mg / kg body weight (10 mg / kg / day for the low-dose group and 20 mg / kg / day for the high-dose group) for 4 consecutive days to establish a rat model of hypothyroidism.

[0054] 5. Model Representation

[0055] Body weight and temperature will be monitored during administration.

[0056] (1) Weight changes

[0057] Compared with the blank control group, rats in all treatment groups (PPT-L and PPT-H) showed a significant decrease in body weight starting from day 3 of administration (P < 0.05), as shown in the results. Figure 1 As shown.

[0058] (2) Changes in body temperature

[0059] Compared with the blank control group, the body temperature of rats in all drug-treated groups was significantly reduced from day 2 of drug administration (P < 0.0001), as shown in the results. Figure 2 As shown.

[0060] (3) Serum biochemical index detection

[0061] Serum samples were collected 4 days after drug administration for biochemical analysis.

[0062] Serum superoxide dismutase (SOD) activity was significantly decreased (P < 0.0001), and malondialdehyde (MDA) level was significantly increased (P < 0.0001), as shown in the results. Figure 3 As shown.

[0063] (4) Serum thyroid hormone level detection

[0064] Serum total triiodothyronine (TT3) and total tetraiodothyronine (TT4) levels were significantly decreased (P < 0.0001), while thyroid-stimulating hormone (TSH) levels were significantly increased (P < 0.0001). The results are as follows: Figure 4 As shown.

[0065] (5) Thyroid tissue pathological examination

[0066] Four days after administration, thyroid tissue was taken for HE staining and pathological examination.

[0067] In the blank control group, the thyroid tissue of rats was divided into many lobules with indistinct boundaries by the membrane connective tissue extending into the gland. The lobules contained a large number of follicles and a small number of parafollicular cells. The interfollicles were loose connective tissue containing abundant capillaries. The follicles were surrounded by a single layer of low cuboidal follicular epithelium. The follicles were round, oval or irregular in shape, and varied greatly in size. No obvious abnormalities were observed.

[0068] In the PPT-treated group, a small amount of follicular epithelial shedding was observed in the follicles of the thyroid tissue (red arrows). The follicular lumen contained colloid, with numerous tiny vacuoles visible (black arrows). Reduced colloid content in the follicular lumen was less common (yellow arrows). Figure 5 As shown.

[0069] The above results indicate that podophyllotoxin treatment caused toxic damage to the thyroid tissue of rats, successfully inducing a rat model of hypothyroidism.

[0070] Example 2: Establishment of a podophyllotoxin-induced hypothyroidism cell model

[0071] 1. Cell selection

[0072] Rat thyroid epithelial cells (PR2036, Wuxi Xinrun Biotechnology Co., Ltd.).

[0073] 2. Cell Culture

[0074] Rat thyroid epithelial cells PR2036 were cultured in a 37°C, 5% CO2 incubator using a dedicated complete culture medium. Cells in the logarithmic growth phase and in good growth condition were used for experiments.

[0075] 3. Preparation of modeling drugs

[0076] Accurately weigh 7.7288 mg of podophyllotoxin powder into a centrifuge tube, add 1 mL of DMSO, and shake until the powder is completely dissolved to obtain a 20 mM podophyllotoxin stock solution. For cell experiments, simply dilute with cell culture medium to obtain the working solution.

[0077] 4. CCK-8 cell viability detection and modeling concentration screening

[0078] Rat thyroid follicular epithelial cells in the logarithmic growth phase and in good growth condition were harvested and processed at a concentration of 0.5 × 10⁻⁶. 4 Inoculate at a density of 100 μL per well in 96-well plates and incubate overnight at 37°C.

[0079] The next day, the old culture medium was discarded, and the cells were treated with seven different concentrations of PPT (0, 0.5, 1, 2.5, 5, 10, and 20 nM). The podophyllotoxin was directly diluted with the culture medium to the working concentration, and the cells were cultured for another 24 hours.

[0080] After the drug treatment was completed, 10 μL of CCK-8 solution was added to each well, and the mixture was incubated at 37°C in the dark for 1 hour. The absorbance (OD) at 450 nm was then measured using a microplate reader. 450 This was used for subsequent cell viability analysis to screen for appropriate drug concentrations in the modeling PPT. The results are as follows: Figure 6 As shown.

[0081] Based on the CCK8 results, the appropriate drug concentration for modeling is 5-20 nM, with 5-10 nM being the most suitable.

[0082] 5. Cell modeling, grouping, and treatment

[0083] Rat thyroid follicular epithelial cells were cultured separately and the cell concentration was adjusted to 5 × 10⁻⁶. 4 Cells / mL were seeded into 96-well culture plates, 100 μL per well, and cultured in a 37°C, 5% CO2 incubator.

[0084] After the cells adhered the following day, they were processed according to the following groups:

[0085] Control group: No treatment performed (blank control)

[0086] PPT-L group: PPT was added to a final concentration of 5 nM.

[0087] PPT-H group: PPT was added to a final concentration of 10 nM.

[0088] After adding the PPT, continue culturing for 24 hours.

[0089] 6. Cell viability assay

[0090] After the reaction was completed, 10 μL of CCK-8 solution was added to each well and incubated in a 37℃, 5% CO2 incubator for 1 hour in the dark. The OD value was measured at 450 nm using a microplate reader, and the measured OD value was used to analyze the effect on cell growth.

[0091] like Figure 7 The results showed that rat thyroid follicular epithelial cells had significantly reduced cell viability after treatment with 5-10 nM PPT for 24 hours (5 nM, P < 0.01; 10 nM, P < 0.0001).

[0092] 7. Detection of thyroid hormones and related indicators in cells

[0093] The levels of total triiodothyronine (TT3), total tetraiodothyronine (TT4), thyroid peroxidase (TPO), and total triglycerides (TG) in cells were measured.

[0094] The results are as follows Figure 8 The results showed that the levels of TT3 (5 nM, P < 0.05; 10 nM, P < 0.001) and TT4 (5 nM, P < 0.01; 10 nM, P < 0.01) and the content of TPO (P < 0.0001) in cells were significantly decreased, while the level of TG (5 nM, P < 0.0001; 10 nM, P < 0.0001) was significantly increased.

[0095] 8. Apoptosis detection

[0096] The proportion of apoptosis was detected by AV / PI flow cytometry.

[0097] Figure 9 The results showed that after 24 hours of exposure to 5 nM and 10 nM PPT, the proportion of apoptosis was significantly increased compared with the control group (P < 0.0001); and the proportion of apoptosis was also significantly increased in the 10 nM PPT-H exposure group compared with the 5 nM PPT-L exposure group (P < 0.0001).

[0098] The above results indicate that podophyllotoxin treatment caused toxic damage to rat thyroid follicular epithelial cells, successfully inducing a hypothyroid cell model.

[0099] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of podophyllotoxin in the preparation of animal models of hypothyroidism.

2. The application according to claim 1, characterized in that, The animal in question is a rodent.

3. The application according to claim 1 or 2, characterized in that, The method for establishing the hypothyroidism animal model includes: administering podophyllotoxin into the animal to induce thyroid tissue damage.

4. The application according to claim 3, characterized in that, The administration method is by gavage; the dosage of the podophyllotoxin is 10-20 mg / kg body weight / day, and the administration cycle is 3-5 days.

5. A method for establishing an animal model of hypothyroidism induced by podophyllotoxin, characterized in that, Includes the following steps: (1) Provide rats; (2) Dissolve podophyllotoxin in a solvent to prepare a podophyllotoxin preparation; (3) The podophyllotoxin preparation was administered to rats by gavage at a dose of 10-20 mg / kg body weight / day for 3-5 consecutive days; (4) Obtain a rat model of hypothyroidism.

6. The method according to claim 5, characterized in that, The rats were SPF-grade male SD rats aged 6-8 weeks, weighing 190-230 g; the solvent in step (2) was a mixed solution of 2% DMSO and 0.5% sodium carboxymethyl cellulose.

7. Application of podophyllotoxin in the preparation of hypothyroid cell models.

8. The application according to claim 7, characterized in that, The method for establishing the hypothyroid cell model includes: diluting podophyllotoxin to a working concentration with cell culture medium and adding it to thyroid follicular epithelial cells to induce cell damage.

9. A method for establishing a podophyllotoxin-induced hypothyroidism cell model, characterized in that, Includes the following steps: (1) Provide rat thyroid follicular epithelial cells; (2) Dilute podophyllotoxin to a working concentration of 5-10 nM with cell culture medium; add it to thyroid follicular epithelial cells and culture for 20-28 hours; (3) Obtain a hypothyroid cell model.

10. The method according to claim 9, characterized in that, The rat thyroid follicular epithelial cells were rat thyroid epithelial cells PR2036.

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