CN115606550A - A method for constructing an animal model of low ovarian reserve induced by autoimmune thyroiditis - Google Patents

Abstract

The invention relates to a method for constructing an animal model with low ovarian reserve function induced by autoimmune thyroiditis, which comprises the steps of taking an animal, injecting a primary immune emulsifier subcutaneously for 2 weeks continuously, 1-2 times per week, and injecting a reinforced immune emulsifier for 5-13 weeks from the 3 rd week and 1-2 times per week. The DOR model caused by EAT established by the invention has obvious thyroid pathological change and ovary reserve function reduction, and can be used for the research of autoimmune thyroiditis combined with low ovary reserve function diseases.

Description

An animal model of low ovarian reserve induced by autoimmune thyroiditis build method

technical field

The invention relates to the field of biotechnology, and more specifically relates to a method for constructing an animal model of low ovarian reserve function induced by autoimmune thyroiditis.

Background technique

Due to factors such as environment and life pressure, the incidence of autoimmune thyroiditis is increasing year by year. According to the results of studies and meta-analysis, women of reproductive age with autoimmune thyroiditis (AIT) have lower ovarian reserve compared with women of reproductive age without autoimmune thyroiditis (AIT). In addition to familial susceptibility to the onset of AIT, some studies have suggested that high iodine is the main inducing factor. Iodine is a strong oxidant that can cause oxidative stress in the thyroid gland.

There are animal models of autoimmune thyroiditis, but there is no animal model of autoimmune thyroiditis-induced low ovarian reserve.

Contents of the invention

In order to solve the above-mentioned technical problems, the present invention provides a method for constructing an animal model of autoimmune thyroiditis-induced low ovarian reserve function, which comprises the following steps:

Animals were taken, and the primary immunoemulsifier was injected subcutaneously for 2 consecutive weeks, 1-2 times a week, and the enhanced immunoemulsifier was injected from the third week for 5-13 weeks, 1-2 times a week;

The animal is fed with sodium iodide solution every day during the injection of the emulsifier;

The primary immune emulsifier is an emulsion with porcine thyroglobulin antigen concentration of 200-800 mg·L ^-1 prepared by mixing porcine thyroglobulin antigen solution and Freund's complete adjuvant;

The enhanced immune emulsifier is an emulsion with porcine thyroglobulin antigen concentration of 200-800 mg·L ^-1 prepared by mixing porcine thyroglobulin antigen solution and Freund's incomplete adjuvant.

Further, the injection time is every Monday and Thursday.

Furthermore, the injection time starts at 9 am every Monday and Thursday.

Further, the animal is a mammal, preferably a mouse.

Furthermore, the mice are Kunming mice, preferably Kunming mice aged 4-7 months.

Further, the injection site is neck, back, inner thigh and/or abdomen.

Further, the injection of boosted immunoemulsifier is for 5 weeks, 7 weeks, 9 weeks, 11 weeks or 13 weeks, preferably 11 weeks.

Further, the injection of fortified immune emulsifier is carried out for 13 weeks, and then the sodium iodide solution is fed until 17 to 19 weeks.

Furthermore, the concentration of the sodium iodide solution is 0.3-1 g·L ^-1 .

Further, the preparation method of the primary immune emulsifier is:

Freund's complete adjuvant and porcine thyroglobulin antigen solution were mixed at a volume ratio of 1:1, and mixed to form a viscous emulsifier at a rotational speed of 1000-5000 rpm, in which the concentration of porcine thyroglobulin antigen was 500 mg·L ^-1 .

Further, the preparation method of the boosted immune emulsifier is:

Freund's incomplete adjuvant and porcine thyroglobulin antigen solution are mixed at a volume ratio of 1:1, and mixed at a speed of 1000 to 5000 rpm to form a viscous emulsifier, in which the concentration of porcine thyroglobulin antigen is 500mg L ^{- 1} .

Furthermore, the porcine thyroglobulin antigen solution is a solution prepared by dissolving porcine thyroglobulin antigen in phosphate buffer solution with a concentration of 0.5-1 mg/ml.

The present invention also provides an animal model of low ovarian reserve function induced by autoimmune thyroiditis, which is a mouse model constructed according to the aforementioned method.

In the present invention, high iodine water feeding combined with antigen immunization twice a week induces 11-13 weeks to establish the DOR of low ovarian reserve function caused by autoimmune thyroiditis EAT with normal thyroid function, and the DOR of low ovarian reserve caused by EAT of hypothyroidism can be established for more than 15 weeks. DOR. High iodine water feeding combined with once-a-week antigen immunization induction can establish DOR caused by EAT with normal thyroid function for 13-15 weeks, and DOR caused by EAT with hypothyroidism can be established after 17 weeks. Antigen immunization induction twice a week combined with high iodine water feeding for 13 weeks established the EAT combined with DOR model with normal thyroid function and established a model relatively early, with obvious thyroid destruction and obvious decline in ovarian reserve. The EAT-induced DOR model established by the invention has obvious thyroid pathological changes and decreased ovarian reserve function, and can be used for the research of autoimmune thyroiditis combined with low ovarian reserve function.

Apparently, according to the above content of the present invention, according to common technical knowledge and conventional means in this field, without departing from the above basic technical idea of the present invention, other various forms of modification, replacement or change can also be made.

The above-mentioned content of the present invention will be further described in detail below through specific implementation in the form of examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Description of drawings

Figure 1 Changes in rectal temperature of mice in each group after modeling

Figure 2 Ovarian organ index in J group and BIW group

Figure 3 Thyroid pathological score in group J

Figure 4 thyroid HE staining in BIW group

Figure 5 HE staining of mouse thyroid in each group

Fig. 6 Ovarian follicles and corpus luteum of mice in group J

Figure 7 Ovarian follicles and corpus luteum of mice in BIW group

Figure 8 HE staining of mouse ovary in J group and BIW group

Figure 9 The levels of serum thyroid antibodies, thyroid function and sex hormones in Elisa mice in group J

Figure 10 BIW group mouse Elisa serum thyroid antibody, thyroid function and sex hormone levels

Figure 11 J group mice serum oxidative stress markers

Figure 12 BIW group mice serum oxidative stress markers

detailed description

Example 1 Constructing an animal model of low ovarian reserve induced by autoimmune thyroiditis

1) Preparation of solution

High iodine water: mix 0.64g of sodium iodide crystals with 1L of pure water to make high iodine water with a concentration of 0.64g·L ^-1 ;

Antigen solution: dissolve porcine thyroglobulin (pTg) antigen (1mg/ml) in phosphate buffered saline (PBS);

Primary immune emulsifier: inhale Freund's complete adjuvant (CFA) and antigen solution into a 50ml centrifuge tube at a volume ratio of 1:1, and place the centrifuge tube on a vortex shaker for high-speed vibration (about 2000 rpm) For 40 minutes, until a viscous emulsifier is formed, so that the final concentration reaches 500mg·L ^-1 , and it is prepared and used immediately;

Enhanced immunoemulsifier: prepare 500 mg·L ^-1 water-in-oil emulsion with incomplete Freund's adjuvant (IFA) and antigen solution at a volume ratio of 1:1, as in the preparation method of primary immunoemulsifier.

2) modeling

Initial immunization: inject a total of 0.2ml of the initial immunoemulsifier into the mouse subcutaneously at multiple points (neck, back, inner thigh, abdomen) for 2 consecutive weeks, twice a week (starting at 9 am every Monday and Thursday);

Booster immunization: From the third week, each mouse was subcutaneously injected with a total of 0.2ml of booster immune emulsifier, and the booster immunization was continued for 11 weeks, twice a week (every Monday and Thursday at 9:00 a.m.), during the modeling period. High iodine water feeding.

Example 2 Constructing an animal model of low ovarian reserve induced by autoimmune thyroiditis

1) Preparation of solution

With embodiment 1.

2) modeling

Initial immunization: inject a total of 0.2ml of the initial immunoemulsifier into the mouse subcutaneously at multiple points (neck, back, inner thigh, abdomen) for 2 consecutive weeks, twice a week (starting at 9 am every Monday and Thursday);

Booster immunization: From the third week, each mouse was subcutaneously injected with a total of 0.2ml of booster immune emulsifier, and the booster immunization was continued for 5 weeks, twice a week (starting at 9:00 am every Monday and Thursday). High iodine water feeding.

Example 3 Constructing an animal model of low ovarian reserve induced by autoimmune thyroiditis

1) Preparation of solution

Same as Example 1

2) modeling

Initial immunization: inject a total of 0.2ml of the initial immunoemulsifier into the mouse subcutaneously at multiple points (neck, back, inner thigh, abdomen) for 2 consecutive weeks, twice a week (starting at 9 am every Monday and Thursday);

Booster immunization: From the third week, each mouse was subcutaneously injected with a total of 0.2ml of booster immune emulsifier, and the booster immunization was continued for 13 weeks, twice a week (every Monday and Thursday at 9:00 a.m.), during the modeling period. High iodine water feeding.

Example 4 Constructing an animal model of low ovarian reserve induced by autoimmune thyroiditis

1) Preparation of solution

With embodiment 1.

2) modeling

Initial immunization: inject a total of 0.2ml of the initial immunoemulsifier into the mouse subcutaneously at multiple points (neck, back, inner thigh, abdomen), for 2 consecutive weeks, once a week (starting at 9 am every Monday or Thursday);

Booster immunization: From the third week, each mouse was subcutaneously injected with a total of 0.2ml booster immune emulsifier, and the booster immunization was continued for 11 weeks, once a week (starting at 9:00 am every Monday or Thursday), during the modeling period. High iodine water feeding.

Example 5 Constructing an Animal Model of Low Ovarian Reserve Function Induced by Autoimmune Thyroiditis

1) Preparation of solution

With embodiment 1.

2) modeling

Initial immunization: inject a total of 0.2ml of the initial immunoemulsifier into the mouse subcutaneously at multiple points (neck, back, inner thigh, abdomen, etc.), for 2 consecutive weeks, once a week (every Monday or Thursday at 9 am);

Booster immunization: From the third week, each mouse was subcutaneously injected with a total of 0.2ml of booster immune emulsifier, and the booster immunization was continued for 5 weeks, once a week (starting at 9 am every Monday or Thursday), during the modeling period. High iodine water feeding.

Example 6 Constructing an animal model of low ovarian reserve induced by autoimmune thyroiditis

1) Preparation of solution

With embodiment 1.

2) modeling

Initial immunization: inject a total of 0.2ml of the initial immunoemulsifier into the mouse subcutaneously at multiple points (neck, back, inner thigh, abdomen, etc.), for 2 consecutive weeks, once a week (every Monday or Thursday at 9 am); Booster immunization: From the third week, each mouse was subcutaneously injected with a total of 0.2ml booster immune emulsifier, and the booster immunization continued for 13 weeks, once a week (every Monday or Thursday at 9:00 a.m.), during the modeling period. High iodine water feeding.

Example 7 Constructing an animal model of low ovarian reserve induced by autoimmune thyroiditis

1) Preparation of solution

With embodiment 1.

2) modeling

Initial immunization: inject a total of 0.2ml of the initial immunoemulsifier into the mouse subcutaneously at multiple points (neck, back, inner thigh, abdomen) for 2 consecutive weeks, twice a week (starting at 9 am every Monday and Thursday);

Booster immunization: From the third week, each mouse was subcutaneously injected with a total of 0.2ml booster immune emulsifier, and the booster immune was continued for 13 weeks, twice a week (starting at 9:00 am every Monday and Thursday). High iodine water feeding was started on the day of the dose until the 17th or 19th week.

The beneficial effects of the present invention will be described below by means of test examples.

Test example 1

1 Experimental materials

1.1 Experimental animals

Kunming mice, female, 8-9 weeks old, 96, body weight 25-30g, provided by Chengdu Dashuo Experimental Animal Co., Ltd. supply. Raised in separate cages in the Gynecology Laboratory of Chengdu University of Traditional Chinese Medicine. The breeding environment is well ventilated, the temperature is maintained at 20-24°C, the relative humidity is 40%-79%, and the light is day and night.

1.2 Experimental drugs and reagents

Table 1 Main experimental drugs and reagents

2 Experimental methods

2.1 Reagent preparation

2.1.1 High iodine water

Mix 0.64g of sodium iodide crystals with 1L of pure water to prepare high iodine water with a concentration of 0.64g·L ^-1 ;

2.1.2 Antigen solution

Porcine thyroglobulin (pTg) antigen (1 mg/ml) was dissolved in phosphate buffered saline (PBS).

2.1.3 Emulsifier for primary immunization Suck Freund's complete adjuvant (CFA) and antigen solution into a 50ml centrifuge tube at a volume ratio of 1:1, and place the centrifuge tube on a vortex shaker at high speed (about 2000 rpm/ minutes) for 40 minutes, until a viscous emulsifier is formed, so that the final concentration reaches 500mg·L-1, and it is prepared and used immediately.

2.1.4 Enhanced immunoemulsifier Prepare incomplete Freund's adjuvant (IFA) and antigen solution at a volume ratio of 1:1, and prepare a 500 mg·L-1 water-in-oil emulsion in the same way as the initial immunoemulsifier preparation method.

2.2 Experimental grouping and modeling administration

2.2.1 Experimental grouping

After adaptive feeding for 1 week, 96 Kunming female mice were numbered according to their body weight and grouped by random number table method, and divided into J control group, BIW control group, J model group, and BIW model group. The J model group and BIW model group were further divided into 7-week group (J1 group, BIW1 group), 9-week group (J2 group, BIW2 group), 11-week group (J3 group, BIW3 group) and 13-week group according to the sampling time window. (J4 group, BIW4 group), 15-week group (J5 group, BIW5 group), 17-week group (J6 group, BIW6 group), 19-week group (J7 group, BIW7 group), each with 7 subgroups, a total of 16 groups , 6 in each group.

2.2.2 Modeling drug administration

2.2.2.1 Dosage and method of administration

(1) Model group

J model group (antigen immunization induction once/week): The prepared PTG antigen and adjuvant were mixed at a ratio of 1:1, fully ground to make emulsion antigen, and placed in a 4°C refrigerator for later use. Initial immunization: Inject 0.2ml of primary immune emulsifier into the mouse subcutaneously at multiple points (neck, back, inner thigh, abdomen, etc.) for 2 consecutive weeks, once a week (starting at 9 am every Monday); From 3 weeks onwards, each mouse was subcutaneously injected with 0.2 ml of booster immune emulsifier, and the booster immunization was continued for 5-13 weeks, once a week (starting at 9 am every Monday). Antigen immunization induction was stopped at 17 and 19 weeks. All rats were fed with high iodine water during the modeling period.

BIW model group (antigen immunization induction 2 times/week): The prepared PTG antigen and adjuvant were mixed at a ratio of 1:1, fully ground to make emulsion antigen, and placed in a 4°C refrigerator for later use. Initial immunization: Inject 0.2ml of primary immune emulsifier into the mouse subcutaneously at multiple points (neck, back, inner thigh, abdomen, etc.) for 2 consecutive weeks, 2 times a week (starting at 9:00 am every Monday and Thursday); Immunization: From the third week, each mouse was subcutaneously injected with 0.2ml of booster immunoemulsifier, and the booster immunization was continued for 5-13 weeks, twice a week (every Monday and Thursday at 9 am). Antigen immunization induction was stopped at 17 and 19 weeks. All rats were fed with high iodine water during the modeling period.

(2) Control group

J control group: subcutaneous injection of 0.2 ml of PBS buffer into mice (neck, back, inner thigh, abdomen, etc.), once a week (at 9 o'clock in the morning every Monday).

BIW control group: the mice were subcutaneously injected (neck, back, inner thigh, abdomen, etc.) with 0.2 ml of PBS buffer solution twice a week (at 9:00 a.m. every Monday and Thursday). During the modeling period, the general conditions of the mice in each group were observed, such as body state, activity, coat color, rectal temperature, body weight, and stool changes. Vaginal smears were collected every other day to observe the estrous cycle of the mice in each group.

2.2.2.2 Modeling and sampling in batches

Table 3 Modeling and material collection in batches

2.3 Specimen collection

2.3.1 Preparation before specimen collection All mice were observed by vaginal smears, and were killed in proestrus.

2.3.2 Collection of blood samples After the mice were anesthetized, cut off the beards on both sides of the mice, and collect blood through the orbital venous plexus blood collection method. The blood samples were collected in PCR tubes, and placed in a 3000rpm centrifuge after standing in a refrigerator at 4°C. After 10 minutes, use a pipette to carefully absorb the upper layer of mouse serum, separate the serum and plasma, and transfer them to marked cryopreservation tubes and place them in a -80°C ultra-low temperature refrigerator for storage.

2.3.3 Collection of tissue samples

The mice were killed by dislocation, and the mice were fixed on an ice board in the supine position, the neck of the mice was disinfected, the trachea was separated and the thyroid was stripped, the fascia and connective tissue were removed, and the thyroid tissue was fixed in 4% paraformaldehyde fixative solution , for HE staining of thyroid tissue. Cut along the midline of the lower abdomen, look for the "Y"-shaped uterus of the rat along the vaginal opening, find the left and right ovaries along the two corners of the uterus, carefully peel off the fat tissue around the uterus and ovaries, and weigh the wet weight of the uterus and ovaries Afterwards, the uterus and left ovary were put into EP tubes respectively, and quick-frozen in liquid nitrogen for testing. One ovary was fixed in 4% paraformaldehyde for HE staining.

2.4 Observation indicators

2.4.1 General Conditions

The survival, mental state, behavioral activity, food intake, hair cover (with or without piloerection, fluffy body hair, and hair loss) and excretion of stools of mice in each group were observed daily. Body weight and rectal temperature were recorded every Friday. During the experiment, the general state of the mice was observed and the changes were recorded. If any animal died, record the number and cause of death.

2.4.2 Estrous cycle

2.4.2.1 Estrous cycle observation

From 8:00 to 9:00 the next morning, the mice were smeared with vaginal exfoliated cells. The method was as follows: Wet a small cotton swab with a pointed tip specially used for tattooing with 0.9% NaCl solution, inserted it into the mouse vagina for about 0.5 cm, and rotated it clockwise for 3- At 4 weeks, take out the cotton swab, rotate and smear the vaginal exfoliation on the slide (do not overlap), place the slide under a COIC microscope (10*10 times) and observe the cell morphology under a light microscope to determine the estrous cycle of the mouse Change observation. The estrous cycle of sexually mature female mice is 4-5 days, which can be divided into preestrus, estrus, postestrus and interestrus according to the morphological changes of cytological smears. Cycles that are too long (≥6d), incomplete, too short, or in the same cycle (≥3d) for a long time are considered cycle disorders.

2.4.2.2 Characteristics of vaginal exfoliated cells

Table 4 Characteristics of vaginal exfoliated cells

2.4.3 Histological observation of thyroid and ovary

2.4.3.1 HE staining method for thyroid and ovary

(1) Embedding: The fixed tissue is dehydrated by a fully automatic dehydrator (dehydration time: 75% alcohol 4h, 85% alcohol 2h, 95% alcohol 1h, 100% alcohol 0.5h, 100% alcohol 0.5h, 100% alcohol 0.5h , 100% alcohol 0.5h, xylene 10min, xylene 10min, paraffin 1h, paraffin 2h, paraffin 3h), embedding;

(2) Slicing: Use a Leica RM2235 microtome to cut the tissue into thin slices with a thickness of 5 μm, flatten the tissue in warm water, remove it on a glass slide, and bake the slice at 60°C for at least 2 hours;

(3) Staining: hematoxylin staining for 10-20min; tap water for 1-3min; hydrochloric acid alcohol differentiation for 5-10s; tap water for 1-3min; put in warm water at 50°C or weak alkaline aqueous solution to turn blue until blue appears Rinse with tap water for 1-3min; put in 85% alcohol for 3-5min; stain with eosin for 3-5min; wash with water for 3-5s; dehydrate with gradient alcohol; transparent with xylene; mount with neutral gum;

(4) Microscopic examination: The images of the mouse ovarian tissue slices were collected using a Pannoramic 250 digital slice scanner produced by 3DHISTECH (Hungary). The thyroid tissue of the mouse was collected by the BA210Digital digital trinocular camera microscope camera system produced by Micro Audi Industrial Group Co., Ltd. to collect images of the slices. Each slice was first observed at 40 times the entire tissue to observe the general lesion, and the area to be observed was selected to collect 100 times and 400 times pictures to observe the specific lesion.

2.4.3.2 Thyroid pathology score Refer to Charveire classification for thyroid histopathology score

Table 5 Charveire taxonomy

2.4.3.3 Follicle count at all levels

Follicle counting at all levels, the number of primordial follicles, primary follicles, secondary follicles, mature follicles, atretic follicles, corpus luteum, etc.

2.4.4 Determination of serum thyroid antibodies, thyroid function, AMH and oxidative stress markers by enzyme-linked immunosorbent assay (ELISA)

2.4.4.1 Rewarming Equilibrate all reagents to room temperature.

2.4.4.2 Adding standard samples Set standard wells and sample wells, and add 50 μL of different concentrations of standard to each standard well.

2.4.4.3 For sample addition, set up blank wells (no samples and enzyme-labeled reagents are added to the blank control wells, and the rest of the steps are the same) and sample wells to be tested. Add 40 μL of sample diluent to the test sample well on the enzyme-labeled plate, and then add 10 μL of the test sample (the final dilution of the sample is 5 times).

2.4.4.4 Enzyme incubation

Add 100 μL of enzyme-labeled reagent to each well, except for blank wells, seal the plate with a sealing film and incubate at 37°C for 60 minutes.

2.4.4.5 Dosing Dilute the 20 times concentrated washing solution with distilled water 20 times before use.

2.4.4.6 Washing Carefully peel off the sealing film, discard the liquid, shake dry, fill each well with washing liquid, let it stand for 1 min, then discard, repeat this 5 times, and pat dry.

2.4.4.7 Color development Add 50 μL of substrate solution A to each well, then add 50 μL of substrate solution B, shake gently to mix, and develop color at 37°C in the dark for 15 minutes.

2.4.4.8 Termination Add 50 μl of stop solution to each well, and stop the reaction from blue to yellow.

2.4.4.9 Determination Measure the absorbance (OD value) of each well sequentially at a wavelength of 450 nm within 15 minutes.

2.5 Statistical analysis

ˉ

Measurement data: represented by mean and standard deviation (X±SD). If the data conforms to normal distribution and homogeneous variance, then use ANOVA single measurement data: use mean and standard deviation (LSD test in factor analysis of variance) to carry out inter-group Multiple comparisons; if the variances are not homogeneous, then use Games-Howelltest test to carry out multiple comparisons between groups. Enumeration data adopts chi-square test. All statistical tests all adopt two-sided test, when P＜0.01 has significant statistical significance, P＜0.05 is The difference was statistically significant, P>0.05 was not statistically significant.

3 Experimental results

During the modeling process, some mice died, and the causes of death may be as follows: induration and suppurative infection during the sustained drug release at the injection site, excessive subcutaneous bleeding caused by injection needle puncture and blood vessels, etc.

Table 6 The number of samples (n) of each group of mice before and after modeling

3.1 General conditions of mice in each group

The mice in the BIW control group and the J control group had normal activities, shiny hair, normal stools and regular estrous cycle. After multi-point subcutaneous injection of mice in the model group, induration appeared at the injection site, which did not subside after the modeling was completed; light and thin stools began to appear in the 4th week; about 60% of the mice began to appear from the 7th week of modeling Hair removal on the neck, lips, back, etc.; intestinal dysfunction, when the perianal area is stimulated during the vaginal smear, the defecation becomes slow and the stool is loose.

3.1.1 Changes in body weight and rectal temperature of mice in each group

Table 7. Statistical results of body weight of mice in group J

Note: Comparison between J model group and J control group: *P<0.05, **P<0.01; comparison between model groups: due to the large number of model groups, in order to avoid confusion caused by too many symbols, only "Δ" is used The serial numbers of the added groups represent the differences, namely: compared with group J1: Δ1P<0.05, ΔΔ1P<0.01; compared with group J2: Δ2P<0.05, ΔΔ2P<0.01; compared with group J3: Δ3P<0.05, ΔΔ3P<0.01; Compared with group J4: Δ4P<0.05, ΔΔ4P<0.01; compared with group J5: Δ5P<0.05, ΔΔ5P<0.01; compared with group J6: Δ6P<0.05, ΔΔ6P<0.01; compared with group J7: Δ7P<0.05, ΔΔ7P<0.01 .

The body weight and rectal temperature of mice in group J conformed to normal distribution and homogeneity of variance, and the LSD test in one-way ANOVA was used.

(1) Body weight: There was no statistically significant difference in the body weight of mice in group J before modeling (P>0.05). Compared with the control group, the weight of the mice in the model group after modeling was significantly reduced in the J1 and J2 groups, and the difference was statistically significant (P<0.05); the body weight of the J3-7 groups was significantly reduced, and the difference was statistically significant (P <0.01); J model group (J1-J7 group) had no significant difference in body weight (P>0.05).

(2) Rectal temperature: There was no statistically significant difference in the rectal temperature of mice in group J before modeling (P>0.05). After modeling, compared with the control group, the model group J1, J2, and J3 had a tendency to decrease, but the difference was not statistically significant (P>0.05); the rectal temperature of the J4 and J5 groups decreased significantly, and the difference There was a statistical difference (P<0.05); the rectal temperature between the J6 group and the J7 group was significantly lower, and the difference was statistically different (P<0.01).

Table 8 BIW group mouse body weight statistical results

Note: Compared with BIW model group and BIW control group, *P<0.05, **P<0.01; compared between model groups, compared with BIW1 group: ^Δ1 P<0.05; ^ΔΔ1 P<0.01; compared with BIW2 group: ^Δ2 P <0.05, ^ΔΔ2 P<0.01; compared with BIW3 group: ^Δ3 P<0.05; ^ΔΔ3 P<0.01;; compared with BIW4 group: ^Δ4 P<0.05, ^ΔΔ4 P<0.01; compared with BIW5 group: ^Δ5 P<0.05, ^ΔΔ5 P<0.01; compared with BIW6 group: ^Δ6 P<0.05, ^ΔΔ6 P<0.05; compared with BIW7 group: ^Δ7 P<0.05, ^ΔΔ7 P<0.01.

The body weight and rectal temperature of the mice in the BIW group conformed to the normal distribution and the variance was homogeneous, and the LSD test in one-way ANOVA was used.

(1) Body weight: before modeling, there was no significant difference in the body weight of mice in each group of BIW (P>0.05). Compared with the control group, the body weight of the mice in the model group after modeling was significantly reduced in the BIW1 group (P<0.05); the body weight of the BIW2-BIW7 group was significantly reduced, and the difference was statistically significant (P<0.01); There was no significant difference in body weight among the model group BIW1-BIW7 (P>0.05).

(2) Rectal temperature: before modeling, there was no significant difference in the rectal temperature of mice in each BIW group (P>0.05). After modeling, compared with the control group, the anal temperature of the mice in the model group had no statistical difference in the BIW1 group (P>0.05); the rectal temperature in the BIW2, BIW3, and BIW4 groups was significantly lower, and the difference was statistically significant (P< 0.05); BIW5 group, BIW6 group, BIW7 group rectal temperature decreased significantly, the difference was statistically significant (P<0.01). The rectal temperature of group J and BIW mice gradually decreased with the prolongation of modeling time, as shown in Figure 1.

3.1.2 Changes in the estrous cycle of mice in each group

Table 9 Changes in the estrous cycle of mice in the J group during the modeling process

Note: The estrous cycle of mice is 4-6d. If the cycle is too long (≥6d), incomplete, too short or in the same cycle for a long time (≥3d), it is considered as cycle disorder. Δproestrus; ◇represents estrus; ¤late estrus; ○interestrus.

The vaginal smears of group J showed that: the estrous cycle of the J control group had no obvious change; the estrous cycle of the J model group had no obvious disturbance in the 1-2 weeks of modeling; the estrous cycle of the model group was disordered in the 3-6 weeks of modeling The rate was 16.67%-33.33%; the rate of estrous cycle disorder in the 7-15 week model group was 33.33%-83.33%; the rate of cycle disorder in the group fed with high iodine water until 16-19 weeks was 80.00%-83.33%.

Table 10 estrous cycle changes in BIW group mice during modeling

Note: The estrous cycle of mice is 4-6d. If the cycle is too long (≥6d), incomplete, too short or in the same cycle for a long time (≥3d), it is considered as cycle disorder. △pre-estrus; ◇represents estrus; ¤late-estrus; ○interestrus.

Vaginal smears in the BIW group showed that: the estrous cycle of the BIW control group had no obvious change; the estrous cycle of the BIW model group had no obvious disturbance in the 1-2 weeks of modeling; the estrous cycle of the BIW model group had The disorder rate was 16.70%-50.00%; the estrous cycle disorder rate in the BIW model group was 50.00%-80.00% in the 7-15 weeks of modeling; in the 16-19 weeks of modeling with high iodine water feeding, the estrous cycle in the BIW model group The cycle disorder rate is 80.00%-83.33%.

3.1.3 Gonad index of mice in each group

The mouse ovary index was calculated according to the following formula: ovarian index=wet weight of bilateral ovaries (mg)/mouse body weight before sacrifice (g)×100%.

Table 11 The Ovarian Organ Index of the mice in each group

Note: Compared with the J control group, ^* P<0.05, ^** P<0.01; compared between the model group, compared with the J1 group: ^Δ1 P<0.05, ^ΔΔ1 P<0.01; compared with the J2 group: ^Δ2 P< 0.05, ^ΔΔ2 P<0.01; compared with J3 group: ^Δ3 P<0.05, ^ΔΔ3 P<0.01; compared with J4 group: ^Δ4 P<0.05, ^ΔΔ4 P<0.01; compared with J5 group: ^Δ5 P<0.05, ^ΔΔ5 P<0.01; compared with J6 group: ^Δ6 P ^<0.05 , ^ΔΔ6 P<0.01; compared with J7 group: ^Δ7 P<0.05, ^ΔΔ7 P<0.01. Comparing BIW model group with BIW control group, ^* P<0.05, ^** P<0.01; comparing model group with BIW1 group: ^Δ1 P<0.05, ^ΔΔ1 P<0.01; comparing with BIW2 group: ^Δ2 P<0.05 , ^ΔΔ2 P<0.01; compared with BIW3 group: ^Δ3 P<0.05, ^ΔΔ3 P<0.01; compared with BIW4 group: ^Δ4 P<0.05, ^ΔΔ4 P<0.01; compared with ^BIW5 group: ^{Δ5 P<0.05, ΔΔ5 P} <0.01; compared with BIW6 group: ^Δ6 P<0.05, ^ΔΔ6 P<0.01; compared with BIW7 group: ^Δ7 P<0.05, ^ΔΔ7 P<0.01.

The index of ovarian viscera of mice in group J conformed to normal distribution and homogeneity of variance, and the LSD test in one-way ANOVA was used.

(1) Ovarian organ index of mice in group J: Compared with the control group, the ovarian index of group J1 decreased, but the difference was not statistically significant (P>0.05); group J2 and group J3 decreased significantly (P<0.05 ). Compared with group J4, group J5, group J6 and group J7, it was significantly lower (P<0.01). Comparison among J model groups: J1 group, J2 group, J3 group, J4 group had no statistically significant difference (P>0.05); J5 group, J6 group, J7 group were compared with J1 group, J2 group, J3 group, Significantly decreased in J4 group (P<0.01).

(2) Inter-group comparison of ovarian organ index in the BIW group: Compared with the control group, the ovarian index in the BIW1 group decreased, but the difference was not statistically significant (P>0.05); the BIW2 and BIW3 groups were significantly lower ( P<0.05); BIW4 group, BIW5 group, BIW6 group, BIW7 group significantly decreased (P<0.01). Comparison between BIW model groups: BIW1 group, BIW2 group, BIW3 group, and BIW4 group had no statistically significant difference (P>0.05); BIW5 group was significantly lower than BIW4 group (P<0.05), , BIW3 group were significantly lower (P<0.01); BIW6 group, BIW7 group were significantly lower than BIW1 group, BIW2 group, BIW3 group, BIW4 group (P<0.01). The ovarian viscera index of the J group and BIW mice showed a gradual decline trend with the prolongation of modeling time, as shown in Figure 2.

3.2 Histological observation of thyroid gland and ovary of mice in each group

3.2.1 HE staining of mouse thyroid in each group

3.2.1.1 Thyroid HE staining in group J

Table 12 Statistical results of thyroid Charveire score in group J

Note: Comparing the model group (J1-7 group) with the J control group, ^* P<0.05, ^** P<0.01; the model group comparison, compared with the J1 group: ^Δ1 P<0.05, ^ΔΔ1 P<0.01; Group comparison: ^Δ2 P<0.05, ^ΔΔ2 P<0.01; comparison with J3 group: ^Δ3 P<0.05, ^ΔΔ3 P<0.01; comparison with J4 group: ^Δ4 P<0.05, ^ΔΔ4 P<0.01; comparison with J5 group: ^Δ5 P <0.05, ^ΔΔ5 P<0.01; compared with J6 group: ^Δ6 P<0.05, ^ΔΔ6 P<0.01; compared with J7 group: ^Δ7 P<0.05, ^ΔΔ7 P<0.01.

The infiltration intensity of thyroid lymphocytes, the change of thyroid follicle structure, and the variance of the total pathological score of mice in group J were tested by Games-Howell test, and the statistical results are as follows:

(1) Thyroid lymphocyte infiltration intensity: Compared with the J control group, no obvious pathological changes were seen in the J1 and J2 groups. Although the thyroid lymphocyte infiltration intensity in the J3 group increased (①③⑥ mice), the difference was not statistically significant There were significant differences (P>0.05); J4 group, J5 group, J6 group, J7 group were significantly higher than that (P<0.01). Comparison between J model groups: J4 and J5 groups were significantly higher than J1 and J2 groups (P<0.05); J6 and J7 groups were significantly higher than J1 and J2 groups (P<0.01) and J3 group increased (P<0.05); the rest of the differences between the groups were not statistically significant.

(2) Changes in the structure of thyroid follicles: Compared with the J control group, the infiltration intensity of thyroid follicle cells in the J3 group increased (①③⑥ mice) and the modeling rate was 60%, but the difference was not statistically significant (P>0.05 ); J4 group, J5 group, J6 group, J7 group significantly increased (P<0.01). Comparison between J model groups: J4, J5, J6 and J7 groups were significantly higher than J1, J2 and J3 groups (P<0.01), and the differences among other groups were not statistically significant.

(3) Changes in the total pathological score of the thyroid gland: Compared with the J control group, the infiltration intensity of thyroid lymphocytes in the J3 group increased, but the difference was not statistically significant (P>0.05); J4, J5, J6, J7 group was significantly higher than that (P<0.01). Comparison between J model groups: J4, J5, J6 and J7 groups were significantly higher than J1, J2 and J3 groups (P<0.01), and the differences among other groups were not statistically significant. The thyroid pathological score in group J gradually increased with the prolongation of modeling time, as shown in Figure 3.

3.2.1.2 HE staining of thyroid in BIW group

Table 13 Statistical results of thyroid Charveire classification score in BIW group

Note: Comparing the model group (BIW1-7 group) with the BIW control group, ^* P<0.05, ^** P<0.01; the comparison between the model group and the BIW1 group: ^Δ1 P<0.05, ^ΔΔ1 P<0.01; compared with BIW2 Group comparison: ^Δ2 P<0.05, ^ΔΔ2 P<0.01; comparison with BIW3 group: ^Δ3 P<0.05, ^ΔΔ3 P<0.01; comparison with BIW4 group: ^Δ4 P<0.05, ^ΔΔ4 P<0.01; comparison with BIW5 group: ^Δ5 P <0.05, ^ΔΔ5 P<0.01; compared with BIW6 group: ^Δ6 P<0.05, ^ΔΔ6 P<0.01; compared with BIW7 group: ^Δ7 P<0.05, ^ΔΔ7 P<0.01.

The infiltration intensity of thyroid lymphocytes, structural changes of thyroid follicles, and variance of total pathological scores in mice in BIW group were tested by Games-Howell test. The statistical results are as follows:

(1) Thyroid lymphocyte infiltration intensity: Compared with the BIW control group, the thyroid lymphocyte infiltration intensity in the BIW1 group did not change, and the thyroid lymphocyte infiltration intensity in the BIW2 group increased (lymphocytes and middle granulocytes), but the difference was not statistically significant (P>0.05); BIW3 group, BIW4 group, BIW5 group significantly increased (P<0.05); BIW6 group, BIW7 group significantly increased (P<0.01). Comparison between BIW model groups: BIW3 group, BIW4 group, BIW5 group were significantly higher than BIW1 group (P<0.01), and the differences among other groups were not statistically significant.

(2) Changes in thyroid follicle structure: Compared with the BIW control group, the thyroid follicle structure of mice ⑥ in BIW1 group was slightly changed, and the thyroid follicle structure of mice ④, ⑤, and ⑥ in BIW2 group was changed. The molding rate was 50%; the scores of BIW1 group and BIW2 group increased, but the difference was not statistically significant (P>0.05); BIW3 group was significantly higher than it (P<0.05), BIW4 group, The BIW7 group was significantly higher than that (P<0.01). Comparison between BIW model groups: BIW6 group and BIW7 group were significantly higher than BIW1 group and BIW2 group; the differences among other groups were not statistically significant (P>0.05).

(3) Changes in the total score of thyroid pathology: Compared with the BIW control group, the scores of the BIW1 group and BIW2 group increased, but the difference was not statistically significant (P>0.05); BIW3 group, BIW4 group, BIW5 group, BIW6 group , BIW7 group significantly increased (P<0.01). Comparison between BIW groups: BIW3 group, BIW4 group, BIW5 group were significantly higher than BIW1 group (P<0.05), BIW6 group, BIW7 group were significantly higher than BIW1 group, BIW2 group (P<0.01), and there were no differences between other groups Statistically significant (P>0.05). The thyroid pathology score in the BIW model group gradually increased with the prolongation of modeling time, as shown in Figure 4.

3.2.1.3 Histomorphological changes of thyroid HE staining in mice in each group

Under the light microscope, the thyroid cells of the mice in the control group were arranged neatly, the follicles were elliptical, the epithelial cells were mostly cuboidal, the staining was light red, the cytoplasm was abundant, the nuclei were round and uniform in size, and the follicular cavity was rich in colloid. No lymphocyte infiltration, follicle destruction. The thyroid follicles with uneven size in the modeled mouse thyroid, the follicular epithelium was low cuboid, the thickness of the colloid was different, and the destruction of the thyroid follicles and the infiltration of lymphocytes could be seen. HE staining of thyroid in each group is shown in Figure 5

3.2.2 HE staining of mouse ovaries in each group

3.2.2.1 HE staining of mouse ovary in group J

Table 14 The number of ovarian follicles and corpus luteum of mice in group J

Note: Comparing J model group with J control group, ^* P<0.05, ^** P<0.01; comparing model group with J1 group: ^Δ1 P<0.05, ^ΔΔ1 P<0.01; comparing with J2 group: ^Δ2 P <0.05, ^ΔΔ2 P<0.01; compared with J3 group: ^Δ3 P<0.05, ^ΔΔ3 P<0.01; compared with J4 group: ^Δ4 P<0.05, ^ΔΔ4 P<0.01; compared with J5 group: ^Δ5 P<0.05, ^ΔΔ5 P <0.01; compared with J6 group: ^Δ6 P<0.05, ^ΔΔ6 P<0.01; compared with J7 group: ^Δ7 P<0.05, ^ΔΔ7 P<0.01.

Except for primary follicles, the counts of follicles and corpus luteum of mice in each group conformed to the normal distribution, and the variance of each group was homogeneous, so the LSD test in one-way variance was used, and the primary follicles were tested by Games-Howell test.

(1) Primordial follicles: Compared with the J control group, the primordial follicles in the J1, J2, and J3 groups slightly decreased, but the difference was not statistically significant (P>0.05); the J4, J5 groups significantly decreased (P<0.05); the number of primordial follicles in J6 group and J7 group was significantly reduced (P<0.01). Comparison among J model groups: Primordial follicles in J7 group were significantly less than those in J1 and J2 groups (P<0.05), and there was no statistical difference among the other groups (P>0.05).

(2) Primary follicles: Compared with the J control group, the primary follicles in the J1 group, J2 group, and J3 group had a slight tendency to decrease, but the difference was not statistically significant (P>0.05). Primary follicles in J7 group decreased significantly (P<0.05). Comparison between J model groups: There was no statistical difference among the groups (P>0.05).

(3) Secondary follicles: Compared with the J control group, the secondary follicles in the J1, J2, and J3 groups had a slight tendency to decrease, but the difference was not statistically significant (P>0.05). Secondary follicles in the group and J7 group decreased significantly (P<0.05). Comparison between J model groups: There was no statistical difference among the groups (P>0.05).

(4) Mature follicles: comparison among groups: no significant difference (P>0.05).

(5) Atretic follicles: Compared with the J control group, the J1 group, J2 group, and J3 group had an increasing trend, and the difference was not statistically significant (P>0.05); the J4 group significantly increased compared with it (P<0.05) ), J5 group, J6 group, J7 group significantly increased (P<0.01); model group comparison: J5 group, J6 group significantly increased compared with J1 group (P<0.05), J5 group, J6 group compared with J2 group Significantly increased (P<0.01), the J7 group was significantly higher than the J2 group (P<0.05), and there was no statistically significant difference among the other groups (P>0.05).

(6) Corpus luteum: Compared with the J control group, the J1 group and J2 group had a decreasing trend, and the difference was not statistically significant (P>0.05); <0.05); J6 group and J7 group were significantly reduced (P<0.01). There was no statistically significant difference between J model groups (P>0.05). The counts of follicles and corpus luteum at all levels in Group J are shown in Figure 6.

3.2.2.2 HE staining of mouse ovary in BIW group

Table 15 The number of ovarian follicles and corpus luteum of mice in BIW group

Note: Comparing BIW model group with BIW control group, ^* P<0.05, ^** P<0.01; comparison between model groups, compared with BIW1 group: ^Δ1 P<0.05, ^ΔΔ1 P<0.01; compared with BIW2 group: ^Δ2 P <0.05, ^ΔΔ2 P<0.01; compared with BIW3 group: ^Δ3 P<0.05, ^ΔΔ3 P<0.01; compared with ^BIW4 group: ^Δ4 P<0.05, ^ΔΔ4 P<0.01; compared with BIW5 group: ^Δ5 P<0.05, ^ΔΔ5 P<0.01; compared with BIW6 group: ^Δ6 P<0.05, ^ΔΔ6 P<0.01; compared with BIW7 group: ^Δ7 P<0.05, ^ΔΔ7 P<0.01.

The follicles and corpus luteum of all levels in the BIW group conformed to the normal distribution, and the variances were homogeneous, and the LSD test in the one-way variance was used.

(1) Primordial follicles: Compared with the BIW control group, the primordial follicles in the BIW1 and BIW2 groups had a slight tendency to decrease, but the difference was not statistically significant (P>0.05); the BIW3 group was significantly less than that (P<0.05); Primordial follicles in BIW4 group, BIW5 group, BIW6 group and BIW7 group decreased significantly (P<0.01). There was no statistically significant difference between the BIW model group (BIW1-7 group) (P>0.05).

(2) Primary follicles: Compared with the BIW control group, the BIW1 group, BIW2 group, and BIW3 group had a slight decrease trend, but the difference was not statistically significant (P>0.05); the BIW4 group and BIW5 group were significantly decreased (P<0.05); BIW6 group and BIW7 group were significantly reduced (P<0.01). There was no statistically significant difference between the BIW model group (BIW1-7 group) (P>0.05).

(3) Secondary follicles: Compared with the BIW control group, the BIW1 group, BIW2 group, BIW3 group, and BIW4 group had a tendency to decrease, but the difference was not statistically significant (P>0.05); BIW5 group, BIW6 group , BIW7 group significantly decreased (P<0.05). . There was no statistically significant difference between BIW1-7 groups (P>0.05).

(4) Mature follicles: There was no statistically significant difference among the groups (P>0.05).

(5) Atretic follicles: Compared with the BIW control group, the BIW1 group and BIW2 group had an increasing trend, and the difference was not statistically significant (P>0.05); the BIW3 group, BIW4 group, and BIW5 group were significantly more P<0.05); BIW6 group and BIW7 group increased significantly (P<0.01); there was no statistically significant difference between model groups (P>0.05).

(6) Corpus luteum: Compared with the BIW control group, the BIW1 group had a decreasing trend, and the difference was not statistically significant (P>0.05); the BIW2 and BIW3 groups significantly decreased (P<0.05); group, BIW6 group, and BIW7 group were significantly reduced (P<0.01). Compared with the BIW1 group, the BIW5, BIW6, and BIW7 groups were significantly less (P<0.05), and there was no significant difference among the other groups (P>0.05). The counts of follicles and corpus luteum at all levels in the ovaries of mice in the BIW group are shown in Figure 7. The HE staining of mouse ovaries in group J and BIW is shown in Figure 8.

3.3 Determination of serum thyroid antibody, thyroid function and reproductive hormone levels by enzyme-linked immunosorbent assay (ELISA)

3.3.1 ELisa results of mouse serum in group J

Table 16 J group mouse serum ELisa results

Note: Comparing the model group (J1-7 group) with the J control group, ^* P<0.05, ^** P<0.01; the model group comparison, compared with the J1 group: ^Δ1 P<0.05, ^ΔΔ1 P<0.01; Group comparison: ^Δ2 P<0.05, ^ΔΔ2 P<0.01; comparison with J3 group: ^Δ3 P<0.05, ^ΔΔ3 P<0.01; comparison with J4 group: ^Δ4 P<0.05, ^ΔΔ4 P<0.01; comparison with J5 group: ^Δ5 P <0.05, ^ΔΔ5 P<0.01; compared with J6 group: ^Δ6 P<0.05, ^ΔΔ6 P<0.01; compared with J7 group: ^Δ7 P<0.05, ^ΔΔ7 P<0.01.

The results of serum ELisa in group J conformed to normal distribution and homogeneity of variance, so the LSD test in one-way ANOVA was used.

(1) TGAb: Compared with the J control group, the concentration of TGAb in groups J1, J2, and J3 increased, but the difference was not statistically significant (P>0.05); the concentration of group J4, J6 increased significantly (P <0.05); the concentration of J5 group and J7 group increased significantly (P<0.01). . Comparison between J model groups: the concentration of J5 group and J7 group was significantly higher than that of J2 group (P<0.01), the concentration of J4 group was significantly higher than that of J2 group (P<0.05), and the difference between the other groups was not statistically significant ( P>0.05).

(2) TPOAb: Compared with the J control group, the concentration of TPOAb in the J1, J2, and J3 groups increased, but the difference was not statistically significant (P>0.05); J4, J5, J6, and J7 groups Concentration was significantly increased (P<0.01); compared among J model groups, the concentration of J4, J5, J6 and J7 groups was significantly higher than that of J1 and J2 groups (P<0.01); It was significantly higher in group J3 (P<0.05), and higher in group J5 than in group J3 (P<0.01); the difference among the other groups was not statistically significant (P>0.05).

(3) FT3: Compared with the J control group, the FT3 levels in the J1, J2, J3, J4, and J5 groups decreased, the difference was not significant, and there was no statistical significance (P>0.05); J6, J7 The level of FT3 in the group decreased significantly (P<0.05). There was no statistically significant difference between the J model group (P>0.05).

(4) FT4: Compared with the J control group, the FT3 levels in the J1, J2, J3, J4, and J5 groups decreased, the difference was not significant, and there was no statistical significance (P>0.05); J6, J7 The level of FT4 in the group decreased significantly (P<0.05). Compared with the J model group, the J6 and J7 groups were significantly lower than the J1 group (P<0.05), and the differences between the other groups were not statistically significant (P>0.05). .

(5) TSH: Compared with the control group, the TSH levels in the J1, J2, J3, J4, and J5 groups were elevated, and the difference was not significant, not statistically significant (P>0.05); TSH level in J7 group was significantly increased (P<0.05). Comparison between J model groups: J6 group was significantly higher than J1 group (P<0.05), J7 group was significantly higher than J1 group and J2 group (P<0.01), and the difference between the other groups was not statistically significant (P> 0.05).

(6) AMH: Compared with the control group, the AMH levels of J1, J2, and J3 groups decreased, and the difference was not statistically significant (P>0.05); the AMH levels of J4, J5, J6, and J7 groups significantly decreased (P<0.01). Compared with J model group, J5 group and J7 group were significantly lower than J1 (P<0.05), and the differences among other groups were not statistically significant (P>0.05). The thyroid antibody and TSH of mice in group J gradually increased with the prolongation of modeling time, while FT3, FT4, and AMH gradually decreased with the prolongation of modeling time, as shown in Figure 9.

3.3.2 BIW group mouse serum ELisa results

Table 17 BIW group mouse serum ELisa results

Note: Comparing BIW model group with BIW control group, ^* P<0.05, ^** P<0.01; comparison between model groups, compared with BIW1 group: ^Δ1 P<0.05, ^ΔΔ1 P<0.01; compared with BIW2 group: ^Δ2 P <0.05, ^ΔΔ2 P<0.01; compared with BIW3 group: ^Δ3 P<0.05, ^ΔΔ3 P<0.01; compared with ^BIW4 group: ^Δ4 P<0.05, ^ΔΔ4 P<0.01; compared with BIW5 group: ^Δ5 P<0.05, ^ΔΔ5 P<0.01; compared with BIW6 group: ^Δ6 P<0.05, ^ΔΔ6 P<0.01; compared with BIW7 group: ^Δ7 P<0.05, ^ΔΔ7 P<0.01.

The results of serum ELisa in the BIW group conformed to the normal distribution and homogeneity of variance, and the LSD test in one-way ANOVA was used.

(1) TGAb: Compared with the BIW control group, the concentration of TGAb in the BIW1 group and the BIW2 group increased, but the difference was not statistically significant (P>0.05); the concentration of the Biw3 group and the Biw4 group increased significantly (P<0.05) ; The concentration of BIW5 group, Biw6 group and Biw7 group increased significantly (P<0.01). Comparison between BIW model groups: the concentration of BIW5 group was significantly higher than that of BIW1 group and BIW2 group (P<0.05), the concentration of Biw6 group and Biw7 group was significantly higher than that of BIW1 group and BIW2 group (P<0.01), and the differences among other groups Not statistically significant (P>0.05).

(2) TPOAb: Compared with BIW control group, the concentration of BIW1 group and BIW2 group increased but the difference was not statistically significant (P>0.05); TPOAb in Biw3 group, Biw4 group, Biw5 group, Biw6 group, Biw7 group The concentration was significantly increased (P<0.01). Compared among the BIW model groups, the concentration of TPOAb in the Biw4, Biw5, Biw6, and Biw7 groups was significantly higher than that in the BIW1 and BIW2 groups (P<0.01), and the concentration in the BIW3 group was significantly higher than that in the BIW1 group (P<0.01). In the BIW2 group (P<0.05), the differences among other groups were not statistically significant (P>0.05).

(3) FT3: Compared with the control group, the FT3 levels in the BIW1, BIW2, BIW3, BIW4, and BIW5 groups decreased, and the difference was not statistically significant (P>0.05); the FT3 levels in the BIW6 group decreased significantly ( P<0.01); FT3 level in BIW7 group decreased significantly (P<0.05). Comparison between BIW model groups: BIW6 group was significantly lower than BIW1 group and BIW2 group (P<0.01), and the difference among other groups was not statistically significant (P>0.05).

(4) FT4: Compared with the control group, the FT4 levels in the BIW1, BIW2, BIW3, BIW4, and BIW5 groups decreased, and the difference was not statistically significant (P>0.05); the FT4 levels in the BIW6 group were significantly decreased (P<0.01); FT4 levels in BIW 7 group decreased significantly (P<0.05). Comparison between BIW model groups: There was no statistically significant difference between groups (P>0.05).

(5) TSH: Compared with the control group, the TSH levels of the BIW1, BIW2, BIW3, and BIW4 groups increased, and the difference was not statistically significant (P>0.05); the TSH level of the BIW5 group was significantly increased (P <0.05); TSH levels in BIW6 and BIW7 groups increased significantly (P<0.01). Comparison between BIW model groups: BIW7 group was significantly higher than BIW1 group (P<0.01), significantly higher than BIW2 group (P<0.05), BIW6 group was significantly higher than BIW1 group (P<0.05), and the other groups The difference was not statistically significant (P>0.05).

(6) AMH: Compared with the control group, the AMH levels of the BIW1 and BIW2 groups decreased, and the difference was not statistically significant (P>0.05); the AMH levels of the BIW3 group decreased significantly (P<0.05); AMH levels in the group, Biw6 group, and Biw7 group decreased significantly (P<0.01). Comparison between BIW model groups: BIW4 group, Biw5 group, Biw6 group, Biw7 group decreased significantly compared with BIW1 group and BIW2 group (P<0.01), and the difference among other groups was not statistically significant (P>0.05). The thyroid antibody and TSH of mice in the BIW group gradually increased with the prolongation of the modeling time, while FT3, FT4, and AMH gradually decreased with the prolongation of the modeling time, as shown in Figure 10.

3.4 Determination of serum oxidative stress markers by enzyme-linked immunosorbent assay (ELISA)

3.4.1 Concentrations of GSH-PX, MDA, ROS, and SOD in serum of mice in group J

The change of GSH-PX, MDA, ROS, SOD concentration in the mouse serum of table 18J group

Note: Compared with J model group and J control group, ^* P<0.05, ^** P<0.01; compared between model groups and J1 group: ^Δ1 P<0.05, ^ΔΔ1 P<0.01; compared with J2 group: ^Δ2 P<0.05 , ^ΔΔ2 P<0.01; compared with J3 group: ^Δ3 P<0.05, ^ΔΔ3 P<0.01; compared with J4 group: ^Δ4 P<0.05, ^ΔΔ4 P<0.01; compared with J5 group: ^Δ5 P<0.05, ^ΔΔ5 P<0.01 ; Compared with J6 group: ^Δ6 P<0.05, ^ΔΔ6 P<0.01 ^; Compared with J7 group: ^Δ7 P<0.05, ^ΔΔ7 P<0.01.

The results of serum ELisa in group J were in line with normal distribution and homogeneity of variance, so the LSD test in one-way ANOVA was used.

(1) GSH-PX: Compared with the J control group, the concentration of GSH-PX in the J1 and J2 groups decreased, but the difference was not statistically significant (P>0.05); the concentration in the J3 group decreased significantly (P<0.05); ; J4 group, J5 group, J6 group, J7 group concentration decreased significantly (P<0.01). Comparison between J model groups: J5 group was significantly lower than J2 group (P<0.05); J6 group was significantly lower than J1 group (P<0.05); J6 group and J7 group were significantly lower than J2 group (P<0.01); There was no statistically significant difference between groups (P>0.05).

(2) MDA: Compared with the J control group, the MDA concentrations in the J1 and J2 groups increased, but the difference was not significant, not statistically significant (P>0.05); the MDA concentrations in the J4 and J5 groups were significantly increased ( P<0.05); The concentration of MDA in J6 group and J7 group was significantly increased (P<0.01). There was no statistically significant difference between model groups in group J (P>0.05).

(3) ROS: Compared with the J control group, the ROS concentration in the J1 group, J2 group, and J3 group increased, but the difference was not significant, and there was no statistical significance (P>0.05); the ROS concentration in the J4 group increased significantly ( P<0.05); ROS concentrations in J5, J6 and J7 groups increased significantly (P<0.01). Comparison between model groups in J group: J4, J5, J6 and J7 groups were significantly higher than J1 group (P<0.01); J4 and J5 groups were significantly higher than J2 group ROS concentration (P<0.05); Compared with J2 and J3 groups, the concentrations of J6 and J7 groups were significantly higher (P<0.01), and the differences among the other groups were not statistically significant (P>0.05).

(4) SOD: Compared with J control group, the concentration of J1 group, J2 group and J3 group decreased but the difference was not statistically significant (P>0.05); the concentration of J4 group decreased significantly (P<0.05); , J6 group, J7 group the concentration decreased significantly (P<0.01). Compared with the J model group, the J5 group was significantly lower than the J1 and J2 groups (P<0.05); the J6 group was significantly lower than the J1 group (P<0.01), and the J2 group was significantly lower (P<0.05); the J7 group was significantly lower than the J1 group (P<0.05); The J1 group significantly decreased (P<0.05); the difference among the other groups was not statistically significant (P>0.05). The oxidative stress markers ROS and MDA in group J gradually increased with the prolongation of the modeling time, and the antioxidant enzyme activities SOD and GSH-PX gradually decreased with the prolongation of the modeling time, as shown in Figure 11.

3.4.2 Concentrations of GSH-PX, MDA, ROS and SOD in serum of mice in BIW group

The change of GSH-PX, MDA, ROS, SOD concentration in the mouse serum of table 19BIW group

Note: Comparing BIW model group with BIW control group, ^* P<0.05, ^** P<0.01; comparison between model groups, compared with BIW1 group: ^Δ1 P<0.05, ^ΔΔ1 P<0.01; compared with BIW2 group: ^Δ2 P <0.05, ^ΔΔ2 P<0.01; compared with BIW3 group: ^Δ3 P<0.05, ^ΔΔ3 P<0.01; compared with ^BIW4 group: ^Δ4 P<0.05, ^ΔΔ4 P<0.01; compared with BIW5 group: ^Δ5 P<0.05, ^ΔΔ5 P<0.01; compared with BIW6 group: ^Δ6 P<0.05, ^ΔΔ6 P<0.01; compared with BIW7 group: ^Δ7 P<0.05, ^ΔΔ7 P<0.01.

The serum oxidative stress markers of mice in the BIW group were normally distributed and had homogeneity of variance, so the LSD test in one-way ANOVA was used.

(1) GSH-PX: Compared with BIW control group, GSH-PX in BIW1 group and BIW2 group decreased, but the difference was not statistically significant (P>0.05); GSH-PX in Biw3 group and Biw4 group decreased significantly (P <0.05), GSH-PX in Biw5 group, Biw6 group and Biw7 group decreased significantly (P<0.01). Comparison between BIW model groups: the concentration of Biw5 group and Biw7 group was significantly lower than that of BIW2 group (P<0.05); the concentration of Biw6 group was significantly lower than that of BIW1 group (P<0.01); the difference between the other groups was not statistically significant (P> 0.05).

(2) MDA: Compared with BIW control group, MDA in BIW1 group and BIW2 group increased but the difference was not statistically significant (P>0.05); MDA in Biw5 group, Biw6 group and Biw7 group increased significantly (P<0.01). . Comparison between BIW model groups: the concentrations of BIW4, Biw5, Biw6, and Biw7 groups were significantly higher than those of BIW1 (P<0.05); the concentrations of Biw6 and Biw7 were significantly higher than those of BIW2 (P<0.01); It was higher than that in BIW2 group (P<0.05); the difference among other groups was not statistically significant (P>0.05).

(3) ROS: Compared with the BIW control group, the concentration of BIW1 group and BIW2 group increased, but the difference was not significant, and there was no statistical significance; the ROS of Biw3 group and BIW4 group increased significantly (P<0.05); , Biw5 ROS in the group, Biw6 group and Biw7 group increased significantly (P<0.01). Comparison between BIW model groups: the concentration of Biw3 group, BIW4 group, Biw5 group, Biw6 group and Biw7 group was significantly higher than that of BIW1 group (P<0.01); the concentration of BIW6 group and BIW7 group was significantly higher than that of BIW2 group (P<0.05); The concentration of BIW6 group was significantly higher than that of BIW3 group (P<0.05); the difference among other groups was not statistically significant (P>0.05).

(4) SOD: Compared with the BIW control group, the SOD concentration of BIW1 group, BIW2 group and Biw3 group decreased, but the difference was not statistically significant (P>0.05); the SOD concentration of Biw4 group and Biw5 group decreased significantly (P< 0.05); Biw6 group, Biw7 group SOD concentration decreased significantly (P<0.01). Comparison between BIW model groups: the concentration of Biw6 group and Biw7 group was significantly lower than that of BIW1 group (P<0.05); the concentration of Biw7 group was significantly lower than that of BIW2 group (P<0.05); the difference between the other groups was not statistically significant (P> 0.05). The oxidative stress markers ROS and MDA gradually increased with the prolongation of modeling time, and the antioxidant enzyme activities SOD and GSH-PX gradually decreased with the prolongation of modeling time, as shown in Figure 12.

4 Summary of model group J group and BIW group modeling situation

Table 20J group and BIW group model summary

Note: Oxidative stress (oxidative stress: OS); ↑: means significantly/significantly increased compared with the control group (P<0.05/P<0.01); ↓: means significantly/significantly decreased compared with the control group (P<0.01) 0.05/P<0.01).

4.1 Experimental autoimmune thyroiditis (EAT) mouse model situation

(1) J model group: The thyroid antibodies (TGAb, TPOAb), thyroid function, follicle counts at all levels, and corpus luteum of the mice in the J1 group and J2 group at 7-9 weeks after modeling were not significantly different from those of the J control group. It was statistically significant; in the 11th week of modeling, 3 mice in the J3 group had thyroid lymphocyte infiltration and follicular structure changes, and the thyroid antibodies (TGAb, TPOAb) in the J3 group increased, that is, 60% of the mice The mice became the EAT model. At this time, the number of corpus luteum was significantly reduced compared with the J control group, and the difference in follicle counts at all levels was not statistically significant. At the 13th week of modeling, the mice in the J4 group all showed thyroid lymphocyte infiltration, follicular structure changes and Thyroid antibodies (TGAb, TPOAb) were significantly increased, that is, the modeling rate of the EAT model was 100%. At this time, the number of primordial follicles, primary follicles, and secondary follicles in the mice was significantly/significantly reduced compared with the J control group, and the atresic follicles were significantly/significantly increased. ; This trend was maintained in the 15-19 weeks of subsequent modeling, indicating that the J model group began to have low ovarian reserve function in the J4 group at the 13th week of modeling; EAT mice developed hypothyroidism, which is the stage of hypothyroid EAT combined with decreased ovarian reserve.

(2) BIW model group: the BIW model group was established in the 7-week-old BIW1 group of mice, only the ⑥ mouse had a slight change in the structure of the thyroid follicles, thyroid lymphocyte infiltration, thyroid antibodies (TGAb, TPOAb), There were no statistically significant differences in thyroid function, follicle counts at all levels, corpus luteum and J control group; 3/6 mice in the BIW2 group had thyroid lymphocyte infiltration and follicular structure changes at the 9th week of modeling, and the thyroid glands in the BIW2 group Antibodies (TGAb, TPOAb) increased, that is, 50% of the mice became EAT models. At this time, the number of corpus luteum was significantly reduced compared with that of the BIW control group, but the difference in follicle counts at all levels was not statistically significant; at the 11th week of modeling The mice in the BIW3 group all had thyroid lymphocyte infiltration, follicular structure changes, and thyroid antibodies (TGAb, TPOAb) significantly increased, and the modeling rate of the EAT model was 100%. At this time, the primary follicles, primary follicles, and secondary follicles of the mice Compared with the BIW control group, the number of atretic follicles was reduced, and the number of atretic follicles increased; this trend was maintained in the 13-19 weeks of subsequent modeling, indicating that the BIW model group began to have low ovarian reserve function at the 11th week of modeling; Compared with the control group, the TSH level of the EAT mice in the BIW5 group was significantly higher than that of the control group, while the FT3 and FT4 levels were lower, but the difference was not statistically significant, indicating that subclinical hypothyroidism appeared at this stage; At 19 weeks, the EAT mice in BIW6 group and BIW7 group showed a significant increase in TSH levels, and a significant decrease in FT3 and FT4 levels, indicating that hypothyroidism combined with decreased ovarian reserve function occurred at this stage.

(3) Comparison between the J model group and the BIW model group: the time when the EAT mice in the BIW model group began to show low ovarian reserve function was 11 weeks after modeling, and the time when the EAT mice in the J model group started to show low ovarian reserve function was the time after model establishment. 13 weeks. In the J model group, the Kunming mice with normal thyroid function and EAT-induced ovarian reserve decreased in 13-15 weeks, and in the BIW model group, the Kunming mice with normal thyroid function and EAT-induced decreased ovarian reserve were established in 15 weeks. 11-13 weeks. During the 17th to 19th week of feeding with high iodine water, the J6 group, J7 group, BIW6 group, and BIW7 group were all DOR models caused by hypothyroidism EAT. Compared with the control group, the number of primordial follicles, primary follicles, secondary follicles and corpus luteum in the BIW model group of 11-19 weeks of modeling and the J model group of 13-19 weeks of modeling were reduced, while atretic follicles were increased. The reason why there is no difference in mature follicles among the groups is that the time of sampling is preestrus (lasting 9-18 hours), and the follicles in this period are in the growth and development stage. On day 3.70±0.34[2], the corpus luteum of the mouse in the previous cycle of proestrus has not completely subsided, so the corpus luteum can still be seen.

4.2 Model selection of decreased ovarian reserve in EAT mice with normal thyroid function

Table 21 Overview of ovarian reserve function induced by EAT model group with normal thyroid function

Note: EAT model group J4, J5 with normal thyroid function compared with J control group: ^* P<0.05, ^** P<0.01; BIW3 group, BIW4 group with normal thyroid function compared with BIW control group Ratio: ^# P<0.05, ^## P<0.01; comparison between EAT model groups with normal thyroid function: compared with J4 group, ^Δ4 P<0.05, ^ΔΔ4 P<0.01; compared with J5 group, ^Δ5 P<0.05 , ^ΔΔ5 P<0.01; compared with BIW3 group, ^▲3 P<0.05, ▲▲ ³ P<0.01; compared with BIW4, ^▲4 P<0.05, ^▲▲4 P<0.01.

(1) The infiltration intensity of thyroid lymphocytes, structural changes of thyroid follicles, thyroid antibody levels, primordial follicles, atretic follicles, and AMH in EAT model groups with normal thyroid function (BIW3, J4, BIW4, and J5) were compared. The variances of each group were homogeneous, and the one-way variance LSD test was used. The results showed that: thyroid lymphocyte infiltration intensity, thyroid follicle structure change, thyroid antibody level, primitive follicle , atretic follicles, AMH differences were not statistically significant.

(2) Although the difference between the EAT model group with normal thyroid function was not statistically significant, compared with the control group in the EAT model group with normal thyroid function, the primordial follicles and AMH in the BIW4 group after 13 weeks of modeling were significantly decreased (P <0.05), the change of thyroid follicle structure, and the level of TPOAb antibody were significantly increased (P<0.05), so this group has the characteristics of relatively early modeling, thyroid destruction, and obvious decline in ovarian reserve function, which can be used for subsequent experiments. Modeling reference for the study.

4.3 Explanation about the control group (taken at 7 weeks) that can be used as a control

Studies have suggested that middle-aged rats in the natural state exhibit irregular estrous cycles from the age of 9-12 months, usually characterized by prolongation of the estrous cycle, and the natural decline of ovarian function begins. During the modeling period of this study, the mice were about 4-7 months old (15-28 weeks old) and did not reach the stage of natural decline. In order to be more rigorous, another three groups of Kunming female mice with different ages and without any treatment were observed to observe serum AMH levels, namely the 16-week-old group (W16 group, corresponding to the 7-week modeling group), and the 20-week-old group ( W20 group, corresponding to the 11-week modeling group), 24-week-old (W24 group, corresponding to the 15-week modeling group). The serum AMH results of the three groups of mice were as follows:

Table 22 The change of AMH concentration in serum of 3 groups of mice (X ± SD)

Note: Compared with W ₁₆ group, ^* P<0.05, ^** P<0.01; compared with W ₂₀ group, ^# P<0.05, ^## P<0.01; compared with W ₂₄ group, ^Δ P<0.05 , ^ΔΔP <0.01

The statistical results showed that there was no statistically significant difference in serum AMH concentration among the groups (P>0.05), so the 16-week-old J control group and BIW control group at the time of modeling and sampling in the experiment could be compared with the model groups.

4.5 Summary

(1) J model group and BIW model group: With the prolongation of modeling time in J model group and BIW model group, the scores of thyroid lymphocyte infiltration intensity, thyroid follicle structure change score, thyroid antibody (TGAb, TPOAb) level, TSH , atretic follicles all have a gradual upward trend, while AMH, FT3, FT4, primordial follicles, primary follicles, secondary follicles, and corpus luteum all have a gradual downward trend. The scores of thyroid lymphocyte infiltration intensity, thyroid follicle structure change score, thyroid antibody (TGAb, TPOAb) levels were obvious, and oxidative stress markers (ROS, MDA) levels and atretic follicles were significantly higher than those in the control group, and the levels of primordial follicles, primary follicles, secondary follicles, corpus luteum, and antioxidant markers (SOD, GSH-PX) were significantly lower than those in the control group. The BIW model group (BIW3-BIW7 group) from 11 weeks to 19 weeks after modeling and the J model group (J4-J7 group) from 13 to 19 weeks after modeling developed autoimmune thyroiditis combined with low ovarian reserve. Hypothyroidism occurred in groups J6, J7, BIW6, and BIW7 in the 17-19 weeks of continuous feeding with high iodine water.

(2) The time of low ovarian reserve in experimental autoimmune thyroiditis (EAT) mice: BIW model group was earlier than J model group, that is, the time for antigen immunization induced twice a week was better than once a week Antigen immune induction;

(3) Thyroid lymphocyte infiltration intensity, changes in thyroid follicle structure, thyroid antibody levels, primitive follicles, and atresia in EAT mice with normal thyroid function combined with low ovarian reserve in BIW3, BIW4, J4, and J5 groups The difference in follicle and AMH was not statistically significant.

5 Conclusion

Experimental autoimmune thyroiditis (EAT) negatively affects ovarian reserve and can be induced by EAT in a DOR model. High iodine water (0.64g·L ^-1 ) feeding combined with antigen immunization induction (500mg·L ^-1 ) twice a week can establish DOR caused by EAT with normal thyroid function for 11-13 weeks, and hypothyroidism can be established for more than 15 weeks EAT leads to DOR. High iodine water (0.64g·L ^-1 ) feeding combined with once-weekly antigen immunization induction (500mg·L ^-1 ) can establish DOR caused by EAT with normal thyroid function for 13-15 weeks, and hypothyroidism can be established after 17 weeks EAT leads to DOR. Antigen immunization induction twice a week combined with high iodine water feeding for 13 weeks established the EAT combined with DOR model with normal thyroid function and established a model relatively early, with obvious thyroid destruction and obvious decline in ovarian reserve.

Claims (10)

1. a method for building an animal model of low ovarian reserve function induced by autoimmune thyroiditis, characterized in that: it comprises the steps of: Animals were taken, and the primary immunoemulsifier was injected subcutaneously for 2 consecutive weeks, 1-2 times a week, and the enhanced immunoemulsifier was injected from the third week for 5-13 weeks, 1-2 times a week; The animal is fed with sodium iodide solution every day during the injection of the emulsifier; The primary immune emulsifier is an emulsion with porcine thyroglobulin antigen concentration of 200-800 mg·L ^-1 prepared by mixing porcine thyroglobulin antigen solution and Freund's complete adjuvant; The enhanced immune emulsifier is an emulsion with porcine thyroglobulin antigen concentration of 200-800 mg·L ^-1 prepared by mixing porcine thyroglobulin antigen solution and Freund's incomplete adjuvant. 2. The construction method according to claim 1, characterized in that: the time of injection is every Monday and Thursday, and preferably the time of injection starts at 9 am every Monday and Thursday. 3. The construction method according to claim 1, characterized in that: the animal is a mammal, preferably a mouse; the mouse is a Kunming mouse, preferably a 4-7 month old Kunming mouse. 4. The construction method according to claim 1, characterized in that: the injection site is neck, back, inner thigh and/or abdomen. 5. The construction method according to claim 1, characterized in that: the injection of the enhanced immunoemulsifier is for 5 weeks, 7 weeks, 9 weeks, 11 weeks or 13 weeks, preferably 11 weeks. 6. The construction method according to claim 1, characterized in that: the injection of the enhanced immunoemulsifier for 13 weeks, followed by feeding with sodium iodide solution for 17 to 19 weeks. 7. The construction method according to claim 1 or 6, characterized in that: the concentration of the sodium iodide solution is 0.3-1 g·L ^-1 . 8. The construction method according to claim 1, characterized in that: the preparation method of the primary immunoemulsifier is: Freund's complete adjuvant and porcine thyroglobulin antigen solution were mixed at a volume ratio of 1:1, and mixed to form a viscous emulsifier at a rotational speed of 1000-5000 rpm, in which the concentration of porcine thyroglobulin antigen was 500 mg·L ^-1 . 9. The construction method according to claim 1, characterized in that: the preparation method of the enhanced immunoemulsifier is: Freund's incomplete adjuvant and porcine thyroglobulin antigen solution are mixed at a volume ratio of 1:1, and mixed at a speed of 1000 to 5000 rpm to form a viscous emulsifier, in which the concentration of porcine thyroglobulin antigen is 500mg L ^{- 1} ; The porcine thyroglobulin antigen solution is a solution prepared by dissolving porcine thyroglobulin antigen in phosphate buffer solution with a concentration of 0.5-1 mg/ml. 10. An animal model of low ovarian reserve induced by autoimmune thyroiditis, characterized in that it is a mouse model constructed according to the aforementioned method.

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